Purification and characterization of a coagulant protein from the venom of Russell's viper.

The coagulant protein from the venom of Russell's viper was purified by means of successive chromatography on Sephadex G-50, DEAE-cellulose and Sephadex G-200. The purified coagulant protein was homogeneous by polyacrylamide gel electrophoresis and ultracentrifugation. The molecular weight was estimated to be about 100 000 by ultracentrifuge analysis and 130 000 by gel filtration. The coagulant protein contains 11.1% carbohydrate which includes 5.1% hexose (galactose: mannose = 1:1), 5% hexosamine (glucosamine), and 1% neuraminic acid (N-acetylneuraminic acid and N-glycolyneuraminic acid). The isoelectric point is pH 6.3. The results of both sodium dodecyl sulfate electrophoresis and gel filtration in 6 M guanidium chloride suggest that it consists of four polypeptide chains. The coagulant protein functions as an enzyme in activating blood coagulation factor X in the presence of Ca2+. N-a-p-Toluenesulfonyl-L-arginine methyl ester hydrolyzing activity in the preparation definitely decreased during purification and it suggests that the clotting activity is not associated with the esterase activity. The clotting activity is inhibited by diisopropyl phosphorofluoridate and by phenylmethylsulfonyl fluoride, suggesting that the coagulant protein is a serine protease. The optimum pH is between pH 7.0 and pH 8.0. At neutral pH the coagulant protein is stable below 50 degrees C, but is rapidly inactivated above 55 degrees C.     

Induction of reaginic (IgE) gonococcal antibodies in the rat by a common antigen of Neisseria gonorrhoeae.

An antigen (ZAB) common to Neisseria gonorrhoeae was prepared by stepwise elution of a crude gonococcal antigen (ZA) from columns of diethylaminoethyl cellulose employing 0.02 M phosphate buffers, pH 7.6, containing increasing concentrations of sodium chloride. Rats immunized with ZAB produced reaginic (IgE) antibody which cross-reacted with ZA prepared from eight gonococcal strains by the passive cutaneous anaphylaxis (PCA) test. Heating of the sera at 56 degrees C for 4 h destroyed the PCA activity. The PCA activity of the anti-ZAB rat serum was removed after absorption with ZAB antigen or with rabbit anti-rat IgE but not after absorption with gonococcal lipopolysaccharide or with heat-killed or formalinized gonococci. Treatment of ZAB with trypsin or heating at 100 degrees C for 30 min destroyed or reduced the antigenic activity respectively. Further purification of ZAB by filtration through Sephadex G-100 gave a preparation (ZAB2) which contained the common antigen as shown by the cross-reactivity of anti-ZAB2 rat serum with seven stains of N. gonorrhoeae. Fraction ZAB2 contained material which had a molecular weight less than 13,700 and was associated with the presence of material absorbing at 260 nm. The results of this study indicate that a low molecular weight antigen, which appears to be protein in nature and associated with nuclei acid, is common to the gonococcus and is the main antigenic component inducing reaginic (IgE) antibody in the rat.     

Molecular characterization of a mucin-type antigen associated with human pancreatic cancer. The DU-PAN-2 antigen

This work describes the molecular characterization of a human pancreatic cancer-associated antigen defined by a murine monoclonal antibody (DU-PAN-2). DU-PAN-2 antigen was isolated from a pancreatic adenocarcinoma cell line (HPAF) or patient's ascitic fluid, and the antigenic activity was monitored by competitive inhibition radioimmunoassay. Affinity chromatography and CsCl/guanidine HCl density gradient centrifugation were employed to remove other populations of mucin-type glycoproteins and noncovalently associated proteins, respectively. Three electrophoretically distinct components were detected by 1% agarose gel electrophoresis and were resolved by chromatography on Sepharose CL-4B. The major fraction (FII) was subjected to carbohydrate and amino acid analyses. The sum of threonine, serine, proline, glycine, and alanine comprised more than 50% of the amino acid residues. The saccharide units, O-glycosidically linked to the peptide via GalNAc, contained fucose, galactose, GlcNAc, GalNAc, and sialic acid. The total carbohydrate content of FI and FII was 80.8% and 77.4% by weight, respectively. The molecular weight of FII antigen showed two species of molecules of 1.45 X 10(6) and 4.59 X 10(6) by analytical sedimentation equilibrium. DU-PAN-2 antigen was susceptible to neuraminidase, pepsin, Pronase, and papain digestion. These results suggest that both protein components and sialic acid residues may play important roles in the binding of DU-PAN-2 antibody.     

