Aminopeptidases in the Acidic Fraction of the Yeast Autolysate

Two aminopeptidases, I and II, were found in the acidic fraction of the yeast autolysate, adsorbed on DEAE-cellose and DEAE-Sephadex A&50. Aminopeptidase I was purified as a single protein with a molecular weight of 200,000. The enzyme required Zn for its activity and hydrolyzed dipeptides, and a polypeptide (glucagon). It also hydrolyzed amides, naphthylamides and the p-nitroanilide of amino acids. The enzyme was strongly inhibited by sulfhydryl reagents. Aminopeptidase II seemed also to be a metal enzyme with a molecular weight of 34,000. The enzyme hydrolyzed the dipeptide and tetrapeptide but not leucine-p-nitroanilide.     

Occurrence and Regulatory Properties of Uridine Diphosphatase in Fully Expanded Leaves of Soybean (Glycine max Merr.) and Other Species

High activities (100-200 micromoles UDP hydrolyzed per milligram chlorophyll per hour) of uridine-5′ diphosphatase (UDPase) have been identified in extracts of fully expanded soybean (Glycine max Merr.) leaves. In desalted crude extracts, UDPase activity was strongly inhibited by low concentrations of Mg:ATP (I₅₀ = 0.3 millimolar). Two forms of the enzyme were resolved by gel filtration on Sephadex G-150. The higher molecular weight form (UDPase I, about 199 kilodaltons by gel filtration) retained ATP sensitivity (I₅₀ = 0.3 millimolar), whereas the major, lower molecular weight form (UDPase II, about 58 kilodaltons) was markedly less sensitive to ATP inhibition (I₅₀ = 2.7-3.0 millimolar). Subsequent purification of UDPase I by ion-exchange chromatography on DEAE cellulose produced a lower molecular weight enzyme (about 74 kilodaltons by gel filtration) that had reduced ATP sensitivity similar to UDPase II. Ion-exchange chromatography of UDPase II did not alter molecular weight or ATP sensitivity. UDPase II, after the DEAE-cellulose step, was specific for nucleoside diphosphates. Maximum reaction velocity decreased in the following sequence; UDP > GDP > CDP. ADP was not a substrate for the enzyme. The reaction catalyzed was hydrolysis of the terminal-P of UDP to form UMP. The enzyme was stimulated by Mg²⁺ and the pH optimum was centered between pH 6.5 and 7.0. In a survey of various species, soybean cultivars had highest activities of apparent UDPase and other species ranged in apparent activity from 0 to 30 micromoles hydrolyzed per milligram chlorophyll per hour.     

Purification and Properties of Leucine Aminopeptidase I–III from Aspergillus oryzae

An aminopeptidase from Aspergillus oryzae 460 was purified from the rivanol precipitable fraction. The partially purified enzyme was not homogeneous in disc electrophoresis, although symmetric profiles were obtained for enzyme protein and activity in Sephadex gel filtration. Its optimum pH is at pH 8.5 for l-leucyl-β-naphthylamide. The enzyme activity was inhibited by metal chelating agents and S-S dissociating agents, but not inhibited by SH reagents. The molecular weight of the enzyme was estimated to be about 26,500 by gel filtration. The enzyme was named leucine aminopeptidase I of Asp. oryzae 460, since it preferentially hydrolyzed oligopeptides that possess leucine as the amino terminal amino acid.     

Partial purification and characterization of dipeptidyl-aminopeptidase III fromDictyostelium discoideum

Dipeptidyl-aminopeptidase III was isolated from cells of the cellular slime moldDictyostelium discoideum in the culmination stage of development. The enzyme was purified 18-fold by precipitation with ammonium sulfate and gel filtration chromatography and was shown to have a molecular weight of 158,000 and a sharp pH optimum at pH 10.2 and to be inhibited by sulfhydryl reagents. The enzyme acted upon the artificial substratearginyl-arginyl-β-naphthylamide, producing arginyl-arginine andβ-naphthylamine but notarginyl-β-naphthylamide. Activity towardarginyl-arginyl-β-naphthylamide was strongly inhibited by physiological concentrations of angiotensin III and, to a lesser extent, by angiotensins I and II and other angiotensin-related peptides but not by enkephalin peptides. Several dipeptides known to inhibit mammalian dipeptidyl-aminopeptidase III also inhibited theDictyostelium enzyme. Incubation of the enzyme preparation with angiotensins resulted in their conversion into a complex mixture of products. Thus dipeptidyl-aminopeptidase III fromDictyostelium closely resembles the mammalian enzyme in many of its characteristics.     

