Localization of angiotensin-converting enzyme,prolyl endopeptidase and other peptidases in cultured neuronal or glial cells

To determine the cellular localization of nervous tissue peptidases, 7 peptidases and 2 lysosomal marker enzyme activities were measured in cultured mouse and rat cells. Neuronal cells of both species exhibited higher activities of angiotensin-converting enzyme (ACE) and prolyl endopeptidase (Pro-EP) than glial cells did. In contrast, arginyl endopeptidase and lysosomal enzymes (acid phosphatase, β-glucuronidase) in the neuronal cell lines were lower than those in the glial cell lines. Other peptidases (alanyl aminopeptidase, arginyl aminopeptidase, leucyl aminopeptidase, dipeptidyl aminopeptidase) activities were not specifically localized in either cell lines. The effects of cellular differentiation on these peptidase activities in the PC 12h cell line and rat glioblasts were also examined using nerve growth factor (NGF) and glia maturation factor (GMF), respectively. Neuron specific peptidase (ACE and Pro-EP) activities were decreased in PC12h cells cultured with NGF, and Pro-EP activity was increased in the glioblast cells cultured with GMF. These results support the idea that some of the peptidases are differentially localized in neuronal or glial cells, and play physiological roles in central or peripheral neural tissues.     

Peptidases in dog-ileum circular and longitudinal smooth-muscle plasma membranes. Their relative contribution to the metabolism of neurotensin

We established the content in neuropeptide-metabolizing peptidases present in highly purified plasma membranes prepared from the circular and longitudinal muscles of dog ileum. Activities were measured by the use of fluorigenic substrates and the identities of enzymes were confirmed by the use of specific peptidase inhibitors. Endopeptidase 24.11, angiotensin-converting enzyme, post-proline dipeptidyl aminopeptidase and aminopeptidases were found in both membrane preparations. Proline endopeptidase was only detected in circular smooth muscle plasma membranes while pyroglutamyl-peptide hydrolase was not observed in either tissue. The relative contribution of these peptidases to the inactivation of neurotensin was assessed. The enzymes involved in the primary inactivating cleavages occurring on the neurotensin molecule were as follows. In both membrane preparations, endopeptidase 24.11 was responsible for the formation of neurotensin-(1-11) and contributed to the formation of neurotensin-(1-10); a recently purified neurotensin-degrading neutral metallopeptidase was also involved in the formation of neurotensin-(1-10). A carboxypeptidase-like activity hydrolysed neurotensin at the Ile12-Leu13 peptide bond, leading to the formation of neurotensin-(1-12). Proline endopeptidase and endopeptidase 24.15 only occurred in circular muscle plasma membranes, yielding neurotensin-(1-7) and neurotensin-(1-8), respectively. In addition, the secondary processing of neurotensin degradation products was catalyzed by the following peptidases. In circular and longitudinal muscle membranes, angiotensin-converting enzyme converted neurotensin-(1-10) into neurotensin-(1-8) and tyrosine resulted from the rapid hydrolysis of neurotensin-(11-13) by bestatin-sensitive aminopeptidases. A post-proline dipeptidyl aminopeptidase activity converted neurotensin-(9-13) into neurotensin-(11-13) in circular muscle plasma membranes. The mechanism of neurotensin inactivation occurring in these membranes will be compared to that previously established for membranes from central origin.     

Changes in the Level of Peptidase Activities in Pea Ovaries during Senescence and Fruit Set Induced by Gibberellic Acid

The activities and changes in the levels of exopeptidase and endopeptidase activities were characterized in unpollinated ovaries of Pisum sativum L. cv Alaska during senescence and early fruit development induced by gibberellic acid (GA₃). Two aminopeptidases and one iminopeptidase were electrophoretically separated. These peptidases were sensitive to inhibitors of sulfhydryl proteases. Carboxypeptidase activity was inhibited by phenylmethyl sulfonyl fluoride. An azocasein-degrading endopeptidase, sensitive to thiol protease inhibitors, was also found. An increase in the specific activity of aminopeptidase during both fruit development and ovary senescence was observed. In contrast, the specific activity of carboxypeptidase and endopeptidase increased only during senescence of the ovary. Changes in exopeptidase activity in senescing ovaries could be mainly the consequence of a greater stability to proteolysis while the rise in endopeptidase activity appeared to be due to new or increased synthesis of the enzyme. These results suggest that endopeptidase, and not amino or carboxypeptidase, plays a key role in the senescence of pea ovaries and that the changes in unpollinated ovaries leading to ovary senescence or fruit development can be controlled by gibberellins.     

