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

The induced acid phosphatase (EC 3.1.3.2) of Euglena gracilis has been solubilized and partially purified. The enzyme has a wide range of substrate specificity and, as predicted from earlier studies on whole cells, the ratio of its activity at pH 5 to its activity at pH 7 in presence of 5 mM fluoride is 1.1 for $\text{p-}\text{nitrophenylphosphate}$ as substrate. The enzyme is competitively inhibited by arsenate and phosphate, but exhibits mixed competitive-non-competitive inhibition with molybdate. The enzyme migrates towards the cathode when electrophoresis is performed on cellulose acetate strips at pH 8.2. Euglena also contains several other acid phosphatases. The two major constitutive acid phosphatases, which remain particle-bound after a variety of extraction procedures, differ in their thermal stability from each other and from the induced phosphatase. The ratio of activity at pH 5 to the activity at pH 7 plus 5 mM fluoride for the mixture of these two constitutive enzymes is 15. These observations establish that the increase in acid phosphatase activity occurring in Euglena in response to phosphate deprivation is due to the synthesis of a separate enzyme. The heat of thermal inactivation of the purified induced enzyme is about four times larger than the heat of denaturation computed from the effect of temperature on the rate of reversion of the induced phosphatase in vivo.

• 1.

  1. Nuclear magnetic resonance (NMR) spectra were measured to demonstrate the specific interaction of substrates (mononucleotides) with bovine pancreatic ribonuclease I (ribonucleate pyrimidinenucleotido-2′-transferase (cyclizing), EC 2.7.7.16) and Taka-Diastase ribonuclease T₁ (ribonucleate guaninenucleotido-2′-transferase (cyclizing), EC 2.7.7.26).

• 2.

  2. The line widths at half height of certain low field signals exhibited by substrates were taken as a measure of the extent of the interaction.

• 3.

  3. The NMR spectrum of ribonuclease T₁ has been measured and interpreted in the light of its known primary structure for the first time.

• 4.

  4. The spectra of 2′(3′)UMP and uridine were measured in the presence of native ribonuclease I and thermally denaturated ribonuclease I, and it was found that 2′(3′)UMP signals underwent narrowing after heat treatment whereas the spectrum of uridine remained almost unchanged, indicating that only 2′(3′)UMP interacts effectively with the enzyme molecule and that this interaction does not occur after denaturation of the enzyme.

• 1.

  1. Ornithine acetyltransferase (proposed name, $\text{α-N-}\text{acetyl-}\text{l}\text{-ornithine:}\text{l}\text{-glutamate}$N-acetyltransferase) which catalyzes the first step in arginine biosynthesis, the formation of N-acetylglutamate from $\text{α-N-}\text{acetyl-}\text{l}\text{-ornithine}$ and l-glutamate, has been isolated from the freshwater alga Chlamydomonas reinhardti.

• 2.

  2. The enzyme has a broad pH optimum between 7.5 and 9. The K_m value of the enzyme at pH 7.5 is 1.3 · 10⁻² M for glutamate and 5.5 · 10 ⁻³ M for $\text{α-N-}\text{acetyl-}\text{l}\text{-ornithine}$. The reaction is reversible; the equilibrium constant expressed as K = [acetylglutamate][ornithine]/[acetylornithine][glutamate] is 0.47.

• 3.

  3. Beside the transferase activity, the enzyme has also a hydrolytic activity. The rate of the hydrolytic reaction for α-N acetylornithine is 1% of that of the acetyltransferase reaction.

• 4.

  4. No specific cofactor has been found. The enzyme is inhibited by p-chloromercuribenzoate, but not by iodoacetate.

• 1.

  1. The experiments described in this report have indicated that the reduction of NAD⁺ by succinate in Thiobacillus novellus is energy-dependent. By blocking the electron transport chain with antimycin A, the endergonic reduction of NAD⁺ by succinate required ATP. The pyridine nucleotide reduction involved the mediation of the flavoprotein system as Atabrine and Amytal inhibited the process.

• 2.

  2. Added mammalian cytochrome $\text{c}$ has been shown to couple with the electron transport chain of T. novellus thus effecting the catalysis of the generation of high-energy intermediates coupled to succinate oxidation in the absence of inorganic phosphate. The non-phosphorylated high-energy compounds thus can be generated either at coupling site II by oxidation of succinate with cytochrome $\text{c}$ as electron acceptor under anaerobic conditions, or at sites II and III under aerobic conditions, or at site III by the oxidation of ferrocytochrome $\text{c}$ involving electron transport to molecular oxygen through the cytochrome oxidase portion of the respiratory chain. In all cases the reduction of NAD⁺ was driven by the generated high-energy intermediates involving reversal of electron transfer from the cytochrome $\text{c}$ level.

