Leucine P-nitroanilide Research Articles

Proteases influence colony aggregation behavior in Vibrio cholerae

Aggregation behavior provides bacteria protection from harsh environments and threats to survival. Two uncharacterized proteases, LapX and Lap, are important for Vibrio cholerae liquid-based aggregation. Here, we determined that LapX is a serine protease with a preference for cleavage after glutamate and glutamine residues in the P1 position, which processes a physiologically based peptide substrate with a catalytic efficiency of 180± 80M-1s-1. The activity with a LapX substrate identified by a multiplex substrate profiling by mass spectrometry screen was 590± 20M-1s-1. Lap shares high sequence identity with an aminopeptidase (termed VpAP) from Vibrio proteolyticus and contains an inhibitory bacterial prepeptidase C-terminal domain that, when eliminated, increases catalytic efficiency on leucine p-nitroanilide nearly four-fold from 5.4± 4.1×104M-1s-1 to 20.3± 4.3×104M-1s-1. We demonstrate that LapX processes Lap to its mature form and thus amplifies Lap activity. The increase is approximately eighteen-fold for full-length Lap (95.7± 5.6×104M-1s-1) and six-fold for Lap lacking the prepeptidase C-terminal domain (11.3± 1.9×105M-1s-1). In addition, substrate profiling reveals preferences for these two proteases that could inform invivo function. Furthermore, purified LapX and Lap restore the timing of the V.cholerae aggregation program to a mutant lacking the lapX and lap genes. Both proteases must be present to restore WT timing, and thus they appear to act sequentially: LapX acts on Lap, and Lap acts on the substrate involved in aggregation.

Characterization of leucine amino peptidase from Streptomyces gedanensis and its applications for protein hydrolysis

The aim of this work was to purify and characterize the extra-cellular leucine amino peptidase (LAP) from Streptomyces gedanensis and also study its applications for protein hydrolysis. The enzyme was purified to homogeneity by ammonium sulfate fractionation and sequential chromatography steps. LAP appeared to be a monomeric enzyme with a molecular weight of ∼75 kDa determined by sodium dodecyl sulfate poly acryl amide gel electrophoresis (SDS-PAGE). The enzyme preferentially hydrolyzed leucine p-nitroanilide followed by Met, Phe, Lys and Arg derivatives. Kinetic studies on the purified enzyme confirmed that it can hydrolyze peptide as well as ester substrates at comparable rates. This amino peptidase was highly resistant to different concentrations of various organic solvents. The characteristics of this amino peptidase, including thermo stability, organic solvent resistance, its activity against various substrates, and also it showed esterase and peptidase activity at comparable rates; identified this amino peptidase as a novel one. The specificity towards aromatic and hydrophobic amino acid residues, the solvent-resistance and thermo stability make this amino peptidase could offer interesting possibilities for various industrial applications including debittering of protein hydrolysates, peptide and ester synthesis.

Changes and induction of aminopeptidase activities in response to pathogen infection during germination of pigeonpea (Cajanas cajan) seeds

Aminopeptidases play important role in the mobilization of storage proteins at the cotyledon during seed germination. It is often referred as inducible component of defense against herbivore attack. However the role of aminopeptidase in response to pathogen attack in germinating seeds is remained to be unknown. An attempt was made to analyze change in the aminopeptidase (EC 3.4.11.1) activity during germination of pigeonpea (Cajanus cajan L.) seeds by infecting the seeds with fungi. Two aminopeptidase activity bands (AP1 and AP2) were detected in control as well as infected pigeonpea seeds. During latter stages of germination in control seeds, AP1 activity was replaced by AP2 activity. However AP1 activity was significantly induced in germinating seeds infected with Fusarium oxysporum f.sp. ciceri and Aspergillus niger var. niger. The estimated molecular weights of AP1 and AP2 were ∼97 and 42.8kDa respectively. The induced enzyme was purified up to 30 fold by gel filtration chromatography. The purified enzyme was preferentially cleaved leucine p-nitroanilide than alanine p-nitroanilide. The enzyme was strongly inhibited by bestatin and 1,10-phenanthroline. Almost 50% of enzyme activity was inhibited by ethylene diamine tetra acetate. The purified enzyme showed broad pH optima ranging from pH 6.0 to 9.0 and optimum at pH 8.5. The induction of aminopeptidase activity during pigeonpea seed germination and in response to pathogen attack indicates significant involvement of these enzymes in primary as well as secondary metabolism of the seeds. These findings could be helpful to further dissect defensive role of aminopeptidases in seed germination which is an important event in plant's life.