Myoglobin as an Inhibitor of Exopeptidases from Lactobacillus sake

The effects of myoglobin on exopeptidases of Lactobacillus sake were determined. Inhibition of the aminopeptidases increased as the myoglobin concentration increased; aminopeptidase 3 was the most affected (90% inhibition). Aminopeptidases 1, 2, and 4 showed similar inhibition levels (around 60%). Myoglobin did not affect tripeptidase activity. Thus, myoglobin could limit amino acid generation in meat systems.     

Effect of neonatal handling on brain enkephalin-degrading peptidase activities.

Neonatal handling decreases neutral endopeptidase 24.11 activity in the amygdala. However, this procedure does not affect aminopeptidase activities in any of the brain areas studied. Neonatal handling has been one of the most commonly used strategies to study the plasticity of the nervous system. The crucial role of the opioids in the control of different aspects of behaviour and development has been well documented. Regarding this subject, the endogenous opioid system might mediate some of the effects induced by neonatal handling. In this work, we have studied the effects of neonatal handling on several enkephalin-degrading peptidases, including soluble and membrane-bound aminopeptidases (puromycin-sensitive and -insensitive) and neutral endopeptidase 24.11 in different rat brain areas. Tyrosine aminopeptidase activities were measured fluorimetrically using tyrosine-beta-naphthylamide as substrate and puromycin as selective inhibitor of one of the membrane-enzymes. Dansyl-D-Ala-Gly-Phe(pNO2)-Gly was the fluorogenic substrate for neutral endopeptidase. The reduced neutral endopeptidase 24.11 activity in the amygdala of neonatal handled rats could reduce enkephalin catabolism in this area and it could be responsible for some of the effects induced by neonatal handling.     

Peptidohydrolases of Soybean Root Nodules : IDENTIFICATION, SEPARATION, AND PARTIAL CHARACTERIZATION OF ENZYMES FROM BACTEROID-FREE EXTRACTS

Nodule extracts prepared from Glycine max var Woodworth possessed endopeptidase, aminopeptidase, and carboxypeptidase activities. Three distinct endopeptidase activities could be resolved by disc-gel electrophoresis at pH 8.8. According to their order of increasing electrophoretic mobility, the first of these enzymes hydrolyzed azocasein and n-benzoyl-l-Leu-beta-naphthylamide, while the second hydrolyzed n-benzoyl-l-Arg-beta-naphthylamine (Bz-l-Arg-betaNA), n-benzoyl-l-Arg-p-nitroanilide (Bz-l-Arg-pNA), and azocasein. The third endopeptidase hydrolyzed Bz-l-Arg-betaNA, Bz-l-Arg-pNA, and hemoglobin. Fractions of these enzymes extracted from electrophoresis gels were shown to have pH optima from 7.5 to 9.8. All of the endopeptidases were completely inhibited by diisopropylphosphorofluoridate, demonstrating that they were serine proteases.Aminopeptidase activity was measured using amino acyl-beta-naphthylamides. Electrophoresis of nodule extracts at pH 6.8 resolved the aminopeptidase activity of nodule extracts into at least four fractions based on mobility and on activities toward amino acyl-beta-naphthylamides. The major activity of two of the aminopeptidases was directed toward l-Leu- and l-Met-beta-naphthylamide, while the other two aminopeptidases exhibited broader specificity and were capable of hydrolyzing a large number of amino acyl-beta-naphthylamides. Two of the aminopeptidases extracted from electrophoresis gels were classified as thiol type enzymes, and all four aminopeptidases had neutral to basic pH optima.     

Maize leaf proteases and their effect on endogenous inorganic pyrophosphatase

Several different proteolytic enzymes are present in leaf and root tissue of maize seedlings. The activity of these enzymes diminishes to a basal level by the time seedling height reaches 20–30 cm. We have partially characterized an endopeptidase with trypsin-like specificity and two aminopeptidases, all from leaf tissue, and compared them to previously reported proteases from maize. Both the endopeptidase and the aminopeptidases degrade the maize leaf enzyme, inorganic pyrophosphatase. Modification of the pyrophosphatase by the peptidases results in the formation of catalytically active, electrophoretically distinct products. The aminopeptidases have little effect on several other maize leaf enzymes, but also modify yeast inorganic pyrophosphatase.     

