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Identification and Characterization of an Arabidopsis thaliana Mutant with Tolerance to N-lauroylethanolamine

Description: 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 ...
Date: December 2015
Creator: Adhikari, Bikash

A Quantitative Radioimmunoassay for Phosphoglucose Isomerase and Its Utilization in Detecting Cross-Reactive Material in Variant Forms of Phosphoglucose Isomerase and in Human Tissues

Description: 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.
Date: May 1979
Creator: Purdy, Kimberly L.

pH Dependence of the Kinetic Parameters for the Oxalacetate Decarboxylation and Pyruvate Reduction Reactions Catalyzed by Malic Enzyme

Description: 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.
Date: August 1985
Creator: Park, Sang-Hoon

Changes in Body Composition, Plasma Alanine, and Urinary Nitrogen in Rats Subjected to Negative Caloric Balance Through Diet, Diet/Exercise, and Exercise

Description: 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).
Date: August 1982
Creator: Ayres, John J. (John Jay)

Purification and Studies of Methylglyoxal Reductase from Sheep Liver

Description: 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.
Date: May 1983
Creator: Lambert, Patricia A.

NAD+-Dependent 15-Hydroxyprostaglandin Dehydrogenase from Swine Kidney: Characterization and Kinetic Mechanism

Description: 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.
Date: December 1979
Creator: Kung-Chao, Diana T.-Y.

Isolation and Characterization of Two Enzyme Proteins Catalyzing Oxido-Reduction at C-9 and C-15 of Prostaglandins from Swine Kidney

Description: 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.
Date: December 1980
Creator: Chang, David Guey-Bin

Identification and Characterization of a Calcium/Phospholipid-Dependent Protein Kinase in P1798 Lymphosarcomas

Description: 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.
Date: May 1984
Creator: Magnino, Peggy E. (Peggy Elizabeth)

The Regulation of HMG-CoA Reductase by Enzyme-Lipid Interactions

Description: 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.
Date: May 1981
Creator: Smith, Vana L.

Studies on Lipoprotein Specificity of Human Plasma Lecithin Cholesterol Acyltransferase

Description: 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.
Date: May 1981
Creator: Jahani, Mehrnoosh

Studies on the Biological Activity of N-nitrosamines

Description: 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.
Date: August 1980
Creator: Barton, Rodney A. (Rodney Alan)

Structural Analyses of a Human Valine Transfer RNA Gene and of a Transfer RNA Pseudogene Cluster

Description: 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.
Date: December 1987
Creator: Lee, Mike Ming-Jen

Regulation of an S6/H4 Kinase in Crude Lymphosarcoma P1798 Preparations

Description: 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.
Date: December 1998
Creator: Taylor, Allison Antoinette

The Nucleotide Sequences of a Mammalian Tyrosine Transfer RNA and a Cluster of Human Transfer RNA Genes

Description: 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.
Date: August 1986
Creator: Johnson, Gary D. (Gary Dean), 1960-

In Vitro Modulation of Rat Liver Glyoxalase II Activity

Description: 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.
Date: August 1988
Creator: Mbamalu, Godwin E.

Development of Enabling Technologies to Visualize the Plant Lipidome

Description: 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.
Date: August 2013
Creator: Horn, Patrick J.

Studies of the Mechanism of Plasma Cholesterol Esterification in Aged Rats

Description: 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 ...
Date: December 1989
Creator: Lee, Sun Min

Studies of the Mechanism of the Catalytic Subunit of cAMP Dependent Protein Kinase

Description: 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.
Date: August 1989
Creator: Yoon, Moon-Young

Kinetic and Chemical Mechanism of Pyrophosphate-Dependent Phosphofructokinase

Description: 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 ...
Date: December 1988
Creator: Cho, Yong Kweon

Mechanism of the Adenosine 3',5'-Monophosphate Dependent Protein Kinase

Description: 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 ...
Date: May 1988
Creator: Kong, Cheng-Te

Alternate Substrates and Isotope Effects as a Probe of the Malic Enzyme Reaction

Description: 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 ...
Date: August 1988
Creator: Gavva, Sandhya Reddy

Studies of Enzyme Mechanism Using Isotopic Probes

Description: 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.
Date: August 1987
Creator: Chen, Cheau-Yun

Physical, Chemical and Catalytic Properties of the Isozymes of Bovine Glucose Phosphate Isomerase

Description: 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.
Date: August 1987
Creator: Cini, John Kenneth

Studies on Hog Plasma Lecithin:cholesterol Acyltransferase: Isolation and Characterization of the Enzyme

Description: 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.
Date: May 1987
Creator: Park, Yong Bok