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

The proteinase content of highly purified preparations of human peripheral blood monocytes was investigated. Monocyte cell lysates exhibited activity at neutral pH against azocasein, ³H-labelled elastin as well as several synthetic substrates used to detect serine proteinases (EC 3.4.21.-) of human polymorphonuclear leucocytes. The cell lysates also contain at least two acid proteinases. The levels of neutral proteinase activity in monocytes was considerably less than that found in polymorphonuclear leucocytes. The effect of inhibitors on the monocyte neutral proteinases showed them to be of the serine type. Monocytes also solubilized and degraded the type IV collagen found in human glomerular basement membrane at neutral and acid pH. The action of the monocyte proteinase on glomerular basement membrane indicated that their properties were similar but not identical to that of the polymorphonuclear leucocyte serine proteinases. Since monocytes infiltrate the glomerulus in certain forms of immunologically mediated glomerulonephritis, it may well be that monocyte serine proteinases make a contribution to the glomerular damage that occurs.

Bacteriophage T4 ghosts were found to possess lysozyme (mucopeptide N-acetylmuramoylhydrolase, EC 3.2.1. 17) activity. This enzyme is probably responsible for the lysis from without, observed at high multiplicity of infection, a process independent of the presence of the e gene product which is also a lysozyme. The ghost lysozyme and e lysozyme differed with respect to their requirements for maximal catalytic activity and to some extent in substrate specificity. The ghost lysozyme was released from phase particle by the action of Triton X-100.

The reaction of phenylglyoxal with Escherichia coli ornithine transcarbamylase (carbamoylphosphate: l-ornithine carbamoyltransferase, EC 2.1.3.3) leads to complete loss of enzymatic activity. The behavior of this reagent towards ornithine transcarbamylase is remarkably similar to that observed with E. coli aspartate transcarbamylase (carbamoylphosphate: l-aspartate carbamoyltransferase, EC 2.1.3.2) and its catalytic subunit (Kantrowitz, E.R. and Lipscomb, W.N. (1976) J. Biol. Chem. 251, 2688–2695). The rate of phenylglyoxal inactivation increases in the order ornithine transcarbamylase, catalytic subunit of aspartate transcarbamylase and aspartate transcarbamylase. For ornithine transcarbamylase, the substrate carbamyl phosphate alone or in combination with the substrate analog norvaline protect the enzyme from phenylglyoxal inactivation. Under similar conditions, carbamyl phosphate or carbamyl phosphate plus succinate will protect the catalytic subunit of aspartate transcarbamylase in an almost identical manner. Using [¹⁴C]phenylglyoxal, the number of arginine residues involved in loss of activity was determined to be approx. three per ornithine transcarbamylase molecule or one arginine per active site. The data suggest that the arginine necessary for activity is involved in the binding of carbamyl phosphate to the enzyme. The similarity in phenylglyoxal reactivities combined with genetic and structural data suggest very strongly that there is an evolutionary relationship between ornithine transcarbamylase and the catalytic subunit of aspartate transcarbamylase.

Guanylate kinase (ATP:(d)GMP phosphotransferase, EC 2.7.4.8) was purified about 200-fold with 4% yield from baker's yeast. The enzyme preparation showed a single band on polyacrylamide gel electrophoresis and the molecular weight of the enzyme was calculated to be 25 000 by gel filtration. With ATP as a phosphate donor, the kinase used only GMP as a phosphate acceptor. $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ values for ATP and GMP were 0.5 and 0.048 mM, respectively. The enzyme reacted optimally at pH 7.5. The enzyme was labile during storage at 4°C and inactivation was prevented by 20% glycerol.

Ca²⁺-dependent ATPase (Ca²⁺-dependent ATP phosphohydrolase, EC 3.6.1.3) present in a subcellular fraction derived from rat pancreatic islet homogenates was examined to determine kinetic parameters and responses to various substances with known effects upon insulin secretion. Experiments demonstrated the presence of a Ca²⁺-ATPase with a $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ ATP of 7 · 10⁻⁵ M and two $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ Ca of 1.3 · 10⁻⁷ M and 5.7 · 10⁻⁶ M. The enzyme had little activity in acidic media while retaining considerable activity in basic media. Optimal activity was obtained at pH 7.5. The enzyme was relatively temperature insensitive ($\text{Q}{}_{10}\text{= 1.49}$), since activity decreased less than 50% with a 15°C decrease in temperature. Studies on the stability of enzyme activity upon storage at −20°C indicated that for intact islets activity was stable for 3 weeks, while in homogenates activity was stable for only 1 week, after which activity rapidly declined in both cases. Certain substances known to either stimulate or inhibit insulin secretion were tested for their ability to alter enzyme activity. Potassium, glibenclamide and cyclic AMP had no effects upon activity. Mannoheptulose significantly suppressed enzyme activity while 2-deoxyglucose did not alter activity. These observations are consistent with the hypothesis that a Ca²⁺-ATPase present in pancreatic islets may act as a modulator of pancreatic islet β cell activity.

