Bacillus subtilis var. amyloliquefaciens biosynthesis of extracellular protease possessing coagulase activity and formed under conditions of limiting the nitrogen sources in the medium

An exocellular protease having the activity of coagulase was synthesized by Bacillus subtilis var. amyloliquefaciens when the growth medium contained no nitrogen sources. The removal of a nitrogen source from the medium was found to induce the synthesis of exoproteases by washed bacterial cells. Protein as a sole source of nitrogen in the medium inhibited rather than induced the biosynthesis of proteases possessing the activity of coagulase by the cells. The production of exoproteases with the coagulase activity was inhibited when a mixture of amino acids was used as a sole nitrogen source.     

Regulation of exocellular proteases in Neurospora crassa: metabolic requirements of the process.

To induce exocellular proteolytic enzyme from carbon-starved exponential-phase cells of Neurospora crassa, both a protein substrate and an activating protease of certain specific properties must be present at the same time. The cells must be capable of protein synthesis, since cycloheximide inhibits the process, but cell growth, as determined by increase in cell mass, does not appear to be required. Both soluble (bovine serum albumin, myoglobin) and insoluble protein substrates (collagen, corn zein) will affect protease induction, although certain soluble, globular proteins (egg white globulin, bovine gamma globulin) will not. In most cases, rates of protease induction are proportional to protein concentration, regardless of the nature of the inducing protein. All activating proteases capable of affecting induction in a manner similar to that of N. crassa exocellular protease were of bacterial origin and were exoproteases. Mammalian proteases and peptidases had little or no effect on the induction process.     

Cell-bound and secreted proteases of Serratia marcescens.

Exoprotease of Serratia marcescens ATCC 25419 is exceptional among members of the family Enterobacteriaceae in that it is secreted in large amounts by viable cells into the culture medium. Labeling of cells with radioactive amino acids revealed no intracellular protein that could be precipitated with antibodies raised against purified exoproteases. With substances known to interfere with the excretion of some proteins--tosyl-L-lysine chloromethyl ketone, phenethyl alcohol, procaine, and sodium azide--and with rifampin, an intracellular form (apparent molecular weight, 52,000) larger than the major exoform (molecular weight, 51,000) was identified. Moreover, the 52,000-molecular-weight form was the main protein in immunoprecipitates of a cysteine-auxotrophic mutant starved for cysteine. Beside the major exoform, protease I, two additional exoproteases, termed II and III, appeared in the medium of stationary cultures. They were precipitated by antibodies against protease I, were identical in the Ouchterlony double-diffusion assay, and exhibited only a small difference, if any at all, in the peptide pattern after partial hydrolysis with protease V8 of Staphylococcus aureus. The amino- and carboxy-terminal amino acid sequences of protease I and II were determined and found to be identical, NH2-Ala-Ala-Thr-Gly-Gly-Tyr-Asp-Ala-Val-Asp and Phe-Ile-Val-COOH, respectively. The microheterogeneity of the isolated exoforms revealed by anion-exchange chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis was also observed in samples pulse-labeled with radioactive amino acids. It remains to be determined whether the different protease forms are the result of processing (modification) reactions or whether they constitute isoenzymes encoded by very similar genes.     

Effect of levorin on the intracellular pool of free amino acids in Ehrlich ascites carcinoma cells

Levorene, a polyenic antibiotic, lowered the concentration of amino acids in the cells of Ehrlich carcinoma. The decrease in the intracellular level of the amino acids was due not only to inhibition of their entrance to the cells but also to their increased leaching from the cells. The effect of levorene on the intracellular level of the neutral amino acids was higher than that on the main amino acids which was associated with different sensitivity of the amino acid transport systems to the antibiotic.     

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.     

