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Identification and Characterization of an Arabidopsis Thaliana Mutant with Tolerance to N-lauroylethanolamime
N-Acylethanolamines (NAEs) are fatty acid derivatives in plants that negatively influence seedling growth. N-Lauroylethanolamine (NAE 12:0), one type of NAE, inhibits root length, increases radial swelling of root tips and reduces root hair numbers in a dose dependent manner in Arabidopis thaliana L. (ecotype Columbia). A forward genetics approach was employed by screening a population of T-DNA “activation-tagged” developed by the Salk Institute lines for NAE resistance to identify potential genes involved in NAE signaling events in Arabidopsis thaliana L. (ecotype Columbia). Seeds of the activation tagged lines were grown at 0, 25, 30, 50, 75 and 100 µM N-lauroylethanolamime (NAE 12:0). Ten plants which displayed NAE tolerance (NRA) seedling phenotypes, compared with wildtype (Columbia, Col-0) seedlings were identified. I focused on one mutant line, identified as NRA 25, where the tolerance to NAE 12:0 appears to be mediated by a single dominant, nuclear gene. Thermal asymmetric interlaced (TAIL) PCR identified the location of the T-DNA insert as 3.86 kbp upstream of the locus At1g68510. Quantitative PCR indicated that the transcript level corresponding to At1g68510 is upregulated approximately 20 fold in the mutant relative to wildtype. To determine whether the NAE tolerance in NRA 25 is associated with overexpression of At1g68510 I created overexpressing lines of At1g68510 with and without GFP fusions behind the 2X35S CaMV promoter. As predicted, results with overexpressing lines of At1g68510 also exhibited enhanced resistance to NAE when compared with the wildtype. Confocal images of the fusion proteins suggest that GFP-At1g68510 is concentrated in the nucleus and this was confirmed by counterstaining with 4', 6-Diamidino-2-phenylindol (DAPI). Futhermore, At1g68510 overexpressing lines and NRA 25 line also exhibited tolerance to abscisic acid (ABA) during seedling germination. The findings suggests that At1g68510 overexpression mediates seedling tolerance to both ABA and NAE, a mechanism independent of fatty acid amide hydrolase ...
Manipulations of Sucrose/Proton Symporters and Proton-pumping Pyrophosphatase Lead to Enhanced Phloem Transport But Have Contrasting Effects on Plant Biomass
Delivery of photoassimilate, mainly sucrose (Suc) from photoautotrophic source leaves provides the substrate for the growth and maintenance of sink tissues such as roots, storage tissues, flowers and fruits, juvenile organs, and seeds. Phloem loading is the energized process of accumulating solute in the sieve element/companion cell complex of source leaf phloem to generate the hydrostatic pressure that drives long-distance transport. In many plants this is catalyzed by Suc/Proton (H+) symporters (SUTs) which are energized by the proton motive force (PMF). Overexpression of SUTs was tested as means to enhance phloem transport and plant productivity. Phloem specific overexpression of AtSUC2 in wild type (WT) tobacco resulted in enhanced Suc loading and transport, but against the hypothesis, plants were stunted and accumulated carbohydrates in the leaves, possibly due to lack of sufficient energy to support enhanced phloem transport. The energy for SUT mediated phloem loading is provided from the PMF, which is ultimately supplied by the oxidation of a small proportion of the loaded photoassimilates. It was previously shown that inorganic pyrophosphate (PPi) is necessary for this oxidation and overexpressing a proton-pumping pyrophosphatase (AVP1) enhanced both shoot and root growth, and augmented several energized processes like nutrient acquisition and stress responses. We propose that AVP1 localizes to the PM of phloem cells and uses PMF to synthesize PPi rather than hydrolyze it, and in doing so, maintains PPi levels for efficient Suc oxidation and ATP production. Enhanced ATP production in turn strengthens the PMF via plasma membrane (PM) ATPase, increasing phloem energization and phloem transport. Phloem-specific and constitutive AVP1 overexpressing lines showed increased growth and more efficiently moved carbohydrates to sink organs compared to WT. This suggested changes in metabolic flux but diagnostic metabolites of central metabolism did not show changes in steady state levels. This research focuses on fundamental aspects ...