Partial purification and characterization of Helicoverpa armigera (Lepidoptera: Noctuidae) active aminopeptidase secreted in midgut

We report the partial purification to apparent homogeneity of a soluble aminopeptidase (EC 3.4.11.1) from midgut of Helicoverpa armigera larvae, which preferentially degraded Leucine p-nitroanilide (LpNA). After midgut isolation, extraction and precipitation of soluble proteins with acetone, proteins were purified in two consecutive steps including gel filtration and ion-exchange chromatographies. Aminopeptidase activity was increased 8.95 fold after gel filtration chromatography. The purified enzyme appeared as single band with a molecular mass of approximately 112 kDa in SDS-PAGE, with a pH optimum of 7.0. Zymogram analysis revealed two enzymatically active proteinases using LpNA as substrate. The optimal temperature of aminopeptidase activity was 50-60 degrees C. The enzyme was characterized as metalloprotease as it was strongly inhibited by 1,10 phenanthroline. Strong inhibition was also being observed using the specific aminopeptidase inhibitor bestatin. Heavy metal ions, EDTA and cysteine strongly inhibited the enzyme, while Ca(+2), Mn(+2) and Mg(+2) somewhat stimulated aminopeptidase activity. Besides LpNA, the purified aminopeptidase also cleaved with decreasing activity ApNA, VpNA and BApNA. Study could be helpful to understand the mechanism of action of N-terminal degrading enzymes and also important is to further study the differential interaction of Bacillus thuringiensis cry insecticidal toxin with midgut receptor of insects.

Purification and properties of major midgut leucyl aminopeptidase of Morimus funereus (Coleoptera, Cerambycidae) larvae

The major leucyl aminopeptidase (LAP) from the midgut of Morimus funereus larvae was purified and characterised. Specific LAP activity was increased 292-fold by purification of the crude midgut extract. The purified enzyme had a pH optimum of 7.5 (optimum pH range 7.0-8.5) and preferentially hydrolysed p-nitroanilides containing hydrophobic amino acids in the active site, with the highest V(max)/K(M) ratio for leucine-p-nitroanilide (LpNA). Among a number of inhibitors tested, the most efficient were 1,10-phenanthroline having a K(i) value of 0.12 mM and cysteine with K(i) value of 0.31 mM, while EGTA stimulated LAP activity. Zn(2+), Mg(2+) and Mn(2+) all showed bi-modal effects on LAP activity (activated at low concentrations and inhibited at high concentrations). The purified LAP (after gel filtration on Superose 6 column) had molecular mass of 400 kDa with an isoelectric point of 6.2. Sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed one band of 67 kDa, suggesting that the enzyme is a hexamer. Six peptide sequences from protein band were obtained using ESI/MS-MS analysis. Comparison of the obtained peptide sequences with the EMBL-EBI sequence analysis toolbox and the BLASTP database showed a high degree of identity with other insect aminopeptidases.

Detection and quantification of leucyl aminopeptidase after native electrophoresis using leucine-p-nitroanilide

A general method for detecting leucyl aminopeptidase activity after native polyacrylamide gel electrophoresis (PAGE) in situ is described. The method is based on diazotization of p-nitroaniline, liberated in the polyacrylamide gel by leucyl aminopeptidase action on leucine-p-nitroanilide (LpNA) and subsequent coupling with a chromogen, 1-naphthylamine, until a pink azo dye product at the position of enzyme activity is obtained. A possible use of this technique for leucyl aminopeptidase detection and quantification is indicated. This method was found to be reproducible with the coefficient of variation below 15% for a 32-fold range, while the colored area of enzyme activity was in linear dependence to enzyme activity. Applications of this method with some other aminoacyl-p-nitroanilides and for detection of kidney bean leucyl aminopeptidase isoforms are demonstrated.

