Isolation and properties of an aminopeptidase from Bacillus licheniformis

An extracellular aminopeptidase, purified 465-fold from culture filtrates of Bacillus licheniformis, was found to be a metalloenzyme consisting of a single peptide chain. Sedimentation equilibrium yielded a molecular weight of 43,270 and two polyacrylamide electrophoretic procedures gave values of 37,500 and 36,000, respectively. The activity of the enzyme was inhibited severely by 1,10-phenanthroline and to a lesser extent by EDTA, cyanide, and fluoride. The addition of Co²⁺ ions greatly stimulated enzymatic activity, but analysis of the purified enzyme revealed the presence of zinc, not cobalt, in stoichiometric quantities. Moreover, the ratio of zinc to protein was found to increase during fractionation, reaching a final value corresponding to 1 g-atom/mol. The aminopeptidase possessed characteristics of a euglobulin, sparingly soluble in water and dilute buffer solutions, but soluble in buffers containing higher concentrations of salts. Both activity and pH optimum were substantially influenced by ionic strength; as the latter was increased over the range from 0.01 to 0.1, activity increased and the pH optimum was shifted to more acidic values. Enzymatic activity was affected by the identity of the buffer, being markedly greater in Tris-HCl than in sodium barbital and strongly inhibited by phosphate. The Bacillus aminopeptidase hydrolyzed substrates with unsubstituted amino groups of the l configuration, including dipeptides, aminoacylnaphthylamides, and amino acid amides.     

Identification of yeast aspartyl aminopeptidase gene by purifying and characterizing its product from yeast cells

Aspartyl aminopeptidase (EC 3.4.11.21) cleaves only unblocked N-terminal acidic amino-acid residues. To date, it has been found only in mammals. We report here that aspartyl aminopeptidase activity is present in yeast. Yeast aminopeptidase is encoded by an uncharacterized gene in chromosome VIII (YHR113W, Saccharomyces Genome Database). Yeast aspartyl aminopeptidase preferentially cleaved the unblocked N-terminal acidic amino-acid residue of peptides; the optimum pH for this activity was within the neutral range. The metalloproteases inhibitors EDTA and 1.10-phenanthroline both inhibited the activity of the enzyme, whereas bestatin, an inhibitor of most aminopeptidases, did not affect enzyme activity. Gel filtration chromatography revealed that the molecular mass of the native form of yeast aspartyl aminopeptidase is approximately 680,000. SDS/PAGE of purified yeast aspartyl aminopeptidase produced a single 56-kDa band, indicating that this enzyme comprises 12 identical subunits.     

Monkey brain arylamidase. II. Further characterization and studies on mode of hydrolysis of physiologically active peptides.

A large-scale purification of monkey brain arylamidase was carried out. Amino acid analyses indicate that the enzyme is rich in acidic amino acids and is poor in cystine. The amino terminal residue was determined to be alanine by dansylation. The enzyme was activated by sulfhydryl compounds. Dithiothreitol was more effective than beta-mercaptoethanol. Bestatin competitively inhibited the enzyme activity and the Ki value was calculated to be 2.5 x 10(-7) M, which was of the same order as that of puromycin. The inhibitions by puromycin and bestatin were reversible. The enzyme hydrolyzed di-, tri-, and oligopeptides including physiologically active peptides. Of physiologically active peptides, enkephalins and Met-Lys-bradykinin, which possess a neutral amino acid at the N-terminal position, were more rapidly hydrolyzed by the enzyme. Peptides such as LH-RH and TRH, which possess a pyrrolidonecarboxylyl group at the N-terminal position, and substance P and bradykinin, which possess a proline residue adjacent to the N-terminal residue, were not hydrolyzed by the enzyme. The Km values for various peptides indicate that the enzyme has higher affinity for oligopeptides than di- and tripeptides. The aminopeptidase activity of the enzyme was also competitively inhibited by puromycin and bestatin. Analyses of the hydrolysis products of various peptides by the dansylation method indicate that the enzyme has both kinin-converting activity and angiotensinase activity.     

An extracellular aminopeptidase from Clostridium histolyticum.