Evidence for Multiple Functions of a Medicago Truncatula Transporter
Legumes play an important role in agriculture as major food sources for humans and as feed for animals. Bioavailable nitrogen is a limiting nutrient for crop growth. Legumes are important because they can form a symbiotic relationship with soil bacteria called rhizobia that results in nitrogen-fixing root nodules. In this symbiosis, rhizobia provide nitrogen to the legumes and the legumes provide carbon sources to the rhizobia. The Medicago truncatula NPF1.7/NIP/LATD gene is essential for root nodule development and also for proper development of root architecture. Work in our lab on the MtNPF1.7/MtNIP/LATD gene has established that it encodes a nitrate transporter and strongly suggests it has another function. Mtnip-1/latd mutants have pleiotropic defects, which are only partially explained by defects in nitrate transport. MtNPF1.7/NIP/LATD is a member of the large and diverse NPF/NRT1(PTR) transporter family. NPF/NRT1(PTR) members have been shown to transport other compounds in addition to nitrate: nitrite, amino acids, di- and tri-peptides, dicarboxylates, auxin, abscisic acid and glucosinolates. In Arabidopsis thaliana, the AtNPF6.3/NRT1.1( CHL1) transporter was shown to transport auxin as well as nitrate. Atchl1 mutants have defects in root architecture, which may be explained by defects in auxin transport and/or nitrate sensing. Considering the pleiotropic phenotypes observed in Mtnip-1/latd mutant plants, it is possible that MtNPF1.7/NIP/LATD could have similar activity as AtNPF6.3/NRT1.1(CHL1). Experimental evidence shows that the MtNPF1.7/NIP/LATD gene is able to restore nitrate-absent responsiveness defects of the Atchl1-5 mutant. The constitutive expression of MtNPF1.7/NIP/LATD gene was able to partially, but not fully restore the wild-type phenotype in the Atchl1-5 mutant line in response to auxin and cytokinin. The constitutive expression of MtNPF1.7/NIP/LATD gene affects the lateral root density of wild-type Col-0 plants differently in response to IAA in the presence of high (1mM) or low (0.1 mM) nitrate. MtNPF1.7/NIP/LATD gene expression is not regulated by nitrate ...
A Quantitative Radioimmunoassay for Phosphoglucose Isomerase and Its Utilization in Detecting Cross-Reactive Material in Variant Forms of Phosphoglucose Isomerase and in Human Tissues
A method for purification and radiolabelling phosphoglucose isomerase was devised in order to develop a sensitive quantitative radioimmunoassay for the detection of the enzyme irrespective of its catalytic activity. For four genetic variants of PGI no difference in the molecular specific activity was observed. In one variant (PGI-Denton), liver and heart tissue extracts, and in mature erythrocytes (as compared to normal erythrocytes), a decreased molecular specific activity was observed which initially may imply that these samples contain cross-reactive material which is not catalytically active.
pH Dependence of the Kinetic Parameters for the Oxalacetate Decarboxylation and Pyruvate Reduction Reactions Catalyzed by Malic Enzyme
Ascaris suum NAD-malic enzyme catalyzes the decarboxylation of oxalacetate and reduction of pyruvate. Thus, the present classification (E.C. for this enzyme should be changed to E.C. In the absence of nucleotide, both the chicken liver NADP-malic enzyme and Ascaris suum NAD-malic enzymes catalyze the decarboxylation of oxalacetate. A study of the pH dependence of kinetic parameters for oxalacetate decarboxylation and pyruvate reduction was carried out for the NAD(P)-malic enzyme with Mg^2+ and Mn^2+ in the presence and absence of nucleotide. In all cases, an enzyme residue is required in its protonated form for reaction while for oxalacetate decarboxylation the β-carboxyl of oxalacetate is required unprotonated. Of a number of inhibitory binding analogs of malate tested, oxalate is the tightest binding inhibitor for Ascaris suum enzyme.
