Isolation and properties of a T-kininogenase from bovine erythrocyte membranes

A kininogenase from bovine erythrocyte membranes has been purified 140-fold by affinity chromatography on pepstatin A-Agarose followed by ion exchange chromatography on CM Cellulose. The purified enzyme showed an apparent molecular weight of 31,000 daltons as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. ItspH optimum is 7.5, and it was totally inhibited by soybean trypsin inhibitor, phenylmethylsulfonylfluoride, aprotinin, pepstatin, and dithiotreitol, suggesting the presence of a disulfide bond(s) whose integrity is(are) essential for maintaining the native three-dimensional structure. The referred enzyme was able to release kinin from a substrate partially purified from rat plasma. The kininogenase was activated by Zn²⁺, Ca²⁺, and cysteine-HCl.     

Influence of associated lipid on the properties of purified bovine erythrocyte acetylcholinesterase

Acetylcholinesterase has been isolated from bovine erythrocyte membranes by affinity chromatography using a m-trimethylammonium ligand. The purified enzyme had hydrophobic properties by the criterion of phase partitioning into Triton X-114. The activity of the hydrophobic enzyme was seen as a slow-moving band in nondenaturing polyacrylamide gels. After treatment with phosphatidylinositol-specific phospholipase C, another form of active enzyme was produced that migrated more rapidly toward the anode in these gels. This form of the enzyme partitioned into the aqueous phase in Triton X-114 phase separation experiments and was therefore hydrophilic. The hydrophobic form bound to concanavalin A in the absence of Triton X-100. As this binding was partially prevented by detergent, but not by alpha-methyl mannoside, D-glucose, or myo-inositol, it is in part hydrophobic. Erythrocyte cell membranes showed acetylcholinesterase activity present as a major form, which was hydrophobic by Triton X-114 phase separation and in nondenaturing gel electrophoresis moved at the same rate as the purified enzyme. In the membrane the enzyme was more thermostable than when purified in detergent. The hydrophobic enzyme isolated, therefore, represents a native form of the acetylcholinesterase present in the bovine erythrocyte cell membrane, but in isolation its stability becomes dependent on amphiphile concentration. Its hydrophobic properties and lectin binding are attributable to the association with the protein of a lipid with the characteristics of a phosphatidylinositol.     

Purification and characterization of carbonic anhydrase from bovine erythrocyte plasma membrane

Carbonic anhydrase (CA) was purified from bovine erythrocyte plasma membrane and characterized in this study. For this purpose, the blood taken from young animals was hemolysed, the membrane fraction was separated, and this fraction was repeatedly washed. The enzyme (CA) was removed from the membrane with buffered TritonX-100 (1%); it could be purified at a factor of 22.8 by affinity chromatography.The CA obtained from erythrocyte membrane has an esterase activity as well as hydratase activity. The Vmax and Km of the enzyme for the substrate (p-nitrophenyl acetate) are 1.948x10(-3) mM/L x dak, and 3.596 mM, respectively. The purification degree of the enzyme was controlled by SDS-PAGE (3-10), which showed two distinct bands. It was determined that the enzyme had activity within the pH range of 4.5-9.5 and that the optimal pH was 7.5. The temperature at which it showed activity was 20-60 degrees C and optimal temperature was 37 degrees C. Molecular weight of CA was found to be 29844 and 61706 Dalton by gel filtration. On the other hand, sulfanilamide and acetazolamide affected the enzyme.     

Properties of bovine erythrocyte acetylcholinesterase solubilized by phosphatidylinositol-specific phospholipase C1