A partial characterization of the cyclic nucleotide phosphodiesterases of Drosophila melanogaster

The cyclic nucleotide phosphodiesterases in crude homogenate, soluble material, and particulate preparations of adult Drosophila melanogaster flies, hydrolyze cyclic AMP with nonlinear kinetics. Cyclic GMP is hydrolyzed by the phosphodiesterases in crude homogenate and soluble material with linear kinetics. Physical separation techniques of gel filtration, velocity sedimentation, and ion-exchange chromatography reveal that Drosophila soluble fraction contains two major forms of cyclic nucleotide phosphodiesterase. Form I hydrolyzes both cyclic AMP and cyclic GMP. Inhibition experiments suggest that the hydrolysis of both cyclic nucleotides by Form I occurs at a single active site. The K_m's for hydrolysis of both substrates are about 4 μm. This form has a molecular weight of about 168,000 as estimated by gel nitration. Form II cyclic nucleotide phosphodiesterase is specific for cyclic AMP as substrate. Gel filtration indicates that this form has a molecular weight of about 68,000. The K_m for cyclic AMP is about 2 μm.     

Characterization and Subcellular Localization of Aminopeptidases in Senescing Barley Leaves

Four aminopeptidases (APs) were separated using native polyacrylamide gel electrophoresis of cell-free extracts and the stromal fractions of isolated chloroplasts prepared from primary barley (Hordeum vulgare L., var Numar) leaves. Activities were identified using a series of aminoacyl-β-naphthylamide derivatives as substrates. AP1, 2, and 3 were found in the stromal fraction of isolated chloroplasts with respective molecular masses of 66.7, 56.5, and 54.6 kilodaltons. AP4 was found only in the cytoplasmic fraction. No AP activity was found in vacuoles of these leaves. It was found that 50% of the l-Leu-β-naphthylamide and 25% of the l-Arg-β-naphthylamide activities were localized in the chloroplasts. Several AP activities were associated with the membranes of the thylakoid fraction of isolated chloroplasts. AP1, 2, and 4 reacted against a broad range of substrates, whereas AP3 hydrolyzed only l-Arg-β-naphthylamide. Only AP2 hydrolyzed l-Val-β-naphthylamide. Since AP2 and AP3 were the only ones reacting against Val-β-naphthylamide and Arg-β-naphthylamide, respectively, several protease inhibitors were tested against these substrates using a stromal fraction from isolated chloroplasts as the source of the two APs. Both APs were sensitive to both metallo and sulfhydryl type inhibitors. Although AP activity decreased as leaves senesced, no new APs appeared on gels during senescence and none disappeared.     

Purification and characterization of an aminopeptidase fromStreptococcus mitis ATCC 903

An aminopeptidase isolated from the cytoplasmic fraction of a cell extract ofStreptococcus mitis ATCC 903 was purified 330-fold by ion-exchange chromatography, gel filtration, and hydroxyapatite chromatography. The partially purified enzyme had a broad substrate specificity. Twelve aminoacyl-ß-naphthylamide substrates were hydrolyzed and also several di-, tri-, tetra-, and pentapeptides and bradykinin. The enzyme hydrolyzed arginine-ß-naphthylamide at the highest rate. Optimal conditions for activity were at pH 7.0–7.2 and at 37–40°C. The molecular weight of the enzyme was estimated to be 93,000. The enzyme was activated by Co²⁺ ions. Hg²⁺ inhibited the activity completely. SDS, EDTA, urea, and pCMB also inhibited activity. Inhibition by EDTA could be completely reversed by dialysis and addition of Co²⁺ ions. Reducing agents, sodium fluoride, and PMSF had no effect on the activity of the enzyme. The isoelectric point of the enzyme was at pH 4.3. High substrate concentrations inhibited activity. Substrate inhibition increased in the presence of high concentrations of Co²⁺ ions.     

