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

• 1.

  1. The amperometrically titratable disulfide and sulfhydryl content of beef-liver catalase is not constant. It changes with enzyme activity, pH and after the action of weak oxidizing agents. As a rule, the ratio disulfide: sulfhydryl increases with increasing enzyme activity. With variation of pH latent sulfhydryl groups are released, and free sulfhydryl groups are masked. The pK of this equilibrium reaction is 7.18. Through the action of oxidizing agents, some sulfhydryl groups can be oxidized to disulfide groups.

• 2.

  2. When the sulfhydryl groups of catalase are blocked by Hg²⁺ or C₆H₅Hg⁺ a loss of enzyme activity occurs. The loss of activity with increasing Hg²⁺ or C₆H₅Hg⁺ concentration may be represented as an equilibrium curve. The change of activity on blocking the prosthetic groups with azide can be expressed in the same way.

• 3.

  3. The dissociation of the proton from the sulfhydryl group was followed by electrometric titration. The dissociation constant was found to be 2.00 · 10⁻⁹ M (at 0°).

The ground plasma (150 000 × g supernatant) of the human intestine contains a uridinediphosphate glucuronate glucuronyltransferase (EC 2.4.1.17) which catalyses exclusively the formation of 17β-glucuronides of oestriol, 17β-oestradiol and testosterone. This enzyme was found in the fractions obtained between 60 and 80% saturation of the ground plasma with ammonium sulphate; it could be separated from two other uridine diphosphate glucuronate glucuronyltransferases which, with oestriol as substrate, were capable of forming the 3-glucuronide and the 16α-glucuronide. The two latter enzymes were present in the fractions obtained at 0–30% and 30–60% saturation of the ground plasma with ammonium sulphate. The low specific activity of the enzyme catalysing the formation of the 3-glucuronide suggests the probability of its origin by microsomal leakage.

The kinetics of the uridinediphosphate glucuronate: 17β-hydroxysteroid glucuronyltransferase are described. The formation of the 17β-glucuronide of oestriol shows a maximum at pH 6.8. The Michaelis-Menten constant was found to be 3.5 · 10⁻⁴M, whereas the activation energy of the enzyme amounted to 12.2 kcal/mole.

• 1.

  1. Some of the properties of a proline-sRNA synthetase from rat liver have been studied by either the hydroxamate assay or the proline-dependent ³²PP_i-ATP exchange.

• 2.

  2. The enzyme was noticeably stabilized by sucrose. Although all preparations were stimulated by mercaptoethanol, some aged preparations had an absolute requirement for added mercaptan for activity.

• 3.

  3. The enzyme required either magnesium, manganese, or calcium for activity. Hydroxamate formation was inhibited by PP_i.

• 4.

  4. [¹⁴C]Proline hydroxamate formation was inhibited by 3,4-dehydroproline; azetidine carboxylic acid; thiazolidine carboxylic acid; 2-methyl-, 2-ethyl-, or 2-propylthiazolidine carboxylic acid; thiazolidine; and mercaptoethylamine. Thiazolidine and mercaptoethylamine were competitive inhibitors of proline.

• 5.

  5. The specific activity of extracts from younger rats was higher than that from older rats. Starvation also increased the activity of the extracts.

Isozymes of alcohol dehydrogenase (alcohol: NAD⁺ oxidoreductase, EC 1.1.1.1) and retinol (vitamin A₁) dehydrogenase (retinol: NAD⁺ oxidoreductase) were studied in retina and liver extracts of rat by starch-gel electrophoresis. The retina demonstrated two zones of retinol dehydrogenase activity and a separate zone of alcohol dehydrogenase activity. The liver had single zones each of retinol and alcohol dehydrogenase activity, neither of which coincided with the three zones from the retina. It is concluded on the basis of the electrophoretic findings supplemented with inhibition, heat inactivation, and pH studies that these five zones represent five different enzymes, and that vitamin A is oxidized in the retina and the liver by specific and distinct enzymes. Liver-type alcohol dehydrogenase is absent from the eyes and eye-type is absent from the liver.

• 1.

  1. Mannitol-i-phosphate dehydrogenase (d-mannitol-i-phosphate: NAD⁺ oxidoreductase, EC 1.1.1.17) was purified by a simple procedure from mannitol-grown Escherichia coli.

• 2.

  2. The active protein was apparently homogeneous in the ultracentrifuge, with a molecular weight of 25 000. At pH 9.0 the $\text{K}{}_{\mathrm{m}}$ for mannitol i-phosphate was $\text{3.1·10}{}^{\mathrm{-4}}\text{M}$, and the $\text{K}{}_{\mathrm{m}}$ for NAD⁺ was $\text{1.4·10}{}^{\mathrm{-4}}\text{M}$. At pH 7.0 the $\text{K}{}_{\mathrm{m}}$ for fructose 6-phosphate was $\text{1.6·10}{}^{\mathrm{-4}}\text{M}$, while that for NADH was $\text{4.2·10}{}^{\mathrm{-6}}\text{M}$. Adenosine phosphates inhibited the enzyme reaction in both directions.

• 3.

  3. In mannitol-grown E. coli, mannitol-i-phosphate dehydrogenase may account for nearly 2% of the total soluble protein.