Separation of rat muscle aminopeptidases.

By means of chromatography on DEAE-Sephadex, two arylamidases (hydrolysing L-arginine 2-naphthylamide) and three dipeptidyl peptidases (hydrolysing dipeptide 2-naphthylamides) were distinguished in extracts of rat muscle. However, the arylamidase from the larger peak also hydrolysed the dipeptide 2-naphthylamides. Glycyl-L-arginine amide, an alternative substrate for dipeptidyl peptidase I, was not hydrolysed by arylamidase. L-Leucine amide was hydrolysed by an enzyme, presumed to be leucine aminopeptidase, from a separate peak, but was also hydrolysed by arylamidase. Arylamidase, dipeptidyl peptidase III and most of the leucine aminopeptidase could be extracted from the muscle with a neutral salt solution, but dipeptidyl peptidase I was extracted only in the presence of Triton X-100; dipeptidyl peptidase II showed an intermediate extraction behaviour.     

An Arylamidase from Flavobacterium meningosepticum

An arylamidase was purified from Flavobacterium meningosepticum by a series of chromatographies on CM-cellulose, DEAE-Sephadex A-50 and Sephadex G-150. The purified enzyme appeared homogeneous on SDS-gel electrophoresis. The molecular weight of the enzyme was estimated to be more than 500,000 dalton by using a column of Sepharose 4B and to be 62,000 when checked by SDS-gel electrophoresis. The enzyme was most active at pH 7.5 toward Leu-β-naphthylamide (Leu-β-NA). It catalyzed the hydrolysis of not only various amino acid-β-naphthylamides but also some peptides, but the hydrolysis rate of the latter substrates was quite low. Cys-di-β-naphthylamide was split by this enzyme at an optimal pH of 6.2. Incubation of oxytocin with the enzyme resulted in a decrease in the biological activity, indicating that this arylamidase possesses an oxytocinase (cystyl aminopeptidase)-like activity.     

Endometrial arylamidase activity in the guinea pig: changes during the oestrous cycle, decidualization and ovarian steroid hormone treatment.

1. As suggested by comparative studies done in various species, amino acid arylamidases (amino peptidases) may play a role in blastocyst implantation. 2. Histochemical studies of the guinea pig endometrium indicate that arylamidase increases in the stroma during pregnancy but is depleted in the vicinity of the blastocyst during implantation. 3. To further explore the possible significance of arylamidases in uterine function, endometrial arylamidase activity was measured in guinea pigs during the reproductive cycle, decidualization and after ovariectomy with and without estrogen (E) and/or progesterone (P) treatment. Arylamidase activity was maximal during pro-oestrus-oestrus (40.0 +/- 10.0 mu/mg protein). 4. Enzyme activity was markedly depleted in decidualized endometrial stroma (12.3 +/- 1.6, P less than 0.01); reduced by ovariectomy (20.5 +/- 2.7); and stimulated by E (29.2 +/- 1.2); P had little effect (21.9 +/- 3.5). 5. The physiological significance of modulation of endometrial arylamidase activity by steroid hormones is discussed.     

Studies on the proteolytic activity of Bacteroides fragilis

The proteolytic activity of the intestinal bacterium Bacteroides fragilis NCDO 2217 was cell-bound during exponential growth, but was progressively released from the cells in stationary phase. Proteins hydrolysed included casein, trypsin, chymotrypsin, azocasein and the proteins in azosoya bean flour. Collagen, azocoll, elastin, gelatin, ovalbumin and bovine serum albumin were either weakly degraded or completely refractory to proteolysis. Arylamidase activity was exhibited against leucine p-nitroanilide (LPNA), leucine beta-naphthylamide, glycyl-proline p-nitroanilide and valyl-alanine p-nitroanilide. The bacterium grew with ammonia, peptone or casein as sole nitrogen source. Azocasein- and LPNA-hydrolysing activities were consistently higher when grown on casein. Cell-bound protease activity increased concomitantly with growth rate in both carbon- and nitrogen-limited continuous culture. Leucine arylamidase activity was also growth-rate-dependent, being 3-fold greater at D = 0.18 h-1 compared to D = 0.03 h-1. Extracellular proteolytic activity was only detected at low growth rates, accounting for about 25% of total protease activity.     