Purification and properties of a new ribonuclease from Aspergillus saitoi.

From a commercial digestive produced from Aspergillus saitoi, a ribonuclease [EC 3.1.4.23] having a molecular weight of 12,500 has been isolated in addition to the RNase reported previously, which had a molecular weight of 38,000. The enzyme was found to be homogeneous by chromatography on DEAE-cellulose, disc electrophoresis on polyacrylamide gel, and ultracentrifugation. The NH2-terminal amino acid was identified as glutamic acid. The amino acid composition indicated the presence of about 13 tyrosyl residues, 3 histidyl residues, and 2 half-cystine residues. The pH optimum of the RNase was 4.5, using RNA as a substrate. The enzyme was stable on heating at 70 degrees for 5 min from pH 2 to 10. It hydrolysed RNA completely to mononucleotides via 2', 3'-cyclic nucleotides. The rates of release of nucleotides and 2', 3'-cyclic nucleotides were in the order: guanylic acid is greater than adenylic acid is greater than cytidylic acid is greater than uridylic acid.     

Agglutinin from Limulus polyphemus. Purification with formalinized horse erythrocytes as the affinity adsorbent.

We have purified an agglutinin from the hemolymph of Limulus polyphemus about 1500-3000-fold by adsorption to formalinized horse erythrocytes, elution with N-acetylneuraminic acid and subsequent fractionation on Sephadex G-200. Recovery was in the range of 50 percent. On ultracentrifugation the agglutinin behaves as an homogenous protein with a molecular weight of about 460 000. On polyacrylamide gel electrophoresis of the dissociated protein in sodium dodecylsulfate we found a single prominent diffuse band with an apparent molecular weight of 22 000 plus or minus 2000. This band contained carbohydrate as determined by periodic acid-Schiff staining. The intensity of staining compared with standards suggested a carbohydrate content of less than 4 percent. The protein contains a preponderance of acidic amino acids and has an isoelectric point of 4.83.5 residues per 1000 of glucosamine were detected on amino acid analysis. Agglutination of formalinized horse erythrocytes by the purified protein is inhibited not only by N-acetylneuraminic acid but also by D-glucuronic acid; but not by a number of other monosaccharides. D-Glucuronic acid may be used in place of N-acetylneuraminic acid as the eluting sugar in the purification procedure.     

MRC OX-2 antigen: a lymphoid/neuronal membrane glycoprotein with a structure like a single immunoglobulin light chain.

The MRC OX-2 antigen is a rat cell surface glycoprotein of mol. wt. 41 000-47 000 found on neurones, thymocytes, B cells, follicular dendritic cells and endothelium. We now report the amino sequence for this antigen as deduced from the nucleotide sequence of cDNA clones detected by use of an oligonucleotide probe. The sequence contains 248 amino acid residues of which 202 residues are likely to be outside the cell with two domains that show homology with immunoglobulins. The N-terminal domain fits best with Ig V domains and Thy-1 antigen while the C-terminal part is like an Ig C domain. Thus the structure overall is similar to an Ig light chain or the T cell receptor beta chain. Three glycosylation sites are identified on each of the MRC OX-2 antigen domains.     

Isolation and characterization of a hemagglutinin from Amphitrite ornata, a polychaetous annelid.