Purification and properties of an aminopeptidase from the mid-gut gland of scallop ( Patinopecten yessoensis)

An aminopeptidase was isolated from the mid-gut gland of Patinopecten yessoensis. The enzyme was purified from an acetone-dried preparation by extracting, ammonium sulfate precipitation, Hi-Load Q column chromatography, isoelectric focusing, and POROS HP2 and HQ column chromatography. The molecular weight of the enzyme was estimated to be 61 kDa by SDS-polyacrylamide gel electrophoresis and 59 kDa by gel permeation chromatography. The isoelectric point of the enzyme was 5.2 and the optimum pH was 7.0 toward leucine p-nitroanilide (Leu- pNA). The enzyme was inhibited by o-phenanthroline. The activity of the enzyme treated with o-phenanthroline was completely recovered by adding excess Zn 2+. Relative hydrolysis rates of amino acid- pNAs and amino acid-4-methylcoumaryl-7-amides (amino acid-MCAs) indicated that the enzyme preferred substrates having Ala or Met as an amino acid residue. The enzyme had a K m of 32.2 μM and k cat of 29.5 s −1 with Ala- pNA and a K m of 11.1 μM and k cat of 9.49 s −1 with Ala-MCA. The enzyme sequentially liberated amino acids from the amino-termini of Ala–Phe–Tyr–Glu.

Purification, characterization, and genetic analysis of a leucine aminopeptidase from Aspergillus sojae

Extracellular leucine aminopeptidase (LAP) from Aspergillus sojae was purified to protein homogeneity by sequential fast protein liquid chromatography steps. LAP had an apparent molecular mass of 37 kDa, of which approximately 3% was contributed by N-glycosylated carbohydrate. The purified enzyme was most active at pH 9 and 70 °C for 30 min. The enzyme preferentially hydrolyzed leucine p-nitroanilide followed by Phe, Lys, and Arg derivatives. The LAP activity was strongly inhibited by metal-chelating agents, and was largely restored by divalent cations like Zn 2+ and Co 2+. The lap gene and its corresponding cDNA fragment of the A. sojae were cloned using degenerated primers derived from internal amino acid sequences of the purified enzyme. lap is interrupted by three introns and is transcribed in a 1.3-kb mRNA that encodes a 377-amino-acid protein with a calculated molecular mass of 41.061 kDa. The mature LAP is preceded by a leader peptide of 77 amino acids, predicted to include an 18-amino-acid signal peptide and an extra sequence of 59 amino acids. Two putative N-glycosylation sites are identified in Asn-87 and Asn-288. Southern blot analysis suggested that lap is a single-copy gene in the A. sojae genome. The deduced amino acid sequence of A. sojae LAP shares only 11–33.1% identity with those of LAPs from 18 organisms.

Purification and characterization of aminopeptidase B from Escherichia coli K-12.

Aminopeptidase B, which is one of the four cysteinylglycinases of Escherichia coli K-12, was purified to electrophoretic homogeneity and its enzymatic characteristics were observed. Aminopeptidase B was activated by various divalent cations such as Ni2+, Mn2+, Co2+, and Cd2+, and lost its activity completely on dialysis against EDTA. This indicates that aminopeptidsase B is a metallopeptidase. It was stabilized against heat in the presence of Mn2+ or Co2+. The activity of aminopeptidase B, which was saturated with one of above divalent cations, was enhanced on the addition of a very small amount of a second divalent cation. Alpha-glutamyl p-nitroanilide, leucine p-nitroanilide, and methionine p-nitroanilide were good substrates for aminopeptidase B, while native peptides, cysteinylglycine and leucylglycine, were far better substrates. The kcat/Km for cysteinylglycine was much bigger than those for leucylglycine or leucine p-nitroanilide.

