Aminopeptidase Activity of Haloalkalophilic Bacteria of the Genus <Emphasis Type="Italic">Halomonas</Emphasis> Isolated from the Soda-Saline Lakes in the Badain Jaran Desert

Three strains of haloalkaliphilic bacteria were isolated from microbial mats of soda-saline lakes of the Badain Jaran Desert, Inner Mongolia (China). Based on the data on ribosomal phylogeny, they were identified as members of the genus Halomonas. These bacteria were moderate alkaliphiles and extreme halophiles. The peptidases secreted by these bacteria were shown to have narrow substrate specificity. They hydrolyzed proteins and para-nitroanilide substrates and showed maximal activity in the hydrolysis of L-leucyl-p-nitroanilides (LpNA). The maximum activity of the peptidases occurred at alkaline pH values (8–10) and elevated salinity (50–100 g/L); the enzymes were thermostable (up to 50°С). The results of inhibitor analysis and substrate specificity examination of extracellular enzymes indicated them to belong to the class of aminopeptidase-like metallopeptidases.     

Purification and properties of dipeptidyl peptidase IV from Streptococcus mitis ATCC 9811

Dipeptidyl peptidase IV (EC 3.4.14.—) from Streptococcus mitis ATCC 9811 was purified to a specific activity of 56.2 units/mg protein by a series of column chromatographic techniques. The purified enzyme was apparently homogeneous as judged by disc gel electrophoresis. Gel filtration on a calibrated column indicated an apparent molecular weight of 120,000 for the native enzyme. Gel electrophoresis of the denatured enzyme in the presence of sodium dodecyl sulfate in a constant acrylamide concentration resulted in the appearance of a single component for which a molecular weight of 53,000 was calculated. The purified enzyme has an optimum pH between 6.0 and 8.7 and an isoelectric point of 4.0. The K_m value toward glycylprolyl-p-nitroanilide is about 6.0 × 10^?5m. Substrate specificity studies indicated that the purified enzyme hydrolyzes specifically N-terminal X-proline from X-Pro-p-nitroanilides. Inhibition of this enzyme was achieved with Hg²⁺, Pb²⁺, Zn²⁺, EDTA, and diisopropyl phosphorofluoridate, but not with N-ethyl-maleimide and sulfhydryl inhibitors.     

Aminopeptidase activity from germinated jojoba cotyledons

One major and two minor aminopeptidase activities from germinated jojoba (Simmondsia chinensis) cotyledon extracts were separated by ammonium sulfate precipitation and chromatofocusing. None of the activities were inhibited by 1,10 phenanthroline.

The major aminopeptidase, purified 260-fold, showed a pH optimum of 6.9 with leucine-p-nitroanilide as substrate, a molecular weight estimated at 14,200 by electrophoretic analysis, and an isoelectric point of 4.5 according to the chromatofocusing pattern. Activity was inhibited by p-chloromercuribenzoate, slightly stimulated by 1,10 phenanthroline and 2-mercaptoethanol, and not influenced by Mg²⁺ or diethyl pyrocarbonate. Inhibition by p-chloromercuribenzoate was prevented by the presence of cysteine in the assay. Leucine-p-nitroanilide and leucine-β-naphthylamide were the most rapidly hydrolyzed of 11 carboxy-terminal end blocked synthetic substrates tested. No activity on endopeptidase or carboxypeptidase specific substrates was detected. The major aminopeptidase showed activity on a saline soluble, jojoba seed protein preparation and we suggest a possible physiological role for the enzyme in the concerted degradation of globulin reserve proteins during cotyledon senescence.

