Characterization of proteolytic enzymes of the midgut and excreta of the biting fly Stomoxys calcitrans

Proteolytic enzymes were characterized in the midgut and the excreta of the stable fly Stomoxys calcitrans (L) with proteins, synthetic substrates, and inhibitors. Inhibition studies suggested trypsinlike activity in sugar-fed fly midguts, whereas excreta and blood-fed fly guts exhibited other proteases. Trypsinlike activity in midguts removed 20 and 30 h after a blood meal increased from 20% to 50% of the total proteolytic enzymes present. Trypsinlike activity was inhibited with human sera, trypsin-specific inhibitors, and a protein isolated from the stable fly thorax. When human albumin and globulin fractions were incubated with trypsinlike enzymes isolated from the midgut and excreta, the albumin fraction was less inhibitory than the globulin fractions and was readily hydrolyzed by the proteolytic enzymes. These results may indicate that the proteolytic enzymes produce an abortive complex with the globulin fractions of the sera. Such a complex may explain the temporary inhibition of proteolysis by the blood meal. Soybean trypsin inhibitor fed to stable flies caused 50% inhibition in proteolytic activity in the midguts of sugar-fed stable flies and 25% inhibition in the midguts of blood-fed stable flies. Complete inhibition of proteolytic enzyme activity was achieved only in vitro. pH profiles of proteolytic enzyme activity isolated from the excreta of blood-fed stable flies indicated that several proteolytic enzymes were excreted.     

Experimental study of the immunotherapy of burns

Experiments were conducted on rats. The influence of the serum of a burn convalescent on the toxic properties, the level of proteolytic enzymes activity, and morphological changes following burn were studied. After the burn the blood serum and the extracts of the organs acquired toxic properties; there was an increase in the activity of proteolytic enzymes, and marked morphological changes developed Administration of the serum of aburn convalescent promoted detoxication, diminished the activity of proteolytic enzymes distinctly, and decreased the morphological disturbances.     

大鵟消化系统蛋白水解酶种类和活性分析

采用蛋白水解酶复性电泳(G-PAGE)技术对大(Buteo hemilasius)消化系统5种器官腺胃、胰脏、十二指肠、空肠、大肠蛋白水解酶的种类和性质进行了研究,以期为研究野生鸟类的分类地位、系统演化提供基础资料,结果表明,①受pH值的影响和制约,大消化系统蛋白水解酶的活性在碱性、中性与酸性条件下递减;②在酸性条件下,45 ku蛋白水解酶存在于除腺胃外的各受检器官;③pH 7.0时,腺胃、胰脏酶谱相似,均含有683、5、342、0 ku的蛋白水解酶;④pH 8.0时,空肠和十二指肠的蛋白水解酶种类最多、活性最强,分别检出8种和7种蛋白水解酶。总之,pH值对蛋白水解酶的活性有明显的制约作用,46、41ku蛋白水解酶随着pH值的增高而失去活性,为酸性蛋白水解酶,250、2064、5 ku蛋白水解酶随着pH值的增高活性逐渐增强,为碱性蛋白水解酶。十二指肠和空肠的蛋白水解酶种类多、活性强,可能为蛋白质消化的主要场所。     

Effect of pasteurization on the activity of proteinases in salmon roe

We studied the dependence of activity and stability of proteolytic enzymes in salmon roe on pH and temperature. The activity of proteolytic enzymes in roe was primarily determined by proteinases. These enzymes were active at acid pH and had an optimum of 3.6. A study of subclasses of proteolytic enzymes in salmon roe and the published data suggest that the activity of proteinases may be related to the presence of aspartyl proteinases (cathepsin D). Serine proteinases and metalloenzymes were not found in roe. The activity of cysteine proteinases was low. The proposed conditions of pasteurization favored the complete inactivation of salmon roe at pH 6.0-6.4.     

Effect of Pasteurization on the Activity of Proteinases in Salmon Roe

We studied the dependence of activity and stability of proteolytic enzymes in salmon roe on pH and temperature. The activity of proteolytic enzymes in roe was primarily determined by proteinases. These enzymes were active at acid pH and had an optimum of 3.6. A study of subclasses of proteolytic enzymes in salmon roe and the published data suggest that the activity of proteinases may be related to the presence of aspartyl proteinases (cathepsin D). Serine proteinases and metalloenzymes were not found in roe. The activity of cysteine proteinases was low. The proposed conditions of pasteurization favored the complete inactivation of salmon roe at pH 6.0–6.4.     

