The glyoxalase system mediates the conversion of methylglyoxal, a toxic ketoaldehyde, to D-lactic acid. The system is composed of two enzymes, glyoxalase I (Glo-I) and glyoxalase II (Glo-II), and exhibits an absolute requirement for a catalytic quantity of glutathione (GSH). Glo-I catalyzes the isomerization of a hemithioacetal, formed non-enzymatically from methylglyoxal and GSH, to the corresponding a -D-hydroxyacid thioester, s-D-lactoylglutathione (SLG). Glo-II catalyzes the irreversible breakdown of SLG to D-lactate and GSH. We have observed that ATP or GTP significantly inhibits the Glo-II activity of tissue homogenates from various sources. We have developed a rapid, one step chromatography procedure to purify Glo-II such that the purified enzyme remains "sensitive" to inhibition by ATP or GTP (Glo-II-s). Studies indicate that inhibition of Glo-II-s by nucleotides is restricted to ATP, GTP, ADP, and GDP, with ATP appearing most effective. Kinetics studies have shown that ATP acts as a partial non-competitive inhibitor of Glo-II-s activity, and further suggest that two kinetically distinguishable forms of the enzyme exist. The sensitivity of pure Glo-II-s to nucleotide inhibition is slowly lost on storage even at -80° C. This loss is accelerated at higher temperatures or in the presence of ATP. Kinetics studies on the resultant "insensitive" enzyme (Glo-II-i) show that a significant reduction of the affinity of the enzyme for the substrate, SLG, occurs and further suggest that only one form of the enzyme is kinetically distinguishable after "de-sensitization". Tryptophan fluorescence studies of the two enzyme preparations suggest that a subtle conformational change in the enzyme has occurred during de-sensitization. We have also observed that Glo-II-i is "resensitized" to nucleotide inhibition after incubation in the presence of a reagent that reduces disulfide bonds. The resensitized enzyme exhibits an increased KM value similar to that of the original Glo-II-s. Kinetics studies show that ATP or GTP again ...
Resonance energy transfer was employed to study the conformational changes of actomyosin during ATP hydrolysis. To calibrate the technique, the parameters for resonance energy transfer were defined. With conformational searching algorithms to predict probe orientation, the distances measured by resonance energy transfer are highly consistent with the atomic models, which verified the accuracy and feasibility of resonance energy transfer for structural studies of proteins and oligonucleotides. To study intramyosin distances, resonance energy transfer probes were attached to skeletal myosin's nucleotide site, subfragment-2, and regulatory light chain to examine nucleotide analog-induced structural transitions. The distances between the three positions were measured in the presence of different nucleotide analogs. No distance change was considered to be statistically significant. The measured distance between the regulatory light chain and nucleotide site was consistent with either the atomic model of skeletal myosin subfragment-1 or an average of the three models claimed for different ATP hydrolysis states, which suggested that the neck region was flexible in solution. To examine the participation of actin in the powerstroke process, resonance energy transfer between different sites on actin and myosin was measured in the presence of nucleotide analogs. The efficiencies of energy transfer between myosin catalytic domain and actin were consistent with the actoS1 docking model. However, the neck region was much closer to the actin filament than predicted by static atomic models. The efficiency of energy transfer between Cys 374 and the regulatory light chain was much greater in the presence of ADP-AlF4, ADP-BeFx, and ADP-vanadate than in the presence of ADP or no nucleotide. These data detect profound differences in the conformations of the weakly and strongly attached crossbridges which appear to result from a conformational selection that occurs during the weak binding of the myosin head to actin. The resonance energy transfer data exclude a number ...
Recently, plastidial carbonic anhydrase (CA, EC 18.104.22.168) cDNA clones encoding functional CA enzymes were isolated from a nonphotosynthetic cotton tissue. The role of CA in photosynthetic tissues have been well characterized, however there is almost no information for the role of CA in nonphotosynthetic tissues. A survey of relative CA transcript abundance and enzyme activity in different cotton organs revealed that there was substantial CA expression in cotyledons of seedlings and embryos, both nonphotosynthetic tissues. To gain insight into the role(s) of CA, I examined CA expression in cotyledons of seedlings during post-germinative growth at different environmental conditions. CA expression in cotyledons of seedlings increased from 18 h to 72 h after germination in the dark. Seedlings exposed to light had about a 2-fold increase in CA activities when compared with seedlings kept in the dark, whereas relative CA transcript levels were essentially the same. Manipulation of external CO2 environments [zero, ambient (350 ppm), or high (1000 ppm)] modulated coordinately the relative transcript abundance of CA (and rbcS) in cotyledons, but did not affect enzyme activities. On the other hand, regardless of the external CO2 conditions seedlings exposed to light exhibited increase CA activity, concomitant with Rubisco activity and increased chlorophyll content. Our data revealed that steady-state levels of CA and rbcS transcripts are regulated at the transcriptional level in response to external CO2 conditions, while CA and Rubisco activities are modulated at the post-transcriptional level by light. Thus CA expression in cotyledons during post-germinative growth may be to “prime” cotyledons for the transition at the subcellular level for the transition from plastids to chloroplasts, where it provides CO2 for Rubisco during photosynthesis. Furthermore, CA expression increased during embryo maturation similar to oil accumulation. Specific sulfonamide inhibitors of CA activity significantly reduced the rate of [14C]-acetate incorporation into total lipids ...
The relatively high level of palmitic acid (22 mol%) in cottonseeds may be due in part to the activity of a palmitoyl-acyl carrier protein (ACP) thioesterase (PATE). In embryo extracts, PATE activity was highest at the maximum rate of reserve accumulation (oil and protein). The cotton FatB mRNA transcript abundance also peaked during this developmental stage, paralleling the profiles of PATE enzyme activity and seed oil accumulation. A cotton FatB cDNA clone was isolated by screening a cDNA library with a heterologous Arabidopsis FatB probe (Pirtle et al., 1999, Plant and Cell Physiology 40: 155-163). The predicted amino acid sequence of the cotton PATE preprotein had 63% identity to the Arabidopsis FatB thioesterase sequence, suggesting that the cotton cDNA clone probably encoded a FatB-type thioesterase. When acyl-CoA synthetase-minus E. coli mutants expressed the cotton cDNA, an increase in 16:0 free fatty acid content was measured in the culture medium. In addition, acyl-ACP thioesterase activity assays in E. coli lysates revealed that there was a preference for palmitoyl-ACP over oleoyl-ACP in vitro, indicating that the cotton putative FatB cDNA encoded a functional thioesterase with a preference for saturated acyl-ACPs over unsaturated acyl-ACPs (FatA). Overexpression of the FatB cDNA in transgenic cotton resulted in elevated levels of palmitic acid in transgenic somatic embryos compared to control embryos. Expression of the anti-sense FatB cDNA in transgenic cotton plants produced some plants with a dwarf phenotype. These plants had significantly smaller mature leaves, all with smaller cells, suggesting that these plants may have less palmitic acid available for incorporation into extraplastidial membrane lipids during cell expansion. Thus manipulation of FatB expression in cotton directly influenced palmitic acid levels. Collectively, data presented in this dissertation support the hypothesis that there indeed is a palmitoyl-ACP thioesterase in cotton, encoded by the isolated FatB cDNA, which plays ...
Transgenic tobacco plants were developed containing a partial PLD clone in antisense orientation. The PLD isoform targeted by the insertion was identified. A PLD clone was isolated from a cDNA library using the partial PLD as a probe: Nt10B1 shares 92% identity with PLDβ1 from tomato but lacks the C2 domain. PCR analysis confirmed insertion of the antisense fragment into the plants: three introns distinguished the endogenous gene from the transgene. PLD activity was assayed in leaf homogenates in PLDβ/g conditions. When phosphatidylcholine was utilized as a substrate, no significant difference in transphosphatidylation activity was observed. However, there was a reduction in NAPE hydrolysis in extracts of two transgenic plants. In one of these, a reduction in elicitor- induced PAL expression was also observed.