Changes in Body Composition, Plasma Alanine, and Urinary Nitrogen in Rats Subjected to Negative Caloric Balance Through Diet, Diet/Exercise, and Exercise
Male Fischer rats (n=43) were used in a diet-diet/ exercise design to investigate the apparent protein sparing effects of exercise. The animals were divided into five groups: INITIAL (baseline), SEDENTARY (control), DIET, DIET/EXERCISE, and EXERCISE. Carcasses were analyzed for body composition, the blood for plasma alanine concentration and the urine for urea nitrogen concentration. The results showed no significant differences between groups in urinary urea nitrogen, plasma alanine, body weight, or carcass weights. The EXERCISE group had a significant increase in percent protein and a significant decrease in percent fat and grams of fat when compared to all other groups (p <.05).
Purification and Studies of Methylglyoxal Reductase from Sheep Liver
The objectives of these investigations were (1) the purification of MG reductase from sheep liver and (2) studies of some of its characteristics. MG reductase was purified 40 fold and showed a single band on SDS-PAGE. Molecular weight estimations with SDS-PAGE showed a molecular weight of 44,000; although gel filtration with Sephadex G-150 gave a molecular weight of 87,000 indicating that the enzyme might be a dimer. The Km for MG is 1.42 mM and for NADH it is 0.04 mM. The pH optimum for the purified enzyme is pH 7.0. Isoelectric focusing experiments showed a pI of 9.3. In vivo experiments involving rats treated with 3,3',5-triiodothyronine (T_3) and 6-n-propyl-2-thiouracil (PTU) indicated that MG reductase was depressed by T_3 and elevated by PTU.
NAD+-Dependent 15-Hydroxyprostaglandin Dehydrogenase from Swine Kidney: Characterization and Kinetic Mechanism
Cytoplasmic 15-hydroxyprostaglandin dehydrogenase from swine kidney was purified to specific activity of 1.2 U per mg protein, by chromatographic techniques. Native molecular weight of enzyme was estimated at 45,000. Enzyme was inhibited by sulfhydryls, diuretics, and various fatty acids. Substrate studies indicated NAD+ specificity and ability to catabolize prostaglandins, except prostaglandin B and thromboxane B. Initial velocity studies gave intersecting plots conforming to a sequential mechanism. 15-keto-prostaglandin exhibited linear noncompetitive production inhibition with respect to either prostaglandin or NAD+; NAD yielded linear competitive production inhibition with respect to NADH. Results, and those of dead-end inhibition and alternated substrate studies, are consistent with an ordered Bi-Bi mechanism: NAD+ is added first, then prostaglandin; then 15-keto-rostaglandin is released, then NADH.
Isolation and Characterization of Two Enzyme Proteins Catalyzing Oxido-Reduction at C-9 and C-15 of Prostaglandins from Swine Kidney
Two swine kidney proteins (PI 4.8 and 5.8) both possessing 9-prostaglandin ketoreductase (9-PGKR) and 15-hydroxyprostaglandin dehydrogenase (15-PGDH) activities were purified to homogeneity. Purification increased specific activities in parallel. Molecular weight, subunit size, amino acid composition, coenzyme and substrate specificity and antigenicity of both proteins were similar. Gel filtration and SDS-polyacrylamide gel electrophoresis molecular weights of 29,500 and 29,000, respectively, suggested a single subunit. Although a variety of prostaglandins served as substrates, the best for 15-PGDH was PGB, while PGA_1-GSH showed the lowest Km for 9-PGKR. Rabbit antibody against the PI 5.8 protein crossreacted with both purified renal enzymes and with extracts from rat spleen, lung, heart, aorta, and liver.
Identification and Characterization of a Calcium/Phospholipid-Dependent Protein Kinase in P1798 Lymphosarcomas
Calcium/phospholipid-dependent protein kinase (PKC) was partially purified from P1798 lymphosarcoma. Phospholipid-dependence was specific for phosphatidylserine. PKC phosphorylated Histone 1, with an apparent K_m of 14.1 μM. Chlorpromazine, a lipid-binding drug, inhibited PKC activity by 100%. Further studies were undertaken to establish analytical conditions which could be applied to the study of PKC in intact cells. The conditions included (1) determining optimum cell concentration for measuring PKC activity, (2) recovering PKC into the soluble fraction of cell extracts, (3) evaluating calcium and phospholipid requirements of PKC in this fraction, and (4) inhibiting PKC in this fraction. Final studies involved treatment of intact cells with potential activators. Both phytohaemagglutinin and a phorbol ester increased PKC activation.
