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

The cyclic nucleotide phosphodiesterase (3′ : 5′-cyclic nucleotide 5′-nucleotidohydrolase, EC 3.1.4.17) systems of many tissues show multiple physical and kinetic forms. In contrast, the soluble rat uterine phosphodiesterase exists as a single enzyme form with non-linear Lineweaver-Burk kinetics for cyclic AMP (app. K_m of approx. 3 and 20 μM) and linear kinetics for cyclic GMP (app. K_m of approx. 3 μM) since the two hydrolytic activities are not separated by a variety of techniques. In uterine cytosolic fractions, cyclic AMP is a non-competitive inhibitor of cyclic GMP hydrolysis (K_i approx. 32 μM). Also, cyclic GMP is a non-competitive inhibitor of cyclic AMP hydrolysis (K_i approx 16 μM) at low cyclic GMP/cyclic AMP substrate ratios. However, cyclic GMP acts as a competitive inhibitor of cyclic AMP phosphodiesterase (K_i approx 34 μM) at high cyclic GMP/cyclic AMP substrate ratios. When a single hydrolytic form of uterine phosphodiesterase, separated initially by DEAE anion-exchange chromatography, is treated with trypsin (0.5 μg/ml for 2 min) and rechromatographed on DEAE-Sephacel, two major forms of phosphodiesterase are revealed. One form elutes at 0.3 M NaOAc⁻ and displays anomalous kinetics for cyclic AMP hydrolysis (app. K_m of 2 and 20 μM) and linear kinetics for cyclic GMP (app. K_m approx. 5 μM), kinetic profiles which are similar to those of the uterine cytosolic preparations. A second form of phosphodiesterase elutes at 0.6 M NaOAc⁻ and displays a higher apparent affinity for cyclic AMP (app. K_m approx. 1.5 μ) without appreciable cyclic GMP hydrolytic activity. These data provide kinetic and structural evidence that uterine phosphodiesterase contains distinct catalytic sites for cyclic AMP and cyclic GMP. Moreover, they provide further documentation that the multiple forms of cyclic nucleotide phosphodiesterase in mammalian tissue smay be conversions from a single enzyme species.

A glutathione-dependent thioltransferase (thiol : disulfide oxidoreductase) has been partially purified (70-fold) from anterior pituitary cytosol, and characterized. Purification was effected by differential centrifugation, precipitation between 30 and 60% (NH₄)₂SO₄, and sequential chromatography on Sepharose 6B, DEAE-cellulose, and CM-cellulose. Enzyme activity, monitored by the disappearance of NADPH, was associated with a protein of molecular weight 170 000 both by gel filtration and by polyacrylamide gel electrophoresis in SDS. There was apparent charge heterogeneity after the gel filtration step, and only the major DEAE-cellulose peak was further purified on CM-cellulose. When SDS-polyacrylamide gel electrophoresis was carried out in the presence of mercaptoethanol, the two predominant bands seen in its absence were converted to five major bands, all of different apparent molecular weights from the originals. Isoelectric focusing yielded two major peaks of enzyme activity, at pI 7.0 and pI 4.5–5.0. These peaks were shown to be interconvertible upon reelectrofocusing. Both low- and high-molecular weight disulfides could be reduced. The pH optimum was sharp, at pH 8.2. The K_m values for glutathione and cystine (the standard assay disulfide) were 0.57 and 0.062 mM, respectively, each in the presence of saturating concentrations of the other substrate. N-Ethylmaleimide at 0.1 and 1.0 mM inhibited enzyme activity non-competitively, suggesting a non-catalytic role of enzyme thiol(s) for maintenance of optimal activity.

