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At least 19 recordsLinked to original sources
PubMed1979

The study of Escherichia coli proteases. Intracellular serine protease of E. coli-an analogue of bacillus proteases.

Two serine proteases in extracts of Escherichia coli grown to stationary phase were purified to homogeneity using affinity chromatography on gramicidin S-Sepharose 4B. One enzyme was closely related to, if not identical with, the 'trypsin-like' protease II of E. coli. The other was capable of cleaving the subtilisin chromogenic substrate N-carbobenzoxy-L-alanyl-L-alanyl-L-leucine-p-nitroanilide and resembled the intracellular serine proteases of Bacillus spp. The amino acid composition of this E. coli protease was similar to that of the Bacillus licheniformis enzyme. These data indicate a relationship between proteolytic enzymes of evolutionary distant Gram-negative Enterobacteriaceae and Gram-positive spore-forming Bacillus.

Amino Acids↗
PubMed1978

On the appearance of Bacillus subtilis intracellular serine protease in the cell membrane and culture medium. Comparison of the enzyme and other Bacillus subtilis serine proteases.

While about 80% of the cell-bound intracellular serine protease of Bacillus subtilis A-50 have been recovered in the soluble fraction upon disruption of cells, the rest of the enzyme was found to be associated with the membrane fraction. Soluble cytoplasmic intracellular serine protease, as well as membrane-bound serine protease liberated by non-ionic detergent treatment, have been isolated in a pure state and shown to be identical. The same protease might also be found extracellularly, due presumably to cell lysis or altered membrane permeability. Intracellular serine protease of Bacillus subtilis A-50 was clearly related to Bacillus subtilis serine proteases W1 and bacillopeptidase F described as extracellular enzymes.

Amino Acids↗
PubMed1978

Susceptibilities of various myofibrillar proteins to muscle serine protease.

The ability of serine protease of skeletal muscle to degrade native myofibrillar proteins, such as myosin, actin, troponin, tropomyosin, alpha-actinin, and M-protein from rabbit skeletal muscle was studied. The amino acids or peptides liberated from these proteins by the protease were determined fluorometrically using o-phthalaldehyde. The order of their susceptibilities at a molar ratio of the serine protease to substrate of 1:100 was: myosin greater than tropnin greater than tropomyosin greater than actin. Alpha-Actinin and M-protein were not degraded. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the myosin heavy chain was degraded into two fragments, having molecular weights of 100,000 and 88,000, whereas the light chains were scarcely degraded. The serine protease degraded troponin-T rapidly and troponin-I slowly, but did not degrade troponin-C. Tropomyosin was degraded rapidly into two components with molecular weights of 21,500 and 19,000. Actin was degraded slowly, but no liberated fragment could be detected.

Actinin↗
PubMed1975

Production and possible function of serine protease during sporulation of Bacillus subtilis.

The production of extracellular protease during sporulation in Bacillus subtilis 168 was investigated. Two proteases are produced, an alkaline serine protease and a neutral metalloprotease. In vivo inhibition of the serine protease with phenylmethylsulfonylfluoride indicated that the metalloprotease was degraded by the serine protease during sporulation. The experiments with phenylmethylsulfonylfluoride also show that the serine protease is necessary for the sequential process of sporulation and that it is required continuously for the first 2 to 3 h of the 8-h process.

Alkaline Phosphatase↗
PubMed1979

Purification, characterization and localization of serine protease of Morris hepatoma 8999.

1. A serine protease of hepatoma 8999, isolated in the mitochondrial fraction, was purified and crystallized. The purified enzyme was apparently homogeneous on ultracentrifugal analysis and polyacrylamide disc gel electrophoresis. The ratio of absorbance at 280 nm and 260 nm, A280/A260, was 1.90 and its absorption coefficient, A280 1% was 10.5 cm-1 estimated from dry weight measurements. Its S20, w value was 2.23 S and its molecular weight was estimated to be 24000 +/- 1000. The enzyme contained twice as much lysine, arginine and histidine as chymotrypsinogen did, but had a very similar amino acid composition to serine protease from skeletal muscle. Its isoelectric point was pH 10.6. 2. The substrate specificity of the enzyme was the same as that of chymotrypsin A. Its Km and kcat values for N-acetyl-L-tyrosine ethyl ester, N-acetyl-L-phenylalanine ethyl ester and N-acetyl-L-tryptophan ethyl ester were 0.35 mM and 10.69 s-1, 0.38 mM and 10.7 s-1, and 0.11 mM and 11.8 s-1, respectively. Its activity was completely inhibited by phenylmethylsulfonyl fluoride and partially inhibited with tosylphenylalanine chloromethyl ketone. 3. The enzyme was shown to be located in different granules from the intracellular particules (light and heavy mitochondrial fraction) by sucrose density gradient centrifugation, and it was stained in mast cells of the hepatoma 8999 by the immunofluorescent technique. 4. Serine protease is present in different amounts in various organs of rat and the enzyme from hepatoma 8999 gave a single band that fused completely with those of the enzymes from skeletal muscle, heart, liver and kidney, respectively, on Ouchterlony double-diffusion analysis using antiserum to the crystalline enzyme of hepatoma 8999, but the enzyme from small intestine did not react with the antiserum.

