Purification and characterization of acid trehalase from the yeast suc2 mutant

Acid trehalase was purified from the yeast suc2 deletion mutant. After hydrophobic interaction chromatography, the enzyme could be purified to a single band or peak by a further step of either polyacrylamide gel electrophoresis, gel filtration, or isoelectric focusing. An apparent molecular mass of 218,000 Da was calculated from gel filtration. Polyacrylamide gel electrophoresis of the purified enzyme in the presence of sodium dodecyl sulfate suggested a molecular mass of 216,000 Da. Endoglycosidase H digestion of the purified enzyme resulted after sodium dodecyl sulfate gel electrophoresis in one distinct band at 41,000 Da, representing the mannose-free protein moiety of acid trehalase. The carbohydrate content of the enzyme was 86%. Amino acid analysis indicated 354 residues/molecule of enzyme including 9 cysteine moieties and only 1 methionine. The isoelectric point of the enzyme was estimated by gel electrofocusing to be approximately 4.7. The catalytic activity showed a maximum at pH 4.5. The activity of the enzyme was not inhibited by 10 mM each of HgCl2, EDTA, iodoacetic acid, phenanthrolinium chloride or phenylmethylsulfonyl fluoride. There was no activation by divalent metal ions. The acid trehalase exhibited an apparent Km for trehalose of 4.7 +/- 0.1 mM and a Vmax of 99 mumol of trehalose min-1 X mg-1 at 37 degrees C and pH 4.5. The acid trehalase is located in the vacuoles. The rabbit antiserum raised against acid trehalase exhibited strong cross-reaction with purified invertase. These cross-reactions were removed by affinity chromatography using invertase coupled to CNBr-activated Sepharose 4B. Precipitation of acid trehalase activity was observed with the purified antiserum.     

Human brain hypoxanthine guanine phosphoribosyltransferase: structural and functional comparison with erythrocyte hypoxanthine guanine phosphoribosyltransferase

A rapid and simple method, based on GMP Sepharose affinity chromatography, was used for the purification of human brain hypoxanthine guanine phosphoribosyltransferase. A single protein band was detected by polyacrylamide gel electrophoresis of the native purified enzyme. A subunit molecular weight of 25,000 was estimated by SDS gel electrophoresis. The Km values for hypoxanthine and phosphoribosyl pyrophosphate were 50 and 111 microM, respectively. The Ki values for GMP and IMP with phosphoribosyl pyrophosphate were 21 and 37 microM, respectively. The purified enzyme from human brain did not differ significantly from the human erythrocyte one in amino acid composition. The brain and erythrocyte hypoxanthine guanine phosphoribosyltransferases showed complete immunochemical identity on Ouchterlony double diffusion.     

Purification and characterization of pyruvate decarboxylase from sweet potato roots.

Pyruvate decarboxylase [2-oxo acid carboxy-lyase, EC 4.1.1.1] was isolated from sweet potato roots and was partially purified from healthy and diseased tissues. There was no appreciable difference in properties between the enzymes from healthy and diseased tissues. The molecular weight of the enzyme was found to be 240,000 by polyacrylamide gel electrophoresis. Since sodium dodecyl sulfate polyacrylamide gel electrophoresis gave a molecular weight of 60,000 for the monomeric form of the enzyme, it is likely that sweet potato pyruvate decarboxylase contains 4 single polypeptide chains. The optimal pH of the decarboxylation reaction was 6.1--6.6. The Lineweaver-Burk double reciprocal plot curved upward, and the Hill coefficient was more than 1, with low concentrations of pyruvate. The enzyme was localized in the cytosol fraction. The activity of the enzyme increased in response to black-rot fungus infection, but decreased in response to cutting.     

Purification of N-hydroxy-2-acetylaminofluorene reductase from rabbit liver cytosol

N-Hydroxy-2-acetylaminofluorene reductase was purified from rabbit liver cytosol by fractionation with ammonium sulfate, and chromatography with DEAE-cellulose, Sephadex G-200 and hydroxylapatite. The purified enzyme was homogeneous by the criterion of sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 34,000 by the electrophoresis and by gel filtration on Sephadex G-200. The enzyme required cysteine, glutathione, dithiothreitol, 2-mercaptoethanol, NADPH or NADH as an electron donor. The enzyme activity was inhibited by p-chloromercuribenzoic acid, N-ethylmaleimide, cupric sulfate or disulfiram, but little by oxygen.     