γ-Cyclodextrin was found to be hydrolyzed by human salivary and pancreatic α-amylases (1,4-α-d-glucan glucanohydrolase, EC 3.2.1.1) at appreciable rates. The optimum pH for the enzyme reactions at 37°C in the presence of 0.1 M NaCl was at around pH 5, which was remarkably different from the optimum pH (pH 6.9) of the enzymes for starch. The K_m value (2.9 mg/ml) of pancreatic α-amylase for γ-cyclodextrin was smaller than that (5.3 mg/ml) of salivary α-amylase at pH 5.3, while the V value of the former was 3.7-times larger than that of the latter. The hydrolyses of γ-cyclodextrin by both enzymes took place via the multiple attack mechanism. The degrees of multiple attack by salivary and pancreatic α-amylases for γ-cyclodextrin at pH 5.3 were 2.0 and 1.1, respectively. The distribution of maltodextrins produced by hydrolysis of γ-cyclodextrin by salivary α-amylase was suggested to be independent of the substrate concentration, while that produced by pancreatic α-amylase was presumably dependent on the substrate concentration.

An extracellular aminopeptidase (Alteromonas aminopeptidase III) of a marine pseudomonad, designated Alteromonas B-207, was purified to homogeneity. The enzyme was found to have a native molecular weight of 63 000 by exclusion chromatography and a subunit weight of 33 000 by SDS-polyacrylamide gel electrophoresis. Cross-linking with dimethyladipimidate prevented dissociation of the dimer. The enzyme has a pH optimum of 9.3, a temperature optimum of 70°C and is stable at 60°C for approx. 1 h. It has high specificity for l-leucyl-β-naphthylamide and, of the peptides tested, it shows a preference for l-Leu, Gly and l-Pro over l-α-Asp and l-Lys. The enzyme is inhibited by 1,10-phenanthroline, added to the enzyme-substrate reaction mixture, and by EDTA when the enzyme is dialyzed against 10⁻³ M soln. Activity can be partially restored to EDTA-dialyzed enzyme by removal of the EDTA and incubation of the enzyme with Zn²⁺. Atomic absorption data also implicate zinc as a required metal. Sulfhydryl- and serine- inhibitors have no effect on the enzyme.

A reproducible purification procedure of native tyrosine hydroxylase (l-tyrosine, tetrahydropteridine : oxygen oxidoreductase (3-hydroxylating), EC 1.14.16.2) from the soluble fraction of the bovine adrenal medulla has been established. This procedure accomplished a 90-fold purification with a recovery of 30% of the activity. This purified enzyme served for studying the kinetic properties of tyrosine hydroxylase using $\text{(6R)-}\text{l}\text{-erythro-1′,2′-}\text{dihydroxypropyltetrahydropterin}$ [$\text{(6R)-}\text{l}\text{-erythro-}\text{tetrahydrobiopterin}$] as cofactor, which is supposed to be a natural cofactor. Two different K_m values for tyrosine, oxygen and natural $\text{(6R)-}\text{l}\text{-erythro-}\text{tetrahydrobiopterin}$ itself were obtained depending on the concentration of the tetrahydrobiopterin cofactor. In contrast, when unnatural $\text{(6S)-}\text{l}\text{-erythro-}\text{tetrahydrobiopterin}$ was used as cofactor, a single K_m value for each tyrosine, oxygen and the cofactor was obtained independent of the cofactor concentration. The lower K_m value for $\text{(6R)-}\text{l}\text{-erythro-}\text{tetrahydrobiopterin}$ was close to the tetrahydrobiopterin concentration in tissue, indicating a high affinity of the enzyme to the natural cofactor under the in vivo conditions. Tyrosine was inhibitory at 100 μM with $\text{(6R)-}\text{l}\text{-erythro-}\text{tetrahydrobiopterin}$ as cofactor, and the inhibition by tyrosine was dependent on the concentrations of both pterin cofactor and oxygen. Oxygen at concentrations higher than 4.8% was also inhibitory with $\text{(6R)-}\text{l}\text{-erythro-}\text{tetrahydrobiopterin}$ as cofactor.