Amino Acids↗
PubMed1978

The serine protease from rat liver and hepatoma 8999. Location and role in mitochondrial protein degradation.

1. Hepatoma 8999 showed extremely high activity of serine protease, but similar activities of other lysosomal proteases to those of normal rat liver. 2. Serine protease from rat liver formed a single immunoprecipitation band against antiserum to purified protease from hepatoma 8999. 3. The serine proteases in rat liver and hepatoma 8999 were restricted to the inner membranes of the mitochondrial fraction. 4. Polyacrylamide gel electrophoresis with sodium dodecylsulfate showed that hepatoma 8999 mitochondria contained less of the slowest moving protein component than rat liver mitochondrial protein. This component was found to be the best substrate for mitochondrial serine protease in both liver and hepatoma 8999. 5. The role of serine protease in mitochondrial protein degradation is discussed on the basis of these results.

Animals↗
PubMed1979

Sporulation and the production of serine protease and cephamycin C by Streptomyces lactamdurans.

Streptomyces lactamdurans, producer of the antibiotic cephamycin C, excretes at least two proteases. Physiological studies indicate that antibiotic synthesis and serine protease formation are coordinately regulated. Both are produced only after culture growth ends, and they appear with essentially identical kinetics. In addition, strains which produce superior levels of cephamycin C form equally superior levels of the serine protease. Genetic evidence reveals that the syntheses of the antibiotic and serine proteases are associated with sporulation. Mutants which fail to produce aerial hyphae (bald mutants) also fail to synthesize the antibiotic and serine proteases.

Cephalosporins↗
PubMed1978

Intracellular serine protease of Bacillus subtilis: sequence homology with extracellular subtilisins.

Intracellular serine protease was isolated from stationary-grown Bacillus subtilis A-50 cells and purified to homogeneity. The molecular weight of the enzyme is 31,000 +/- 1,000, with an isoelectric point of 4.3. Its amino acid composition is characteristically enriched in glutamic acid content, differing from that of extra-cellular subtilisins. The enzyme is completely inhibited with phenylmethylsulfonyl fluoride and ethylenediaminetetraacetic acid. Intracellular protease possesses negligible activity towards bovine serum albumin and hemoglobin, but has 5- to 20-fold higher specific activity against p-nitroanilides of benzyloxycarbonyl tripeptides than subtilisin BPN'. Esterolytic activity of the enzyme is also higher than that of subtilisin BPN'. The enzyme is sequence homologous with secretory subtilisins throughout 50 determined NH2-terminal residues, indicating the presence of duplicated structural genes for serine proteases in the B. subtilis genome. The occurrence of two homologous genes in the cell might accelerate the evolution of serine protease not only by the loosening of selective constrainst, but also by creation of sequence variants by means of intragenic recombination. Three molecular forms of intracellular protease were found, two of them with NH2-terminal glutamic acid and one minor form, three residues longer, with asparagine as NH2 terminus. These data indicate the possible presence of an enzyme precursor proteolytically modified during cell growth.

Amino Acid Sequence↗
PubMed1978

Serine protease in mice with hereditary muscular dystrophy.

The activities of serine protease, cathepsin B1, ornithine aminotransferase, and aldolase in skeletal muscles of mice with hereditary muscular dystrophy and their normal litter mates were studied. In dystrophic muscle, the specific and total activities of serine protease were much higher than in normal muscle, and the specific activities, but not the total activities, of cathepsin B1 and ornithine aminotransferase were twice those in normal muscle, and several new fragments, which are normally formed by limited proteolysis, were found in dystrophic muscle. When myofibrillar proteins of normal and dystrophic muscles were incubated with highly purified serine protease, their myosin, alpha-actinin and tropomyosin disappeared completely.