PURIFICATION OF L-GLUTAMIC ACID DECARBOXYLASE FROM CATFISH BRAIN

—L-Glutamic acid decarboxylase (GAD) from brain of the channel catfish (Ictalurus punctatus) has been purified to homogeneity by a combination of ammonium sulfate fractionation, gel filtration, calcium phosphate gel and preparative polyacrylamide gel electrophoresis. The purity of the enzyme preparation was established by showing that on both 7.5% regular and 3.7–15% gradient polyacrylamide gel electrophoresis the enzyme migrated as a single protein band which contained all the enzyme activity. The molecular weight of the purified GAD was estimated by gel filtration and gradient polyacrylamide gel to be 84,000 ± 2000 and 90,000 ± 4000, respectively. SDS-polyacrylamide gel electrophoresis revealed three major proteins with molecular weights of 22,000 ± 2000, 40,000 ± 5000 and 90, 000 ± 6000 which may represent a monomer, dimer, and tetramer. Antibodies against the purified enzyme were obtained from rabbit after four biweekly injections with a total of 80 μg of the enzyme. A double immunodiffusion test using these antibodies and a crude extract from catfish brains showed only a single, sharp precipitin band which still retained the enzyme activity, suggesting that the precipitin band was indeed a GAD-anti-GAD complex. In an enzyme inhibition study, a maximum inhibition of 60–70% was obtained at a ratio of GAD protein/anti-GAD serum of about 1:1.6. Furthermore, the precipitate from the GAD-anti-GAD incubation mixture also contained the enzyme activity, suggesting that the antibody was specific to GAD and that the antigen used was homogeneous. Advantages and drawbacks of the purification procedures described here and those used for mouse brain preparations are also discussed.     

Beta-glucuronidase of rat preputial gland. Crystallization, properties, carbohydrate composition, and subunits.

Rat preputial gland beta-glucuronidase [ED 3.2.1.31] was purified by ammonium sulfate precipitation, ethanol fractionation, gel filtration on Sephadex G-200 and crystallization. The purified enzyme appeared homogeneous on electrophoresis in polyacrylamide gel, and on analytical ultracentrifugation and had a molecular weight of approximately 320,000, and a sedimentation coefficient of 12S. SDS polyacrylamide gel electrophoresis indicated that the enzyme consisted of subunits with molecular weight of 79,000, so the native enzyme appeared to be a tetramer. The Km with p-nitrophenyl beta-D-glucosiduronic acid as substrate was about 0.53 mM. The enzyme had a single pH optimum at 4.5. The enzyme had a very low content of sulphur-containing amino acid and contained 5.7 per cent carbohydrate, consisting of mannose, glucose, fucose, galactose, and glucosamine in a ratio of 44;9;6;2;41. Sialic acid was not detected in the crystallized enzyme.     

Purification and characterization of a human kidney neutral endopeptidase that hydrolyzes succinyl trialanine-4-nitroanilide and biologically active peptides

An enzyme hydrolyzing succinyl trialanine-4-nitroanilide was extracted from human kidney homogenate and purified by means of gel filtration on Sepharose CL-4B, anion-exchange chromatography on DEAE-Sepharose CL-6B and affinity chromatography on carbobenzoxy-L-Ala-L-Ala-D-Ala-polylysine-agarose. The purified enzyme consists of a single peptide, and its molecular weight was estimated to be about 125 000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme cleaved the substrate at the bond between succinyl dialanine and alanine-4-nitroanilide and showed a Km value of 2.1 mM at the optimal pH of 8.0. The activity was increased by Ca2+ and Mg2+, but was inhibited by phosphoramidon and ethylenediaminetetraacetic acid. The enzyme cleaved the oxydized insulin B chain, angiotensinogen tetradecapeptide, angiotensin I, angiotensin II, angiotensin III, [Sar1,Ala8]-angiotensin II, bradykinin, des-Pro2-bradykinin, Leu5-enkephalin, Met 5-enkephalin, [D-Ala2,Met5]-enkephalinamide and [D-Ala2-Met5]-enkephalin, but did not cleave [D-Ala2,D-Leu5]-enkephalin. The bonds on the amino side of the hydrophobic amino acids of the peptides were cleaved by the enzyme.     