An Assessment of Proteolytic Enzymes in Tetrahymena thermophila

Cellular extracts of Tetrahymena thermophila were found to contain substantial levels of proteolytic activity. Protein digestion occurred over broad ranges of pH, ionic strength, and temperature and was stimulated by treatment with thiol reductants, EDTA and sodium dodecyl sulfate. Incubation at temperatures ≥60° C or with high concentrations of chaotropic reagents such as 10 M urea or 6 M guanidine-HCl caused an apparent irreversible loss of activity. Activity was also strongly diminished by increasing concentrations of divalent cations. Several peptide aldehydes, p-hydroxymercuribenzoate, and alkylating reagents such as iodoacetate, N-tosyl-L-lysine chloromethyl ketone, N-tosyl-L-phenylalanine chloromethyl ketone, N-methylmaleimide, and trans-epoxysuccinyl-L-leucylamido-(4-guanidino)-butane were potent inhibitors of proteolytic activity. Aprotinin diminished activity by approximately 40% while benzamidine, 3,4-dichloroisocoumarin, and trypsin inhibitors from soy bean, lima bean, and chicken egg caused relatively modest inhibition of proteolytic activity. Phenylmethanesulfonyl fluoride had no apparent effect. Electrophoretic separation of proteins on SDS-polyacrylamide gels copolymerized with gelatin substrate revealed that at least eight active proteolytic enzymes were present in cell extracts ranging in apparent molecular weight from 45,000 to 110,000. Five of these apparent proteases were detected in 70% ammonium sulfate precipitates. Gelatinase activity was not detectable when extracts were pretreated with iodoacetate or E-64, indicating that all of the enzymes observed in activity gels were sensitive to thiol alkylation. Cellular extracts of T. thermophila appeared to contain multiple forms of proteolytic enzymes which were stimulated by thiol reductants and inhibited by thiol modifying reagents. Accordingly, the proteolytic enzymes present in cell extracts appear to be predominantly cysteine proteinases.     

Hymenolepis diminuta: interactions of the isolated brush border membrane with proteolytic enzymes

Proteins of the isolated brush border membrane of Hymenolepis diminuta were hydrolyzed in vitro by chymotrypsin, papain, pepsin, subtilopeptidase A (= subtilisin Carlsberg), and trypsin. Neither proteolytic nor amidase activity was demonstrable in the isolated membrane using proteinaceous (casein and hemoglobin) or chromogenic (benzoyl-arginine-p-nitroanilide and succinyl-alanyl-alanyl-propyl-phenylalanine p-nitroanilide) substrates, and the membrane preparation did not inhibit the proteolytic and amidase activities of these enzymes. Thus, the isolated tegumental membrane of H. diminuta is not inherently resistant to the action of proteolytic enzymes, and it does not inhibit proteolytic activity. In control incubations containing only buffer, the alkaline phosphatase activity of the brush border membrane decreased in a time dependent manner, but in the presence of chymotrypsin, subtilopeptidase A, and trypsin, the membrane retained greater alkaline phosphatase activity (pepsin and papain could not be tested for this effect on alkaline phosphatase activity). A similar time dependent decrease in activity was also noted for each of the proteolytic enzymes in control assays, but subtilopeptidase A and papain retained greater activity in the presence of the isolated membrane preparation when these assays were compared to controls.     

An assessment of proteolytic enzymes in Tetrahymena thermophila.

Cellular extracts of Tetrahymena thermophila were found to contain substantial levels of proteolytic activity. Protein digestion occurred over broad ranges of pH, ionic strength, and temperature and was stimulated by treatment with thiol reductants, EDTA and sodium dodecyl sulfate. Incubation at temperatures > or = 60 degrees C or with high concentrations of chaotropic reagents such as 10 M urea or 6 M guanidine-HCl caused an apparent irreversible loss of activity. Activity was also strongly diminished by increasing concentrations of divalent cations. Several peptide aldehydes, p-hydroxymercuribenzoate, and alkylating reagents such as iodoacetate, N-tosyl-L-lysine chloromethyl ketone, N-tosyl-L-phenylalanine chloromethyl ketone, N-methylmaleimide, and trans-epoxysuccinyl-L-leucylamido-(4-guanidino)-butane were potent inhibitors of proteolytic activity. Aprotinin diminished activity by approximately 40% while benzamidine, 3,4-dichlorosocoumarin, and trypsin inhibitors from soy bean, lima bean, and chicken egg caused relatively modest inhibition of proteolytic activity. Phenylmethanesulfonyl fluoride had no apparent effect. Electrophoretic separation of proteins on SDS-polyacrylamide gels copolymerized with gelatin substrate revealed that at least eight active proteolytic enzymes were present in cell extracts ranging in apparent molecular weight from 45,000 to 110,000. Five of these apparent proteases were detected in 70% ammonium sulfate precipitates. Gelatinase activity was not detectable when extracts were pretreated with iodoacetate or E-64, indicating that all of the enzymes observed in activity gels were sensitive to thiol alkylation. Cellular extracts of T. thermophila appeared to contain multiple forms of proteolytic enzymes which were stimulated by thiol reductants and inhibited by thiol modifying reagents. Accordingly, the proteolytic enzymes present in cell extracts appear to be predominantly cysteine proteinases.     

