Biochimica et Biophysica Acta (BBA) - Enzymology and Biological Oxidation最新文献

A procedure has been developed for the preparation of essentially pure dihydrofolate reductase (5,6,7,8-tetrahydrofolate: NAD(P)⁺ oxidoreductase, EC 1.5.1.3) from calf-thymus glands. The following criteria of purity were observed: The preparation exhibited only a single protein component by sedimentation ultracentrifugation, sedimentation equilibrium, sucrose gradient centrifugation, and Sephadex chromatography; with a mean mol. wt. of 33 500.

Electrophoresis on acrylamide gel showed only a single boundary. The pH-activity curve, relative substrate specificity, Michaelis constants of the substrates, and the SH character of the enzyme have been determined. Monovalent cations and Mg²⁺ enhanced the enzyme activity. Other divalent cations and sulfhydryl reagents were inhibitory. No stimulation of enzyme activity could be obtained by organic mercurials or urea.

• 1.

  1. Allantoinases (allantoin amidohydrolase, EC 3.5.2.5) from Streptococcus allantoicus, Arthrobacter allantoicus, Escherichia coli, Pseudomonas fluorescens, Pseudomonas acidovorans, frog liver, goldfish liver, Phaseolus hysterinus and Glycine hispida were studied.

• 2.

  2. The enzymes were purified 2.5- to 37-fold by (NH₄)₂SO₄ precipitation and DEAE-cellulose chromatography.

• 3.

  3. The pH optimum curve, Michaelis constant, activation energy, stability and specificity were determined and compared.

• 4.

  4. The enzymes from S. allantoicus, A. allantoicus and E. coli were aspecific; the others degraded (+)-allantoin 4–22 times faster than (−)-allantoin.

• 5.

  5. The allantoinases from Ph. hysterinus and G. hispida were activated by acid pretreatment below pH 5.4.

Amino acid composition of crystalline ATP: l-arginine phosphotransferase

• 1.

  1. ATP: l-arginine phosphotransferase (EC 2.7.3.3) has been crystallized from lobster muscle. Its specific activity was found to be 210–230 μmoles of P transferred per min per mg protein.

• 2.

  2. From the amino acids analysis performed with a yield of 97.56%, the lobster enzyme was shown to have the following composition: Asp 38, Glu 46, Arg 19, Lys 31, Thr 21, Ser 20, His 8, Pro 11, Try 2, Tyr 11, Phe 20, Gly 28, Ala 28, Val 22, Ile 20, Leu 33, Met 8, Cyś 6, amide-N 23.

• 3.

  3. The calculated molecular weight is 42 150 and the specific volume 0.731 ml/g.

• 1.

  1. The oxidation of sulfite, measured by the uptake of oxygen, was employed to detect the formation of free radicals in the iodination of tyrosine by myeloperoxidase in the presence of added H₂O₂.

• 2.

  2. The uptake of oxygen was dependent on sulfite, tyrosine and myeloperoxidase. Tyrosine could be replaced by monoiodotyrosine, diiodotyrosine, 4-hydroxy-3,5-diiodophenylacetic acid or 4-hydroxy-3,5-diiodobenzaldehyde but not by thyroglobulin, phenylacetic acid, phenylalanine or iodide. Iodide was inhibitory.

• 3.

  3. The oxidation of sulfite under these conditions was affected by pH and had an optimal range between pH 5 and 6.5.

• 4.

  4. Sulfite and bisulfite inhibited completely the iodination of tyrosine and of thyroglobulin by myeloperoxidase in the presence of added H₂O₂. Sulfate was without effect.

• 5.

  5. The iodination of tyrosine by myeloperoxidase in the presence of added H₂O₂ showed the same pH optimum as the oxidation of sulfite.

• 6.

  6. A concentrated solubilized preparation of beef thyroid tissues, which catalyzed the peroxidation of $\text{o-}\text{dianisidine}$ and the iodination of tyrosine, could not substitute effectively for myeloperoxidase in the oxidation of sulfite. However, the iodination of tyrosine by this enzyme preparation was inhibited by sulfite and bisulfite.

• 7.

  7. These results are discussed in relation to a free radical reaction mechanism involved in the synthesis of iodotyrosines by myeloperoxidase and by the beef thyroid preparation.

Kinetic and inhibitor studies have been carried out on lipoyl dehydrogenase (NADH:lipoamide oxidoreductase, EC 1.6.4.3) from Saccharomyces cerevisiae. The results obtained are similar in many respects to those reported for the enzyme prepared from pig heart although some differences have been observed. The overall picture of activity with various electron donors and acceptors is qualitively the same with the two enzymes, the yeast enzyme showing considerably greater activity using K₃Fe(CN)₆ and oxidized 3-acetylpyridine-adenine dinucleotide as electron acceptors. Inhibition of the enzyme by arsenite and copper ions is less marked than that of pig-heart lipoyl dehydrogenase, though again the results are qualitively very similar.