Production of Cell-Wall-Bound Proteinases in Bifidobacterium adolescentis 94-BIM

Bifidobacterium adolescentis 94-BIM was found to produce cell-wall-bound proteolytic enzymes active at acidic, neutral, and alkaline pH values. The solubilization of proteinases with 0.5% Triton X-100 substantially improved the yield of the enzymes. The most active accumulation of cell-bound proteinases was observed in the third hour of cultivation at rates of 156.7, 179.5, and 111.1 U/(mg h), measured at pH 2.5, 7.0, and 9.0, respectively. It is suggested that the cell-wall-bound proteinases of B. adolescentis 94-BIM are the precursors of the enzymes secreted into the medium.     

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.     

Study of the physiological and biochemical characteristics of bifidobacteria at the late stages of population development

An investigation of the physiological and biochemical characteristics of the Bifidobacterium bifidum no. 1, B. adolescentis MC-42, and B. adolescentis 94-BIM strains showed that bifidobacteria with a higher growth rate produced greater amounts of the end fermentation products, acetate and lactate. The growth of the strains in batch cultures was found to be inhibited by acidic fermentation products. The growth of B. bifidum no. 1 in a batch mode lasted 100 h at a population density of 10(6) CFU/ml and the growth of B. adolescentis MC-42 and 94-BIM lasted 96-120 h at population densities from 10(4) to 10(7) CFU/ml. Analysis of the bifidobacterial populations by light and electron microscopy showed that they represent conglomerates of cells with lysed cytoplasm in the cell center and intact cytoplasm in the apical parts of the cells. The maximum production of extra-cellular and cell-bound proteinases was observed in the logarithmic growth phase. By the 120th h of cultivation, the metabolic activity of cells, the production of proteinases, and the protein content of bifidobacterial cultures considerably decreased. In the first, second, and third subcultures of 96-h-old bifidobacterial cells on fresh nutrient media, the population density of bifidobacteria and their normal physiological and biochemical characteristics were restored after 48 to 72 h of cultivation.     

Studies on the interaction between Streptomyces pepsin inhibitor and several acid proteinases by means of a zinc(II)-dye complex as a probe.

The zinc(II) complex of pyridine-2-azo-p-dimethylaniline is bound to several acid proteinases, at pH 5.0, accompanied by a change is the visible absorption spectrum. Streptomyces pepsin inhibitor, which was discovered by Satoi and Murao (Satoi, S. and Murao, S. (1970) Agric. Biol. Chem. 34, 1265-1267 and Satoi, S. and Murao, S. (1971) Agric. Biol. Chem. 35, 1482-1487), is also bound to acid proteinases. Spectrophotometric studies with ten acid proteinases from different sources have revealed that in several acid proteinases, zinc(II)-pyridine-2-azo-p-dimethylaniline is released from the enzyme by the inhibitor, while some acid proteinase forms a quaternary complex, zinc(II)-pyridine-2-azo-p-dimethylaniline-inhibitor-enzyme. It is speculated that zinc(II)-pyridine-2-azo-p-dimethylaniline is bound to two catalytic carboxylate groups in the active site of the acid proteinases and the inhibitor is bound mainly to the substrate-binding site of the enzymes. The binding of the inhibitor may overlap the catalytic site completely or partially. The degree of overlapping is characteristic of the kind of acid proteinases.     

Patterns of growth and beta-galactosidase production by bifidobacteria

We investigated the patterns of growth and beta-galactosidase formation in the strains Bifidobacterium adolescentis GO-13, MS-42, 91-BIM, and 94-BIM, and B. bifidum No. 1, LVA-3, 791 on media with various carbon sources. The synthesis of beta-galactosidase was shown to be associated with exponential growth of the cultures involved. The maximum specific rate of beta-galactosidase synthesis of 0.20 U mg(-1) h(-1) was observed in B. bifidum LVA-3 after 3-6 h of cultivation. This value for B. adolescentis 91-BIM and 94-BIM was lower and amounted to 0.03-0.08 U mg(-1) h(-1). On the medium with lactose, the highest specific growth rates for B. bifidum LVA-3 and B. bifidum No.1 were 0.38 and 0.60 h(-1), respectively, after 3-6 h of cultivation. For B. adolescentis 91-BIM and 94-BIM, this parameter peaked at 12-15 h of cultivation at 0.13 and 0.22 h(-1), respectively. The hydrolytic activity of beta-galactosidase in the growth medium decreased during the stationary growth phase of the tested cultures.     

