Proline Iminopeptidase Research Articles

Protease and Peptidase Activity of Micrococcus Species

Nine different strains or species of micrococci were examined for intracellular protease and peptidase activity. Highest activities of leucine, alanine, and methionine aminopeptidases and proline iminopeptidase were found in Micrococcus sp. ATCC 398. Lysine aminopeptidase and dipeptidase activities were maximum in Micrococcus freudenreichii ATCC 407. Highest intracellular protease activity appeared in Micrococcus sp. LL3 isolated from Cheddar cheese. All strains except Micrococcus sp. ATCC 398 preferentially hydrolyzed the β-casein component during growth in skim milk at 30°C, and after longer incubation whole casein (αs1 and β) was completely hydrolyzed. Micrococcus sp. ATCC 398 failed to grow in skim milk under our experimental conditions. This pattern of proteolysis also was observed when skim milk was incubated with cell-free extracts of all micrococci tested as well as during growth of Micrococcus caseolyticus ATCC 13548 in ultrafiltered milk (five-fold concentration).

A novel assay method for aminopeptidase P and partial purification of two types of the enzyme in Escherichia coli.

A new assay for aminopeptidase P was established by coupling with proline iminopeptidase using Gly-Pro-chromogen (e.g. Gly-Pro-β-naphthylamide, Gly-Pro-p-nitroanilide, or .Gly-Pro-4-methyl coumarin amide) as the substrate. With each substrate, a linear relationship was established between the enzyme amounts and color development or fluorescence due to the chromogen released. This assay method did not suffer from interference by materials in culture broth. By using this assay method, aminopeptidase P was partially purified from Escherichia coli HB101 by chromatographies on DEAE-Sephadex and high performance liquid chromatography (HPLC). On the chromatogram with a DEAE-Sephadex column, two peaks of aminopeptidase P were observed and were named APP-I and APP-II. APP-I was further purified by HPLC using DEAE-5PW and Phenyl-5PW columns. Optimum pHs of APP-I and APP-II were 8.0 and 9.0, respectively. In contrast to APP-I which was stable around pH 10, APP-II was stable at pH 8 to 9. After incubation for 30min at pH 8.0, fifty percent of the remaining activity of APP-I and APP-II were observed at 60°C and 50°C. APP-I and APP-II were activated 3-fold by the addition of 5 and 30μM Mn2+. They were inhibited by EDTA, and reactivated by adding Mn2+. The molecular weights of APP-I and APP-II were 350, 000 and 210, 000, respectively. Each enzymes released the ammo terminal amino acid when proline is at the penultimate position. The velocity of hydrolysis by the enzymes was not significantly different for most X-Pro bonds (X=amino acid) of peptides except for Pro-Pro bond. APP-II hydrolyzed penta-(Pro-Pro-Gly) at a much higher rate than APP-I, suggesting the aminopeptidase P reported by Yaron and Mlynar (BBRC, 32, 658 (1968)) to be APP-II.

Proteolytic activity of crude cell-free extract of Lactobacillus casei and Lactobacillus plantarum.

Crude cell-free extracts of some strains of each L. casei and L. plantarum were assayed for their amino-, imino- and endopeptidase as well as the caseinolytic activity. L-alanine-, L-phenylalanine- and L-leucine-p-nitroanilide but not L-glutamic acid-p-nitroanilide, were hydrolyzed by all the strains indicating an amino-peptidase activity. The activity of proline iminopeptidase was very low compared to that of the aminopeptidase. L. casei could be distinguished from L. plantarum by its high endopeptidase activity against succinyl-phenylalanine- and glutaryl-phenylalanine-p-nitroanilide. The caseinolytic activity of cell-free extract of L. casei ATCC 393 was about one seventh the caseinolytic activity of intact cells, suggesting that the bulk of the cellular proteinase activity is located in the cell wall. It appears that a metallo aminopeptidase and a probable cysteine one are involved in the hydrolysis of amino acid-p-nitroanilide, whereas the endopeptidase activity appears to be related to a cysteine enzyme. Incubation of gels with L-leucine-p-nitroanilide after electrophoresis allowed the revealing of 2 zones of aminopeptidase activity in a strain of L. casei and only one in two others, but in L. plantarum it did not allow the revealing of any. The high proteolytic activities of L. casei compared to those of L. plantarum may explain its more frequent occurrence in cheese and its probable role in the ripening of many cheese varieties.

