Biochimica et Biophysica Acta (BBA) - Specialized Section on Enzymological Subjects最新文献

The results in this paper show that extracts of baker's yeast contain a pyruvate carboxylase which catalyzes the carboxylation of pyruvate to form oxaloacetate and the exchange of oxaloacetate and pyruvate. The carboxylation reaction requires GSH, MgCl₂, ATP and is stimulated by acetyl-CoA. In the exchange reaction the transfer of the β-COOH group of oxaloacetate to pyruvate does not require ATP and is not stimulated by acetyl-CoA. Both reactions are inhibited by low concentrations of avidin and p-chloromercuribenzoate.

l-Rhamnulokinase (ATP:l-rhamnulose phosphotransferase, EC 2.7.1.5.) has been purified 34-fold from extracts of Escherichia coli K40. The purified enzyme catalyzes the phosphorylation of l-rhamnulose, but not of l-rhamnose, in the presence of Mg²⁺ and adenosine 5′-triphosphate. Uridine triphosphate, cytidine 5′-triphosphate, guanosine 5′-triphosphate, and thymidine triphosphate also can act as phosphoryl donors, but less well than adenosine 5′-triphosphate.

The product of the phosphorylation of l-rhamnulose by the enzyme has been isolated and characterized by periodic acid oxidation studies as l-rhamnulose i-phosphate.

• 1.

  1. The properties of an extracellular peptidase (peptidase B) of Penicillium janthinellum which was separated by paper electrophoresis from a preparation of peptidase A, have been studied.

• 2.

  2. Unlike peptidase A, peptidase B will not activate trypsinogen.

• 3.

  3. Peptidase B is active on a number of peptides, none of which are substrates for peptidase A. Peptidase B is especially active on Cbz-l-glutamyl-l-phenylalanine, Cbz-l-glutamyl-l-tyrosine and Cbz-l-tyrosyl-l-glutamic acid, and while inactive on Cbz-glycyl-l-phenylalanine, it promotes hydrolysis of the free dipeptide glycyl-l-phenylalanine.

• 4.

  4. The hydrolysis of Cbz-l-glutamyl-l-tyrosine by peptidase B takes place optimally at pH 4.7. The extent of inhibition imposed by a range of anions on this hydrolysis has been recorded, and the effect of selected inhibitors on the kinetics of the reaction studied. The inhibition imposed by salts of the fatty acid series is proportional to the molecular weight of the inhibitor.

• 5.

  5. None of a series of metal salts tested increased the activity of the enzyme, and ferric, cadmium and barium ions were inhibitory.

• 6.

  6. The enzyme is not inhibited by sulfhydryl reagents.

• 7.

  7. A value for K_m of 5·10⁻⁴ M of Cbz-l-glutamyl-l-tyrosine was obtained.

Some previous studies have seemed to prove the evidence of an active peptide occurring from trypsin (EC 3.4.4.4) hydrolysate. It had been obtained either by peptic hydrolysis of acetyltrypsinogen or by tryptic autolysis.

In the present paper, the different experiments are tried again and discussed. With both methods, the enzymatic activity finally obtained is related with the presence of unhydrolysed trypsin in the active fraction. Spectral analysis of this fraction shows that an important part of the enzyme is denatured.

• 1.

  1. The oxidation of NADH by fumarate has been demonstrated in both intact and digitonin fragments of beef-heart mitochondria and this oxidation is accompanied by a concomitant phosphorylation of ADP.

• 2.

  2. P:2e ratios of 0.8 and 0.7 have been shown for the intact mitochondria and digitonin fragments, respectively.

• 3.

  3. Low concentrations of antimycin and 2-n-heptyl-4-hydroxyquinoline-N-oxide increase the P:2e ratios by 0.1 while higher concentrations of these inhibitors decrease the ATP synthesis.

• 4.

  4. Hexylguanidine inhibits the phosphorylation reaction when added prior to phosphate acceptor whereas neither Synthalin (decamethylene diguanidine) nor phenylethylbiguanide have any appreciable effect on the P:2e ratio.

• 1.

  1. An enzymic assay of xanthine dehydrogenase (xanthine: NAD oxidoreductase) from Drosophila melanogaster is reported which is linear with respect to enzyme concentration and is sensitive enough to measure activity in single flies. The assay is based on the change in absorbance at 340 mμ as nicotinamide-adenine dinucleotide is reduced or at 395 mμ as thio-nicotinamide-adenine dinucleotide is reduced with either xanthine or hypoxanthine as substrate. Tests of realiability have been performed on single flies from various inbred and non-inbred stocks, and significant stock differences have been demonstrated.

• 2.

  2. A method of purification has been devised resulting in a 530-fold purification of the enzyme.

• 3.

  3. Using this purified preparation, Michaelis constants for hypoxanthine, xanthine, nicotinamide-adenine dinucleotide, and thio-nicotinamide-adenine dinucleotide are shown to be 2.0·10⁻⁵ M, 2.36·10⁻⁵ M, 2.5·10⁻⁴ M and 2.0·10⁻⁵ M, respectively.

• 4.

  4. Stoichiometric studies indicate the reduction of 1 mole of nicotinamide-adenine dinucleotide for each mole of xanthine converted to uric acid, and the reduction of 2 moles of thio-nicotinamide-adenine dinucleotide for each mole of hypoxanthine converted to uric acid.