Chromogranin A-processing proteinases in purified chromaffin granules: contaminants or endogenous enzymes?

It was the purpose of this study to define the chromogranin A-processing proteinases present in highly purified preparations of bovine chromaffin granules. The most active enzyme had a pH optimum of 5.0 and was inhibited by pepstatin. It could be identified immunologically as a cathepsin D-like enzyme and subcellular fractionation established its lysosomal origin. After removal of this enzyme the remaining activity at pH 5.0 was mainly due to a cathepsin B-like proteinase. The presence of this enzyme could also be attributed to lysosomal contamination. In the presence of calcium, a further proteolytic activity became apparent at pH 5.0. This enzyme which was inhibited by rho-chloromercuriphenylsulfonic acid was localized in chromaffin granules. A trypsin-like peptidase, most active at pH 8.2, was enriched in a membrane wash of chromaffin granules. Subcellular fractionation indicated that this enzyme is preferentially bound to the membranes of very dense particles probably representing a subpopulation of chromaffin granules. This study establishes that the most active chromogranin A-degrading proteinases present in highly purified chromaffin granules are attributable to lysosomal contamination. Two enzymes with low activity (a Ca2+ activated proteinase and a trypsin-like enzyme) are, apparently, true constituents of chromaffin granules.     

Patterns of growth and β-galactosidase production by Bifidobacteria

We investigated the patterns of growth and β-galactosidase production in the strains Bifidobacterium adolescentis GO-13, MS-42, 91-BIM, and 94-BIM and b. bifidum No.1, LVA-3, 791 on media with various carbon sources. The synthesis of β-galactosidase was shown to be associated with exponential growth of the cultures involved. The maximum specific rate of β-galactosidase synthesis of 0.20 U mg^?1 h^?1 was observed in B. bifidum LVA-3 after 3–6 h of cultivation. This value for B. adolescentis 91-BIM and 94-BIM was lower and amounted to 0.03–0.08 U mg^?1h^?1. On the medium with lactose, the highest specific growth rates for B. bifidum LVA-3 and B. bifidum No.1 were 0.38 and 0.60 h^?1, respectively, after 3–6 h of cultivation. For B. adolescentis 91-BIM and 94-BIM, this parameter peaked at 12–15 h of cultivation at 0.13 and 0.22 h^?1, respectively. The hydrolytic activity of β-galactosidase in the growth medium decreased during the stationary growth phase of the tested cultures.     

On the Nature of the Proteinases Secreted by the Aleurone Layer of Barley Grain

The nature of the proteinases which are secreted by barley aleurone layers in response to gibberellic acid was studied by constructing pH vs. activity curves for the hydrolysis of gelatin by incubation media and aleurone layer extracts. The results indicate that the aleurone layers release several different proteinases. The main component is a labile sulphydryl enzyme with an optimum pH of 3.9. The other enzymes include two sulphydryl proteinases with pH optima between pH 5 and 6.5 and a metal-activated enzyme active at pH 7.0. No differences could be demonstrated between the proteinases released by and retained in the aleurone layers.     

Investigation of the Physiological and Biochemical Characteristics of Bifidobacteria at the Late Stages of Their Development

An investigation of the physiological and biochemical characteristics of the Bifidobacterium bifidumno. 1, B. adolescentisMC-42, and B. adolescentis94-BIM strains showed that bifidobacteria with a higher growth rate produced greater amounts of the end fermentation products, acetate and lactate. The growth of the strains in batch cultures was found to be inhibited by acidic fermentation products. The growth of B. bifidumno. 1 in a batch mode lasted 100 h at a population density of 10⁶CFU/ml and the growth of B. adolescentisMC-42 and 94-BIM lasted 96–120 h at population densities from 10⁴to 10⁷CFU/ml. Analysis of the bifidobacterial populations by light and electron microscopy showed that they represent conglomerates of cells with a lysed cytoplasm in the cell center and an intact cytoplasm in the apical parts of the cells. The maximum production of extracellular and cell-bound proteinases was observed in the logarithmic growth phase. By the 120th h of cultivation, the metabolic activity of cells, the production of proteinases, and the protein content of bifidobacterial cultures considerably decreased. In the first, second, and third subcultures of 96-h-old bifidobacterial cells on fresh nutrient media, the population density of bifidobacteria and their normal physiological and biochemical characteristics were restored after 48 to 72 h of cultivation.     

