Degradation of ureidoglycolate in French bean (Phaseolus vulgaris) is catalysed by a ubiquitous ureidoglycolate urea-lyase

A ureidoglycolate-degrading activity was analysed in different tissues of French bean (Phaseolus vulgaris L.) plants during development. Activity was detected in all the tissues analysed, although values were very low in seeds before germination and in cotyledons. After radicle emergence, the activity increased due to high activity present in the axes. The highest levels of specific activity were found in developing fruits, from which the enzyme was purified and characterised. This is the first ureidoglycolate-degrading activity that has been purified to homogeneity from a ureide legume. The enzyme was purified 280 fold, and the specific activity for the pure enzyme was 4.4 units mg⁻¹, which corresponds to a turnover number of 1,055 min⁻¹. The native enzyme has a molecular mass of 240 kDa and consists of six identical or similar-sized subunits each of 38 kDa. The activity of the purified enzyme was completely dependent on manganese and asparagine. The enzyme exhibited hyperbolic, Michaelian kinetics for ureidoglycolate with a K _m value of 3.9 mM. This enzyme has been characterised as a ureidoglycolate urea-lyase (EC 4.3.2.3).     

Allantoate amidinohydrolase (Allantoicase) from Chlamydomonas reinhardtii: its purification and catalytic and molecular characterization

An allantoate-degrading enzyme has been purified to electrophoretic homogeneity for the first time from a photosynthetic organism, the unicellular green algae Chlamydomonas reinhardtii. The purification procedure included a differential protein extraction followed by conventional steps such as ammonium sulfate fractionation, gel filtration, anion exchange chromatography, and preparative electrophoresis. Under the routine assay conditions (7 mM allantoate), specific activity for the purified enzyme was 185 U/mg, which rose to 225 U/mg under kinetic considerations (saturating substrate). Therefore, a turnover number of 4.5 x 10(4) min(-1) can be deduced for the 200-kDa protein. The enzyme is a true allantoicase (EC 3.5.3.4) that catalyzes the degradation of allantoate to (-)ureidoglycolate and (+)ureidoglycolate to glyoxylate. The enzyme exhibited hyperbolic kinetic for allantoate and ureidoglycolate with K(m) values of 2 and 0.7 mM, respectively. V(max) of the reaction with allantoate as substrate was nine times higher than that with ureidoglycolate. The native enzyme has a molecular weight of 200 kDa and consists of six identical or similar-sized subunits of 34 kDa each, organized in two trimers of 100 kDa. Each subunit has five cysteine residues, four of which are involved in disulfide bonds, with a total of 12 disulfide bonds in the 200-kDa protein. Allantoate inhibits competitively the reaction with ureidoglycolate as substrate. In addition, buffers and group-specific reagents affect the activity in the same manner irrespective of the substrate used. Those results suggest that both substrates use the same active site. The effect of group-specific reagents suggest that the amino acids histidine, tyrosine, and cysteine are essentials for the allantoicase activity with both substrates.     

Ureidoglycolate amidohydrolase from developing French bean fruits (Phaseolus vulgaris [L.].)

Ureidoglycolate is an intermediate of allantoin catabolism in ureide-transporting legumes. This report describes the first purification of ureidoglycolate degrading activity (UGDA) from plant tissue in which the enzyme has been separated from urease. The enzyme from developing fruits of Phaseolus vulgaris has been purified 48-fold to give a preparation free of allantoinase and urease activity. UGDA was inhibited by EDTA while the Vmax was increased in the presence of Mn2+. The Km values for ureidoglycolate in the presence and the absence of Mn2+ were 2.0 and 5.4 mM, respectively. In the absence of Mn2+ UGDA was heat labile at 40 degrees C, but in the presence of Mn2+ the activity was stable up to temperatures of 60 degrees C. The Mr of UGDA was determined to be 300,000 by gel filtration chromatography and the pH optimum ranged from pH 7.0 to 8.5. Ammonia was determined to be the nitrogen-containing product of UGDA by a microdiffusion assay. This enzyme should therefore be described as ureidoglycolate amidohydrolase. The activity was shown to be associated with peroxisomes by fractionation of a crude extract on a sucrose density gradient. The products of ureidoglycolate degradation are glyoxylate, ammonia, and presumably carbon dioxide, which can be readily utilized by pathways of metabolism that are known to be present in this organelle.     