The efficiency of human cytomegalovirus pp65(495-503) CD8+ T cell epitope generation is determined by the balanced activities of cytosolic and endoplasmic reticulum-resident peptidases

Control of human CMV (HCMV) infection depends on the cytotoxic activity of CD8(+) CTLs. The HCMV phosphoprotein (pp)65 is a major CTL target Ag and pp65(495-503) is an immunodominant CTL epitope in infected HLA-A*0201 individuals. As immunodominance is strongly determined by the surface abundance of the specific epitope, we asked for the components of the cellular Ag processing machinery determining the efficacy of pp65(495-503) generation, in particular, for the proteasome, cytosolic peptidases, and endoplasmic reticulum (ER)-resident peptidases. In vitro Ag processing experiments revealed that standard proteasomes and immunoproteasomes generate the minimal 9-mer peptide epitope as well as N-terminal elongated epitope precursors of different lengths. These peptides are largely degraded by the cytosolic peptidases leucine aminopeptidase and tripeptidyl peptidase II, as evidenced by increased pp65(495-503) epitope presentation after leucine aminopeptidase and tripeptidyl peptidase II knockdown. Additionally, with prolyl oligopeptidase and aminopeptidase B we identified two new Ag processing machinery components, which by destroying the pp65(495-503) epitope limit the availability of the specific peptide pool. In contrast to cytosolic peptidases, silencing of ER aminopeptidases 1 and 2 strongly impaired pp65(495-503)-specific T cell activation, indicating the importance of ER aminopeptidases in pp65(495-503) generation. Thus, cytosolic peptidases primarily interfere with the generation of the pp65(495-503) epitope, whereas ER-resident aminopeptidases enhance such generation. As a consequence, our experiments reveal that the combination of cytosolic and ER-resident peptidase activities strongly shape the pool of specific antigenic peptides and thus modulate MHC class I epitope presentation efficiency.     

Neurotensin-Metabolizing Peptidases in Rat Fundus Plasma Membranes

The mechanisms by which neurotensin (NT) was inactivated by rat fundus plasma membranes were characterized. Primary inactivating cleavages occurred at the Arg8-Arg9, Pro10-Tyr11, and Ile12-Leu13 peptidyl bonds. Hydrolysis at the Arg8-Arg9 bond was fully abolished by the use of N-[1(R,S)-carboxy-2-phenylethyl]-alanyl-alanyl-phenylalanine-p- aminobenzoate, a result indicating the involvement at this site of a recently purified soluble metallopeptidase. Hydrolysis of the Pro10-Tyr11 bond was totally resistant to N-benzyloxycarbonyl-prolyl-prolinal and thiorphan, an observation suggesting that the peptidase responsible for this cleavage was different from proline endopeptidase and endopeptidase 24.11 and might correspond to a NT-degrading neutral metallopeptidase recently isolated from rat brain synaptic membranes. The enzyme acting at the Ile12-Leu13 bond has not yet been identified. Secondary cleavages occurring on NT degradation products were mainly generated by bestatin-sensitive aminopeptidases and post-proline dipeptidyl aminopeptidase. The content in NT-metabolizing peptidases present in rat fundus plasma membranes is compared with that previously established for purified rat brain synaptic membranes.     

Neuropeptide-hydrolysing activities in synaptosomal fractions from dog ileum myenteric, deep muscular and submucous plexi. Their participation in neurotensin inactivation

The mapping of neuropeptidases in synaptosomal fractions prepared from dog ileum myenteric, deep muscular and submucous plexus was established by means of fluorigenic substrates and specific inhibitors. Endopeptidase 24.11, angiotensin-converting enzyme and aminopeptidases were found in all tissues, the highest amounts being recovered in the submucous preparation. Post-proline dipeptidyl aminopeptidase was obtained in high quantities whatever the tissue source while proline endopeptidase was detected in low amounts and pyroglutamyl-peptide hydrolase was never detectable. The above peptidases were examined for their putative participation in the inactivation of neurotensin by monitoring the effect of specific inhibitors on the formation of the metabolites of labeled neurotensin separated by HPLC. Endopeptidases 24.11, 24.15 and 24.16 were respectively responsible for the formation of neurotensin(1-11), neurotensin(1-8) and neurotensin(1-10) that are devoid of biological activity. The secondary attacks occurring on neurotensin degradation products were the following: cleavage of neurotensin(1-10) into neurotensin(1-8) by angiotensin-converting enzyme; conversion of neurotensin(9-13) into neurotensin(11-13) by post-proline dipeptidyl aminopeptidase; hydrolysis of neurotensin(11-13) into free tyrosine by aminopeptidase(s).     

