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.     

Peptidases A,B, C,D and S in the American mink: polymorphism and chromosome localization

Summary An electrophoretic analysis of peptidases was carried out in a population of American mink. Based on substrate and tissue specificities, as well as subunit composition, homologies were established between mink peptidases A, B, C, D and S and human peptidases. Polymorphism for peptidases B and D was demonstrated for minks of three coat colour types. Breeding data indicated that the peptidase variations are under the control of allele pairs at distinct autosomal loci designated as PEPB and PEPD, respectively. Using a panel of American mink-Chinese hamster hybrid clones, the gene for PEPB was assigned to mink chromosome 9.     

Biochemical genetics of Fundulus heteroclitus (L.). IV. Spatial variation in gene frequencies of Idh-A,Idh-B, 6-Pgdh-A,and Est-S

The spatial variation in gene frequencies of four unlinked polymorphic loci was studied in the teleost Fundulus heteroclitus. Three loci (Idh-A, Idh-B, and Est-S) exhibit significant north-south clinal variation in allelic frequencies along the Atlantic Coast of North America, while a fourth locus (6-Pgdh-A) shows a modest clinal variation. These data, together with our previous data for Ldh-B, Mdh-A, Gpi-B, and Pgm-A, reveal a pattern of low gene diversity in the colder northern extremes of the species range and high gene diversity in warmer southern latitudes.This work was supported by Grants DEB76-19877 and DEB79-12216 from the National Science Foundation and by Grant P60-80-04 from the State of Maryland. REC and RVB were supported by NIH Training Grant GM07231 to the Department of Biology.Contribution No. 1104 from the Department of Biology, The Johns Hopkins University.     

Cysteine peptidases, secreted by Trichomonas gallinae, are involved in the cytopathogenic effects on a permanent chicken liver cell culture

Trichomonas gallinae, the aetiological agent of avian trichomonosis, was shown to secrete soluble factors involved in cytopathogenic effect on a permanent chicken liver (LMH) cell culture. The present study focused on the characterization of these molecules. The addition of specific peptidase inhibitors to the cell-free filtrate partially inhibited the monolayer destruction, which implied the presence of peptidases in the filtrate and their involvement in the cytopathogenic effect. One-dimensional substrate (gelatin) SDS-PAGE confirmed the proteolytic character of the filtrate by demonstrating the proteolytic activity within the molecular weight range from 38 to 110 kDa. In addition, the proteolytic activity was specifically inhibited by addition of TLCK and E-64 cysteine peptidase inhibitors implying their cysteine peptidase nature. Furthermore, variations in the intensity and the number of proteolytic bands were observed between cell-free filtrates of low and high passages of the same T. gallinae clonal culture. Two-dimensional substrate gel electrophoresis of concentrated T. gallinae cell-free filtrate identified at least six proteolytic spots. The mass spectrometric analysis of spots from 2-D gels identified the presence of at least two different Clan CA, family C1, cathepsin L-like cysteine peptidases in the cell-free filtrate of T. gallinae. In parallel, a PCR approach using degenerated primers based on the conserved amino acid sequence region of cysteine peptidases from Trichomonas vaginalis identified the coding sequences for four different Clan CA, family C1, cathepsin L-like cysteine peptidases. Finally, this is the first report analyzing molecules secreted by T. gallinae and demonstrating the ubiquity of peptidases secreted by this protozoon.     

Soluble peptidase isozymes of the Japanese medaka (Oryzias latipes): Tissue distributions and substrate specificities

Peptidases catalyze the hydrolysis of di- and tripeptidases to their constituent amino acids. Five isozymes (PEP A, B, C, D, and S) were shown to be the products of independent genetic loci by several criteria including distinct adult tissue and substrate specificities, non-cross-reacting immunochemical properties, and independent genetic variation at three of the loci. Four of the peptidases had at least one substrate against which they contributed over 95% of the activity. These substrates were used for isozyme-specific assays. In adult tissues, three of the peptidases had higher activities in liver and intestine than in other tissues (PEP A, B, and S). PEP C had a 10-fold higher specific activity in brain than in other tissues.     

