A calcium-stimulated serine peptidase from a true-branching cyanobacterium, <Emphasis Type="Italic">Westiellopsis ramosa</Emphasis> sp. nov.

Unbranched heterocytous cyanobacteria produce a number of serine peptidases. We have characterized several peptidases in the cell-free extracts of a true-branched N₂-fixing cyanobacterium, Westiellopsis ramosa sp. nov. Upon substrate-gel zymography of intact filaments and heterocytes, five peptidase bands were resolved, whereas in vegetative cells, a single band was discernible. No band was detected in \({\text{NO}}_{3}^{ - } /{\text{NH}}_{4}^{ + }\)-grown cultures suggesting that the peptidases were present under diazotrophic conditions with much of them confined to heterocytes. Using salt precipitation and chromatography, a caseinolytic peptidase, called Wrp49, was purified which also demonstrated fibrinolytic activity. In SDS-PAGE, the purified peptidase was resolved into 17 and 27 kDa fragments. The enzyme in its native state exhibited M_r ≈ 49 kDa, and digested gelatin in a substrate gel at a corresponding position. The enzyme showed amidolytic activity on a plasmin specific substrate, D-Val-Leu-Lys p-nitroanilide. Moreover, a trypsin specific substrate, N-benzoyl-DL-Arg p-nitroanilide was hydrolyzed at an apparent K_m = 0.195 mM and V_max = 5 × 10^?7 M s^?1. The enzyme was stable in a wide pH and temperature range. While Ca²⁺ stimulated the activity; phenylmethane sulfonyl fluoride, leupeptin, EDTA and chelants were inhibitory. The activity of the EDTA-inactivated enzyme was completely restored upon adding Ca²⁺, suggesting that both compounds competed with each other in modulating the enzyme activity. The enzyme showed similarities with a Ca²⁺ stimulated subtilisin-like serine peptidase of Anabaena variabilis ATCC 29413, but also presented several unique features of metallopeptidases, such as the chelant’s response. Moreover, the N-terminal sequence (MTVENLARTGVGPGWR) did not match with any of the known peptidases.     

Partial purification and characterization of intracellular car☐ypeptidase ofCandida albicans

Intracellular car☐ypeptidase was partially purified from the yeast form ofCandida albicans H-317 by acid dialysis, ammonium sulfate fractionation, gel filtration, and ion-exchange chromatography. Peptidase activity was measured with an enzyme-coupled colorimetric assay. Fractionation by native polyacrylamide gel electrophoresis and Sephacryl S-200 gel filtration indicated the presence of a single peak of car☐ypeptidase, with the isoelectric point at pH 4.6 and a molecular weight of 100,000. The pH optimum and apparentK_m usingN-carbobenzoxy-l-phenylalanine-l-leucine (N-Cbz-l-Phe-l-Leu) as substrate were 6.5 and 2 × 10⁻⁴M, respectively. The best substrates for the enzyme wereN-Cbz-Ala-X peptides, withN-Cbz-Ala-Leu giving the highest rate of hydrolysis. Substrates that gave 7% or less of the control (N-Cbz-Phe-Leu) rate of hydrolysis were Gly-Leu, Gly-Phe, Ile-Phe, Ile-Met, Glu-Phe, Glu-Tyr, Val-Phe, and Pro-Phe. There was no detectable hydrolysis of the followingN-Cbz peptides; Gly-Met, Gly-Val, Gly-Tyr, Gly-Ile, or Ile-Val. Enzyme activity was inhibited by phenylmethylsulfonyl fluoride,p-chloromercuribenzoate, benzyloxy-carbonyl-l-phenylalanine chloromethyl ketone, and tosyl-l-phenylalanine chloromethyl ketone, but was not affected by EDTA, tosyl-l-lysine chloromethyl ketone, pepstatin A, leupeptin, bestatin, or antipain. Although secretory proteinases are thought to play a role in the pathogenesis of this organism, the role of this intracellular car☐ypeptidase has yet to be determined.     