The properties of acetylcholinesterase solubilized from bovine erythrocyte membrane by phosphatidylinositol (PI)-specific phospholipase C of Bacillus thuringiensis or with a detergent, Lubrol-PX, were studied. The activity of Lubrol-PX-solubilized acetylcholinesterase was broadly distributed in the fractions having Ve/Vo = 1.0-2.0 in gel filtration on a Sepharose 6B column. The intermediary fractions (Ve/Vo = 1.3-1.7) were collected as "the middle active Sepharose 6B eluate" and characterized on the basis of enzymology and protein chemistry. When this eluate was treated with PI-specific phospholipase C, the major activity peak was obtained in the later fractions with Ve/Vo = 1.75-2.0 on the same column chromatography. Lubrol-solubilized and phospholipase C-treated acetylcholinesterase preparations were different in the thermostability, the elution profiles of chromatography on Mono Q, butyl-Toyopearl and phenyl-Sepharose columns, and the affinity to phospholipid micelles. On treatment with PI-specific phospholipase C, Lubrol-solubilized acetylcholinesterase became more thermostable. The phospholipase C-treated enzyme was eluted at lower NaCl concentration from the Mono Q column than the Lubrol-solubilized enzyme. The most important difference was observed in the hydrophobicity of these two enzyme preparations. The Lubrol-solubilized enzyme shows high affinity to phospholipid micelles and hydrophobic adsorbents such as butyl-Toyopearl and phenyl-Sepharose. However, this hydrophobicity was lost when acetylcholinesterase was solubilized from bovine erythrocyte membrane by PI-specific phospholipase C. The presence of myo-inositol was confirmed in the purified preparation of acetylcholinesterase by gas chromatography (GC)-mass spectrometry (MS).(ABSTRACT TRUNCATED AT 250 WORDS)     

A small hydrophobic domain that localizes human erythrocyte acetylcholinesterase in liposomal membranes is cleaved by papain digestion

A small hydrophobic domain in isolated human erythrocyte acetylcholinesterase is responsible for the interaction of this enzyme with detergent micelles and the aggregation of the enzyme on removal of detergent. Papain has been shown to cleave this hydrophobic domain and to generate a fully active hydrophilic enzyme that shows no tendency to interact with detergents or to aggregate [Dutta-Choudhury, T.A., & Rosenberry, T.L. (1984) J. Biol. Chem. 259, 5653-5660]. We report here that the intact enzyme could be reconstituted into phospholipid liposomes while the papain-disaggregated enzyme showed no capacity for reconstitution. More than 80% of the enzyme reconstituted into small liposomes could be released by papain digestion as the hydrophilic form. Papain was less effective in releasing the enzyme from large liposomes that were probably multilamellar. In a novel application of affinity chromatography on acridinium resin, enzyme reconstituted into small liposomes in the presence of excess phospholipid was purified to a level of 1 enzyme molecule per 4000 phospholipid molecules, a ratio expected if each enzyme molecule was associated with a small, unilamellar liposome. Subunits in the hydrophilic enzyme form released from reconstituted liposomes by papain digestion showed a mass decrease of about 2 kilodaltons relative to the intact subunits according to acrylamide gel electrophoresis in sodium dodecyl sulfate, a difference similar to that observed previously following papain digestion of the soluble enzyme aggregates. The data were consistent with the hypothesis that the same hydrophobic domain in the enzyme is responsible for the interaction of the enzyme with detergent micelles, the aggregation of the enzyme in the absence of detergent, and the incorporation of the enzyme into reconstituted phospholipid membranes.     

Purification, characterization, and amino acid composition of rabbit pulmonary bleomycin hydrolase

Bleomycin (BLM) hydrolase, a protective enzyme that inactivates the antitumor antibiotic BLM, was purified (6000-fold) to homogeneity from rabbit lungs by DEAE-Sephacel, phenyl-Sepharose chromatography, BLM-Sepharose affinity chromatography, and Mono Q fast protein liquid chromatography. The enzyme had a molecular mass of 250,000 daltons as demonstrated by Superose gel permeation chromatography and polyacrylamide gel electrophoresis (PAGE) under native conditions. Sodium dodecyl sulfate-PAGE revealed a single band of 50,000 daltons, suggesting a pentameric structure. The Km and Vmax for BLM A2 were 1.3 mM and 5.9 mumol mg-1 h-1, respectively. BLM hydrolase activity was labile, had a half-life of 25 min at 56 degrees C, 10 h at 37 degrees C, and 5 days at 4 degrees C, and was stabilized by 2 mM dithiothreitol. The enzyme had a pH optimum of 7.0-7.5 and was inhibited by N-ethylmaleimide, leupeptin, puromycin, and divalent cations such as Cu2+, Cd2+, Zn2+, and Co2+ but was unaffected by chelating agents. On the basis of Mono P chromatofocusing chromatography, three isoforms of BLM hydrolase (apparent pI's of 5.3, 4.5, and 4.3) were present in rabbit pulmonary cytosol. The elution profiles of BLM hydrolase from phenyl-Sepharose and Mono P chromatofocusing indicated that this enzyme is hydrophobic and acidic. This was confirmed by amino acid composition analysis, which demonstrated that 48% of the total amino acids of bleomycin hydrolase were hydrophobic and 37% were acidic.     