THE EFFECTS OF PHENYLALANINE ON AMINO ACID METABOLISM AND PROTEIN SYNTHESIS IN BRAIN CELLS IN VITRO1

Incubation of brain cell suspensions with 14 mM-phenylalanine resulted in rapid alterations of amino acid metabolism and protein synthesis. Both thc rate of uptake and the final intracellular concentration of several radioactively-labelled amino acids were decreased by high concentrations oi phenylalanine. By prelabelling cells with radioactive amino acids, phenylalanine was also shown to effect a rapid loss of the labelled amino acids from brain cells. Amino acid analysis after the incubation of the cells with phenylalanine indicated that several amino acids were decreased in their intracellular concentrations with effects similar to those measured with radioisotopic experiments (large neutral > small and large basic > small neutral > acidic amino acids). Although amino acid uptake and efflux were altered by the presence of 14 mwphenylalanine, little or no alteration was detected in the resulting specific activity of the intracellular amino acids. High levels of phenylalanine did not significantly altcr cellular catabolism of either alanine, lysine, leucine or isoleucine. As determined by the isolation of labcllcd aminoacyl-tRNA from cells incubated with and without phenylalanine, there was little or no alteration in the level of this precursor for radioactive alanine and lysine. There was, however, a detectable decrease in thc labelling of aminoacyl-tRNA for leucine and isoleucine. Only aftcr correcting for the changes of the specific activity of the precursors and thcir availability to translational events, could the effects of phenylalanine on protein synthesis be established. An inhibition of the incorporation into protein for each amino acid was approximately 20%.     

Proteases involved in generation of beta- and alpha-amylases from a large amylase precursor in Bacillus polymyxa.

The genes for extracellular neutral protease (Npr) and intracellular serine protease (Isp) were cloned from Bacillus polymyxa in order to elucidate the process involved in the generation of multiple beta-amylases and an alpha-amylase from a large amylase precursor. The npr gene was composed of 1,770 bp and 570 amino acids, while the isp gene was composed of 978 bp and 326 amino acids. Both proteases produced by E. coli cleaved the amylase precursor to generate beta- and alpha-amylases. Furthermore, several other proteases produced the same products from the precursor. A 130-kDa amylase precursor has two large domain structures responsible for the generation of beta- and alpha-amylases. The junction region of approximately 200 amino acids may be exposed on the surface of the molecule and susceptible to proteolytic enzymes, which results in the formation of multiple amylases.     

Tempe fermentation: some aspects of formation of gamma-linolenic acid, proteases and vitamins

During a tempe fermentation the concentrations of linoleic, and alpha-linolenic acids (ALA) decreased while the concentration of oleic acid increased. During fatty acid synthesis Rhizopus sp. produced only gamma-linolenic acid (GLA) instead of ALA. The amount of GLA in tempe were influenced by varying external parameters.The proteolytic capacity of 36 strains of the genus Rhizopus isolated from Indonesian tempe or tempe inocula was examined. There was a distinct increase in the amount of free amino acids during tempe fermentation. Fermentations with mixed populations of bacteria and Rhizopus yielded a lower level of free amino acids, but an increase in total amount of amino acids. In comparison to intracellular, and extracellular proteases the proteases of the cell wall fraction are most responsible for proteolytic capacity of the different Rhizopus strains.Two isolated strains of Citrobacter freundii were found to be the best vitamin B(12) producers during the soaking of soybeans. In the solid substrate fermentation the Rhizopus molds formed vitamin B(6), riboflavin, and nicotinic acid. The addition of bacteria to the solid substrate fermentation resulted in a strong increase of active vitamin B(12) in tempe. In the presence of the Rhizopus mold, the vitamin B(12) formation by C. freundii was three times higher than that of a fermentation without the mold.     

Protease activation of the entomocidal protoxin of Bacillus thuringiensis subsp. kurstaki

Two isolates of Bacillus thuringiensis subsp. kurstaki were examined which produced different levels of intracellular proteases. Although the crystals from both strains had comparable toxicity, one of the strains, LB1, had a strong polypeptide band at 68,000 molecular weight in the protein from the crystal; in the other, HD251, no such band was evident. When the intracellular proteases in both strains were measured, strain HD251 produced less than 10% of the proteolytic activity found in LB1. These proteases were primarily neutral metalloproteases, although low levels of other proteases were detected. In LB1, the synthesis of protease increased as the cells began to sporulate; however, in HD251, protease activity appeared much later in the sporulation cycle. The protease activity in strain LB1 was very high when the cells were making crystal toxin, whereas in HD251 reduced proteolytic activity was present during crystal toxin synthesis. The insecticidal toxin (molecular weight, 68,000) from both strains could be prepared by cleaving the protoxin (molecular weight, 135,000) with trypsin, followed by ion-exchange chromatography. The procedure described gave quantitative recovery of toxic activity, and approximately half of the total protein was recovered. Calculations show that these results correspond to stoichiometric conversion of protoxin to insecticidal toxin. The toxicities of whole crystals, soluble crystal protein, and purified toxin from both strains were comparable.     