Chemical mechanism of lysophosphatidylcholine: lysophosphatidylcholine acyltransferase from rabbit lung. pH-dependence of kinetic parameters.

Lysophosphatidylcholine: lysophosphatidylcholine acyltransferase is an enzyme that catalyses two reactions: hydrolysis of lysophosphatidylcholine and transacylation between two molecules of lysophosphatidylcholine to give disaturated phosphatidylcholine. Following the kinetic model previously proposed for this enzyme [Martín, Pérez-Gil, Acebal & Arche (1990) Biochem. J. 266, 47-53], the values of essential pK values in free enzyme and substrate-enzyme complexes have now been determined. The chemical mechanism of catalysis was dependent on the deprotonation of a histidine residue with pK about 5.7. This result was supported by the perturbation of pK values by addition of organic solvent. Very high and exothermic enthalpy of ionization was measured, indicating that a conformational re-arrangement in the enzyme accompanies the ionization of the essential histidine residue. These results, as well as the results from previous studies, enabled the proposal of a chemical mechanism for the enzymic reactions catalysed by lysophosphatidylcholine: lysophosphatidylcholine acyltransferase from rabbit lung.     

pH dependence of penicillin amidase enantioselectivity for charged substrates.

The pH dependence of E (enantiomeric ratio or enantioselectivity, a quantitative measure for enzyme stereospecificity) was studied for penicillin amidase catalysed hydrolysis of charged enantiomeric substrates. Theoretical analysis shows that a pH dependence can only be observed around the pK values of groups in the active site whose ionisation control the enzyme activity. For charged substrates that may perturb these pK values, a pH dependence of E is also expected. This was experimentally verified around these pK values. The S'(1)-stereospecificity of penicillin amidase was studied for the hydrolysis of the enantiomeric phenylacetyl-S/R-Phe and for the racemic phenylacetyl-S,R-PhG. The S(1)-stereospecificity was investigated for the hydrolysis of the enantiomeric S/R-PhG-NH(2). The observed pH modulation of E (more than 3-fold for the studied substrates in the pH range 4.5-9) was found to be a result of compensatory effects for binding and catalysis. The ratios k(cat, S)/k(cat,R) and K(m,S)/K(m,R) for the hydrolysis of the enantiomeric phenylacetyl-Phe were found to decrease from 1000 to 10 and from 0.1 to 0.01, respectively in the pH range 5-8. The dependence was stronger for the S'(1)- than for the S(1)-subsite. This is probably due to the stronger influence of the substrate carboxyl group in the S'(1)-subsite than that of the substrate amino group in the S(1)-subsite on the pK of the N-terminal Ser B1 that is essential for the activity. The observed pH dependence of E was used to discuss the importance of ground-state interactions for discrimination between enantiomers and for enzyme catalysis in general. The experimental results conform to the split site model according to which a better binding must not be fundamentally inhibitory.     

The pH-dependence of pepsin-catalysed reactions

1. The pH-dependence of the pepsin-catalysed hydrolysis of three peptide substrates was studied by using a method for the continuous monitoring of the formation of ninhydrin-positive products. 2. Two peptide acid substrates, N-acetyl-l-phenylalanyl-l-phenylalanine and N-acetyl-l-phenylalanyl-l-phenylalanyl-glycine, show apparent pK(a) values of 1.1 and 3.5 in the plots of k(0)/K(m) versus pH. By contrast a neutral substrate, N-acetyl-l-phenylalanyl-l-phenylalanine amide, shows apparent pK(a) values of 1.0 and 4.7. 3. Together with the data of the preceding paper (Knowles, Sharp & Greenwell, 1969), these results are taken to indicate that the rate of pepsin-catalysed hydrolysis is controlled by the ionization of two groups, which on the free enzyme have apparent pK(a) values of 1.0 and 4.7. It is apparent that the anions of peptide acid substrates are not perceptibly bound to the enzyme, resulting in apparent pK(a) values of 3.5 for the dependence of k(0)/K(m) for these materials.     

