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P D Weitzman

Publications and source records attributed to P D Weitzman.

At least 19 recordsLinked to original sources
PubMed1991

Hydrogenosomal succinate thiokinase in Tritrichomonas foetus and Trichomonas vaginalis.

Succinate thiokinase displays a diversity of nucleotide specificity and molecular size throughout Nature. Eukaryotes and Gram-positive bacteria possess distinct 'small' (dimeric) thiokinase enzymes which are specific for adenine (ADP) or guanine (GDP) nucleotides, whereas Gram-negative bacteria contain a single 'large' (tetrameric) enzyme which utilizes both nucleotides. Succinate thiokinase activities, both ADP- and GDP-dependent, were shown to be hydrogenosomal in Tritrichomonas foetus and Trichomonas vaginalis. Surprisingly, the 'small' enzyme was found in T. foetus whereas T. vaginalis contained a 'large' enzyme.

Adenosine Diphosphate↗
PubMed1988

Models of proteolysis of oligomeric enzymes and their applications to the trypsinolysis of citrate synthases.

A simple statistical approach was used to generate predictive models of the proteolysis of multisubunit enzymes in order to correlate the loss of enzyme activity with the loss of native subunit. The models were applied to the trypsinolysis of the citrate synthases of pig heart, Bacillus megaterium and Escherichia coli. With the dimeric citrate synthases (pig heart and B. megaterium) trypsinolysis of one of the subunits appears to destroy the activity of the whole enzymic molecule. The hexameric E. coli citrate synthase behaves like a trimer of dimeric units, each of the dimers behaving similarly to the B. megaterium and pig heart enzymes. Palmitoyl-CoA is required for the trypsinolysis of pig heart citrate synthase, and at relatively high concentrations of this compound trypsinolysis of one subunit leaves the other subunit fully active. Palmitoyl-CoA is not required for the trypsinolysis of the other citrate synthases, and high concentrations of this metabolite do not affect the correlation of proteolysis with inactivation of these enzymes.

Animals↗
PubMed1988

A new spectrophotometric assay for citrate synthase and its use to assess the inhibitory effects of palmitoyl thioesters.

We have demonstrated that citrate synthase may be assayed by a simple, discontinuous, spectrophotometric procedure based on the measurement of oxaloacetate utilization with 2,4-dinitrophenylhydrazine. The assay is applicable both to the purified enzyme and to cell extracts, and has the advantage that it can be used in the presence of high concentrations of thiols and thioesters. We have used this new assay in part of our investigations into the inhibitory effects of palmitoyl thioesters on diverse citrate synthases. Both palmitoyl-CoA and palmitoyl thioglycollate inhibit citrate synthases from pig heart, Bacillus megaterium and Escherichia coli, the E. coli enzyme showing the greatest sensitivity to these effectors. With palmitoyl-CoA the extent of inhibition is time-dependent, but the enzymes can be protected from the effect by the substrates oxaloacetate and acetyl-CoA. Using the dinitrophenylhydrazine assay, we have shown that the thioester bond is essential for inhibition; that is, if the palmitoyl thioesters are cleaved to give a mixture of palmitate and a thiol compound, the inhibitions of pig heart and B. megaterium citrate synthases are eliminated and that of the E. coli enzyme is markedly decreased.

Acyl Coenzyme A↗
PubMed1988

Physiological roles of animal succinate thiokinases. Specific association of the guanine nucleotide-linked enzyme with haem biosynthesis.

The discovery of two distinct succinate thiokinases in mammalian tissues, one (G-STK) specific for GDP/GTP and the other (A-STK) for ADP/ATP, poses the question of their differential metabolic roles. Evidence has suggested that the A-STK functions in the citric acid cycle in the direction of succinyl-CoA breakdown (and ATP formation) whereas one role of the G-STK appears to be the re-cycling of succinate to succinyl-CoA (at the expense of GTP) for the purpose of ketone body activation. A third metabolic participation of succinyl-CoA is in haem biosynthesis. This communication shows that in chemically induced hepatic porphyria, when the demand for succinyl-CoA is increased, it is the level of G-STK only which is elevated, that of A-STK being unaffected. The results implicate G-STK in the provision of succinyl-CoA for haem biosynthesis, a conclusion which is further supported by the observation of a high G-STK/A-STK ratio in bone marrow.