Biochemical characterisation of an allantoate-degrading enzyme from French bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis>): the requirement of phenylhydrazine

In tropical legumes like French bean (Phaseolus vulgaris) or soybean (Glycine max), most of the atmospheric nitrogen fixed in nodules is used for synthesis of the ureides allantoin and allantoic acid, the major long distance transport forms of organic nitrogen in these species. The purpose of this investigation was to characterise the allantoate degradation step in Phaseolus vulgaris. The degradation of allantoin, allantoate and ureidoglycolate was determined “in vivo” using small pieces of chopped seedlings. With allantoate and ureidoglycolate as substrates, the determination of the reaction products required the addition of phenylhydrazine to the assay mixture. The protein associated with the allantoate degradation has been partially purified 22-fold by ultracentrifugation and batch separation with DEAE-Sephacel. This enzyme was specific for allantoate and could not use ureidoglycolate as substrate. The activity was completely dependent on phenylhydrazine, which acts as an activator at low concentrations and decreases the affinity of the enzyme for the substrate at higher concentrations. The optimal pH for the activity of the purified protein was 7.0 and the optimal temperature was 37°C. The activity was completely inhibited by EDTA and only manganese partially restored the activity. The level of activity was lower in extracts obtained from leaves and fruits of French bean grown with nitrate than in plants actively fixing nitrogen and, therefore, relying on ureides as nitrogen supply. This is the first time that an allantoate-degrading activity has been partially purified and characterised from a plant extract. The allosteric regulation of the enzyme suggests a critical role in the regulation of ureide degradation.     

Catalysis, stereochemistry, and inhibition of ureidoglycolate lyase

Ureidoglycolate lyase (UGL, EC 4.3.2.3) catalyzes the breakdown of ureidoglycolate to glyoxylate and urea, which is the final step in the catabolic pathway leading from purines to urea. Although the sequence of enzymatic steps was worked out nearly 40 years ago, the stereochemistry of the uric acid degradation pathway and the catalytic properties of UGL have remained very poorly described. We now report the first direct investigation of the absolute stereochemistry of UGL catalysis. Using chiral chromatographic analyses with substrate enantiomers, we demonstrate that UGL catalysis is stereospecific for substrates with the (S)-hydroxyglycine configuration. The first potent competitive inhibitors for UGL are reported here. These inhibitors are compounds which contain a 2,4-dioxocarboxylate moiety, designed to mimic transient species produced during lyase catalysis. The most potent inhibitor, 2,4-dioxo-4-phenylbutanoic acid, exhibits a KI value of 2.2 nM and is therefore among the most potent competitive inhibitors ever reported for a lyase enzyme. New synthetic alternate substrates for UGL, which are acyl-alpha-hydroxyglycine compounds, are described. Based on these alternate substrates, we introduce the first assay method for monitoring UGL activity directly. Finally, we report the first putative primary nucleotide and derived peptide sequence for UGL. This sequence exhibits a high level of similarity to the fumarylacetoacetate hydrolase family of proteins. Close mechanistic similarities can be visualized between the chemistries of ureidoglycolate lyase and fumarylacetoacetate hydrolase catalysis.     

Identification of the ureidoglycolate hydrolase gene in the DAL gene cluster of Saccharomyces cerevisiae.

This report describes the isolation of the genes encoding allantoicase (DAL2) and ureidoglycolate hydrolase (DAL3), which are components of the large DAL gene cluster on the right arm of chromosome IX of Saccharomyces cerevisiae. During this work a new gene (DAL7) was identified and found to be regulated in the manner expected for an allantoin pathway gene. Its expression was (i) induced by allophanate, (ii) sensitive to nitrogen catabolite repression, and (iii) responsive to mutation of the DAL80 and DAL81 loci, which have previously been shown to regulate the allantoin degradation system. Hybridization probes generated from these cloned genes were used to analyze expression of the allantoin pathway genes in wild-type and mutant cells grown under a variety of physiological conditions. When comparison was possible, the patterns of mRNA and enzyme levels observed in various strains and physiological conditions were very similar, suggesting that the system is predominantly regulated at the level of gene expression. Although all of the genes seem to be controlled by a common mechanism, their detailed patterns of expression were, at the same time, highly individual and diverse.     