Purification and characterization of acetylcholinesterase from the Lake Van fish (Chalcalburnus tarichii Pallas, 1811)

In this study, acetylcholinesterase (AChE; EC 3.1.1.7) was purified from plasma and erythrocytes in the Lake Van fish (Chalcalburnus tarichii P.1811) by affinity chromatography. Enzymatic activity was spectrophotometrically measured according to Ellman's method, at 412 nm. Then, the optimal pH and temperature of the enzyme was determined. According to the results, the optimal pH and the optimum temperature were 8.0 and 25 degrees C, respectively. In order to control the purification of the enzyme, sodium dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was done. SDS-PAGE showed a single band for enzyme. The purification rates for plasma AChE and erythrocyte AChE are 3251.6 and 8500, respectively.     

Isolation and characterization of pig kidney mitochondrial ferredoxin:NADP+ oxidoreductase

A two-step affinity chromatography procedure, using 2',5'-ADP-agarose and adrenodoxin-Sepharose 4B affinity supports, was used to purify mitochondrial ferredoxin:NADP+ oxidoreductase (EC 1.18.1.2, formerly EC 1.6.7.1) from pig kidney. The 450:270 nm absorbance ratio of the enzyme was 0.128, and it had a specific activity of 16,305 nmol/min/mg for the reduction of cytochrome c. The mitochondrial enzyme was a monomer which contained one molecule of FAD and had calculated molecular masses of 51,500 and 48,000 daltons when determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and high performance liquid chromatography gel exclusion chromatography, respectively. The porcine enzyme had a Km for NADPH of 0.94 microM and it expressed maximal activity when coupled with its homologous ferredoxin, although it was also active with the heterologous ferredoxin from bovine adrenal. The purified ferredoxin:NADP+ oxidoreductase supported the in vitro reduction of membrane-bound adrenal mitochondrial P-450, and it was demonstrated from immunologic studies that the enzyme shares some common epitopes with bovine adrenodoxin:NADP+ oxidoreductase.     

Acetylcholinesterase: characterization of native and proteolytically derived forms and identification of structural protein components.

The assymmetric 18S and 14S forms of acetylcholinesterase (EC 3.1.1.7) from Electrophorus electricus purified by affinity chromatography on N-methylacridinium Sepharose 2B were subjected to trypsin or collagenase proteolysis and changes in the enzyme composition and structure were monitored by sucrose gradient sedimentation, gel chromatography, and sodium dodecyl sulphate - polyacrylamide gel electrophoresis. A distinction between autolytic and tryptic degradation products is described and the generation of two new forms of acetylcholinesterase from the 18S and 14S enzyme by collagenase proteolysis is reported. The species derived from the 18S form of acetylcholinesterase has a sedimentation coefficient of 21.1S and a Stokes radius of 12.9 nm while the 14S form gives rise to a 17.3S species with a Stokes radius of 11.1 nm. The proteolytically sensitive component ('tail') of the asymmetric forms of acetylcholinesterase is identified with a subunit of 45 000 daltons on sodium dodecyl sulphate - polyacrylamide electrophoresis gels.     

Purification of protein methylase II from human erythrocytes

Protein methylase II (S-adenosylmethionine:protein-carboxyl O-methyltransferase, EC. 2.1.1.24) which methyl esterifies free carboxyl groups of protein substrate using S-adenosyl-L-methionine as the methyl donor, has been purified from human erythrocytes approximately 13000-fold with a yield of 12%. The purified enzyme was over 95% pure as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A bulk of hemoglobin present in the erythrocyte lysate, which severely limited the use of affinity chromatography for purification, was effectively removed by ammonium sulfate precipitation and by the subsequent salt washing of the precipitates followed by molecular sieve chromatography on Sephadex G-75. This preparation can be further purified by affinity chromatography, in which S-adenosyl-L-homocysteine is covalently linked to Sepharose-4B, followed by Sephadex G-75 chromatography to yield an enzyme with an activity of 27000 pmol methyl group transferred/mg/min at 37 degrees C.     