Acyl Coenzyme A↗
PubMed1988

Two distinct succinate thiokinases in both bloodstream and procyclic forms of Trypanosoma brucei.

Two succinate thiokinase activities specific for either adenine or guanine nucleotides have been found in Trypanosoma brucei. Key glycolytic and citric acid cycle enzymes were measured to show repression of glycolysis and derepression of the citric acid cycle in the procyclic form, relative to the bloodstream form. A marked rise in adenine-linked succinate thiokinase activity accompanied a rise in activity of citric acid cycle enzymes. However, guanine-linked succinate thiokinase was found to increase only slightly in activity. These results implicate the adenine-linked enzyme as an essential component of the citric acid cycle, whereas the guanine-linked enzyme appears to be under separate control. This communication also reports for the first time the occurrence of citrate synthase activity in the bloodstream (long slender) form of T. brucei.

Adenine Nucleotides↗
PubMed1988

Purification and partial characterization of 4-aminobutyrate:2-oxoglutarate aminotransferase from sheep brain and locust ganglia.

We report here the first purification to homogeneity of 4-aminobutyrate: 2-oxoglutarate aminotransferase (EC 2.6.1.19) (GABA-T) from an invertebrate source (locust) and its initial comparison with that of GABA-T from mammalian brain (sheep). The enzyme from both organisms was found to be a dimer of similar-sized subunits, with a native Mr of approx. 97,000. The pI of GABA-T from the locust was 6.7 and that of the sheep enzyme was 5.5. Michaelis constants for 4-aminobutyric acid (GABA) and 2-oxoglutarate were respectively 0.79 +/- 0.16 mM and 0.27 +/- 0.08 mM for the locust enzyme and 2.2 +/- 0.24 mM and 0.22 +/- 0.11 mM for the sheep enzyme. 5-(Aminomethyl)-3-isoxazolol (muscimol) was a competitive inhibitor of both enzymes, whereas 5-amino-1,3-cyclohexadienylcarboxylic acid (gabaculine) acted as a potent suicide substrate. However, 3-aminopropane-1-sulphonic acid, diaminobutyric acid, 1,2,3,4-tetrahydro-1-methyl-3-pyridinecarboxylic acid (isoguvacine), beta-(aminomethyl)-4-chlorobenzenepropanoic acid (baclofen), bicuculline and picrotoxin did not inhibit either enzyme at concentrations below 100 mM. Polyclonal antisera raised against GABA-T from the sheep failed to cross-react with the enzyme from locust in either an Ouchterlony immunodiffusion plate or a competitive enzyme-linked immunosorbent assay. The purification procedures differed considerably. Ion-exchange chromatography, which was found suitable for the purification of GABA-T from the sheep, was ineffective with locust enzyme, which was finally purified by hydrophobic-interaction chromatography and chromatofocusing.

4-Aminobutyrate Transaminase↗
PubMed1987

GABA binding to receptor sites in locust supraoesophageal ganglia.

