Intracellular Peptidase of Bac. subtilis

A peptidase was isolated from the cells of amylase-producing Bac. subtilis by means of cell lysis with egg white lysozyme, followed by freezing and thawing, salting out, dialysis and ion-exchanger column chromatography. The enzyme required manganese ion to show the enzyme activity. Also the enzyme was stable in the presence of magnesium ion. The enzyme hydrolyzed various synthetic peptides by stepwise removal of the amino terminal amino acid of peptides and thus the peptidase was found to be aminopeptidase.     

Biochemical properties of an extracellular trehalase from <Emphasis Type="Italic">Malbranchea pulchella</Emphasis> var. Sulfurea

The thermophilic fungus Malbranchea pulchella var. sulfurea produced good amounts of extracellular trehalase activity when grown for long periods on starch, maltose or glucose as the main carbon source. Studies with young cultures suggested that the main role of the extracellular acid trehalase is utilizing trehalose as a carbon source. The specific activity of the purified enzyme in the presence of manganese (680 U/mg protein) was comparable to that of other thermophilic fungi enzymes, but many times higher than the values reported for trehalases from other microbial sources. The apparent molecular mass of the native enzyme was estimated to be 104 kDa by gel filtration and 52 kDa by SDS-PAGE, suggesting that the enzyme was composed by two subunits. The carbohydrate content of the purified enzyme was estimated to be 19 % and the pi was 3.5. The optimum pH and temperature were 5.0–5.5 and 55° C, respectively. The purified enzyme was stimulated by manganese and inhibited by calcium ions, and insensitive to ATP and ADP, and 1 mM silver ions. The apparent K_M values for trehalose hydrolysis by the purified enzyme in the absence and presence of manganese chloride were 2.70±0.29 and 2.58±0.13 mM, respectively. Manganese ions affected only the apparent V_max, increasing the catalytic efficiency value by 9.2-fold. The results reported herein indicate that Malbranchea pulchella produces a trehalase with mixed biochemical properties, different from the conventional acid and neutral enzymes and also from trehalases from other thermophilic fungi.     

Purification and characterization of an aminopeptidase from bovine leukocytes

An aminopeptidase was purified about 4,000-fold from the clarified homogenate of bovine leukocytes by a series of column chromatographies on DEAE-cellulose, hydroxyapatite, Sephadex G-150, and DEAE-Toyopearl. The purified enzyme had a specific activity of 3.8 mumol X min-1 X mg-1 with arginine beta-naphthylamide (Arg-2-NNap) as substrate, and a minute amount of contaminating protein was found to be present by gel electrophoresis. The molecular weight of the enzyme was estimated to be 94,000 by gel filtration on Sephadex G-150. The enzyme had a broad substrate specificity and a pH optimum between 6.5 and 7.0 for the hydrolysis of alpha-aminoacyl beta-naphthylamides. It hydrolyzed beta-naphthylamides of basic, aliphatic, and aromatic amino acids, and also catalyzed the liberation of amino-terminal phenylalanine from phenylalanyl peptides. The enzyme was inhibited by bestatin, puromycin, 1,10-phenanthroline, sulfhydryl reagents, and a variety of heavy metal ions. Only the cobaltous ion stimulated the enzyme and the values of both Km and Vmax for Arg-2-NNap increased. In gross properties the present enzyme resembles porcine liver aminopeptidase reported previously (Kawata, S., et al. (1982) J. Biochem. 92, 1093-1101) very closely.     

Homocarnosinase: a hog kidney dipeptidase with a broader specificity than carnosinase.

A dipeptidase was isolated from hog kidney; it is the first enzyme described that has the capacity to cleave homocarnosine. It was purified to apparent homogeneity and split carnosine, anserine, and several other dipeptides in addition to homocarnosine. Homocarnosinase had a molecular weight of 57,000 as determined by sodium dodecyl sulfate-gel electrophoresis; it appeared to consist of a single polypeptide chain and did not contain sulfhydryl groups or serine residues essential to its activity. The enzyme was activated by Co²⁺ and by Mn²⁺, cobaltous ions being much more effective than manganous ions. Its isoelectric point was 5.6 and no evidence of isozymes was seen during isoelectric focusing. Homocarnosinase had a broader specificity, higher solubility, lower stability, and different metal ion sensitivity than hog kidney carnosinase (EC 3.4.13.3). Carnosinase was present in most tissues of the rat, whereas homocarnosinase was detected only in kidney, uterus, lung, and liver.     