Partial purification and characterization of dipeptidyl peptidase II (DPP II) from guinea pig testes

Depeptidyl peptidase (DPP II) was partially purified from guinea pig testes by (NH₄)₂SO₄ precipitation, Con A-Sepharose 4B chromatography, and Sephadex G-200 chromatography to a specific activity of 27.4 μmol Ala₃ hydrolyzed min^?1 mg^?1 protein. Chromatography on a calibrated G-200 column yielded a molecular weight of 135,000 daltons for the enzyme. Sodium dodecyl sulfate polyacrylamide electrophoresis showed an enrichment of a broad doublet at 64–66,000 daltons. The enzyme had optimal activity toward hydrolysis of L-alanyl-alanyl-alanine at pH 4.5 and showed sensitivity to cations of increasing size with Tris producing the most inhibition of those tested. The enzyme was moderately inhibited by serine proteinase inhibitors. Thin-layer chromatography revealed the dipeptidase nature of the enzyme's activity on tripeptides and dipeptidyl arylamides. A doublet of activity occurred when nitrocellulose electroblots of nondenaturing gel electrophoresis of the (NH₄)₂SO₄ fraction were reacted with the specific DPP II substrate, lysyl-alanyl-4-methoxy-2-napthylamide. Analytical isoelectric focusing of the G-200 fraction followed by fluorescent enzyme activity detection that used cellulose triacetate overlay membranes impregnated with the specific DPP II substrate, lysyl-alanyl-7-amino-4-trifluoromethylcou-marin, revealed multiple isoforms focusing at pI = 4.8–5.6. Two prominent bands focused at pI = 4.9 and pI = 5.1. The properties of guinea pig testicular DPP II are compared and contrasted with similar dipeptidyl peptidases from other sources.     

Electrophoretic heterogeneity of mouse erythrocyte peptidases

Starch gel electrophoresis in conjunction with a specific staining method revealed the occurrence of five distinct peptidases in mouse red blood cells. These enzymes can be distinguished on the basis of substrate specificity and electrophoretic mobility. They have been designated peptidases A, B, C, D, and E to correspond with the nomenclature adopted for human peptidases with which the mouse enzymes appear to be homologous. Genetically determined variants of peptidase C are described. The phenotype Pep C1 occurs in C57BL/Gr mice and the phenotype Pep C2 in CBA/Gr and Strong A/Gr mice. These phenotypes and the presumed heterozygote, Pep C2-1, appear to be due to the occurrence of codominant autosomal alleles which have been designated Pep-C ¹ and Pep-C ². F₁ and F₂ crosses show segregation in the expected Mendelian ratios. F₂ embryos and their placentae show the same electrophoretic pattern for peptidase C. The occurrence of a separate locus controlling the structure of each distinct peptidase is postulated.     

Purification and properties of dipeptidyl peptidase IV from Streptococcus mitis ATCC 9811

Dipeptidyl peptidase IV (EC 3.4.14.—) from Streptococcus mitis ATCC 9811 was purified to a specific activity of 56.2 units/mg protein by a series of column chromatographic techniques. The purified enzyme was apparently homogeneous as judged by disc gel electrophoresis. Gel filtration on a calibrated column indicated an apparent molecular weight of 120,000 for the native enzyme. Gel electrophoresis of the denatured enzyme in the presence of sodium dodecyl sulfate in a constant acrylamide concentration resulted in the appearance of a single component for which a molecular weight of 53,000 was calculated. The purified enzyme has an optimum pH between 6.0 and 8.7 and an isoelectric point of 4.0. The K_m value toward glycylprolyl-p-nitroanilide is about 6.0 × 10^?5m. Substrate specificity studies indicated that the purified enzyme hydrolyzes specifically N-terminal X-proline from X-Pro-p-nitroanilides. Inhibition of this enzyme was achieved with Hg²⁺, Pb²⁺, Zn²⁺, EDTA, and diisopropyl phosphorofluoridate, but not with N-ethyl-maleimide and sulfhydryl inhibitors.     

Apparent differences in tryptophan hydroxylation by cockroach (periplaneta americana) nervous tissue and rat brain