The binding of 4-amino-n-[2,3-3H]butyric acid (GABA) to receptor sites in the supraoesophageal ganglia of the locust Schistocerca gregaria is reported. Binding is saturable with a Kd of 30 nM and a Bmax of 150 fmol/mg protein. Binding is sodium-independent with a pH optimum of 6.8 and the pharmacological properties of the site suggest a receptor rather than an uptake or transport protein. The assay is being utilised in a comparative study of the binding sites of the GABA receptor and the enzyme 4-aminobutyrate: 2-oxoglutarate amino-transferase (EC 2.6.1.19, GABA-T). GABA binds to at least 4 proteins in the nervous system of vertebrates: the GABAA and GABAB receptors, GABA-T the enzyme involved in the GABA shunt, and the GABA transport system. In the invertebrates the status of these GABA-binding proteins is less well established. There are reports of a GABA receptor complex resembling the GABAA receptor; GABA-T activity has been reported and we have recently purified the enzyme from locust ganglia; it is assumed that GABA uptake systems are present in invertebrates. Proteins with different functions which specifically bind the same ligand are interesting from an evolutionary point of view. Are they distinct gene products or is the sub-unit of the receptor which binds GABA an enzyme which has lost the ability to bind pyroxidal phosphate? Do either receptor or enzyme differ significantly from their mammalian counterparts?

Animals↗
PubMed1987

Patterns of diversity of citric acid cycle enzymes.

The citric acid cycle performs a dual role in cell metabolism, acting as a source of both 'energy' and biosynthetic starting materials. The widespread occurrence of the cycle throughout Nature is an excellent example of the unity of biochemistry, but closer examination reveals that there is considerable diversity in the citric acid cycle of different organisms with respect to metabolic role, molecular enzymology and mode of regulation. Two enzymes of the cycle--citrate synthase and succinate thiokinase--have been found to exhibit particularly striking patterns of diversity in structure and catalytic and regulatory function. Some of these patterns show a correlation with the taxonomic groupings of the organisms and with their physiological characteristics. Comparative enzyme studies have a contribution to make to an ultimate understanding of the cycle and its cellular operation, and there are substantial benefits to be gained from interactive studies on both prokaryotic and eukaryotic systems.

Animals↗
PubMed1986

Distinct physiological roles of animal succinate thiokinases. Association of guanine nucleotide-linked succinate thiokinase with ketone body utilization.

Two distinct succinate thiokinases have recently been shown to exist in animal tissues, one specific for guanine nucleotide and the other for adenine nucleotide. Their physiological roles have here been investigated by comparing the levels of the two enzymes in liver and brain of normal and diabetic rats. A marked rise in the level of brain guanine nucleotide-linked succinate thiokinase in the diabetic condition is consistent with an enhanced utilization of ketone bodies and hence with the associated elevated demand for succinyl-CoA for the activation of acetoacetate. Taken together with the reported mitochondrial values of the ATP/ADP and GTP/GDP ratios, the results are interpreted to indicate that the adenine nucleotide-linked enzyme functions as a component of the citric acid cycle whereas the guanine nucleotide-linked enzyme functions in the opposite metabolic direction to produce succinyl-CoA from succinate.

Adenosine Triphosphate↗
PubMed1986

Organization of citric acid cycle enzymes into a multienzyme cluster.

The possibility that some of the enzymes of the citric acid cycle may be loosely associated into a multienzyme cluster has been investigated using extracts prepared by gentle disruption of cells. Gel filtration and sucrose density gradient centrifugation have shown that five sequential enzymes of the cycle specifically associate into a cluster: fumarase, malate dehydrogenase, citrate synthase, aconitase and isocitrate dehydrogenase. Ultrasonication destroys the abilities of the enzymes to associate. The cluster could catalyse the sequence of reactions leading from fumarate to oxoglutarate and has been found in extracts of several bacterial species as well as rat liver mitochondria.

Acinetobacter↗
PubMed1986

Occurrence of two distinct succinate thiokinases in animal tissues.

Although succinate thiokinase from mammalian sources has hitherto been described as showing substrate specificity for guanine nucleotide, a range of mammalian tissues has here been found to display succinate thiokinase activity with both guanine and adenine nucleotides as substrates. Evidence is presented for the existence of two distinct succinate thiokinases and this is confirmed by their separation by affinity chromatography. Each enzyme is specific for one nucleotide and is inhibited by the non-substrate nucleotide. The physiological roles of the two enzymes is yet to be established.

