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

A collagenolytic enzyme from the land planarian Bipalium kewense has been purified by preparative isoelectric focusing. The enzyme has a molecular weight of 47 000 ± 2 000 and appears to be dimeric. It has an isoelectric point of 4.6 ± 0.1 and a high content of acidic amino acids. The amino acid composition of the Bipalium collagenase is similar to that of human skin fibroblast collagenases but clearly different from previously reported collagenolytic proteases from other invertebrates, Uca pugilator and Hypoderma lineatum. In its action on guinea-pig collagen, the enzyme produces distinct products, at low incubation temperatures, different from those produced by vertebrate and other invertebrate collagenolytic enzymes. These products have glycine as their N-terminal amino acids. As determined by viscosity measurements, the Bipalium collagenase is more active on invertebrate, earthworm, collagen than it is on the vertebrate, Type I guinea-pig skin, collagen. The Bipalium collagenase differs from both bacterial and vertebrate collagenases as well as from invertebrate, collagenolytic serine proteases.

Glutathione S-transferase (RX: glutathione R-transferase, EC 2.5.1.18) from human placenta has been purified to homogeneity. This enzyme, transferase π, is an acidic protein (isoelectric point at pH 4.8) composed of two subunits. The molecular weights for the dimer and monomer were determined by independent methods as 47 000 and 23 400, respectively. These properties are not significantly different from those of glutathione S-transferase ϱ from human erythrocytes. Antibodies to transferase π reacted with the enzyme from erythrocytes but not with the basic transferases α-ϵ and the neutral transferase μ isolated from human liver. Antibodies to the latter enzymes did not react with the transferase from placenta. Further similarities between transferases π and ϱ appear in amino acid compositions, kinetic constants and substrate specificities. Both the placental and the erythrocyte enzyme have considerably higher activity with ethacrynic acid than any other of the human glutathione S-transferases. The glutathione S-transferase could be distinguished from two additional acidic glutathione-dependent enzymes, glyoxalase I and selenium-dependent glutathione peroxidase. It is concluded that transferase π from placenta is identical with or very closely related to transferase ϱ from erythrocytes.

Cultured rat hepatoma cells were homogenized and subjected to subcellular fractionation by analytical sucrose density centrifugation to determine the localization of γ-glutamyltransferase ((5-glutamyl)-peptide : amino-acid 5-glutamyltransferase, EC 2.3.2.2). The activity was exclusively localized to the plasma membrane. Diazotized sulphanilic acid was used as a non-penetrant membrane reagent which inactivates ectoenzymes. With both intact and sonicated cells, only 70–75% inhibition of γ-glutamyltransferase activity was observed. At least 12% of the total cell complement of γ-glutamyltransferase activity is highly resistant to inactivation by diazotized sulphanilic acid even after Triton X-100 solubilization. The enzyme was purified from hepatoma cells and its properties compared with enzyme from normal liver. Apart from the striking increase in V^app there were only minor differences between the enzymes from the two sources. In contrast to the complete abolition of transpeptidase activity of the purified hepatoma enzyme by diazotized sulphanilic acid, the hydrolytic activity of this preparation was only slightly inhibited.

During development of the mushroom carpophore of the basidiomycete Coprinus cinereus, and through the operation of endogenous control mechanisms, the enzyme NADP-linked glutamate dehydrogenase (l-glutamate: NADP⁺ oxidoreductase (deaminating), EC 1.4.1.4) increases greatly in activity in the developing cap, while remaining at a barely detectable level in the stipe and parental mycelium. This behaviour can be reproduced in vegetative mycelium which, after growth in a rich medium, is transferred to a medium lacking in nitrogen source and containing 100 mM pyruvate as sole carbon source. Such treatment immediately causes induction of activity of NADP-linked glutamate dehydrogenase. Only glucose, fructose, dihydroacetone, acetate and propan-1-ol share with pyruvate the ability to induce this enzyme activity. A mutant mycelium which is known to lack the enzyme acetyl-CoA synthetase failed to show induction of glutamate dehydrogenase activity on acetate medium, although normal induction occurred on medium containing either glucose or pyruvate. It is concluded that induction requires synthesis of acetyl-CoA and that this latter compound is the probable intracellular regulator. Inclusion of as little as 2 mM NH₄Cl in the transfer medium is sufficient to prevent enzyme induction. Some other nitrogen sources are also able to prevent induction but all seem to operate through the formation of ammonium which is excreted into the medium. Other compounds, like alanine or glutamate are unable either to promote or prevent induction. External concentrations of ammonium which are able to prevent induction do not correlate with elevated internal ammonium levels, so it is concluded that, perhaps through some membrane-reaction, the external level of ammonium determines whether induction will occur. The regulation mechanism is therefore interpreted as one in which the enzyme is induced by elevated intracellular levels of acetyl-CoA providing external levels of ammonium are low.

Sucrase-isomaltase immunoprecipitated from brush border of an intestinal transplant lacking pancreatic proteases was found to be a single, high molecular weight protein. Elastase digestion converted this protein into two subunits which co-migrated on electrophoresis with those normally found on the microvillus membrane. The high molecular weight from had full sucrase and isomaltase activities.

