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K F Tipton

Publications and source records attributed to K F Tipton.

At least 199 records · Page 11Linked to original sources
PubMed1981

The activation of ox-brain NAD+-dependent isocitrate dehydrogenase by magnesium ions.

Two independent methods were used to assess the dependence of the activity of ox brain NAD+-dependent isocitrate dehydrogenase on the concentration of magnesium ions. The results indicated the complex between magnesium and isocitrate to be the true substrate for the enzyme. Free isocitrate is neither a substrate nor an inhibitor of the enzyme but free magnesium ions inhibit competitively with respect to the magnesium-isocitrate complex. The inhibition of the enzyme by ATP and citrate appears to be largely explicable in terms of their effects on the concentration of the complex between Mg2+ and isocitrate. The dependence of the activation of the enzyme by ADP on the concentration of magnesium ions suggests that free ADP, rather than its complex with Mg2+, is the activator.

Adenosine Diphosphate↗
PubMed1981

Kinetic comparisons between soluble and membrane-bound glutaminase preparations from pig brain.

The kinetic properties of membrane-bound glutaminase from pig brain have been compared with those of the soluble enzyme that had been preincubated with either Tris/HCl buffer or Tris/phosphate/borate buffer. The two preparations of the soluble enzyme were similar in the dependence of their activities on pH and in their inhibition by borate and some glutamine analogues. They differed markedly in their responses to phosphate and their inhibition by glutamate. The membrane-bound enzyme differed from the other preparations in showing a sigmoid dependence of initial velocity on glutamine concentration at higher pH values and being activated by borate ions. The apparently cooperative behaviour that has been previously reported for soluble preparations of glutaminase may have resulted from failure to measure the true initial rate of the reaction. The Km values for glutamine and the inhibitor and activator constants for glutamate and phosphate respectively were such as to indicate that changes in the concentrations of these compounds might affect the glutaminase activity in vivo. None of the enzyme preparations was inhibited significantly by ammonium ions at concentrations up to 50 mM. Considerable differences were found between the three preparations in their activation by compounds containing sulphate or sulphonate groups.

Animals↗
PubMed1981

The effect of ligands on the irreversible inhibition of the NAD+-dependent isocitrate dehydrogenase from ox brain.

The effects of ligands on the irreversible inhibition of the NAD+-dependent isocitrate dehydrogenase from ox brain were studied. Isocitrate in the presence of Mg2+ ions was found to protect against denaturation at 40 degrees C and this protection was enhanced by ADP which also protected on its own. None of the substrates or the activator ADP afforded protection against inhibition by diethylpyrocarbonate. Inactivation by this compound and by elevated temperatures did not obey first-order kinetics with respect to time. Inhibition by iodoacetate appeared to obey first-order kinetics both with respect to time and to inhibitor concentration. Protection against inhibition was afforded by isocitrate, NAD+ and ADP. The dependence of the extent of protection on the concentration of the magnesium-isocitrate complex was sigmoid at both pH 6.5 and pH 7.5 indicating the apparent homotropic cooperativity seen in initial-rate kinetic experiments to be a reflection of cooperative binding of this substrate. ADP reduced the affinity of the enzyme for this substrate without affecting the degree of this cooperativity. The dependence of the extent of protection by ADP upon its concentration obeyed Michaelis-Menten kinetics. The significance of these observations in terms of the kinetic and allosteric mechanism followed by this enzyme is discussed in the light of previous kinetic studies.

Adenosine Diphosphate↗
PubMed1981

Kinetic studies with rat-brain succinic-semialdehyde dehydrogenase.