Characterization of proteolytic activities of rumen bacterial isolates from forage-fed cattle.

The proteolytic activities of eight strains of ruminal bacteria isolated from New Zealand cattle were characterized with respect to their cellular location, response to proteinase inhibitors and hydrolysis of artificial proteinase substrates. The Streptococcus bovis strains had predominantly cell-bound activity, which included a mixture of serine and cysteine-type proteinases which had high activity against leucine p-nitroanilide (LPNA). The Eubacterium strains had a mainly cell-associated activity with serine and metallo-type proteinases which showed high activity against the chymotrypsin substrate, N-succinyl alanine alanine phenylalanine proline p-nitroanilide (NSAAPPPNA) and some LPNA activity. A Butyrivibrio strain, C211, had a cell-bound mixture of cysteine and metallo-proteinase activities and strongly hydrolysed NSAAPPPNA and LPNA while the high activity Butyrivibrio-like strain, B316, had a cell-bound, mainly serine proteinase activity which strongly hydrolysed NSAAPPPNA. A Prevotella-like strain, C21a, had a mixture of cysteine, serine and metallo-proteinase activities which were cell-bound and hydrolysed LPNA. The activities of these strains did not match those of the bacterial fraction of rumen fluid, which contained activities mainly of the cysteine type with specificity towards the substrate N-succinyl phenylalanine p-nitroanilide. The contribution of these strains to proteolysis in the rumen is discussed.

Occurrence of Proteoyltic and Lectin-Degrading Activities in the Bark of Robinia pseudoacacia

Various endo- and exopeptidase activities were examined in an extract of the bark of Robinia pseudoacacia L. The crude extract hydrolysed azocasein, gelatin and benzyloxycarbonyl-Phe-Ala (Z-Phe-Ala) at acidic pH and leucine p-nitroanilide (LPA), alanine p-nitroanilide (APA) and N-benzoyl-arginine ρ-nitroanilide (BAPA) at neutral to weakly alkaline pH. The extract also hydrolysed lectin, which is one of the predominant bark proteins, to smaller polypeptides at acidic pH. The proteolytic activities against azocasein and Z-Phe-Ala remained at relatively high levels during the season when the amount of lectin in the bark decreased rapidly.

Purification and characterization of an aminopeptidase from Lactobacillus casei subsp. rhamnosus S93.

An aminopeptidase of broad specificity was extracted by cell lysis of a selected strain of Lactobacillus casei subsp. rhamnosus during the late exponential phase. The enzyme was purified 195-fold from crude extract by using an f.p.l.c. system. Native and SDS/PAGE of the purified enzyme showed a single protein band of 89 kDa. The maximum aminopeptidase activity was observed at pH 7.0 and 39 degrees C. The enzyme hydrolysed a range of nitroanilide-substituted amino acids, as well as dipeptides, and accounted for most of the aminopeptidase activity found in cell-free extracts. The enzyme activity was inhibited by metal chelators such as EDTA and 1,10-phenanthroline. Cobalt ions only stimulated aminopeptidase activity and were also able to re-activate the enzyme previously inhibited by metal chelators. The Km and Vmax. values of the aminopeptidase for leucine p-nitroanilide were 0.06 mM and 12.6 mmol/min per mg of protein respectively. This enzyme was stable over the pH range of 5-9 and below 45 degrees C.