Effects of divalent cations on encapsulation and release in the GroEL-assisted folding

Chaperonin GroEL assists protein folding in the presence of ATP and magnesium. Recent studies have shown that several divalent cations other than magnesium induce conformational changes of GroEL, thereby influencing chaperonin-assisted protein folding, but little is known about the detailed mechanism for such actions. Thus, the effects of divalent cations on protein encapsulation by GroEL/ES complexes were investigated. Of the divalent cations, not only magnesium, but also manganese ions enabled the functional refolding and release of 5,10-methylenetetrahydroforate reductase (METF) by GroEL. Neither ATP hydrolysis nor METF refolding was observed in the presence of zinc ion, whereas only ATP hydrolysis was induced by cobalt and nickel ions. SDS-PAGE and gel filtration analyses revealed that cobalt, nickel and zinc ions permit the formation of stable substrate-GroEL-GroES cis-ternary complexes, but prevent the release of METF from GroEL.     

Isolation and characterization of proline peptidase mutants of Salmonella typhimurium

The proline requirement of Salmonella typhimurium strain proB25 can be satisfied by either of the peptides Leu-Pro or Gly-Pro-Ala. A mutant derivative of strain proB25 isolated by penicillin selection in medium containing Leu-Pro as proline source fails to use either Leu-Pro or Gly-Pro-Ala as a source of proline. This strain is a double mutant that lacks two aminoacyl-proline-specific peptidases. One of these enzymes (peptidase Q) catalyzes the rapid hydrolysis of Leu-Pro but does not hydrolyze Gly-Pro-Ala or poly-l-proline. Mutations at a site (pepQ) near metE lead to loss of this activity. The other peptidase (peptidase P) catalyzes the hydrolysis of Gly-Pro-Ala and poly-l-proline but is only weakly active with Leu-Pro as substrate. This enzyme is similar to aminopeptidase P previously described in Escherichia coli (16). Mutations at a locus (pepP) near serA lead to loss of this enzyme.     

Proline-specific dipeptidyl peptidase from the blue blowfly Calliphora vicina hydrolyzes in vitro the ecdysiostatic peptide trypsin-modulating oostatic factor (Neb-TMOF)

To elucidate the mechanisms of inactivation of the ecdysiostatic peptide trypsin-modulating oostatic factor (Neb-TMOF) in the blue blowfly Calliphora vicina, we investigated its proteolytic degradation. In homogenates and membrane and soluble fractions, this hexapeptide (sequence: NPTNLH) was hydrolyzed into two fragments, NP and TNLH, suggesting the involvement of a proline-specific dipeptidyl peptidase. The dipeptidyl peptidase activity was highest in the late larval stage. It was purified 240-fold from soluble fractions of pupae of mixed age and classified on the basis of several catalytic properties as an invertebrate homologue of mammalian dipeptidyl peptidase IV (EC 3.4.14.5). Fly dipeptidyl peptidase IV has a molecular mass of 200 kDa, showed a pH optimum of 7.5–8.0 with the chromogenic substrate Gly-Pro-4-nitroanilide, and cleaved other chromogenic substrates with penultimate Pro or, with lower activity, Ala. It liberated Xaa-Pro dipeptides from the N-terminus of several bioactive peptides including substance P, neuropeptide Y, and peptide YY but not from bradykinin, indicating that the peptide bond between the two proline residues was resistant to cleavage. Fly dipeptidyl peptidase belongs to the serine class of proteases as the mammalian enzyme does; the fly enzyme, however, is not inhibited by several selective or nonselective inhibitors of its mammalian counterpart. It is suggested that dipeptidyl peptidases exert a regulatory role for the clearance not only of TMOF in flies but for other bioactive peptides in various invertebrates. Arch. Insect Biochem. Physiol. 37:146–157, 1998. © 1998 Wiley-Liss, Inc.     

