Cleavage of di- and tripeptides by Prevotella ruminicola

The final step in the conversion of protein to amino acids by the common Gram-negative rumen bacterium, Prevotella (formerly Bacteroides) ruminicola , is the cleavage of di- and tripeptides. Dipeptidase and tripeptidase activities were predominantly cytoplasmic, and toluene treatment increased the rate of Ala2 and Ala3 hydrolysis by whole cells, suggesting that transport limited the rate of hydrolysis of extracellular di- and tripeptides. The hydrolysis of Ala2 and Ala3 by whole cells was not affected by protonophores, ionophores or dicyclohexylcarbodiimide, but Ala2 hydrolysis by EDTA-treated cells was inhibited by the Ca2+/H+ ionophore, tetronasin. Ala3 hydrolysis was not affected by protonophores or ionophores in EDTA-treated cells. The dipeptidase of strain M384 was inhibited > 99% by 1,10-phenanthroline and 39% by EDTA but not other protease inhibitors, consistent with the enzyme being a metalloprotease. Tripeptidase was insensitive to protease inhibitors, except for a 33% inhibition by EDTA. Cleavage of tripeptides occurred at the bond adjacent to the N-terminal amino acid. Distinct di-, tri- and oligopeptidase peaks were obtained by anion-exchange liquid chromatography of disrupted cells. Banding patterns on native PAGE using activity staining also indicated that P. ruminicola M384 had separate single dipeptidase and tripeptidase enzymes which hydrolysed a range of peptides. The dipeptidase of strain M384 was different from other strains of P. ruminicola: strains GA33 and B(1)4 had activities which ran at the same R(f); strain GA33 had another band of lower activity; strain 23 had two bands different from those of the other strains. The tripeptidases ran at the same R(f) for the different strains. Dipeptidase activity of all strains was inhibited by 1,10-phenanthroline on gels. Gel permeation chromatography indicated that the M(r) of the dipeptidases from strains M384 and B(1)4 were 115,000 and 114,500 respectively, and 112,500 and 121,500 for the corresponding tripeptidases. Thus the metabolism of small peptides by P. ruminicola involves separate permeases and intracellular peptidases for di- and tripeptides.     

Purification and Properties of a Type II-like Dipeptidyl Peptidase from the Ruminal Peptidolytic Bacterium,Prevotella albensis M384

A dipeptidyl peptidase which hydrolyses the synthetic dipeptidyl peptidase (DPP) substrate, Ala₂- p -nitroanilide, was purified 193-fold from the ruminal peptidolytic bacterium, Prevotella albensis M384. The enzyme was a homodimer of molecular mass 91 kDa. Its activity against Ala₂- p -nitroanilide had optimal pH and temperature of 7.2 and 40°C respectively. Enzyme activity was inhibited by the serine protease inhibitors, PMSF and dichloroisocoumarin, but not by inhibitors of other categories of proteases. Synthetic substrates for DPP-1 (GlyArg- p -nitroanilide, GlyArg-4-methoxy-naphthylamide), DPP-3 (ArgArg-4-methoxynaphthylamide) and DPP-4 (GlyPro-4-methoxynaphthylamide) or for leucine or alanine aminopeptidase were not hydrolysed, nor were di- or tripeptides. N-Acetyl-Ala₂- p -nitroanilide was not hydrolysed. Oligopeptides with Ala, Ile, Ser or Val adjacent to the N-terminal amino acid were all hydrolysed, while peptides with basic or acidic residues in the same position were not. The purified DPP from P. albensis is therefore most similar in its catalytic properties to mammalian DPP-2.     

Breakdown of different peptides byPrevotella (Bacteroides) ruminicola and mixed microorganisms from the sheep rumen

Several di-, tri-, and oligopeptides were incubated individually in vitro with rumen fluid from two sheep receiving a mixed grass hay/concentrate diet and with washed cells ofPrevotella (formerlyBacteroides)ruminicola M384 andP. ruminicola B₁4. The rates of breakdown of most peptides were similar in the rumen fluid from the two sheep. Acidic and proline-containing peptides tended to be more slowly degraded than neutral or basic peptides. The dipeptide at the N-terminus of higher peptides was observed as an early product of hydrolysis, confirming that a dipeptidyl aminopeptidase type of activity was present. The relative rates of breakdown of dipeptides byP. ruminicola were different from that of rumen fluid, but the hydrolysis of higher peptides followed a similar pattern, and dipeptides from the N-terminus were detected as early products.     