Aminopeptidase a from Rhynchosciara americana (Diptera) larval midguts: Properties and midgut distribution

AbstractL‐aspartic acid α‐(β‐naphthylamide) (AspβNA) hydrolase activity is restricted mostly to the midgut caeca of Rhynchosciara americana larvae. The membrane‐bound activity is solubilized in detergent and, after electrophoretic separation, proved to be identical to leucine p‐nitroanilide (LpNA) hydrolases previously described. Differential centrifugation of midgut caeca homogenates, followed by assays of enzyme markers and aminopeptidase, suggests that the soluble AspβNA hydrolase is associated with the cell glycocalyx. Soluble aminopeptidases from R. americana midgut caeca are resolved into three fractions by gel electrophoresis. The slow migrating fraction hydrolyzes AspβNA well and displays a low activity on LpNA and proline β‐naphthylamide (ProβNA). Thus, this enzyme is an aminopeptidase A (EC 3.4.11.7). It has a pH optimum of 7.5, Mr 117,000 (gel filtration), and is competitively inhibited by aspartate hydroxamate (Ki 0.1 mM). Nevertheless, this enzyme, in contrast to the vertebrate enzyme, is not activated by calcium ions. The aminopeptidase A seems to have a charge variant that displays an intermediate migration and is not resolved from an aminopeptidase N (enzyme very active on LpNA). These two activities are not resolved by either gel filtration or ion‐exchange chromatography. The aminopeptidases N with intermediate and high migration, previously reported to be charge variants, were shown in this paper to differ in substrate specificities and in the strength with which they associate to the cell glycocalyx. © 1994 Wiley‐Liss, Inc.

Functional analysis of a leucine aminopeptidase fromSolanum tuberosum L.

A protein encoded by a potato cDNA homologous to a leucine aminopeptidase (LAP) from bovine lens (Hildmann et al. 1992) was expressed in Escherichia coli cells and biochemically characterized by hydrolysis of leucine p-nitroanilide. Activity was highest under alkaline conditions with an optimum at about pH 10. Maximal activities were measured at 65 degrees C. Apart from leucine p-nitroanilide the enzyme could also efficiently hydrolyze the p-nitroanilides of arginine and methionine. Complete inhibition of the enzyme was achieved by incubating bacterial extracts with bestatin and EDTA, which classifies the enzyme as a metalloprotease belonging to the same group as the homohexameric LAPs from mammals. Protein blots showed low constitutive expression of the LAP in all organs of potato plants: buds, flowers, tubers, roots and leaves. An increase in steady-state protein that was paralleled by an increase in total LAP activity was observed in leaf extracts after supplying jasmonic acid via the petioles. Plants containing the cDNA in antisense orientation behind the constitutive Cauliflower Mosaic Virus 35S promoter showed nearly complete reduction of the corresponding mRNA in leaves. However, in these plants LAP activities were only decreased by about 20% as compared to non-transgenic potato plants, while after feeding with jasmonic acid the activity of transgenic plants was reduced to about 5% of that of non-transgenic plants also induced by jasmonic acid. There was no phenotypic difference between wild-type and LAP antisense plants.

Characteristics of the peptidase activity contained in Taiwan cobra ( Naja naja atra) venom

D. Boumrah, C. J. Suckling and M. J. Dufton. Characteristics of the peptidase activity contained in Taiwan cobra ( Naja naja atra) venom. Toxicon 31, 1293–1303, 1993.—Cobra venoms ( Naja species) contain a little-understood peptidase activity which shows specificity towards small peptides containing glycine and non-polar aromatic/aliphatic residues. We have examined the ability of whole cobra venom to degrade several types of peptide with emphasis on the action of Taiwan cobra ( Naja naja atra) venom on l-alanylglycylglycine and glycylglycyl- l-phenylalanine. These are competing substrates, and it proved possible to generate inhibitors of the degradation of glycylglycyl- l-phenylalanine by synthesizing l-alanylglycylglycine analogues in which the peptide bond between the second and third residues had been replaced by different linkages. These analogues were themselves resistant to hydrolysis. The peptidase activity can also be inhibited by bestatin, captopril and chloromethyl ketones. Kinetic analyses suggested that even the best substrate discovered was of poor efficacy, so the natural peptide substrate remains to be identified. In unsuccessful attempts to devise a reliable chromogenic assay, it was found that the venom had activity against N-blocked amino acid p-nitrophenol esters, but not against leucine p-nitroanilide or ester substrates for trypsin-like and chymotrypsin-like enzymes.