Membrane-bound aminopeptidase P from bovine lung. Its purification, properties, and degradation of bradykinin.

The membrane-bound form of aminopeptidase P (aminoacylprolyl-peptide hydrolase) (EC 3.4.11.9) was purified to apparent homogeneity from bovine lung microsomes. The enzyme was solubilized using phosphatidylinositol-specific phospholipase C (Bacillus thuringiensis), indicating that bovine lung amino-peptidase P is attached to membranes via a glycosylphosphatidylinositol anchor. The enzyme was purified 1900-fold with a yield of 25% by chromatography on decyl-agarose, omega-aminodecyl-agarose, a second decylagarose column, DEAE-Sephacel, and an ultrafiltration step. Native gradient polyacrylamide gel electrophoresis revealed a single stained protein band whose position in the gel corresponded to cleavage of the Arg1-Pro2 bond of bradykinin. The Mr was 360,000 by gel permeation chromatography and 95,000 by reducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The substrate specificity of aminopeptidase P was determined using approximately 50 peptides with proline in the second position. The enzyme could hydrolyze lower NH2-terminal homologs of bradykinin, including Arg-Pro-Pro, which was used as the routine substrate in a rapid fluorescence assay performed in the absence of added Mn2+. Some peptides having NH2-terminal amino acids other than arginine were also cleaved. Aminopeptidase P appeared to favor peptides that had 2 proline residues or proline analogs in positions 2 and 3 of the substrate. In general, tripeptides having a single proline residue in position 2 were poor substrates. Aminopeptidase P was inhibited by a series of peptides, 3-8 residues long, having an NH2-terminal Pro-Pro sequence. The enzyme was also inhibited by metal-chelating agents, 2-mercaptoethanol (4 mM), p-chloromercuribenzenesulfonic acid, and NaCl at concentrations greater than or equal to 0.25 M. The purified enzyme had a pH optimum of 6.5-7.0 and was most stable in the basic pH range. A role for membrane-bound aminopeptidase P in the pulmonary inactivation of circulating bradykinin is proposed.     

Purification and initial characterization of lipase from the scutella of corn seedlings

The lipase from the scutella of corn (Zea mays) MO-17 seedlings was purified 272-fold to apparent homogeneity as evidenced by sodium dodecyl sulfate polyacrylamide gel electrophoresis and double immunodiffusion. The procedure involved isolation of the lipid bodies, extraction with diethyl ether, DE-52 ion exchange chromatography, and sucrose density gradient centrifugation. The enzyme had an approximate molecular weight of 270,000 daltons after sucrose density gradient centrifugation, and 65,000 daltons after sodium dodecyl sulfate polyacrylamide gel electrophoresis. The lipase contained no cysteine and its molecular weight in sodium dodecyl sulfate was not reduced by β-mercaptoethanol. The amino acid composition as well as a biphasic partition using Triton X-114 revealed the enzyme to be a hydrophobic protein. Rabbit γ-globulin containing antibodies raised against the purified lipase formed one precipitin line with the lipase in a double diffusion test, and precipitated all the lipase activity from a solution.     

Purification and detection of multiple forms of histidine decarboxylase in rat gastric mucosa (author's transl)]

A specific histidine decarboxylase from rat gastric mucosa has been obtained at high purity and good yield (purification about 600-fold). The purification procedure included double (NH4)2SO4 fractionation, ion-exchange chromatography, preparative isoelectric focusing in a granulated gel and gel filtration. Only the specific histidine enzyme was obtained by that procedure; DOPA decarboxylase, a non-specific enzyme, was absent in our final preparation. Each step of the purification was visualized by polyacrylamide gel electrophoresis and analytical isoelectric focusing. The purified enzyme was apparently homogenous by criteria of electrophoresis and gel filtration and has a molecular weight of 94 000. Several protein bands appeared after isoelectric focusing and the enzyme activity was localized in 3 distinct peaks. The gastric enzyme consists of 3 active forms which could be distinguished by their isoelectric points: 5.4, 5.75 and 6. Moleculare weights estimated by SDS polyacrylamide gel electrophoresis were 97 000, 93 000 and 90 000, and no subunits were observed. Pyridoxal phosphate was required as a coenzyme and resolution of the holoenzyme agreed with a portion of the coenzyme tightly bound to the apoenzyme. The purified enzyme was stable at low ionic strength, near neutral pH; concentrated reducing agents inhibit the enzyme.     