The Regulation of HMG-CoA Reductase by Enzyme-Lipid Interactions
The temperature-dependent catalytic activity of rat liver 3-hydroxy-3 -methylglutaryl coenzyme A reductase (HMG-CoA reductase) displays the nonlinear Arrhenius behavior characteristic of many membrane-bound enzymes. A two-conformer equilibrium model has been developed to characterize this behavior. In the model, HMG-CoA reductase undergoes a conformational change from a low specific activity to a high specific activity form. This conformation change is apparently driven by a temperature-dependent phase transition of the membrane lipids. It has been found that this model accurately describes the data from diets including rat chow, low-fat, high-carbohydrate, and diets supplemented with fat, cholesterol or cholestyramine. The effects characterized by the model are consistent with the regulation of HMG-CoA reductase by enzyme-lipid interactions.
Studies on Lipoprotein Specificity of Human Plasma Lecithin Cholesterol Acyltransferase
Huian plasma high-density lipoprotein (HDL) were isolated by a procedure employing polyanion precipitation and column chromatography. Lipid and protein composition of the HDL isolated by this method was found to be similar to another HDL preparation isolated by ultracentrifugation. However, minor differences were noted, including a higher phospholipid and apoproteinE content and lower triglyceride content of the HDL isolated by column chromatography. Four subfraction of HDL were obtained following chromatography on an anion exchange column. The subfraction four had the highest esterified to free cholesterol ratio, the second highest phospholipid to unesterified cholesterol, and the lowest molecular weight. In addition it was consistently coincided with lecithin: cholesterol acyltransferase (LCAT) activity and found to be the best substrate for the enzyme.
Studies on the Biological Activity of N-nitrosamines
Two aspects of the biological activity of N-nitrosamines were studied. First, the effect of ascorbate on the mutagenicity of N-nitrosopiperidines was studied in the Ames Salmanella/ mammalian microsome mutagenicity test. The addition of ascorbate significantly enhanced the mutagenicity of these compounds. This enhancement was selective for N-nitrosamines suggesting a possible role of ascorbate in N-nitrosamine induced carcinogenicity. Second, the technique of velocity sedimentation in alkaline sucrose density gradients was applied to the detection of N-nitrosamine induced DNA damage in Balb/c 3T3 cells. This technique detected N-nitrosamine induced DNA damage when the cells were made permeable before treatment. This technique compares favorably with other test systems used to evaluate N-nitrosamines and should be useful in further studies of N-nitrosamines.
Structural Analyses of a Human Valine Transfer RNA Gene and of a Transfer RNA Pseudogene Cluster
Two different cloned human DNA segments encompassing transfer RNA gene and pseudogene clusters have been isolated from a human gene library harbored in bacteriophage lambda Charon 4-A. One clone (designated as λhVal7) encompassing a 20.5-kilobase (Kb) human DNA insert was found to contain a valine transfer RNA_AAC gene and several Alu-like elements by Southern blot hybridization analysis and DNA sequencing with the dideoxyribonucleotide chain-termination method in the bacteriophage M13mp19 vector. Another lambda clone (designated as λhLeu8) encompassing a 14.3-Kb segment of human DNA was found to contain a methionine elongator transfer RNA_CAT pseudogene and other as yet unidentified transfer RNA pseudogenes.
Regulation of an S6/H4 Kinase in Crude Lymphosarcoma P1798 Preparations
Purified S6/H4 kinase (Mr 60,000) requires autophosphorylation for activation. A rabbit anti-S6/H4 kinase peptide (SVIDPVPAPVGDSHVDGAAK) antibody recognized both the S6/H4 kinase holoenzyme and catalytic domain. Immunoreactivity with p60 kinase protein, and S6/H4 kinase activity were precisely correlated in fractions obtained from ion exchange chromatography of P1798 lymphosarcoma extracts. An enzyme which catalyzed the MgATP-dependent phosphorylation and activation of S6/H4 kinase coeluted with immunoreactivity from Mono 5, but not Mono Q chromatography. Since S6/H4 kinase is homologous with rac-activated PAK65, the observation that phosphorylation is also required for activation suggests a complex mechanism for in vivo activation of the S6/H4 kinase.