An aminopeptidase was isolated from the culture filtrate of Clostridium histolyticum and purified to homogeneity. Absence of endopeptidase activity in the purified preparation was demonstrated. Gel filtration on a calibrated column indicates an apparent molecular weight of 340000 for the native enzyme. Gel electrophoresis of the denatured enzyme in the presence of dodecylsulfate in constant acrylamide concentration and in a concentration gradient, resulted in the appearance of a single component for which a molecular weight of 51000 and 59000 respectively, was calculated. From mobilities of crosslinked and denatured protein species a molecular weight of 56000 was obtained for the monomer. Specificity studies show that the enzyme cleaves all types of N-terminel amino acid residues including proline and hydroxyproline from small peptides and from polypeptides. The peptide bond formed between an N-terminal amino acid residue and proline is not cleaved by the enzyme. The combined action of aminopeptidase-P and clostridal aminopeptidase leads to complete hydrolysis of the proline-rich nonapeptide bradykinin. Low rates of hydrolysis was observed for charged residues, and amides of amino acids. Kinetic studies with five tripeptides of the general structure X-Gly-Gly, where X stands for Leu, Phe, Val, Ala, or Pro, show a decrease in Km with the increasing size of the hydrophobic side chain of X. The highest Kcat values are observed with proline and alanine. In the series Pro-Gly, Pro-Gly-Pro, Pro-Gly-Pro-Pro, the last peptide is the best substrate, indicating an active site complementary to at least four amino acid residues. The enzymatic activity is dependent on the presence of divalent cations, maximal activation being reached with Mn2+ and Co2+. The optimal pH for the Mn2+ and Co2+- activated enzyme is 8.6 and 8.2 respectively. The optimal temperature is 40 degrees C. Inhibition of the aminopeptidase was achieved with Zn2+, Cu2+ and p-mercuribenzoate, but not with diisopropylphosphofluoridate.     

Aminopeptidase PC from the hepatopancreas of the Kamchatka crab Paralithodes camtshatica

Homogeneous aminopeptidase PC was isolated with yield 67% and purification degree 237 from the hepatopancreas of the Kamchatka crab Paralithodes camtshatica by ion-exchange chromatography on DEAE-Sepharose, hydrophobic chromatography on Phenyl-Sepharose, and gel-filtration on Sephadex G-150. The enzyme is a homodimer with a molecular mass 220 kD (110 x 2). Aminopeptidase PC has pI = 4.1. It hydrolyzes Leu-pNA optimally at pH 6.0 and at the optimum temperature 36-40 degrees C; in the presence of Ca2+ the enzyme is stable at pH 5.5-8.0. Aminopeptidase PC is activated by Ca2+, Mg2+, and Fe2+; it is completely inhibited by EDTA, o-phenanthroline, and bestatin. The enzyme contains four Zn atoms per molecule and is therefore a metalloaminopeptidase. The aminopeptidase PC can effectively cleave N-terminal Arg and Lys residues as well as Leu, Phe, and Met residues. Km and kcat values for hydrolysis of Leu-pNA were 0.075 mM and 0.19 sec-1 and for hydrolysis of Arg-pNA 0.078 mM and 0.48 sec-1, respectively. D-Amino acid residues cannot be cleaved. Thus, aminopeptidase PC of the Kamchatka crab has a mixed substrate specificity which is characteristic of some microbe aminopeptidases. Its N-terminal sequence ESVEIELPEGLSPLV is 46% coincident with that of yeast vacuolar aminopeptidase YSCA.     

Human placental dipeptidyl aminopeptidase III: hydrolysis of enkephalins and its stimulation by cobaltous ion

The degradation of enkephalin and related peptides by highly purified dipeptidyl aminopeptidase III (EC 3.4.14.4) was studied. The enzyme releases the N-terminal dipeptide units from substrates greater in length than the tetrapeptide. The enzyme exhibits an optimum of pH 7.5, Km of 81 microM and Vmax of 0.043 mumole/min for Leu-enkephalin. Its activity was markedly stimulated by Co2+, with both the Km and Vmax being increased. Among the enkephalin-related peptides examined, des-Tyr1-Leu-enkephalin was the most rapidly hydrolyzed with Co2+, but only slight stimulation was observed with Co2+.     