Extracts of the marine polychaetous annelid, Amphitrite ornata, agglutinate rat, rabbit, chicken and human erythrocytes and in other work have been shown to inhibit the growth of Ehrlich ascites tumors in mice. Fractionation of extracts on Sephadex G-100 gave three active fractions with molecular weights of 30 000, 54 000 and 100 000. The 30 000 dalton fraction (B) was purified 72-fold by ammonium sulfate precipitation, gel filtration and preparative disc gel electrophoresis. The purified hemagglutinin, amphitritin, was homogenous on analytical disc gel electrophoresis at four different pH values and gave a sharp boundary in sedimentation velocity ultracentrifugation. The three fractions showed paralled specificity toward rat and chicken erythrocytes, the former giving the higher titer. The purified agglutinin was active toward human blood groups A, B and O and exhibited 4-fold higher activity toward group A. The hemagglutinin titer against rat red blood cells was lowered only by N-acetylgalactosamine, the terminal sugar residue of the group A determinant. None of the saccharides tested inhibited agglutination of chicken erythrocytes. Hemagglutinin activity was insensitive to dialysis or treatment with EDTA. The activity was not affected by digestion with trypsin or pronase, but was destroyed by phenol extraction. Analytical disc gel electrophoresis showed one protein band with high anodal mobility at pH 8.5, which was not affected by proteolytic enzymes but was removed by phenol. Activity was unaffected by heating at 70 degrees C for 30 min but was destroyed by similar treatemtn at 85 degrees C. Activity was at a maximum at pH 7-9 and decreased reversibly down to pH 4 at which point it was irreversibly inactivated. The higher molecular weight agglutinin (A1) could be dissociated to give amphitritin by treatment with 6M urea of precipitation in 55% (NH4)2SO4. This dissociation was not reversed by dialysis. Amphitritin is a glycoprotein with a molecular weight determined by gel filtration of 30 000 and by approach to equilibrium sedimentation of 32 000. Amino acid analysis showed a preponderance of aspartic and glutamic acids and relatively large amounts of glycine, proline, alanine, valine and cysteine. The carbohydrate moeity which represented 12.8% of the molecule, contained mannose, galactose, glucosamine and sialic acid. Amphitritin is the first hemagglutinin to be isolated from a polychaetous annelid.     

Quantitative analysis of N-sulfated, N-acetylated, and unsubstituted glucosamine amino groups in heparin and related polysaccharides

A colorimetric procedure for quantitative determination of free and substituted glucosamine amino groups in heparin and related polysaccharides has been developed. The total content of hexosamine amino groups is determined by a modification of the method of Tsuji et al. (1969, Chem. Pharm. Bull. 17, 1505-1510); this method involves acid hydrolysis under conditions effecting complete removal of N-acetyl and N-sulfate groups, deaminative cleavage with nitrous acid, and colorimetric analysis of the resultant anhydromannose residues by reaction with 3-methyl-2-benzothiazolinone hydrazone (MBTH). N-sulfated glucosamine residues are cleaved selectively by treatment with nitrous acid at pH approximately 1.5 (J. E. Shively, and H.E. Conrad, 1976, Biochemistry 15, 3932-3942) and quantitated by the MBTH reaction. Under carefully controlled conditions, deamination at pH approximately 1.5 is highly specific for N-sulfated glucosamine residues, but an excess of reagent causes some cleavage of residues with unsubstituted amino groups as well. Deaminative cleavage at pH approximately 4.5 results in preferential degradation of unsubstituted glucosamine residues, but some cleavage (5-8%) of N-sulfated residues also occurs. However, analysis of the content of N-sulfated residues by the specific pH 1.5 procedure allows appropriate corrections to be made. From the value for total hexosamine content and the sum of N-sulfated and unsubstituted residues, the content of N-acetylated residues is calculated by difference. The modified deamination procedures, in combination with product analysis by the MBTH reaction, have been applied to several problems commonly encountered in the analysis and characterization of heparin.     

Proteinase inhibitors from lonchocarpus capassa (appleleaf) seed

Four proteinase inhibitors (DE-1 to DE-4) were purified from L. capassa seed by chromatographic procedures involving Sephadex G-50 and DEAE-cellulose. They comprise each 80 amino acids (MW ca 10 000) including fourteen half-cystine residues. The partial amino acid sequence of inhibitor DE-4 was determined; 60 of the 80 residues have been sequenced. The MW, cystine content and partial sequence of DE-4 resemble those of the Bowman-Birk-type proteinase inhibitors. The properties of inhibitors DE-1 and DE-4 are very similar. Each contains a potent inhibitor for porcine trypsin but they inhibit bovine α-chymotrypsin only weakly.     

Purification and properties of pig brain guanine deaminase.