Tryptophan hydroxylation in cockroach (Periplaneta americana) nervous tissue was measured and compared to the hydroxylation of tryptophan in rat brain. Tryptophan hydroxylation in both tissues requires a pterine cofactor, and is inhibited by p-chlorophenylalanine. The molecular weight of the protein responsible for hydroxylation of tryptophan in cockroach nervous tissue obtained from gel filtration was estimated to be 54,000.The pH optima and enzyme kinetics differed greatly between the two hydroxylases. Hydroxylation of tryptophan by the enzyme obtained from cockroach tissues incubated with dimethyltetrahydropterine had a pH optimum of about 5.8–5.9 and a K_m in crude enzyme preparations of 2.6 × 10⁻⁶ M and is activity was substrate inhibited above 10⁻⁴ M tryptophan. Hydroxylation of tryptophan by the enzyme obtained from rat brain incubated with dimethyltetrahydropterine had a pH optimum of about 6.5–7.0, a K_m of about 6.7 × 10⁻⁴ M and exhibited no substrate inhibition at tryptophan concentrations up to 2 × 10⁻³ M.When incubated with biopterin, the presumed natural cofactor, the hydroxylase from cockroach tissues had a K_m of about 6.8 × 10⁻⁵ M and no substrate inhibition occurred at tryptophan concentrations up to 2 × 10⁻³ M. Under the same conditions rat hydroxylase had a K_m of 1.1 × 10⁻⁵M and substrate inhibition occurred above 10⁻⁴ M tryptophan.Unlike the mammalian situation, administration of tryptophan peripherally did not change the 5-hydroxytryptamine concentration in cockroach nervous tissue, but did increase tryptophan levels. The low V_max values of the cockroach hydroxylase and the inability of administered tryptophan to elevate 5-hydroxytryptamine levels suggest that in the cockroach hydroxylation of tryptophan itself may be the limiting factor in the biosynthesis of 5-hydroxytryptamine.     

Separation of peptide transport and hydrolysis in trimethionine uptake by Saccharomyces cerevisiae.

Intact cells of Saccharomyces cerevisiae 139 hydrolyzed amino acid-p-nitroanilide by an activity similar to that of aminopeptidase II, as well-characterized external peptidase in yeast. In contrast, trimethionine, a model peptide used in transport assays, was not hydrolyzed by this aminopeptidase II-like activity, and the peptidase activity toward this substrate was localized in the soluble fraction of the yeast. We conclude that this tripeptide is taken up by S. cerevisiae intact and rapidly hydrolyzed inside the cell.     

Further characterization of a neurotensin-degrading neutral metalloendopeptidase from rat brain

A peptidase inactivating neurotensin at the Pro¹⁰-Tyr¹¹ peptidyl bond, leading to the biologically inactive fragments neurotensin_1–10 and neurotensin_11–13 was purified from rat brain homogenate. The peptidase was characterized as a 70 kDa monomer and could be classified as a metaliopeptidase with respect to its sensitivity to o-phenanthroline, EDTA and divalent cations. The enzyme was also strongly inhibited by dithiothreitol but appeared totally insensitive to thiol-blocking agents, acidic and serine protease inhibitors. Experiments performed with a series of highly specific peptidase inhibitors clearly indicated that the peptidase was a novel enzyme distinct from previously purified cerebral peptidases. The enzyme displayed a rather high affinity for neurotensin (Km = 2.3 itM). Studies on its specificity indicated that: (i) neurotensin_9–13 was the shortest neurotensin fragment with full inhibitory potency of [³H]neurotensin degradation. Shortening the C-terminal end of the neurotensin molecule progressively led to inactive analogs; (ii) the peptidase exhibited a strong stereospecificity towards the residues in positions 8, 9 and 11. By contrast, neither introduction of a steric hindrance in position 11 nor amidation of the C-terminal end of the neurotensin molecule affected the ability of the corresponding analog to inhibit [³H]neurotensin degradation; (iii) Pro-Phe was the most potent dipeptide to compete for [³H]neurotensin degradation; (iv) the peptidase could not be described as an exclusive “neurotensinase” activity since, in addition to the neurotensin natural analogs (neuromedin N and xenopsin), non related natural peptides such as angiotensins I and II, dynorphins 1–8 and 1–13, atriopeptin III and bradykinin potently inhibited [³H]neurotensin degradation. Most of these peptides behaved as substrates for the enzyme.     

Purification and properties of d-xylulose reductase from Pachysolen tannophilus

d-Xylulose reductase (EC 1.1.1.9) from Pachysolen tannophilus IFO 1007 was purified by Sephadex G-100 gel chromatography with three columns and DEAE cellulose chromatography. The purified enzyme was entirely homogeneous on disc gel electrophoresis. It was most active at pH 9.1–10.0 and 55°C, and stable at pH 7–9 and below 25 °C. Its activity was stimulated by NH₄Cl,NaCl,MgCl₂,KCl, glutathione, cysteine and glycine, and inhibited remarkably by SH inhibitor such as lead acetate, HgCl₂ and AgNO₃. It oxidized xylitol, sorbitol, ribitol and glycerine but not mannitol, inositol, arabitol and erythritol. Its K_m values of enzyme against xylitol, sorbitol and ribitol were 1.1 × 10⁻² M, 3.0 × 10⁻² M and 5.0 × 10⁻² M, respectively. Its molecular weight was determined to be 120,000 by Sephadex G-200 column chromatography, and that of its subunit was 40,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis.     