A novel luminescence resonance energy transfer (LRET) nanocircuit assay involving a donor and two acceptors in tandem was developed to study the dynamic interaction of skeletal muscle contraction proteins. The donor transmits energy relayed to the acceptors distinguishing myosin subfragment-1 (S1) lever arm orientations. The last acceptor allows the detection of S1's bound near or in between troponin complexes on the thin filament. Additionally, calcium related changes between troponin T and myosin were detected. Based on this data, the troponin complex situated every 7 actin monomers, hinders adjacently bound myosins to complete their power stroke; whereas myosins bound in between troponin complexes undergo complete power strokes.
In order to understand the structural changes in myosin S1, fluorescence polarization and computational dynamics simulations were used. Dynamics simulations on the S1 motor domain indicated that significant flexibility was present throughout the molecular model. The constrained opening versus closing of the 50 kDa cleft appeared to induce opposite directions of movement in the lever arm. A sequence called the "strut" which traverses the 50 kDa cleft and may play an important role in positioning the actomyosin binding interface during actin binding is thought to be intimately linked to distant structural changes in the myosin's nucleotide cleft and neck regions. To study the dynamics of the strut region, a method of fluorescent labeling of the strut was discovered using the dye CY3. CY3 served as a hydrophobic tag for purification by hydrophobic interaction chromatography which enabled the separation of labeled and unlabeled species of S1 including a fraction labeled specifically at the strut sequence. The high specificity of labeling was verified by proteolytic digestions, gel electrophoresis, and mass spectroscopy. Analysis of the labeled S1 by collisional quenching, fluorescence polarization, and actin-activated ATPase activity were consistent with predictions from structural models of the probe's location. Although the fluorescent intensity of the CY3 was insensitive to actin binding, its fluorescence polarization was notably affected. Intriguingly, the mobility of the probe increases upon S1 binding to actin suggesting that the CY3 becomes displaced from interactions with the surface of S1 and is consistent with a structural change in the strut due to cleft motions. Labeling the strut reduced the affinity of S1 for actin but did not prevent actin-activated ATPase activity which makes it a potentially useful probe of the actomyosin interface. The different conformations of myosin S1 indicated that the strut is not as flexible as several other key regions of myosin ...
The study of root nodule symbiosis between nitrogen-fixing bacteria and leguminous plant species is important because of the ability to supplement fixed nitrogen fertilizers and increase plant growth in poor soils. Our group has isolated a mutant called nip in the model legume Medicago truncatula that is defective in nodule symbiosis. The nip mutant (numerous infections with polyphenolics) becomes infected by Sinorhizobium meliloti but then accumulates polyphenolic defense compounds in the nodule and fails to progress to a stage where nitrogen fixation can occur. Analysis of the transcriptome of nip roots prior to inoculation with rhizobia was undertaken using Affymetric Medicago Genome Array microarrays. The total RNA of 5-day old uninoculated seedlings was analyzed in triplicate to screen for the NIP gene based on downregulated transcript levels in the mutant as compared to wild type. Further microarray data was generated from 10 days post inoculation (dpi) nip and wild type plants. Analysis of the most highly downregulated transcripts revealed that the NIP gene was not identifiable based on transcript level. Putative gene function was assigned to transcripts with altered expression patterns in order to characterize the nip mutation phenotypically as inferred from the transcriptome. Functional analysis revealed a large number of chaperone proteins were highly expressed in the nip mutant, indicating high stress in the mutant prior to infection by rhizobia. Additionally, a database containing the information regarding the nip expression profile at both 0 days post inoculation (dpi) and 10 dpi were created for screening of candidate genes as predicted from sequence in the genomic region containing NIP.
Several key flexure sites exist in the muscle crossbridge including the actomyosin binding site which play important roles in the actomyosin crossbridge cycle. To distinguish between these sources of flexibility, a new single molecule assay was developed to observe the swiveling of rod about a single myosin. Myosins attached through a single crossbridge displayed mostly similar torsional characteristics compared to myosins attached through two crossbridges, which indicates that most of the torsional flexibility resides in the myosin subfragment-2, and thus the hinge between subfragment-2 and light meromyosin should contribute the most to this flexibility. The comparison of torsional characteristics in the absence and presence of ADP demonstrated a small but significant increase in twist rates for the double-headed myosins but no increase for single-headed myosins, which indicates that the ADP-induced increase in flexibility arises due to changes in the myosin head and verifies that most flexibility resides in myosin subfragment-2.
The lipid composition of cotton (Gossypium hirsutum, L) fibers was determined. Fatty acid profiles revealed that linolenate and palmitate were the most abundant fatty acids present in fiber cells. Phosphatidylcholine was the predominant lipid class in fiber cells, while phosphatidylethanolamine, phosphatidylinositol and digalactosyldiacylglycerol were also prevalent. An unusually high amount of phosphatidic acid was observed in frozen cotton fibers. Phospholipase D activity assays revealed that this enzyme readily hydrolyzed radioactive phosphatidylcholine into phosphatidic acid. A profile of expressed sequence tags (ESTs) for genes involved in lipid metabolism in cotton fibers was also obtained. This EST profile along with our lipid metabolite data was used to predict lipid metabolic pathways in cotton fiber cells.
N-Acylethanolamines (NAEs) are endogenous lipid metabolites that occur in a variety of dry seeds, and their levels decline rapidly during the first few hours of imbibition (Chapman et al., 1999, Plant Physiol., 120:1157-1164). Biochemical studies supported the existence of an NAE amidohydrolase activity in seeds and seedlings, and efforts were directed toward identification of DNA sequences encoding this enzyme. Mammalian tissues metabolize NAEs via an amidase enzyme designated fatty acid amide hydrolase (FAAH). Based on the characteristic amidase signature sequence in mammalian FAAH, a candidate Arabidopsis cDNA was identified and isolated by reverse transcriptase-PCR. The Arabidopsis cDNA was expressed in E. coli and the recombinant protein indeed hydrolyzed a range of NAEs to free fatty acids and ethanolamine. Kinetic parameters for the recombinant protein were consistent with those properties of the rat FAAH, supporting identification of this Arabidopsis cDNA as a FAAH homologue. Two T-DNA insertional mutant lines with disruptions in the Arabidopsis NAE amidohydrolase gene (At5g64440) were identified. The homozygous mutant seedlings were more sensitive than the wild type to exogenously applied NAE 12:0. Transgenic seedlings overexpressing the NAE amidohydrolase enzyme showed noticeably greater tolerance to NAE 12:0 than wild type seedlings. These results together provide evidence in vitro and in vivo for the molecular identification of Arabidopsis NAE amidohydrolase. Moreover, the plants with altered NAE amidohydrolase expression may provide new tools for improved understanding of the role of NAEs in germination and seedling growth.
The purpose of this research was to study the hybridization of molecular beacons under different conditions and designs. Data collected suggest that the inconsistency found in the emission intensity of several of these probes may be caused by 3 important factors: length of the probe, nucleotide sequence and, the formation of an alternative complex structure such as a dimer. Of all three factors, dimer formation is the most troublesome, since it reduces the emission of the reporter molecules. A new probe design was used to reduce dimer formation. The emission signal of the improved probe was several folds stronger than those probes with the early design. In this research, dimer formation is detected, furthermore a new probe with a different design was tested. If dimer formation can be reduced molecular beacons can be integrated into more complex hybridization systems providing an important tool in research and diagnosis of genetic disorders.