Types and localization of aminopeptidases in different human blood cells

1. Erythrocytes, polymorphonuclears, monocytes and lymphocytes isolated from human peripheral blood, were shown to possess in their cytosols, granules and microsomal fractions, aminopeptidases capable of hydrolysing arginyl-, leucyl-, methionyl-, phenylalanyl- and alanyl-2-naphthylamide. 2. In different cell compartments enzymes of different pI were responsible for these activities. 3. Chloride activated arginine aminopeptidase, broad specificity aminopeptidase and dipeptidyl peptidase III were found in cytosols of all examined cells. 4. In granules at least two aminopeptidases, a basic or neutral one, and an acidic one inactive at pH 4.4, could be discerned, whereas in microsomal fractions a broad specificity aminopeptidase preferring methionine was detected. 5. There is a considerable degree of similarity in the pattern of aminopeptidases within different blood cells. This may suggest that their functions are correlated to the physiological role of a particular cell compartment, rather than to that of a distinct cell type.     

Differentiation of endopeptidases and aminopeptidases by high-performance liquid chromatography of reaction products from chromogenic peptide p-nitroanilines as substrates

A method for differentiating endopeptidases and aminopeptidases on the basis of substrate specificity is presented. Various synthetic chromogenic substrates, succinyl-(Ala)3-p-nitroaniline, succinyl-(Ala)2-p-nitroaniline, (Ala)3-p-nitroaniline, and (Ala)2-p-nitroaniline, were incubated with various peptidases and the incubation mixtures were directly analyzed by high-performance liquid chromatography to determine the splitting patterns of these substrates by the enzymes. The substrates and hydrolyzed products containing the chromophore were separated on a reverse-phase column under isocratic conditions, and the chromophore was specifically detected in the effluent fractions by absorbance measurement at 314 nm. Endopeptidases, leucine aminopeptidase, and dipeptidyl aminopeptidase showed different patterns of cleavage of the substrates. This simple and rapid high-performance liquid chromatographic procedure is suitable for identifying the above activities in different fractions obtained during separation and purification studies. The same approach was applied to the simultaneous determination of three types of endopeptidase activities in rat tissues based on the ability of the enzymes to hydrolyze different sites in succinyl-(Ala)3-p-nitroaniline.     

Comparative peptide specificity of cell wall, membrane and intracellular peptidases of group N streptococci

Solubilized cell walls of group N streptococci contain two electrophoretically distinct peptidases, one of which hydrolysed trileucine only, while the second hydrolysed a wide range of di- and tripeptides. Neither enzyme possessed leucine aminopeptidase or endopeptidase activity. Four and three peptidases, respectively, were separated in intracellular extracts of Streptococcus lactis subsp. lactis and Strep, lactis subsp. cremoris produced by osmotic lysis of spheroplasts. In contrast with the cell-wall extracts, two of the peptidases had broad specificites, though only one of these hydrolysed trileucine. Purified membranes of Strep. lactis subsp. lactis contained only one electrophoretically distinct peptidase of very narrow specificity. There were small differences between the numbers of peptides hydrolysed by cell wall preparations from milk-grown or broth-grown cells.     

Comparative peptide specificity of cell wall, membrane and intracellular peptidases of group N streptococci

Solubilized cell walls of group N streptococci contain two electrophoretically distinct peptidases, one of which hydrolysed trileucine only, while the second hydrolysed a wide range of di- and tripeptides. Neither enzyme possessed leucine aminopeptidase or endopeptidase activity. Four and three peptidases, respectively, were separated in intracellular extracts of Streptococcus lactis subsp. lactis and Strep. lactis subsp. cremoris produced by osmotic lysis of spheroplasts. In contrast with the cell-wall extracts, two of the peptidases had broad specificites, though only one of these hydrolysed trileucine. Purified membranes of Strep. lactis subsp. lactis contained only one electrophoretically distinct peptidase of very narrow specificity. There were small differences between the numbers of peptides hydrolysed by cell wall preparations from milk-grown or broth-grown cells.     