Atrioactivase, a specific peptidase in bovine atria for the processing of pro-atrial natriuretic factor. Purification and characterization

A seryl protease which catalyzes conversion of proatrial natriuretic factor (ANF) to the active circulating form, ANF(99-126), was purified from a particulate fraction of bovine atria. The enzyme was solubilized with 1.6 M KCl. The molecular mass of the purified enzyme was 580 kDa on gel filtration, whereas by sodium dodecyl sulfate-polyacrylamide gel electrophoresis a cluster of six bands with molecular masses around 30 kDa was observed. The purified enzyme produced ANF(99-126) from partially purified bovine pro-ANF by the selective cleavage of the arginyl peptide bond in the -Pro97-Arg98-Ser99-sequence in pro-ANF. The enzyme was localized mainly in the microsomal fraction rather than the granule fraction. It is likely that the enzyme selectively cleaves the Arg98-Ser99 peptide bond in pro-ANF during the process of secretion.     

Purification and properties of one component of acid phosphatase produced by Aspergillus niger.

One component, the i form, of acid phosphatase (orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2) produced by Aspergillus niger was purified from the mycelial extract. The purified enzyme was homogenous on Sephadex G-200 gel filtration, disc electrophoresis and heat inactivation. The purified enzyme was studied and the following results were obtained: 1. The enzyme catalyzed the hydrolysis of a wide variety of phosphomonoesters, but not that of bis(p-nitrophenyl)phosphate, adenosine 3',5'-cyclic monophosphate, fructose 1,6-diphosphate, adenosine 5'-diphosphate or adenosine 5'-triphosphate. 2. Fluoride, orthophosphate, arsenate, borate, molybdate and (+)-tartrate acted as inhibitors. This enzyme was inactivated by N-bromosuccinimide and 2-hydroxy-5-nitrobenzyl bromide, and was not affected by p-chloromercuribenzoate, N-acetylimidazole, p-diazobenzenesulfonic acid and tetranitromethane. From these results, tryptophan was estimated to play an important role in the enzyme activity. 3. The apparent molecular weight was 310000 by Sephadex G-200 gel filtration. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate suggested that the molecular weight of the subunit was approximately 89000. 4. The purified enzyme contained 29% carbohydrate consisting of glucosamine, mannose and galactose. The amino acid composition of this enzyme was not specific compared with other known acid phosphatases.     

Characterization of lysosomal acid lipase purified from rabbit liver

Lysosomal acid lipase from rabbit liver was solubilized with digitonin and purified 25,000-fold by Bio-Gel A-1.5 m, DEAE Bio-Gel A and phenyl Sepharose column chromatographies, preparative slab gel electrophoresis and finally Affi-Gel Blue affinity column chromatography. The purified enzyme gave a single protein band on polyacrylamide gel electrophoresis both in the presence and absence of sodium dodecyl sulfate. The molecular weight of the acid lipase was estimated to be 42,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis and 40,000 by gel filtration on Bio-Gel A-0.5 m. The enzyme was a hydrophobic glycoprotein with an isoelectric point of 5.15-5.90. The purified enzyme hydrolyzed tri-, di-, and monoolein and cholesterol oleate, with apparent Vmax values of 5.41, 56.1, 21.7, and 3.25 mumol/min/mg protein, and Km values of 50, 70, 200, and 40 microM, respectively. It hydrolyzed 4-methylumbelliferyl esters with fatty acids of different lengths in the order, medium length chains greater than long chains much greater than short chains. It did not hydrolyze dipalmitoylphosphatidylcholine. Its activity was inhibited by micromolar concentrations of p-chloromercuriphenyl sulfonic acid and p-bromophenacyl bromide and millimolar concentrations of Cu2+ and diethylpyrocarbonate. The activities of the enzyme towards the five substrates listed above showed almost identical thermal stabilities, mobilities on polyacrylamide gel electrophoresis and inhibition by several inhibitors. These findings support the idea that one enzyme is involved in the hydrolysis of both acylglycerols and cholesterol esters in lysosomes.     