The Nucleotide Sequences of a Mammalian Tyrosine Transfer RNA and a Cluster of Human Transfer RNA Genes
Tyrosine tRNA was isolated from bovine liver and its nucleotide sequence was determined using in vitro 32p_ labeling techniques. Several important structural features of the tRNA are: the presence of gal-Q in the first position of the anticodon, acp3U at position 20, and a pair of adjacent N,N-dimethylguanosines (residues 26 and 27). A human DNA fragment harbored in a lambda phage clone was isolated, and restriction enzyme analysis revealed the presence of three tRNA genes in a 6.0-kb BamHI subfragment. Portions of the 6.0-kb DNA fragment containing the tRNA genes were sequenced by the method of Maxam and Gilbert and analyzed for transcriptional activity in vitro using homologous cytoplasmic extracts. A threonine tRNAUGU gene exhibited high transcriptional activity dependent on its 5'- flanking sequence. The enhanced transcription is not completely inhibited by alpha-amanitin. The value of studying tRNA structure in concert with the cognate tRNA. genes is discussed.
In Vitro Modulation of Rat Liver Glyoxalase II Activity
Glyoxylase II (Glo II, E.C. catalyzes the hydrolysis of S-D-Lactoylglutathione (SLG) to D-Lactate and glutathione. This is the rate limiting step in the conversion of methylglyoxal to D-Lactate. The purpose of the present study was to determine whether or not a relationship exists between some naturally occuring metabolites and in vivo modulation of Glo II. We have observed a non-competitive inhibition (~ 45%) of Glo II in crude preparation of rat liver by GTP (0.3 mM). A factor (apparently protein),devoid of Glo II,when reconstituted with the purified Glo II, enhanced Glo II activity. This coordinate activation and inhibition of Glo II suggest a mechanism whereby SLG levels can be modulated in vivo.
Development of Enabling Technologies to Visualize the Plant Lipidome
Improvements in mass spectrometry (MS)-based strategies for characterizing the plant lipidome through quantitative and qualitative approaches such as shotgun lipidomics have substantially enhanced our understanding of the structural diversity and functional complexity of plant lipids. However, most of these approaches require chemical extractions that result in the loss of the original spatial context and cellular compartmentation for these compounds. To address this current limitation, several technologies were developed to visualize lipids in situ with detailed chemical information. A subcellular visualization approach, direct organelle MS, was developed for directly sampling and analyzing the triacylglycerol contents within purified lipid droplets (LDs) at the level of a single LD. Sampling of single LDs demonstrated seed lipid droplet-to-droplet variability in triacylglycerol (TAG) composition suggesting that there may be substantial variation in the intracellular packaging process for neutral lipids in plant tissues. A cellular and tissue visualization approach, MS imaging, was implemented and enhanced for visualizing the lipid distributions in oilseeds. In mature cotton seed embryos distributions of storage lipids (TAGs) and their phosphatidylcholine (PCs) precursors were distribution heterogeneous between the cotyledons and embryonic axis raising new questions about extent and regulation of oilseed heterogeneity. Extension of this methodology provides an avenue for understanding metabolism in cellular (perhaps even subcellular) context with substantial metabolic engineering implications. To visualize metabolite distributions, a free and customizable application, Metabolite Imager, was developed providing several tools for spatially-based chemical data analysis. These tools collectively enable new forms of visualizing the plant lipidome and should prove valuable toward addressing additional unanswered biological questions.