Characteristics of protease synthesis in Bacteroides splanchnicus NCTC 10825

Studies to determine the physiological and nutritional characteristics of protease synthesis by Bacteroides splanchnicus NCTC 10825 showed that the proteases were constitutive and cell-associated during exponential growth in batch culture. As growth slowed and the bacteria entered the stationary phase, proteases that had accumulated intracellularly were released into the culture media. In continuous cultures, [dilution rate (D)=0.03 h−1 to D=0.29 h−1], protease activity was completely cell-bound and maximal during nitrogen-limited growth at high dilution rates. The proteases hydrolysed a relatively restricted range of protein substrates including casein, azocasein and gelatine (comparative maximum rates of hydrolysis were 1.0, 4.1 and 2.7 units mg−1 protein respectively). B. splanchnicus proteases exhibited arylamidase activities against leucine p-nitroanilide, valylalanine p-nitroanilide and glycylproline p-nitroanilide. Inhibition experiments indicated that the bacterium produced a mixture of serine, thiol and, possibly, metalloproteases. Protease activities were affected by reducing agents and divalent metal ions. Mercaptoethanol at 1 mm was slightly stimulatory; however, dithiortheitol and dithioerythritol (each 10 mm) respectively inhibited protease activities by 91% and 100%. Calcium ions (5 mm) stimulated protease activity by 30%, whereas Mn2+ and Mg2+ had little or no effect. Protease and arylamidase activities had neutral to alkaline pH optima. Together, these results show that with respect to the types of protease formed and the physiology of the process, B. splanchnicus proteolysis is similar in many respects to that occurring in species belonging to the B. fragilis group.

Identification and characterization of aminopeptidases from Aplysia californica.

Aminopeptidase activities were identified in extracts of kidney, ovotestis, head ganglia, heart and haemolymph of Aplysia californica. These enzyme preparations hydrolysed [3H][Leu]enkephalin at the Try-1-Gly-2 bond as determined by h.p.l.c. analysis of cleavage products. In all these tissues, enkephalin-degrading aminopeptidase activities were present both in membrane-bound and cytosolic fractions. The bivalent-cation-chelating agent, 1,10-phenanthroline, inhibited kidney membrane aminopeptidase activity with an IC50 of 30 microM, suggesting that this enzyme is a metalloproteinase. The aminopeptidase inhibitor amastatin was the most potent inhibitor of [Leu]enkephalin degradation (IC50 25 nM) by membrane-bound aminopeptidase, and bacitracin, bestatin and puromycin were about 100-1000 times less potent. In contrast with membrane-bound aminopeptidase, the cytosolic form is sensitive to puromycin. Angiotensin-converting enzyme inhibitor had no effect on [Leu]enkephalin degradation by kidney membranes, while the neutral endopeptidase inhibitors were poor inhibitors of the enzymes in this preparation. The Km values of the aminopeptidase in the kidney membranes and cytosolic fractions for the [Leu]enkephalin substrate were 2.4 and 7.4 microM respectively. The aminopeptidase present in the kidney membranes also hydrolysed endogenous Phe-Met-Arg-Phe-amide peptide at the Phe-1-Met-2 bond as well as synthetic alanine p-nitroanilide and leucine p-nitroanilide. When used in a competition assay, these substrates inhibited hydrolysis of [3H][Leu]enkephalin, suggesting that the same enzyme degraded all these substrates. Taken together, these results suggest that Aplysia tissues contain both a membrane-bound aminopeptidase related to the mammalian aminopeptidase N and a cytosolic puromycin-sensitive aminopeptidase.