Purification and Partial Characterization of Arginine Decarboxylase from Brassica campestris

Arginine decarboxylase (EC 4.1.1.19) has been purified and characterized from Brassica campestris cv B-9. The enzyme was purified 1120 fold and the recovery was 9%. The mol wt of the enzyme determined by gel filtration was 240 kD with identical subunits of 60 kD. The pH and temperature optima for the enzyme were 8.0 and 30°C respectively. The Km was 0.31mM. Polyamines inhibited the enzyme activity significantly. Immunodiffusion with ADC-specific antibodies showed cross reactivity against purified ADC from Brassica.     

四季豆幼苗中尿囊酸酰胺水解酶的一些特性

酰脲代谢在许多固氮豆科植物氮素代谢中起重要作用;尿囊酸的酰胺水解酶(EC3.5.3.9)分解尿囊酸成为脲基乙醇酸和CO2、NH3,脲基乙醇酸的酰胺水解酶进一步分解脲基乙醇酸产生乙醛酸和CO2、NH3.该文首次报告测定四季豆尿囊酸降解酶(分解尿囊酸的酶)的方法,酶反应基质需要盐酸苯肼存在.在四季豆干种子、幼苗根、茎和叶,均可测出尿囊酸降解酶活力.从四季豆幼苗分离出两个尿囊酸降解酶.一个分子量大于200 kD,另一个分子量为13.5 kD;小分子量的尿囊酸降解酶(没有脲基乙醇酸酰胺水解酶或脲酶活力)用于性质研究.酶反应产物分析表明,该酶是尿囊酸的酰胺水解酶.该酶反应的最适pH为8.5.Mn2 是该酶的金属辅助因子.Km为76μmol/L,Vmax为16.7 nKat/mg(=1 002 nmol min1mg1).乙醛酸和乙醇酸抑制该酶活力.赖氨酸残基和色氨酸残基是酶活力的必需基团;巯基和酪氨酸残基不是酶活力的必需基团.     

A Xyloglucan-Specific Endo-1,4-[beta]-Glucanase Isolated from Auxin-Treated Pea Stems

A xyloglucan-specific endo-1,4-[beta]-glucanase was isolated from the apoplast fraction of auxin-treated pea (Pisum sativum) stems, in which both the rate of stem elongation and the amount of xyloglucan solubilized were high. The enzyme was purified to apparent homogeneity by sequential cation-exchange chromatographies, affinity chromatography, and gel filtration. The purified enzyme gave a single protein band on sodium dodecyi sulfate-polyacrylamide gel electrophoresis, and the molecular size was determined to be 77 kD by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 70 kD by gel filtration. The isoelectric point was about 8.1. The enzyme specifically cleaved the 1,4-[beta]-glucosyl linkages of the xyloglucan backbone to yield mainly nona- and heptasaccharides but did not hydrolyze carboxymethylcellulose, swollen cellulose, and (1->3, 1->4)-[beta]-glucan. By hydrolysis, the average molecular size of xyloglucan was decreased from 50 to 20 kD with new reducing chain ends in the lower molecular size fractions. This suggests that the enzyme has endo-1,4-[beta]-glucanase activity against xyloglucan. In conclusion, a xyloglucan-specific endo-1,4-[beta]-glucanase with an activity that differs from the activities of cellulase and xyloglucan endotransglycosylase has been isolated from elongating pea stems.     

Purification of turnip peroxidase and its kinetic properties

Peroxidase from turnip roots was purified using metal affinity chromatography up to a specific activity of 337 units/mg protein with 3.02 RZ and 63.5% recovery. After purification, the enzyme showed 2-3 bands on sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of the purified enzyme was found to be 37-39 kD with matrix assisted laser desorption ionization mass spectrometer (MALDI-MS). The enzyme showed maximum activity in phosphate buffer, pH 6.0, and lowest activity in borate buffer at the same pH. The Km of the enzyme was found to be 7.07 x 104 mM. Turnip peroxidase also contains an iron moiety which is found to be about 0.28%. The enzyme showed 50% inhibition of its specific activity with ethylene diamine tetraacetic acid (EDTA).     

Identification of protease from Euphorbia amygdaloides latex and its use in cheese production

In this research, protease enzyme was purified and characterized from milk of Euphorbia amygdaloides. (NH4)2SO4 fractionation and CM-cellulose ion exchange chromatography methods were used for purification of the enzyme. The optimum pH value was determined to be 5, and the optimum temperature was determined to be 60 degrees C. The V(max) and K(M) values at optimum pH and 25 degrees C were calculated by means of Linewearver-Burk graphs as 0.27 mg/L min(-1) and 16 mM, respectively. The purification degree was controlled by using SDS-PAGE and molecular weight was found to be 26 kD. The molecular weight of the enzyme was determined as 54 kD by gel filtration chromatography. These results show that the enzyme has two subunits.In the study, it was also researched whether purified and characterized protease can be collapsed to milk. It was determined that protease enzyme can collapse milk and it can be used to produce cheese.     