Molecular and Functional Characterization of Medicago Truncatula Npf17 Gene
Legumes are unique among plants for their ability to fix atmospheric nitrogen with the help of soil bacteria rhizobia. Medicago truncatula is used as a model legume to study different aspects of symbiotic nitrogen fixation. M. truncatula, in association with its symbiotic partner Sinorhizobium meliloti, fix atmospheric nitrogen into ammonia, which the plant uses for amino acid biosynthesis and the bacteria get reduced photosynthate in return. M. truncatula NPF1.7 previously called MtNIP/LATD is required for symbiotic nitrogen fixing root nodule development and for normal root architecture. Mutations in MtNPF1.7 have defects in these processes. MtNPF1.7 encodes a member of the NPF family of transporters. Experimental results showing that MtNPF1.7 functioning as a high-affinity nitrate transporter are its expression restoring chlorate susceptibility to the Arabidopsis chl1-5 mutant and high nitrate transport in Xenopus laevis oocyte system. However, the weakest Mtnip-3 mutant allele also displays high-affinity nitrate transport in X. laevis oocytes and chlorate susceptibility to the Atchl1-5 mutant, suggesting that MtNPF1.7 might have another biochemical function. Experimental evidence shows that MtNPF1.7 also functions in hormone signaling. Constitutive expression of MtNPF1.7 in several species including M. truncatula results in plants with a robust growth phenotype. Using a synthetic auxin reporter, the presence of higher auxin in both the Mtnip-1 mutant and in M. truncatula plants constitutively expressing MtNPF1.7 was observed. Previous experiments showed MtNPF1.7 expression is hormone regulated and the MtNPF1.7 promoter is active in root and nodule meristems and in the vasculature. Two potential binding sites for an auxin response factors (ARFs) were found in the MtNPF1.7 promoter. Chromatin immunoprecipitation-qRT-PCR confirmed MtARF1 binding these sites. Mutating the MtARF1 binding sites increases MtNPF1.7 expression, suggesting a mechanism for auxin repression of MtNPF1.7. Consistent with these results, constitutive expression of an ARF in wild-type plants partially phenocopies Mtnip-1 mutants’ phenotypes.
Functional Characterization of Mtnip/latd’s Biochemical and Biological Function
Symbiotic nitrogen fixation occurs in plants harboring nitrogen-fixing bacteria within the plant tissue. The most widely studied association is between the legumes and rhizobia. In this relationship the plant (legumes) provides the bacteria (rhizobia) with reduced carbon derived from photosynthesis in exchange for reduced atmospheric nitrogen. This allows the plant to survive in soil, which is low in available of nitrogen. Rhizobia infect and enter plant root and reside in organs known as nodules. In the nodules the bacteria fix atmospheric nitrogen. The association between the legume, Medicago truncatula and the bacteria Sinorhizobium meliloti, has been studied in detail. Medicago mutants that have defects in nodulation help us understand the process of nitrogen fixation better. One such mutant is the Mtnip-1. Mtnip-1 plants respond to S. meliloti by producing abnormal nodules in which numerous aberrant infection threads are produced, with very rare rhizobial release into host plant cells. The mutant plant Mtnip-1 has an abnormal defense-like response in root nodules as well as defects in lateral root development. Three alleles of the Mtnip/latd mutants, Mtnip-1, Mtlatd and Mtnip-3 show different degrees of severity in their phenotype. Phylogenetic analysis showed that MtNIP/LATD encodes a protein belonging to the NRT1(PTR) family of nitrate, peptide, dicarboxylate and phytohprmone transporters. Experiments with Mtnip/latd mutants demonstrats a defective nitrate response associated with low (250 μM) external nitrate concentration rather than high (5 mM) nitrate concentration. This suggests that the mutants have defective nitrate transport. To test if MtNIP/LATD was a nitrate transporter, Xenopus laevis oocytes and Arabidopsis thaliana mutant plants Atchl1-5, defective in a major nitrate transporter AtNRT1.1(CHL1), were used as surrogate expression systems. Heterologous expression of MtNIP/LATD in X. laevis oocytes and Atchl1-5 mutant plants conferred on them the ability to take up nitrate from external media with high affinity, thus demonstrating that MtNIP/LATD ...
Studies of the Mechanism of Plasma Cholesterol Esterification in Aged Rats
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 ...
Studies of the Mechanism of the Catalytic Subunit of cAMP Dependent Protein Kinase
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.
Kinetic and Chemical Mechanism of Pyrophosphate-Dependent Phosphofructokinase
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 ...