     

A Nonproteolytic "Trypsin-like" Enzyme: Purification and Properties of Arachain

By the use of the proteolytic substrates benzoyl-dl-arginine-p-nitroanilide and benzoyl-l-arginine ethyl ester the enzyme arachain has been purified 325-fold from acetone powders of ungerminated peanuts. The pH optimum for the hydrolysis of benzoyl-dl-arginine-p-nitroanilide was 8.1 in tris buffer, and for benzoyl-l-arginine ethyl ester was 7.5 using N - 2 - hydroxyethylpiperazine - N′ - 2 - ethanesulfonic acid buffer. The purest fraction showed one main band with one to three minor bands on disc gel electrophoresis. The major protein component had an S_20,w of 6.20. The energy of activation for the hydrolysis of benzoyl-dl-arginine-p-nitroanilide was calculated to be 16 kilocalories. The Michaelis constant for benzoyl-dl-arginine-p-nitroanilide was 10 micromolar and for benzoyl-l-arginine ethyl ester was 110 micromolar. The enzyme showed essentially no activity with casein, dimethyl casein, or bovine serum albumin as substrates. A large number of peptides were hydrolyzed by the enzyme, only l-leucyl-l-tyrosine being resistant of the peptides tested. The results suggest that arachain is not a “trypsin-like” protease but is a peptide hydrolase.     

Purification and Characterization of an X-Prolyl-Dipeptidyl Peptidase from Lactobacillus sakei

An X-prolyl-dipeptidyl peptidase has been purified from Lactobacillus sakei by ammonium sulfate fractionation and five chromatographic steps, which included hydrophobic interaction, anion-exchange chromatography, and gel filtration chromatography. This procedure resulted in a recovery yield of 7% and an increase in specificity of 737-fold. The enzyme appeared to be a dimer with a subunit molecular mass of approximately 88 kDa. Optimal activity was shown at pH 7.5 and 55°C. The enzyme was inhibited by serine proteinase inhibitors and several divalent cations (Cu²⁺, Hg²⁺, and Zn²⁺). The enzyme almost exclusively hydrolyzed X-Pro from the N terminus of each peptide as well as fluorescent and colorimetric substrates; it also hydrolyzed X-Ala at the N terminus, albeit at lower rates. K_m s for Gly-Pro- and Lys-Ala-7-amido-4-methylcoumarin were 29 and 88 μM, respectively; those for Gly-Pro- and Ala-Pro-p-nitroanilide were 192 and 50 μM, respectively. Among peptides, β-casomorphin 1-3 was hydrolyzed at the highest rates, while the relative hydrolysis of the other tested peptides was only 1 to 12%. The potential role of the purified enzyme in the proteolytic pathway by catalyzing the hydrolysis of peptide bonds involving proline is discussed.     

Purification and Characterization of a Fibrinogen-Degrading Protease in Bacteroides fragilis Strain YCH46

A novel fibrinogenolytic protease was purified from Bacteroides fragilis strain YCH46. The protease was extracted from cells by ultrasonic treatment and was purified 425-fold with a recovery of 2.1% by sequential procedures using azocasein as a substrate. The purified protease showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an estimated molecular weight of 100 kDa, which was consistent with the value obtained by gel filtration, indicating a monomeric native structure. Its optimal pH, K_m, and V_max for azocasein were 7.5, 0.2%, and 286 U/min/mg, respectively. The protease activity was completely inhibited by addition of 1 mM Hg²⁺, Cu²⁺, Zn²⁺, diisopropyl fluorophosphate, N-ethylmaleimide or p-chloromercuribenzoate but not by the inhibitors of metalloprotease or aspartic protease, suggesting that the enzyme is a serine-thiol-like protease. The protease hydrolyzed azocasein, casein, fibrinogen, gelatin, and azocoll, but not bovine serum albumin, ovalbumin, fibrin, fibronectin, immunoglobulins, transferrin, hemoglobin or types I, III, and IV collagen. The enzyme also hydrolyzed the chromogenic substrates alanyl-alanine p-nitroanilide, L -valyl-alanine p-nitroanilide, alanyl-alanyl-valyl-alanine p-nitroanilide, and glycyl-proline p-nitroanilide, but was inert toward L -alanine p-nitroanilide, alanyl-alanyl-alanine p-nitroanilide, and N-α-benzoyl-DL -arginine p-nitroanilide. The protease completely hydrolyzed the α-chain of fibrinogen at 37 C within 10 hr and at the same time the time required for clotting of protease-treated fibrinogen by thrombin was prolonged. The fibrinogenolytic activity of a crude extract of B. fragilis was stronger than that of other species of the Bacteroides fragilis group tested: B. ovatus, B. distasonis, B. eggerthii, B. uniformis, and B. thetaiotaomicron. These results suggest that the fibrinogenolytic protease is an important biological factor in Bacteroides infection.     

