Peptide utilization by nitrogen-starved Neurospora crassa

Peptides ranging in size from a mean number of 30 residues down to dipeptides supported growth of a leucine auxotroph when used as both a nitrogen and leucine source. Under nitrogen-limiting conditions, the peptides induced extracellular peptidohydrolytic activity, hydrolyzing peptides to monomer amino acids. Growth of a leu-2 mutant of Neurospora crassa on those peptides transportable by the oligopeptide transport system did not result in induction of hydrolytic activity, whereas growth of a leu-2; gltR mutant on these same peptides resulted in induction of peptidohydrolytic activity. The induced extracellular proteolytic activity was shown to be analogous to that inducible by growth on proteins, e.g., bovine serum albumin.     

Protease and peptidase activities in growing and sporulating cells and dormant spores of Bacillus megaterium.

Peptidase and protease activities on many different substrates have been determined in several stages of growth of Bacillus megaterium. Extracts of log-phase cells, sporulating cells, and dormant spores of B. megaterium each hydrolyzed 16 different di- and tripeptides. The specific peptidase activity was highest in dormant spores, and the activity in sporulating cells and log-phase cells was about 1.2-fold and 2- to 3-fold lower, respectively. This peptidase acticity was wholly intracellular since extracellular peptidase activity was not detected throughout growth and sporulation. In contrast, intracellular protease activity on a variety of common protein substrates was highest in sporulating cells, and much extracellular activity was also present at this time. The specific activity of intracellular protease in sporulating cells was about 50- and 30-fold higher than that in log-phase cells and dormant spores, respectively. However, the two unique dormant spores proteins known to be the major species degraded during spore germination were degraded most rapidly by extracts of dormant spores, and slightly slower by extracts from log-phase or sporulating cells. The specific activities for degradation of peptides and proteins are compared to values for intracellular protein turnover during various stages of growth.     

A peptidase activity exhibited by human serum pseudocholinesterase

The identity of a peptidase activity with human serum pseudocholinesterase (PsChE) purified to apparent homogeneity was demonstrated by co-elution of both peptidase and PsChE activities from procainamide-Sepharose and concanavalin-A--Sepharose affinity chromatographic columns; comigration on polyacrylamide gel electrophoresis; co-elution on Sephadex G-200 gel filtration and coprecipitation at different dilutions of an antibody raised against purified PsChE. The purified enzyme showed a single protein band on gel electrophoresis under non-denaturing conditions. SDS gel electrophoresis under reducing conditions, followed by silver staining, also gave a single protein band (Mr approximately equal to 90,000). Peptidase activity using different peptides showed the release of C-terminal amino acids. Blocking the carboxy terminal by an amide or ester group did not prevent the hydrolysis of peptides. There was no evidence for release of N-terminal amino acids. Potent anionic or esterase site inhibitors of PsChE, such as eserine sulphate, neostigmine, procainamide, ethopropazine, imipramine, diisopropylfluorophosphate, tetra-isopropylpyrophosphoramide and phenyl boronic acid, did not inhibit the peptidase activity. An anionic site inhibitor (neostigmine or eserine) in combination with an esterase site inhibitor (diisopropylfluorophosphate) also did not inhibit the peptidase. However, the choline esters (acetylcholine, butyrylcholine, propionylcholine, benzoylcholine and succinylcholine) markedly inhibited the peptidase activity in parallel to PsChE. Choline alone or in combination with acetate, butyrate, propionate, benzoate or succinate did not significantly inhibit the peptidase activity. It appeared that inhibitor compounds which bind to both the anionic and esteratic sites simultaneously (like the substrate analogues choline esters) could inhibit the peptidase activity possibly through conformational changes affecting a peptidase domain.     

Isolation and characterization Salmonella typhimurium mutants lacking a tripeptidase (peptidase T).