Proteolysis of isolated mitochondria by myocardial lysosomal enzymes.

1. Solubilized mitochondria and lysosomal fractions were obtained from guinea-pig heart by differential centrifugation and selective membrane disruption. 2. Mitochondria incubated at 37 degrees C in the presence of lysosomal enzymes underwent proteolysis. The rate of protein degradation was inversely dependent on pH. 3. The use of proteinase inhibitors showed that at low pH the major enzyme involved in mitochondrial digestion was cathepsin D. 4. At neutral pH carboxyl proteinases were still active, but thiol proteinases accounted for most of the protein breakdown. 5. The role of lysosomal enzymes as mediators of mitochondrial damage in ischaemic myocardium is discussed.     

Extracellular metalloproteinases in Phytomonas serpens

The detection of extracellular proteinases in Phytomonas serpens, a trypanosomatid isolated from tomato fruits, is demonstrated in this paper. Maximal production occurred at the end of the logarithmic phase of growth. These enzymes exhibited selective substrate utilization in SDS-PAGE, being more active with gelatin; hemoglobin and bovine serum albumin were not degraded. Three proteinases were detected in SDS-PAGE-gelatin, with apparent molecular masses between 94 and 70 kDa. The proteolytic activity was completely blocked by 1,10-phenanthroline and strongly inhibited by EDTA, whereas a partial inhibition was observed with trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane (E-64) and soybean trypsin inhibitor; phenylmethylsulfonyl fluoride weakly inhibited the enzymes. This inhibition profile indicated that these extracellular proteinases belong to the metalloproteinase class.     

Purification and characterization of aspartic proteinase from sunflower seeds

Aspartic proteinases were purified from sunflower seed extracts by affinity chromatography on a pepstatin A-EAH Sepharose column and by Mono Q column chromatography. The final preparation contained three purified fractions. SDS-PAGE showed that one of the fractions consisted of disulfide-bonded subunits (29 and 9 kDa), and the other two fractions contained noncovalently bound subunits (29 and 9 kDa). These purified enzymes showed optimum pH for hemoglobinolytic activity at pH 3.0 and were completely inhibited by pepstatin A like other typical aspartic proteinases. Sunflower enzymes showed more restricted specificity on oxidized insulin B chain and glucagon than other aspartic proteinases. The cDNA coding for an aspartic proteinase was cloned and sequenced. The deduced amino acid sequence showed that the mature enzyme consisted of 440 amino acid residues with a molecular mass of 47,559 Da. The difference between the molecular size of purified enzymes and of the mature enzyme was due to the fact that the purified enzymes were heterodimers formed by the proteolytic processing of the mature enzyme. The derived amino acid sequence of the enzyme showed 30-78% sequence identity with that of other aspartic proteinases.     

Purification and Characterization of Extracellular Proteinases of Aspergillus oryzae

The extracellular proteinases of Aspergillus oryzae EI 212 were separated into two active fractions by (NH4)2SO4 and ethanol fractionation followed by diethylaminoethyl-Sephadex A-50 and hydroxyapatite chromatography. The molecular weight was estimated by gel filtration to be about 70,000 and 35,000 for proteinases I and II, respectively. Optimum pH for casein and hemoglobin hydrolysis was 6.5 at 60 C for proteinase I and 10.0 at 45 C for proteinase II, and for gelatin hydrolysis it was 6.5 at 45 C for both enzymes. The enzymes were stable over the pH range 6 to 8 at 30 C for 60 min. The enzyme activity for both the proteinases was accelerated by Cu2+ and inhibited by Fe2+, Fe3+, Hg2+, and Ag+. Halogenators (e.g., N-chlorosuccinimide) and diisopropyl fluorophosphate inhibited proteinase II. Sulfhydryl reagents such as p-chloromercuribenzoate and iodoacetate inhibited proteinase I. Sulfhydryl compounds accelerated the action of both enzymes.     

