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

Reversible inactivation of homogeneous nitrate reductase (NAD(P)H: nitrate oxidoreductase, EC 1.6.6.2) from the green alga Ankistrodesmus braunii has been carried out by aerobic incubation of the enzyme with reduced pyridine nucleotide. The involvement of superoxide radicals in the inactivation process is inferred from the fact that it does not take place in the absence of oxygen or in the presence of superoxide dismutase. On the other hand, cyanide also causes the inactivation of the enzyme under reducing conditions. The inactivation of A. braunii nitrate reductase takes place in two steps; the first is the one-electron reduction of the enzyme probably involving the molybdenum centers, and the second, and rate-limiting step, results from the interaction of the reduced enzyme with a nucleophylic agent such as superoxide or cyanide. The mean potential value, at pH 7.5, of the inactivation process, measured by reductive titration with dithionite in the presence of cyanide, was −50 mV. Inactive nitrate reductase, previously dialyzed to remove the inactivating agents, can be immediately reactivated by treatment with ferricyanide in a process requiring the removal of only one electron. This process showed a mean potential value, measured by oxidative titration with ferricyanide, of +230 mV at pH 7.5, independent of the system used to inactivate the enzyme.

The NADP-linked malate dehydrogenase (decarboxylating) (l-malate: NADP⁺ oxidoreductase (oxaloacetate-decarboxylating), EC 1.1.1.40), known as ‘malic’ enzyme, has been isolated and purified to apparent homogeneity from the mango fruit, Mangifera indica, by means of extraction, gel chromatography with Sephadex G-200 and anion-exchange chromatography with DEAE-Sephacel. A 16-fold purification with a 49% yield was obtained. The enzyme was physically characterized and its homogeneity determined by polyacrylamide gel electrophoresis, isoelectric focusing, sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis and analytical chromatography. An average molecular weight of 258 000 was obtained for the enzyme as well as a Stokes radius of 54.0 · 10⁻⁸ cm and a diffusion coefficient of 3.96 · 10⁻⁷ cm² · s⁻¹. A frictional ratio, $\text{f/f}{}_0$ of 1.28 indicated the globular character of the enzyme. The native enzyme consists of four subunits with an average molecular weight of 64 900. The enzyme thus appears to be an oligomeric protein with an apparently homogeneous quarternary structure. The pH optimum for the decarboxylation reaction varied between 6.8 and 7.5, depending on the type of buffer and increased with increasing malate concentration. No cooperativity could be detected between the malate binding sites in the presence of Mn²⁺ as cofactor. It would seen as if Mn²⁺ elicits a positive allosteric effect on the enzyme. Increasing Mn²⁺ concentrations lead to an increase in the $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value for l(-)malate from 666 μM at 1.0 mM Mn²⁺ to 1.08 mM at 5.0 mM Mn²⁺. The $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value determined at pH 7.1 for Mn²⁺ was 14.3 μM. An approximate $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value of 16 μM was found for NADP⁺ with some indication of cooperativity between the nucleotide binding sites. The enzyme activity was much more sensitive to regulation when Mg²⁺ served as cation. The allosteric activator, succinate, removed the sigmoidicity observed in the velocity-malate saturation curve and lowered to Hill coefficient from 1.5 to 1.0 Differences were found in the response of the enzyme to certain metabolites depending on whether Mg²⁺ or Mn²⁺ served as cofactor. It appears as if Mg²⁺ and Mn²⁺ stabilize two structurally distinct forms of the enzyme which vary in catalytic and regulatory properties.

Glutamine synthetase (l-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2) from pumpkin leaf cytosol is an oligomer that consists of eight identical monomers. The enzyme has a partial specific volume of 0.720 ml/g. The pH optimum in the presence of Mg²⁺ is 7.2. It is shown by means of electron microscopy that the enzyme consists of elongated monomers, which are arranged with point 42 symmetry at the vertices of two squares. These squares are twisted about the 4-fold axis at 40° relative to each other.

An extract of chick pectoral muscle was prepared in which the level of carnosine synthetase (l-histidine; β-alanine ligase (AMP-forming), EC 6.3.2.11) activity was approx. 10-times that of previous preparations. In affinity chromatography studies, this material was applied to a Cibracon blue-agarose column, and elution of carnosine synthetase by carnosine was attempted. Results indicated that the elution was not specific as the eluate contained large amounts of myosin. An (NH₄)₂SO₄ fraction (21–30% satn.) of the crude extract was prepared which, in comparison to the crude extract, had a higher specific activity, was more stable on storage at 4°C and had much lower myosin content. On affinity chromatography of this fraction apparently homogeneous carnosine synthetase was eluted with carnosine, and the specific activity of the preparation was 1700-times that of the fresh crude extract. Amino acid analysis of the preparation indicated that it had a very high histidine content (141 per 1000 residues). On analysis of the preparation by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS), a polypeptide of $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ 119 000 was observed, whereas gel permeation chromatography of the native enzyme indicated an $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ of 250 000, suggesting that the native enzyme is a dimer.