Localization of cytosolic NADP-dependent isocitrate dehydrogenase in the peroxisomes of rat liver cells: biochemical and immunocytochemical studies.

Two types of NADP-dependent isocitrate dehydrogenases (ICDs) have been reported: mitochondrial (ICD1) and cytosolic (ICD2). The C-terminal amino acid sequence of ICD2 has a tripeptide peroxisome targeting signal 1 sequence (PTS1). After differential centrifugation of the postnuclear fraction of rat liver homogenate, approximately 75% of ICD activity was found in the cytosolic fraction. To elucidate the true localization of ICD2 in rat hepatocytes, we analyzed the distribution of ICD activity and immunoreactivity in fractions isolated by Nycodenz gradient centrifugation and immunocytochemical localization of ICD2 antigenic sites in the cells. On Nycodenz gradient centrifugation of the light mitochondrial fraction, ICD2 activity was distributed in the fractions in which activity of catalase, a peroxisomal marker, was also detected, but a low level of activity was also detected in the fractions containing activity for succinate cytochrome C reductase (a mitochondrial marker) and acid phosphatase (a lysosomal marker). We have purified ICD2 from rat liver homogenate and raised a specific antibody to the enzyme. On SDS-PAGE, a single band with a molecular mass of 47 kD was observed, and on immunoblotting analysis of rat liver homogenate a single signal was detected. Double staining of catalase and ICD2 in rat liver revealed co-localization of both enzymes in the same cytoplasmic granules. Immunoelectron microscopy revealed gold particles with antigenic sites of ICD2 present mainly in peroxisomes. The results clearly indicated that ICD2 is a peroxisomal enzyme in rat hepatocytes. ICD2 has been regarded as a cytosolic enzyme, probably because the enzyme easily leaks out of peroxisomes during homogenization. (J Histochem Cytochem 49:1123-1131, 2001)     

Isolation of lysophosphatidic acid phosphatase from developing peanut cotyledons

The soluble fraction of immature peanut (Arachis hypogaea) was capable of dephosphorylating [(3)H]lysophosphatidic acid (LPA) to generate monoacylglycerol (MAG). The enzyme responsible for the generation of MAG, LPA phosphatase, has been identified in plants and purified by successive chromatography separations on octyl-Sepharose, Blue Sepharose, Superdex-75, and heparin-agarose to apparent homogeneity from developing peanuts. This enzyme was purified 5,048-fold to a final specific activity of 858 nmol min(-1) mg(-1). The enzyme has a native molecular mass of approximately 39 kD determined by gel filtration and migrates as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a subunit molecular mass of 39 +/- 1.5 kD. The K(m) values for oleoyl-, stearoyl-, and palmitoyl-sn-glycerol-3-phosphate were determined to be 28.6, 39.3, and 47.9 microM, respectively. The LPA phosphatase was specific to LPA and did not utilize any other substrate such as glycerol-3-phosphate, phosphatidic acid, or p-nitrophenylphosphate. The enzyme activity was stimulated by the low concentrations of detergents such as Triton X-100 and octylglucoside. Cations had no effect on the enzyme activity. Fatty acids, sphingosine, and sphingomyelin at low concentrations stimulated the enzyme activity. The identification of LPA phosphatase in plants demonstrates the existence of MAG biosynthetic machinery in plants.     

Characterization of plant beta-ureidopropionase and functional overexpression in Escherichia coli