Dominance of iminopeptidase activity in the human oral bacteriumTreponema denticola ATCC 35405

Treponema denticola ATCC 35405, a human oral spirochete associated with periodontal disease, was shown to contain three enzymes (I, II, and III) with proline iminopeptidase activity. II and III were considered to be true iminopeptidases, whereas enzyme I was found to be a benzoylarginine peptidase with iminopeptidase activity. Enzyme III, the dominant proline iminopeptidase ofT. denticola in terms of its activity towardN-l-prolyl-2-naphthylamine, was considered to be a sulfhydryl peptidase: 0.167 μM p-chloromercuribenzoic acid totally inactivated the enzyme, and 1.0 mM dithiothreitol restored 92% of activity. The activity of this enzyme was not affected by metal chelators. Chemical modification of enzyme III suggests that tyrosyl (or histidyl) and carboxyl groups may be necessary for its activity. The hydrolysis ofN-l-prolyl-2-naphthylamine was found to be very characteristic ofT. denticola ATCC 35405; out of 24 differentN-l-aminoacyl-2-naphthylamines tested, only the proline derivative was hydrolyzed at a high rate. The substrate specificity of the enzymes discovered indicates that they may be important for the nutrition ofT. denticola. The iminopeptidase activity may be related to the pathogenicity of this organism in periodontal disease.

Benzoylarginine peptidase and iminopeptidase profiles ofTreponema denticola strains isolated from the human periodontal pocket

Seven clinical isolates and the ATCC strain 35405 ofTreponema denticola, obtained from human periodontal pockets, were studied for peptidase activity with several chromogenic compounds as substrates. The cell sonicates of all strains hydrolyzed phenylazobenzyloxycarbonyl-l-prolyl-l-leucyl-glycyl-l-prolyl-d-arginine (a collagenase substrate), azocasein, and the 2-naphthylamines ofl-proline,l-hydroxyproline,l-pyrrolidine, and benzoyl-l-arginine, but the rates of hydrolysis varied considerably from strain to strain. Fast protein liquid chromatography on gel and anion exchange columns revealed further biochemical differences between the strains. The ATCC strain consistently produced several proline iminopeptidases, whereas four of the clinical isolates yielded high and three yielded low iminopeptidase activity. The ATCC strain and six clinical isolates displayed high benzoylarginine peptidase activity. The use ofN-l-prolyl-2-naphthylamine as substrate revealed more differences between the strains than other substrates. The substrate specificity of the enzymes discovered suggests that they may be important for the nutrition of the organism or in the protection of the organism against chemical defense factors present in the gingival pocket.

Occurrence of an ascamycin dealanylating enzyme, Xc-aminopeptidase, in mammalian cell membranes and susceptibility to ascamycin.

An ascamycin dealanylating enzyme (Xc-aminopeptidase) has been isolated from Xanthomonas citri and characterized as a proline iminopeptidase of molecular weight of 38,000 (H. OSADA & K. ISONO, Biochem. J. 233: 459 approximately 463, 1986). Immunological studies have shown that all the mammalian cells tested possess a closely-related enzyme(s) on their cell membranes. This enzyme is more active in transformed cells than in nontransformed cells. A decreased ratio of ID50 (ascamycin/dealanylascamycin based on [35S]methionine uptake) in transformed cells compared with the nontransformed cell can be explained on the basis of the conversion of ascamycin to dealanylascamycin by the enzyme. It is suggested that ascamycin cannot be transported through mammalian cell membranes but dealanylascamycin can permeate; a similar situation exists in prokaryotic cells.