Protease activation of the entomocidal protoxin of Bacillus thuringiensis subsp. kurstaki.

Two isolates of Bacillus thuringiensis subsp. kurstaki were examined which produced different levels of intracellular proteases. Although the crystals from both strains had comparable toxicity, one of the strains, LB1, had a strong polypeptide band at 68,000 molecular weight in the protein from the crystal; in the other, HD251, no such band was evident. When the intracellular proteases in both strains were measured, strain HD251 produced less than 10% of the proteolytic activity found in LB1. These proteases were primarily neutral metalloproteases, although low levels of other proteases were detected. In LB1, the synthesis of protease increased as the cells began to sporulate; however, in HD251, protease activity appeared much later in the sporulation cycle. The protease activity in strain LB1 was very high when the cells were making crystal toxin, whereas in HD251 reduced proteolytic activity was present during crystal toxin synthesis. The insecticidal toxin (molecular weight, 68,000) from both strains could be prepared by cleaving the protoxin (molecular weight, 135,000) with trypsin, followed by ion-exchange chromatography. The procedure described gave quantitative recovery of toxic activity, and approximately half of the total protein was recovered. Calculations show that these results correspond to stoichiometric conversion of protoxin to insecticidal toxin. The toxicities of whole crystals, soluble crystal protein, and purified toxin from both strains were comparable.     

Biochemical Characterization of Secreted Proteases During Growth in Tetrahymena pyriformis WH-14: Comparison of Extracellular with Intracellular Proteases

Tetrahymena pyriformis strain WH-14 secreted large quantities of intracellular proteases into its culture medium during growth. Extracellular enzymes were purified to homogeneity from cell-free medium by ammonium sulfate precipitation, CM-Sephadex column chromatography, gel filtration, and DEAE-cellulose column chromatography. The DEAE-cellulose eluates were separated into four peaks (P-I, P-II, P-III, and P-IV), each of which exhibited a different specific activity toward azocasein and α-N-benzoyl-DL-arginine-ρ-nitroanilide (Bz-Arg-Nan). These four forms of the protease showed similarity in amino acid composition, molecular weight (21,000–24,000), and antigenic reactivity. They had pH optima at neutral range. P-I showed the highest specificity to azocasein whereas P-IV was most effective toward the synthetic substrates. The Km values for hydrolysis of Bz-Arg-Nan were 2.4, 1.6, 1.3, and 1.4 mM for P-I, P-II. P-III, and P-IV, respectively, and the corresponding Kcat/Km values were 5.0, 9.4, 28.5, and 114.3 S^-1.M^-1. These properties of secreted proteases were compared with those of intracellular proteases purified by the same procedure except for the initial Triton X-100 extraction. There were similarities in specific activity toward two substrates, molecular weight, Km, pH optima, and antigenic reactivity between the proteases from two sources, providing evidence that the intracellular proteases may be secreted into the extracellular medium without modification.     

The effect of transport system A and N amino acids and of nerve and epidermal growth factors on the induction of ornithine decarboxylase activity

The induction of ornithine decarboxylase (EC 4.1.1.17) (ODC) by amino acids and by the peptide hormones nerve growth factor (NGF) and epidermal growth factor (EGF) in salts-glucose media has been studied. Only those neutral amino acids taken into the cell via one of the Na+ dependent transport systems stimulate ODC activity. Asparagine and the nonmetabolizable alpha-amino-isobutyric acid (AIB) were used as representatives of this class of inducing amino acids, and their intracellular concentrations were related to the levels of ODC induced. A threshold intracellular concentration of asparagine or AIB has to be attained before ODC can be induced. Further slight increases in intracellular concentrations of asparagine or AIB produce disproportionately large increases of ODC, resulting in a sigmoidal curve of ODC induction. These results, and the fact that the decrease in ODC levels caused by valine is associated with a concurrent decrease in the intracellular level of the inducing amino acid, suggest that the intracellular amino acid level is causally related to the induction of ornithine decarboxylase. Glutamic acid, EGF, and NGF do not induce ODC except in the presence of an inducing amino acid. They act synergistically with the inducing amino acid and produce higher ODC levels at the same intracellular concentration of the inducing amino acid.     