Isolation and characterization of a new aminopeptidase from bovine lens

An aminopeptidase has been purified to homogeneity from bovine lens tissue by gel filtration and DEAE-cellulose chromatography. This enzyme has a molecular weight of 96,000 under both native and denaturing conditions. The purified enzyme hydrolyzed a variety of synthetic substrates as well as di-, tri-, and higher molecular weight peptides. Significantly this enzyme is capable of hydrolyzing arginine, lysine, and proline aminoacyl bonds. The pH optimum for activity and stability was 6.0. Both a reduced sulfhydryl group and a divalent metal ion are essential for activity. The native enzyme contains 1.6 mol of zinc and 1.0 mol of copper/mol of enzyme. No activation was seen upon incubation with either magnesium or manganese; however, heavy metal ions were inhibitory. Bestatin and puromycin were effective inhibitors and no endopeptidase activity could be detected in the purified preparation. This enzyme is clearly distinct from the lens leucine aminopeptidase, but rather, is identical to a cytosolic aminopeptidase III isolated from other tissues. Evidence is presented which argues that this enzyme may be the major lens aminopeptidase under in vivo conditions.     

Rat liver mitochondrial intermediate peptidase (MIP): purification and initial characterization.

A number of nuclearly encoded mitochondrial protein precursors that are transported into the matrix and inner membrane are cleaved in two sequential steps by two distinct matrix peptidases, mitochondrial processing peptidase (MPP) and mitochondrial intermediate peptidase (MIP). We have isolated and purified MIP from rat liver mitochondrial matrix. The enzyme, purified 2250-fold, is a monomer of 75 kDa and cleaves all tested mitochondrial intermediate proteins to their mature forms. About 20% of the final MIP preparation consists of equimolar amounts of two peptides of 47 kDa and 28 kDa, which are apparently the products of a single cleavage of the 75 kDa protein. These peptides are not separable from the 75 kDa protein, nor from each other, under any conditions used in the purification. The peptidase has a broad pH optimum between pH 6.6 and 8.9 and is inactivated by N-ethylmaleimide (NEM) and other sulfhydryl group reagents. The processing activity is divalent cation-dependent; it is stimulated by manganese, magnesium or calcium ions and reversibly inhibited by EDTA. Zinc, cobalt and iron strongly inhibit MIP activity. This pattern of cation dependence and inhibition is not clearly consistent with that of any known family of proteases.     

Purification and characterization of an X-prolyl-dipeptidyl peptidase from Lactobacillus sakei

An X-prolyl-dipeptidyl peptidase has been purified from Lactobacillus sakei by ammonium sulfate fractionation and five chromatographic steps, which included hydrophobic interaction, anion-exchange chromatography, and gel filtration chromatography. This procedure resulted in a recovery yield of 7% and an increase in specificity of 737-fold. The enzyme appeared to be a dimer with a subunit molecular mass of approximately 88 kDa. Optimal activity was shown at pH 7.5 and 55 degrees C. The enzyme was inhibited by serine proteinase inhibitors and several divalent cations (Cu(2+), Hg(2+), and Zn(2+)). The enzyme almost exclusively hydrolyzed X-Pro from the N terminus of each peptide as well as fluorescent and colorimetric substrates; it also hydrolyzed X-Ala at the N terminus, albeit at lower rates. K(m) s for Gly-Pro- and Lys-Ala-7-amido-4-methylcoumarin were 29 and 88 microM, respectively; those for Gly-Pro- and Ala-Pro-p-nitroanilide were 192 and 50 microM, respectively. Among peptides, beta-casomorphin 1-3 was hydrolyzed at the highest rates, while the relative hydrolysis of the other tested peptides was only 1 to 12%. The potential role of the purified enzyme in the proteolytic pathway by catalyzing the hydrolysis of peptide bonds involving proline is discussed.     