Presence of X-Prolyl-Dipeptidyl-Peptidase in Lactic Acid Bacteria

Prolyl-dipeptidyl-peptidase activity was detected in cell extracts of 21 lactic acid bacteria tested. Using disc electrophoresis and various substrates it was possible to distinguish it from proline iminopeptidase and proline endopeptidase. Generally the activity was high and was greater than that of proline iminopeptidase or proline endopeptidase at neutral pH and at 25°C.

Purification and Properties of a Proline Iminopeptidase from Propionibacterium acnes

Proline iminopeptidase was extracted from the cells of a strain of Propionibacterium acnes and purified. The molecular weight was estimated to be about 120,000 by SDS-polyacrylamide gel electrophoresis. The enzyme showed the highest activity at 50°C–55°C and its optimum pH was found at 7.5–8.0. The enzyme activity was inhibited by p-chloromercuribenzoate, indicating that this peptidase is a SH-enzyme. Especially prolyl-glycyl-glycine but also prolyl-proline bonds were hydrolyzed by this enzyme, glycyl-proline was not split.

Proline iminopeptidase from Bacillus coagulans: purification and enzymatic properties.

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

Proline iminopeptidase: chromatographic demonstration of a single form in human cultured skin fibroblasts, liver and kidney using a fluorimetric assay

Proline iminopeptidase: chromatographic demonstration of a single form in human cultured skin fibroblasts, liver and kidney using a fluorimetric assay

The purification and characterization of a proline dipeptidase from guinea pig brain.

A proline dipeptidase (EC 3.4.13.9) from guinea pig brain was purified to over 90% homogeneity by a combination of ammonium sulfate fractionation, DEAE-cellulose chromatography, calcium phosphate-cellulose chromatography, chromatofocusing, and gel filtration on Sephadex G-200. A purification factor of 2718-fold was obtained with a yield of 7%. The purified enzyme was found to have an apparent molecular weight of 132,000 and to consist of two dissimilar subunits of molecular weights 64,000 and 68,000. The substrate specificity of the enzyme is not that of a strict proline dipeptidase. Although it preferentially hydrolyzes proline dipeptides (Leu-Pro) it also hydrolyzes prolyl dipeptides (Pro-Leu) and dipeptides not containing proline (Leu-Leu). The purified enzyme preparation exhibited weak aminoacylproline aminopeptidase activity against Arg-Pro-Pro but it did not exhibit any post-proline dipeptidyl aminopeptidase, post-proline cleaving endopeptidase, proline iminopeptidase, prolyl carboxypeptidase or carboxypeptidase P activities when tested with a large variety of peptides and arylamides. With all of the proline and prolyl dipeptides examined the enzyme exhibited biphasic kinetics (two distinct slopes on Lineweaver-Burk plots). However, with Leu-Leu as substrate normal Michaelis-Menten kinetics were obeyed.

Proline Iminopeptidase from Bacillus megaterium: Purification and Characterization1

Proline iminopeptidase [EC 3.4.11.5] was purified about 1,700-fold from cell free extract of Bacillus megaterium by a series of column chromatographies on DEAE-Toyopearl, PCMB-T-Sepharose and hydroxyapatite, and gel filtration on Toyopearl FW-55. The purified enzyme still contained a minor contaminant as judged by disc gel electrophoresis. The enzyme was most active at pH 7.0 with Pro-beta-naphthylamide (Pro-2-NNap) as the substrate, and hydrolyzed Pro-X (X = amino acid, peptide, amide, and arylamide) bonds when the proline residue was at the amino terminal. The enzyme was completely inactivated by p-chloromercuribenzoate (PCMB), but was not inhibited by metal chelators, diisopropylphosphorofluoridate (DFP) and phenylmethanesulfonyl fluoride (PMSF). The enzyme inactivated with PCMB was reactivated by adding 2-mercaptoethanol. From this result and the chromatographic profile on PCMB-T-Sepharose, the enzyme seems to be a sulfhydryl enzyme. The isoelectric point of the enzyme was 4.0. The molecular weight of the enzyme was estimated to be 58,000 by gel filtration on Toyopearl and 60,000 by sodium dodecyl sulfate (SDS) gel electrophoresis, suggesting that the enzyme is a monomer.

