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

p-Guanidinobenzoate derivatives were prepared and their inhibitory effects on trypsin, plasmin, pancreatic kallikrein, plasma kallikrein, thrombin, C1r̄ and C1 esterase were examined. Among the various inhibitors tested, 6′-amidino-2-naphthyl-4-guanidinobenzoate dihydrochloride, 4-(β-amidinoethenyl)phenyl-4-guanidinobenzoate dimethanesulfonate and 4-amidino-2-benzoylphenyl-4-guanidinobenzoate dimethanesulfonate were the most effective inhibitors of trypsin, plasmin, pancreatic kallikrein, plasma kallikrein and thrombin and they strongly inhibited the esterolytic activities of C1r̄ and C1 esterase, and then strongly inhibited complement-mediated hemolysis.

Purified bovine testicular hyaluronidase (hyaluronate 4-glycanohydrolase, EC 3.2.1.35) was inactivated by butane-2,3-dione in either borate or Hepes buffer, pH 8.3. The presence of borate enhanced the inactivation process which followed pseudo-first-order kinetics with a calculated second-order rate constant of 13.54 M⁻¹ · min⁻¹. Using kinetic data it was estimated that the modification of 1 mol arginine per mol enzyme was sufficient for inactivation to occur, whereas amino acid analysis indicated that 4 mol arginine had been modified. The inactivation process was partially prevented by using either competitive inhibitors or substrates of the enzyme, thus indicating that the essential arginine residue is close to the active site of hyaluronidase. A full kinetic analysis of the enzyme with either hyaluronic acid or chondroitin 6-sulphate as substrate showed that the activity of hyaluronidase was uncompetitively activated by either protons or NaCl. The product obtained by reduction of the carboxyl groups of hyaluronic acid to the corresponding alcohol groups was a competitive inhibitor. The possibility that the microenvironment of hyaluronic acid was responsible for the observed kinetic effects of pH and ionic strength was dispelled. It is concluded that these data are compatible with a mechanism that involves an ionic interaction between a carboxyl group on the substrate and an arginine residue on the enzyme.

Fructose-1,6-bisphosphate aldolase (d-fructose-1,6-bisphosphate d-glyceraldehyde-3-phosphate lyase, EC 4.1.2.13) partitions between the microsomes and the cytosol when a rat liver homogenate is fractionated by differential centrifugation. Gel electrophoresis and immunodiffusion indicate that the one isozyme present in the liver of the young adult rat is found in both fractions. The association of the aldolase with membranes is differentially sensitive to a variety of metabolites and inorganic salts. In the absence of cellular salts, 1 mM fructose 1,6-bisphosphate or glucose 1,6-bisphosphate elutes 50% of the enzyme from the microsomes. About 9 mM P_i or citrate is necessary to produce the same effect. With other metabolites or inorganic salts higher concentrations are required. The fraction of total enzyme which partitions with the microsomes when a homogenate is submitted to high speed centrifugation, correlates inversely with the level of fructose 1,6-bisphosphate in the supernatant solution and this concentration is higher when the tissue concentration in the homogenate is greater. The K_m for fructose 1,6-bisphosphate of 3 · 10⁻⁴ for aldolase bound to microsomes is decreased to 6 · 10⁻⁶ M when the enyme is dissociated from the membranes with salt. These observations appear relevant to the ongoing discussion regarding the physiological relevance of the subcellular localization of glycolytic enzymes.

Stress-dependent variations in the properties of the rat muscle aldolase (d-fructose-1,6-bisphosphate d-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13) have been linked to the corresponding changes in the levels of proteolytic activities in rat muscle. Whole-body X-irradiation of rat was shown to result in loss of muscle aldolase activity towards fructose 1,6-bisphosphate by 50% while fructose 1-phosphate activity remained unchanged (Pote, M.S. and Altekar, W. (1980) Ind. J. Biochem. Biophys. 17, 255–262). Incubation of muscle extract of irradiated rat with that from control rat or rabbit muscle aldolase caused similar changes in aldolase activity. The changes are attributed to the action of catheptic enzymes possessing latency characteristics and capable of using aldolase as a substrate; the time course of their increase after irradiation corresponds to that of loss in muscle aldolase activities. Exposure of rats to stress resulted in an increase in the ‘free’ proteolytic activity, and the concomitant loss of ‘bound’ activity in muscle lysosomes indicates labilization of lysosomal membrane. The observed degradation of aldolase in vivo by muscle lysosomes is shown to be due to the action of cathepsin B (EC 3.4.22.1) present in the proteolytic enzymes released into cytosol under stress. Inactivation of rabbit muscle aldolase and rat muscle aldolase by rat muscle cathepsin B is inhibited by leupeptin, antipain and iodoacetamide, but not by pepstatin. Inactivation is shown to be due to the release of C-terminal tyrosine if aldolase required for its catalytic activity. Cathepsin B acts as a rate-limiting enzyme in the degradation of aldolase. Such a proteolytic modification of aldolase in vivo could be relevant not only to the regulation of aldolase activity for glycolysis in muscle but also to the degradation of aldolase during stress conditions related to tissue damage and the maintenance of normal aldolase levels in the blood.