Purification and Characterization of Aminopeptidase from Lactobacillus casei ssp. casei LLG

An aminopeptidase of broad specificity was purified to homogeneity from Lactobacillus casei ssp. casei LLG by fast protein liquid chromatography. The enzyme was purified to 153-fold over the crude extract with a recovery of 9%. The purified enzyme appeared as a single band on native PAGE and SDS-PAGE and had a molecular weight of 87 kDa and an isoelectric point of 4.73. Purity of the enzyme was confirmed by the activity staining on substrate-coated blotted membrane. The enzyme hydrolyzed a range of nitroanilide-substituted amino acids, as well as dipeptides, and accounted for all of the aminopeptidase and some of the dipeptidase activities found in cell-free extracts. Kinetic studies indicated that the enzyme has a low affinity for leucine p-nitroanilide (Michaelis-Menton constant, .22mM) but could hydrolyze the substrate at high rates (maximal velocity, 16.3 mmol/min per mg of protein). The aminopeptidase was shown to be a metal-dependent enzyme, because both EDTA and 1,10-phenanthroline caused complete inhibition of the aminopeptidase activity. Although Co2+ and Ni2+ could partially restore the activity lost by the treatment of 1,10-phenanthroline, EDTA caused an irreversible inhibition of the enzyme activity. Higher concentration of substrate and hydrolysis products also inhibited the activity of the enzyme. The enzyme showed optimal activity at pH 7.0 and at 39°C and was fairly stable over the pH range of 5 to 9 and below 45°C.

Comparative Studies on Peptidases of Lactobacillus casei Subspecies

The objective of this study was to isolate strains of lactobacilli containing peptidases of high specific activities and wide spectrum specificities for the enzyme modification and accelerated ripening of Cheddar cheese. Six selected strains of Lactobacillus casei (ssp. casei, ssp. pseudoplantarum, and ssp. rhamnosus) were evaluated for their amino-, di-, tri- and carboxypeptidase activities using 30 different synthetic peptides and peptide derivatives. All six strains showed similar substrate specificities towards various peptides, except for phenylalanine-proline, which was only hydrolyzed by Lactobacillus casei ssp. casei strains. Analysis of variance revealed significant differences among strains with respect to their specific activities. Overall profiles showed that Lactobacillus casei subspecies contained high amino- and dipeptidase with relatively weak tripeptidase activities. Lactobacillus casei ssp. casei strains LLG and ATCC 393 had the highest di- and tripeptidase activities respectively, whereas Lactobacillus casei ssp. rhamnosus strain S93 contained the highest activities for leucine-, lysine-, alanine-, and valine- and methionine-aminopeptidases, and proline-iminopeptidase. The dipeptidases of all six strains exhibited 8 to 10 times higher affinity for alanine-X dipeptides. Highest activities for amino- and dipeptidases were observed with leucine p-nitroanilide (256 μmol/min per mg of protein) and alanine-methionine (4976 absorbance units/min per mg of protein) as substrates. Lactobacillus casei ssp. casei strains showed strong activities against bitter peptides (phenylalanine-proline, proline-phenylalanine, proline-isoleucine) and sulfur-containing amino and peptide derivatives (methionine p-nitroanilide, cysteine p-nitroanilide, and alanine-methionine), which are of great importance in removing bitterness from and in ripening of cheeses. None of the strains showed any detectable carboxypeptidase activity when tested on seven carboxyl end-substituted peptides.

Buccal absorption. III. Simultaneous diffusion and metabolism of an aminopeptidase substrate in the hamster cheek pouch.

The simultaneous diffusion and metabolism of the D- and L-isomers of the aminopeptidase substrate, leucine-p-nitroanilide (LPNA), were examined in vitro in the hamster cheek pouch. L-LPNA was completely hydrolyzed during diffusion across the cheek pouch, whereas D-LPNA crossed the cheek pouch intact. The metabolic barrier appeared to be localized in the epithelium of the cheek pouch. Addition of an aminopeptidase inhibitor, bestatin, to both diffusion cell reservoirs resulted in decreased hydrolysis of L-LPNA. The experimental results were analyzed with a mathematical model which was developed to describe the simultaneous diffusion and metabolism processes. Using this model it was estimated that the rate of diffusion of L-LPNA across the cheek pouch was less than the capacity of the tissue to hydrolyze L-LPNA.