Specificity of alkaline elastase Bacillus on the oxidized insulin A- and B-chains

The substrate specificity of alkaline elastase Bacillus from alkalophilic Bacillus sp. Ya-B was investigated using oxidized insulin A- and B-chains. Under time-limited cleavage, the initial cleavage site of the enzyme on the oxidized insulin A-chain and B-chain was at the leucine13-tyrosine14 bond and the leucine15-tyrosine16 bond, respectively. When the cleavage was completed, three major cleavage sites and three minor cleavage sites on the A-chain, and five major cleavage sites and four minor cleavage sites on the B-chain were found. However, most of the peptides produced after complete hydrolysis of the A- or B-chain by the enzyme were composed of four to six amino acid residues. The results suggest that this enzyme cleaves the oxidized insulin A- and B-chains in a block-cutting manner.     

A study of the enzymatic profile of soil isolates of Nocardia asteroides

In this study, using the API-ZYM system, we have reported the enzyme profile of 42 soil strains and 2 clinical strains of Nocardia asteroides isolated locally. Of the 19 enzymes tested, only 7 were demonstrable in over 90% of the soil isolates. These included alkaline phosphatase, esterase lipase, leucine arylamidase, acid phosphatase, phosphohydrolase, α-glucosidase and β-glucosidase. In addition, β-galactosidase activity was demonstrated in all the strains by the O-nitrophenyl-β-D-galactopyranoside (ONPG) test. The enzymes which were not demonstrable in >95% of the strains included valine arylamidase, cystine arylamidase, trypsin, chymotrypsin, α-galactosidase, β-glucoronidase, N-acetyl-β-glucosaminidase, α-mannosidase and α-fucosidase. With the exception of valine arylamidase, which was lacking in all but one isolate, the enzyme profiles of the soil isolates were comparable with the clinical isolates of N. asteroides reported in previous studies. The reasons for this difference in the two sets of isolates is not clear. The study reinforces the view that specific differences in the enzymatic profiles of Nocardia species could be used for their rapid identification. However, more extensive studies are needed to establish the reproducibility of this method. To the best of our knowledge, this is the first study of the enzymatic profile of soil isolates of N. asteroides originating from a single geographic region. This revised version was published online in June 2006 with corrections to the Cover Date.     

Arylamidase Activity of Salmonella Species

Arylamidase activity in cell extracts of sonically cell treated suspensions of 23 Salmonella strains, including 12 strains of S. typhimurium, was investigated. All cultures hydrolyzed five of nine different neutral and basic substrates. Activity against aspartyl-, cystyl- histidinyl-, and isoleucyl-β-naphylamide was negligible. Alanyl-β-naphthylamide was the preferred substrate for the Salmonella species; however, specific activities ranged widely. Of several gram-negative organisms surveyed, all except Proteus vulgaris hydrolyzed alanyl-β-naphthylamide at the fastest rate. The most preferred substrate for the Proteus culture was glycyl-β-naphthylamide. No relationship could be shown between virulence and arylamidase activity for the Salmonella strains.     