Subcellular localisation of di- and tripeptidases in guinea pig and rat enterocytes.

Enterocytes were isolated from rat and guinea pig jejunum and subcellular fractions were prepared by density gradient centrifugation. Gradient fractions were assayed for principal organelle marker enzymes and for di- and tripeptidases. The hydrolases showed a dual localisation with both brush border and cytosol components. In the rat, approximately equal portions of dipeptidase activities were found in the two fractions but, in the guinea pig, three times more activity were found in the two fractions but, in the guinea pig, three times more activity was found in the soluble than in the brush border fractions. Cytosol components in the rat were markedly inhibited by p-hydroxymercuribenzoate. In both species tripeptidase, leucyl-2-naphthylamidases and gamma-glutamyltransferase activities were found predominantly in the brush border fractions.     

Distribution,properties, and functions of midgut carboxypeptidases and dipeptidases from Musca domestica larvae

Dipeptidase and carboxypeptidase A activities were determined in cells and luminal contents of the fore-, mid-, and hind-midgut of Musca domestica larvae. Dipeptidase activity was found mainly in hind-midgut cells, whereas carboxy-peptidase activity was recovered in major amounts in both cells and in luminal contents of hind-midguts. The subcellular distribution of dipeptidase and part of the carboxypeptidase A activities is similar to that of a plasma membrane enzyme marker (aminopeptidase), suggesting that these activities are bound to the microvillar membranes. Soluble carboxypeptidase A seems to occur both bound to secretory vesicles and trapped in the cell glycocalyx. Based on density-gradient ultracentrifugation and thermal inactivation, there seems to be only one molecular species of each of the following enzymes (soluble in water or solubilized in Triton X-100): membrane-bound dipeptidase (pH optimum 8.0; Km 3.7 mM GlyLeu, M_r 111,000), soluble carboxypeptidase (pH optimum 8.0; Km 1.22 mM N-carbobenzoxy-glycyl-L-phenylalanine (ZGlyPhe), M_r45,000) and membrane-bound carboxypeptidase (pH optimum 7.5, Km 2.3 mM ZGlyPhe, M_r58,000). The results suggest that protein digestion is accomplished sequentially by luminal trypsin and luminal carboxypeptidase, by membrane-bound carboxypeptidase and aminopeptidase, and finally by membrane-bound dipeptidase.     

Phenotypic Characterization of Polysaccharidases Produced by Four Prevotella Type Strains

Four ruminal Prevotella type strains, P. ruminicola JCM8958^T, P. bryantii B₁4^T, P. albensis M384^T, and P. brevis ATCC19188^T, were characterized for polysaccharide-degrading activities with the reducing sugar release assay and zymogram analyses. Carboxymethylcellulase, xylanase, and polygalacturonate (PG)-degrading enzyme activities were determined in cultures grown on oat spelt xylan, xylose, arabinose, cellobiose, and glucose as sole growth substrates. P. ruminicola and P. albensis showed carboxymethylcellulase induction patterns. When xylan was supplied as a sole growth substrate, xylanase activities produced by P. bryantii and P. albensis were at least 18- and 11-fold higher, respectively, than during growth on other carbohydrates, suggesting that the regulation of the xylanases was highly specific to xylan. All strains constitutively produced PG-degrading enzymes. The corresponding activity of P. bryantii was more than 40-fold higher than in other strains. Zymogram analyses routinely detected the presence of high-molecular-weight (100–170 kDa) polysaccharide-degrading enzymes in ruminal Prevotella. Characteristics of the polysaccharide-degrading activities showed diversity of ruminal Prevotella species. Received: 29 November 1999 / Accepted: 1 February 2000     

Identification of slowly metabolized sugars and sugar derivatives that could be used to establish new or modified microbial species in the rumen

