Chromosomal effects on peptidase activities inDrosophila melanogaster

The peptidase system inDrosophila melanogaster (dipeptidase-A, -B, and -C and leucine aminopeptidases G and P) was used as a model to study the effects of modifier genes on activity of enzymes with similar functions. A screen of X, second, and third chromosome substitution isogenic lines revealed the presence of activity modifiers for peptidases on all three chromosomes. Correlation analyses indicated that covariation between some of the peptidase activities is independent of genetic background, while others are associated with variable second chromosomes. Chromosome-specific effects onK _m ,V _max, and specific activity of partially purified peptidases were also detected. Moreover, a repeatable technique using anion-exchange column chromatography allowed the characterization of possibly two putative peptidic enzymes, glycyl-l-isoleucine-ase andl-leucyl-l-proline-ase, whose kinetic properties differ from the dipeptidases and the leucine aminopeptidases. These findings confirm the existence of activity modifiers for peptidases, much like other enzymes inDrosophila melanogaster. These studies were supported by grants from the National Institutes of Health (GM42-115-01A1), the Whitaker Foundation of the Research Corporation (C-2560), and the National Science Foundation (USE 8951018) to Kazuo Hiraizumi.     

Role of protein degradation in the survival of carbon-starved Escherichia coli and Salmonella typhimurium.

When an Escherichia coli K-12 culture was starved for glucose, 50% of the cells lost viability in about 6 days. When a K-12 mutant lacking five distinct peptidase activities, CM89, was starved in the same manner, viability was lost much more rapidly; 50% of the cells lost viability in about 2 days, whereas a parent strain lacking only one peptidase activity lost 50% viability in about 4 days. Compared with the wild-type strain and with its parent strain CM17, CM89 was defective in both protein degradation and protein synthesis during carbon starvation. Similar results were obtained with glucose-starved Salmonella typhimurium LT2 and LT2-derived mutants lacking various peptidase activities. An S. typhimurium mutant lacking four peptidases, TN852, which was deficient in both protein degradation and synthesis during carbon starvation (Yen et al., J. Mol. Biol. 143:21-33, 1980), was roughly one-third as stable as the isogenic wild type. Isogenic S. typhimurium strains that lacked various combinations of three of four peptidases and that displayed protein degradation and synthesis rates intermediate between those of LT2 and TN852 (Yen et al., J. Mol. Biol. 143:21-33, 1980) displayed corresponding stabilities during carbon starvation. These results point to a role for protein degradation in the survival of bacteria during starvation for carbon.     

Genetic characterization of dipeptidase activity modifiers inDrosophila melanogaster from natural populations

An examination of Drosophila melanogaster from natural populations revealed genetic variation for dipeptidase-A (DIP-A) and dipeptidase-B (DIP-B) activities within sets of lines that differed from one another only in the second or the third chromosome. Analyses of diallel crosses indicate that both activities are inherited additively, and coordinate control of expression is suggested by the significant positive correlation between the two activities. Electrophoresis and thermal denaturation studies failed to detect structural differences among lines with different levels of DIP-A activity. No characteristic level of activity could be associated with any DIP-A allozyme. Mapping experiments revealed the presence of activity modifiers that are in tight linkage with the structural gene, as well as those that manifest their effects from a distance. The maximum genetic distance between a high-activity effect on DIP-A and the structural gene was determined to be 0.029 map unit. These results are in accordance with the prevalence of activity modifiers for various enzymes in Drosophila melanogaster.     

Dipeptidase-C inDrosophila melanogaster: Genetic,ontogenetic, and tissue-specific variation