N-Acylethanolamines (NAEs) are enriched in seed-derived tissues and are believed to be formed from the membrane phospholipid, N-acylphosphatidylethanolamine (NAPE) via the action of phospholipase D (PLD). In an effort to identify a functional NAPE-PLD in cotton seeds and seedlings, we have screened a cotton seedling cDNA (cotyledon mRNA from 48 h dark grown seedlings) library with a 1.2 kb tobacco partial cDNA fragment encoding the middle third of a putative PLDβ/γ (genbank accession, AF195614) isoform. Six plaques were isolated from the Uni-ZAP lambda library, excised as pBluescript SK(-) phagemids and subjected to nucleotide sequence analysis. Alignment of derived sequences with Arabidopsis PLD family members indicated that the cDNAs represent six different PLD gene products -three putative PLD β isoforms and three putative PLD δ isoforms. The PLD β isoforms, designated Ghpldβ1a, GHpldβ1b and a truncated Ghpldβ1b isoform. Both the full-length PLD β proteins contained characteristic HKxxxxD catalytic domains, a PC-binding domain, a PIP2-binding domain and a C2 domain. In addition both cotton PLD β isoforms had a N-terminal "SPQY" rich domain which appeared to be unique to these PLDs. The three PLD δ isoforms, designated Ghpldδ1a, Ghpldδ1b and Ghpldδ1b-2 encode full-length PLDδ proteins, and like the above PLDs, contained the characteristic catalytic and regulatory domains. The expression of Ghpldδ1b showed hydrolytic and transphosphatidylation activity toward radiolabelled phosphatidylcholine (PC) but it appears Ghpldδ1b does not utilize NAPE as a substrate to produce NAEs nor does it seem to be suppressed by NAEs.
Commonly, when it is desirable to replace an essential gene with an allelic series of mutated genes, or genes with altered expression patterns, the complementing constructs are introduced into heterozygous plants, followed by the selection of homozygous null segregants. To overcome this laborious and time-consuming step, the newly developed two-component system utilizes a site-specific recombinase to excise a wild-type copy of the gene of interest from transformed tissues. In the first component (the first vector), a wild-type version of the gene is placed between target sequences recognized by FLP recombinase from the yeast 2 μm plasmid. This construct is transformed into a plant heterozygous for a null mutation at the endogenous locus, and progeny plants carrying the excisable complementing gene and segregating homozygous knockout at the endogenous locus are selected. The second component (the second vector) carries the experimental gene along with the FLP gene. When this construct is introduced, FLP recombinase excises the complementing gene, leaving the experimental gene as the only functional copy. The FLP gene is driven by an egg apparatus specific enhancer (EASE) to ensure excision of the complementing cDNA in the egg cell and zygote following floral-dip transformation. The utility of this system is being tested using various experimental derivatives of the essential sucrose-proton symporter, AtSUC2, which is required for photoassimilate transport.
An understanding of the potential roles as lipid mediators of a family of bioactive metabolites called N-acylethanolamines (NAEs) depends on their accurate identification and quantification. The levels of 18C unsaturated NAEs (e.g. NAE18:2, NAE 18:3, etc.) in wild-type seeds (about 2000 ng/g fw) generally decreased by about 80% during germination and post-germinative growth. In addition, results suggest NAE-degradative fatty acid amide hydrolase (FAAH) expression does not play a major role in normal NAE metabolism as previously thought. Seedlings germinated and grown in the presence of abscisic acid (ABA), an endogenous plant hormone, exhibited growth arrest and secondary dormancy, similar to the treatment of seedlings with exogenous Nlauroylethanolamine (NAE12:0). ABA-mediated growth arrest was associated with higher levels of unsaturated NAEs. Overall, these results are consistent with the concept that NAE metabolism is activated during seed germination and suggest that the reduction in unsaturated NAE levels is under strict temporal control and may be a requirement for normal seed germination and post-germinative growth.
To elaborate on the function(s) of the ENOD8 gene in the nodules of M. truncatula, several different experimental approaches were used. A census of the ENOD8 genes was first completed indicating that only ENOD8.1 (nt10554-12564 of GenBank AF463407) is highly expressed in nodule tissues. A maltose binding protein-ENOD8 fusion protein was made with an E. coli recombinant system. A variety of biochemical assays were undertaken with the MBP-ENOD8 recombinant protein expressed in E. coli, which did not yield the esterase activity observed for ENOD8 protein nodule fractions purified from M. sativa, tested on general esterase substrates, α-naphthyl acetate, and p-nitrophenylacetate. Attempts were also made to express ENOD8 in a Pichia pastoris system; no ENOD8 protein could be detected from Pichia pastoris strains which were transformed with the ENOD8 expression cassette. Additionally, it was shown that the ENOD8 protein can be recombinantly synthesized by Nicotiana benthamiana in a soluble form, which could be tested for activity toward esterase substrates, bearing resemblance to nodule compounds, such as the Nod factor. Transcription localization studies using an ENOD8 promoter gusA fusion indicated that ENOD8 is expressed in the bacteroid-invaded zone of the nodule. The ENOD8 protein was also detected in that same zone by immunolocalization. Confocal immunomicroscopy with an affinity-purified anti-ENOD8 oligopeptide antibody showed that the ENOD8 protein localizes at the interface between the plant and the bacteroid-differentiated rhizobia, in the symbiosome membrane or symbiosome space. This suggests a possible link between ENOD8 protein and bacteroid differentiation, nitrogen fixation, or plant defense. These possible functions for ENOD8 could be tested with an ENOD8-RNAi transgenic line devoid of detectable ENOD8 proteins.
Numerous troponin T (TnT) isoforms are generated by alternative RNA splicing primarily in its NH2-terminal hypervariable region, but the functions of these isoforms are not completely understood. In this dissertation work, calcium and terbium binding behavior of several forms of TnT were investigated by spectroscopic and radioactive techniques. Chicken breast muscle TnT binds calcium and terbium through its NH2-terminal Tx motif (HEEAH)n with high affinity (10-6 mM) and fast on-rate (106 - 107 M-1 s-1). Chicken leg muscle TnT and a human cardiac TnT NH2-terminal fragment, which both lack the Tx motif on their NH2-terminal regions, do not have affinities for calcium in the physiological range. Computational predictions on TnT N47 suggest that the TnT NH2-terminal region might fold into an elongated structure with at least one high affinity metal ion binding pocket comprised primarily of the Tx motif sequence and several lower affinity binding sites. In addition, calcium binding to TnT N47 might alter its conformation and flexibility. Luminescence resonance energy transfer measurements and other experimental observations are consistent with the computational predictions suggesting the computational simulated atomic model is reasonable. TnT mutations are responsible for 15% of familiar hypertrophic cardiomyopathy (FHC) cases with a phenotype of relatively mild hypertrophy, but a high incidence of sudden death. Detection of those genetic mutations would facilitate the clinical diagnosis and initiation of treatment at an early stage. This dissertation also investigated a novel hybridization proximity assay (HYPA) combining molecular beacon and luminescence resonance energy transfer (LRET) technologies. Experimental results suggest that a shared stem probe design produces a more consistent response upon hybridization, whereas the internally labeled probe was less consistent, but can yield the highest responses. Using the optimally designed molecular probes, the HYPA provides a detection of alterations in nucleic acid structure of as little as a single nucleotide. ...
The industrial uses of cottonseed oil are limited by its fatty acid composition. Genetic modification of cotton lipid profiles using seed-specific promoters could allow cotton growers to produce valuable new oils in the seed without adverse effects on fiber quality and yield, therefore making this crop more commercially profitable. Transgenic cotton callus harboring a diverged fatty acid desaturase gene (FADX) from Momordica charantia was characterized for production of alpha-eleostearic acid (conjugated double bonds: 18:3 D9 cis, 11 trans, 13 trans), not normally found in cotton. Gas chromatography (GC) in conjunction with mass spectrometry (MS) confirmed production of alpha-eleostearic acid in the transgenic cotton tissues. A second series of transformation experiments introduced the cotton fatty acid thioesterase B (FATB) cDNA, fused to the seed-specific oleosin promoter into cotton to promote the over-expression of FATB, to generate cotton with increased palmitate in the cottonseed. PCR amplification, as well as fatty acid analysis by gas chromatography, confirmed introduction of the FATB cDNA in transgenic tissues. Collectively, these results demonstrate the feasibility of manipulating the fatty acid composition in cotton via transgenic approaches and form the basis for continued efforts to create novel oils in cottonseed.