Rat kidney L-alpha-hydroxy acid oxidase: isolation of enzyme with one flavine coenzyme per two subunits.

L-alpha-Hydroxy acid oxidase (listed as EC 1.4.3.2, L-amino acid: O2 oxidoreductase) has been purified 100-fold from rat kidney to apparent homogeneity by gel electrophoresis. A subunit molecular weight of 47,500 was found by sodium dodecyl sulfate gel electrophoresis, but in contrast to previous reports, the enzyme has been found to have a molecular weight of ca. 200,000 by Sephadex gel filtration and by dodecyl sulfate gel electrophoresis of the enzyme cross-linked with dimethyl suberimidate. A somewhat higher value was found by sedimentation equilibrium, but a tetrameric structure for the active enzyme is definitely established. The enzyme was found to contain the FMN coenzyme at a concentration of one FMN/102,000 daltons or one flavine/two subunits, a highly unusual finding. This ratio was determined from spectroscopic analysis of the FMN in lyophilized samples of the enzyme and by titration of the coenzyme with the flavine specific enzyme inactivator 2-hydroxy-3-butynoate. The enzyme has the same specific activity as a crystalline sample of the enzyme reported to have twice as much flavine/milligram.     

Proline iminopeptidase from Bacillus coagulans: purification and enzymatic properties

Proline iminopeptidase [EC 3.4.11.5] was purified about 2,700-fold from cell-free extract of Bacillus coagulans by a series of column chromatographies on DEAE-Toyopearl, PCMB-T-Sepharose, and hydroxyapatite, and gel filtration on Sephadex G-150. The purified enzyme was homogeneous as judged by disc gel electrophoresis. The enzyme was most active at pH 7.3 with Pro-beta-naphthylamide (Pro-2-NNap) as the substrate, and hydrolyzed Pro-X (X = amino acid including proline, peptide, amide, and arylamide) bonds when the proline residue was at the amino terminus. Pro-D-amino acid bonds were also susceptible to the enzyme. The enzyme was completely inhibited by p-chloromercuribenzoate (PCMB) and partially by proline but not by metal chelators, diisopropylphosphorofluoridate (DFP), or phenylmethanesulfonyl fluoride (PMSF). The enzyme inactivated with PCMB was reactivated by incubation with 2-mercaptoethanol. These results and the chromatographic profile on PCMB-T-Sepharose suggest that the enzyme is a sulfhydryl enzyme. The isoelectric point of the enzyme was 4.0, and the molecular weight of the enzyme was estimated to be 40,000 by gel filtration on Sephadex G-100 and 35,000 by sodium dodecyl sulfate (SDS) gel electrophoresis, indicating that the enzyme exists as a monomer.     

Characterization of an intracellular oligopeptidase from Lactobacillus paracasei.

An intracellular oligopeptidase from Lactobacillus paracasei Lc-01 has been purified to homogeneity by Fast Flow Q Sepharose, hydroxyapatite, and Mono Q chromatography. The molecular mass of the enzyme was determined to be 140 kDa by gel filtration and approximately 30 kDa by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and SDS-capillary electrophoresis. The pI of the enzyme was at pH 4.5. The enzyme expressed maximum activity at pH 8.0 and 40 degrees C. Oligopeptidase activity on bradykinin was inhibited strongly by 1,10-phenantroline and EDTA and partly by p-chloromercuribenzoic acid but not by phosphoramidon or phenylmethylsulfonyl fluoride. Marked inhibition by beta-casein fragment 58 to 72 was demonstrated. The enzyme showed neither general aminopeptidase nor caseinolytic activity, and it degraded only oligopeptides between 8 and 13 amino acids. The enzyme readily hydrolyzed the Phe-Ser and Pro-Phe bonds of bradykinin; the Phe-His bond of angiotensin I; the Pro-Gln, Gln-Phe, and Phe-Gly bonds of substance P; and the Pro-Tyr bond of neurotensin. Weak activity toward the Ala-Tyr and Pro-Ser bonds of alpha(s1)-casein fragment 157 to 164, was observed. The N-terminal amino acid sequence of the oligopeptidase showed a high degree of homology to the lactacin B inducer from Lactobacillus acidophilus.     

