Enzyme & protein最新文献

Several classes of extracellular matrix (ECM)-degrading proteases have been shown to play an important role in tumor invasion and metastasis. Among them, the matrix metalloproteinases (MMPs) and the plasminogen activator (PA)-plasmin system have been the focus of numerous studies. However, few of those have examined the interaction of these two classes of proteases during tumor progression and their specific roles in this complex process have remained unclear. In this article, comparative information on the structure, function, and regulation of these two classes of proteases is reviewed and their interaction on various levels is discussed. This review shows that MMPs and the PA-plasmin system closely cooperate to achieve optimal degradation of the ECM during the invasive and metastatic process.

Urokinase plasminogen activator (uPA) is a multifunctional protein involved in both extracellular proteolysis and signal transduction. uPA usually mediates its actions while attached to a membrane-bound receptor, termed uPAR. In this study, uPA and its receptor were measured at both protein and mRNA levels in breast cancer. At both levels, concentrations of uPA were significantly correlated with those for uPAR. uPA levels also correlated significantly with cathepsin B and cathepsin D but not with cathepsin L, MMP-8 or MMP-9 levels. Irrespective of the cut-off point used (e.g., median, tertile or quartile values), uPA was a significant prognostic marker for breast cancer.

Extracellular proteolysis is required for matrix degradation and the regulation of cytokine activity during angiogenesis, and this is dependent on a cohort of proteases and protease inhibitors produced by endothelial and nonendothelial cells. The plasminogen activator (PA)/plasmin system has been extensively investigated in these processes, and descriptive studies have demonstrated that urokinase-type PA (uPA), uPA receptor (uPAR) and PA inhibitor-1 (PAI-1) are expressed by endothelial cells during angiogenesis in vivo. In vitro studies have led to the notion that normal capillary morphogenesis is dependent on a protease-antiprotease equilibrium. These findings are discussed in the context of recent observations on uPA-, uPAR-, PAI-1 and plaminogen-deficient mice, in which developmental and physiological angiogenesis appear to occur normally. This has led to a reevaluation of the role of the PA/plasmin system during angiogenesis. In particular, these observations raise the possibility that the role of this system may be limited to situations in which endothelial cells encounter and must degrade fibrin in order to form new capillary sprouts.

Neutral cholesterol ester hydrolase from rat liver microsomes was inactivated in a dose and time-dependent manner by classical sulphydryl-reacting reagents such as p-hydroxymercuribenzoic acid, 5,5'-dithio-bis-(2-nitrobenzoic acid), N-ethylmaleimide, or iodoacetate. The concentrations at which half-maximal inhibition of the native microsomal cholesterol ester hydrolase occurred (IC50) were 15, 68, and 370 mumol/l and 68 mmol/l, respectively. Only partial reactivation of the enzyme was observed under excess dithiothreitol or mercaptoethanol treatment. The stimulation of cholesterol ester hydrolase by the metal ions Ca2+ and Mg2+ was dependent on the integrity of the thiol groups. Solubilization of cholesterol ester hydrolase from membranes preserved its sensitivity towards sulphydryl reagents and thiols, as well as its ability to be activated by Ca2+ and Mg2+. Dithiothreitol, mercaptoethanol, and Ca2+ and Mg2+ provided total protection of the enzyme against inactivation by thiol-reacting reagents. The results indicate that one or more thiol groups are either at the active centre of the native and solubilized forms of rat liver microsomal cholesterol ester hydrolase or are sufficiently near, to interfere with the catalysis when they are reacted.

Stromelysin-1 was one of the first proteinases found to be associated with cancer. In this review we describe the role of stromelysin-1 in normal mammary gland involution. When stromelysin-1 is overexpressed in transgenic mice the mammary gland undergoes precocious involution and is predisposed to forming a reactive stroma resembling that of a wound site or a tumor. Stromelysin-1 may act as an oncogene because transgenic mice expressing an active form of the enzyme develop mammary tumors. These observations suggest that stromelysin-1 and other matrix metalloproteinases may be useful targets for therapeutic intervention in cancer.