Trypanosoma brucei: The peptidases of bloodstream trypanosomes

Peptidases (EC.3.4.11 and EC.3.4.13.9) from bloodstream Trypanosoma brucei were examined by starch gel electrophoresis and substrate specificities and relative activities of six peptidases (S,B,E,A,F,D,) determined. The substrate specificities corresponded closely to those of the peptidases of human blood cells and tissues although human peptidase C appeared not to have a T. brucei equivalent.     

Action de la chlorhexidine sur l'expression de la virulence de Candida albicans

Candida albicans is an opportunistic yeast. Its pathogenicity is linked to the susceptibility of the host surface as well as to particular factors of the strain: adhesion, filamentous growth and secretion of proteolytic enzymes. Chlorhexidine digluconate is an antiseptic with fungicidal properties. The action of the antiseptic on the growth of the yeast shows a minimal inhibitory concentration (MIC) at 50 μg·mL⁻¹ and a minimal fungicidal concentration (MFC) at 100 μg·mL⁻¹. The consequences of antiseptic treatment are studied using two indicators of pathogenicity: filamentation and the secretion of acid proteinase. Concerning the morphological indicator, a complete inhibition of filamentation for antiseptic concentration far below the MIC is observed; concerning the biochemical indicator, the results show a maintenance of proteolytic activity for concentration equal to the MIC and a disappearance of this activity between the MIC and the MFC. Concerning the proteolytic activity, Skim Milk (Difco) was substituted for bovine serum albumin with success. This mixture of casein allows to use a cheaper and more easily available substrate.     

Characterization of an antifungal glycolipid secreted by the yeast Sympodiomycopsis paphiopedili

An antifungal glycolipid was purified from the culture liquid of the ustilaginomycetous yeast Sympodiomycopsis paphiopedili by column and thin-layer chromatography. According to nuclear magnetic resonance and mass-spectroscopy experiments it was a cellobioside containing 2,15,16-trihydroxypalmitic acid as an aglycon. The minimal effective concentrations leading to ATP leakage and growth inhibition were 45 and 160 μg ml⁻¹ for Cryptococcus terreus and Candida albicans, respectively.     

Purification and characterization of a juvenile hormone binding protein from hemolymph of the silkworm,Bombyx mori

A juvenile hormone binding protein (JHBP) has been isolated from Bombyx mori hemolymph by gel filtration, ion-exchange chromatography, chromatofocusing and hydroxyapatite column chromatography. Gel electrophoresis indicates that the isolated protein is homogeneous in the presence or absence of a denaturing agent. The JHBP in question has a relative molecular mass of 32 kDa, determined by denaturing gel electrophoresis. Chromatofocusing analysis indicated that the JHBP is an acidic protein with pI 4.9. The protein exhibits a dissociation constant of 9.0 × 10⁻⁸ M for JH I, 1.14 × 10⁻⁷ M for JH II and 3.9 × 10⁻⁷ M for JH III, and thus its affinity for JH analogues is in the order of JHI >JHII >JHIII. Its amino acid composition indicates that the protein consists of 297 residues of 18 kinds of amino acids. The sequence of the N-terminus of the polypeptide chain was determined for 34 of the first 36 residues: Asp-Gln-Asp-Ala-Leu-Leu-Lys-Pro-?-Lys-Leu-Gly-Asp-Met-Gln-Ser-Leu-Ser-Ser-Ala-Thr-Gln-Gln-Phe-Leu-Glu- Lys-Thr-Ser-Lys-Gly-Ile-Pro-?-Tyr-His-.     