Production of fibrous materials, containing an immobilized proteolytic enzyme and urea]

Immobilization of cellulose and polyacrylic acid on a grafted copolymer increased significantly the stability of proteolytic enzymes to inactivation by urea. Materials containing immobilized proteolytic enzymes and urea and displaying a combined biological activity were obtained.     

Degradation of proteinaceous substrates by xylotrophic basidiomycetes

The ability of various xylotrophs to produce extracellular proteolytic enzymes has been studied, with emphasis on medium-related factors regulating their secretion. Direct measurement of proteolytic activity in the culture liquid and postelectrophoresis determination of protease activity in polyacrylamide gel copolymerized with gelatin demonstrated that the secreted enzymes are quantitatively and qualitatively diverse. Activity levels of extracellular proteolytic enzymes strongly depend on pH and contents of protein and carbohydrate in the medium. All secreted proteases notably differed in molecular weight (of 51 kDa or higher and in excess of 95 kDa) and belonged mostly to two classes of proteolytic enzymes (serine proteases and metalloproteinases).     

Degradation of proteinaceous substrates by xylotrophic basidiomycetes

The ability of various xylotrophs to produce extracellular proteolytic enzymes has been studied, with emphasis on medium-related factors regulating their secretion. Direct measurement of proteolytic activity in the culture liquid and postelectrophoresis determination of protease activity in polyacrylamide gel copolymerized with gelatin demonstrated that the secreted enzymes are quantitatively and qualitatively diverse. Activity levels of extracellular proteolytic enzymes strongly depend on pH and contents of protein and carbohydrate in the medium. All secreted proteases notably differed in molecular weight (of 51 kDa or higher and in excess of 95 kDa) and belonged mostly to two classes of proteolytic enzymes (serine proteases and metalloproteinases).     

Some properties of proteolytic enzymes from Tribolium confusum

The digestive tract of Tribolium confusum Duv. larvae was studied for proteolytic enzymes properties. The pH optima are determined for the enzymes effect on various substrates. Proteases were partially purified by gel chromatography on Sephadex G = 100 and investigated for thyol compound influence on their activity. The activity of the enzymes is shown to increase considerably with addition of cystein, glutathione, 2-mercaptoethanol. dithiotreitol and EDTA. Dithiotreitol produces the strongest restoring effect and in concentration of 10(-6) M it activates the enzyme almost twice. Storage for 48 h at 4 degrees C induced a 2.5-fold decrease in the proteolytic enzyme activity; SH-groups in the catalytic action of enzymic solutions is shown. The maximum proteolytic activity is found in extracts from 14-day insects.     

东北虎幼体消化系统蛋白水解酶的初步研究

蛋白水解酶在许多生命活动中是必需的物质(Vassalli and Pepper,1994)。蛋白质的酶解修饰(Xuet al.,1999)、细胞迁移、组织再生与修复、消化系统对蛋白质的消化等均与蛋白水解酶有关(Baimbridgeet al.,1992),且蛋白水解酶功能失调会导致许多疾病(Teichertet al.,1989)。东北虎(     

Characterization of Proteinases Excreted by Bacillus subtilis Marburg Strain during Sporulation

S ummary . This study has characterized 3 proteolytic enzymes during sporulation by Bacillus subtilis Marburg strain when grown in nutrient broth. A method of purification is described which permits the separation of 2 different proteinases: one belonging to the metal enzyme group and the other to the serine enzyme group. The third enzyme, probably an esterase, showed a high esterolytic activity, but only low proteolytic activity. Determination of the 3 enzymes in a mixture was accomplished by using specific substrates and inhibitors. They were excreted simultaneously between the end of the growth phase until the appearance of the prespores. During this entire period, 20% of the total proteolytic activity was due to the metal proteinase; 80% of the proteolytic activity and 15% of the esterolytic activity was due to the serine proteinase; 85% of the esterolytic activity was the result of the esterase. These findings will contribute to a more complete phenotypic characterization of those mutants of sporulation that appear to be involved in the production of extracellular hydrolytic enzymes.     