Membrane-associated carbonic anhydrase purified from bovine lung

We found carbonic anhydrase activity associated with particulate fractions of homogenates of rat, rabbit, human, and bovine lungs. These membrane-associated carbonic anhydrases were remarkably stable in solutions containing sodium dodecyl sulfate (SDS). The bovine enzyme was dissolved with SDS and purified by affinity chromatography and gel filtration. The purified enzyme contains glucosamine, galactose, and sialic acid; it is at least 20% carbohydrate. The apparent molecular weight by SDS-polyacrylamide gel electrophoresis (52,000) may be higher than the actual molecular weight due to the presence of carbohydrate. The enzyme contains cystine, an amino acid that is absent in bovine erythrocyte carbonic anhydrase. Dithiothreitol greatly accelerated the rate of inactivation of the membrane-associated enzyme in SDS, so disulfide bonds appear to stabilize this enzyme. The specific CO2-hydrating activity was about half that of the erythrocyte enzyme. Acetazolamide inhibits the membrane-associated enzyme (Ki = 10 nM) nearly as well as the erythrocyte enzyme (Ki = 3 nM). Antibody to bovine erythrocyte carbonic anhydrase did not inhibit the membrane-associated enzyme. Other investigators have accumulated a good deal of evidence for carbonic anhydrase on the luminal surface of pulmonary capillaries. The enzyme described here appears to be a new isozyme whose properties are consistent with such a localization.     

Isolation and Some Properties of a beta-d-Xylosidase from Clostridium acetobutylicum ATCC 824

A beta-d-xylosidase from C. acetobutylicum ATCC 824 was purified by column chromatography on CM-Sepharose, hydroxylapatite, Phenyl Sepharose, and Sephadex G-200. The enzyme had an apparent molecular weight of 224,000 as estimated by gel filtration. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the enzyme consisted of two subunits of 85,000 and one subunit of 63,000 daltons. It exhibited optimal activity at pH 6.0 to 6.5 and 45 degrees C. the enzyme had an isoelectric point of 5.85. It hydrolyzed p-nitrophenylxyloside readily with a K(m) of 3.7 mM. The enzyme hydrolyzed xylo-oligosaccharides with chain lengths of 2 to 6 units by cleaving a single xylose from the chain end. It showed little or no activity against xylan, carboxymethyl cellulose, and other p-nitrophenylglycosides.     

Purification and characterization of heparinase from Flavobacterium heparinum

Heparinase (EC 4.2.2.7) isolated from Flavobacterium heparinum was purified to homogeneity by a combination of hydroxylapatite chromatography, repeated gel filtration chromatography, and chromatofocusing. Homogeneity was established by the presence of a single band on both sodium dodecyl sulfate and acid-urea gel electrophoretic systems. Amino acid analysis shows that the enzyme contains relatively high amounts of lysine residues (9%) consistent with its cationic nature (pI 8.5) but contains only 4 cysteine residues/polypeptide. The molecular weight of heparinase was estimated to be 42,900 +/- 1,000 daltons by gel filtration and 42,700 +/- 1,200 daltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is very specific, acting only on heparin and heparan monosulfate out of 12 similar polysaccharide substrates tested. It has an activity maximum at pH 6.5 and 0.1 M NaCl and a stability maximum at pH 7.0 and 0.15 M NaCl. The Arrhenius activation energy was found to be 6.3 kcal/mol. However, the enzyme is very sensitive to thermal denaturation and loses activity very rapidly at temperatures over 40 degrees C. Kinetic studies of the heparinase reaction at 37 degrees C gave a Km of 8.04 X 10(-6) M and a Vm of 9.85 X 10(-5) M/min at a protein concentration of 0.5 microgram/ml. By adapting batch procedures of hydroxylapatite and QAE (quaternary aminoethyl)-Sephadex chromatography, gram quantities of heparinase that is nearly free of catalytic enzyme contaminants can be purified in 4-5 h.     