New isozyme systems for maize (Zea mays L.): Aconitate hydratase,adenylate kinase,NADH dehydrogenase,and shikimate dehydrogenase

Electrophoretic variation and inheritance of four novel enzyme systems were studied in maize (Zea mays L.). A minimum of 10 genetic loci collectively encodes isozymes of aconitate hydratase (ACO; EC 4.2.1.3.), adenylate kinase (ADK; EC 2.7.4.3), NADH dehydrogenase (DIA; EC 1.6.99.—), and shikimate dehydrogenase (SAD; EC 1.1.1.25). At least four loci are responsible for the genetic control of ACO. Genetic data for two of the encoding loci,Aco1 andAco4, demonstrated that at least two maize ACOs are active as monomers. Analysis of organellar preparations suggests that ACO1 and ACO4 are localized in the cytosolic and mitochondrial subcellular fractions, respectively. Maize ADK is encoded by a single nuclear locus,Adk1, governing monomeric enzymes that are located in the chloroplasts. Two cytosolic and two mitochondrial forms of DIA were electrophoretically resolved. Segregation analyses demonstrated that the two cytosolic isozymes are controlled by separate loci,Dia1 andDia2, coding for products that are functional as monomers (DIA1) and dimers (DIA2). The major isozyme of SAD is apparently cytosolic, although an additional faintly staining plastid form may be present. Alleles atSad1 are each associated with two bands that cosegregate in controlled crosses. Linkage analyses and crosses with B-A translocation stocks were effective in determining the map locations of six loci, including the previously described but unmapped locusAcp4. Several of these loci were localized to sparsely mapped regions of the genome.Dia2 andAcp4 were placed on the distal portion of the long arm of chromosome 1, 12.6 map units apart.Dia1 was localized to chromosome 2, 22.2 centimorgans (cM) fromB1. Aco1 was mapped to chromosome 4, 6.2 cM fromsu1. Adk1 was placed on the poorly marked short arm of chromosome 6, 8.1 map units fromrgd1. Less than 1% recombination was observed betweenGlu1 (on chromosome 10) andSad1. In contrast to many other maize isozyme systems, there was little evidence of gene duplication or of parallel linkage relationships for these allozyme loci. This work was supported by grants from Pioneer Hi-Bred International, Inc., of Johnston, Iowa, the National Institute of Health (Research Grant GM11546), and the United States Department of Agriculture (Competitive Research Grant 83-CRCR-1-1273). This is Paper No. 11372 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh.     

Biochemical genetics of Fundulus heteroclitus (L.). III. Inheritance of isocitrate dehydrogenase (Idh-A and Idh-B), 6-phosphogluconate dehydrogenase (6-Pgdh-A), and serum esterase (Est-S) polymorphisms

Starch gel electrophoresis has shown that natural populations of Fundulus heteroclitus have variants at four enzyme-coding loci: Idh-A, Idh-B, 6-Pgdh-A, and Est-S. Analysis of the phenotypic distribution of the F₁ generation suggests that each of the variants segregates as autosomally inherited codominant alleles. Tissue specificity and intracellular localization were also determined for the IDH and 6PGDH isozymes.This work was supported by Grants DEB 76-19877 and DEB 79-12216 from the National Science Foundation and by Grant P60-80-04 from the State of Maryland. RVB and REC were supported by NIH Training Grant GM07231 to the Department of Biology.Contribution No. 1103 from the Department of Biology, The Johns Hopkins University.     

Purification of Two Dipeptidyl Aminopeptidases II from Rat Brain and Their Action on Proline-Containing Neuropeptides