Animals↗
PubMed1978

Immunofluorescent localization of a serine protease in rat small intestine.

An intracellular serine protease, which is believed to initiate the degradation of several intracellular pyridoxal phosphate-dependent enzymes, was localized by immunofluorescence in atypical mast cells of the lamina propria and in intraepithelial cells of the rat small intestine. Some mucus-secreting goblet cells also contained the protease antigen. Atypical mast cells containing the enzyme were present in large numbers beneath the epithelium of bronchioles. All atypical mast cells also contained low levels of the chymotrypsin-like protease of normal mast cells. Both enzymes were consistently present in normal connective tissue mast cells. Amino acid content, molecular weight, and lack of immunologic crossreactivity indicate that the two enzymes are similar but not identical. The cell-specific localization of the intestinal serine protease makes it unlikely that the enzyme has any general role in the degradation of pyridoxal phosphate-dependent enzymes. The function of the enzyme in mast cells, atypical mast cells, and intestinal goblet cells is not known.

Animals↗
PubMed1978

Modes of proteolysis of cystathionase and ornithine aminotransferase by serine protease from the rat small intestine.

The mechanisms of proteolysis of two apo-forms of pyridoxal enzymes, cystathionase [EC 4.2.1.15] and ornithine aminotransferase [EC 2.6.1.13] by serine protease from the rat small intestine were compared. The apo-forms of these two pyridoxal enzymes are susceptible to the serine protease, whereas the holo-forms of the enzymes are not. Pyridoxal phosphate, the coenzyme of these two enzymes, prevented their inactivation by the serine protease. The difference in susceptibility of the apo- and holo-forms to the serine protease was due to the difference in their conformations. The time course of inactivation of cystathionase by the protease was apparently biphasic. During the first phase of inactivation, disappearance of the band corresponding to the native protomer, with a molecular weight of 47,000, was accompanied by accumulation of new material with a molecular weight of 39,000. In the late stage of proteolysis, extensive degradation of the large molecular weight intermediate was observed. This large intermediate product was isolated and found to compete with intact cystathionase as a substrate for the protease. Proteolysis of cystathionase was accompanied by both dissociation of the enzyme molecule and loss of its antigenicity. Limited proteolysis of ornithine aminotransferase apoenzyme by the serine protease resulted in formation of a large molecular weight product similar to the native apoenzyme, but with nicks in the molecule. Dodecylsulfate-polyacrylamide gel electrophoresis showed that the apoenzyme is degraded to intermediate forms (molecular weight 41,500 and 15,000) and later, to stable forms (molecular weight 25,500 and 13,500).

Animals↗
PubMed1978

Abnormal expression of a serine protease in human dystrophic muscle.

The activities of serine protease in muscles from normal persons and from patients with progressive muscular and neuromuscular diseases have been determined. A significant increase in the level of serine protease was found in muscle of patients with Duchenne-type muscular dystrophy and with Becker-type muscular dystrophy, but the activity was not increased in muscle of a patient with amyotrophic lateral sclerosis.

Adult↗
PubMed2025

Hatching of whipworm eggs induced by bacterial contact is serine-protease dependent.

Whipworms (Trichuris spp) are ubiquitous parasites of humans and domestic and wild mammals that cause chronic disease, considerably impacting human and animal health. Egg hatching is a critical phase in the whipworm life cycle that marks the initiation of infection, with newly hatched larvae rapidly migrating to and invading host intestinal epithelial cells. Hatching is triggered by the host microbiota; however, the physical and chemical interactions between bacteria and whipworm eggs, as well as the bacterial and larval responses that result in the disintegration of the polar plug and larval eclosion, are not completely understood. Here, we examined hatching in the murine whipworm, Trichuris muris, and investigated the role of specific bacterial and larval structures and molecules in this process. Using scanning and transmission electron microscopy, we characterised the physical interactions of both fimbriated (Escherichia coli, Salmonella typhimurium and Pseudomonas aeruginosa) and non-fimbriated (Staphylococcus aureus) bacteria with the egg polar plugs during the induction/initiation stage, and visualised the effects of structural changes in the polar plugs, leading to larval eclosion. Further, we found that protease inhibitors blocked whipworm hatching induced by both fimbriated and non-fimbriated bacteria in a dose-dependent manner, suggesting the partial involvement of bacterial enzymes in this process. In addition, we identified the minimal egg developmental timing required for whipworm hatching, and transcriptomic analysis of T. muris eggs through embryonation revealed the specific upregulation of serine proteases (S01A family) in fully embryonated eggs containing 'hatch-ready' L1 larvae. Finally, we demonstrated that inhibition of serine proteases with the serine-protease inhibitor Pefabloc ablated T. muris egg hatching induced by bacteria. Collectively, our findings unravel the temporal and physicochemical bacterial-egg interactions leading to whipworm hatching and indicate serine proteases of both bacterial and larval origin mediate these processes.

