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

• 1.

  1. Succinate dehydrogenase (succinate: (acceptor) oxidoreductase, EC 1.3.99.1) reacts immediately with competitive inhibitors to form spectrally detectable enzyme-inhibitor complexes, which are converted to succinate-reduced enzyme on the subsequent addition of succinate.

• 2.

  2. The enzyme-inhibitor complexes formed with malonate, fumarate, maleate and methylene succinate differ from that with oxaloacetate in spectral characteristics, the latter showing a wide, diffuse band from 500 to 750 mμ.

• 3.

  3. Pyrophosphate, a strong competitive inhibitor of succinate dehydrogenase, but which is not in any obvious way structurally related to succinate, has no effect on the spectrum.

• 4.

  4. Dissociation constants of the enzyme-inhibitor complexes determined by spectral titration are in good agreement with kinetically determined inhibitor constants.

• 5.

  5. The changes in spectrum associated with the formation of the enzyme-inhibitor complexes are explainable in terms of changes in polarity near the flavin prosthetic group and by the formation of charge-transfer complexes between the electron-donating inhibitor and the electron-accepting enzyme.

• 6.

  6. The apparent incomplete reoxidation by fumarate of dithionite-reduced enzyme, reported by previous workers, can be explained by the formation of the complex between oxidized enzyme and fumarate.

• 1.

  1. Crude sisal extract has been fractionated into seven protein components by chromatography on DEAE-cellulose.

• 2.

  2. Each component was shown to be essentially homogeneous on rechromatography on DEAE-cellulose and on gel filtration on Sephadex G 100.

• 3.

  3. Five of the components were shown to have proteolytic activity toward casein and four had amidase activity toward α-benzoyl-l-arginine amide.

• 4.

  4. Four of the active components were found to be inhibited by metal binding agents and di-isopropylphosphorofluoridate.

• 5.

  5. None of the active components appeared to depend on a sulphydryl group for activity.

• 6.

  6. The components differed in their specific activities, pH optima, and molelcular weights as indicated by gel filtration on Sephadex G 100.

• 1.

  1. An enzyme catalysing the conversion of α,β-dihydroxyisovalerate and α,β-dihydroxy-β-methylvalerate to α-ketoisovalerate and α-keto-β-methylvalerate has been partially purified from green gram (Phaseolus radiatus), and its characteristics studied.

• 2.

  2. A natural inhibitor, heat stable and inorganic in nature, was observed in the crude extracts.

• 3.

  3. The observed K_m values for α-β-dihydroxyisovalerate and α,β-dihydroxy-β-methylvalerate were 2.4 · 10⁻³ M and 9 · 10⁻⁴ M, respectively.

• 4.

  4. The enzyme required the presence of a divalent metal ion (Mg²⁺, Mn²⁺ or Fe²⁺) for maximal activity. Heavy metals like Ag⁺ and Hg²⁺ were inhibitory.

• 5.

  5. The optimal activity was around pH 8.0 and the optimum temperature at 52°. The activation energy is found to be 12 600 cal/mole.

• 6.

  6. The enzyme was inhibited by p-hydroxymercuribenzoate, N-ethylmaleimide and sulphydryl compounds like cysteine, glutathione, 2-mercaptoethanol and 2,3-dimercaptopropanol. The inhibition by p-hydroxymercuribenzoate could not be reversed by any of the sulfhydryl compounds tested.

Electron transport enzymes, RNA, DNA, and iodinating activity have been assayed in subcellular fractions derived from calf tissue.

About 0.1 μequiv DPNH is oxidized per min by mitochondria prepared from 1 g thyroid in 0.25 M sucrose and assayed at 25°. Cytochrome c reductase system and cytochrome oxidase (EC 1.9.3.1) activities, confined largely to the “mitochondrial” fractions, are more than 10 times greater. DPNH oxidase system is antimycin A-sensitive; cytochrome c reductase system is antimycin A-insensitive. Reduced pyridine nucleotide-dichlorophenol-indophenol reductase (“DT diaphorase”), TPN+-specific isocitrate dehydrogenase (EC 1.1.1.42), and glucose-6-phosphate dehydrogenase (EC 1.1.1.49) are present in the soluble portion in cell and have activities comparable to cytochrome c reductase system and cytochrome oxidase. There is ample basis for formation of large amounts of TPNH within the cell, and its rate of oxidation appears to be low.

RNA was distributed throughout all cell fractions without marked concentration in “microsomes”, and almost 70% was in the cell supernatant.

Iodinating activity was most intense in fractions that may be classified as heavy microsomes, and its distribution did not coincide with the distribution of cytochrome c reductase system or cytochrome oxidase.

Electron-transport enzymes, iodinating activity, and nucleic acid content varied in parallel following physiologic alterations in rat-thyroid function.

The enzyme “DT diaphorase”, which mediates transfer of electrons from TPNH or DPNH to dichlorophenol-indophenol is abundant in the thyroid. In common with the enzyme described by Ernster and coworkers¹ in liver, the enzyme is largely present in the soluble portion of the cell, and is inhibited by bishydroxycoumarin, but not by antimycin A. Addition of FAD markedly augments activity.

The enzyme appears to play little role in oxidative metabolism and could not be related to iodination of tyrosine by thyroid cell particles or a soluble iodinating enzyme derived from the particles.