For identification of compounds that could potentially be used to sustain a population of new or genetically modified organisms in the rumen, the rates of metabolism of several sugars and sugar derivatives were measured in ovine rumen fluid in vitro. Several sugars and sugar alcohols, including sorbitol, xylitol, dulcitol, arabinose, ribose, and maltitol, were degraded slowly and were therefore identified as candidates for use in this new manipulation strategy. None of the rumen bacteria,Bacteroides amylophilus WP91,Bacteroides ruminicola M384,Butyrivibrio fibrisolvens JW11,Lactobacillus casei LB17,Megasphaera elsdenii J1,Selenomonas ruminantium Z108, orStreptococcus bovis C277, grew on the above sugar alcohols, and onlyB. ruminicola M384 andL. casei LB17 grew significantly on the pentoses. The non-rumen strain,Escherichia coli ML308, grew rapidly on dulcitol and sorbitol; this suggests a possible role forE. coli in manipulation of rumen fermentation.     

Glutathione-degrading enzymes of microvillus membranes

Microvillus membranes from rat kidney, jejunum, and epididymis have been purified by the Ca precipitation method. The membranes exhibit enrichment in specific activities of gamma-glutamyl transpeptidase, aminopeptidase M, and a dipeptidase. The latter has been characterized and shown to be the principal activity responsible for the hydrolysis of S derivatives of Cys-Gly (including cystinyl-bis-glycine (Cys-bis-Gly) and 5-hydroxy-6-S-cysteinylglycyl-1-7,9-trans-11,14-cis-eicosatetraenoic acid (leukotriene D4)). A method is described for the simultaneous purification of papain-solubilized forms of the three enzymes from renal microvilli. Dipeptidase (Mr = 105,000) appears to be a zinc metalloprotein composed of two Mr = 50,000 subunits. The enzyme is severalfold more effective in the hydrolysis of dipeptides than aminopeptidase M. Dipeptidase, in contrast to aminopeptidase M, is inhibited by thiol compounds; Cys-Gly, in particular, is a potent inhibitor (Ki = 20 microM). The inhibition of dipeptidase by thiols has been employed to probe the relative significance of dipeptidase and aminopeptidase M in the metabolism of glutathione and its derivatives at the membrane surface.     

Purification and properties of human pancreas dipeptidase

Dipeptidase [EC 3.4.13] was purified from human pancreas; the activity was followed with L-Leu-L-Leu as a substrate. Polyacrylamide gel electrophoresis showed that the final preparation was homogeneous. The molecular weight of the dipeptidase was estimated to be 135,000 by gel filtration. From the result of SDS-polyacrylamide gel electrophoresis, it was found that the enzyme consisted of two subunits with equal molecular weights of 68,000. By atomic absorption analysis, the dipeptidase was shown to be a zinc metalloenzyme containing one atom of zinc for each subunit. Cu2+ and Hg2+ (1 mM) inhibited the enzyme by 50%. o-Phenanthroline strongly inhibited the enzyme. The dipeptidase hydrolyzed dipeptides such as L-Ala-L-Ala, L-Met-L-Met, L-Ala-L-Leu, L-Leu-Gly, and L-Leu-L-Leu but did not hydrolyze tripeptides, Bz-amino acids, CBz-amino acids, or L-amino acid beta-naphthylamides. The dipeptidase from human pancreas was immunologically distinct from human liver dipeptidase.     

Antibiotic resistance patterns and plasmids of ruminal strains of Bacteroides ruminicola and Bacteroides multiacidus

Summary Plasmids were recovered and are described from three ruminal Bacteroides strains — 23, 223/M2/7 and 46/5(2). Although all three were originally isolated as Bacteroides ruminicola, 46/5(2) is shown here to be a strain of Bacteroides multiacidus, a species not previously described from the rumen. An 11.7 kbp plasmid present in strain 46/5(2) gave the same digestion pattern with Sal I and Sma I as a plasmid in B. multiacidus subgroup b type strain P208-58. Strains 46/5(2) and P208-58 both showed resistance to tetracycline, as did B. ruminicola strain 223/M2/7. B. multiacidus P208-58, and, to a lesser degree, B. ruminicola 23, showed resistance to ampicillin. Four other strains of B. ruminicola and one of B. multiacidus in which plasmids were not detected were sensitive to both antibiotics. It has still to be established whether these resistance traits are plasmid or chromosomally coded.     