Animals↗
PubMed1978

Selective cleavage of peptide bonds by a serine protease from rat skeletal muscle.

The selective cleavage of peptide bonds by a serine protease from skeletal muscle (SK-protease) was examined using glucagon and neurotensin as substrates. Among the peptide bonds cleaved in these substrates, the most susceptible were Phe-Thr-Ser, Tyr-Leu, Trp-Leu, and Tyr-Ile. These results indicate that the SK-protease hydrolyzed the carboxyl side of aromatic amino acid residues under the experimental conditions. When the amino acid on the carboxyl side of aromatic amino acid residues was serine, threonine or glutamic acid, these peptide bonds, such as Phe-Thr, Tyr-Ser, and Tyr-Glu, were not susceptible to another serine protease from small intestine (SI-protease) under the same experimental conditions. The peptide bond between the arginines of Pro-Arg-Arg-Pro in neurotensin was hydrolyzed by the SI-protease, but not by the SK-protease. Thus the specificity of the SK-protease differs from that of the SI-protease. These results suggest that the specificity of the hydrolytic action of the SK-protease is more like that of bovine chymotrypsin A than like that of porcine chymotrypsin C and of the SI-protease.

Amino Acids↗
PubMed1978

Amino acid sequence alignment of bacterial and mammalian pancreatic serine proteases based on topological equivalences.

The three-dimensional structures of the bacterial serine proteases SGPA, SGPB, and alpha-lytic protease have been compared with those of the pancreatic enzymes alpha-chymotrypsin and elastase. This comparison shows that approximately 60% (55-64%) of the alpha-carbon atom positions of the bacterial serine proteases are topologically equivalent to the alpha-carbon atom positions of the pancreatic enzymes. The corresponding value for a comparison of the bacterial enzymes among themselves is approximately 84%. The results of these topological comparisons have been used to deduce an experimentally sound sequence alignment for these several enzymes. This alignment shows that there is extensive tertiary structural homology among the bacteria and pancreatic enzymes without significant primary sequence identity (less than 21%). The acquisition of a zymogen function by the pancreatic enzymes is accompanied by two major changes to the bacterial enzymes' architecture: an insertion of 9 residues to increase the length of the N-terminal loop, and one of 12 residues to a loop near the activation salt bridge. In addition, in these two enzyme families, the methionine loop (residues 164-182) adopts very different comformations which are associated with their altered substrate specificities.

Amino Acid Sequence↗
PubMed1976

Production, purification and characterization of thermomycolase, the extracellular serine protease of the thermophilic fungus Malbranchea pulchella var. sulfurea.

The thermophilic fungus Malbranchea pulchella produces a single extracellular, alkaline, serine protease when grown at 45 degrees C, on 2% casein as sole carbon source. The growth-associated production of protease in submerged cultures was inhibited by addition of glucose, amino acids, or yeast extract. A simple four-step purification which yields homogeneous protease in 78% yield is described. The protease has an isoelectric point of 6.0, a pH optimum of 8.5, and is completely inhibited by serine protease inhibitors. A specificity study with small synthetic ester substrates indicated that the protease preferentially hydrolyzed bonds situated on the carboxyl side of aromatic or apolar amino acid residues which are not beta-branched, positively charged or of the D configuration. Peptidase substrates and others such as N-acetyl-L-tyrosine-ethyl ester were not hydrolyzed. The protease was stable over a broad range of pH (6.5-9.5 at 30 degrees C, 20 h), and was particularly thermostable (t1/2 = 110 min at 73 degrees C, pH 7.4) in the presence of Ca2+ (10 mM). Macromolecules and Ca2+ also provide protection against the significant autolysis which occurs at pure protease concentrations greater than 0.01 mg/mo, as well as against surface denaturation which is enhanced by the presence of a silicone antifoam agent. Hence the stability of protease in submerged cultures is rationalized.

Calcium↗