Proteolytic enzymes in Chlamydomonas I. A survey on the aminopeptidase pattern in asynchronous vegetative cells of Chlamydomonas reinhardii

The supernatant of a crude extract from vegetative cells ofChlamydomonas reinhardii contains three different types of aminopeptidases.They are similar in their substrate specificities to the relativealanine specific aminopeptidases, the relative leucine specificaminopeptidases and the specific proline iminopeptidases describedin many other systems. Relative alanine specific aminopeptidasewhich also cleaves N-terminal Lys and Leu residues has a molecularweight of 92,000 daltons and is inhibited by zinc and manganeseions.Relative leucine specific aminopeptidase shows high activitywith N-terminal Phe besides Leu, and is capable of cleavingTyr, Pro, and to a minor degree Ala. It has a molecular weightof 76,000 daltons. No effects on its activity were detectedin the presence of divalent cations or chelating agents. Theiminopeptidase specifically splits N-terminal Pro and has amolecular weight of about 255,000 daltons. All the enzymes showoptimal activity at pH 8.0–8.5. The two aminopeptidases can be separated from the iminopeptidaseby ammonium sulfate solubilization and from each other by subsequentfractionation on DEAE-cellulose. Relative leucine specific activityappeared as a single enzyme in all the fractionation techniquesused, but it gave two distinct bands when crude extracts wererun on native polyacrylamide gels. Therefore, this enzyme mayexist in multiple molecularforms. (Received October 17, 1978; )     

The proteolytic system of Penicillium roqueforti. V. -- Purification and properties of an alkaline aminopeptidase.

An alkaline aminopeptidase was isolated from the culture medium of Penicillium roqueforti. The enzyme was purified by ammonium sulfate precipitation, filtration on Bio-Gel P-100, chromatography on D.E.A.E.-cellulose and hydroxylapatite, filtration on Bio-Gel P-150 and electrofusing. The purified preparation was homogeneous on polyacrylamide gel electrophoresis at pH 8.5. The molecular weight of the enzyme was estimated to be about 35,000 daltons. The isoelectric point is 4.5. The optimum pH for L-leucine-p-nitroanilide hydrolysis is 8.0. At 35 degrees C the enzyme is stable between pH 6.0 and 7.0. Ethylenediamine tetraacetic acid and a sulfhydryl reagent (p-hydroxymercuribenzoate) inhibit the activity, but the enzyme is insensitive to diisopropylfluorophosphate. Hydrolysis of synthetic peptides shows that the enzyme releases apolar amino acids. Dipeptides are poorly hydrolyzed and Gly in penultimate or N-terminal position causes poor activity. The enzyme is able to cleave the N-terminal Arg-Pro bond of bradykinin.     

An aminopeptidase activity in bovine pituitary secretory vesicles that cleaves the N-terminal arginine from beta-lipotropin60-65

Secretory vesicles isolated from the neural and intermediate lobes of the bovine pituitary contained a membrane-bound aminopeptidase activity which cleaved arginine from beta-LPH60-65 (Arg-Tyr-Gly-Gly-Phe-Met) and Arg-MCA. Neither methionine enkephalin (Tyr-Gly-Gly-Phe-Met) nor Substance P, which has an N-terminal arginine followed by a proline, could serve as substrates for this aminopeptidase activity; nor could cathepsin B-like or chymotrypsin-like enzyme activities be detected in the vesicle preparations. Maximal enzyme activity was at pH 6.0, and the activity was inhibited by EDTA, stimulated by Co2+ and Zn2+, but was unaffected by leupeptin, pepstatin A, phenylmethylsulfonyl fluoride and p-chloromercuribenzenesulfonate, suggesting that the enzyme is a metalloaminopeptidase. The presence of this aminopeptidase activity in secretory vesicles suggests that it may be involved in peptide prohormone processing.     

Blood digestion in the mosquito, Anopheles stephensi Liston (Diptera: Culicidae): partial characterization and post-feeding activity of midgut aminopeptidases

Aminopeptidase activity was partially characterized from midguts of Anopheles stephensi Liston which had been dissected 30 h after blood feeding. In crude midgut homogenate supernatants the aminopeptidases showed optimum activity at pH 8.0 and preferentially hydrolyzed alanine- and leucine-terminal amino acid substrates. Methionine, proline, lysine, and arginine terminal substrates were hydrolysed, but not glutamic acid. Activity was stimulated by Mg2+, EDTA, and low Ca2+ concentrations, while Mn2+, Tris, 1,10 phenanthroline, and higher Ca2+ concentrations were inhibitory. Supernatants from midguts homogenized in 1% Triton X-100 showed a two-fold increase in activity. Differential centrifugation of midgut homogenates demonstrated 45% of the total activity in a putative microvillar pellet and 32% in a soluble fraction. More than 92% of the total activity was solubilized after homogenization in Triton X-100. Activity in homogenate supernatants was restricted to one major peak (Mr = 552,000) with a higher molecular weight shoulder. Three distinct peaks of aminopeptidase activity were observed following Triton X-100 treatment: a minor high molecular weight peak (Mr = 552,000), and two major peaks at Mr = 123,000 and Mr = 32,000 respectively. The activity of aminopeptidase increased after a blood meal, in parallel to the post-feeding changes in trypsin activity, indicating its important role in secondary digestion of blood meal proteins.     