The kinetic theory of the substrate reaction during irreversible inhibition of enzyme activity described previously has been applied to a study on the kinetics of the course of inactivation of yeast alcohol dehydrogenase (YADH) by o-phthaldehyde (OPTA). The microscopic constants for the reaction of the inactivators with the free enzyme and with the enzyme-substrate complexes were determined. The inactivation is a monophasic pseudo-first-order reaction with OPTA. The apparent rate constant A is independent of the OPTA concentration, indicating that the inactivation is a noncomplexing inhibition. The marked protective effect of substrates on the inactivation of YADH by OPTA has been observed. This result suggests that the modification of the enzyme by OPTA may occur at the active site.

The enzyme porphobilinogen deaminase (PBG deaminase, EC 4.3.1.8) catalyzes the condensation of four molecules of PBG to give the linear tetrapyrrol, hydroxymethylbilane. It has been shown that this enzyme forms stable mono-, di-, tri- and tetrapyrrole-enzyme covalent complexes. When the enzyme, partially purified in the absence or presence of phenylmethylsulfonyl fluoride (PMSF) and preincubated with PBG, was applied on DEAE-cellulose columns, three peaks with PBG deaminase activity were detected. Using Ehrlich's reagent, it was found that the active peaks corresponded to mono-, di- and tri-pyrrylmethane-enzyme complexes. Therefore, the mechanism of action of PBG deaminase from Saccharomyces cerevisiae also involves the sequential addition of four PBG units, leading to the formation of the enzyme-substrate intermediate complexes, as has already been described for the same enzyme from other sources.

Pig platelet acidic carboxypeptidases hydrolyzed only N-blocked dipeptides with bulky aromatic and aliphatic hydrophobic amino acids. The optimum hydrolysis of these substrates was at pH 5.0. The main acidic carboxypeptidase in pig platelet lysate was lysosomal carboxypeptidase A (1CPA), which hydrolyzed Cbz-Phe-Ala at the highest rate. A lower activity of this enzyme was found on Cbz-Glu-Tyr and Cbz-Glu-Phe. 1CPA also hydrolyzed Cbz-Glu-Tyr at pH 3.5. No activity of lysosomal carboxypeptidase B in platelet lysate was detectable using Bz-Gly-Arg. Pig platelet acidic carboxypeptidase hydrolyzed Cbz-Pro-Phe and Cbz-Pro-Ala, which are specific substrates of lysosomal prolylcarboxypeptidase, more slowly. The incubation of platelet lysate with plasma did not influence the rate of hydrolysis of Cbz-Glu-Tyr, whereas no hydrolysis of Cbz-Pro-Phe was observed.

In order to evaluate tubular damage in diabetic patients, the activity of renal proximal tubule derived enzymes excreted in 24-hour urine were recorded in 5 groups as follows: (i) 48 noninsulin-independent diabetic patients with normal renal function and a urinary albumin excretion rate within the normal range; (ii) 45 noninsulin-dependent diabetic patients with normal renal function and a high urinary albumin level; (iii) 26 noninsulin-dependent diabetic patients with renal failure; (iv) 40 patients with essential hypertension and normal renal function, and (v) 48 normal control subjects. Regardless of whether cases were noninsulin-dependent diabetics with normal or high urinary albumin excretion rate or cases with renal dysfunction, urinary dipeptidyl aminopeptidase IV and N-acetyl-beta-D-glucosaminidase excretions were significantly higher than in healthy subjects, and urinary gamma-glutamyl transpeptidase excretion was significantly lower than in healthy subjects. No significant changes in urinary enzyme excretions showed specific variations in the essential hypertensive patients. These results suggest that there is tubular damage in the early stages of noninsulin-dependent diabetic patients with normal renal function and normal urinary albumin excretion rate. Detection of urinary excretion of dipeptidyl aminopeptidase IV, N-acetyl-beta-D-glucosaminidase and gamma-glutamyl transpeptidase may be especially useful for the early diagnosis of diabetic nephropathy.