Salmonella typhimurium contains an enzyme, peptidase T, that hydrolyzes a variety of tripeptides. Specificity studies with a peptidase activity stain after gel electrophoresis of crude cell extracts showed that peptidase T hydrolyzes tripeptides containing N-terminal methionine, leucine, or phenylalanine. Little or no activity could be detected against dipeptides, N-blocked or C-blocked tripeptides, and tetrapeptides. Analysis of reaction products by high-pressure liquid chromatography showed that peptidase T removes the N-terminal amino acid from tripeptides. Mutants lacking peptidase T were isolated by screening microcultures grown in the wells of plastic microtitration plates for hydrolysis of Met-Ala-Ser or Met-Gly-Gly. Mutations (pepT) that eliminate this enzyme were found to be phage P22 cotransducible with purB at approximately 25 map units on the S. typhimurium map. Comparison of the growth properties of mutant and wild-type strains suggests that peptidase T does not function in utilization of tripeptides to provide amino acids during growth.     

Compartmentation of free amino acids for protein biosynthesis. Influence of diurnal changes in hepatic amino acid concentrations of the composition of the precursor pool charging aminoacyl-transfer ribonucleic acid.

To investigate further the mechanisms by which amino acids are segregated for protein biosynthesis, the distribution of a pulse of [3H]valine was monitored in hepatic amino acid pools at seven intervals in the diurnal cycle of meal-fed rats. Although each condition was characterized by a unique balance between intracellular and extracellular valine, in every case the specific radioactivity of valyl-tRNA at steady state was higher that that of intracellular valine but below the extracellular value. Further, the specific radioactivity of the valyl-tRNA could be accurately predicted if extracellular and intracellular valine were combined in proportions specified by the transmembrane concentration gradient. These observations not only substantiate our earlier conclusions that the amino acids used for protein synthesis do not originate exclusively from either the intracellular or extracellular pools, but also strengthen our theory that the membrane transport system is the physical basis for such compartmentation. On the basis of these data we present a method for measuring the specific radioactivity of the precursor pool for protein biosynthesis in cases where the actual isolation of the aminoacyl-tRNA is not technically feasible, and also suggest a theoretical basis for interpreting the unequal distribution of both total and [3H]valine between intracellular and extracellular fluids.     

Peptide utilization in yeast studies on methionine and lysine auxotrophs ofSaccharomyces cerevisiae

A study was made of the growth response and cellular peptidase activity of several amino acid auxotrophs ofSaccharomyces cerevisiae to peptides containing the required amino acid. A methionine-requiring auxotroph grew on, and contained intra-cellular peptidase activity toward Met-Met, Met-Met-Met, and Met-Gly-Met-Met. In contrast, Gly-Met-Gly did not support the growth of this mutant nor did three lysine-requiring strains utilize any lysine-containing peptides tested, although cell-free extracts from the respective mutants contained the necessary peptidase activity. The absence of a transport system of relatively high affinity for these peptides is suggested as the reason for their inability to satisfy the nutritional requirements of the cells.     

Genetics of the proteolytic system of lactic acid bacteria

Abstract The proteolytic system of lactic acid bacteria is of eminent importance for the rapid growth of these organisms in protein-rich media. The combined action of proteinases and peptidases provides the cell with small peptides and essential amino acids. The amino acids and peptides thus liberated have to be translocated across the cytoplasmic membrane. To that purpose, the cell contains specific transport proteins. The internalized peptides are further degraded to amino acids by intracellular peptidases. The world-wide economic importance of the lactic acid bacteria and their proteolytic system has led to an intensive research effort in this area and a considerable amount of biochemical data has been collected during the last two decades. Since the development of systems to genetically manipulate lactic acid bacteria, data on the genetics of enzymes and processes involved in proteolysis are rapidly being generated. In this review an overview of the latest genetic data on the proteolytic system of lactic acid bacteria will be presented. As most of the work in this field has been done with lactococi, the emphasis will, inevitably, be on this group of organisms. Where possible, links will be made with other species of lactic acid bacteria.     