From the soluble and membrane fractions of rat brain homogenate, two enzymes that liberate dipeptides of the type Xaa-Pro from chromogenic substrates were purified to homogeneity. The two isolated dipeptidyl peptidases had similar molecular and catalytic properties: For the native proteins, molecular weights of 110,000 were estimated; for the denatured proteins, the estimate was 52,500. Whereas the soluble peptidase yielded one band of pI 4.2 after analytical isoelectric focusing, two additional enzymatic active bands were detected between pI 4.2 and 4.3 for the membrane-associated form. As judged from identical patterns after neuraminidase treatment, both peptidases contained no sialic acid. A pH optimum of 5.5 was estimated for the hydrolysis of Gly-Pro- and Arg-Pro-nitroanilide. Substrates with alanine instead of proline in the penultimate position were hydrolyzed at comparable rates. Acidic amino acids in the ultimate N-terminal position of the substrates reduced the activities of the peptidases 100-fold as compared with corresponding substrates with unblocked neutral or, especially, basic termini. The action of the dipeptidyl peptidase on several peptides with N-terminal Xaa-Pro sequences was investigated. Tripeptides were rapidly hydrolyzed, but the activities considerably decreased with increasing chain length of the peptides. Although the tetrapeptide substance P 1-4 was still a good substrate, the activities detected for the sequential liberation of Xaa-Pro dipeptides from substance P itself or casomorphin were considerably lower. Longer peptides were not cleaved. The peptidases hydrolyzed Pro-Pro bonds, e.g., in bradykinin 1-3 or 1-5 fragments, but bradykinin itself was resistant. The enzymes were inhibited by serine protease inhibitors, like diisopropyl fluorophosphate or phenylmethylsulfonyl fluoride, and by high salt concentrations but not by the aminopeptidase inhibitors bacitracin and bestatin. Based on the molecular and catalytic properties, both enzymes can be classified as species of dipeptidyl peptidase II (EC 3.4.14.2) rather than IV (EC 3.4.14.5). However, some catalytic properties differentiate the brain enzyme from forms of dipeptidyl peptidase II of other sources.     

Trypsin-like proteins of the fungi as possible markers of pathogenicity

Sequences of peptidases with conserved motifs around the active site residues that are characteristic of trypsins (similar to trypsin peptidases, STP) were obtained from publicly-available fungal genomes and related databases. Among the 75 fungal genomes, 29 species of parasitic Ascomycota contained genes encoding STP and their homologs. Searches of non-redundant protein sequences, patented protein sequences, and expressed sequence tags resulted in another 18 STP sequences in 10 fungal species from Ascomycota, Basidiomycota, and Zygomycota. A comparison of fungi species containing STP sequences revealed that almost all are pathogens of plants, animals or fungi. A comparison of the primary structure of homologous proteins, including the residues responsible for substrate binding and specificity of the enzyme, revealed three groups of homologous sequences, all presumably from S1 family: trypsin-like peptidases, chymotrypsin-like peptidases and serine peptidases with unknown substrate specificity. Homologs that are presumably functionally inactive were predicted in all groups. The results in general support the hypothesis that the expression of trypsin-like peptidases in fungi represents a marker of fungal phytopathogenicity. A phylogenetic tree was constructed using peptidase and homolog amino acid sequences, demonstrating that all have noticeable differences and almost immediately deviate from the common root. Therefore, we conclude that the changes that occurred in STP of pathogenic fungi in the course of evolution represent specific adaptations to proteins of their respective hosts, and mutations in peptidase genes are important components of life-style changes and taxonomic divergence.     

Equine peptidases: Correspondence with human peptidases and polymorphism for erythrocyte peptidase a

Equine erythrocyte peptidases were compared to the six human erythrocyte peptidases, A, B, C, D, E, and F, regarding substrate specificity, relative activity, and electrophoretic mobility. Five equine erythrocyte peptidases appeared homologous to human peptidases A, B, D, E, and F. In contrast to human, equine peptidase C was absent in red cells, although it was weakly active in white cells. On the other hand, an equine peptidase, probably homologous to human peptidase S, was weakly active in red cells as well as present in white cells. Polymorphism for equine erythrocyte peptidase A is reported.     

Degradation of proline peptides in peptidase-deficient strains of Salmonella typhimurium.