Purification and characterization of a beta-galactosidase from peach (Prunus persica)

A beta-galactosidase (EC 3.2.1.23) from peach (Prunus persica cv Mibackdo) was purified and characterized. The purified peach beta-galactosidase was 42 kDa in molecular mass and showed high enzyme activity against a the beta-galactosidase substrate, rho-nitrophenyl-beta-D-galactopyranoside. The Km and Vmax values of the enzyme activity of the peach beta-galactosidase were 5.16 and 0.19 mM for rho-nitrophenyl-beta-D-galactopyranoside mM/h, respectively. The optimum pH of the enzyme activity was pH 3.0, but it was relatively stable from pH 3.0-10.0. The temperature optimum was 50 degrees C. The enzyme activities were not improved in the buffers that contained Ca2+, Cu2+, Zn2+, and Mg2+, which indicates that the purified peach beta-galactosidase did not require these cations as co-factors. However, the enzyme was completely inhibited by Hg2+. The purified protein was cross-reacted with an antibody against the persimmon fruit beta-galactosidase. A further comparison of the N-terminal amino acid sequence of the purified protein showed high homologies to those of beta-galactosidase in apple (87%), persimmon (80%), and tomato (87%). Therefore, enzymatic, immunological, and molecular evidences in this study indicate that the purified 42-kDa protein is a peach beta-galactosidase.     

Identification of a kallikrein-like latent serine protease in human erythrocyte membranes

We have discovered and characterized a kallikrein-like latent serine protease in intact human erythrocytes and ghosts. The enzyme is activatable by trypsin. The solubilized enzyme has esterolytic activity with a pH optimum of 9; but the membrane-associated activity increases almost linearly up to pH 10. The activated enzyme releases kinin from bovine low molecular weight kininogen. Enzyme activity is inhibited by TosLysCH2Cl , phenylmethylsulfonyl fluoride, aprotinin and amiloride, and weakly by soybean or lima bean trypsin inhibitor. It is inhibited by Co2+, Zn2+ and Mn2+ but is stimulated by Fe2+, deoxycholate and phospholipase A2. An erythrocyte membrane protein (Mr = 88,000) with an active site serine residue was identified with [14C]-diisopropylphosphorofluoridate labeling. Consistent with the finding of tryptic activation of the latent erythrocyte serine protease, trypsin treatment reduced the density of labeling of this protein and revealed a lower molecular weight form (Mr = 64,000). Possible relationships between the activity of this newly identified serine protease and events such as erythrocyte membrane ion fluxes might be of interest.     

Purification and partial characterization of an intracellular aminopeptidase from Streptococcus salivarius subsp. thermophilus strain ACA-DC 114.

An intracellular aminopeptidase from Streptococcus salivarius subsp. thermophilus strain ACA-DC 114, isolated from traditional Greek yoghurt, was purified by chromatography on DEAE-cellulose and Sephadex G-100. The enzyme had a molecular weight of 89,000. It was active over a pH range 4.5-9.5 and had optimum activity on L-lysyl-4-nitroanilide at pH 6.5 and 35 degrees C with Km = 1.80 mmol/l; above 55 degrees C the enzyme activity declined rapidly. The aminopeptidase was capable of degrading substrates by hydrolysis of the N-terminal amino acid; it had very low endopeptidase and no carboxypeptidase activity. The enzyme was strongly inactivated by EDTA. Serine and sulphydryl group reagents had no effect on enzyme activity.     

Cleavage of the Arg1-Pro2 bond of bradykinin by a human lung peptidase: isolation, characterization, and inhibition by several beta-lactam antibiotics

An aminopeptidase P (EC 3.4.11.9) that cleaves the Arg1-Pro2 bond of bradykinin has been isolated for the first time from human lung and purified 473-fold. The enzyme also catalyzes the cleavage of arginine from des-[Arg9]-bradykinin and the hydrolysis of several X-proline dipeptides including L-arginyl-L-proline, L-leucyl-L-proline, and L-alanyl-L-proline. Purified enzyme was routinely assayed (after initial identification with des-[Arg9]-bradykinin) with L-leucyl-L-proline. The molecular weight, in nondenaturing buffers, is 188,000 +/- 8500 Da. The pH optimum was 8.0 with arginyl-proline, and was 6.8 with leucyl-proline. Chelating agents do not inactivate the enzyme, but rather only remove loosely bound cations that stimulate the enzyme. Manganese is the principal cation that stimulates the enzyme. The enzyme is inhibited by several beta-lactam antibiotics, cephalexin and oxacillin being the most effective of those tested. The antibiotic inhibition is time and temperature dependent, and it is not fully reversible by exhaustive dialysis of the antibiotic-treated enzyme.     