In this experiment, fluorescent N- (1-pyrenyl) iodoacetamide modified the two reactive thiols, SH1 (Cys 707) and SH2 (Cys 697) on myosin to detect SH1-SH2 a -helix melting. The excimer forming property of pyrene is well suited to monitor the dynamics of the SH1 and SH2 helix melting, since the excimer should only form during the melted state. Decreased anisotropy of the excimer relative to the monomeric pyrene fluorescence is consistent with the disordering of the melted SH1-SH2 region in the atomic model. Furthermore, nucleotide analogs induced changes in the anisotropy of the excimer, suggesting that the nucleotide site modulates the flexibility of SH1-SH2 region.
Mechanistic details on the regulation of striated muscle contraction still need to be determined, particularly the specific structural locations of the elements comprising the thick and thin filaments. Of special interest is the location of the regulatory component, troponin, on the actin filament and how its presence influences the behavior of myosin binding to the thin filament. In the present study: (1) Luminescence resonance energy transfer was used to monitor potential conformational changes in the reconstituted thin filament between the C-terminal region of troponin T and myosin subfragment 1; (2) Location of troponin in previously derived atomic models of the acto-myosin complex was mapped to visualize specific contacts; and (3) Shortened tropomyosin was engineered and protein binding and ATPase assays were performed to study the effect of myosin binding close to the troponin complex. Analysis of the results suggest the following: (1) Irrespective of calcium levels, the C-terminal region of troponin T is located close to myosin loop 3 and a few actin helices that may perturb strong acto-myosin interactions responsible for force production. (2) Atomic models indicate myosin subfragment 1 cannot attain the post- powerstroke state due to the full motion of the lever arm being sterically hindered by troponin. (3) A shortened tropomyosin with five actin binding modules (instead of the native seven in muscle cells) binds actin contiguously in a head-to-tail manner and serves to increase the periodicity of troponin complexes on the actin filament. Such behavior eliminates the structure of the actin filament being responsible for the binding location of tropomyosin. (4) Differential behavior of myosin subfragment 1 i.e. (a) binding adjacent to troponin and (b) binding further away from troponin, is apparent as tropomyosin and troponin appear to govern the regions or "target zones" where myosin can bind productively along the actin filament. Physiologically, myosins ...
Prostanoids are oxygenated derivatives of arachidonic acid with a wide range of physiological effects in vertebrates including modulation of inflammation and innate immune responses. Nonsteroidal anti-inflammatory drugs (NSAIDs) act through inhibition of cyclooxygenase (COX) conversion of arachidonic acid to prostanoids. In order to better understand the potential of environmental NSAIDS for interruption of normal levels COX products in fishes, we developed an LC/MS/MS-based approach for tissue analysis of 7 prostanoids. Initial studies examining muscle, gut and gill demonstrated that prostaglandin E2 (PGE2) was the most abundant of the measured prostanoids in all tissues and that gill tissue had the highest and most consistent concentrations of PGE2. After short-term 48-h laboratory exposures to concentrations of 5, 25, 50 and 100 ppb ibuprofen, 50.0ppb and 100.0 ppb exposure concentrations resulted in significant reduction of gill tissue PGE2 concentration by approximately 30% and 80% respectively. The lower exposures did not result in significant reductions when compared to unexposed controls. Measured tissue concentrations of ibuprofen indicated that this NSAID had little potential for bioaccumulation (BCF 1.3) and the IC50 of ibuprofen for inhibition of PGE2 production in gill tissue was calculated to be 0.4 µM. Short-term laboratory exposure to ibuprofen did not result in significant alteration of concentrations of PGE2 at environmentally relevant concentrations.
Faithful segregation of chromosomes is ensured by the spindle checkpoint. If a kinetochore does not correctly attach to a microtubule the spindle checkpoint stops cell cycle progression until all chromosomes are attached to microtubules or tension is experienced while pulling the chromosomes. The C. elegans gene, san-1, is required for spindle checkpoint function and anoxia survival. To further understand the role of san-1 in the spindle checkpoint, an RNAi screen was conducted to identify genetic interactions with san-1. The kinetochore gene hcp-1 identified in this screen, was known to have a genetic interaction with hcp-2. Interestingly, san-1(ok1580);hcp-2(ok1757) had embryonic and larval lethal phenotypes, but the phenotypes observed are less severe compared to the phenotypes of san-1(ok1580);hcp-1(RNAi) animals. Both san-1(ok1580);hcp-1(RNAi) and san-1(ok1580);hcp-2(RNAi) produce eggs that may hatch; but san-1(ok1580):hcp-1(RNAi) larvae do not survive to adulthood due to defects caused by aberrant chromosome segregations during development. Y54G9A.6 encodes the C. elegans homolog of bub-3, and has spindle checkpoint function. In C.elegans, bub-3 has genetic interactions with san-1 and mdf-2. An RNAi screen for genetic interactions with bub-3 identified that F31F6.3 may potentially have a genetic interaction with bub-3. This work provided genetic evidence that hcp-1, hcp-2 and F31F6.2 interact with spindle checkpoint genes.
Familial Hypertrophic cardiomyopathy (HCM) causes ventricle walls to thicken and often leads to sudden death especially in adults. Mutations in the subfragment 2 (S2) of β-cardiac myosin are implicated in the genetic disorder. This S2 region is a coiled-coil rod region resulting from the dimeric form of myosin II. It has been proposed that an elastic quality allows normal S2 to absorb force during the powerstroke according to the sliding filament model. To test the flexibility of single molecules of S2 against levels of physiological force, the Gravitational Force Spectrometer (GFS) is being developed. This novel system employs a standard microscope on an equatorial mount that allows the spectrometer to be rotated freely in space. Stationary glass beads are attached to a microscope slide where the molecule is tethered between the stationary bead and a smaller mobile bead. The GFS is oriented so that the force of gravity can act on the mobile bead and so impart a small force to the tethered subfragment. Additionally, a video system in conjunction with ImageJ software makes a distance measurement of the molecule possible with a resolution of around 11 nm. The S2 can be stretched parallel or perpendicular to the coiled coil to elucidate different structural properties of the rod. This study is the first to show structural evidence that S2 in vertebrate skeletal myosin uncoils proportionally to physiological force loads. Because of this, the usefulness and promise of the novel GFS is highlighted, and the biological role of S2's flexibility can be directly commented on. If the dimer undergoes uncoiling at physiological force loads as shown, then it is reasonable to think that this might occur in nature in response to the stress of the powerstroke on a single molecule. This unwinding could be to absorb force as a mechanism to ...
Phloem transport is along hydrostatic pressure gradients generated by differences in solute concentration between source and sink tissues. Numerous species accumulate raffinose-family oligosaccharides (RFOs) in the phloem of mature leaves to accentuate the pressure gradient between source and sinks. In this study, metabolic engineering was used to generate RFOs at the inception of the translocation stream of Arabidopsis thaliana, which transports predominantly sucrose. To do this, three genes, GALACTINOL SYNTHASE, RAFFINOSE SYNTHASE and STACHYOSE SYNTHASE, were expressed from promoters specific to the companion cells of minor veins. Two transgenic lines homozygous for all three genes (GRS63 and GRS47) were selected for further analysis. Sugars were extracted and quantified by high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD), and 21-day old plants of both lines had levels of galactinol, raffinose, and stachyose approaching 50% of total soluble sugar. All three exotic sugars were also identified in phloem exudates from excised leaves of transgenic plants whereas levels were negligible in exudates from wild type leaves. Differences in starch accumulation or degradation between wild type and GRS63 and GRS47 lines were not observed. Similarly, there were no differences in vegetative growth between wild type and engineered plants, but engineered plants flowered earlier. Finally, since the sugar composition of the phloem translocation stream is altered in these plants, we tested for aphid feeding. When green peach aphids were given a choice between WT and transgenic plants, WT plants were preferred. When aphids were reared on only WT or only transgenic plants, aphid fecundity was reduced on the transgenic plants. When aphids were fed on artificial media with and without RFOs, aphid reproduction did not show differences, suggesting the aphid resistance is not a direct effect of the exotic sugars.