Dip-A, Dip-B, and Dip-C constitute structural genes for three peptidic enzymes in Drosophila melanogaster distinct from the leucine aminopeptidases. Their ontogenetic and tissue distributions of activities suggest the involvement of these enzymes in a general metabolic role, such as the regulation of amino acid and oligopeptide pools to make amino acids available for protein synthesis. Screening of chromosome substitution isogenic lines for DIP-C activity indicated that, like DIP-A and DIP-B, unlinked activity modifiers exist for Dip-C. The developmental profiles of dipeptidase activities are very similar, except in the pupal stage, during which DIP-C activity is markedly low compared to the other two enzymes. Intercorrelations of dipeptidase activities vary ontogenetically, which is consistent with the need for coordinate expression of these enzymes during certain developmental stages. Tissue-specific expression of dipeptidases in larvae and adults are also similar, although the relative levels of DIP-A activity differ from those of DIP-B and DIP-C in certain organs and body parts. Some of the differences among chromosome substitution lines for dipeptidase activities appear to be systemic, while others are developmental stage-specific and tissue-specific. Second- and third-chromosome variants for DIP-C activity differed in their tissue distribution. This is consistent with the presence of temporal and spatial variants in natural populations for other Drosophila enzymes.     

Genetic,ontogenetic, and tissue-specific variation of dipeptidases in Drosophila melanogaster

Three dipeptidases in Drosophila melanogaster are under independent genetic control and their structural genes have been localized, Dip-A to 2R and Dip-B and Dip-C to 3R (Voelker and Langley, 1978; Ohnishi and Voelker, 1981). These enzymes were characterized with respect to their substrate specificities, genetic variability (electrophoretic mobility and quantitative activity level), ontogeny (activity and isozyme pattern), and tissue localization. The dipeptide substrate specificities of DIP-A and DIP-B overlap each other considerably, but do not overlap with DIP-C. In natural populations, DIP-B and DIP-C are essentially monomorphic electrophoretically whereas DIP-A is polymorphic for three allozymes. Both DIP-A and DIP-B show quantitative genetic variation of activity level within an allozyme class. All three enzymes are expressed at all stages in the life cycle, but DIP-A and DIP-B activities vary considerably according to developmental stage and sex of adult. The tissue localizations of DIP-A and DIP-B activities show similar patterns and a nearly ubiquitous occurrence of both enzymes, but with particularly high values in larval and adult midguts and in the adult female reproductive system. These results suggest a general metabolic role for the enzymes, such as regulation of the concentrated pools of amino acids and oligopeptides found in Drosophila tissues.This work was supported by Public Health Service Grant GM 11546.Paper No. 7066 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh.     

Comparative peptide specificity of cell wall, membrane and intracellular peptidases of group N streptococci

Solubilized cell walls of group N streptococci contain two electrophoretically distinct peptidases, one of which hydrolysed trileucine only, while the second hydrolysed a wide range of di- and tripeptides. Neither enzyme possessed leucine aminopeptidase or endopeptidase activity. Four and three peptidases, respectively, were separated in intracellular extracts of Streptococcus lactis subsp. lactis and Strep, lactis subsp. cremoris produced by osmotic lysis of spheroplasts. In contrast with the cell-wall extracts, two of the peptidases had broad specificites, though only one of these hydrolysed trileucine. Purified membranes of Strep. lactis subsp. lactis contained only one electrophoretically distinct peptidase of very narrow specificity. There were small differences between the numbers of peptides hydrolysed by cell wall preparations from milk-grown or broth-grown cells.     

Comparative peptide specificity of cell wall, membrane and intracellular peptidases of group N streptococci

Solubilized cell walls of group N streptococci contain two electrophoretically distinct peptidases, one of which hydrolysed trileucine only, while the second hydrolysed a wide range of di- and tripeptides. Neither enzyme possessed leucine aminopeptidase or endopeptidase activity. Four and three peptidases, respectively, were separated in intracellular extracts of Streptococcus lactis subsp. lactis and Strep. lactis subsp. cremoris produced by osmotic lysis of spheroplasts. In contrast with the cell-wall extracts, two of the peptidases had broad specificites, though only one of these hydrolysed trileucine. Purified membranes of Strep. lactis subsp. lactis contained only one electrophoretically distinct peptidase of very narrow specificity. There were small differences between the numbers of peptides hydrolysed by cell wall preparations from milk-grown or broth-grown cells.     

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.     