PURIFICATION AND COMPARISON OF 2'',3''-CYCLIC NUCLEOTIDE 3''-PHOSPHOHYDROLASES FROM BOVINE BRAIN AND SPINAL CORD

The membrane-bound enzyme 2′,3′-cyclic nucleotide 3′-phosphohydrolase has been purified from acetone powders of bovine white matter and spinal cord. Affinity chromatography on AMP-Sepharose has been used as the final step in the chromatographic purifications. The yield was about 3 mg of purified enzyme per 100 g of tissue in each instance. The enzymes from the two sources were indistinguishable by chromatography, gel electrophoresis, and amino acid analysis; the enzyme from spinal cord, however, has shown a specific activity of 225 units/mg compared to 342 units/mg for the enzyme from white matter. Both proteins had a molecular weight of 100,000 by gel filtration and 50,000 by sodium dodecyl sulfate-gel electrophoresis under reducing conditions. The properties of the enzyme, including amino acid composition determined on the purified soluble protein and on the protein purified by sodium dodecyl sulfate-gel electrophoresis, were those of a basic hydrophobic protein.     

Affinity purification and properties of cathepsin-E-like acid proteinase from rat spleen.

A unique acid proteinase different from cathepsin D was purified from rat spleen by a method involving precipitation at pH 3.5, affinity chromatography on pepstatin-Sepharose 4B and concanavalin A-Sepharose 4B, chromatography on Sephadex G-100 and DEAE-Sephacel, and isoelectric focusing. A purification of 4200-fold over the homogenate was achieved and the yield was 11%. The purified enzyme appeared to be homogeneous on electrophoresis in polyacrylamide gels. The isoelectric point of the enzyme was determined to be 4.1-4.4. The enzyme hydrolyzed hemoglobin with a pH optimum of about 3.1. The molecular weight of the enzyme was estimated to be about 90000 by gel filtration on Sephadex G-100. In sodium dodecylsulfate polyacrylamide gel electrophoresis, the purified enzyme showed a single protein band corresponding to a molecular weight of about 45000. The hydrolysis of bovine hemoglobin by the enzyme was much higher than that of serum albumin. Various synthetic and natural inhibitors of the enzyme were tested. The enzyme was inhbited by Zn2+, Fe3+, Pb2+, cyanide, p-chloromercuribenzoate, iodoacetic acid and pepstatin, whereas 2-mercaptoethanol, phenylmethyl-sulfonyl fluoride and leupeptin showed no effect.     

Purification and properties of maize polyamine oxidase: a flavoprotein

Polyamine oxidase was purified from maize shoots to homogeneityby the criteria of polyacrylamide gel electrophoresis and ultracentrifugation.The purified yellow enzyme showed absorption maxima at 278,380 and 460 nm. The molecular weight estimated by gel filtrationwas about 65,000 and the sedimentation coefficient was 5.95S. Sodium dodecylsulfate gel electrophoresis yielded a singleband at a molecular weight of 65,000. The enzyme contained 1mole of FAD per mole of enzyme. Amino acid composition and kineticproperties of the enzyme are presented. (Received April 30, 1980; )     

Purification and NH2-terminal amino acid sequence of human thymidylate synthase in an overproducing transformant of mouse FM3A cells

Human thymidylate synthase [EC 2.1.1.45] was purified to homogeneity and its NH2-terminal amino acid sequence was determined taking advantage of the following facts: i) The source of the enzyme was a transformant of mouse FM3A mutant cells which lacks mouse thymidylate synthase but overproduces human thymidylate synthase. ii) The enzyme could be purified on two kinds of affinity column, Cibacron blue dye-bound agarose and methotrexate-bound Sepharose. iii) The enzyme could finally be separated from a trace of impurities by electrophoresis on polyacrylamide gel containing sodium dodecyl sulfate. The purified human thymidylate synthase had a subunit with a molecular weight of 33,000, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The enzyme was subjected to Edman degradation and the NH2-terminal 24 amino acids were sequenced by successive use of a high-sensitivity gas-phase protein sequencer and high performance liquid chromatography to be as follows: Pro-Val-Ala-Gly-Ser-Glu-Leu-Pro-Arg-Arg-Pro-Leu-Pro-Pro-Ala-Ala-Gln-Glu- Arg-Asp -Ala-Glu-Pro-Arg-.