Proline iminopeptidase from apricot seeds. Part II. Substrate specificity of a proline iminopeptidase from apricot seeds.

Proline iminopeptidase from apricot seeds. Part II. Substrate specificity of a proline iminopeptidase from apricot seeds.

Purification and properties of a proline iminopeptidase from apricot seeds.

A proline iminopeptidase was purified about 18,000-fold from apricot seeds (Prunus armeniaca LINN.) by a five-step procedure comprised of extraction from seeds, ammonium sulfate fractionation, DEAE-cellulose chromatography, CM-Sepharose chromatography, and rechromatography on CM-Sepharose. The purified enzyme had a molecular weight of 220,000 by gel filtration and 55,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This indicates that the native enzyme may be composed of four identical subunits. The isoelectric point was 6.2 as determined by gel electrofocusing. The pH optimum for L-proline beta-naphthylamide was between pH 7.5 and 8.0, and the enzyme was stable in the pH 6.5-to-8.0 region and up to 40 degrees C. The enzyme was specific for L-proline beta-naphthylamide among various amino acid beta-naphthylamides, and it also hydrolzyed L-prolylglycine and L-prolylglycylglycine. The enzyme was strongly inhibited by p-chloromercuribenzoate, 5,5'-dithiobis(2-nitrobenzoic acid), N-ethylmaleimide, and heavy metal ions, but was not activated significantly by thiol compounds. Moreover, the enzyme was inactivated by diethyl pyrocarbonate, p-bromophenacyl bromide, and photooxidation, but was not affected by diisopropyl fluorophosphate, phenylmethanesulfonyl fluoride, bestatin, puromycin, or metal chelating agents. No activation of the enzyme was observed on addition of metal ions. These results suggest that the enzyme is not classifiable as a metalloenzyme, and that cysteine and histidine residues may participate in the enzyme activity.

Biochemical markers of bone turnover in paget's disease

The two most commonly used biochemical markers of bone turnover are the serum alkaline phosphatase and the urinary excretion of peptide-bound hydroxyprollne, both of which are increased in Paget's disease. Serum alkaline phosphatase is assumed to be derived from osteoblosts during the process of bone formation, whereas small peptides containing hydroxyproline are excreted in the urine following the degradation of bone collagen. The alkaline phosphatase is probably the more useful measurement for diagnosis and for following response to treatment, whereas hydroxyproline, although very sensitive, presents technical difficulties in collection and measurement. Several other biochemical changes in Paget s disease indicate abnormal bone metabolism. These include increased urinary excretion of hydroxylysine and its glycosides derived from collagen, as well as the release into the circulation and subsequent urinary excretion of fragments of pro-collagen indicative of increased collagen formation. Proteins specific to bone, such as osteocalcin, are increased in serum, as are various enzymes possibly derived from bone cells, including acid phosphatase and proline iminopeptidase. Treatment of Paget's disease results in a fall in urinary hydroxyproline before alkaline phosphatase. This indicates that drug treatment, whether with diphosphonates, calchonin or mithramycin, has a primary action to Inhibit bone resorption, with a subsequent adaptive reduction fn bone formation rate.

Proline specific endo- and exopeptidases.

Peptidases which are specific for proline residues have been described and include endopeptidases (post-proline cleaving enzyme and proline specific endopeptidase), N-terminal exopeptidases (post-proline dipeptidyl aminopeptidase, proline iminopeptidase, aminopeptidase P), C-terminal exopeptidases (prolylcarboxypeptidase, and carboxypeptidase P) and dipeptidases (prolyl dipeptidase and proline dipeptidase). The properties, distinguishing charcteristics, and possible significance of these proline specific endo- and exopeptidases are discussed. In addition, reference is made to a series of enzymes which can hydrolyze proline containing peptide bonds, but which are not specific for proline.