Purification and Characterization of Dipeptidase Hydrolyzing L-Cysteinylglycine from Radish Cotyledon

Dipeptidase activity was detected in the soluble fraction of radish (Raphanus sativus L.) cotyledon, and the purified enzyme had a specific activity of 7.32 nkat/mg protein for hydrolyzing L-cysteinylglycine. The dipeptidase was found to be a hexameric metalloenzyme, composed of homological 55 kDa-subunits. This is the first glutathione catabolism-related dipeptidase isolated from higher plants.     

Peptidase-deficient mutants of Escherichia coli.

Mutant derivatives of Escherichia coli K-12 deficient in several peptidases have been obtained. Mutants lacking a naphthylamidase, peptidase N, were isolated by screening for colonies unable to hydrolyze L-alanine beta-naphthylamide. Other mutants were isolated using positive selections for resistance to valine peptides. Mutants lacking peptidase A, a broad-specificity aminopeptidase, were obtained by selection for resistance to L-valyl-L-leucine amide. Mutants lacking a dipeptidase, peptidase D, were isolated from a pepN pepA strain by selection for resistance to L-valyl-glycine. Starting with a pepN pepA pepD strain, selection for resistance to L-valyl-glycyl-glycine or several other valine peptides produced mutants deficient in another aminopeptidase, peptidase B. Mutants resistant to L-valyl-L-proline lack peptidase Q, an activity capable of rapid hydrolysis of X-proline dipeptides. Using these selection procedures, a strain (CM89) lacking five different peptidases has been isolated. Although still sensitive to valine, this strain is resistant to a variety of valine di- and tripeptides. The ability of this strain to use peptides as sources of amino acids is much more restricted than that of wild-type E. coli strains. Strains containing only one of the five peptidases missing in CM89 have been constructed by transduction. The peptide utilization profiles of these strains show that each of the five peptidases can function during growth in the catabolism of peptides.     

Diversity of Extracellular Proteolytic Activities Among Prevotella Species from the Rumen

The current research was aimed at comparing proteolytic activities among ruminal Prevotella spp. Growth rates of Prevotella sp. 2202, Prevotella ruminicola D31d, P. brevis GA33, P. albensis M384, and P. bryantii B₁4 varied with N source, and no one N source produced the fastest growth in all species. Proteolytic activity was greatest with casein compared with peptides, AA, and NH₄Cl in all species. Proteolytic activity of Prevotella sp. 2202, P. brevis GA33, and P. bryantii B₁4 was modulated by N source. With gelatin co-polymerized SDS-PAGE, the extracellular activities of the Prevotella spp. showed wide variation in number, size, and type of proteases. Prevotella sp. 2202 and P. albensis M384 produced metalloproteases of low molecular weight (40 kDa). P. ruminicola D31d produced one cysteine protease (100–200 kDa) and two metalloproteases (90–100 kDa). P. brevis GA33 generated a diffuse clearing zone (95–160 kDa) containing serine, cysteine, and metalloproteases. P. bryantii B₁4 produced a metalloprotease greater than 200 kDa in size. The molecular sizes provided are estimations and served only to differentiate among the bacterial species in this study. Large variations in proteolytic activities among species and the known genetic diversity of the Prevotella taxon suggested that targeting this bacterial assemblage for genetic manipulation in order to alter the bacterial impact on ruminal protein degradation would be difficult. Received: 12 January 1999 / Accepted: 19 May 1999     

Factors affecting glucose uptake by the ruminal bacterium Bacteroides ruminicola

Summary Glucose uptake by whole cells of Bacteroides ruminicola B₁4 is constitutive. Potassium concentrations between 10 and 150 mm stimulated uptake over fourfold, while sodium had little effect on uptake. The involvement of potassium in glucose uptake by B. ruminicola was supported by strong inhibition of uptake by the ionophores valinomycin, lasalocid, and monensin. The electron transport inhibitor antimycin A had little effect on uptake, but menadione and acriflavine inhibited uptake by 30 and 48%, respectively. Potent inhibitors of uptake included oxygen, p-chloromercuribenzoate, HgCl₂, and o-phenanthroline. Sodium arsenate decreased uptake by 40%, suggesting that a high-energy phosphate compound and possibly a binding protein may be involved in glucose uptake. The protonophores carbonyl cyanide m-chlorophenylhydrazone and 2,4-dinitrophenol inhibited glucose uptake by 37 and 22%, respectively. Little change in uptake activity was observed at extracellular pH values between 4.0 and 8.0. Excess (10 mm) cellobiose, maltose, and sucrose inhibited glucose uptake less than 15%. High levels (0.15% w/v) of p-coumaric acid and vanillin decreased uptake by 32 and 37%, respectively, while 0.15% ferulic acid decreased uptake by 15%.     