Sequence analysis of a gene product synthesized by Xanthomonas sp. during growth on natural rubber latex

Xanthomonas sp. secretes an extracellular protein (Mr approximately 70+/-5 kDa) during growth on purified natural rubber [poly(1,4-cis-isoprene)] but not during growth on water-soluble carbon sources such as glucose or gluconate. A 1.3 kbp DNA fragment coding for an internal part of the structural gene of the 70 kDa protein was amplified by nested polymerase chain reaction (PCR) using amino acid sequence information obtained after Edman degradation of selected trypsin-generated peptides of the purified 70 kDa protein. The PCR product was used as a DNA probe to clone the complete structural gene from genomic DNA of Xanthomonas sp. The sequenced DNA contained a 2037 bp open reading frame which coded for a polypeptide of 678 amino acids (Mr 74.6 kDa) and which included the features of the N-terminal signal peptidase cleavage site (Mr approximately 72.9 kDa for the mature protein). Analysis of the amino acid sequence revealed the presence of two heme binding motifs (CXXCH) and a approximately 20 amino acids long sequence that is conserved in the Paracoccus denitrificans and Pseudomonas aeruginosa diheme cytochrome c peroxidases (CCPs). This region includes a histidine residue (H519 in Xanthomonas sp. and H265 and H271 in the Pseudomonas strains, respectively) that is essential for activity in CCPs and that is also conserved in other bacterial oxidases. Blast analysis confirmed the relatedness of the 70 kDa protein to heme-containing oxidases and suggested that it is a member of a new family of relatively large (approximately 500 to approximately 1000 amino acids) extracellular proteins with so far unknown function being only far related in amino acid sequence to P. denitrificans and P. aeruginosa CCPs.     

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.     

Intracellular peptide hydrolysis by Pseudomonas putida and Pseudomonas maltophilia.

Amino acids liberated by peptidase hydrolysis of di- and oligopeptides by Pseudomonas putida were measured by trinitrobenzenesulphonate assay and high voltage electrophoresis or paper chromatography followed by ninhydrin spray. Intact bacteria or periplasmic contents released by lysozyme treatment did not hydrolyse peptides. Subcellular fractionation showed that glycylmethionine peptidase activity was cytoplasmic. This enzyme had a Km of 2 mM, and was stimulated fivefold by I mM-Co2+. Crude peptidase extract did not cleave peptides with D-residues, acylated N-terminal amino groups or N-methylated peptide bonds but otherwise showed a wide specificity. Di- or tripeptides with blocked C-terminus were hydrolysed. Leucylleucine (12 mM) and leucylglycylglycine (10 mM) did not compete with glycylmethionine (1-2 mM) and glycylmethionylglycine (1-0 mM), respectively, for hydrolysis. Pseudomonas maltophilia also contained peptidase activity (0-84 mumol amino acid released from glycylmethionylglycine/min/mg protein). Peptidases of both P. putida and P. maltophilia were constitutive.     

Utilization of dipeptides by Lactococcus lactis ssp. cremoris

Different strains of Lactococcus lactis ssp. cremoris hydrolyze peptides at different rates while the cell-free extracts of these strains all show the same or much higher rates of hydrolysis. These observations indicate that the uptake of peptides is the rate-limiting step in peptide hydrolysis. Utilization of leucyl-leucine by non-growing cells is competitively inhibited by the structurally related dipeptide alanyl-alanine. After hydrolysis of peptides, the amino acids are released into the medium and only a small fraction is accumulated and/or incorporated. This hydrolysis is independent of the synthesis of proteases indicating that the synthesis of proteases and peptidases are regulated differently. The specific growth rate of L. lactis ssp. cremoris E8 depends upon the amino acid source in the medium. No significant differences have been observed in the intracellular peptidase activities and the rates of peptide uptake between L. lactis ssp. cremoris E8 cells grown in different media, indicating that this growth rate is determined by the availability of amino acids in free amino acids or peptides.     

Peptide utilization in Pseudomonas aeruginosa: evidence for membrane-associated peptidase.

A methionine auxotroph of Pseudomonas aeruginosa grew on methionine-containing peptides as a source of the required amino acid. Amino-terminus-blocked peptides would not serve as growth substrates, despite the fact that peptidases active on these blocked peptides were readily detectable in cell extracts. No evidence was found for extracellular enzymes capable of degrading the oligopeptides investigated. The degradative enzymes were not found in the periplasmic space of the cellular envelope. A high proportion of cellular peptidase activity was associated with the particulate (membrane) fraction of the cell lysate.     