Effect of membrane perturbing treatments on the membrane-bound peptidases ofStreptococcus cremorisHP

SummaryThe effects of solubilization, treatment with organic solvents and storage under alkaline conditions on membrane-associated peptidases of intact cells ofStreptococcus cremorisHP were studied. Differences in the response of the peptidase activities towards these membrane perturbing treatments were observed. Pyrrolidonecarboxylylpeptidase (PCP) and an endopeptidase (P50) showed 50% irreversible inhibition at the same concentration of each solvent tested. An amino- and proline iminopeptidase activity and the endopeptidase P37were in this respect much more sensitive to the action of the solvents. Within a homologous series of n-alkanols irreversible inhibition of PCP showed a dependence on the hydrophobicity of the solvent molecules. Only P37activity was increased considerably upon solubilization of the enzyme. Similar levels of activation were found upon treatment of cells with 3% (v/v) n-butanol at 25 °C or storage at 30 °C at an alkaline pH. Optimal activity of P50during n-butanol treatment was at 25 °C using a concentration of 5% (v/v), but no activation was observed upon solubilization. The results are discussed in terms of enzyme–lipid interaction and accessibility of the enzymes in situ. It is concluded that the enzymes apparently occupy different positions within the membrane although they may together constitute a functional peptide-hydrolysing unit.

Isolation of aminopeptidases fromHistoplasma capsulatum

Two aminopeptidases (arylamidases) were isolated and partially purified from Histoplasma capsulatum. The larger molecular weight enzyme was a proline iminopeptidase and hydrolyzed primarily a synthetic substrate, L-prolyl-beta-napthylamide. The other aminopeptidase was less substrate specific and hydrolyzed rapidly the following amino acid beta-napthylamides (beta NA): L-arginyl-beta NA greater than L-lysyl-beta NA greater than -L-4-methoxy-leucyl-beta NA greater than L-leucyl-beta NA greater than L-phenylalanyl-beta NA greater than L-alanyl-beta NA. The proline iminopeptidase was purified 1420 fold while the leucine aminopeptidase was purified 650 fold with good recovery.

Serum levels of proline imino-peptidase in normal adults and children

Serum proline imino-peptidase activities in 98 normal individuals aged 1 month to 75 years are examined. Mean enzyme activity in sera of individuals ages less than 1 year is 66.6 U; 2 to 6 years, 46.2 U; 7 to 11 years, 52.8 U; 12 to 16 years, 48.8 U; 17 to 19 years, 34.1 U; 20 to 29 years, 28.8 U; 30 to 39 years, 30.5 U; 40 to 59 years, 35.6 U; and more than 60 years, 31.6 U. Serum enzyme activities were markedly elevated in infants aged less than 1 year, then decreased with age up to 20 years. Serum proline imino-peptidase activity in 44 normal adults aged 20 to 59 years ranged from 19.9 to 43.1 U and averaged 31.5 ± 5.8 U. There is no statistical difference in serum activities in normal adults between males and females.

Serum proline imino-peptidase activity in normal adult subjects and in patients with paget's disease of bone

The activity of proline imino-peptidase (EC 3.4.1.4) may provide a measure of collagen degradation, and a method for its estimation in human serum is described. The mean value of this enzyme activity in 48 normal adults of 4.7 mU/1 (S.D. 0.9) did not change with sex, age or dietary collagen. In 8 patients with liver disease associated with elevated levels of plasma alkaline phosphatase activity the mean value was normal (4.5 mU/1, S.D. 1.5). However, in 25 patients with untreated Paget's disease of bone, values were significantly increased (mean 7.4 mU/1; S.D. 2.8; p < 0.001) and were positively correlated with plasma alkaline phosphatase activity ( r = 0.75; p < 0.001) and with urine total hydroxyproline excretion ( r = 0.68; p < 0.001). In patients given disodium ethane-1-hydroxy-1, 1-diphosphonate for 3–6 months, serum proline imino-peptidase activity decreased to normal values.