Soluble di- and aminopeptidases in Escherichia K-12. Dispensible enzymes.

As part of a study of the peptidase content of Escherichia coli K-12, two peptidase-deficient amino acid auxotrophs isolated and characterized by Miller as pepD- (strain CM17) and pepD- pepN- pepA- pepB- pepQ- (strain CM89) were examined for the presence of several peptidases previously obtained from strain K-12 in this laboratory. The soluble fraction of each mutant was found to lack the broad-specificity strain K-12 dipeptidase DP and the strain CM89 fraction also lacked activity characteristic of the strain K-12 aminopeptidases AP, L, and OP; like strain CM17, strain CM89 contained the tripeptide-specific aminopeptidase TP. Strain CM89 (but not CM17) appeared to contain little if any activity attributable to the ribosome-bound aminopeptidase I of strain K-12. Whereas loss of DP, AP, OP, and aminopeptidase I activity may be attributed to the pepD-, pepB-, pepN-, and pepA- mutations, respectively, the reason for the loss of L activity remains uncertain. Grown responses of strain CM89 in liquid media containing di- or tripeptides were in accord with absence of enzymes catalyzing rapid hydrolysis of dipeptides. In synthetic liquid media supplemented with the required amino acids per se or with peptone, cultures of both CM strains grew more slowly than strain K-12 and produced smaller cell-yields than those produced by strain K-12.     

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.     

Purification of Peptidases of Aspergillus oryzae and Some Properties of the Purified Peptidases

The purification of Aspergillus oryzae peptidases was attempted by the fractional precipitation with acetone, ammonium sulphate, and by starch zone electrophoresis. We, thus, achieved a great success in the separation of dipeptidase free from aminopolypeptidase and proteinase as well as in the separation of aminopolypeptidase free from dipeptidase and proteinase.

The specific activity (C₀) of the former (leucylglycine hydrolysis) was 7000 and that of the latter (leucylglycylglycine hydrolysis) 22000.

The leucylglycine dipeptidase was remarkably activated by Zn⁺⁺, and Co⁺⁺. Some other enzyme properties were also found and are discussed.     

Selected low-cohesion variants of Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus lack distinct antigens recognized by human antibodies

Actinobacillus actinomycetemconitans OMZ 346 A and Haemophilus aphrophilus OMZ 384 A, isolated on a synthetic selective and differentiating agar, show the highly cohesive and wall adherent growth in liquid medium which is typical for all primary oral isolates of these species. From each of them a low cohesion variant, OMZ 346 F and OMZ 384 F, respectively, was obtained by selection for cells growing in suspension. Screening of Western blots of these four strains with several human sera revealed the loss of a 4000 M_r antigen in both F strains. Human antibodies bound to the 400 M_r band material on preparative Western blots of the A strains were cluted with 4M magnesium chloride. These antibodies showed no cross-reaction between the 4000 M_r material of the two closely related species.     

1H nuclear magnetic resonance studies of histidine-containing di- and tripeptides. Estimation of the effects of charged groups on the pKa value of the imidazole ring.

The nmr titration curves of chemical shifts versus pH were observed for the protons of various histidine-containing di- and tripeptides. With these results, the macroscopic pK_a values and the chemical shifts intrinsic to each ionic species were determined by a computer curve-fitting based on a simple acid dissociation sequence. The pK_a value of the imidazole ring in N-acetyl-L -histidine methylamide was assumed to represent the intrinsic (or unperturbed) pK_a of the imidazole rings of histidine having peptide linkages at both the CO and NH sides. The pK_a values of the imidazole rings observed for most di- and tripeptides were reasonably reproduced by simple calculations using the intrinsic value and the perturbations due to the CO₂^? and NH₃⁺ groups located at various positions. Some other factors affecting the pK_a value of the imidazole ring are also discussed.