Purification of glycosylphosphatidylinositol-anchoring aminopeptidase in from the plasma membrane of larval midgut epithelial cells of the silkworm,Bombyx mori

• 1.1. Aminopeptidase N was selectively released from larval midgut of silkworm, Bombyx mori, by phosphatidylinositol-specific phospholipase C, and purified to a homogeneous state by ion exchange, gel filtration. Con A-Sepharose and 4-aminobenzyl phosphonic acid-agarose column chromatographies.
• 2.2. The purified aminopeptidase N preparation showed 190.8 U/mg of specific activity. Its molecular weight was estimated to be around 100 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
• 3.3. Purified aminopeptidase N molecule preferentially hydrolyzed Leu-, Ala- and Met-p-nitroanilide as substrates. Especially, Leu-p-nitroanilide proved to be the best substrate for aminopeptidase N from larval midgut of silkworm.
• 4.4. By treatment with phosphatidylinositol-specific phospholipase C, two other hydrolases, alkaline phosphatase and alkaline phosphodiesterase I, were also solubilized from silkworm midgut.

     

Arylamidase Activity of Salmonella Species

Arylamidase activity in cell extracts of sonically cell treated suspensions of 23 Salmonella strains, including 12 strains of S. typhimurium, was investigated. All cultures hydrolyzed five of nine different neutral and basic substrates. Activity against aspartyl-, cystyl- histidinyl-, and isoleucyl-β-naphylamide was negligible. Alanyl-β-naphthylamide was the preferred substrate for the Salmonella species; however, specific activities ranged widely. Of several gram-negative organisms surveyed, all except Proteus vulgaris hydrolyzed alanyl-β-naphthylamide at the fastest rate. The most preferred substrate for the Proteus culture was glycyl-β-naphthylamide. No relationship could be shown between virulence and arylamidase activity for the Salmonella strains.     

Neutral peptidases in the stroma of pea chloroplasts

One endopeptidase (EP1) and at least three aminopeptidases (AP1, AP2, and AP3) were discovered in the stroma of chloroplasts isolated from pea seedlings (Pisum sativum L.), and purified over 100-fold. EP1 requires added Mg²⁺ or Ca²⁺ for activity, may have an additional tightly bound metal atom, and is inhibited by sulfhydryl reagents but not by serine residue-directed inhibitors. It is reversibly inhibited by dithiothreitol. Its specificity is for the bond between two adjacent Ala or Gly residues. Its molecular mass is 93 kilodaltons, estimated on a gel filtration column. Aminopeptidase activities were detected with the aid of different amino acyl-β-naphthylamides as substrates. They were resolved into at least three individual proteins by gel filtration and DEAE-cellulose chromatography, having apparent molecular masses of 269,000 (AP1), 84,000 (AP2), and 42,000 (AP3) daltons, respectively. Each has a unique specificity for substrates, with AP1 hydrolyzing only the Prolyl-β-naphthylamide. None of the APs require added divalent cations for activity, but the possibility of a tightly bound metal function was suggested in AP2 and AP3 (not AP1) from effects of inhibitors. A probable sulfhydryl residue function was indicated for all three, from inhibition by p-hydroxymercuribenzoate and Zn²⁺. All these peptidases had pH optima at 7.7.     

Purification and characterization of an alpha-glucosidase from germinating millet seeds

An α-glucosidase (α-d-glucoside glucohydrolase, EC 3.2.1.20) was isolated from germinating millet (Panicum miliaceum L.) seeds by a procedure that included ammonium sulfate fractionation, chromatography on CM-cellulofine/Fractogel EMD SO₃, Sephacryl S-200 HR and TSK gel Phenyl-5 PW, and preparative isoelectric focusing. The enzyme was homogenous by SDS-PAGE. The molecular weight of the enzyme was estimated to be 86,000 based on its mobility in SDS-PAGE and 80,000 based on gel filtration with TSKgel super SW 3000, which showed that it was composed of a single unit. The isoelectric point of the enzyme was 8.3. The enzyme readily hydrolyzed maltose, malto-oligosaccharides, and α-1,4-glucan, but hydrolyzed polysaccharides more rapidly than maltose. The K_m value decreased with an increase in the molecular weight of the substrate. The value for maltoheptaose was about 4-fold lower than that for maltose. The enzyme preferably hydrolyzed amylopectin in starch, but also readily hydrolyzed nigerose, which has an α-1,3-glucosidic linkage and exists as an abnormal linkage in the structure of starch. In particular, the enzyme readily hydrolyzed millet starch from germinating seeds that had been degraded to some extent.     