N-Acylethanolamines (NAEs) are endogenous plant lipids hydrolyzed by fatty acid amide hydrolase (FAAH). When wildtype Arabidopsis thaliana seeds were germinated and grown in exogenous NAE 12:0 (35 µM and above), growth was severely reduced in a concentration dependent manner. Wildtype A. thaliana seeds sown on exogenous abscisic acid (ABA) exhibited similar growth reduction to that seen with NAE treatment. AtFAAH knockouts grew and developed similarly to WT, but AtFAAH overexpressor lines show markedly enhanced sensitivity to ABA. When low levels of NAE and ABA, which have very little effect on growth alone, were combined, there was a dramatic reduction in seedling growth in all three genotypes, indicating a synergistic interaction between ABA and NAE. Notably, this synergistic arrest of seedling growth was partially reversed in the ABA insensitive (abi) mutant abi3-1, indicating that a functional ABA signaling pathway is required for the full synergistic effect. This synergistic growth arrest results in an increased accumulation of NAEs, but no concomitant increase in ABA levels. The combined NAE and ABA treatment induced a dose-dependent increase in ABI3 transcript levels, which was inversely related to growth. The ABA responsive genes AtHVA22B and RD29B also had increased expression in both NAE and ABA treatment. The abi3-1 mutant showed no expression of ABI3 and AtHVA22B, but RD29B expression remained similar to wildtype seedlings, suggesting an alternate mechanism for NAE and ABA interaction. Taken together, these data suggest that NAE metabolism acts through ABI3-dependent and independent pathways in the negative regulation of seedling development.
Fatty acid amide hydrolase (FAAH) terminates the endocannabinoid signaling pathway that regulates numerous neurobehavioral processes in animals by hydrolyzing a class of lipid mediators, N-acylethanolamines (NAEs). Recent identification of an Arabidopsis FAAH homologue (AtFAAH) and several studies, especially those using AtFAAH overexpressing and knock-out lines suggest that a FAAH-mediated pathway exists in plants for the metabolism of endogenous NAEs. Here, I provide evidence to support this concept by identifying candidate FAAH cDNA sequences in diverse plant species. NAE amidohydrolase assays confirmed that several of the proteins encoded by these cDNAs indeed catalyzed the hydrolysis of NAEs in vitro. Kinetic parameters, inhibition properties, and substrate specificities of the plant FAAH enzymes were very similar to those of mammalian FAAH. Five amino acid residues determined to be important for catalysis by rat FAAH were absolutely conserved within the plant FAAH sequences. Site-directed mutation of each of the five putative catalytic residues in AtFAAH abolished its hydrolytic activity when expressed in Escherichia coli. Contrary to overexpression of native AtFAAH in Arabidopsis that results in enhanced seedling growth, and in seedlings that were insensitive to exogenous NAE, overexpression of the inactive AtFAAH mutants showed no growth enhancement and no NAE tolerance. However, both active and inactive AtFAAH overexpressors displayed hypersensitivity to ABA, suggesting a function of the enzyme independent of its catalytic activity toward NAE substrates. Yeast two-hybrid screening identified Arg/Ser-rich zinc knuckle-containing protein as a candidate protein that physically and domain-specifically interacts with AtFAAH and its T-DNA knock-out Arabidopsis was hypersensitive to ABA to a degree similar to AtFAAH overexpressors. Taken together, AtFAAH appears to have a bifurcating function, via NAE hydrolysis and protein-protein interaction, to control Arabidopsis growth and interaction with phytohormone signaling pathways. These studies help to functionally define the group of enzymes that metabolize NAEs in plants, and further will ...
The stability of myosin subfragment 2 was analyzed using gravitational force spectroscopy. The region was found to destabilize under physiological force loads, indicating the possibility that subfragment 2 may uncoil to facilitate actin binding during muscle contraction. As a control, synthetic cofilaments were produced to discover if the observations in the single molecule assay were due to the lack of the stability provided by the thick filament. Statistically, there was no difference between the single molecule assay data and the synthetic cofilament assay data. Thus, the instability of the region is due to intrinsic properties within subfragment 2.
The model legume, Medicago truncatula, is able to enter into a symbiotic relationship with soil bacteria, known as rhizobia. This relationship involves a carbon for nitrogen exchange in which the plant provides reduced carbon from photosynthesis in exchange for reduced, or “fixed” atmospheric nitrogen, which allows the plant to thrive in nitrogen depleted soils. Rhizobia infect and enter plant root organs, known as nodules, where they reside inside the plant cell in a novel organelle, known as the symbiosome where nitrogen fixation occurs. the symbiosome is enriched in plant proteins, however, little is known about the mechanisms that direct plant proteins to the symbiosome. Using the M. truncatula ENOD8 (MtENOD8) protein as a model to explore symbiosome protein targeting, 3-cis domains were identified within MtENOD8 capable of directing green fluorescent protein (GFP) to the symbiosome, including its N-terminal signal peptide (SP). the SP delivered GFP to the vacuole in the absence of nodules suggesting that symbiosome proteins share a common targeting pathway with vacuolar proteins. a time course analysis during nodulation indicated that there is a nodule specific redirection of MtENOD8-SP from the vacuole to the symbiosome in a MtNIP/LATD dependent manner. GFP expression by the MtENOD8 promoter revealed spatial discrepancy between promoter activity and protein localization. in situ localization of MtENOD8 mRNA showed localization to infected cells, where the protein is found, suggesting mRNA cell-to-cell movement. Expression of MtENOD8 in Arabidopsis showed that the SP did not direct GFP to the vacuole indicating that vacuolar targeting of MtENOD8’s SP may be legume specific. Taken together, the research presented here indicates that the MtENOD8 symbiosome protein has evolved redundant domains for targeting, which has part of a common pathway with vacuolar proteins. Observed spatial discrepancy between the MtENOD8 promoter and protein shows additional mechanisms of gene regulation through cell-to-cell mRNA ...
Non-steroidal anti-inflammatory drugs (NSAIDs) are among the most abundant environmental pharmaceutical contaminants. In this study, a proteomic analysis was conducted to identify proteins differentially expressed in gill tissue of zebrafish (Danio rerio) after a 14-day exposure to the NSAIDs ibuprofen or naproxen. A total of 104 proteins with altered expression as indicated by 2-dimensional electrophoresis were analyzed by liquid chromatography with ion trap mass spectrometry (MS/MS). A total of 14 proteins fulfilled our requirements for identification which included consistency among replicate gels as well as successful MS/MS ion searches with the MASCOT database. The most prominent feature of the differential protein expression observed after NSAID exposure was an up-regulation of proteins belonging to the globin family which are involved in the transport of oxygen from gills and availability of heme molecules required for synthesis of cyclooxygenase. Differential expression was observed at exposure concentrations as low as 1-10 µg/L indicating that altered gene expression may occur in fish subjected to environmentally realistic levels of NSAID exposure.