Comparison of the peptidase activity in the oncosphere excretory/secretory products of Taenia solium and Taenia saginata

We compared the peptidase activities of the excretory/secretory (E/S) antigens of oncospheres of Taenia solium and related, but nonpathogenic, Taenia saginata. Taenia solium and T. saginata oncospheres were cultured, and the spent media of 24-, 48-, 72-, and 96-hr fractions were analyzed. Activities for serine peptidases (chymotrypsin-, trypsin-, and elastase-like), cysteine peptidases (cathepsin B-, cathepsin L-, and calpaine-like), and aminopeptidase (B-like peptidases) were tested fluorometrically with peptides coupled to 7-amino-4-methylcoumarin. In both species, the E/S antigens showed cysteine, serine, and aminopeptidase activities. Although no particular peptidase had high activity in T. solium, and was absent in T. saginata, or vice versa, different patterns of activity were found. A chymotrypsin-like peptidase showed the highest activity in both parasites, and it had 10 times higher activity in T. solium than in T. saginata. Trypsin-like and cathepsin B-like activities were significantly higher in T. solium. Minimal levels of cathepsin B were present in both species, and higher levels of elastase-like and cathepsin L-like activity were observed in T. saginata. Taenia solium and T. saginata have different levels and temporal activities of proteolytic enzymes that could play a modulator role in the host specificity for larval invasion through penetration of the intestinal mucosa.     

The effects of boric acid-induced oxidative stress on antioxidant enzymes and survivorship in Galleria mellonella

Larvae of the wax moth, Galleria mellonella (L.), were reared from first instar on a diet supplemented with 156, 620, 1,250, or 2,500 ppm boric acid (BA). The content of malondialdehyde (MDA, an oxidative stress indicator), and activities of the antioxidant enzymes [superoxide dismutase (SOD), catalase (CAT), glutathione S-transferase (GST), and glutathione peroxidase (GPx)] were determined in the fat body and hemolymph in the 7th instar larvae and newly emerged pupae. Relative to control larvae, MDA was significantly increased in larval hemolymph, larval and pupal fat body, but decreased in the pupal hemolymph. Insects reared on diets with 156- and 620-ppm BA doses yielded increased SOD activity but 1,250- and 2,500-ppm doses resulted in decreased SOD activity in larval hemolymph. SOD activity was significantly increased but CAT was decreased in the larval fat body. High dietary BA treatments led to significantly decreased GST activity. However, they increased GPx activity in larval hemolymph. Dietary BA also affected larval survival. The 1,250- and 2,500-ppm concentrations led to significantly increased larval and pupal mortality and prolonged development. In contrast, the lowest BA concentration increased longevity and shortened development. We infer that BA toxicity is related, at least in part, to oxidative stress management.     

Branchial carbonic anhydrase activity and ninhydrin positive substances in the Pacific white shrimp, Litopenaeus vannamei, acclimated to low and high salinities

The Pacific white shrimp, Litopenaeus vannamei, acclimated to 30 ppt salinity, was transferred to either low (15 and 5 ppt), or high (45 ppt) salinity for 7 days. Hemolymph osmolality, branchial carbonic anhydrase activity, and total ninhydrin-positive substances (TNPS) in abdominal muscle were then measured for each condition. Hemolymph osmotic concentration was regulated slightly below ambient water osmolality in shrimp acclimated to 30 ppt. At 15 and 5 ppt, shrimp were strong hyper-osmotic regulators, maintaining hemolymph osmolality between 200 and 400 mOsm above ambient. Shrimp acclimated to 30 ppt and transferred to 45 ppt salinity were strong hypo-osmotic and hypo-ionic regulators, maintaining hemolymph osmolality over 400 mOsm below ambient. Branchial carbonic anhydrase (CA) activity was low (approximately 100 micromol CO(2) mg protein(-1) min(-1)) and uniform across all 8 gills in shrimp acclimated to 30 ppt, but CA activity increased in all gills after exposure to both low and high salinities. Anterior gills had the largest increases in CA activity, and levels of increase were approximately the same for low and high salinity exposure. Branchial CA induction appears to be functionally important in both hyper- and hypo-osmotic regulations of hemolymph osmotic concentrations. Abdominal muscle TNPS made up between 19 and 38% of the total intracellular osmotic concentration in shrimp acclimated to 5, 15, and 30 ppt. TNPS levels did not change across this salinity range, over which hemolymph osmotic concentrations were tightly regulated. At 45 ppt, hemolymph osmolality increased, and muscle TNPS also increased, presumably to counteract intracellular water loss and restore cell volume. L. vannamei appears to employ mechanisms of both extracellular osmoregulation and intracellular volume regulation as the basis of its euryhalinity.     