Biochemical Characterization of an Acetylcholine-hydrolyzing Enzyme from Bean Seedlings

An acetylcholine hydrolyzing enzyme was prepared and purified (40 times) from dwarf bean hypocotyl hooks. The purity of the enzyme was proved by polyacrylamide gel electrophoresis. The molecular weight of the enzyme was determined to be 65,000 daltons. Enzyme activity was the highest at pH 8.0 and between 30 and 36 C. The enzyme had an apparent affinity constant (K_m) for acetylcholine of 460/micromolar. The affinity for substrate analogs increased from butyrylthiocholine to propionylthiocholine to acetylthiocholine. The enzyme activity was inhibited by choline, neostigmine, physostigmine, manganese, and calcium. Magnesium had no influence on the enzyme activity. We conclude that the enzyme from dwarf beans is an acetylcholinesterase (EC 3.1.1.7).     

Purification and characterization of a membrane-associated phosphatidylserine synthase from Bacillus licheniformis

A CDP-diacylglycerol-dependent phosphatidylserine synthase was solubilized from Bacillus licheniformis membranes and purified to near homogeneity. The purification procedure consisted of CDP-diacylglycerol-Sepharose affinity chromatography followed by substrate elution from blue dextran-Sepharose. The purified preparation showed a single band with an apparent relative molecular mass of 53 000 daltons when subjected to sodium dodecyl sulfate--polyacrylamide gel electrophoresis. Proteolytic digestion of the enzyme yielded a smaller (41 000 daltons) active form. The preparation was free of any phosphatidylglycerophosphate synthase, phosphatidylserine decarboxylase, CDP-diacylglycerol hydrolase, and phosphatidylserine hydrolase activities. The utilization of substrates and the formation of products occurred with the expected stoichiometry. Radioisotopic exchange patterns between related substrate and product pairs suggest a sequential Bi-Bi reaction as opposed to the ping-pong mechanism exhibited by the well-studied phosphatidylserine synthase of Escherichia coli [Larson, T. J., & Dowhan, W. (1976) Biochemistry 15, 5212-5218]. The B. licheniformis enzyme was also found to be markedly dissimilar to the E. coli enzyme with regard to association with detergent micelles, affinity for ribosomes, and antigenicity.     

Purification and properties of alkaline phosphatase from the mucosa of rat small intestine

Alkaline phosphatase [orthophosphoric monoester phosphohydrolase, EC 3.1.3.1] was purified from the mucosa of rat small intestine by butanol extraction, ethanol fractionation, gel filtration, with controlled-pore glass-10 and DEAE-cellulose column chromatography. On the gel filtration, the enzyme activity was separated into three peaks; A in the void volume, B and C at lower molecular weight positions. Enzyme A was purified to homogeneity. The activity of enzymes A, B, and C was detected even on sodium dodecyl sulfate-polyacrylamide gel electrophoresis at the position of the protein of enzyme A, which had a molecular weight of 110,000 daltons. Enzymatic properties such as pH optimum, Km value for the substrate, heat inactivation and inhibition by amino acids were the same in all three enzymes. Based on these findings, together with the elution positions on gel filtration, enzyme A was regarded as an aggregate, and enzymes B and C as dimer and monomer molecules, respectively.     

露花叶片PEP羧化酶的纯化及某些分子特性

经硫酸铵分部沉淀,DEAE-纤维素(DE52),DEAE-Sephadex A-50,SephacrylS-200和二次羟基磷灰石等柱层析,从露花叶片中分离得到纯化63.9倍、电泳均一的磷酸烯醇式丙酮酸羧化酶。此酶的天然分子量经聚丙烯酰胺梯度凝胶电泳测定为260kD,经SephadexG-200凝胶过滤法测定为240kD。用SDS-聚丙烯酰胺梯度凝胶电泳测得酶的亚基分子量为115kD,表明此酶是个二同聚体。此酶的等电点为PI=5.6。免疫双扩散的结果表明此酶与高梁PEPG的抗原决定簇呈部分同一性。     

Purification and characterization of a Ca2+/calmodulin-dependent protein kinase from rat brain