A mutant strain of Salmonella typhimurium that lacks two proline-specific peptidases (peptidases P and Q) could not complete the degradation of proline peptides formed as intermediates in starvation-induced protein breakdown. The wild-type strain produced free proline as the product of degradation of proline-labeled proteins. The pepP pepQ mutant, however, produced a mixture of small proline peptides. In the absence of peptidase Q only, peptidase P could complete the degradation of most of the proline peptide intermediates formed. In the absence of peptidase P only, about 50% of the proline-labeled, acid-soluble products were proline peptides. These results are consistent with in vitro specificity data indicating that peptidase Q hydrolyzes X-Pro dipeptides only, whereas peptidase P attacks both X-Pro dipeptides and longer peptides with X-Pro at their N-termini. A mutant strain lacking four broad-specificity peptidases (peptidases N, A, B, and D), but containing peptidases P and Q, also produced proline peptides as products of protein breakdown. This observation suggests that broad-specificity peptidases are required to generate the X-Pro substrates of peptidases P and Q. A strain lacking six peptidases (N, A, B, D, P, and Q) was constructed and produced less free proline from protein breakdown than either the pepP pepQ strain or the pepN pepA pepB pepD strain. These observations suggest that the degradation of peptide intermediates involves the sequential removal of N-terminal amino acids and requires both broad-specificity aminopeptidases (peptidases N, A, and B) and the X-Pro-specific aminopeptidase, peptidase P.     

Multiple intracellular peptidases in Neurospora crassa.

Neurospora crassa possesses multiple intracellular peptidases which display overlapping substrate specificities. They were readily detected by an in situ staining procedure for peptidases separated in polyacrylamide gels, within which the auxilliary enzyme, l-amino acid oxidase, was immobilized. Eleven different intracellular peptidases were identified by electrophoretic separation and verified by their individual patterns of substrate specificities. Most peptide substrates tested were hydrolyzed by several different peptidases. The multiple intracellular peptidases may play overlapping roles in several basic cell processes which involve peptidase activity. The amount of peptidase activity for leucylglycine present in crude extracts of cells grown under widely different conditions was relatively constant, suggesting that this enzyme may be constitutive, although alterations in the amounts of individual peptidase isozymes may occur. A single enzyme, designated peptidase II, was partially purified and obtained free from the other peptidase species. Peptidase II was found to be an aminopeptidase with activity toward many peptides of varied composition and size. It was more active with tripeptides than homologous dipeptides and showed strong activity toward methionine-containing peptides. This enzyme, with a molecular weight of about 37,000, was thermolabile at 65 degrees C and was strongly inhibited by p-hydroxymercuribenzoate, Zn(2+), Co(2+), and Mn(2+), but was insensitive to the serine protease inhibitor phenylmethylsulfonyl fluoride. Peptidase II apparently possesses an essential sulfhydryl group and may be a metalloenzyme.     

Cysteine peptidases in the tomato trypanosomatid Phytomonas serpens: influence of growth conditions, similarities with cruzipain and secretion to the extracellular environment

We have characterized the cysteine peptidase production by Phytomonas serpens, a tomato trypanosomatid. The parasites were cultivated in four distinct media, since growth conditions could modulate the synthesis of bioactive molecules. The proteolytic profile has not changed qualitatively regardless the media, showing two peptidases of 38 and 40 kDa; however, few quantitative changes were observed including a drastic reduction (around 70%) on the 40 and 38 kDa peptidase activities when parasites were grown in yeast extract and liver infusion trypticase medium, respectively, in comparison with parasites cultured in Warren medium. The time-span of growth did not significantly alter the protein and peptidase expression. The proteolytic activities were blocked by classical cysteine peptidase inhibitors (E-64, leupeptin, and cystatin), being more active at pH 5.0 and showing complete dependence to reducing agents (dithiothreitol and l-cysteine) for full activity. The cysteine peptidases were able to hydrolyze several proteinaceous substrates, including salivary gland proteins from Oncopeltus fasciatus, suggesting broad substrate utilization. By means of agglutination, fluorescence microscopy, flow cytometry and Western blotting analyses we showed that both cysteine peptidases produced by P. serpens share common epitopes with cruzipain, the major cysteine peptidase of Trypanosoma cruzi. Moreover, our data suggest that the 40 kDa cysteine peptidase was located at the P. serpens cell surface, attached to membrane domains via a glycosylphosphatidylinositol anchor. The 40 kDa peptidase was also detected in the cell-free culture supernatant, in an active form, which suggests secretion of this peptidase to the extracellular environment.     