Mechanism of the addition half of the O-acetylserine sulfhydrylase-A reaction

O-Acetylserine sulfhydrylase (OASS) catalyzes the last step in the cysteine biosynthetic pathway in enteric bacteria and plants, substitution of the beta-acetoxy group of O-acetyl-l-serine (OAS) with inorganic bisulfide. The first half of the sulfhydrylase reaction, formation of the alpha-aminoacrylate intermediate, limits the overall reaction rate, while in the second half-reaction, with bisulfide as the substrate, chemistry is thought to be diffusion-limited. In order to characterize the second half-reaction, the pH dependence of the pseudo-first-order rate constant for disappearance of the alpha-aminoacrylate intermediate was measured over the pH range 6.0-9.5 using the natural substrate bisulfide, and a number of nucleophilic analogues. The rate is pH-dependent for substrates with a pK(a) > 7, while the rate constant is pH-independent for substrates with a pK(a) < 7 suggesting that the pK(a)s of the substrate and an enzyme group are important in this half of the reaction. In D(2)O, at low pD values, the amino acid external Schiff base is trapped, while in H(2)O the reaction proceeds through release of the amino acid product, which is likely rate-limiting for all nucleophilic reactants. A number of new beta-substituted amino acids were produced and characterized by (1)H NMR spectroscopy.     

A single protein research integrated advanced biochemistry laboratory course; spectroscopic determination of tyrosyl side chain pKa

The unique bio-analytical properties of the amino acid tyrosine (Tyr) are the focus of this experiment from the research oriented biochemistry laboratory course at our university. In the present study pK(a(1)), pK(a(2)), and pK(a(3)) values for free Tyr were estimated to be 2.30, 9.40, and 9.97, respectively, when free Tyr was titrated with 1mM NaOH and 1mM HCl using a pH meter. Spectrophotometric analysis of the phenolic side chain pK(a(3)) revealed a value of 10.14, which was consistent with the pK(a)s estimated from the pH meter. The results from this experiment will allow students to compare the free Tyr properties with those present in a protein.     

Characterization of leucine amino peptidase from Streptomyces gedanensis and its applications for protein hydrolysis

The aim of this work was to purify and characterize the extra-cellular leucine amino peptidase (LAP) from Streptomyces gedanensis and also study its applications for protein hydrolysis. The enzyme was purified to homogeneity by ammonium sulfate fractionation and sequential chromatography steps. LAP appeared to be a monomeric enzyme with a molecular weight of ～75 kDa determined by sodium dodecyl sulfate poly acryl amide gel electrophoresis (SDS-PAGE). The enzyme preferentially hydrolyzed leucine p-nitroanilide followed by Met, Phe, Lys and Arg derivatives. Kinetic studies on the purified enzyme confirmed that it can hydrolyze peptide as well as ester substrates at comparable rates. This amino peptidase was highly resistant to different concentrations of various organic solvents. The characteristics of this amino peptidase, including thermo stability, organic solvent resistance, its activity against various substrates, and also it showed esterase and peptidase activity at comparable rates; identified this amino peptidase as a novel one. The specificity towards aromatic and hydrophobic amino acid residues, the solvent-resistance and thermo stability make this amino peptidase could offer interesting possibilities for various industrial applications including debittering of protein hydrolysates, peptide and ester synthesis.     

Monkey brain arylamidase. II. Further characterization and studies on mode of hydrolysis of physiologically active peptides.

A large-scale purification of monkey brain arylamidase was carried out. Amino acid analyses indicate that the enzyme is rich in acidic amino acids and is poor in cystine. The amino terminal residue was determined to be alanine by dansylation. The enzyme was activated by sulfhydryl compounds. Dithiothreitol was more effective than beta-mercaptoethanol. Bestatin competitively inhibited the enzyme activity and the Ki value was calculated to be 2.5 x 10(-7) M, which was of the same order as that of puromycin. The inhibitions by puromycin and bestatin were reversible. The enzyme hydrolyzed di-, tri-, and oligopeptides including physiologically active peptides. Of physiologically active peptides, enkephalins and Met-Lys-bradykinin, which possess a neutral amino acid at the N-terminal position, were more rapidly hydrolyzed by the enzyme. Peptides such as LH-RH and TRH, which possess a pyrrolidonecarboxylyl group at the N-terminal position, and substance P and bradykinin, which possess a proline residue adjacent to the N-terminal residue, were not hydrolyzed by the enzyme. The Km values for various peptides indicate that the enzyme has higher affinity for oligopeptides than di- and tripeptides. The aminopeptidase activity of the enzyme was also competitively inhibited by puromycin and bestatin. Analyses of the hydrolysis products of various peptides by the dansylation method indicate that the enzyme has both kinin-converting activity and angiotensinase activity.     