Proteinase B (EC 3.4.22.9) was purified from commercial baker's yeast and from wild type strains of Saccharomyces cerevisiae and Saccharomyces carlsbergensis. For large scale purification a procedure was developed involving hydrophobic chromatography on octyl-Sepharose 4B and gel filtration on Sephadex G-100. A rapid purification of small amounts of proteinase B was achieved by affinity chromatography on the nitrated proteinase B inhibitor, immobilized on CH-Sepharose according to Bünning and Holzer (Bünning, P. and Holzer, H. (1977). J. Biol. Chem. 252, 5316–5323). The enzyme prepared from all three sources appeared to be homogeneous and exhibited a molecular weight of 33 000 in SDS-polyacrylamide gel electrophoresis. Homogeneity and molecular weight were confirmed for the enzyme from baker's yeast by ultracentrifugation studies. Polyacrylamide gel electrophoresis without SDS and electrofocusing however, indicated microheterogeneity of the proteinase B activity. The aminoterminal residue of the enzyme was found to be glycine. Proteinase B turned out to be a glycoprotein, containing 8–9% neutral sugars and 1.5% amino sugars. The enzyme is blocked by p-hydroxymercuribenzoate and by the serine proteinase inhibitors DFP and PMSF. Among the proteinase inhibitors from microbial origin, chymostatin and antipain were the most powerful inhibitors of proteinase B.

When homogenates of rat liver and hepatomas were centrifuged at 78 000 × g, over 90% of liver N-acetylglucosaminyltransferase assayed with β-galactosidase- and β-N-acetylhexosaminidase-treated asialofetuin as acceptor was recovered in the particulate fraction, while as much as 24% of hepatoma transferase was in the supernatant fraction. The particulate transferase solubilized by 0.2% sodium deoxycholate emerged from a DEAE-cellulose column at 0.04 M NaCl (transferase A). The supernatant fractions from all the hepatomas tested contained a second N-acetylglucosaminyltransferase eluted from the column at 0.02 M NaCl (transferase B). Transferase B was absent from liver supernatant fraction. The activities of these transferases toward various acceptors and the effect of β-N-acetylhexosaminidase on their products suggest that both transferases are UDP-N-acetylglucosamine : α-mannoside β-N-acetylglucosaminyltransferase. Although ovalbumin and glycopeptide V, which was isolated from pronase digest of ovalbumin, were good acceptors, transferase A utilized ovalbumin and glycopeptide V with apparent K_m values of 0.44 and 0.33 mM, respectively, whereas the corresponding values for transferase B were 4.5 and 0.050 mM.

Two periplasmic aminopeptidases were selectively released from Alteromonas B-207 when its outer membrane and peptidoglycan layer were systematically removed. Neither enzyme was detected in cytoplasmic materials. The first enzyme (aminopeptidase II) was isolated and purified 160-fold from the supernatant of osmotically shocked cells. The second enzyme (aminopeptidase I) was obtained from the peptidoglycan fraction of lysozymetreated mureinoplasts and purified 15-fold. The two enzymes are distinguished by molecular weights, subunit structures, temperature profiles, pH optima, effects of EDTA and substrate specificities. Aminopeptidase I has a molecular weight of approx. 450 000 and appears to be a tetramer; while aminopeptidase II is a stable monomer with a molecular weight of 81 000–88 000. Aminopeptidase II has higher pH and temperature optima than does aminopeptidase I. Aminopeptidase II has broader peptide specificity than aminopeptidase I. This is particularly evident when amino acid β-naphthylamides are used as substrates. Aminopeptidase I shows its greatest activity against l-α-Asp-β-naphthylamide and l-Ala-β-naphthylamide, whereas aminopeptidase II shows a decided preference for l-Leu-β-naphthylamide. Aminopeptidase I appears to be more sensitive to EDTA than aminopeptidase II, but both enzymes apparently require Zn²⁺.

Autolysis of a washed heavy particle fraction from rat gastrocnemius muscle was largely prevented when the animals were injected with Compound ⁴⁸/80 to degranulate mast cells. The ‘myofibrillar protease’ activity appears to be due therefore to the proteolytic enzyme from mast cells. Alkaline proteinase activity (casein substrate) in rat heart preparations was also largely depleted by Compound ⁴⁸/80. Autolytic activity was similarly affected in the heavy particle fraction but much less so in the homogenate.