Adenosine Diphosphate↗
PubMed1986

Separation of isoenzymes of citrate synthase and isocitrate dehydrogenase by fast protein liquid chromatography.

Fast protein liquid chromatography (FPLC) has been shown to be a rapid and effective method of separating isoenzymes of citrate synthase and isocitrate dehydrogenase in extracts of Pseudomonas aeruginosa and Acinetobacter calcoaceticus. The advantages of FPLC over conventional methods of fractionation are discussed and it is suggested that this may be a valuable and more general technique for isoenzyme resolution.

Acinetobacter↗
PubMed1986

Molecular size diversity of citrate synthases from Pseudomonas species.

Two forms of citrate synthase (EC 4.1.3.7) have been found in several species of Pseudomonas, a 'large' form (Mr congruent to 250,000) which is generally inhibited by NADH and reactivated by AMP, and a 'small' form (Mr congruent to 100,000) which is insensitive to these nucleotide effectors. Other species of Pseudomonas were found to contain either the 'large' or the 'small' form. Gel filtration and ion-exchange with the technique of fast protein liquid chromatography were used to resolve the enzymes. Where both citrate synthases were present, there did not appear to be an equilibrium between the two forms. The results reveal a new and complex diversity of citrate synthase within the genus Pseudomonas.

Chromatography, Gel↗
PubMed1983

Modulation of isocitrate dehydrogenase activity in Acinetobacter calcoaceticus by acetate.

The addition of acetate to a culture of Acinetobacter calcoaceticus grown in medium containing limiting succinate as the sole carbon and energy source leads to an increase in the specific activity of isocitrate dehydrogenase. This is in contrast to similar studies with several other microorganisms in which acetate induces an ATP-dependent phosphorylation and concomitant decrease in the specific activity of this enzyme.

Acetates↗
PubMed1983

Citrate synthase from a Gram-positive bacterium. Purification and characterization of the Bacillus megaterium enzyme.

Citrate synthase was purified to homogeneity from a Gram-positive bacterium (Bacillus megaterium) for the first time. The Mr of the native enzyme was determined to be 84 000 (S.E.M. +/- 5000). Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and gel filtration in guanidinium chloride revealed a single protein species of Mr 40 300 (S.E.M. +/- 4400), indicating a dimeric enzyme. This dimeric structure was confirmed by cross-linking the native enzyme with dimethyl suberimidate and with glutaraldehyde, followed by electrophoretic analysis. The enzyme follows Michaelis-Menten kinetics with respect to both substrates, acetyl-CoA and oxaloacetate, and is sensitive to non-specific inhibition by a range of adenine nucleotides. In both molecular and catalytic properties the citrate synthase closely resembles the enzyme from eukaryotic sources and contrasts markedly with the larger, hexameric, enzyme from Gram-negative bacteria.

Amino Acids↗
PubMed1983

Affinity chromatography of acyl-CoA utilizing enzymes on Procion Red-agarose.

The dye Procion Red HE3B immobilized on agarose and available as Matrex Gel Red A is shown to bind citrate synthase and succinate thiokinase from a number of diverse organisms. Salt-gradient elution removes the enzymes in high yields and with substantial purification. The elution profiles follow a pattern similar to that of the molecular size variations of the enzymes.

Chemical Phenomena↗
PubMed1983

Reversible effects of cross-linking on the regulatory cooperativity of Acinetobacter citrate synthase.

Citrate synthase was purified from Acinetobacter calcoaceticus and treated with the cleavable cross-linking reagent dithiobis(succinimidyl propionate). Cross-linking of the enzyme resulted in the abolition of the sigmoidal responses to inhibition by NADH and re-activation by AMP displayed by the native enzyme. Inhibition and re-activation were still observed but without any cooperativity. Cleavage of the disulphide bonds in the cross-links by treatment with dithiothreitol restored the sigmoidal characteristics of both inhibition and re-activation.

Acinetobacter↗