The effect of amino acids on growth and phosphate metabolism in a prototrophic yeast strain.

The addition of casamino acids to a log. phase culture of a prototrophic yeast strain under conditions in which their catabolism is repressed caused stimulation in growth rate. The neutral amino acids and arginine were the principal contributors to this stimulation effect. An early response of the cells to the addition of amino acids was the accumulation of low molecular weight polyphosphates. This accumulation was shown to correlate to the basicity of a given amino acid rather than to its effect on growth rate. A role for the polyphosphates in intracellular buffering is therefore suggested.     

Turnover of proteins in asporogenicBacillus megaterium. Evidence for a gradual decrease of the turnover rate

The rate of protein turnover in asporogenicBacillus megaterium decreases continuously during incubation in a sporulation medium. The capability of equilibration of external amino acids with amino acids in the metabolic pool of non-growing cells was retained for at least 5 h. Leucine, while repressing the synthesis of the exocellular protease, does not significantly influence the course of protein degradationin vivo. Transfer of non-growing cells after 4 h to a fresh sporulation medium does not influence the rate of protein degradation. The gradual decrease of the rate of protein turnover in non-growing cells of the asporogenic variant is thus not an artifact caused by a decreased uptake of amino acids by cells or by conditions under which the protein turnover is determined.     

Regulation of Exocellular Proteases in Neurospora crassa: Role of Neurospora Proteases in Induction

Cells of Neurospora crassa strain 74A, grown on sucrose for 12 h and transferred to a medium containing protein as sole carbon source, would not produce exocellular protease in significant amounts. When a filtrate from a culture induced to make protease by normal growth on a medium containing protein as principal carbon source was added to an exponential-phase culture in protein medium, exocellular protease was made in amounts similar to those made during normal induction. The material in the culture filtrate that participated in the induction process was identified as protease by its heat lability, molecular weight, and the dependence of induction rate on units of proteolytic activity added to the exponential-phase culture. Induction of the formation of exocellular protease by exponential-phase cells appears to require a protein substrate, added proteolytic activity, and protein synthesis. The protease produced by induced exponential-phase cells was as efficient in promoting induction as normally induced enzyme, whereas constitutive intracellular enzyme was only 50% as efficient. The bacterial protease thermolysin was able to induce exocellular protease at 90.7% of the rate observed with added N. crassa exocellular protease.     

Cloning, high-level expression and enzymatic properties of an intracellular serine protease from Bacillus sp. WRD-2

A gene, isp-B, encoding an intracellular serine protease from a newly isolated Bacillus sp. WRD-2 was cloned and characterized. Nucleotide sequence analysis showed an open reading frame of 960 bp encoding a polypeptide comprised of 319 amino acids. The primary structure of the enzyme predicted the structural features characteristic of other intracellular serine proteases, including active sites, Ser, His and Asp, as well as no signal sequence. The predicted amino acid sequence showed more than 60% homology with the intracellular serine proteases from Bacillus species. When expressed in E. coli, the recombinant enzyme (rISP-B) was overproduced in the cytoplasm as soluble and active form. The purified enzyme was completely inhibited by phenylmethylsulfonyl fluoride, EDTA and antipain. The enzyme showed maximum activity at pH 8.0 and 45 degrees C. It was stable atpH from 7.5 to 11.0 and below 50 degrees C.     