O-Acetylserine Sulfhydrylase (OASS) is a pyridoxal phosphate enzyme that catalyzes the reaction of O-acetyl-Lserine with sulfide to give L-cysteine. OASS is present as two isoforms, designated -A and -B. The kinetic mechanism of OASS-A is well known and there is also much known concerning the acid-base chemistry of the enzyme. However, little is known concerning the location of the rate determining steps, the sequencing of chemical steps that occur at the active site, or the nature of the rate determining transition states. The studies performed to help elucidate these aspects of the OASS-A mechanism included determination of the thermodynamics of both half reactions, along with studies utilizing substrate analogs of OAS halting the reaction at specific points along the reaction pathway allowing the identification of reaction intermediates. The free energy change of the first half reaction was shown to be -5.7 Kcal/mole while the second half reaction was shown to be, for all intents and purposes, irreversible. Intermediates along the reaction pathway that have been previously identified include the internal Schiff base and the a-aminoacrylate. The external Schiff base was identified using the analogs cysteine, alanine, and glycine while the geminal diamine was identified using the analog serine. Formation of the external aldimine was shown to be pH dependent with a pK of 8.1 ± 0.3 most likely representing a general base that accepts a proton from the a-amine of cysteine to facilitate a nucleophilic attack on C4r of the PLP imine. Formation of the geminal diamine was also shown to be pH dependent with two pK values having an average value of 8.1. One of the groups most likely represents the general base which accepts a proton from the a-amine of cysteine while the second group likely interacts with the amino acid side chain to orientate the amino acid ...
Two highly homologous synthetic peptides MLC(3-13) (K-R-A-K-A-K-T-TK-K-R-G) and MLC(5-13) (A-K-A-K-T-T-K-K-R-G) corresponding to the amino terminal amino acid sequence of smooth muscle myosin light chain were utilized as substrates for protein kinase C purified from murine lymphosarcoma tumors to determine the role of the primary amino acid sequence of protein kinase C substrates in defining the lipid (phosphatidyl serine and diacylglycerol) requirements for the activation of the enzyme. Removal of the basic residues lysine and arginine from the amino terminus of MLC(3-13) did not have a significant effect on the Ka value of diacylglycerol. The binding of effector to calcium-protein kinase C appears to be random since binding of one effector did not block the binding of the other.
Bacterial toxins have been shown to modify animal cell proteins in vivo with ADPR. Animal cells also contain endogenous enzymes that can modify proteins. Indirect evidence for the existence in vivo of rat liver proteins modified by ADPR on arginine residues has been reported previously. Presented here is direct evidence for the existence of ADP-ribosylarginine in rat liver proteins. Proteins were subjected to exhaustive protease digestion and ADP-ribosyl amino acids were isolated by boronate chromatography.
Three genomic clones encompassing human DNA segments (designated LhX-3, LhX-4, and LhX5) were isolated from an X chromosome-specific library and subjected to analysis by physical mapping and DNA sequencing. It was found that these three clones are very rich in repetitive DNA sequence elements and retropseudogenes.
A complete initial velocity study of the 6-phosphogluconate dehydrogenase from Candida utilis in both reaction directions suggests a rapid equilibrium random kinetic mechanism with dead-end E:NADP:(ribulose 5-phosphate) and E:NADPH:(6- phosphogluconate) complexes. Initial velocity studies obtained as a function of pH and using NAD as the dinucleotide substrate for the reaction suggest that the 2'-phosphate is critical for productive binding of the dinucleotide substrate. Primary deuterium isotope effects using 3-<i-6-phosphogluconate were obtained for the 6-phosphogluconate dehydrogenase reaction using NADP and various alternative inucleotide substrates.
To define the overall kinetic and chemical mechanism of adenosine 3',5'-monophosphate dependent protein kinase catalytic subunit, the mechanism in the direction of MgADP phosphorylation was determined, using studies of initial velocity in the absence and presence of dead-end inhibitors. The kinetic mechanism was determined as a function of uncomplexed Mg^2+ (Mg_f) at pH 7.2 and as a function of pH at low (0.5 mM) Mg_f. At pH 7.2 data are consistent with a random kinetic mechanism in the direction of MgADP phosphorylation with both pathways allowed: the pathway in which MgADP binds to enzyme prior to phosphorylated peptide (PSP) and that in which PSP binds before MgADP. One or the other pathway predominates, depending on Mg_f concentration. At 0.5 mM Mg_f, the mechanism is steady-state ordered with the pathway where PSP binds first preferred; at 10 mM Mg_f, the mechanism is equilibrium ordered, and the pathway in which MgADP binds first preferred. This change in mechanism to equilibrium ordered is due to an increase in affinity of enzyme for MgADP and a decrease in affinity for PSP. There is also a pH-dependent change in mechanism at 0.5 mM Mg_f. At pH 6 the mechanism is equilibrium ordered with the pathway where PSP binds first preferred. At pH 7.6 the mechanism is ordered with MgADP binding first. The log V/E_t vs. pH profile is pH-independent, suggesting only the correctly protonated form of each substrate binds to enzyme. The log V/K_MgADP vs. PH profile gives a pK of 7, likely that of a general acid, which must be protonated for activity. The pK_iPSP vs. pH profile gives a pK of 6.5, likely reflecting the peptide phosphoryl group, which must be unprotonated for activity.
N-Acylphosphatidylethanoiamine (NAPE) is synthesized in the microsomes of cotton seedlings by a mechanism that is possibly unique to plants, the ATP-, Ca2+-, and CoA-independent acylation ofphosphatidylethanolamine (PE) with unesterified free fatty acids (FFAs), catalyzed by NAPE synthase. A photoreactive free fatty acid analogue, 12-[(4- azidosalicyl)amino]dodecanoic acid (ASD), and its 125I-labeled derivative acted as substrates for the NAPE synthase enzyme.
Recently, three distinct isoforms of phospholipase D (PLD) were identified in Arabidopsis thaliana. PLD α represents the well-known form found in plants, while PLD β and γ have been only recently discovered (Pappan et al., 1997b; Qin et al., 1997). These isoforms differ in substrate selectivity and cofactors required for activity. Here, I report that PLD β and γ isoforms were active toward N-acylphosphatidylethanolamine (NAPE), but PLD α was not. The ability of PLD β and γ to hydrolyze NAPE marks a key difference from PLD α. N-acylethanolamines (NAE), the hydrolytic products of NAPE by PLD β and γ, inhibited PLD α from castor bean and cabbage. Inhibition of PLD α by NAE was dose-dependent and inversely proportional to acyl chain length and degree of unsaturation. Enzyme kinetic analysis suggested non-competitive inhibition of PLD α by NAE 14:0. In addition, a 1.2-kb tobacco (Nicotiana tabacum L.) cDNA fragment was isolated that possessed a 74% amino acid identity to Arabidopsis PLD β indicating that this isoform is expressed in tobacco cells. Collectively, these results provide evidence for NAE producing PLD activities and suggest a possible regulatory role for NAE with respect to PLD α.
O-Acetyl-L-serine sulfhydrylase-A (OASS-A) forms acetate and L-cysteine from O-acetyl-L-serine (OAS) and sulfide. One molecule of the cofactor pyridoxal 5'- phosphate (PLP) is bound in each holoenzyme protomer.
A number of protein kinases have been shown to undergo autophosphorylation, but few have demonstrated a coordinate increase or decrease in enzymatic activity as a result. Described here is a novel S6 kinase isolated from human placenta which autoactivates through autophosphorylation in vitro. This S6/H4 kinase, purified in an inactive state, was shown to be a protein of Mr of 60,000 as estimated by SDS-PAGE and could catalyze the phosphorylation of the synthetic peptide S6-21, the histone H4, and myelin basic protein. Mild digestion of the inactive S6/H4 kinase with trypsin was necessary, but not sufficient, to activate the kinase fully
The kinetic mechanism of activation of the NAD-malic enzyme by fumarate and the transition state structure for the oxidation malate for the NAD-malic enzyme reaction have been studied. Fumarate exerts its activating effect by decreasing the off-rate for malate from the E:Mg:malate and E:Mg:NAD:malate complexes. The activation by fumarate results in a decrease in K_imalate and an increase in V/K_malate by about 2-fold, while the maximum velocity remains constant. A discrimination exists between active and activator sites for the binding of dicarboxylic acids. Activation by fumarate is proposed to have physiologic importance in the parasite. The hydride transfer transition state for the NAD-malic enzyme reaction is concerted with respect to solvent isotope sensitive and hydride transfer steps. Two protons are involved in the solvent isotope sensitive step, one with a normal fractionation factor, another with an inverse fractionation factor. A structure for the transition state for hydride transfer in the NAD-malic enzyme reaction is proposed.