A di- and tripeptide transport system can supply Listeria monocytogenes Scott A with amino acids essential for growth.

Listeria monocytogenes takes up di- and tripeptides via a proton motive force-dependent carrier protein. This peptide transport system resembles the recently cloned and sequenced secondary di- and tripeptide transport system of Lactococcus lactis (A. Hagting, E. R. S. Kunji, K. J. Leenhouts, B. Poolman, and W. N. Konings, J. Biol. Chem. 269:11391-11399, 1994). The peptide permease of L. monocytogenes has a broad substrate specificity and allows transport of the nonpeptide substrate 5-aminolevulinic acid, the toxic di- and tripeptide analogs, alanyl-beta-chloroalanine and alanyl-alanyl-beta-chloroalanine, and various di- and tripeptides. No extracellular peptide hydrolysis was detected, indicating that peptides are hydrolyzed after being transported into the cell. Indeed, peptidase activities in response to various synthetic substrates were detected in cell extracts obtained from L. monocytogenes cells grown in brain heart infusion broth or defined medium. The di- and tripeptide permease can supply L. monocytogenes with essential amino acids for growth and might contribute to growth of this pathogen in various foods where peptides are supplied by proteolytic activity of other microorganisms present in these foods. Possible roles of this di- and tripeptide transport system in the osmoregulation and virulence of L. monocytogenes are discussed.     

Chemotaxis to oligopeptides by Pseudomonas aeruginosa.

A number of peptides were evaluated as chemoattractants for Pseudomonas aeruginosa. Several strains recognized tri-, tetra-, penta-, and hexapeptides in a capillary tube assay. Tripeptides altered at the carboxyl terminus were good attractants, whereas tripeptides altered at the amino terminus did not serve as chemoattractants. Methionine-containing peptides were relatively poor attractants. Arginine-containing peptides gave the best responses. Reduced responses to larger peptides suggest that porin penetration is required. No extracellular peptidase activity was detected. We conclude that oligopeptides are good attractants and that specificity for chemotactic recognition of oligopeptides exists.     

Protein turnover in pulmonary macrophages. Utilization of amino acids derived from protein degradation.

Conditions were defined under which rates of protein synthesis and degradation could be estimated in alveolar macrophages isolated from rabbits by pulmonary lavage and incubated in the presence of plasma concentrations of amino acids and 5.6 mM-glucose. Phenylalanine was validated as suitable precursor for use in these studies: it was not metabolized appreciably, except in the pathways of protein synthesis and degradation; it entered the cells rapidly; it maintained a stable intracellular concentration; and it was incorporated into protein at measurable rates. When extracellular phenylalanine was raised to a concentration sufficient to minimize dilution of the specific radioactivity of the precursor for protein synthesis with amino acid derived from protein degradation, the specific radioactivity of phenylalanyl-tRNA was only 60% of that of the extracellular amino acid. This relationship was unchanged in cells where proteolysis increased 2.5-fold after uptake and degradation of exogenous bovine serum albumin. In contrast, albumin prevented the decrease in phenylalanine incorporation observed in macrophages deprived of an exogenous source of amino acids. These observations suggested that macrophages preferentially re-utilized amino acids derived from the degradation of endogenous, but not from exogenous (albumin), protein. However, when the extracellular supply of amino acids was restricted, substrates derived from albumin catabolism could support the protein-synthetic pathway.     

Utilization of 15N-labelled yeast hydrolysate in Lactococcus lactis IL1403 culture indicates co-consumption of peptide-bound and free amino acids with simultaneous efflux of free amino acids

Lactococcus lactis subsp. lactis IL1403 was grown in medium containing unlabelled free amino acids and ¹⁵N-labelled yeast hydrolysate to gain insight into the role of peptides as a source of amino acids under conditions where free amino acids are abundant. A mathematical model was composed to estimate the fluxes of free and peptide-derived amino acids into and out of the intracellular amino acid pool. We observed co-consumption of peptides and free amino acids and a considerable efflux of most free amino acids during growth. We did not observe significant differences between the peptide consumption patterns of essential and non-essential amino acids, which suggests that the incorporation of a particular amino acid is more dependent on its availability in a readily assimilated form than the organism’s auxotrophy for it. For most amino acids the contribution of peptide-bound forms to the formation of biomass was initially between 30 and 60 % with the remainder originating from free amino acids. During the later stages of fermentation we observed a decrease in the utilization of peptide-bound amino acids, thus indicating that the more readily assimilated peptides are gradually exhausted from the medium during growth.     