Studies on the role of exogenous proteolytic enzymes in digestion processes in fish

Studies were carried out on the proteolytic activity of the fry of common carp, rainbow trout, grass carp, and whitefish, as well as on the activity of digestive organs of adult common carp and rainbow trout. Activity of exogenous enzymes in relation to endogenous ones was assessed on the basis of the proteolytic value of fish food and the activity of digestive organs. It was found that the share of proteolytic enzymes of natural food in the digestion process in fish was high. Beginning from a weight of 50–100 g for common carp and 10 g for rainbow trout, the relation between the daily enzymatic ration and the weight of fish indicates the cooperation of an approximately constant amount of exogenous enzymes.     

Preparations of extracellular proteinases from Aspergillus ochraceus 513 and Aspergillus alliaceus 7dN1

Preparations of extracellular proteolytic enzymes with high anticoagulant activity resembling protein C activators were isolated from the culture liquids of Aspergillus ochraceus 513 and Aspergillus alliaceus 7 dN1 by precipitation with ammonium sulfate and subsequent purification from ammonium ions by gel filtration on a column with Sephadex G-25. The pH and temperature activity optima and stability of the proteolytic enzymes from A. ochraceus 513 and A. alliaceus 7 dN1 were determined.     

Extracellular proteolytic enzymes of Azospirillum brasilensis strain Sp7 and regulation of their activity by a homologous lectin

It was found that Azospirillum brasilensis strain Sp7 is able to produce extracellular proteolytic enzymes. The enzymes were active within a broad range of pH values, with two peaks of activity being located in the acid and alkaline pH areas; required calcium ions; and exhibited substrate specificity with respect to azogelatin. Zymography allowed at least four proteolytic enzymes with molecular weights of 32, 45, 52, and 174 kDa to be detected in A. brasilense Sp7 culture liquid. It was shown that the lectin from A. brasilense Sp7 can inhibit proteolytic enzymes.     

Lysosomal proteolysis: effects of aging and insulin

Age-related characteristics of the effect of insulin on the activity of lysosomal proteolytic enzymes were studied. The relationship between the insulin effect on protein degradation and insulin degradation was analyzed. The effect of insulin on the activities of lysosomal enzymes was opposite in young and old rats (inhibitory in 3-month-old and stimulatory in 24-month-old animals). The activities of proteolytic enzymes were regulated by insulin in a glucose-independent manner: similar hypoglycemic effects of insulin in animals of different ages were accompanied by opposite changes in the activities of lysosomal enzymes. The inhibition of lysosomal enzymes by insulin in 3-month-old rats is consistent with a notion on the inhibitory effect of insulin on protein degradation. An opposite insulin effect in 24-month-old rats (i.e., stimulation of proteolytic activity by insulin) may be partly associated with attenuation of the degradation of insulin, resulting in disturbances in signaling that mediates the regulatory effects of insulin on protein degradation.     

Biosynthesis of alpha-amylase and protease by Streptomyces olivaceus 142. II. Biosynthesis of protease.

Streptomyces olivaceus 142 produces proteolytic enzymes de novo, mainly in the stationary phase of growth. The highest activity of the enzymes was observed in media containing maltose or fructose. In media supplemented with glucose, glycerol or starch the activity was lower. The synthesis of proteases is subject to catabolic repression. The proteolytic activity is reduced by phosphate buffer.     

Screening for Milk-clotting Enzyme from Basidiomycetes

Screening tests for aspartic proteinases with milk-clotting activity were done on basidiomycetes. Crude enzymes from 6 strains had a high ratio of milk-clotting activity to caseinolytic activity. These enzymes showed acidic pH optimum for proteolytic activity and were inhibited considerably by pepstatin, a specific aspartic proteinase inhibitor. Among them, the crude enzyme from Laetiporus sulphureus was more heat-labile than the other enzymes.