Pyrimidine bases are rapidly catabolized in growing plant tissues. The final enzyme of the catabolic pathway, beta-ureidopropionase (beta-UP; EC 3.5.1.6), was partially purified from the shoots of etiolated maize (Zea mays) seedlings. The enzyme had a K(m) for beta-ureidopropionate (the substrate derived from uracil) of 11 microM. Only one enantiomer of racemic beta-ureidoisobutyrate (derived from thymine) was processed with a K(m) of 6 microM. The enzyme was inactivated by dialysis against 1,10-phenanthroline and activity could be partially restored by addition of Zn(2+). Maize beta-UP was very sensitive to inactivation by iodoacetamide. This could be prevented by addition of substrate, indicating the presence of an active site Cys. The enzyme was strongly inhibited by short chain aliphatic acids and aryl propionates, the most potent inhibitor of which was 2-(2, 6-dinitrophenoxy)-propionate (I(50) = 0.5 microM). A gene for Arabidopsis beta-UP encodes a polypeptide of 405 amino acids and has about 55% homology with the enzymes from other eukaryotic organisms. Several highly conserved residues link the plant beta-UP with a larger class of prokaryotic and eukaryotic amidohydrolases. An Arabidopsis cDNA truncated at the N terminus by 14 residues was cloned and overexpressed in Escherichia coli. The recombinant enzyme (43.7 kD) was soluble, functional, and purified to homogeneity with yields of 15 to 20 mg per 30 g fresh weight of E. coli cells. The recombinant enzyme from Arabidopsis and the native enzyme from maize had molecular masses of approximately 440 kD, indicating the enzyme is a decamer at pH 7.     

花生根瘤菌类菌体氢酶的分离提纯及特性

花生根瘤菌类菌体经超声波破碎,ＴｒｉｔｏｎＸ－１００溶解,正已烷－硫酸铵处理后,再经ＤＥＡＥ－纤维素和Ｓｅｐｈａｃｒｙｌ凝胶柱层析等纯化步骤,获得凝胶电泳纯的膜结合态氢酶,比活为７１．４μｍｏｌＨ２ｍｇ－１Ｐｒｏｔｍｉｎ－１,为类菌体吸Ｈ２活性的２１１倍。纯化的氢酶分子量为１１０ｋＤ。经ＳＤＳ－ＰＡＧＥ后,呈现两个蛋白带,分子量分利为６５ｋＤ和３５ｋＤ。纯酶的Ｎｉ含量为０．６２ｍｏｌＮｉ／ｍｏｌ氢酶。在磷酸缓冲液中其活性的最适ｐＨ为６．５。ＤＣＩＰ、亚甲蓝、铁氰化钾、细胞色素Ｃ均可作为氢酶的电子受体,其中以ＤＣＩＰ为最适。     

嗜碱芽孢杆菌Bacillus sp.F26过氧化氢酶的分离纯化及性质研究

从一株低度嗜盐、兼性嗜碱芽孢杆菌Bacillus sp．F26中纯化得到一种碱性过氧化氢酶,并对该酶进行了性质研究。纯化过程经硫酸铵沉淀、阴离子交换层析、凝胶过滤层析及疏水层析四步最终获得电泳纯的目标酶(纯化58．5倍)。该过氧化氢酶的分子量为140kD,由两个大小相同的亚基组成。天然酶分子在408nm处显示特征吸收峰(Soret band)。吡啶血色素光谱显示了酶分子以原卟啉Ⅸ(protoheme Ⅸ)作为辅基。计算获得酶的表观米氏常数为32．5mmol／L。该过氧化氢酶不受连二亚硫酸钠的还原作用影响,但被氰化物、叠氮化物和3．氨基．1,2,4-三唑(单功能过氧化氢酶的专一抑制剂)强烈抑制。以邻联茴香胺、邻苯二胺和二氨基联苯胺作为电子供体测定酶活时,该酶不显示过氧化物酶活性。同时,酶的N-端序列比对结果说明,该过氧化氢酶与单功能过氧化氢酶亚群有一定的相似性,而与双功能过氧化氢酶亚群及猛过氧化氢酶亚群均没有同源性。因此,本文将纯化的碱性过氧化氢酶定性为单功能过氧化氢酶。此外,该酶具有热敏感的特点,且酶活在pH5～9的范围内不受pH影响,此后,活性随着pH的升高而升高,并在pH 11处有明显的酶活高峰。20℃、pH 11条件下的酶活半衰期达49h。在pH 11的高碱条件下表现出最高活力和一定的稳定性,这在已报道的过氧化氢酶中还未见描述。同时,该酶也显示了良好的盐碱稳定性,0．5mol／L NaCl、pH 10．5条件下的酶活半衰期达90h。另一方面,本文所研究的过氧化氢酶是第一个来源于嗜碱微生物的同源二聚体单功能过氧化氢酶,也是第一个来源于天然碱湖的单功能过氧化氢酶,它能部分地反映出细胞抗氧化体系对相应环境的适应情况。     

A radiometric assay for kynurenine 3-hydroxylase based on the release of 3H2O during hydroxylation of L-[3,5-3H]kynurenine.