Peptidase mutants of Salmonella typhimurium

Six peptidase activities have been distinguished electrophoretically in cell extracts of Salmonella typhimurium with the aid of a histochemical stain. The activities can also be partially separated by chromatography on diethylaminoethyl-cellulose. These peptidases show overlapping substrate specificities. Mutants (pepN) of the parent strain leu-485 lacking one of these enzymes (peptidase N) were obtained by screening for colonies that do not hydrolyze the chromogenic substrate l-alanyl-beta-naphthylamide. The absence of this broad-specificity peptidase in leu-485 pepN(-) mutants allowed the selection of mutants unable to use l-leucyl-l-alaninamide as a leucine source. These mutants (leu-485 pepN(-)pepA(-)) lack a broad-specificity peptidase (peptidase A) similar to aminopeptidase I previously described in Escherichia coli. Mutants (pepD) lacking a dipeptidase (peptidase D) have been isolated from a leu-485 pepN(-)pepA(-) parent by penicillin selection for mutants unable to use l-leucyl-l-glycine as a leucine source. Mutants (pepB) lacking a fourth peptidase (peptidase B) have been isolated from a leu-485 pepN(-)pepA(-)pepD(-) strain by penicillin selection for failure to utilize l-leucyl-l-leucine as a source of leucine. Single recombinants were obtained by transduction for each of the peptidases missing in a leu-485 pepN(-)pepA(-)pepD(-)pepB(-) strain. The growth response of these recombinants to leucine peptides shows that all of these peptidases can function in the catabolism of peptides and that they display overlapping substrate specificities in vivo.     

Proteins of the kidney microvillar membrane. Biosynthesis of endopeptidase-24.11, dipeptidylpeptidase IV and aminopeptidases N and A in pig kidney slices.

An organ culture employing slices of renal-cortex tissue from piglets of the Yucatan strain was used to study the biogenesis of four microvillar peptidases: endopeptidase-24.11 (EC 3.4.24.11), dipeptidyl peptidase IV (EC 3.4.14.5), aminopeptidase N (EC 3.4.11.2) and aminopeptidase A (EC 3.4.11.7). The viability of the culture system was confirmed by the preservation of ultrastructural integrity and by an unchanged uptake of [3H]alanine into cells during the period of the experiments. After labelling with [35S]methionine, treatment with Mg2+ yielded two fractions, one containing microvilli and another, the Mg2+ pellet, containing intracellular and basolateral membranes. The labelled forms of the peptidases, isolated by immunoprecipitation, were analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and fluorography. The Mg2+ pellet contained the earliest detectable forms of the enzymes. In each case, a polypeptide of lower Mr than the mature form and sensitive to treatment with endo-beta-N-acetylglucosaminidase H was the first form to be detected. These high-mannose forms were followed, about 30 min after the pulse, by a complex glycosylated form of higher Mr. Only the latter form was observed in microvilli and then only after 90 min of the chase period. A quantitative study of dipeptidyl peptidase IV showed that the forms observed in the Mg2+ pellet were precursors of those in the microvillar fraction. No labelled forms were observed in the cytosol. All four peptidases were thus synthesized within membrane compartments and glycosylated in two steps before assembly in microvilli.     

The Distribution of Acidic and Neutral Peptidases in Barley and Wheat Kernels

The activity of barley and wheat peptidases which hydrolyze alpha-N-benzoyl-dl-arginine-p-nitronnilide (BAPA) and alpha-N-benzoyl-l-arginine ethyl ester (BAEE) has been measured in proximal and distal portions of ungerminated grain and in these tissues during 6 and 7 day incubations. The proximal portion of ungerminated barleys contained the major part of both the acidic (BAPA-ase and acidic BAEE-ase) and neutral (neutral BAEE-ase) peptidases. In ungcrminated wheat these acidic and neutral peptidases were nearly evenly distributed between the proximal and dislal portions. Commercial wheat embryo was very high in acidic peptidase but contained no neutral peptidase. During the germination of both wheat and barleys, acidic and neutral peptidase activity in the seedlings increased with time. No such consistent increase was observed for aleurone and starchy endosperm tissue for any of these grains. Aleurone and starchy endosperm tissue incubated in the absence of the proximal portion of the kernel showed reduced peptidase activities.     