Guanine deaminase (guanine aminohydrolase, EC 3.5.4.3) from pig brain was purified to homogeneity by column chromatography and ammonium sulphate fractionation. Homogeneity was established by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulphate (SDS). The molecular weight of 110 000 was determined by gel filtration and sucrose density gradient centrifugation. SDS polyacrylamide gel electrophoresis indicated subunits of a molecular weight of 50 000. The amino acid composition, the isoelectric point and the number of -SH groups were determined. 5.5'-Dithiobis-(2-nitrobenzoic acid) reacts with about seven -SH groups in the native enzyme, but upon denaturation with SDS, 10 -SH groups react with this former reagent. Using electrolytic reduction, 44 half-cystines were determined in accordance with the number of cysteic acid residues determined by amino acid analysis after performic acid oxidation. The Km values determined for substrates of the enzyme were 1.1 . 10(-5) M for guanine in 0.1 M Tris. HCl buffer (pH 8.0) and 3.3 . 10(-4) M for 8-azaguanine in 0.1 M phosphate buffer, pH 6.4. The pKa values determined for ionizable groups of the active site of the enzyme were near pH 6.2 and pH 8.2. The chemical and kinetic evidence suggests that cysteine and histidine may be essential for the catalysis.     

Changes in the structure of thyroglobulin following the administration of thyroid-stimulating hormone.

In each of three separate experiments, female guinea pigs in groups of 20 were given 4 units of thyroid-stimulating hormone (TSH) each day for 3 days, while controls were given saline. Na125I was injected on the 3rd day, and the animals were killed 22 hours later. The pooled throids of each group were homogenized, and thyroglobulin was purified by one of the following methods: gel filtration on Sephadex G-200 followed by density gradient ultracentrifugation, two sequential filtrations on 4 percent agarose, or filtration on 4 percent agarose followed by Sephadex G-200. TSH administration was associated with the folling changes in thyroglobulin: (1) an increase in the ratio of tri-iodothyronine to thyroxine; (2) a decrease in dissociation of the 19 S to the 12 S form; (3) an alteration in its pattern on gel electrophoresis in sodium dodecyl sulfate-urea; and (4) changes in its amino acid composition, with significant increases in the content of lysine (by 15 percent), isoleucine (by 15 percent), and methionine (by 7 percent) relative to leucine. Over-all, there were no significant changes in the content of iodine, fucose, hexosamine, or sialic acid. These data show that TSH can alter the composition of thyroglobulin independently of its effects on iodine content. We suggest that these changes may stem from alterations in the subunit composition of thyroglobulin. There were also small but significant variations in amino acid composition among the three preparations of thyroglobulin from saline-treated animals and among the three from the TSH-treated. This finding shows that thyroglobulin can be heterogeneous in its protein portion as well as in its iodine content.     

Purification and characterization of a β-amylase from soya beans

1. β-Amylase obtained by acidic extraction of soya-bean flour was purified by ammonium sulphate precipitation, followed by chromatography on calcium phosphate, diethylaminoethylcellulose, Sephadex G-25 and carboxymethylcellulose. 2. The homogeneity of the pure enzyme was established by criteria such as ultracentrifugation and electrophoresis on paper and in polyacrylamide gel. 3. The pure enzyme had a nitrogen content of 16·3%, its extinction coefficient, E^1%_1cm., at 280mμ was 17·3 and its specific activity/mg. of enzyme was 880 amylase units. 4. The molecular weight of the pure enzyme was determined as 61700 and its isoelectric point was pH5·85. 5. Preliminary examinations indicated that glutamic acid formed the N-terminus and glycine the C-terminus. 6. The amino acid content of the pure enzyme was established, one molecule consisting of 617 amino acid residues. 7. The pH optimum for pure soya-bean β-amylase is in the range 5–6. Pretreatment of the enzyme at pH3–5 decreases enzyme activity, whereas at pH6–9 it is not affected.     

Physicochemical characterization of pituitary follicle-stimulating hormone

The physiochemical characteristics of the follicle-stimulating hormone (FSH) from whole sheep pituitary glands have been studied. The hormone behaves as a single protein in electrophoresis, diffusion, and ultracentrifugation. It has an isoelectric point at pH 4.5 and a molecular weight of 67,000 and contains 1.23 per cent hexose and 1.51 per cent hexosamine. The amino acid composition has also been determined in large part. The stability of the hormone to acid and heat has been investigated.     

Studies on fungal polysaccharide. XVII. A new glucuronan "protuberic acid" produced by a fungus Kobayashia nipponica.