Pathway for degradation of peptides generated by proteasomes: a key role for thimet oligopeptidase and other metallopeptidases

The degradation of cellular proteins by proteasomes generates peptides 2-24 residues long, which are hydrolyzed rapidly to amino acids. To define the final steps in this pathway and the responsible peptidases, we fractionated by size the peptides generated by proteasomes from beta-[14C]casein and studied in HeLa cell extracts the degradation of the 9-17 residue fraction and also of synthetic deca- and dodecapeptide libraries, because peptides of this size serve as precursors to MHC class I antigenic peptides. Their hydrolysis was followed by measuring the generation of smaller peptides or of new amino groups using fluorescamine. The 14C-labeled peptides released by 20 S proteasomes could not be degraded further by proteasomes. However, their degradation in the extracts and that of the peptide libraries was completely blocked by o-phenanthroline and thus required metallopeptidases. One such endopeptidase, thimet oligopeptidase (TOP), which was recently shown to degrade many antigenic precursors in the cytosol, was found to play a major role in degrading proteasome products. Inhibition or immunodepletion of TOP decreased their degradation and that of the peptide libraries by 30-50%. Pure TOP failed to degrade proteasome products 18-24 residues long but degraded the 9-17 residue fraction to peptides of 6-9 residues. When aminopeptidases in the cell extract were inhibited with bestatin, the 9-17 residue proteasome products were also converted to peptides of 6-9 residues, instead of smaller products. Accordingly, the cytosolic aminopeptidase, leucine aminopeptidase, could not degrade the 9-17 residue fraction but hydrolyzed the peptides generated by TOP to smaller products, recapitulating the process in cell extracts. Inactivation of both TOP and aminopeptidases blocked the degradation of proteasome products and peptide libraries nearly completely. Thus, degradation of most 9-17 residue proteasome products is initiated by endoproteolytic cleavages, primarily by TOP, and the resulting 6-9 residue fragments are further digested to amino acids by aminopeptidases.     

Genetically defined peptidases of maize I. Biochemical characterization of allelic and nonallelic forms

A number of biochemical properties have been investigated for both allelic and nonallelic forms of maize peptidases. Four aminopeptidases exist in maize (LAP-A, LAP-B, LAP-C, and LAP-D) and are the products of four diallelic loci. The aminopeptidases fall into two biochemical groups on the basis of these studies. LAP-A and LAP-D have comparatively low apparent K _m (K _app) values for arginine-naphthylamide derivatives and high velocities for arginine-naphthylamide and lysine-naphthylamide. LAP-B and LAP-C, on the other hand, have lower K _app values for leucine-naphthylamide and higher velocities for nonpolar amino acid-naphthylamides than for arginine-naphthylamide. LAP-A and LAP-D are also relatively more heat stable than LAP-B and LAP-C and have somewhat higher molecular weights (71,500) than LAP-B and LAP-C (63,500). In determining molecular weights of the peptidases, use was made of their differential substrate specificities toward amino acid-naphthylamides. Some properties of genetically defined maize endopeptidase are also presented. Maize endopeptidase is inhibited by the sulfhydryl reagents N-ethylmaleimide and p-chloromercuribenzoate (pCMB), and by tosyl lysine chloromethyl ketone. Maize aminopeptidase activity is inhibited by N-ethylmaleimide, pCMB, and EDTA (ethylenediamine tetraacetic acid).This research was supported by U.S. Atomic Energy Commission Contract AT(38-1)-770, and in part by Grant No. GM-22733 from the National Institute of General Medical Sciences, U.S. Public Health Service, to J. G. S.Paper No. 4740 of the Journal Series of the North Carolina Agricultural Experiment Station, Raleigh, North Carolina.     

Decay of proteinase and peptidase activities of human and rabbit erythrocytes during cellular aging

Variations in activity of the membrane-bound and cytosolic proteinases and peptidases were analyzed in human and rabbit erythrocytes at various stages of their life-span. The patterns observed with human erythrocytes were the following. (a) The acidic endopeptidase activity associated with the membranes undergoes a substantial decline during cellular aging, with an estimated half-life of 65 days. Concomitantly it appears to become progressively more latent. (b) All cytosolic proteinase and peptidase activities described previously (Pontremoli, S., Melloni, E., Salamino, F., Sparatore, B., Michetti, M., Benatti, U., Morelli, A. and De Flora, A. (1980) Eur. J. Biochem. 110, 421–430) decline exponentially throughout the erythrocyte life-span, with the exception of dipeptidyl aminopeptidase III. The calculated half-lives were: 60 days for the neutral endopeptidase; 87 days for the total acidic endopeptidase activity which is accounted for by three distinct enzymes; 49 days for aminopeptidase B and 133 days for a second aminopeptidase with broad substrate specificity; 84 days for dipeptidyl aminopeptidase II. The results obtained with the rabbit erythrocytes were: (a) no significant decline of leucine aminopeptidase, dipeptidyl aminopeptidase II and III activities in the transition from reticulocytes to mature erythrocytes; (b) very limited decline of aminopeptidase B activity; (c) a pronounced age-dependent decay, in increasing order, of neutral endopeptidase, aminopeptidase A, carboxypeptidase and acidic endopeptidase activities.     