A soluble Ca2+/calmodulin-dependent protein kinase has been purified from rat brain to near homogeneity by using casein as substrate. The enzyme was purified by using hydroxylapatite adsorption chromatography, phosphocellulose ion-exchange chromatography, Sepharose 6B gel filtration, affinity chromatography using calmodulin-Sepharose 4B, and ammonium sulfate precipitation. On sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gels, the purified enzyme consists of three protein bands: a single polypeptide of 51 000 daltons and a doublet of 60 000 daltons. Measurements of the Stokes radius by gel filtration (81.3 +/- 3.7 A) and the sedimentation coefficient by sucrose density sedimentation (13.7 +/- 0.7 S) were used to calculate a native molecular mass of 460 000 +/- 29 000 daltons. The kinase autophosphorylated both the 51 000-dalton polypeptide and the 60 000-dalton doublet, resulting in a decreased mobility in NaDodSO4 gels. Comparison of the phosphopeptides produced by partial proteolysis of autophosphorylated enzyme reveals substantial similarities between subunits. These patterns, however, suggest that the 51 000-dalton subunit is not a proteolytic fragment of the 60 000-dalton doublet. Purified Ca2+/calmodulin-dependent casein kinase activity was dependent upon Ca2+, calmodulin, and ATP X Mg2+ or ATP X Mn2+ when measured under saturating casein concentrations. Co2+, Mn2+, and La3+ could substitute for Ca2+ in the presence of Mg2+ and saturating calmodulin concentrations. In addition to casein, the purified enzyme displayed a broad substrate specificity which suggests that it may be a "general" protein kinase with the potential for mediating numerous processes in brain and possibly other tissues.     

Purification and characterization of bovine lung calmodulin-dependent cyclic nucleotide phosphodiesterase. An enzyme containing calmodulin as a subunit

A rabbit lung cyclic nucleotide phosphodiesterase (PDE) prepared by successive chromatography on DEAE-cellulose and G-200 Sephadex columns in the presence of EGTA was activated by Ca2+ and contained calmodulin (CaM), suggesting that the enzyme exists as a stable CaM X PDE complex (Sharma, R. K., and Wirch, E. (1979) Biochem. Biophys. Res. Commun. 91, 338-344). An enzyme with similar properties was demonstrated to exist in bovine lung extract. C1, a monoclonal antibody previously shown to react with the 60-kDa subunit of bovine brain PDE isozymes (Sharma, R. K., Adachi, A.-M., Adachi, K., and Wang, J. H.) (1984) J. Biol. Chem. 259, 9248-9254), cross-reacted with the lung enzyme. Purification of the lung enzyme by C1 antibody immunoaffinity chromatography rendered the enzyme dependent on exogenous CaM for Ca2+ stimulation. Further purification was achieved by CaM affinity chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the purified enzyme showed a predominant polypeptide of Mr 58,000 and a minor band of about 50,000. The purified enzyme could be reconstituted into a PDE X CaM complex upon incubation with CaM in the presence of either Ca2+ or EGTA. The reconstituted protein complex did not dissociate in buffers containing 0.1 mM EGTA. Analysis of the purified and reconstituted lung phosphodiesterase by Sephacryl S-300 gel filtration indicated that the lung enzyme is a dimeric protein and that the reconstituted enzyme contained two molecules of calmodulin. Analysis of the reconstituted phosphodiesterase by sodium dodecyl sulfate-polyacrylamide gel electrophoresis also showed it to contain equimolar calmodulin and the enzyme subunit. The CaM antagonists, fluphenazine, compound 48/80, and calcineurin at concentrations abolishing CaM stimulation of bovine brain PDE had little effect on the activity of reconstituted bovine lung phosphodiesterase.     

Purification of human lung angiotensin-converting enzyme by high-performance liquid chromatography: properties and N-terminal amino acid sequence

Angiotensin-converting enzyme from the human lung was purified to apparent homogeneity, using high-performance liquid chromatography following trypsin treatment of the detergent-extract. A 1,750-fold purification was achieved with a 26% yield. The specific activity of the enzyme was 105 units per mg protein with the substrate hippuryl-L-histidyl-L-leucine (HHL) at 37 degrees C, and the Km value for HHL was 1.9 mM. The molecular weight was estimated to be 170,000 by sodium dodecyl sulfate gel electrophoresis, and the isoelectric point was about 4.8, by chromatofocusing. The N-terminal amino acid sequence was (NH2)-X-X-Pro-Gly-Leu-Glu-Pro-Gly-X-Phe-Ser-Ala-Arg-Glu-Ala-Gly-Ala. This is highly homologous to the corresponding sequences of the enzymes from bovine and rabbit lung and from pig, bovine, and mouse kidney, but significantly different from that of the human kidney enzyme.