Kinetic and physical properties of Co2+ enolase

The activation of yeast enolase by cobaltous ion in 0.1 M KCl is characterized by an activation constant of 1 microM and an inhibition constant of 18 microM. Measurements of binding of Co2+ to the apoenzyme show that a maximum of four Co2+ ions are bound per dimer in the presence or absence of substrate although binding is far tighter in the presence of substrate. Ultraviolet spectral titrations show evidence for a conformational change due exclusively to the binding of the first two ions of Co2+. Both visible and EPR spectra confirm that the environment of the first pair of cobalt ions ("conformational sites") is markedly different from that of the second pair in the "catalytic" sites. Cobalt at the conformational site appears to be a tetragonally distorted octahedral complex while the second pair of metal ions appears to be in a more regular tetrahedral symmetry. Addition of either Mg2+ or substrate to the enzyme with only one pair of cobalt ions per dimer causes striking changes in the metal ion environment. The conformational metal sites appear sufficiently shielded from solvent to be inaccessible to oxidation by H2O2, in contrast to the second pair of cobaltous ions whose ready oxidation by H2O2 inactivates the enzyme. Comparison of kinetic and binding data suggests that only one site of the dimeric enzyme can be active, since activity requires more than two metals bound per dimer and inactivation results from the binding of the fourth ion per dimer.     

The metalloenzymic nature of collagenase-like peptidase of the rat testis.

Collagenase-like peptidase, an enzyme degrading synthetic collagenase substrate (PZ-pentapeptide), was purified from rat testes and its properties were examined. Its activity was strongly inhibited by chelating agents, such as EDTA and 1,10-phenanthroline. By chelation and exhaustive dialysis it was possible to obtain this enzyme in its inactive, metal-free form. The activity of the metal-free enzyme was partly recovered by treatment with zinc or manganese ions, while a combined zinc and manganese treatment resulted in complete recovery of enzyme activity.     

Pentose Metabolism by Candida utilis

Xylose isomerase, an enzyme isomerizing xylose to xylulose, was produced adaptively when Candida utilis, a fodder yeast, was grown in a medium containing xylose. This experiment was carried out in order to isolate and purify the enzyme, and to clarify properties of the enzyme. As a result, it was revealed that the enzyme could be solubilized by plasmolysis, and was purified by dialysis, salting out with ammonium sulfate, precipitating with acetone, and adsorbing to calcium phosphate gel. The enzyme requires some divalent metal ions for its action and among them manganese ion is the most effective. The enzyme is inhibited particularly by ethylenediamine-tetraacetic acid and has somewhat thermotolerant character.     

Peripheral inactivation of neurotensin. Isolation and characterization of a metallopeptidase from rat ileum

A peptidase that inactivated neurotensin by cleaving the peptide at the Pro10-Tyr11 bond, generating the biologically inactive fragments neurotensin(1-10) and neurotensin(11-13) was purified from whole rat ileum homogenate. The purified enzyme behaved as a 70-75-kDa monomer as determined by SDS-PAGE analysis in reducing or non-reducing conditions and gel permeation on Ultrogel AcA34. The peptidase was insensitive to thiol-blocking agents and acidic and serine protease inhibitors but could be strongly inhibited by 1,10-phenanthroline, EDTA, dithiothreitol and heavy metal ions such as zinc, copper and cobalt. Zinc was the only divalent cation able potently to reactivate the apoenzyme. This enzyme could be distinguished from endopeptidases EC 3.4.24.15 and EC 3.4.24.11, angiotensin-converting enzyme, proline endopeptidase, aminopeptidase and pyroglutamyl-peptide hydrolase since it was not affected by micromolar concentrations of their specific inhibitors. The peptidase displayed a high affinity for neurotensin (1.6 microM). Studies concerning the specificity of the enzyme towards the sequence of neurotensin established the following. (a) Neurotensin(9-13) was the shortest partial sequence that fully inhibited tritiated neurotensin degradation; shortening the C-terminal part of the neurotensin molecule led to inactive fragments. (b) Amidation of the C-terminal end of the peptide did not prevent the recognition by the peptidase. (c) There existed a strong stereospecificity of the peptidase for the residues in positions 8, 9 and 11 of the neurotensin molecule. (d) Pro-Xaa dipeptides (where Xaa represented aromatic or hydrophobic residues) were the most potent inhibitors of tritiated neurotensin degradation while all the Xaa-Pro dipeptides tested were totally ineffective. (e) The neurotensin-related peptides: neuromedin N, xenopsin and [Lys8-Asn9]neurotensin(8-13), as well as angiotensins I and II and dynorphins(1-8) and (1-13) were as potent as neurotensin in inhibiting [3H]neurotensin hydrolysis.     