Identification in Tetrahymena pyriformis of 3-hydroxy-3-methyl glutaryl coenzyme a lyase: its purification and properties

The major HMG-CoA utilizing enzyme activity in T. pyriformis has been determined to be HMG-CoA lyase. The enzyme was purified 32-fold to a specific activity of 431 units/mg from a mitochondrial fraction. Sephacryl S-200 chromatography gave an estimated molecular weight of 50,000 daltons for the HMG-CoA lyase. SDS gel electrophoresis revealed two bands stained by Coomassie Blue--a major band of 50,000 daltons and a minor band of 25,000 daltons. The latter is believed to be an impurity in the preparation. The enzyme has a pH optimum of 9.0, is stimulated slightly by sulfhydryl reagents, and requires a divalent cation for maximum activity. The KM for HMG-CoA is 15 microM.     

Isolation and characterization of an aminopeptidase from Lactobacillus acidophilus R-26

An intracellular aminopeptidase (alpha-aminoacyl-peptide hydrolase (cytosol), EC 3.4.11.1) isolated from cell extracts of Lactobacillus acidophilus R-26 was purified 634-fold to homogeneity. This enzyme, which was responsible for all of the N-terminal exopeptidase and amidase activities observed in crude extracts, had no detectable endopeptidase or esterase activity. Although a broad range of L-amino acid peptide, amide and p-nitroanilide derivatives possessing free alpha-amino termini are attacked, the enzyme favored substrates with hydrophobic N-terminal R groups. The native enzyme, which was found to be a tetramer of molecular weight 156000, contained 4 mol of tightly bound Zn2+. The catalytically inactive native zinc metalloenzyme was capable of being activated by either Zn2+, Co2+, Ni2+ or Mn2+. The shape of the log Vmax versus pH plot indicates that two active-center ionizable groups (pKES1 = 5.80; pKES2 = 8.00) may be involved in catalysis. Methylene-blue-sensitized photooxidation of the enzyme resulted in the complete loss of activity, while L-leucine, a competitive inhibitor, partially protected against this inactivation. Amino-acid analysis indicated that this photooxidative loss of activity corresponds to the modification of one histidine residue per monomer of protein.     

Sequential hydrolysis of proline-containing peptides with immobilized aminopeptidases

Proline-containing polypeptides are shown to be sequentially degraded by two aminopeptidases. Clostridial aminopeptidase (EC 3.4.11-) cleaves off any N-terminal amino acid residue including proline from polypeptide chains, but does not cleave the N-terminal secondary peptide bonds involving a prolyl nitrogen. Aminopeptidase P (EC 3.4.11.9) cleaves exclusively such secondary bonds. The two enzymes were immobilized by coupling them covalently to porous amino glass beads. Highly stable preparations were obtained with unchanged pH optimum and thermal stability. The applicability of clostridial aminopeptidase to sequence determination was demonstrated by the time-dependent hydrolysis of enkephalin and Substance P octapeptide. Sequential hydrolysis with the two immobilized enzymes was demonstrated with the proline-containing (Pro-Gly-Pro)10, [Asn1, Val5]angiotensin II, bradykinin, Substance P and tuftsin. Absence of endopeptidase activities was demonstrated by resistance of cytochrome c to hydrolysis and by the ordered release of amino acids during the sequential degradation by immobilized clostridial aminopeptidase and aminopeptidase P.     

Isolation and properties of leucine aminopeptidase from Aspergillus oryzae]

Homogenious leucine aminopeptidase is purified from "oryzine"--mixture of enzymes produced by surface culture of Asperigillus oryzae using treatment with activated characoal, followed by DEAE-cellulose and hydroxylapatite chromatographies, Biogel P-100 gel-filtration and polyacrylamide-gel electrophoresis. The enzyme has pH optimum 9.0 and the molecular weight 37500 as estimated by gil-filtration through Sephadex G-100 (superfine) and SDS-polyacrylamide gel electrophoresis. Leucine aminopeptidase from Asp. oryzae has a broad substrate specificity, therefore, cleaving with the highest rate the peptides carrying N-terminal leucine. The enzyme is completely inhibited with EDTA and beta-mercaptoethanol, and it is a metalloenzyme.     

