Phosphoenolpyruvate carboxylase from the Crassulacean plant Bryophyllum fedtschenkoi has been purified to homogenetity by DEAE-cellulose treatment, (NH4)2SO4 fractionation,, and chromatography on DEAE-cellulose and hydroxyapatite. Poly(ethylene glycol) is required in the extraction medium to obtain maximum enzyme activity. The purified enzyme has a specific activity of about 26 units/mg of protein at 25 degrees C. It gives a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, corresponding to a mol.wt. of 105,000, and gives a single band on non-denaturing gel electrophoresis at pH8.4. Cross-linking studies at pH8.0 indicate that the subunit structure is tetrameric but that the dimer may also be an important unit of polymerization. Gel filtration results at pH6.7 confirm that the native enzyme is tetrameric with a concentration-dependent dissociation to a dimer. The kinetic behaviour is characterized by (i) relatively small variations in maximum velocity between pH5.5 and 9.0 with a double optimum, (ii) a reversible temperature-dependent inactivation between 30 and 45 degrees C, (iii) inhibition by malate, which is pH-sensitive, and (iv) almost Michaelis-Menten behaviour with phosphoenolpyruvate as the varied ligand but sigmoidal behaviour under suitable conditions with malate as the varied ligand. The findings are related to other studies to the possible role phosphoenolpyruvate carboxylase in controlling a circadian rhythm of CO2 fixation.
1. 4-Iodoacetamido salicylic acid is an irreversible inhibitor of ox liver glutamate dehydrogenase.2 . The rate of this inactivation is analysed in terms of a preliminary reversible formation of an enzyme/inhibitor complex.3. Absorption and fluorescence spectral measurements give a 1 :1 equivalence of modification and inactivation. 4. a-Oxoglutarate protects against this inactivation and this was used to derive a binding constant of 4.3 mM for this substrate in the absence of coenzyme.5. The rate a t which the response of the enzyme to the allosteric inhibitor GTP is lost is greater than the rate of loss of activity. This was used to provide information about the nature of the subunit interactions in the enzyme.6. The response of glutamate dehydrogenase to the activator ADP is not affected by inactivation.The activity of glutamate dehydrogenase is affected by a variety of small molecules [l]. Among these, GTP is an allosteric inhibitor [ Z ] while the family of dicarboxylic acids and related molecules are competitive inhibitors of a-oxoglutarate [3]. Among these latter are derivatives of salicyclic acid as these possess a pair of negative charges with approximately the same separation as is found in a-oxoglutarate. Thus 4-iodoacetamido salicylic acid is a competitive inhibitor of the enzyme [4]. I n addition, in the presence of NADH, it leads to irreversible inhibition. This was thought to be the result of the alkylation of an amino acid residue at, or near the active site. This paper reports the effect of modification of glutamate dehydrogenase by iodoacetamido salicylate on the properties of the enzyme.A preliminary report of this work has already been published [5].
NATERIALS AND METHODS4-Iodoacetamido salicylic acid was obtained from Koch-Light Laboratories Ltd, (Colnbrook, Bucks.). Sephadex (3-25 (medium) was obtained from Pharmacia, (Uppsala, Sweden). The origin of glutamate dehydrogenase and all other reagents was as described previously [el.Phosphate buffer, pH 7.6 and 0.1 ionic strength, was made up in deionised glass distilled water, using Enzyme. Glutamate dehydrogenase or L-glutamate: EC 1.4.1.3).
NAD(P)H oxidoreductase (deaminating) (the "Analar" sodium salts (British Drug Houses, Poole, Dorset) as also was Tris-C1 buffer, pH 9 and ionic strength 0.01, to which sodium chloride was added to give an ionic strength of 0.1.The preparation of solutions of glutamate dehydrogenase (from ox liver) and the estimation of their concentrations were as described previously [el.Other solutions were made up gravimetrically in phosphate buffer, except for NADH which was made up in Tris-C1. The concentrations of NADH and GTP solutions were checked spectrophotometrically .The pH of solutions was measured using an EIL direct reading pH meter.Absorbancies were measured using a Hilger and Watts spectrophotometer, while spectra were recorded on a Unicam SP 800 recording spectrophotometer.The activities of samples of the enzyme were measured by following the decrease in absorbance at 340 nm on a Hilger-Gilford kinetic spe...
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