Apparent dipeptidyl peptidase activities of acylamino acid-releasing enzymes

An acylamino acid-releasing enzyme purified from porcine liver showed peptidase activity above pH 8. Of the non-acylated peptides tested, this peptidase activity was only exerted on peptides with Gly or Ala at their N-termini. These results are consistent with the previous observations for similar enzymes from sheep red blood cells (Witheiler, J. & Wilson, D.B. (1972) J. Biol. Chem. 247, 2217-2221) and beef liver (Gade, W. & Brown, J.L. (1978) J. Biol. Chem. 253, 5012-5018). The pH dependence of the peptidase activity showed that only peptides with uncharged N-terminal amino acids such as glycyl- or alanyl-peptides act as substrates for the enzyme. These results suggest that the peptidase activity seen for the acylamino acid-releasing enzyme is an intrinsic activity of the enzyme that is triggered by misrecognition of uncharged smaller N-terminal amino acids in non-acylated peptides as acyl groups at higher pHs.     

Metal ion effects on target sites of modification in metallocarboxypeptidase B

Native carboxypeptidase B and its Co2+-substituted derivative were oxidized by the active-site-directed agent m-chloroperbenzoic acid. The following results were obtained a) In the cobalt enzyme there was a decrease in both the peptidase and the esterase activities, whereas in the zinc enzyme only the peptidase activity decreased. Peptide or ester pseudo-substrates protected the cobalt enzyme but not the zinc enzyme against inactivation. b) Upon oxidation and formation of Co3+, cleavage of peptide bonds occurred in the cobalt enzyme but not in the zinc enzyme. Both enzymes retained their original metal content. c) Following oxidation of the enzymes, amino acid analysis revealed a modification of a methionyl residue in the zinc enzyme only; the cobalt enzyme, on the other hand, showed a modification of a histidyl residue. d) Peptide mapping of the enzymes after cleavage by cyanogen bromide indicated that two methionyl peptides were missing in the oxidized zinc enzyme. These peptides point to Met-64 as the site of modification. The peptide map of the oxidized cobalt enzyme was similar to that of the unmodified native (i.e., zinc) enzyme. These studies indicate that the specific metal ion present in the enzyme imposes certain structural and functional differences on the active site, leading to differing reactivities of specific amino acid residues and to a different alignment of the active-site-directed reagent in the two enzymes.     

Rat osseous plate alkaline phosphatase: mechanism of action of manganese ions

Polidocanol-solubilized osseous plate alkaline phosphatase was modulated by manganese ions in a similar way as by zinc ions. For concentrations up to 1.0 nm, the enzyme was stimulated by manganese ions, showing site-site interactions (n = 2.2). However, larger concentrations (> 0.1 m) were inhibitory. Manganese ions could play the role of zinc ions stimulating the enzyme synergistically in the presence of magnesium ions (K _d = 7.2 m; V = 1005.5 U mg^–1). Manganese ions could also play the role of magnesium ions, stimulating the enzyme synergistically in the presence of zinc ions (K _d = 2.2 m; V = 1036.7 U mg^–1). However, manganese ions could not substitute for zinc and magnesium at the same time since ion assymetry is necessary for full activity of the enzyme. A steady-state kinetic model for the modulation of enzyme activity by manganese ions is proposed.     

Enzymatic Conversion of d-Glucose to d-Fructose

Glucose isomerizing enzyme was partially purified after investigation on the properties of crude enzyme extract. The crude extract was partly inactivated by the contact with air. The addition of manganese was effective to improve the stability. Magnesium was essential to the enzyme action and cobalt accelerated the reaction.

The maximal activity was observed at pH about 7.6 and 50°C was optimal for the incubation time of 30 minutes. The enzyme solution reacted with d-xylose as well as d-glucose. The activity of the enzyme was inhibited at high glucose concentrations.

An enzyme which catalyzes the conversion of d-glucose to d-fructose has been demonstrated in cell-free extracts of Streptomyces phaeochromo genus grown in the presence of D-xylose. The enzyme preparation reacts with d-glucose and d-xylose, but not with other sugars tested. It appears to require magnesium for the maximal activity and the addition of cobaltous ion remarkably intensifies the heat tolerance of the enzyme. The maximal activity occurs at about pH 9.3~9.5. Equilibrium is reached when about 52% fructose is present in the reaction mixture. The enzyme has half-maximal activity when the concentration of d-glucose is about 0.3 M at pH 9 and 60°C.