Functional expression and characterization of the cytoplasmic aminopeptidase P of Caenorhabditis elegans.

Aminopeptidase P (AP-P; X-Pro aminopeptidase; EC 3.4.11.9) cleaves the N-terminal X-Pro bond of peptides and occurs in mammals as both cytosolic and plasma membrane forms, encoded by separate genes. In mammals, the plasma membrane AP-P can function as a kininase, but little is known about the physiological role of the cytosolic enzyme. The C. elegans genome contains a single gene encoding AP-P (W03G9.4), analysis of which predicts regions displaying high levels of amino-acid sequence homology between the predicted gene product and mammalian cytoplasmic AP-P, with the absolute conservation of key catalytic residues. The sequence of an EST (yk91g4), comprising the open reading frame of W03G9.4, confirmed the predicted genomic structure of the gene and the prediction that W03G9.4 codes for a nonsecreted protein with a molecular mass of 68 kDa. Nematodes transformed with a promoter reporter construct, W03G9.4:GFP, showed high levels of fluorescence in the intestine of larvae and adult hermaphrodites, indicating that the intestine is a major site of W03G9.4 expression. yk91g4 tagged with a hexahistidine and DLYDDDDK peptide epitope was expressed in Escherichia coli to yield, after affinity purification, a recombinant protein with a molecular mass of 71 kDa. The recombinant W03G9.4 removed the N-terminal amino acid from bradykinin (RPPGFSPFR), a Caenorhabditis elegans neuropeptide (KPSFVRFamide) and Lem Trp 1 (APSGFLGVRamide), but did not display activity towards angiotensin I (NRVYIHPFHL), des-Arg bradykinin and AF1 (KNEFIRFamide). The activity towards bradykinin was inhibited by EDTA and 1, 10 phenanthroline, as expected for a metalloenzyme, and also by apstatin (IC50, 1 microM), a selective inhibitor of mammalian AP-P. A Km of 45 microM and an optimum pH of 7-8 was observed with bradykinin as the substrate. The activity of the nematode AP-P, like its mammalian counterparts, was strongly influenced by metal ions, with Co2+, Mn2+ and Zn2+ all inhibiting the hydrolysis of bradykinin. We conclude that W03G9.4 codes for a cytoplasmic AP-P with very similar enzymatic properties to those of mammalian AP-P, and we suggest that the enzyme has a physiological role in the intracellular hydrolysis of proline-containing peptides absorbed from the lumen of the intestine.     

An Aminopeptidase from Mouse Brain Cytosol That Cleaves N-Terminal Acidic Amino Acid Residues

An aminopeptidase with specificity directed toward peptides with acidic N-terminal amino acid residues has been isolated from mouse brain cytosol. Purification by ion-exchange chromatography and gel filtration resulted in an enzyme that hydrolyzed aspartyl-phenylala-nine methyl ester at a rate of 13.2 μu,mol/min/mg protein at pH 7.5, an increase in specific activity of 1000-fold over that of brain homogenate. Its apparent molecular weight, determined by gel filtration, is ?450,000. Dipeptides with N-terminal aspartyl residues are cleaved preferentially to glutamic-containing analogs, and a neutral amino acid (or histidine) is necessary in the adjacent position. For pep-tides of the form aspartyl-X, relative activity was 100, 81, 71, 66, 19, or 0, where X was alanine, serine, leucine, phenylalanine, histidine, or proline, respectively. Tripep-tides were more rapidly hydrolyzed than dipeptides; however, activity tended to decline with increasing chain length. The acidic aminopeptidase can account for almost all of the activity of brain cytosol toward the N-terminal aspartyl residue of angiotensin II, aspartyl-phenylalanine methyl ester or aspartyl-alanine, and the N-terminal glu-tamyl residue of adrenocorticotropin(5-10). The enzyme was unaffected by bestatin or amastatin. It was inhibited by o-phenanthroline and EDTA. The latter effect could be reversed completely by Zn²⁺ and partially by Mn²⁺ or Mg²⁺; Co²⁺ and Fe²⁺ had no effect; Ca²⁺ was inhibitory. These properties distinguish the brain acidic aminopeptidase from aminopeptidase A isolated from human serum or pig kidney and the aspartyl aminopeptidase of dog kidney.