Identification of amino acids in the leader peptide of Methanococcus voltae preflagellin that are important in posttranslational processing

Archaeal flagellins are made initially as preproteins with short, positively charged leader peptides. Analysis of all available archaeal preflagellin sequences indicates that the -1 position is always held by a glycine while the -2 and -3 positions are almost always held by charged amino acids. To evaluate the importance of these and other amino acids in the leader peptides of archaeal flagellins for processing by a peptidase, Methanococcus voltae mutant FlaB2 preflagellin genes were generated by PCR and the proteins tested in a methanogen preflagellin peptidase assay that detects the removal of the leader peptide from preflagellin. When the -1 position was changed from glycine to other amino acids tested, no cleavage was observed by the peptidase, with the exception of a change to alanine at which poor, partial processing was observed. Amino acid substitutions at the -2 lysine position resulted in a complete loss of processing by the peptidase, while changes at the -3 lysine resulted in partial processing. A mutant preflagellin with a leader peptide shortened from 12 amino acids to 6 amino acids was not processed. When the invariant glycine residue present at position +3 was changed to a valine, no processing of this mutant preflagellin was observed. The identification of critical amino acids in FlaB2 required for proper processing suggests that a specific preflagellin peptidase may cleave archaeal flagellins by recognition of a conserved sequence of amino acids.     

Peptidase activity in Tetrahymena.

This report strongly suggests that two compartments in Tetrahymena thermophila contain peptidase activity: the cytoplasm and the outer cell surface. Determinations of amino acid concentrations in the extracellular medium upon incubation of cells with peptides suggest that the surface-bound peptidase activity hydrolyses di- and tri-phenylalanine equally fast on a molar basis. Growth experiments designed to characterize the in vivo peptidase specificities showed that both T. thermophila and T. pyriformis can use L-leucyl-L-leucine, but not L-leucyl-D-leucine as a leucine donor. These results are independent of whether the cells form food vacuoles or not.     

Correlation between the fatty acid composition and the activity of extracellular enzymes from Bacillus caldolyticus

Summary Bacillus caldolyticus, grown at 70°C, produces a highly active extracellular amylase and protease. Both enzymes are formed either within the membrane, or at its inner surface. The activity of both extracellular enzymes was found to decline drastically when brain-heart infusion was omitted from the medium. A simultaneous increase of both enzymes inside the cell was observed. The shifting in extra- and intra-cellular activity was caused by changes in membrane composition due to the increase of anteiso-odd and n-even, and the decrease of iso-odd fatty acids. Membrane composition and enzymic activity could be influenced by the addition of either leucine or iso-leucine as precursors for the synthesis of branched-chain fatty acids: In presence of leucine the anteiso-odd and n-even fatty acids returned to their normal level, while the iso-odd fatty acids increased. Simultaneously the extracellular protease activity increased, and the intracellular activity declined. Growth in amylose-medium supplied with leucine lead to a decrease of both the intra- and extracellular amylase, and changes in the fatty acid composition of the membrane which could not be restored by transfer of the organism to complete media. Addition of iso-leucine first lead to a sharp decrease of extracellular protease and a drastic increase of intracellular protease activity, accompanied by an increase of anteiso-odd and n-even fatty acids, and a decrease of iso-odd compounds. After the second growth in presence of iso-leucine the intra- and extra-cellular protease activity was reversed, and thus showed a return to the starting situation. The reversal is accompanied by the preferential incorporation of fatty acids with a higher melting point into the membrane. Extracellular amylase activity was found to increase after the first growth with iso-leucine, and to decline sharply after the second culture with iso-leucine, together with a very high intracellular amylase activity at that point. Extra- and intra-cellular amylase activity both declined upon growth in complete medium, while the fatty acid distribution remained different from the initial composition.