Presence of additional peptidases in Streptococcus thermophilus CNRZ 302 compared to Lactococcus lactis

Streptococcus thermophilus is widely used in the dairy industry but little is known about its peptidase system. The aim of this study was to determine the biochemical and genetic characteristics of this system, and to compare it to the well known system of Lactococcus lactis . We separated the intracellular proteins of Strep. thermophilus CNRZ 302 and L. lactis NCDO 763 by ion-exchange chromatography and we detected the activity of the different types of peptidases. In both L. lactis and Strep. thermophilus strains, we showed 13 different peptidase activities with biochemical homologies between both species. Streptococcus thermophilus also possessed two peptidases which we did not find in L. lactis : an aminopeptidase and an oligopeptidase. We performed Southern blot experiments and among the eight peptidase genes tested, only the genes encoding the general aminopeptidases, pepC and pepN , were homologous between the L. lactis and Strep. thermophilus strains. Besides biochemical and genetic similarities, the peptidase systems of Strep. thermophilus and L. lactis thus differed by the presence of additional peptidases in Strep. thermophilus .     

The efficiency of human cytomegalovirus pp65(495-503) CD8+ T cell epitope generation is determined by the balanced activities of cytosolic and endoplasmic reticulum-resident peptidases

Control of human CMV (HCMV) infection depends on the cytotoxic activity of CD8(+) CTLs. The HCMV phosphoprotein (pp)65 is a major CTL target Ag and pp65(495-503) is an immunodominant CTL epitope in infected HLA-A*0201 individuals. As immunodominance is strongly determined by the surface abundance of the specific epitope, we asked for the components of the cellular Ag processing machinery determining the efficacy of pp65(495-503) generation, in particular, for the proteasome, cytosolic peptidases, and endoplasmic reticulum (ER)-resident peptidases. In vitro Ag processing experiments revealed that standard proteasomes and immunoproteasomes generate the minimal 9-mer peptide epitope as well as N-terminal elongated epitope precursors of different lengths. These peptides are largely degraded by the cytosolic peptidases leucine aminopeptidase and tripeptidyl peptidase II, as evidenced by increased pp65(495-503) epitope presentation after leucine aminopeptidase and tripeptidyl peptidase II knockdown. Additionally, with prolyl oligopeptidase and aminopeptidase B we identified two new Ag processing machinery components, which by destroying the pp65(495-503) epitope limit the availability of the specific peptide pool. In contrast to cytosolic peptidases, silencing of ER aminopeptidases 1 and 2 strongly impaired pp65(495-503)-specific T cell activation, indicating the importance of ER aminopeptidases in pp65(495-503) generation. Thus, cytosolic peptidases primarily interfere with the generation of the pp65(495-503) epitope, whereas ER-resident aminopeptidases enhance such generation. As a consequence, our experiments reveal that the combination of cytosolic and ER-resident peptidase activities strongly shape the pool of specific antigenic peptides and thus modulate MHC class I epitope presentation efficiency.     

Localization of post-proline cleaving peptidases in Tenebrio molitor larval midgut

Two soluble post-proline cleaving peptidase activities, PPCP1 and PPCP2, were demonstrated in Tenebrio molitor larval midgut with the substrate benzyloxycarbonyl-L-alanyl-L-proline p-nitroanilide. Both activities were serine peptidases. PPCP1 was active in acidic buffers, with maximum activity at pH 5.3, and was located mainly in the more acidic anterior midgut lumen. The dynamics of PPCP1 activity and the total activity of soluble digestive peptidases in the course of food digestion were similar, suggesting that the enzyme participates in protein digestion. PPCP2 is a nondigestive soluble tissue enzyme evenly distributed along the midgut. An increase in the activity of PPCP2 was observed in buffers of pH 5.6-8.6 and was maximal at pH 7.4. The sensitivity of PPCP2 to inhibitors and the effect of pH are similar to prolyl oligopeptidases with a cysteine residue near the substrate binding site.     