A simple procedure is described that gives an approximately 100-fold purification of rat brain succinic-semialdehyde dehydrogenase with a high yield. The enzyme exhibits a relatively low Km value for succinic semialdehyde (2.5 microM) and is inhibited by high concentrations of that substrate in an uncompetitive manner with respect to NAD+ (Ki = 150 microM). p-Hydroxybenzaldehyde was shown to give competitive inhibition with respect to succinic semialdehyde and uncompetitive inhibition with respect to NAD+. Initial rate studies in the presence of a fixed concentration of this inhibitor allowed a more accurate estimation of the kinetic parameters for the uninhibited reaction. The results of these studies, together with analysis of the dead-end inhibition by AMP and the effects of NAD+ and 3-acetylpyridine--adenine dinucleotide as alternative acceptors in the reaction, were consistent with the enzyme-catalysed reaction obeying a compulsory-order mechanism in which NAD+ was the first substrate to bind to the enzyme and NADH was the last product to dissociate from it.

Aldehyde Oxidoreductases↗
PubMed1980

Purification of glutamate dehydrogenase from ox brain and liver. Evidence that commercially available preparations of the enzyme from ox liver have suffered proteolytic cleavage.

1. A rapid procedure, involving ion-exchange chromatography on DEAE-cellulose and affinity chromatography on GTP-Sepharose, was used to purify glutamate dehydrogenase from ox brain and liver. 2. Preparations purified in this way differed from those of the ox liver enzyme that were obtained from commercial suppliers in their mobilities on polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. This difference appears to result from the occurrence of limited proteolysis during the preparation of the latter enzyme samples. 3. N-Terminal sequence analysis showed the presence of four amino acid residues in the enzyme prepared in this study that were not present in those obtained from the commercial sources and which have not been reported in previous studies on the sequence of the ox liver enzyme. 4. A preliminary examination of the enzyme prepared in this way indicated that the Michaelis constants for the substrates are similar to those obtained from the commercial preparation, but that the response to allosteric effectors was modified.

Amino Acid Sequence↗
PubMed1980

The effect of pH on the allosteric behaviour of ox-brain NAD+-dependent isocitrate dehydrogenase.

The sigmoid dependence of the initial rate of the reaction catalysed by NAD+-dependent isocitrate dehydrogenase on the concentration of isocitrate is greatly reduced as the pH is decreased. The apparent pK for this process is 7.3. At pH values of 6.0 and 6.5 the enzyme exhibits hyperbolic kinetics with respect to isocitrate at relatively high concentrations of NAD+, but the dependence becomes sigmoid at lower NAD+ concentrations. The allosteric activators ADP and citrate have only small effects on the activity of the enzyme at pH 6.5 but in the latter case the effects are increased by decreasing the concentration of NAD+. The dependence of initial velocity on the NAD+ concentration is hyperbolic at all pH values in the pH range 6--8.5. NADH is a competitive inhibitor of enzyme activity with respect to NAD+, and its presence induces a sigmoid dependence of initial velocity on isocitrate concentration at pH 6.5 and in the presence of high concentrations of NAD+. Kinetic studies at pH 6.5 indicate that the apparent maximum velocity of the reaction with respect to NAD+ is insensitive to changes in the isocitrate concentration and that with isocitrate as the substrate is relatively insensitive to changes in the NAD+ concentration under conditions where the behaviour appears to be hyperbolic. threo-Ds-Isocitrate is the true substrate for the enzyme and the corresponding Ls isomer is neither a substrate nor an inhibitor.

Adenosine Diphosphate↗
PubMed1978

The purification and properties of pig-liver catechol-O-methyl transferase.

A procedure utilising affinity chromatography is described for the large-scale purification of pig-liver catechol-O-methyl transferase. The enzyme prepared by this method appears to be homogeneous by polyacrylamide gel electrophoretic criteria and gel chromatography. It is stable for prolonged periods when stored at -5 degrees C in 20% (v/v) glycerol. The enzyme has a molecular weight of about 23000 and does not appear to be a compound of subunits, or to associate to any appreciable degree. The pH optimum of the enzyme's activity is approximately pH 7.1--7.4, it does not catalyse the methylation of benzimidazole and has a Km of 0.64 mM and 0.056 mM towards 3,4-dihydroxyphenylacetic acid and S-adenosyl-L-methionine, respectively. Amino acid analysis showed the presence of five cysteine residues.

Amino Acids↗