Substrate specificities of Cl- -activated arginine aminopeptidases from human and rat origin

The substrate specificities of four Cl^?-activated arginine aminopeptidases purified from the livers and inflammatory exudates of the rat, human fetal livers, and human erythrocytes were studied using peptides and N-l-aminoacyl-2-naphthylamides as substrates. With 2-naphthylamide substrates, these aminopeptidases showed similar substrate specificity; only the derivatives of Arg and Lys were measurably hydrolyzed. Di- and tripeptides with Arg or Lys as the N-terminal residue were readily split by the enzymes from the livers and inflammatory exudates of the rat and human fetal livers but oligopeptides were not hydrolyzed. Arg- and Lys-peptides were also hydrolyzed by the erythrocyte enzyme but this enzyme additionally split several other peptides, oligopeptides being hydrolyzed at internal bonds. The following properties were similar for all four arginine aminopeptidases: Dipeptides were preferred over tripeptides both in substrate binding and catalysis. The rat and human liver, rat exudate, and human erythrocyte enzymes revealed similar K_m values for the best substrates, the values increasing in the following order: ArgPhe, ArgTrp, ArgLys < ArgVal, ArgGly, Arg-2-naphthylamide < ArgGlyGly. The k_cat values were also similar for the four arginine aminopeptidases. Arg-2-naphthylamide was by far the most rapidly hydrolyzed substrate by all enzymes followed by ArgPhe and ArgTrp. With peptide substrates the highest Cl^? activation (10–20%) was found with ArgPhe and ArgTrp. With Arg-2-naphthylamide, however, the activating effect of 0.2 m Cl^? was severalfold. The hydrophobicity of the C-terminal residue of the substrate seemed to play an important role both in the Cl^? effect and substrate catalysis. Substrate binding, however, also depended on the charged groups of the substrate. Evidently Arg-2-naphthylamide and the peptides were hydrolyzed at the same active center but the mechanisms involved in the hydrolyses of chromogenic substrates and peptides may be different. It was also concluded that the less specific Cl^?-activated enzyme from human erythrocytes does not belong to the same group of Cl^?-activated arginine aminopeptidases that show a narrow substrate specificity.     

Purification and partial characterization of barley leucine aminopeptidase

A peptidase acting on Leu-Gly-Gly and Leu-Tyr at pH 8 to 10 was purified about 670-fold from germinated grains of barley (Hordeum vulgare L.). Gel electrophoretic analyses indicated a purity of about 90%. The purified enzyme is remarkably similar to mammalian leucine aminopeptidases (EC 3.4.1.1) both in chemical and in enzymatic properties. It has a sedimentation constant of 12.7S and a molecular weight of about 260,000. The enzyme has a high activity on leucine amide and di- and tripeptides with N-terminal leucine or methionine; leucyl-β-naphthylamide, in contrast, is hydrolyzed very slowly. The enzyme also liberates N-terminal amino acids from the insulin B chain. The pH optima for the hydrolysis of different substrates depend on the buffers used; highest reaction rates are generally obtained at pH 8.5 to 10.5. Mg²⁺ and Mn²⁺ ions stabilize (and probably activate) the enzyme. In contrast to mammalian leucine aminopeptidases, the barley enzyme is inactivated in the absence of reducing sulfydryl compounds.     