Formation of binary complex between 6-phosphogluconate dehydrogenase (6-phospho-d-gluconate:NADP⁺ 2-oxidoreductase (decarboxylating), EC 1.1.1.44) from Candida utilis and 6-phosphogluconate was investigated by means of ultraviolet difference spectroscopy. The formation of the enzyme-substrate complex induces in the difference spectrum a positive peak the wavelength and extinction coefficient of which agree well with a tyrosine ionization. Titrimetric studies indicate that the formation of the binary complex is not coupled to a proton release from the protein. These data support an intramolecular proton transfer from a tyrosine to other functional group. This proton transfer could be correlated to the conformational change induced by substrate in 6-phosphogluconate dehydrogenase.

Aminopeptidase A (l-α-aspartyl(l-α-glutamyl)-peptide hydrolase, EC 3.4.11.7) was found in human placenta, partially purified from it and briefly characterized in comparison with the placental leucine aminopeptidase. The aminopeptidase A could be separated from leucine aminopeptidase after trypsin digestion followed by Sephacryl S-300 chromatography. The angiotensinase (EC 3.4.99.3) activity of aminopeptidase A in human placenta was confirmed by a biological method.

Two forms of β-glucosidase (β-d-glucoside glucohydrolase, EC 3.2.1.21) from the culture filtrate of Macrophomina phaseolina were separated and partially purified by (NH₄)₂SO₄ precipitation, ion-exchange chromatography (DE-52) and gel filtration. The final preparation was purified 103-fold and 88-fold for β-glucosidase-I and β-glucosidase-II, respectively. Polyacrylamide gel electrophoresis of the purified enzymes imparted a single band at pH 8.3. The two forms differ from each other with respect to molecular weight (323 600 for β-glucosidase I and 220 000 for β-glucosidase II)pH optima, temperature optima, electrophoretic mobility and substrate specificity. The two forms of β-glucosidase may also be differentiated by inhibition experiments using inhibitors like glucono-δ-lactone and nojirimycin. Of the two inhibitors tested nojirimycin is more potent for β-glucosidase-I than that for β-glucosidase-II. The energy of activation for the two enzymes is also different (12.02 kcal/mol for glucosidase I and 10.0 kcal/mol for glucosidase II).

A perchloric acid extract of eggs of Hemileuca oliviae inhibits bovine trypsin, kallikrein and papain, as well as the native proteolytic activity (pH 7.0) of the developing embryo. Specificity is indicated by the lack of inhibition of other proteases. The amount of inhibitory activity changes during embryological development, reaching a maximum around 23 days, when the larva is fully developed. The inhibitory activity was lost by dialysis and was destroyed by ashing (450°C, 18 h) but was unaffected by exposure to 97°C for 3 min. The presence of two protease inhibitors was detected in the perchloric acid extract. The principal component has a molecular weight of approx. 9000 and its heat sensitivity is affected by pH. At the present time the role of these inhibitors in the developing embryo is unknown. Some trypsin-like native protease activity occurs in the egg during embryogenesis and may thus be the target enzyme in vivo.

A comparison of the inactivation of bovine carbonic anhydrase B (carbonate hydro-lyase, EC 4.2.1.1) by ^.OH, (SCN)₂^$\text{•}$ and Br₂^$\text{•}$ shows that the enzyme contains one or more essential tryptophan residues. Direct oxidation of histidine and tyrosine residues by the radicals is less important in causing inactivation of the enzyme. The effectiveness of all these radicals in inactivating carbonic anhydrase decreases with increasing pH in the region where the activity-linked ionizable group dissociates. Differences between the rates of reaction of Br₂^$\text{•}$ and (SCN)₂^$\text{•}$ with the holo- and apo-enzyme and between the resulting transient product spectra indicate that access to the reactive tyrosine and tryptophan residues is diminished by the presence of Zn²⁺ in the active site region.

Reactions of the inorganic radical anions, Br₂^$\text{•}$ and (SCN)₂^$\text{•}$, with bovine carbonic anhydrase (carbonate hydrolyase, EC 4.2.1.1) have been studied by pulse radiolysis. Reaction is almost completely inhibited by the binding of Br⁻, SCN⁻ and ClO₄⁻ to an electrophilic site at the active centre of the enzyme. Dissociation constants for anion binding calculated from the reduction in free radical reactivity agree well with inhibition constants for these anions. The anions OCN⁻ and CN⁻, although potent inhibitors of carbonic anhydrase activity, have relatively little effect on the reactivity of radical anions with the enzyme. Reaction of radical anions occurs mainly with tryptophan and tyrosine residues in the hydrophobic core of the enzyme, through a channel at the active site. This channel is closed by the anions in accord with their position in the lyotropic series.