The studies described in this dissertation examine the effects of F-2,6-P2 and AMP or phosphorylation on the kinetic mechanism of d-PFK. The effect of varied pH on the activation by F-2,6-P2 is also described.
Initial velocity studies of O-acetylserine sulfhydrylase-B (OASS-B) from Salmonella typhimurium using both natural and alternative substrates suggest a Bi Bi ping pong kinetic mechanism with double substrate competitive inhibition. The ping pong mechanism is corroborated by a qualitative and quantitative analysis of product and dead-end inhibition. Product inhibition by acetate is S-parabolic noncompetitive, indication of a combination of acetate with E followed by OAS. These data suggest some randomness to the OASS-B kinetic mechanism. The pH dependence of kinetic parameters was determined in order to obtain information on the acid-base chemical mechanism for the OASS-B reaction. A mechanism is proposed in which an enzyme general base accepts a proton from α-amine of O-acetylserine, while a second enzyme general base acts by polarizing the acetyl carbonyl assisting in the β-elimination of the acetyl group of O-acetylserine. The ε-amine of the active site lysine acts as a general base to abstract the α-proton in the β-elimination of acetate. At the end of the first half reaction the ε-amine of the active site lysine that formed the internal Schiff base and the general base are protonated. The resulting α-aminoacrylate intermediate undergoes a Michael addition with HS‾ and the active site lysine donates its proton to the α-carbon to give cysteine and regenerate enzyme to start the second half reaction. In addition, substrate specificity, stereochemistry of the internal Schiff base at C4', and sequence around active site lysine of O-acetylserine sulfhydrylase-A have been determined. The [4'-^3H]pyridoxamine generated by reduction of the internal Schiff base with sodium [^3H]borohydride retained most of its tritium after incubation with apoaspartate aminotransferase. These results agree with the hypothesis put forth by Dunathan (Dunathan, 1971; Dunathan and Voet, 1974) that a single surface (Re face) of the active site PLP is accessible to solvent. The sequence around the active site ...
Phloem vascular tissues transport sugars synthesized by photosynthesis in mature leaves by a process called phloem loading in source tissues and unloading in sink tissues. Phloem loading in source leaves is catalyzed by Suc/H+ symporters (SUTs) which are energized by proton motive force. In Arabidopsis the principal and perhaps exclusive SUT catalyzing phloem loading is AtSUC2. In mutant plants harboring a T-DNA insertion in each of the functional SUT-family members, only Atsuc2 mutants demonstrate overtly debilitated phloem transport. Analysis of a mutant allele (Atsuc2-4) of AtSUC2 with a T-DNA insertion in the second intron showed severely stunted phenotype similar to previously analyzed Atsuc2 null alleles. However unlike previous alleles Atsuc2-4 produced viable seeds. Analysis of phloem specific promoters showed that promoter expression was regulated by Suc concentration. Unlike AtSUC2p, heterologous promoter CoYMVp was not repressed under high Suc conc. Further analysis was conducted using CoYMVp to test the capacity of diverse clades in SUT-gene family for transferring Suc in planta in Atsuc2 - / - mutant background. AtSUC1 and ZmSUT1 from maize complemented Atsuc2 mutant plants to the highest level compared to all other transporters. Over-expression of the above SUTs in phloem showed enhanced Suc loading and transport, but against expectations, plants were stunted. The implications of SUT over-expression to enhance phloem transport and loading are discussed and how it induces a perception of phosphate imbalance is presented.
The study was performed to determine factors influencing the esteriflcation of plasma cholesterol in young and aged rats. The distribution of LCAT activity was determined following gel nitration chromatography and ultracentrifugation of whole plasma respectively. When rat plasma was fractionated on a Bio-Gel A-5 Mcolumn, LCAT activity was found to be associated with the HDL fraction. A similar result was observed upon 24 hr density gradient ultracentrifugation of the plasma. However, following prolonged 40 hr preparative ultracentrifugation, the majority of the LCAT activity was displaced into the lipoprotein-free infranatant fraction (d> 1.225 g/ml). The dissociation of LCAT from the HDL fraction occured to a smaller extent in aged rat plasma than in young rat plasma. Plasma incubation (37°C) experiments followed by the isolation of lipoproteins and the subsequent analysis of their cholesterol content revealed that in vitro net esteriflcation of free cholesterol (FC) by LCAT as well as the fractional ufilization of HDL-FC as substrate were lower in the plasma of the aged animal as compared to that of the young animal despite the fact that the total pool of FC was higher in the former. The net transfer of FC from lower density lipoproteins (d<1.07 g/ml) to HDL provided the FC (in addition to HDL-FC) for esteriflcation in the plasma of both young and aged rats, and this process was not substantially affected by aging. Substrate specificity studies indicated that HDL from young rats was a better substrate for LCAT than the HDL from aged rats. The HDL isolated from the plasma of aged rats was enriched with apo E and had a considerably higher molecular weight than the HDL from young rat plasma. The ratio of phosphatidyl choline/sphingomyelin was lower in the HDL of aged rats. These data suggest that the decreased plasma cholesterol esteriflcation in aged rats ...
The kinetic mechanism of the cAMP-dependent protein kinase has been determined to be random in the direction of MgADP phosphorylation by using initial velocity studies in the absence and presence of the product, phospho-Serpeptide (Leu-Arg-Arg-Ala-Ser[P]-Leu-Gly) , and dead-end inhibitors. In contrast to the kinetic parameters obtained in the direction of Serpeptide phosphorylation, the only kinetic parameters affected by Mg^2+ are the dissociation constants for E:phospho-Serpeptide and E:MgADP, which are decreased by about 4-fold. The dead-end analog MgAMPCP binds with an affinity equal to that of MgADP in contrast to MgAMPPCP, which binds weaker than MgATP. The ratio of the maximum velocities in the forward and reverse reactions is about 200, and the Haldane relationship gives a K-eq of (7.2 ± 2) x 10^2. The latter can be compared to the K-eq obtained by direct measurement of reactant concentrations (2.2 ± 0.4) x 10^3 and 31-P NMR (1 ± 0.5) x 10^3. Data for the pH dependence of kinetic parameters and inhibitor dissociation constants for the cAMP dependent protein kinase are consistent with a mechanism in which reactants selectively bind to an enzyme with the catalytic base unprotonated and an enzyme group required protonated for Ser-peptide binding. Preferentially MgATP binds fully ionized and requires an enzyme residue (probably lysine) to be protonated. The maximum velocity and V/K-MgATP are pH independent. The V/K for Serpeptide is bell-shaped with estimated pK values of 6.2 and 8.5. The dependence of 1/K-i for Leu-Arg-Arg-Ala-Ala-Leu-Gly is also bell-shaped, giving pK values identical with those obtained for V/K-Serpeptide, while the K-i for MgAMPPCP increases from a constant value of 650 μM above pH 8 to a constant value of 4 mM below pH 5.5. The K-i for uncomplexed Mg^2+ obtained from the Mg^2+ dependence of V and V/K-MgATP is apparently pH independent.