A new and highly sensitive fluorescence assay for collagenase-like peptidase activity.

A highly sensitive fluorescence assay for collagenase-like peptidase (CL-peptidase) has been developed using a newly synthesized substrate, (succinyl-Gly-Pro-Leu-Gly-Pro)-4-methylcoumaryl-7-amide (Suc-GPLGP-MCA). Suc-GPLGP-MCA was hydrolyzed at the Leu-Gly bond by CL-peptidase, (Gly-Pro)-4-methylcoumaryl-7-amide liberated by the enzyme was immediately hydrolyzed to Gly-Pro and 7-amino-4-methylcoumarin (AMC) by an excess of an auxiliary enzyme, X-prolyl dipeptidyl-aminopeptidase, and the fluorescence intensity of the AMC was measured at 460 nm with excitation at 380 nm. When assayed by this method, CL-peptidase partially purified from chick embryo showed a pH optimum at 8.0 and a K_m value of 4.0 × 10^?4m toward Suc-GPLGP-MCA. Under the optimum condition, the reaction proceeded linearly up to 4 h. The CL-peptidase activity was found in normal human sera by this method and the mean and standard deviation of the activity was 0.59 ± 0.10 nmol/min/ml of serum (n = 10). This assay was also applicable for the CL-peptidase in human liver and kidney. The results suggest that the CL-peptidase assayed by this new substrate may be different from the “PZ-peptidase” which cleaves a synthetic substrate for collagenase-like peptidase, 4-phenylazobenzyloxycarbonyl (PZ)-Pro-Leu-Gly-Pro-d-Arg (PZ-peptide). The new peptide, Suc-GPLGP-MCA, was found not to be a substrate for specific collagenase from tadpole.     

Prolidase from bovine intestine: purification and characterization

Prolidase [iminodipeptidase, EC 3.4.13.9] was highly purified from the cytosol fraction of bovine small intestine by a series of column chromatographies on DEAE-Toyopearl, Sephadex G-150, PCMB-T-Sepharose and hydroxyapatite. The purified enzyme appeared homogeneous as judged by disc gel electrophoresis. The enzyme was most active at pH 7.2 with Gly-Pro as substrate. It was stable between pH 5.5 and 8.5 for 30 min at 30 degrees C and retained half of the activity after 15 min at 40 degrees C. It was completely inactivated by p-chloromercuribenzoate (PCMB) but not inhibited by diisopropylphosphorofluoridate (DFP), phenylmethane sulfonylfluoride (PMSF) and metal chelators. Its amino acid composition was determined. Its molecular weight was estimated to be 116,000 by gel filtration on Sephadex G-150 and 56,000 by sodium dodecyl sulfate (SDS) gel electrophoresis, suggesting that it is a dimer. It hydrolyzed dipeptides represented as X-Pro (X = amino acid).     

Purification of glycosylphosphatidylinositol-anchoring aminopeptidase in from the plasma membrane of larval midgut epithelial cells of the silkworm,Bombyx mori

• 1.1. Aminopeptidase N was selectively released from larval midgut of silkworm, Bombyx mori, by phosphatidylinositol-specific phospholipase C, and purified to a homogeneous state by ion exchange, gel filtration. Con A-Sepharose and 4-aminobenzyl phosphonic acid-agarose column chromatographies.
• 2.2. The purified aminopeptidase N preparation showed 190.8 U/mg of specific activity. Its molecular weight was estimated to be around 100 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
• 3.3. Purified aminopeptidase N molecule preferentially hydrolyzed Leu-, Ala- and Met-p-nitroanilide as substrates. Especially, Leu-p-nitroanilide proved to be the best substrate for aminopeptidase N from larval midgut of silkworm.
• 4.4. By treatment with phosphatidylinositol-specific phospholipase C, two other hydrolases, alkaline phosphatase and alkaline phosphodiesterase I, were also solubilized from silkworm midgut.