An enzyme catalyzing the reduction of leupeptin acid to leupeptin was partially purified from a cell extract of Streptomyces roseus MA839-A1, a leupeptin producer. The enzyme was tentatively named leupeptin acid reductase. The molecular weight was estimated to be 320 000 by chromatography on Sepharose 6B. The reductase eluted with leupeptin acid synthetase both in molecular sieve chromatography and in affinity chromatography. The main properties of the reductase were: (1) ATP and NADPH were required for activity. ATP could not be replaced by GTP, ADP or AMP. NADPH could not be replaced by NADH. (2) Michaelis constants for ATP and NADPH were 4.2 · 10⁻⁵ M and 1.3 · 10⁻⁶ M, respectively. (3) The enzyme was inhibited by leupeptin, the reaction product, and antipain. Both inhibitors have an l-argininal residue at the C-terminal structure. (4) The enzyme did not catalyze the conversion of leupeptin to leupeptin acid. Leupeptin acid reductase and leupeptin acid synthetase were found in the 10 000 × g pellet of the cell homogenate. The reductase was not released as readily from the pellet as the synthetase either by washing or by repeated freeze-thawing. Synthesis of leupeptin from acetyl-CoA, l-leucine and l-arginine in vitro was accomplished by combining leucine acyltransferase and the enzyme complex consisting of leupeptin acid synthetase and leupeptin acid reductase.

Using a novel fluorimetric assay for pyridoxal phosphate phosphatase, human polymorphonuclear leucocytes were found to exhibit both acid and alkaline activities. The neutrophils were homogenised in isotonic sucrose and subjected to analytical subcellular fractionation by sucrose density gradient centrifugation. The alkaline pyridoxal phosphate phosphatase showed a very similar distribution to alkaline phosphatase and was located solely to the phosphasome granules. Fractionation experiments on neutrophils treated with isotonic sucrose containing digitonin and inhibitor studies with diazotised sulphanilic acid and levamisole further confirmed that both enzyme activities had similar locations and properties. Acid pyridoxal phosphate phosphatase activity was located primarily to the tertiary granule with a partial azurophil distribution. Fractionation studies on neutrophils homogenised in isotonic sucrose containing digitonin and specific inhibitor studies showed that acid pyridoxal phosphate phosphatase and acid phosphatase were not the result of a single enzyme activity. Neutrophils were isolated from control subjects, patients with chronic granulocytic leukaemia and patients in the third trimester of pregnancy. The specific activities (munits/mg protein) of alkaline pyridoxal phosphate phosphatase and alkaline phosphatase varied widely in the three groups and the alterations occurred in a parallel manner. The specific activities of acid pyridoxal phosphate phosphatase and of acid phosphatase were similar in the three groups. These results, together with the fractionation experiments and inhibition studies strongly suggest that pyridoxal phosphate is a physiological substrate for neutrophil alkaline phosphatase.

The pyrimidine-3 gene of Neurospora crassa codes for a bifunctional enzyme catalysing the first two steps of the pyrimidine biosynthetic pathway. Difficulties have been experienced in purification due to the lability of the enzyme. The enzyme loses carbamoyl-phosphate synthetase (carbon-dioxide: ammonia ligase (ADP-forming, carbamate-phosphorylating), EC 6.3.4.16) activity and undergoes a change in apparent molecular weight from the native 650 000 to 100 000 of the only detectable fragment. Attempts have been made therefore to stabilize the enzyme so as to minimise these effects. Elastinal, a protease inhibitor, reduces the effects, as do certain ultra-violet-sensitive mutant strains which lack a minor protease. The nature of the loss of carbamoyl-phosphate synthetase suggests an instability in the tertiary structure of the enzyme which can be reduced by the use of glycerol. Glycerol also exhibits a protease-inhibiting effect in this system. Although a range of protease inhibitors and use of uvs mutants can reduce the rate of decay of carbamoyl-phosphate synthetase activity, only glycerol can stabilize the native molecular weight. Our results support the hypothesis that the loss of carbamoyl-phosphate synthetase activity and change in molecular weight of the enzyme is a three-step sequence of proteolysis, conformational shift and cleavage of a further non-covalent bond.

Aspartase (l-aspartate ammonia-lyase, EC 4.3.1.1) of Escherichia coli is composed of four subunits of seemingly identical molecular weight (Suzuki, S., Yamaguchi, J. and Tokushige, M. (1973) Biochim. Biophys. Acta 321, 369–381). The subunit arrangement of the enzyme was studied by two distinct methods, cross-linking of subunits with a bifunctional reagent, dimethyl suberimidate, and statistical classification of negatively stained electron microscopic images. In the former method, the densitometric patterns of the cross-linked aspartase were analyzed quantitatively after separating each component by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and the results were compared with the theoretical distribution. In the latter method, a number of electron microscopic images were classified into several groups according to their characteristic appearance. The results obtained by these two methods are compatible with the possibility that the enzyme has a tetrameric structure consisting of two pairs of dimers, in which the two pairs of rod-shape subunits meet perpendicularly, being typical of D₂ symmetry.

The degradation of bovine myelin basic protein by bovine brain cathepsin D (EC 3.4.23.5) was studied over a pH range of 2.75–6.0. Throughout this pH range pepstatin, an inhibitor of cathepsin D, prevented the degradation. The degradation at a pH away from the optimum of pH 3.5 was predictably slower, but also resulted in more restricted cleavage. Above pH 4.5 bovine basic protein peptide 1–42 was not degraded further to peptide 1–36 as occurs at pH 3.5. Additionally, at pH 5.5 another fragment of basic protein, peptide 1–91, persisted indicating that under certain conditions basic protein as well as basic protein peptide 43ndash;169 may be cleaved in the molecular region of basic protein around the phenylalanyl-phenylalanine residues at position 88ndash;89. The small amount of peptides 1–91 and 92–169 detected at pH 5.5 suggests that the bond between residues 91 and 92 in intact basic protein is a minor cleavage site. The options and variation in cleavage around residues 88–92 of basic protein presumably result from pH-dependent changes in conformation in this region but could also be due to changes in conformation of cathepsin D. These results indicate that local tissue changes such as pH may affect not only the velocity of the reaction but also the nature of the product formed by the degradation of basic protein by brain cathepsin D.