• 3.

  3. The energy-dependent reduction of NAD⁺ by succinate involving reversal of electron transfer from the cytochrome $\text{c}$ level was sensitive to 2,4-dinitrophenol, dicumarol and arsenate. It was also inhibited by atabrine and Amytal. Antimycin A was effective in partial inhibition of the reversal of electron transfer. In addition malonate and cyanide were found to be potent inhibitors. The mechanism for the generation and utilization of energy for the reversed electron flow, is discussed.

Further purification of propionyl-CoA carboxylase (EC 6.4.1.3) from bovine-liver mitochondria is reported. On the basis of the present and earlier investigations it is concluded that binding of acyl-CoA substrates by the carboxylase occurs through the CoA moiety. The relative maximum velocities of propionyl-CoA, propionyl-dephospho-CoA, and propionyl-pantetheine carboxylation were 1.0, 0.29, and 0.014, respectively; the $\text{K}{}_{\mathrm{m}}$ values for the same substrates were 2.6·10⁻⁴, 2.8·10⁻³, and 2.4·10⁻² M, respectively. Enzymatic carboxylation of $\text{propionyl}\text{-N-}\text{acetylcysteamine}$ could not be demonstrated. Coenzyme A and 3′-AMP were found to inhibit the carboxylation reaction competitively with respect to propionyl-CoA, whereas, inhibition by 2′-AMP and 5′-AMP was of a mixed type. The binding of acyl-CoA substrate to the carboxylase appears to involve the 3′-phosphate, adenine, and pantoyl moieties of the subsrate. Propionyl-CoA carboxylase is protected from $\text{p-}\text{chloromercuribenzoate}$ inhibition by propionyl-CoA, whereas, ATP-MgCl₂ facilitates this inhibition.

• 1.

  1. Inorganic pyrophosphate metabolism in cardiac tissue homogenates has been investigated.

• 2.

  2. A sub-cellular distribution study indicated that the predominant PP_i-metabolizing activity is Mg²⁺-stimulated soluble-fraction phosphohydrolase which is maximally active in the range pH 7–7.5.

• 3.

  3. Relatively small amounts of activity also were detected in particulate fractions at pH 7.3 in the presence of Mg²⁺. Activity was essentially absent from all fractions at pH 5.6 without added Mg²⁺.

• 4.

  4. PP_i-glucose phosphotransferase activity¹ could not be detected in any sub-cellular fraction or in homogenates.

• 5.

  5. Catalytically, the soluble fraction activity resembles a number of other PP_i phosphohydrolases with respect to (a) absolute requirement for Mg²⁺, (b) alkaline pH optimum (pH 7.3 for the heart enzyme), and (c) marked sensitivity to Ca²⁺ inhibition.

• 6.

  6. Alloxan diabetes produced an approx. 33% drop in PP_i phosphohydrolase activity. Insulin administration, adrenalectomy, or cortisone treatment did not produce statistically significant changes in levels of enzymic activity.

• 7.

  7. Inhibition due to Ca²⁺ was reversed by EDTA or additional Mg²⁺, but not by supplemental PP_i. Citrate inhibited the system both in the presence and absence of Ca²⁺.

• 8.

  8. A feedback mechanism for retardation of calcification in the vascular system of and near the heart is suggested based on the observations that (a) heart PP_i phosphohydrolase is extremely sensitive to Ca²⁺ inhibition, and (b) calcification of the aorta is inhibited by PP_i (ref. 2).

• 1.

  1. The effect of different inorganic anions on the catalytic activity of native and subunit aspartate transcarbamylase (carbamoylphosphate: l-aspartate carbamoltransferase, EC 2.1.3.2) has been investigated at pH 7.0 and 25°.

• 2.

  2. Several inorganic anions were found to inhibit both native and subunit aspartate transcarbamylase. The order of effectiveness for the best inhibitors was: $\text{PP}{}_{\mathrm{i}}\text{> F}{}^{\mathrm{-}}\text{> P}{}_{\mathrm{i}}\text{> SO}{}_4{}^{\mathrm{2-}}$.

• 3.

  3. For each ion except F⁻, a strict competitive relationship was observed between the anion inhibitors and the substrate carbamyl phosphate.

• 4.

  4. The concentration of l-aspartate also greatly influenced the magnitude of the inhibition. The inhibition increased with increasing concentration of l-aspartate.

• 5.

  5. The effect of F⁻ was shown to be due to a displacement of the pH curve along the pH axis. F⁻ inhibited native asparatate transcarbamylase below pH 8 and activated it above this pH.

• 6.

  6. Possible mechanisms of inhibition are discussed, and it is suggested that an inhibitor-l-aspartate complex is formed at the active site.