Identification and characterization of a dense cluster of placenta-specific cysteine peptidase genes and related genes on mouse chromosome 13

Genes encoding novel murine cysteine peptidases of the papain family C1A and related genes were cloned and mapped to mouse chromosome 13, colocalizing with the previously assigned cathepsin J gene. We constructed a <460-kb phage artificial chromosome (PAC) contig and characterized a dense cluster comprising eight C1A cysteine peptidase genes, cathepsins J, M, Q, R, -1, -2, -3, and -6; three pseudogenes of cathepsins M, -1, and -2; and four genes encoding putative cysteine peptidase inhibitors related to the proregion of C1A peptidases (trophoblast-specific proteins alpha and beta and cytotoxic T-lymphocyte-associated proteins 2alpha and -beta). Because of sequence homologies of 61.9-72.0% between cathepsin J and the other seven putative cysteine peptidases of the cluster, these peptidases are classified as "cathepsin J-like". The absence of cathepsin J-like peptidases and related genes from the human genome suggests that the cathepsin J cluster arose by partial and complete gene duplication events after the divergence of primate and rodent lineages. The expression of cathepsin J-like peptidases and related genes in the cluster is restricted to the placenta only. Clustered genes are induced at specific time points, and their expression increases toward the end of gestation. The specific expression pattern and high expression level suggest an essential role of cathepsin J-like peptidases and related genes in formation and development of the murine placenta.     

An Insoluble Colored Substrate for Dextranase Assay

An assay of dextranase (EC 3.2.1.11) was developed by using Sephadex G-200 coupled with Remazol Brilliant Blue (RBB) as an insoluble substrate. The assay procedure included incubation of the suspension of the colored substrate in a buffer containing the enzyme under study, removal of a residual insoluble substrate, and measurement of the absorbance of supernatant fluid containing colored soluble hydrolysis products at 595 nm. The procedure was examined in the screening of dextranase-forming bacilli from the microbial collection of the Institute of Biology, Ufa Research Center, RAS.     

Substrate mapping and inhibitor profiling of falcipain-2, falcipain-3 and berghepain-2: implications for peptidase anti-malarial drug discovery

The Plasmodium falciparum cysteine peptidases FP-2 (falcipain-2) and FP-3 (falcipain-3), members of the papain-like CAC1 family, are essential haemoglobinases and are therefore potential anti-malarial drug targets. To facilitate a rational drug discovery programme, in the current study we analysed the synthetic substrate and model inhibitor profiles of FP-2 and FP-3 as well as BP-2 (berghepain-2), an orthologue from the rodent parasite Plasmodium berghei. With respect to substrate catalysis, FP-2 exhibited a promiscuous substrate profile based around a consensus non-primeside motif, FP-3 was somewhat more restricted and BP-2 was comparatively specific. Substrate turnover for FP-2 was driven by a basic or acidic P1 residue, whereas for FP-3 turnover occurred predominately through a basic P1 residue only, and for BP-2, turnover was again mainly through a basic P1 residue for some motifs and surprisingly a glycine in the P1 position for other motifs. Within these P1 binding elements, additional recognition motifs were observed with subtle nuances that switched substrate turnover on or off through specific synergistic combinations. The peptidases were also profiled against reversible and irreversible cysteine peptidase inhibitors. The results re-iterated the contrasting kinetic behaviour of each peptidase as observed through the substrate screens. The results showed that the substrate and inhibitor preferences of BP-2 were markedly different from those of FP-2 and FP-3. When FP-2 and FP-3 were compared to each other they also displayed similarities and some significant differences. In conclusion, the in vitro data highlights the current difficulties faced by a peptidase directed anti-malarial medicinal chemistry programme where compounds need to be identified with potent activity against at least three peptidases, each of which displays distinct biochemical traits.