Co‐ordination of osmotic stress responses through osmosensing and signal transduction events in fishes

This review centres upon the molecular regulation of osmotic stress responses in fishes, focusing on how osmosensing and signal transduction events co‐ordinate changes in the activity and abundance of effector proteins during osmotic stress and how these events integrate into osmotic stress responses of varying magnitude. The concluding sections discuss the relevance of osmosensory signal transduction to the evolution of euryhalinity and present experimental approaches that may best stimulate future research. Iterating the importance of osmosensing and signal transduction during fish osmoregulation may be pertinent amidst the increased use of genomic technologies that typically focus solely on changes in the abundances of gene products, and may limit insight into critical upstream events that occur mainly through post‐translational mechanisms.     

Peptidases affecting recombinant protein production by Streptomyces lividans

The influence of peptidases on human interleukin-3 (rhIL-3) production by a recombinant Streptomyces lividans strain was investigated. The bacterium produced several general peptidases and tripeptidyl peptidases compromising the authenticity of rhIL-3. The level of peptidases depended on growth morphology. Growing S. lividans as compact pellets successfully reduced peptidase activity. Maximum general peptidase activity in pellet culture was delayed after maximum rhIL-3 concentration was achieved. The activity of the tripeptidyl peptidase was product (rhIL-3) associated.     

Enzyme activities in the plasma of rainbow trout, Salmo gairdneri Richardson; the effects of nutritional status and salinity

The levels of lactate dehydrogenase, hydroxy butyric dehydrogenase, glutamic oxalacetic transaminase, glutamic pyruvic transaminase, glutamate dehydrogenase, alkaline phosphatase and leucine amino peptidase were determined in the plasma of rainbow trout. The protein concentration and the amount of alkaline phosphatase were reduced in starving trout. Fed trout showed reduced lactate dehydrogenase activity. There was a significant correlation between the condition factor, most of the enzyme activities and the protein concentration. At 10 parts per thousand salinity the activities of alkaline phosphatase and leucine amino peptidase were significantly elevated, while lactate dehydrogenase activity was significantly decreased.     

Hemolymph and tissue-bound peptidase-resistant analogs of the insect allatostatins

While neuropeptides of the allatostatin family inhibit in vitro production of juvenile hormone, which modulates aspects of development and reproduction in the cockroach, Diploptera punctata, they are susceptible to inactivation by peptidases in the hemolymph, gut, and bound to internal tissues. Patterns of peptidase cleavage were investigated in two allatostatin analogs in which sterically bulky components were incorporated into the active core region to block peptidase attack. The results were used to design and synthesize the first pseudopeptide analog of an insect neuropeptide resistant to degradation by both hemolymph and tissue-bound peptidases. This pseudotetrapeptide allatostatin mimetic analog represents a valuable tool to neuroendocrinologists studying mechanisms by which the natural peptides operate and the physiological consequences of challenging an insect with an allatostatin that is not readily degraded via peptidase enzymes. Disruption of critical physiological processes modulated by neuropeptides such as the allatostatins via peptidase-resistant mimetic analogs could form the basis for novel pest insect management strategies in the future.     

Hemolymph levels of methyl farnesoate increase in response to osmotic stress in the green crab, Carcinus maenas

The salinity of estuarine environments can vary widely, exposing resident organisms to considerable osmotic stress. The green crab Carcinus maenas is well known for its ability to osmoregulate in response to such stress. Therefore, we tested the relationship between osmoregulation and hemolymph levels of methyl farnesoate (MF), a compound previously shown to rise in response to various types of environmental stresses. When crabs were transferred from 100% seawater to dilute (hypo-osmotic) seawater, hemolymph osmolality dropped rapidly, reaching an acclimation level 48 h after transfer. Hemolymph levels of MF also rose in these animals after a delay of 6 h, and reached a maximum level at 48 h. MF levels remained elevated as long as the crabs were maintained in dilute seawater, and quickly returned to basal levels when the animals were returned to full strength seawater. In most (but not all) animals, MF levels were elevated when hemolymph osmolality fell below the isosmotic point (approx. 800 mOsm/kg). These data suggest that MF may have a role in osmoregulation by this species. In addition, the elevation of MF by hypo-osmotic seawater suggests an experimental strategy for manipulating MF levels in crustaceans.     

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.