A water-soluble glucuronan "protuberic acid", [alpha] 22-D minus 83.6 degrees, was isolated and purified from Kobayashia Nipponica, and its physicochemical properties were investigated. The purified protuberic acid was homogeneous as shown by zone electrophoresis, gel filtration over Sepharose 4B, and ultracentrifugation. The sedimentation coefficient was 1.8 S and its intrinsic viscosity was 1.1 dl/g. By gel filtration the molecular weight was estimated to be about 170 000. The results of periodate oxidation, methylation analysis, and partial acid hydrolysis indicated that this acidic polysaccharide has a linear structure of mainly 1, 4-linkages and containing an acid-labile linkage. Reduced protuberic acid, [alpha] 22-D minus 44 degrees, is also described.     

Unique human glycoprotein, alpha1-microglycoprotein: isolation from the urine of a cancer patient and its characterization.

A human glycoprotein was isolated from the urine of a patient with plasma cell leukemia. It appears pure and homogeneous when examined by immunoelectrophoresis, sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gel electrophoresis, gel filtration in 6 M guanidine hydrochloride (Gdn.HCl), and NH2-terminal amino acid sequence analysis. It has a brown color due to a tightly (most likely covalently) bound chromophore group(s) and migrates to the alpha1 region in immunoelectrophoresis. A molecular weight (mol wt) of 27 000 was obtained for the glycoprotein by gel filtration in 6 M Gdn.HCl. Its approximate mol wt determined by Na-DodSO4-polyacrylamide gel electrophoresis is 29 000 on 5% and 7.5% and 10% gels. Amino acid and hexosamine analyses showed that it is a glycoprotein and indicated that it contains four half-cystine residues per molecule. Based on the above observations we designated it "alpha1-microglycoprotein" (alpha1-MGP). Isoelectric focusing of alpha1-MGP showed a significant charge heterogeneity, although only a single NH2-terminal amino acid sequence was obtained for alpha1-MGP, i.e., Gly-Pro-Val-Pro-( )-Pro-Pro-Asx-Asx-Ile-Glx-Val-Glx-Glx-Asx-Phe-Phe-Ile-(Ser or Ala)-Arg. The alpha1-MGP was found in significant concentrations in the urine of many patients with neoplastic diseases.     

The acid lability of the glycosidic bonds of L-iduronic acid residues in glycosaminoglycans.

Heparan sulphate, heparin and dermatan sulphate were hydrolysed in 0.5M-H2SO4 at 100 degrees C. At intervals portions of the hydrolysate were removed and treated with HNO2 at pH 4.0 to cleave the glycosidic bonds of the N-unsubstituted hexosamine residues and to convert both free and combined hexosamines into anhydrohexoses. These hydrolysis/deamination mixtures were reduced with NaB3H4 and analysed by radiochromatography for alpha-L-iduronosylanhydrohexose, beta-D-glucuronosylanhydrohexose, and the free uronic acids and anhydrohexose. These data gave a kinetic profile of the cleavage of the alpha-L-iduronosyl and the beta-D-glucuronosyl bonds in these glycosaminoglycans. The beta-D-glucuronosyl bonds showed the expected resistance to acid hydrolysis, but the alpha-L-iduronosyl bonds were found to be as labile to acid as some neutral sugar glycosides. This unusual lability of alpha-D-iduronosyl-anhydromannitol and beta-D-glucuronosylanhydromannitol. The procedures used to follow the kinetics of glycosaminoglycan hydrolysis can also be sued to obtain quantitative analyses of L-iduronic acid, D-glucuronic acid and hexosamine in these polymers.     

Purification, characterization, and quantitation of the antigen employed in the immunodiffusion test for diagnosis of equine infectious anemia.

Equine infectious anemia (EIA) antigen extracted from the spleen of horses infected with EIA virus was purified by pH treatment, (NH4)2SO4 fractionation and affinity chromatography. The homogeneity of the antigen was indicated by sedimentation rate and sedimentation equilibrium experiments. A S20,w of 0.51 was determined and a molecular weight of 7600 was calculated from sedimentation equilibrium analysis. The amino acid composition of the pure antigen indicated the antigen is an acidic protein. Employing radical immunodiffusion (RID) and pure antigen a method for quantitating antigen content of antigen containing preparations was developed.     