Purification and properties of 5′-nucleotidase from alkalophilic Bacillus No. C-3

5′-Nucleotidase (EC 3. 1. 3. 5) from alkalophilic Bacillus no. C-3 was purified to homogeneity. The molecular weight of the enzyme was 80,000 by gel filtration. The optimum pH for the activity was 9.5, and the enzyme was stable at pH 9.5–10.5 in a buffer containing 10 mM 2-mercaptoethanol. Substrate specificity study revealed that the enzyme acted on 5′-AMP strongly, on several 5′-nucleotides and ADP to a certain extent, but not on 3′-nucleotides, 2′-nucleotides, p-nitrophenyl phosphate, or ATP. The K_m value for 5′-AMP was 3.0 × 10⁻⁴ M. The enzyme required no divalent cation for its activity. The enzyme was inhibited by borate and arsenite ions but not by 1 mM EDTA.     

Properties of spinach chloroplast fructose-1,6-diphosphatase

Photosynthetic fructose-1,6-diphosphatase (FDPase) fractions I and II, earlier purified from spinach leaves, show a similar amino acid composition, with the exception of a higher glutamic acid content in the latter. In both fractions glutamic and aspartic acids are the main amino acids. pH activity profiles of fractions I and II are similar, with optima at 8·65–8·70, both showing a high specificity for fructose- 1,6-diphosphate. These two fractions are Mg²⁺-dependent for activity, with an Optimum Mg²⁺ concentration of 10 mM in standard conditions, which shifts to 5 mM when the MG²⁺/EDTA ratio is increased to 10; Mn²⁺ and Co²⁺ are slightly active. EDTA enhances FDPase activity slightly, with an optimum at 0·4–0·8 mM. Cysteine has no activating effect, and acts as an inhibitor above 10 mM. Both I and II have an optimum substrate concentration of 4 mM, and the substrate inhibits at concns above this value. Kinetic velocity curves are sigmoidal, with the concave zone located in the range of physiological substrate concns. (Hill coefficient 1·75 for both). This suggests a strong regulatory role of fructose-1,6-diphosphate. K_m values are 1·4 × 10⁻³ M (fraction I) and 1·1 × 10⁻³ M (fraction II). The highest activity rate occurs at 60°, in accordance with the high thermostability of both fractions; the activation energies are 14·3 kcal/mol (fraction I) and 13·0 kcal/mol (fraction II).     

Identification of a17β-estradiol binding protein inCandida albicans andCandida (Torulopsis) glabrata

The presence of a corticosterone binding system inCandida albicans has been confirmed, and a specific binding system for17β-estradiol has been identified inC. albicans andC. glabrata. The dissociation constants (approximately 6 × 10⁻⁸M and approximately 2 × 10⁻⁸M, respectively) are in the range of those encountered in mammalian systems. Competition for binding sites was exhibited only by estrogenic steroids. Unlike the situation in mammalian systems, neither diethylstilbestrol nor tamoxifen was competitive. More detailed studies withC. albicans indicated that viable, intact fungal cells also incorporated17β-estradiol in a specific manner.     

Biochemical characterization of VQ-VII, a cysteine peptidase with broad specificity, isolated from Vasconcellea quercifolia latex

The latex from Vasconcellea quercifolia (“oak leaved papaya”), a member of the Caricaceae family, contains at least seven cysteine endopeptidases with high proteolytic activity, which helps to protect these plants against injury. In this study, we isolated and characterized the most basic of these cysteine endopeptidases, named VQ-VII. This new purified enzyme was homogeneous by bidimensional electrophoresis and MALDI-TOF mass spectrometry, and exhibited a molecular mass of 23,984 Da and an isoelectric point >11. The enzymatic activity of VQ-VII was completely inhibited by E-64 and iodoacetic acid, confirming that it belongs to the catalytic group of cysteine endopeptidases. By investigating the cleavage of the oxidized insulin B-chain to establish the hydrolytic specificity of VQ-VII, we found 13 cleavage sites on the substrate, revealing that it is a broad-specificity peptidase. The pH profiles toward p-Glu-Phe-Leu-p-nitroanilide (PFLNA) and casein showed that the optimum pH is about 6.8 for both substrates, and that in casein, it is active over a wide pH range (activity higher than 80 % between pH 6 and 9.5). Kinetic enzymatic assays were performed with the thiol peptidase substrate PFLNA (K _m = 0.454 ± 0.046 mM, k _cat = 1.57 ± 0.07 s^?1, k _cat/K _m = 3.46 × 10³ ± 14 s^?1 M^?1). The N-terminal sequence (21 amino acids) of VQ-VII showed an identity >70 % with 11 plant cysteine peptidases and the presence of highly conserved residues and motifs shared with the “papain-like” family of peptidases. VQ-VII proved to be a new latex enzyme of broad specificity, which can degrade extensively proteins of different nature in a wide pH range.     