The mode of degradation of myofibrils and their constituent proteins by cathepsin L (EC 3.4.22.15) of rabbit skeletal muscle was studied. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis showed that cathepsin L degraded myosin heavy chain, α-actinin, actin, troponin T and troponin I assembled in myofibrils and produced mainly fragments of 160 000 and 30 000 daltons in the acidic pH region. This degradation was most intense around pH 4. Degradation of myosin in the isolated state by cathepsin L resulted in the disappearance of the heavy chain and the decrease of light chains 1, 2 and 3, producing fragments of 160 000, 92 000, 83 000 and 60 000 daltons. The degradation of the heavy chain was most severe at pH 4.2. Cathepsin L degraded actin into fragments of 40 000, 37 000 and 30 000 daltons. This action was most intense at pH 4.7. Tropomyosin was not degraded. Troponin T and troponin I were degraded into fragments of 30 000 and 13 000 daltons at pH 3.7-6.7, which were degraded further into smaller fragments. Troponin C was not degraded. α-Actinin was degraded into several fragments, the major one of which showed an $\text{M}{}_{\mathrm{<mtext>r</mtext>}}$ of 80 000. This degradation was most intense at pH 3.0–3.5.

A human mammary epithelial cell line (SKBr3) has been identified in which fatty acid synthetase constitutes up to 28%, by weight of the cytosolic proteins. The enzyme has been purified to near homogeneity from this cell line and some of its properties studied. In common with fatty acid synthetases from other animal tissues, the enzyme is a 480 000 dalton dimer of similar molecular weight subunits, it synthesizes predominantly palmitic acid and is inactive in the absence of free coenzyme A. The kinetic properties and amino acid composition of the enzyme are also similar to those of fatty acid synthetases from various tissues of other animals. Appreciable structural resemblance between human and rodent fatty acid synthetases is indicated by studies on the immunological cross-reactivities of these enzymes.

Two plasma proteins, α₂-plasmin inhibitor and plasma fibronectin, are cross-linked to fibrin by plasma transglutaminase (R-glutaminyl-peptide : amine γ-glutamyl-yltransferase, EC 2.3.2.13, fibrin stabilizing factor) when blood coagulation takes place. The cross-linking reactions of these proteins were analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS) using these radioactively labeled proteins. Both proteins were cross-linked exclusively to the α-chain of fibrin, and each of these cross-linking reactions proceeded independently without being influenced by the other cross-linking reaction. The cross-linking of fibronectin to the α-chain proceeded steadily at a rate similar to that of the cross-linked polymerization of the α-chain. In contrast, the cross-linking reaction of α₂-plasmin inhibitor to fibrin proceeded markedly faster than that of fibrin polymerization but did not proceed further after reaching a certain relatively low level of cross-linking. Most of the cross-linked α₂-plasmin inhibitor molecules at this stage of the fibrin cross-linking process were in the form of complex with the α-chain monomer. The complex with the α-chain monomer was gradually transformed to a complex with the α-chain polymer as the cross-linking polymerization of the α-chain proceeded. The rate of the transformation was the same as that for the disappearance of the α-chain monomer, indicating that whether the α-chain was cross-linked to α₂-plasmin inhibitor or not, the α-chain underwent cross-linking polymerization at the same rate.

Previous studies have shown that a neutral metallo-endopeptidase purified from rat kidney degrades the B chain of insulin, glucagon, ACTH and, at a markedly slower rate, the A chain of insulin. In contrast the enzyme does not attack native insulin, oxytocin, vasopressin, ribonuclease, albumin or denatured hemoglobin. The current studies demonstrate that the neutral peptidase also degrades the isolated C-peptide of proinsulin and cleaves certain peptide bonds in and near the C-peptide moiety of native proinsulin. Time courses of the formation of fluorescamine-reactive material during digestion of proinsulin and isolated C-peptide with the peptidase were identical. However, structural analysis of the peptidase-digested proinsulin showed that the enzyme does not convert proinsulin to insulin but that the peptidase cleaves one bond, Tyr₂₆-Thr₂₇, in the B chain moiety and five bonds in the C-peptide moiety, producing four split proinsulins. One of the split proinsulins is des-octacosapeptide (27–54) porcine proinsulin or des-tetracosapeptide (27–50) bovine proinsulin. Each is a derivative of the insulin molecule having an extension of nine residues (ten residues in the case of the derivative from bovine proinsulin) at the N-terminus of A chain and lacking four residues at the C-terminus of B chain. This two-chain derivative retains full immunoreactivity with insulin antibodies and exhibits 2.4-times more biological activity (promotion of glycogenesis in primary cultured hepatocytes) than proinsulin and about two-thirds the activity of insulin.