Leucine Aminopeptidase in Eggs of the Soybean Cyst Nematode Heterodera glycines

Supernatant from a sonicated macerate of eggs of Heterodera glycines hydrolyzed L-leucine β-naphthylamide and L-leucine 7-amido-4-methylcoumarin. Rate of substrate hydrolysis was influenced by pH and increased with the duration of incubation. A Michaelis-Menten constant of 0.15 mM was obtained. Rate of substrate hydrolysis was decreased by freezing egg supernatant for 26 days or heating above 60 C for 5 minutes. When egg supernatant was incubated with six different substrates, L-leucine β-naphthylamide was hydrolyzed most readily and L-valine β-naphthylamide the least readily. The rate of substrate hydrolysis by egg supernatant was not increased by pretreatment of eggs with 3 mM zinc chloride for up to 14 days.     

Effect of Cl− on the function of the Cl−-activated arginine aminopeptidase purified from human erythrocytes

The Cl^?-activated arginine aminopeptidase was purified from human erythrocytes using electrofocusing in granulated gel, gel permeation chromatography, and affinity chromatography. The purified enzyme showed a molecular weight of 105,000 ± 3000 and was homogenous according to several criteria. A subunit structure was revealed during sodium lauryl sulfate electrophoresis, the main form being of M_r 24,500 ± 1300. The enzyme was considered to be a tetramer consisting of four monomers of equal molecular weight. Cl^? affected the hydrolysis of peptides and synthetic substrates differently, the Cl^? activation being less marked with peptide substrates. The catalysis obeyed regular Michaelis-Menten kinetics and Cl^? affected both the K_m and V values. Arg-Phe and bradykinin showed no cooperativity in the hydrolysis of Arg-2-naphthylamide catalyzed by the Cl^?-activated arginine aminopeptidase. Cl^? affected the enzyme structure reflected by changes in the uv-absorption spectra in the presence and without added Cl^?.     

Purification and Characterization of an Aminopeptidase, LPAase 2, from Euonymus Leaves

An aminopeptidase, LPAase 2, from the leaves of Euonymus alatusf. ciliato-dentatus was purified about 240-fold by a combinationof DEAE-cellulose and Sephadex G-100 column chromatographies.The molecular weight of LPAase 2 was estimated to be about 62,000,and the optimum pH for the hydrolytic activity against leucinep-nitroanilide(LPA) was 7.6. LPAase 2 hydrolyzed LPA, leucine-rß-naphthylamide(leucine-NA), phenylalanine-NA and tyrosine-NA. It was inhibitedstrongly by p-chloromercuribenzoate (PCMB), iodoacetic acidand heavy metal ions, but was not affected by thiol compoundsand metal-chelating reagents. Therefore, a sulfhydryl groupcould be involved in the active site of LPAase 2. None of themetal ions tested promoted LPAase 2 activity. The propertiesof LPAase 2 were compared with those of aminopeptidases reportedfor other plants. (Received November 24, 1983; Accepted April 16, 1984)     

Peptidases from pig reproductive tract: purification and properties of aminopeptidases from uterine secretions, allantoic fluid, and amniotic fluid

In ovariectomized sows, aminopeptidase is secreted into the uterine lumen under the influence of progesterone. The enzyme also accumulates in allantoic and amniotic fluids of pregnant animals. We have purified the predominant form of this enzyme from uterine flushings, allantoic fluid, and amniotic fluid by the following steps: ammonium sulfate precipitation, Sepharose 6B chromatography, ion-exhange chromatography on diethylaminoethyl cellulose, and affinity chromatography usingl-leucylglycine immobilized on agarose. The overall procedure gave approximately 974-, 110-, and 230-fold purifications of the allantoic, uterine, and amniotic enzymes, respectively. The enzymes from all three sources are glycoproteins with pI's around 4 and molecular weights of about 480,000. They may be dissociated into six apparently identical subunits of molecular weight 80,000 as judged by sodium dodecyl sulfate gel electrophoresis. With l-leucyl-β-naphthylamide as substrate the pH optimum and apparent K_m value for each enzyme were 7.1 and 14 μm, respectively. However, the uterine and allantoic aminopeptidases exhibited V values of 0.35 μmol of substrate hydrolyzed/min/mg of protein, whereas the V for the amniotic enzyme was at least sixfold greater. The amniotic enzyme also differed from the other two in pH and temperature stability. The activity of all three enzymes was stimulated by Co²⁺ and inhibited by Cu²⁺, Fe³⁺, and chelating agents, while iodoacetate and mercaptoethanol had no effect on catalysis. The effect of Co²⁺ on the allantoic enzyme was investigated in further detail. The stimulation of peptidase activity by Co²⁺ was shown to be a complex process but consistent with Co²⁺ replacing another metal at the active site and at some other additional site on the enzyme. The function of the aminopeptidases in the pregnant uterus is unknown.     