Identification of novel secreted proteases during extracellular proteolysis by dermatophytes at acidic pH

The dermatophytes are a group of closely related fungi which are responsible for the great majority of superficial mycoses in humans and animals. Among various potential virulence factors, their secreted proteolytic activity attracts a lot of attention. Most dermatophyte-secreted proteases which have so far been isolated in vitro are neutral or alkaline enzymes. However, inspection of the recently decoded dermatophyte genomes revealed many other hypothetical secreted proteases, in particular acidic proteases similar to those characterized in Aspergillus spp. The validation of such genome predictions instigated the present study on two dermatophyte species, Microsporum canis and Arthroderma benhamiae. Both fungi were found to grow well in a protein medium at acidic pH, accompanied by extracellular proteolysis. Shotgun MS analysis of secreted protein revealed fundamentally different protease profiles during fungal growth in acidic versus neutral pH conditions. Most notably, novel dermatophyte-secreted proteases were identified at acidic pH such as pepsins, sedolisins and acidic carboxypeptidases. Therefore, our results not only support genome predictions, but demonstrate for the first time the secretion of acidic proteases by dermatophytes. Our findings also suggest the existence of different pathways of protein degradation into amino acids and short peptides in these highly specialized pathogenic fungi.     

Biochemical and immunochemical studies of proteolytic fragments of exotoxin A from Pseudomonas aeruginosa

Limited proteolysis of Pseudomonas aeruginosa exotoxin A by four proteases (chymotrypsin, Staphylococcal serine proteinase, pepsin A and subtilisin) resulted in the formation of polypeptides having a molecular mass of approximately 25 kDa. They possessed both enzymatic activity and residual antigenicity. Their N-terminal sequence analysis showed that the different proteases cleaved exotoxin A in a very restricted area within domain Ib (amino acids 365-404). As a result, the polypeptides contained a large portion (13-34 amino acids) of domain Ib linked to the adjacent C-terminal domain III (amino acids 405-613). The major fragment derived from subtilisin cleavage, at a final yield of 35% (S-fragment; residues 392-613; 24201 Da; pI 4.7) possessed the same level of ADP-ribosyltransferase activity as uncleaved exotoxin A (by mass), and a 37-fold higher NAD-glycohydrolase activity. Polyclonal antibodies from rabbits against exotoxin A completely inhibited the ADP-ribosyltransferase activity of both exotoxin A and the S-fragment, but not the NAD-glycohydrolase activity of the S-fragment. Antibodies against the S-fragment neutralized the ADP-ribosyltransferase activity of exotoxin A. These data determine the primary proteolytic cleavage site of exotoxin A, suggest that some residues in the amino acid sequence 392-404 of exotoxin A seem to have a role in binding or positioning elongation factor 2 (EF-2) and show that antibodies recognize the EF-2-binding site but not the NAD(+)-binding site.     

Behavioral study of chemoreception in the sea starMarthasterias glacialis: Structure-activity relationships of lactic acid,amino acids,and acetylcholine

Behavioral responses of Marthasterias glacialis to low molecular compounds were studied under laboratory conditions. Feeding postures, stomach eversions and locomotion of initially inactive animals can be released with very dilute solutions of lactic acid, neutral 2 and 3 carbon amino acids, L isomers of 4 to 6 carbon neutral amino acids, L-arginine, acetylcholine iodide, and several of their analogues. Hunger was induced by temporary withdrawal of food. Responsiveness to feeding stimuli was controlled with L-cysteine and L-leucine. The lowest behavioral thresholds for the most effective feeding stimuli were 3 X 10(-11) mol/l for both enantiomers of lactic acid, 10(-8) mol/l for L-proline and both enantiomers of cysteine and 10(-7) mol/l for acetylcholine iodide and some of the effective neutral amino acids. The behavioral threshold values for chemical stimuli differed by a factor between 30 and 100 in different sea stars. The test concentration was 3 X 10(-7) mol/l, the level at which L-cysteine elicited a complete feeding response from all the animals. Structure-activity comparison of substances less effective than the control stimulus was thus possible. The behavioral threshold of fully effective substances was determined later. The independence of receptor mechanisms for different substances can be inferred as: L-cysteine controlled responsiveness is not always accompanied by responsiveness to neutral amino acids. Autotomized marthasterias arms crawled after stimulation with lactic acid, cysteine, and acetylcholine iodide but did not respond to the feeding stimuli betaine and L-proline. An animal became inactive if electric shocks were paired with L-proline or L-cysteine emanating from an 'electric' food model.(ABSTRACT TRUNCATED AT 250 WORDS)