     

Purification and Partial Characterization of a Prolyl-Dipeptidyl Aminopeptidase from Lactobacillus helveticus CNRZ 32

X-prolyl-dipeptidyl aminopeptidase, which hydrolyzed Gly-Pro-p-nitroanilide (relative activity [RA] = 100%) and Arg-Pro-p-nitroanilide (RA, 130%), was purified to homogeneity from the cell extract of Lactobacillus helveticus CNRZ 32. The enzyme also hydrolyzed Ala-Pro-Gly (RA, 11%) and Ala-Ala-p-nitroanilide (RA, 2%) but was not active on Ala-Leu-Ala, dipeptides, and endopeptidase and carboxypeptidase substrates. The enzyme was purified 145-fold by streptomycin sulfate precipitation, ammonium sulfate fractionation, and a series of column chromatographies on DEAE-cellulose, arginine-Sepharose 4B, and glycyl-prolyl-AH-Sepharose 4B. The purified enzyme appeared as a single band on native polyacrylamide gel and sodium dodecyl sulfate-polyacrylamide gel electrophoreses and had a molecular weight of 72,000. Optima for activity by the purified enzyme were pH 7.0 and 40°C. The enzyme was incubated at 40°C for 15 min with various metal ions. It was activated by Mg²⁺ (2.5 mM), Ca²⁺ (0.1 to 2.5 mM), Na⁺ (10 to 50 mM), and K⁺ (10 to 50 mM) and was inhibited by Hg²⁺ (0.1 to 2.5 mM), Cu²⁺ (0.1 to 2.5 mM), and Zn²⁺ (0.1 to 2.5 mM). Enzyme activity was partially inhibited by EDTA (1.0 mM, 20 h at 40°C), 1,10-phenanthroline (1.0 mM, 15 min at 40°C), phenylmethylsulfonyl fluoride (1.0 mM), N-ethylmaleimide (1.0 mM), and iodoacetate (1.0 mM). It was completely inhibited by diisopropyl fluorophosphate (1.0 mM, 2 h at 40°C) and p-chloromercuribenzoate (1.0 mM, 15 min at 40°C). The enzyme was not affected by dithioerythritol (1.0 to 10 mM).     

Properties of γ-Glutamyltransferase from Lentinus edodes

An enzyme preparation catalyzing p-nitroaniline release from γ-glutamyl-p-nitroanilide was obtained in a 200-fold purified state from fruit bodies of an edible mushroom, Lentinus edodes. Analysis of the final preparation by differential centrifugation revealed that the enzyme was still bound with subcellular particles. The enzyme catalyzed both the hydrolysis and transfer of the γ-glutamyl moiety from γ-glutamyl-p-nitroanilide, but exhibited essentially no activity of glutaminase, glutamine aminotransferase, glutamine synthetase or γ-glutamyl cyclotransferase. With γ-glutamyl-p-nitroanilide the activity was maximal at about pH 7.6. The enzyme activity increased with an increasing concentration of Tris-HCl buffer, but not with phosphate buffer which was inhibitory. An apparent Michaelis constant of 4 mm was obtained in 0.5 m Tris-HCl buffer at pH 7.6. S-Alkylcysteine sulfoxide served as the best glutamyl acceptor. A serine-borate mixture, pCMB, Cu²⁺, Hg²⁺ and Zn²⁺ were potent inhibitors. All the experimental results, including the insoluble nature of the enzyme, allowed us to classify the Lentinus enzyme in the family of γ-glutamyl transferase.     