Development of leucine aminopeptidase activity during daylily flower growth and senescence

The possibility that exopeptidases, i.e. aminopeptidases and carboxypeptidases, in addition to the previously studied endopeptidase might also be developmentally regulated in daylily petals was examined. The level of leucine aminopeptidase and endopeptidase activities changed after the flower was fully open while that of carboxypeptidase activity remained relatively unchanged throughout senescence. Leucine aminopeptidase activity seemed to increase after the flower was fully open and peaked several hours earlier than endopeptidase did. Taken together, it is postulated that leucine aminopeptidase might play a role in protein turnover during flower opening and in the initiation of protein hydrolysis associated with petal senescence while the endopeptidase could be responsible for the breakdown of the bulk of proteins at the later stages. The drop in leucine aminopeptidase activity associated with the onset of daylily petal senescence was effectively halted by a cycloheximide treatment of cut daylily flowers for 24 h which was previously shown to prolong the vase life of the flowers and prevent protein loss from the petals. Apart from both being developmentally regulated in daylily petals, the leucine aminopeptidase activity and the previously studied endopeptidase are different in several aspects. They appear to have different pH optima, 8 for leucine aminopeptidase and 6.2 for endopeptidase. Unlike the endopeptidase activity, no new leucine aminopeptidase isozymes appeared during petal senescence, and the leucine aminopeptidase did not appear to belong to the cysteine class of proteolytic enzymes.     

Comparison of metal‐bound and unbound structures of aminopeptidase B proteins from Escherichia coli and Yersinia pestis

Protein degradation by aminopeptidases is involved in bacterial responses to stress. Escherichia coli produces two metal‐dependent M17 family leucine aminopeptidases (LAPs), aminopeptidase A (PepA) and aminopeptidase B (PepB). Several structures have been solved for PepA as well as other bacterial M17 peptidases. Herein, we report the first structures of a PepB M17 peptidase. The E. coli PepB protein structure was determined at a resolution of 2.05 and 2.6 Å. One structure has both Zn²⁺ and Mn²⁺, while the second structure has two Zn²⁺ ions bound to the active site. A 2.75 Å apo structure is also reported for PepB from Yersinia pestis. Both proteins form homohexamers, similar to the overall arrangement of PepA and other M17 peptidases. However, the divergent N‐terminal domain in PepB is much larger resulting in a tertiary structure that is more expanded. Modeling of a dipeptide substrate into the C‐terminal LAP domain reveals contacts that account for PepB to uniquely cleave after aspartate.     

Purification and characterization of an endopeptidase from Lactococcus lactis subsp. cremoris Wg2.

An endopeptidase has been purified to homogeneity from a crude cell extract of Lactococcus lactis subsp. cremoris Wg2 by a procedure that includes diethyl-aminoethane-Sephacel chromatography, phenyl-Sepharose chromatography, hydroxylapatite chromatography, and fast protein liquid chromatography over an anion-exchange column and a hydrophobic-interaction column. Gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated a molecular mass of the purified enzyme of 70,000 Da. The endopeptidase can degrade several oligopeptides into various tetra-, tri-, and dipeptides. The endopeptidase has no aminopeptidase, carboxypeptidase, dipeptidase, or tripeptidase activity. It is optimally active at pH 6.0 to 6.5 and in the temperature range of 30 to 38 degrees C. The enzyme is inactivated by the chemical agents 1,10-phenanthroline, ethylenedinitrilotetraacetate, beta-mercaptoethanol, and phenylmethylsulfonyl fluoride and is inhibited by Cu2+ and Zn2+. The ethylenedinitrilotetraacetate- or 1,10-phenanthroline-treated enzyme can be reactivated by Co2+. Immunoblotting with specific antibodies raised against the purified endopeptidase indicated that the enzyme is also present in other Lactococcus spp., as well as in Lactobacillus spp. and Streptococcus salivarius subsp. thermophilus.