Mechanism of the Adenosine 3',5'-Monophosphate Dependent Protein Kinase
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 ...
Alternate Substrates and Isotope Effects as a Probe of the Malic Enzyme Reaction
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 ...
Studies of Enzyme Mechanism Using Isotopic Probes
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.
Physical, Chemical and Catalytic Properties of the Isozymes of Bovine Glucose Phosphate Isomerase
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.
Studies on Hog Plasma Lecithin:cholesterol Acyltransferase: Isolation and Characterization of the Enzyme
Lecithin:cholesterol acyltransferase (LCAT) was isolated from hog plasma and basic physicochemical properties and functionally important regions were investigated. Approximately one milligram of the enzyme was purified to apparent homogeneity with approximately a 20,000-fold increase in specific activity. In the plasma, hog LCAT was found to associate with high-density lipoproteins (HDL) probably through hydrophobic interactions with apolipoprotein A-I. HDL was the preferred lipoprotein substrate of the enzyme as its macromolecular substrate. The enzyme was found to contain 4 free sulfhydryl groups; at least one of these appeared to be essential for catalytic activity. The enzyme had a tendency to aggregate at high concentrations. More than half of the tryptophan and none of the tyrosine residues of the enzyme were shown to be exposed to the aqueous environment based on fluorescence and absorbance studies, respectively.
Purification, Characterization and Receptor Binding of Human Colony-Stimulating Factor-1
Human colony-stimulating factor-1 (CSF-1) was purified from the serum-free conditioned medium of a human pancreatic carcinoma cell line. The four-step procedure included chromatography on DEAE Sepharose, Con A Sepharose and HPLC on phenyl column and reverse-phase C-3 column. The purity of human CSF-1 was demonstrated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS—PAGE) as a single diffuse band with a molecular weight (Mr) of 42,000-50,000 and was further confirmed by a single amino-terminal amino acid residue of glutamate. Under reducing conditions, purified CSF-1 appeared on SDS-PAGE as a single protein band with a Mr of 21,000-25,000 and concurrently lost its biological activity, indicating that human CSF-1 consists of two similar subunits and that the intact quaternary structure is essential for biological activity. When treated with neuraminidase and endo-8~D~N—acetylglucosaminidase D, the Mr of CSF-1 was reduced to 36,000-40,000 and to a Mr of 18,000-20,000 in the presence of mercaptoethanol.
Analysis of Human Transfer RNA Gene Heteroclusters
Two phage lambda clones encompassing human tRNA genes have been isolated from a human gene library harbored in bacteriophage lambda Charon-UA. One of the clones (designated as hLeuU) containing a 20-kb human DNA fragment was isolated and found to contain a cluster of four tRNA genes. An 8.2-kb Hindlll fragment encompassing the four tRNA genes was isolated from the 20-kb fragment and subcloned into pBR322 for restriction mapping and DNA sequence analysis. The four tRNA genes are arranged as two tandem pairs with the first pair containing a proline tRNAAGQ gene and a leucine tRNAAAQ gene and the second pair containing another proline tRNAAGG gene and a threonine tRNAuQU gene. The two pairs are separated about 3 kb from each other, and the leucine tRNAAAG gene is of opposite polarity from the other three tRNA genes. The tRNA transcription units were sequenced by a unidirectional deletion dideoxyribonucleotide chain-termination method in the M13mpl8 and 19 vectors. The coding regions of the four tRNA genes contain characteristic internal split promoter sequences and do not encode intervening sequences nor the CCA trinucleotide found in mature tRNAs. The proline t R N A A G G gene is separated from the leucine t R N A A A Q gene by a 725-bp intergenic region and the second proline t R N A A G Q is 315 bp downstream of the threonine t R N A U G U gene. The coding sequences of the two proline tRNA genes are identical. The 3'-flanking regions near the 3*-ends of these four tRNA genes have typical RNA polymerase III termination sites of at least four c o n s e c u t i v e T nt. There is no homology between the 5'-flanking regions of these genes. All four tRNA genes are potentially ...