A rapid and sensitive assay for kynurenine 3-hydroxylase (KH) has been developed. This radiometric assay is based on the enzymatic synthesis of tritiated water from L-[3,5-3H]kynurenine during the hydroxylation reaction. Radiolabeled water is quantified following selective adsorption of the isotopic substrate and its metabolite with activated charcoal. The assay is suitable for detecting 0.1 pmol enzyme activity per minute per milligram protein in tissues displaying low levels of the enzyme. The amount of water produced in the reaction, as calculated from the tritium released, was stoichiometric with the 3-hydroxykynurenine product detected by HPLC. Rat liver KH was characterized by cofactor specificity and kinetic parameters. NADPH was preferred over NADH as coreductant in the reaction. Tetrahydrobiopterin was not a cofactor. The tissue distribution of KH activity in the rat suggested that the majority of active enzyme is located in liver and kidney. Detectable amounts were found in several other tissues, including brain which had low but significant levels of activity in every region assayed.     

d-Aspartate N-methyltransferase catalyzes biosynthesis of N-methyl-d-aspartate (NMDA), a well-known selective agonist of the NMDA receptor,in mice

N-Methyl-d-aspartate (NMDA), which is a selective agonist for the NMDA receptor, has recently been shown to be present in various biological tissues. In mammals, the activity of d-aspartate N-methyltransferase (DDNMT), which produces NMDA from d-aspartate, has been detected only in homogenates prepared from rat tissues. Moreover, the enzymatic properties of DDNMT have been poorly studied and its molecular entity has not yet been identified. In this report, we show for the first time that the activity of DDNMT is present in mouse tissues and succeed in obtaining a partially purified enzyme preparation from a mouse tissue homogenate with a purification fold of 1900 or more, and have characterized the enzymatic activity of this preparation. The results indicate that DDNMT, which is highly specific for d-aspartate and is S-adenosyl-l-methionine-dependent, is a novel enzyme that clearly differs from the known methylamine-glutamate N-methyltransferase (EC 2.1.1.21) and glycine N-methyltransferase (EC 2.1.1.20).     

Extracellular alkaline proteinase of Colletotrichum gloeosporioides

The main proteinase of the filamentous fungus Colletotrichum gloeosporioides causing anthracnoses and serious problems for production and storage of agricultural products has molecular mass of 57 kD and was purified more than 200-fold to homogeneity with the yield of 5%. Maximal activity of the proteinase is at pH 9.0-10.0, and the enzyme is stable at pH 6.0-11.5 (residual activity not less than 70%). The studied enzyme completely kept its activity to 55 degrees C, with a temperature optimum of 45 degrees C. The purified C. gloeosporioides proteinase is stable at alkaline pH values, but rapidly loses its activity at pH values lower than 5.0. Addition of bovine serum albumin stabilizes the enzyme under acidic conditions. Data on inhibitor analysis and substrate specificity of the enzyme allow its classification as a serine proteinase of subtilisin family. It is demonstrated that the extracellular proteinase of C. gloeosporioides specifically effects plant cell wall proteins. It is proposed that the studied proteinase--via hydrolysis of cell wall--provides for penetration of the fungus into the tissues of the host plant.     

Isolation and characterization of a new aminopeptidase from bovine lens

An aminopeptidase has been purified to homogeneity from bovine lens tissue by gel filtration and DEAE-cellulose chromatography. This enzyme has a molecular weight of 96,000 under both native and denaturing conditions. The purified enzyme hydrolyzed a variety of synthetic substrates as well as di-, tri-, and higher molecular weight peptides. Significantly this enzyme is capable of hydrolyzing arginine, lysine, and proline aminoacyl bonds. The pH optimum for activity and stability was 6.0. Both a reduced sulfhydryl group and a divalent metal ion are essential for activity. The native enzyme contains 1.6 mol of zinc and 1.0 mol of copper/mol of enzyme. No activation was seen upon incubation with either magnesium or manganese; however, heavy metal ions were inhibitory. Bestatin and puromycin were effective inhibitors and no endopeptidase activity could be detected in the purified preparation. This enzyme is clearly distinct from the lens leucine aminopeptidase, but rather, is identical to a cytosolic aminopeptidase III isolated from other tissues. Evidence is presented which argues that this enzyme may be the major lens aminopeptidase under in vivo conditions.