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

• 1.

  1. A kinetic investigation has been made of the effect of various phosphagens on the reverse reaction catalysed by creatine kinase (ATP: creatine phosphotransferase, EC 2.7.3.2.).

• 2.

  2. The results indicate that phosphoglycocyamine functions as a substrate of the enzyme with a maximum velocity only 0.18% of that obtained with phospho-creatine. Phosphotautocyamine and phosphoarginine were found not to act as substrates although they could combine with the enzyme as inhibitors.

• 3.

  3. Values have been obtained for the tru kinetic constants associated with the reaction of each phosphagen with both the free enzyme and the enzyme-MgADP complex.

• 1.

  1. The oxidation of cysteine to cysteinesulfinate by rat-liver preparations in the presence of hydroxylamine has been studied. This compound is added to the assay system in order to inhibit the enzymatic destruction of the reaction product.

• 2.

  2. The optimum assay conditions for the enzyme system are reported.

• 3.

  3. The reaction is catalysed by a heat-labile factor found in the soluble fraction and is stimulated by TPNH, ferrous ions and mitochondria or microsomes. The stimulating activity in the particulate fractions is heat-labile.

• 4.

  4. The enzyme system appears to be specific for l-cysteine, as very little sulfinate formation was detected when d-cysteine, l-cystine, glutathione or cysteamine was used as substrate.

• 5.

  5. Among different rat tissues studied, only liver contained significant activity.

• 6.

  6. The enzyme system is inhibited by heavy-metal reagents (EDTA, cyanide, o-phenanthroline) and by the sulfhydryl reagents p-hydroxymercuribenzoate and iodoacetate, but not by arsenite.

Reactions of pyridinium oximes with the alkyl phosphate dimethoate and its derivatives. Effects upon cholinesterases

• 1.

  1. Dimethoate (Rogor), a dimethoxydithioalkyl phosphate, does not inhibit the enzymes acetylcholinesterase (EC 3.1.1.7) and cholinesterase (EC 3.1.1.8). An isomerization product (the dithiol derivative) and an oxidation product (the O analogue) may be responsible for observed inhibitory effects. These derivatives are relatively weak inhibitors of cholinesterases.

• 2.

  2. An attempt was made to reactivate the cholinesterases inhibited by these dimethoate derivatives. All the pyridinium oximes employed produced a stronger inhibition of the enzymes, if the concentrations of alkyl phosphate and oxime were high enough.

• 3.

  3. A reaction scheme with a possible explanation for these effects is discussed.

• 1.

  1. OH- has been shown to produce a 2- to 3-fold increase in the apparent activity in vitro of rat-liver microsomal glucose 6-phosphatase (EC 3.1.3.9) and the related enzymatic activities, inorganic pyrophosphatase, inorganic pyrophosphate-glucose phosphotransferase and adenosine-5′-triphosphate-glucose phosphotransferase.

• 2.

  2. Optimal activation is achieved by pre-treatment of liver microsomes with ammonium or amino acid buffers at pH 9.5–9.8. Inactivation is observed above pH 10.

• 3.

  3. This activation is as great or greater than that produced by deoxycholate or Triton X-100 treatment and the thermal instability introduced by these reagents is avoided.

• 4.

  4. Alteration of the microsomal membranes by treatment with base is accompanied by a decrease in turbidity but does not result in a solubilization of the enzyme. The essential protein-lipid binding is apparently retained and the enzyme, so treated, is relatively stable at 30°.

• 5.

  5. NH₄OH pretreatment of microsomes results in a lowering of the apparent Michaelis constant for glucose 6-phosphatase, similar to that caused by digitonin, deoxycholate and Triton X-100.

• 1.

  1. A peroxidase was purified from beef thyroid tissues by column chromatography on ion-exchange CM-Sephadex. Some of the properties of the purified enzyme were studied.

• 2.

  2. By means of electrophoresis on polyacetate strips the purified enzyme was found to consist of one protein component. This single component demonstrated peroxidase activity when stained with o-dianisidine in the presence of H₂O₂.

• 3.

  3. The molecular weight of the beef thyroid peroxidase was estimated to be approx. 50 000 by sucrose gradient centrifugation using as a reference a purified preparation of dog myeloperoxidase.

• 4.

  4. Protoporphyrin IX was identified as the prosthetic group by spectral analysis of the acid methylethyl ketone extract and the pyridine haemochrome of the enzyme.

• 5.

  5. Spectral properties of the enzyme complexes with CN- and CO, as well as its reduction by sodium dithionite, were distinctively different from those of bovine haemoglobin.

• 6.

  6. Kinetic studies using o-dianisidine as a hydrogen donor showed that the K_m of the enzyme for H₂O₂ was between 1.5 to 2.0 · 10⁻⁵ M.

• 7.

  7. The peroxidase activity of the enzyme was inhibited by SCN-, CN-, N₃-, F-, I-, propylthiouracil and Tapazole but not by p-hydroxymercuribenzoate or ClO₄- as shown by kinetic studies. Thyroid-stimulating hormone did not show any effect.

• 8.

  8. The enzyme catalyzed also the iodination of tyrosine actively in the presence of added H₂O₂ or a H₂O₂-generating system. The iodination of tyrosine by this enzyme occurred optimally at pH 6. Thyroglobulin was iodinated much less actively.

• 9.

  9. The enzymic iodination of tyrosine was inhibited by the inhibitors mentioned above, except iodide, and was not affected by thyroid-stimulating hormone.

Intra- and inter-isozymic molecular relationships of mouse lactate dehydrogenase (EC 1.1.1.27) have been studied using starch-gel electrophoresis.

• 1.

  1. Each isozyme (LDH 2 to LDH ₅) exhibits several narrow bands (sub-bands). These sub-bands present sequential and reversible molecular transformations inside the corresponding isozymic region of migration.

• 2.

  2. Several physicochemical factors, in vitro, determine the direction of these molecular transformations.

• 3.

  3. There are, in cells and tissues, definite specific properties, which are depending on the type of cellular differentiation, and which modify the behavior of the intra-isozymic sub-bands.

• 4.

  4. The enzymatic form migrating towards the cathode (cathodic band) may release, under certain conditions, the isozymes LDH₅, LDH₄, LDH₃. This enzymatic form would appear to be an aggregation between isozymes. There is a relationship between cathodic band formation and the intra-isozymic sub-band transformations.

A hypothesis according to which different molecular conformations of lactate dehydrogenase would account for these results, to a first approximation, is discussed.