Purification and Characterization of Two Benzoyl-l-Tyrosine p-Nitroanilide Hydrolases from Etiolated Leaves of Zea mays L

Two benzoyl-l-tyrosine p-nitroanilide hydrolases (BTPAases I and II) were purified from the etiolated leaves of Zea mays L. and characterized. BTPAase I was electrophoretically homogeneous and consisted of two identical subunits having a molecular weight of 53,000. The molecular weight of BTPAase II was 65,000. The Michaelis constants for substrate, BTPA, were 4 millimolar and 1.3 millimolar for BTPAases I and II, respectively. Based on the action of various inhibitors on both enzyme activities, these enzymes were classified as serine proteases. BTPAase I showed caseinolytic activity at neutral pH and the activity was strongly inhibited by the serine protease inhibitors.     

Methane- and n-butaneboronates: nw derivatives for gas-chromatographic analysis of 3-methoxy-4-hydroxyphenylethylenglycol.

Several methods were described for visualization of proteolytic activity on electrophoregrams obtained with agar, agarose, starch, or acrylamide gels as supporting media. In most of these reports casein or hemoglobin were used as nonspecific substrates (1–3). Recently, colorimetric assays for trypsin, using α-benzoyl-d,l-arginine-p-nitroanilide (4), and for subtilisin, using Z-glycyl-glycyl-l-leucine-p-nitroanilide (5) were introduced for the localization of these proteases after acetate celulose and acrylamide gel electrophoresis. No convenient and simple methods were in practice for detection of leucineaminopeptidase (3), although this enzyme was assayed in solution with specific chromogenic substrates such as l-leucine-p-nitroanilide or l-leucine-β-naphtylamide (6,7).The present report describes the use of p-nitroanilide substrate-l-leucine-p-nitroanilide for detection of leucineaminopeptidase activity after acrylamide gel electrophoresis. The method allows a rapid and sensitive localization of leucineaminopeptidases.     

Aminopeptidase C of Aspergillus niger Is a Novel Phenylalanine Aminopeptidase

A novel enzyme with a specific phenylalanine aminopeptidase activity (ApsC) from Aspergillus niger (CBS 120.49) has been characterized. The derived amino acid sequence is not similar to any previously characterized aminopeptidase sequence but does share similarity with some mammalian acyl-peptide hydrolase sequences. ApsC was found to be most active towards phenylalanine β-naphthylamide (F-βNA) and phenylalanine para-nitroanilide (F-pNA), but it also displayed activity towards other amino acids with aromatic side chains coupled to βNA; other amino acids with nonaromatic side chains coupled to either pNA or βNA were not hydrolyzed or were poorly hydrolyzed. ApsC was not able to hydrolyze N-acetylalanine-pNA, a substrate for acyl-peptide hydrolases.     

An assay of dipeptidyl peptidase IV activity in human serum and serum of pregnant women with glycyl-L-proline-1-naphthylamide and other glycyl-L-proline-arylamides as substrates

The authors described a micromethod for measuring dipeptidyl peptidase IV activity in human serum with glycyl-L-proline-1-naphthylamide as substrate. The method requires less than 20 microliters of serum. The pH optimum for cleaving glycyl-L-proline-1-naphthylamine by the enzyme in human serum in Tris-HCl buffer was 8.0 and Km value was established as 7.2 X 10(-4) mol/l. The advantage of this substrate is the absence of spontaneous hydrolysis during the assay of enzyme activity in contrast to glycyl-L-proline-4-nitroanilide. The Km values of the latter substrates and glycyl-L-proline-2-naphthylamide in the same buffer were 1.0 X 10(-4) mol/l and 2.4 X 10(-4) mol/l, respectively. Glycyl-D-proline-4-nitroanilide was not hydrolyzed by the dipeptidyl peptidase IV present in human serum. The activities of dipeptidyl peptidase IV in the sera from 30 healthy human subjects with glycyl-L-proline-1-naphthylamide as substrate were 176.1 +/- 32.8 nkat/l (mean +/- standard deviation; range 100.2-264.1 nkat/l of serum). In this group men had significantly (P less than 0.01) higher activity of the enzyme than women. The cleaving of glycyl-L-proline-1-naphthylamide and glycyl-L-proline-4-nitro anilide by dipeptidyl peptidase IV in human sera was closely correlated (r = 0.86). During normal pregnancy the activity of dipeptidyl peptidase IV in human serum decreases markedly in the first half of pregnancy. After delivery, the serum enzyme activity returns progressively to initial levels.     