The effects of ligands with various field strengths on the optical absorption spectrum of myeloperoxidase have been investigated. As is the case with other hemoproteins, the Soret peak in the optical absorption spectra at 77 K moves to longer wavelengths when strong-field ligands are present, whereas binding of such ligands as chloride and fluoride, which stabilize the high-spin state, shows the opposite effect. With a ligand of intermediate field strength, such as azide, the optical spectrum is not affected at room temperature, but lowering of the temperature results in the formation of the low-spin form of the enzyme. Similarly, in native myeloperoxidase a spin state equilibrium is found in which the low-spin state is favoured at high ionic strength and displays corresponding changes in the optical spectra. From the ligand- and the temperature-induced changes in the optical spectra of the ferric enzyme it is concluded that the band at 620–630 nm is an α band of the low-spin heme iron species, whereas the bands at 500 and 690 nm are probably ‘charge-transfer’ bands^∗ of the heme with the iron in the high-spin state.

Human uterine cervix at term pregnancy was found to contain an alkaline metallo-proteinase by use of a synthetic substrate, 2,4-dinitrophenyl-l-Pro-l-Gln-Gly-l-Ile-l-Ala-Gly-l-Gln-d-Arg. The enzyme (with a molecular weight of 3.8 · 10⁴) was most active around pH 9.2 toward casein and $\text{Nα-}\text{benzoyl-}\text{dl}\text{-Arg}\text{-p-}\text{nitroanilide}$. [¹⁴C]-Gelatin and proteoglycan subunit were also substrates for the enzyme, but [¹⁴C]collagen was not. In particular, the enzyme digested gelatin 70-times faster than the novel neutral proteinase in the cervix. Although EDTA was a potent inhibitor, 1,10-phenanthroline, human serum, diisopropylfluorophosphate and elastatinal had no effect on the enzyme. Alkaline proteinase in term pregnant cervices was significantly higher than in non-pregnant ones.

(1) The fumarate reductase complex from Vibrio succinogenes contains one FAD molecule, one [4Fe-4S]^3+(3+,2+) and one [2Fe-2S]^2+(2+,1+) cluster per enzyme molecule. Both clusters can be partly reduced by succinate. In the presence of excess Na₂S₂O₄ and fumarate, the [2Fe-2S] cluster is completely oxidized, whereas the other cluster is largely reduced. (2) The [2Fe-2S] cluster is localized in the M_r 31 000 subunit. The EPR spectrum of the reduced cluster in the isolated subunit differs slightly in line width, but not in g-value, from the spectrum of the reduced, intact enzyme complex. This demonstrates that the immediate environment of the cluster is little perturbed by dissociating this subunit from the FAD-containing M_r 79 000 subunit. The temperature dependence of the power-saturation behaviour has, however, greatly decreased in the isolated subunit, the saturation at 11 K of the paramagnetic cluster being much less than in the enzyme complex. Moreover, the temperature dependence of the power-saturation behaviour of this cluster in the enzyme is greater with succinate as reducing agent, than with dithionite. (3) The [4Fe-4S] cluster is located on the M_r 79 000 subunit. This cluster is unstable in air when the subunit has been dissociated from the enzyme complex.

The rates of synthesis and degradation of mitochondrial phosphoenolpyruvate carboxykinase (GTP : oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32) in the liver of tadpoles in the two developmental stages (stages VIII and XXII), and in those treated with 3,5,3′-triiodothyronine, were studied by immunochemical techniques. The rate of synthesis of the enzyme was found to be accelerated at 9 h and 6 days after triiodothyronine administration and also during natural metamorphic climax. No difference was observed in the degradation rate of the enzyme between the tadpoles in the two stages, VIII and XXII.

d-Galactonate dehydratase (d-galactonate hydro-lyase, EC 4.2.1.6) catalyzes the first reaction in the d-galactonate catabolic pathway of non-pathogenic Mycobacteria. As a part of studies concerning the metabolism of d-galactose and related compounds as well as its regulation in saprophytic strains of Mycobacteria, d-galactonate dehydratase has been purified and enzymologically characterized. The enzyme has been purified 325-fold from the crude extracts of galactose-grown Mycobacterium butyricum and its molecular weight of about 270 000 has been determined by Sephadex G-200 filtration. Isolation and analysis procedures are described. The dehydratase reaction is optimal within a pH range of 7.8–8.0. The enzyme is strictly specific for d-galactonate; none of the other sugar acids tested serves as a substrate or inhibits the dehydration of d-galactonate. The K_m value for d-galactonate is 1 mM. The enzyme requires Mg²⁺ or Mn²⁺ for activity. The dehydratase is very sensitive to SH-blockers; the most potent inhibitor is ZnSO₄, which considerably inhibits the enzyme at a concentration of 2.5–5.0 μM. Zinc-inhibited enzyme can be reactivated by chelating agents. The dehydratase is heat-resistant but dithiothreitol renders it more sensitive on heating.