Isolation and characterisation of a Salvia bogotensis seed lectin specific for the Tn antigen

A lectin was isolated and characterised from Salvia bogotensis seeds. Removal of the abundant pigments and polysaccharides, which are present in seeds, was an essential step in its purification. Several procedures were assayed and the best suited, including Pectinex treatment, DEAE-cellulose and affinity chromatography, led to a protein being obtained amounting to 18-20mg/100g seeds having high specific agglutination activity (SAA). The lectin specifically agglutinated human Tn erythrocytes and was inhibited by 37mM GalNAc, 0.019mM ovine submaxillary mucin (OSM) or 0.008mM asialo bovine submaxillary mucin (aBSM). Enzyme-linked lectinosorbent assay (ELLSA) revealed strong binding to aOSM and aBSM, corroborating Tn specificity, whereas no binding to fetuin or asialo fetuin was observed. The lectin's monomer MW (38,702Da), amino acid composition, pI, carbohydrate content, deglycosylated form MW, thermal stability and Ca(2+) and Mn(2+) requirements were determined. Evidence of the existence of two glycoforms was obtained. The lectin's specificity and high affinity for the Tn antigen, commonly found in tumour cells, makes this protein a useful tool for immunohistochemical and cellular studies.     

Demonstration that the leukocyte common antigen CD45 is a protein tyrosine phosphatase

It has been proposed on the basis of amino acid sequence homology that the leukocyte common antigen CD45 represents a family of catalytically active, receptor-linked protein tyrosine phosphatases [Charbonneau, H., Tonks, N. K., Walsh, K. A., & Fischer, E. H. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 7182-7186]. The present study confirms that CD45 possesses intrinsic protein tyrosine phosphatase (PTPase) activity. First, a mouse monoclonal antibody to CD45 (mAb 9.4) specifically eliminated, by precipitation, PTPase activity from a high Mr fraction containing CD45, prepared by gel filtration (Sephacryl S200) of a Triton X-100 extract of human spleen. Second, PTPase activity was demonstrated in a highly purified preparation of CD45 that was eluted with a high pH buffer from an affinity column, constructed from the same antibody. Third, on sucrose density gradient centrifugation, PTPase activity was only found in those fractions that contained CD45 as determined by Western analysis. When CD45 was caused to aggregate, first by reacting it with mAb 9.4 and then adding a secondary, cross-linking anti-mouse mAb, the PTPase activity shifted to the same higher Mr fractions that contained CD45. No shift in CD45 or PTPase was observed following addition of a control IgG2a. On this basis, it is concluded that CD45 is a protein tyrosine phosphatase.     

4-Aminobutyrate:2-oxoglutarate aminotransferase of Streptomyces griseus: purification and properties

4-Aminobutyrate: 2-oxoglutarate aminotransferase of Streptomyces griseus was purified to homogeneity on disc electrophoresis. The relative molecular mass of the enzyme was found to be 100 000 +/- 10 000 by a gel filtration method. The enzyme consists of two subunits identical in molecular mass (Mr 50 000 +/- 1000). The transaminase is composed of 486 amino acids/subunit containing 10 and 12 residues of half-cystine and methionine respectively. The NH2-terminal amino acid sequence of the enzyme was determined to be Thr-Ala-Phe-Pro-Gln. The enzyme exhibits absorption maxima at 278 nm, 340 nm and 415 nm with a molar absorption coefficient of 104 000, 11 400 and 7280 M-1 cm-1 respectively. The pyridoxal 5'-phosphate content was calculated to be 2 mol/mol enzyme. The enzyme has a maximum activity in the pH range of 7.5-8.5 and at 50 degrees C. The enzyme is stable at pH 6.0-10.0 and at temperatures up to 50 degrees C. Pyridoxal 5'-phosphate protects the enzyme from thermal inactivation. The enzyme catalyzes the transamination of omega-amino acids with 2-oxoglutarate; 4-aminobutyrate is the best amino donor. The Michaelis constants are 3.3 mM for 4-aminobutyrate and 8.3 mM for 2-oxoglutarate. Low activity was observed with beta-alanine. In addition to omega-amino acids the enzyme catalyzes transamination with ornithine and lysine; in both cases the D isomer is preferred. Carbonyl reagents and sulfhydryl reagents inhibit the enzyme activity. Chelating agents, non-substrate L and D-2-amino acids, and metal ions except cupric ion showed no effect on the enzyme activity.