Cathepsin C activity as related to some histochemical substrates

Summary Hog kidney homogenate was fractionated initially following the steps presented by De La Haba et al. (1959) for the purification of cathepsin C from beef spleen. The preparation was further fractionated by gel filtration using Sephadex G 200, starch gel and immunoelectrophoresis. The following enzymes were identified in the fractions obtained:Cathepsin C, which liberated ammonia from glycyl-phenylalanine amide and naphthylamine from glycyl-phenylalanyl--naphthylamide, pH optimum at 5.0, was activated by cysteine and inhibited by sulfhydryl reagents.An aminopeptidase, which liberated first of all glycine from glycyl-phenylalanine amide and glycyl-phenylalanyl--naphtylainide and after that ammonia and naphthylamine, respectively, hydrolysed numerous amino acid naphthylamides, pH optimum at 7.0–7.5, was activated by Co⁺⁺ and inhibited by EDTA.A peptidase, which liberated glycine from glycyl-phenylalanine amide and naphthylamide, did not hydrolyse amino acid naphthylamides, maximally active at neutral pH, was inhibited by EDTA.Several esterases, two in gel filtration, 5–6 in starch gel and immunoelectrophoresis, hydrolysing 5-bromoindoxyl acetate. The activities were sensitive to E 600.Both the studies on the characteristics of these activities as well as starch gel and immunoelectrophoretic studies support the view that none of the esterase activities is identical with cathepsin C. Cathepsin C, on the other hand, does not hydrolyse significantly 5-bromoindoxyl acetate and, consequently, this substrate can not be used to demonstrate cathepsin C histochemically.Glycyl-phenylalanyl--naphthylamide is recommended as a new sensitive chromogenic substrate for cathepsin C in biochemical studies in which the role of the aminopeptidase (s) can be adequately excluded but cannot be used in the histochemical demonstration of this enzyme.     

Aminopeptidase activity from germinated jojoba cotyledons

One major and two minor aminopeptidase activities from germinated jojoba (Simmondsia chinensis) cotyledon extracts were separated by ammonium sulfate precipitation and chromatofocusing. None of the activities were inhibited by 1,10 phenanthroline.

The major aminopeptidase, purified 260-fold, showed a pH optimum of 6.9 with leucine-p-nitroanilide as substrate, a molecular weight estimated at 14,200 by electrophoretic analysis, and an isoelectric point of 4.5 according to the chromatofocusing pattern. Activity was inhibited by p-chloromercuribenzoate, slightly stimulated by 1,10 phenanthroline and 2-mercaptoethanol, and not influenced by Mg²⁺ or diethyl pyrocarbonate. Inhibition by p-chloromercuribenzoate was prevented by the presence of cysteine in the assay. Leucine-p-nitroanilide and leucine-β-naphthylamide were the most rapidly hydrolyzed of 11 carboxy-terminal end blocked synthetic substrates tested. No activity on endopeptidase or carboxypeptidase specific substrates was detected. The major aminopeptidase showed activity on a saline soluble, jojoba seed protein preparation and we suggest a possible physiological role for the enzyme in the concerted degradation of globulin reserve proteins during cotyledon senescence.

     

Purification and properties of a low-molecular-weight α-mannosidase from Cellulomonas sp.

Cellulomonas sp. isolated from soil produces a high level of α-mannosidase (α-mannanase) inductively in culture fluid. The enzyme had two different molecular weight forms, and the properties of the high-molecular-weight form were reported previously (Takegawa, K. et al.: Biochim. Biophys. Acta, 991, 431–437, 1989). The low-molecular-weight α-mannosidase was purified to homogeneity by polyacrylamide gel electrophoresis. The molecular weight of the enzyme was over 150,000 by gel filtration. Unlike the high-molecular-weight form, the low-molecular-weight enzyme readily hydrolyzed α-1,2- and α-1,3-linked mannose chains.