The study of two alkaliphilic thermophile bacteria of the <Emphasis Type="Italic">Anoxybacillus</Emphasis> genus as producers of extracellular proteinase

Two strains of alkaliphilic thermophile bacteria of the genus Anoxybacillus from hydrothermal vents of Lake Baikal were detected and characterized. It was demonstrated that proteinases secreted by these bacteria had wide substrate specificity, hydrolyzed proteins and n-nitroanilide substrates, and showed maximal activity at pyroglutamyl-alanine-alanine-leucine n-nitroanilide hydrolysis. We determined maximal activity of the proteinases at alkaline pH values (10.0–10.5), the enzymes were thermostable and were characterized by a wide thermal optimum (55–70°C). The results of inhibitor analysis and substrate specifity examination of extracellular enzymes demonstrated their belonging to the subtilisin-like serine proteinases.     

Substrate activation of porcine pancreatic kallikrein by N alpha derivatives of arginine 4-nitroanilides

Hydrolysis of several N alpha-substituted L-arginine 4-nitroanilides with porcine pancreatic kallikrein was studied under different conditions of pH, temperature, and salt concentration. At high substrate concentrations a deviation from Michaelis-Menten kinetics was observed with a significant increase in the hydrolysis rates of almost all substrates. Kinetic data were analyzed on the assumption that porcine pancreatic kallikrein presents an additional binding site with lower affinity for the substrate. Binding to this auxiliary site gives rise to a modulated enzyme species which can hydrolyze an additional molecule of the substrate through a second catalytic pathway. The values of both Michaelis-Menten and catalytic rate constants were higher for the modulated species than for the free enzyme, suggesting a mechanism of enzyme activation by substrate. Kinetic data indicated similar substrate requirements for binding at the primary and auxiliary sites of the enzyme. Tris(hydroxymethyl)aminomethane hydrochloride and NaCl were shown to alter the kinetic parameters of the hydrolysis of N alpha-acetyl-L-Phe-L-Arg 4-nitroanilide by porcine pancreatic kallikrein but not the enzyme activation pattern (ratio of the catalytic constants for the activated and the free enzyme forms). Similar observations were made when the hydrolysis of D-Val-L-Leu-L-Arg 4-nitroanilide was studied under different pH and temperature conditions.     

Aminopeptidases in the Acidic Fraction of the Yeast Autolysate

Two aminopeptidases, I and II, were found in the acidic fraction of the yeast autolysate, adsorbed on DEAE-cellose and DEAE-Sephadex A&50. Aminopeptidase I was purified as a single protein with a molecular weight of 200,000. The enzyme required Zn for its activity and hydrolyzed dipeptides, and a polypeptide (glucagon). It also hydrolyzed amides, naphthylamides and the p-nitroanilide of amino acids. The enzyme was strongly inhibited by sulfhydryl reagents. Aminopeptidase II seemed also to be a metal enzyme with a molecular weight of 34,000. The enzyme hydrolyzed the dipeptide and tetrapeptide but not leucine-p-nitroanilide.     

Letter: Bacteriophage T4D-induced proteinase

The change of trypsin-like proteolytic activity in Eacherichia coli cells infected with bacteriophage T4D has been investigated. Synthetic α,N-benzoyl-d,l-arginine p-nitroanilide was used as an enzyme substrate. Proteinase activity of the host cell was inhibited 30% eight minutes after infection. Later, the activity of the phage-induced proteinase increased and a maximum (40%) increment was reached 18 minutes after infection. It was demonstrated that the newly formed enzyme had a pH optimum (6.7) which differed from the optima of proteolytic enzymes of the host cell.