S-adenosylmethionine: Protein-carboxyl methyltransferase from erythrocyte

Protein methylase II (S-adenosylmethionine:protein—carboxyl methyltrans-ferase), which modifies free carboxyl residues of protein, was purified from both rat and human blood, and properties of the enzymes were studied. The pH optima for the reaction were dependent on the substrate proteins used; pH 7.0 was found with endogenous substrate, 6.1 with plasma, 6.5 with γ-globulin, and 6.0 with fibrinogen. The molecular weight of the enzymes from both rat and human erythrocytes were identical (25,000 daltons) determined by Sephadex G-75 chromatography. Partially purified enzyme from rat erythrocytes showed three peaks on electrofocusing column at pH 4.9, 5.5 and 6.0. The K_m values of the enzymes from rat and human erythrocytes showed 3.1 × 10^?6m and 1.92 × 10^?6m at pH 6.0, 1.96 × 10^?6m and 1.78 × 10^?6m at pH 7.2, respectively, for S-adenosyl-l-methionine. It is also found that S-adenosyl-l-homocysteine is a competitive inhibitor for protein methylase II with K_i value of 1.6 × 10^?6m.     

Agrobacterium tumefaciens-mediated genetic transformation of mungbean (Vigna radiata L. Wilczek) — a recalcitrant grain legume

Agrobacterium-mediated transformation of Vigna radiata L. Wilczek has been achieved. Hypocotyl and primary leaves excised from 2-day-old in-vitro grown seedlings produced transgenic calli on B₅ basal medium supplemented with 5×10⁻⁶ M BAP, 2.5×10⁻⁶ M each of 2,4-D and NAA and 50 mg l⁻¹ kanamycin after co-cultivation with Agrobacterium tumefaciens strains, LBA4404 (pTOK233), EHA105 (pBin9GusInt) and C58C1 (pIG121Hm) all containing β-glucuronidase (gusA) and neomycin phosphotransferase II (nptII) marker genes. Transformed calli were found resistant to kanamycin up to 1000 mg^.l⁻¹. Gene expression of kanamycin resistance (nptII) and gusA in transformed calli was demonstrated by nptII assay and GUS histochemical analysis, respectively. Stable integration of T-DNA into the genome of transformed calli of mungbean was confirmed by Southern blot analysis. Transgenic calli could not regenerate shoots on B₅ or B₅ containing different cytokinins or auxins alone or in combination. However, for the first time, transformed green shoots showing strong GUS activity were regenerated directly from cotyledonary node explants cultured after co-cultivation with LBA4404 (pTOK233) on B₅ medium containing 6-benzylaminopurine (5×10⁻⁷ M) and 75 mg l⁻¹ kanamycin. The putative transformed shoots were rooted on B₅+indole-3-butyric acid (5×10⁻⁶ M) within 10–14 days and resulted plantlets subsequently developed flowers and pods with viable seeds in vitro after 20 days of root induction. The stamens, pollen grains and T₀ seeds showed GUS activity. Molecular analysis of putative transformed plants revealed the integration and expression of transgenes in T₀ plants and their seeds.     

Purification and Characterization of a Tripeptidase from Lactococcus lactis subsp. cremoris Wg2

A tripeptidase from a cell extract of Lactococcus lactis subsp. cremoris Wg2 has been purified to homogeneity by DEAE-Sephacel and phenyl-Sepharose chromatography followed by gel filtration over a Sephadex G-100 SF column and a high-performance liquid chromatography TSK G3000 SW column. The enzyme appears to be a dimer with a molecular weight of between 103,000 and 105,000 and is composed of two identical subunits each with a molecular weight of about 52,000. The tripeptidase is capable of hydrolyzing only tripeptides. The enzyme activity is optimal at pH 7.5 and at 55°C. EDTA inhibits the activity, and this can be reactivated with Zn²⁺, Mn²⁺, and partially with Co²⁺. The reducing agents dithiothreitol and β-mercaptoethanol and the divalent cation Cu²⁺ inhibit tripeptidase activity. Kinetic studies indicate that the peptidase hydrolyzes leucyl-leucyl-leucine with a K_m of 0.15 mM and a V_max of 151 μmol/min per mg of protein.