Arylamidase activity of neutral proteinase from Saccharomonospora canescens. Comparison with other Zn-proteinases that exhibit the same activity

The arylamidase activity of Zn-proteinase from Saccharomonospora canescens (NPS) was studied with series of peptide nitroanilides of varying amino acid sequence and N-acyl blocking groups. The partial mapping of the enzyme S(1), S(2), S(3), S(4) subsites shows that variations in all positions P(1) to P(4) in the substrate structure affect the catalytic efficiency. The importance of P(4)-S(4) and P(1)-S(1) interactions, which is a characteristic feature of the serine proteinases, is evidenced for the studied Zn-proteinases NPS and serralysin too. The presence of arylamidase activity in the case of Zn-proteinases-astacin EC 3.4.24.21 and serralysin EC 3.4.24.40 is correlated with some specific characteristics of their active site structure: penta-coordinated Zn(2+) and a tyrosyl residue as a fifth ligand to the Zn(2+). It is assumed that this tyrosyl residue plays a role in the productive binding and stabilization of the tetrahedral adduct formed during the reaction of enzyme-catalysed hydrolysis of peptide arylamides of corresponding length and sequence.     

Polypeptide antibiotic 26a from Bacillus subtilis. III. Physicochemical and biological in vitro properties.

Antibiotic 26a, a weakly basic (pK1 3.85 and pK2 7.1) polypeptide compound, has been recovered from the fermentation fluids of bacillus subtilis cultures as hydrochloride salt easily soluble in water and dimethylsulphoxide, sparingly soluble in lower alcohols and insoluble in several organic solvents. At low concentrations 26a was effective against gram-positive bacteria, mainly micrococci and corynebacteria, moderately active against mycobacteria, and inactive against gram-negative bacteria, yeasts and moulds even at 300 microgram/ml concentration. From the viewpoint of elemental analysis, electrometric titration, optical rotation, UV, IR and NMR spectra, amino acid composition, molecular weight and biological observations, 26a can be considered as an antibiotic, if not identical, then closely related to bacitracin family polypeptides.     

A new type of muconate cycloisomerase from Rhodococcus rhodochrous strain 89

Muconate cycloisomerase (MCI) was purified from Rhodococcus rhodochrous 89 grown on phenol. The enzyme appears to contain two different type subunits with molecular masses 35.5 and 37 kD. The N-terminal amino acid sequence of both subunits showed more similarity to corresponding enzymes from gram-negative bacteria than to one from Rhodococcus opacus 1CP. MCI from R. rhodochrous 89, like analogous enzymes from gram-negative bacteria, can convert 2-chloromuconate (2-CM) with the formation of both, 2- and 5-chloromuconolactones (CML) as intermediates. Nevertheless, its unique ability to convert 5-CML to cis- but not to trans-dienelactone sets it apart from all known chloromuconate cycloisomerases from gram-negative and gram-positive bacteria.     