Data obtained from isotope exchange at equilibrium, exchange of inorganic phosphate against forward reaction flux, and positional isotope exchange of 18O from the (βγ-bridge position of pyrophosphate to a (β-nonbridge position all indicate that the pyrophosphate-dependent phosphofructokinase from Propionibacterium freudenreichii has a rapid equilibrium random kinetic mechanism. All exchange reactions are strongly inhibited at high concentrations of the fructose 6-phosphate/Pi and MgPPi/Pi substrate-product pairs and weakly inhibited at high concentrations of the MgPPi/fructose 1,6-bisphosphate pair suggesting three dead-end complexes, E:F6P:Pi, E:MgPPi:Pi, and E:FBP:MgPPi. Neither back-exchange by [32p] nor positional isotope exchange of 18O-bridge-labeled pyrophosphate was observed under any conditions, suggesting that either the chemical interconversion step or a step prior to it limits the overall rate of the reaction. Reduction of the pyridoxal 5'-phosphate-inactivated enzyme with NaB[3H]4 indicates that about 7 lysines are modified in free enzyme and fructose 1,6-bisphosphate protects 2 of these from modification. The pH dependence of the enzyme-reactant dissociation constants suggests that the phosphates of fructose 6-phosphate, fructose 1,6-bisphosphate, inorganic phosphate, and Mg-pyrophosphate must be completely ionized and that lysines are present in the vicinity of the 1- and 6-phosphates of the sugar phosphate and bisphosphates probably directly coordinated to these phosphates. The pH dependence of kinetic parameters suggests that the enzyme catalyzes its reaction via general acid-base catalysis with the use of a proton shuttle. The base is required unprotonated in both reaction directions. In the direction of fructose 6-phosphate phosphorylation the base accepts a proton from the hydroxyl at C-l of F6P and then donates it to protonate the leaving phosphate. The maximum velocity of the reaction is pH independent in both reaction directions while V/K profiles exhibit pKs for binding groups (including enzyme and reactant functional groups) as well as pKs for enzyme catalytic groups. These data suggest that reactants bind only when ...
Isotope partitioning experiments were carried out with the adenosine 3',5'-monophosphate-dependent protein kinase catalytic subunit (cAPK) from bovine hearts to obtain information on the order of addition of reactants and the relative rates of reactant release from enzyme compared to the catalytic step(s). A value of 100% trapping for both ErMgATP-[γ-32P] and E:3H-Serpeptide at low Mgf indicates that MgATP and Serpeptide dissociate slowly from the enzyme compared to the catalytic step(s). The K_Serpeptide for MgATP trapping is 17 μM, while the K_MgATP for Serpeptide trapping is 0.58 mM. The latter data indicate that the off-rate for MgATP from the E:MgATP complex is 14 s^-1 while that for Serpeptide from the E: Serpeptide complex is 64 s^-1. At high Mg^, 100% trapping is obtained for the E:MgATP-[γ-32P] complex but only 40% is obtained for the E:Serpeptide complex. Thus, the off-rate for Serpeptide from the E:MgATP:Serpeptide complex becomes significant at high Mg_f. Data suggest a random mechanism in which MgATP is sticky. The V for the cAPK reaction increases 1.5-1.7 fold in the presence of the R_II in the presence of saturating cAMP at a stoichiometry of R:C of 1:1. No change is obtained with the type-I complex under these conditions. At higher ratio of R:C (up to 100) no further change is observed with the type-II complex but inhibition by the type-I R_2(cAMP)_4 complex competitive vs. Serpeptide is observed. The activiation observed in the presence type-II R_2(cAMP)_4 effects neither the K_m for Serpeptide nor the K_m for MgATP. Both the activating affect of the type-II complex and the inhibitory effect of the type-I complex are dependent on the Mg_f with more type-II activation obtained the higher the Mg_f and more type-I complex required for inhibition the higher the Mg_f. The activation and inhibition are discussed in terms of the mechanism of the ...
Dissociation constants for alternate dirmcleotide substrates and competitive inhibitors suggest that the dinucleotide binding site of the Ascaris suum NAD-malic enzyme is hydrophobic in the vicinity of the nicotinamide ring. Changes in the divalent metal ion activator from Mg^2+ to Mn^2+ or Cd^2+ results in a decrease in the dinucleotide affinity and an increase in the affinity for malate. Primary deuterium and 13-C isotope effects obtained with the different metal ions suggest either a change in the transition state structure for the hydride transfer or decarboxylation steps or both. Deuterium isotope effects are finite whether reactants are maintained at saturating or limiting concentrations with all the metal ions and dinucleotide substrates used. With Cd^2+ as the divalent metal ion, inactivation of the enzyme occurs whether enzyme alone is present or is turning over. Upon inactivation only Cd^2+ ions are bound to the enzyme which becomes denatured. Modification of the enzyme to give an SCN-enzyme decreases the ability of Cd^2+ to cause inactivation. The modified enzyme generally exhibits increases in K_NAD and K_i_metai and decreases in V_max as the metal size increases from Mg^2+ to Mn^2+ or Cd^2+, indicative of crowding in the site. In all cases, affinity for malate greatly decreases, suggesting that malate does not bind optimally to the modified enzyme. For the native enzyme, primary deuterium isotope effects increase with a concomitant decrease in the 13-C effects when NAD is replaced by an alternate dinucleotide substrate different in redox potential. This suggests that when the alternate dinucleotides are used, a switch in the rate limitation of the chemical steps occurs with hydride transfer more rate limiting than decarboxylation. Deuteration of malate decreases the 13-C effect with NAD for the native enzyme, but an increase in 13-C effect is obtained with alternate dinucleotides. These suggest the presence of a ...
The isotope partitioning studies of the Ascaris suum NAD-malic enzyme reaction were examined with five transitory complexes including E:NAD, E:NAD:Mg, E:malate, E:Mg:malate, and E:NAD:malate. Three productive complexes, E:NAD, E:NAD:Mg, and E:Mg:malate, were obtained, suggesting a steady-state random mechanism. Data for trapping with E:14C-NAD indicate a rapid equilibrium addition of Mg2+ prior to the addition of malate. Trapping with 14C-malate could only be obtained from the E:Mg2+:14C-malate complex, while no trapping from E:14C-malate was obtained under feasible experimental conditions. Most likely, E:malate is non-productive, as has been suggested from the kinetic analysis. The experiment with E:NAD:malate could not be carried out due to the turnover of trace amounts of malate dehydrogenase in the pulse solution. The equations for the isotope partitioning studies varying two substrates in the chase solution in an ordered terreactant reaction were derived, allowing a determination of the relative rates of substrate dissociation to the catalytic reaction for each of the productive transitory complexes. NAD and malate are released from the central complex at an identical rate, equal to the catalytic rate.
Glucose phosphate isomerase (GPI) occurs in different bovine tissues as multiple, catalytically active isozymes which can be resolved by polyacrylamide gel electrophoresis and isoelectric focusing. GPI from bovine heart was purified to homogeneity and each of the isozymes was resolved. Four of the five isozymes were characterized with regard to their physical, chemical and catalytic properties in order to establish their possible physiological significance and to ascertain their molecular basis. The isozymes exhibited identical native (118 Kd) and subunit (59 Kd) molecular weights but had different apparent pi values of 7.2, 7.0, 6.8 and 6.6. Structural analyses showed that the amino terminus was blocked and the carboxyl terminal sequence was -Glu-Ala-Ser-Gly for all four isozymes. The most basic isozyme was more stable than the more acidic isozymes (lower pi values) at pH extremes, at high ionic strength, in the presence of denaturants or upon exposure to proteases. Kinetic constants, such as turnover number, Km and Ki values, were identical for all isozymes. Identical amino acid composition and peptide mapping by chemical cleavage at methionine and cysteine residues of the isozymes suggest a postsynthetic modification rather then a genetic origin for the in vivo isozymes. When the most basic isozyme was incubated in vitro under mild alkaline conditions, there was a spontaneous generation of the more acidic isozymes with electrophoretic properties identical to those found in vivo. The simultaneous release in ammonia along with the spontaneous shift to more acidic isozymes and changes in the specific cleavage of the Asn-Gly bonds by hydroxylamine of the acidic isozyme indicates deamidation as the probable molecular basis. In summary the isozymes appear to be the result of spontaneous, postsynthetic modifications involving the addition of an equal number of negative charges and are consistent with the deamidation process.
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