Analysis of a Human Transfer RNA Gene Cluster and Characterization of the Transcription Unit and Two Processed Pseudogenes of Chimpanzee Triosephosphate Isomerase
An 18.5-kb human DNA segment was selected from a human XCharon-4A library by hybridization to mammalian valine tRNAiAc and found to encompass a cluster of three tRNA genes. Two valine tRNA genes with anticodons of AAC and CAC, encoding the major and minor cytoplasmic valine tRNA isoacceptors, respectively, and a lysine tRNAcuu gene were identified by Southern blot hybridization and DNA sequence analysis of a 7.1-kb region of the human DNA insert. At least nine Alu family members were found interspersed throughout the human DNA fragment. The tRNA genes are accurately transcribed by RNA polymerase III in a HeLa cell extract, since the RNase Ti fingerprints of the mature-sized tRNA transcription products are consistent with the DNA sequences of the structural genes. Three members of the chimpanzee triosephosphate isomerase (TPI) gene family, the functional transcription unit and two processed pseudogenes, were characterized by genomic blotting and DNA sequence analysis. The bona fide TPI gene spans 3.5 kb with seven exons and six introns, and is the first complete hominoid TPI gene sequenced. The gene exhibits a very high identity with the human and rhesus TPI genes. In particular, the polypeptides of 248 amino acids encoded by the chimpanzee and human TPI genes are identical, although the two coding regions differ in the third codon wobble positions for five amino acids. An Alu member occurs upstream from one of the processed pseudogenes, whereas an isolated endogenous retroviral long terminal repeat (HERV-K) occurs within the structural region of the other processed pseudogene. The ages of the processed pseudogenes were estimated to be 2.6 and 10.4 million years, implying that one was inserted into the genome before the divergence of the chimpanzee and human lineages, and the other inserted into the chimpanzee genome after the divergence.
Manipulating Sucrose Proton Symporters to Understand Phloem Loading
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.
Cottonseed Microsomal N-Acylphosphatidylethanolamine Synthase: Identification, Purification and Biochemical Characterization of a Unique Acyltransferase
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.
N-Acylethanolamines and Plant Phospholipase D
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.
A Study of the Intrinsic Fluorescence of O-Acetyl-L-Serine Sulfhydrylase-A from Salmonella typhimurium
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.
Autophosphorylation and Autoactivation of an S6/H4 Kinase Isolated From Human Placenta
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
Fumarate Activation and Kinetic Solvent Isotope Effects as Probes of the NAD-Malic Enzyme Reaction
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.
Desensitized Phosphofructokinase from Ascaris suum: A Study in Noncooperative Allostery
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.
Kinetic and Chemical Mechanism of O-Acetylserine Sulfhydrylase-B from Salmonella Typhimurium
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 ...
O-Acetylserine Sulhydralase-A from Salmonella typhimurium LT-2: Thermodynamic Properties and SPectral Identification of Intermediates
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 ...
Application of Synthetic Peptides as Substrates for Reversible Phosphorylation
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.
Identification of Endogenous Substrates for ADP-Ribosylation in Rat Liver
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.
Characterization of a Human 28S Ribosomal RNA Retropseudogene and Other Repetitive DNA Sequence Elements Isolated from a Human X Chromosome-Specific Library
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.
Kinetic and Chemical Mechanism of 6-phosphogluconate Dehydrogenase from Candida Utilis
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.
Dependence of the Kinetic Mechanism of Adenosine 3',5'-Monophosphate Dependent Protein Kinase Catalytic Subunit in the Direction of Magnesium Adenosine 5'-Diphosphate Phosphorylation on pH and the Concentration of Free Magnesium Ions
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.
Proteomic Responses in the Gill of Zebrafish Following Exposure to Ibuprofen and Naproxen
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.
Identification of Three Symbiosome Targeting Domains in the MtENOD8 Protein and Cell-to-cell MtENOD8 mRNA Movement in Nodules
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 ...
The Relationship of Force on Myosin Subfragment 2 Region to the Coiled-Coiled Region of the Myosin Dimer
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.
Functional Characterization of Plant Fatty Acid Amide Hydrolases
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 ...
Interactions of N-Acylethanolamine Metabolism and Abscisic Acid Signaling in Arabidopsis Thaliana Seedlings
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.