青藏高原不同高寒草地类型土壤酶活性及其影响因子

土壤酶活性作为生态系统养分循环的关键因素, 是反映土壤质量和生态系统功能的重要指标, 但是关于高寒草地生态系统中不同草地类型间酶活性的差异研究还很少。因此, 该研究在藏北高寒草地选择高寒草甸、高寒草原、高寒草甸草原、高寒荒漠草原和高寒荒漠5种草地类型进行野外原位调查和采样, 测定了涉及碳(C)、氮(N)和磷(P)循环的14种酶的活性, 并建立了高寒草地酶活性与土壤微生物和土壤理化性质等环境因子的关系。结果表明: C循环酶(蔗糖酶、纤维素酶、β-葡萄糖苷酶、多酚氧化酶和过氧化物酶)和P循环酶(碱性磷酸酶)在不同高寒草地类型间活性差异明显, N循环酶中仅芳香氨基酶和亚硝酸盐还原酶两种酶在不同高寒草地类型间活性差异明显。同时, C、N和P循环酶之间存在一定的相关关系, 其中, 蔗糖酶和碱性磷酸酶、纤维素酶和α-乙酰氨基葡萄糖苷酶活性显著正相关, 多酚氧化酶与亚硝酸还原酶和β-乙酰氨基葡萄糖苷酶活性显著负相关。在测定的19个环境指标中, 土壤有机质(SOM)含量、革兰氏阴性菌数量、土壤N和P含量计量比、革兰氏阳性菌数量、细菌数量、放线菌数量、全氮含量、真菌数量是影响土壤酶活性的关键因子, 且SOM含量的影响最大(解释量为11.9%)。综上所述, 不同高寒草地类型间C循环酶、P循环酶和两种N循环酶(芳香氨基酶和亚硝酸还原酶)活性差异显著, SOM含量、微生物数量和N含量等是影响高寒草地生态系统土壤酶活性的关键因子。     

Degradation of prolylleucylglycinamide (MIF) by mouse brain

Prolylleucylglycinamide (MIF) at 1.0 mM concentration and pH 7.0 was hydrolyzed by mouse brain homogenate at a rate of 140 nmol/mg protein/hr. Nearly all of this activity can be accounted for by the action of two enzymes, both of which cleave Pro and Leu sequentially from the N-terminus of MIF. At pH 7.0 the predominant enzyme is arylamidase, inhibited by puromycin (1mM) and Mn²⁺ (2.5 mM). At pH 8.5, in the presence of Mn²⁺, a second enzyme with a higher potential activity (570 nmol/mg protein/hr) was observed. While the arylamidase is primarily localized in the cytosol, the Mn²⁺-stimulated enzyme is equally divided between soluble and particulate fractions. Because of its ability to cleave leucinamide, its high pH optimum, and its Mn²⁺ dependence, it can be classified as a leucine aminopeptidase (LAP). In its substrate specifically and its preference for Mn²⁺ over Mg²⁺ it resembles the LAP from connective tissue more than that from other sources.     

The effect of pH on the transpeptidation and hydrolytic reactions of rat kidney gamma-glutamyltransferase

The effect of pH upon the transpeptidation and hydrolytic reactions of gamma-glutamyltransferase [5-glutamyl)-peptide:amino-acid 5-glutamyltransferase, EC 2.3.2.2) have been investigated. It was found that the enzyme was irreversibly inactivated below pH 7.5 or above pH 9.4. Transpeptidation was markedly pH-dependent, while hydrolysis was pH-independent. The pH optimum for transpeptidation was found to vary for different acceptors. The ascending limb of the pH-optimum curve is attributed to the pK of the alpha-amino group of the acceptor, while the descending limb of the pH-optimum curve is attributed to an ionisable group in the active site of the enzyme. These observations provide much information about the interaction of the enzyme with the acceptor: (1) the true acceptor for gamma-glutamyltransferase is the deprotonated form of the amino acid; (2) glycylglycine has a similar acceptor activity to methionine, its apparent higher activity being due to the low pK of the alpha-amino group; (3) the enzyme is reversibly inactivated at higher pH by the deprotonation of a group in the active site which is involved in both binding of acceptor and catalysis of transpeptidation (this group is not involved in the hydrolysis reaction); (4) at pH 8.5, the normal pH for assay, only 47% of the enzyme is active, while at pH 7.4 gamma-glutamyltransferase is 93% in the active form.