Electrostatic effects and the dynamics of enzyme reactions at the surface of plant cells. 3. Interplay between limited cell-wall autolysis, pectin methyl esterase activity and electrostatic effects in soybean cell walls

Soybean cell walls display a process of autolysis which results in the release of reducing sugars from the walls. Loosening and autolysis of cell wall are involved in the cell-wall growth process, for autolysis is maximum during both cell extension and cell-wall synthesis. Autolysis goes to completion within about 50 h and is an enzymatic process that results from the activity of cell wall exo- and endo-glycosyltransferases. The optimum pH of autolysis is about 5. Increasing the ionic strength of the bulk phase where cell-wall fragments are suspended, results in a shift of the pH profile towards low pH. This is consistent with the view that at 'low' ionic strength, the local pH in the cell wall is lower than in the bulk phase. One of the main ideas of the model proposed in a preceding paper, is that pectin methyl esterase reaction, by building up a high fixed charge density, results in proton attraction in the wall. Low pH must then activate the wall loosening enzymes involved in autolysis and cell growth. This view may be directly confirmed experimentally. The pH of a cell-wall suspension, initially equal to 5, was brought to 8 for 20 min, then back to 5. Under these conditions, the rate of cell-wall autolysis was enhanced with respect to the rate of autolysis obtained with cell-wall fragments kept at pH 5. The pH response of the multienzyme plant cell-wall system basically relies on opposite pH sensitivities of the two types of enzymes involved in the growth process. Pectin methyl esterase, which generates the cell-wall Donnan potential, is inhibited by protons, whereas the wall-loosening enzymes involved in cell growth are activated by protons.     

Strongyloides ransomi: proteolytic enzymes from larvae

The filariform larvae of Strongyloides ransomi can infect their hosts by penetration through skin. In this report, homogenates of these organisms were prepared and their proteolytic enzymes examined. Homogenates prepared in 0.2 M citrate, pH 4.0, contain two thiol-dependent proteinases with molecular weights of approximately 32,000 and 28,000. These proteinases have an acidic pH optimum and show substrate preferences and inhibitor susceptibilities similar to the vertebrate acidic cysteinyl proteinases. Homogenates prepared in 0.1 M Tris, pH 7.5, contain multiple proteolytic enzymes, active against both Azocoll and synthetic substrates. These enzymes do not require thiols for activity and they have an alkaline pH optimum. The enzymes are inhibited by both chelating agents and heavy metals, but not by serine-proteinase inhibitors. Extracts prepared in 0.1 M Tris-HCl, pH 7.5, contain endogenous proteinase inhibitors.     

Neutral proteinases of human spleen. Purification and criteria for homogeneity of elastase and cathepsin G.

1. Human spleen was found to contain proteinases active against azo-casein at neutral and alkaline pH values. 2. The activity was stimulated by high ionic strength and some detergents. 3. Optimal extraction of the proteinases from the tissue was achieved with 1.0M-NaCl containing 0.1% Brij 35 and 0.1% trisodium EDTA. 4. The proteinases were efficiently adsorbed to insoluble material in the absence of salt in the initial stages of purification. 5. Two distinct proteinases were separated by chromatography on DEAE-cellulose, an elastase and a chymotrypsin-like enzyme designated cathepsin G. 6. Both enzymes were highly purified by further column chromatography. 7. The molecular weights of the enzymes were estimated by gel chromatography and sodium dodecyl sulphate-gel electrophoresis. 8. It was shown by isoelectric focusing and gel electrophoresis that both enzymes are cationic proteins that occur in multiple forms.     

The use of benzyloxycarbonyl[125I]iodotyrosylalanyldiazomethane as a probe for active cysteine proteinases in human tissues.

The ability of benzyloxycarbonyl-(125I)Tyr-Ala-CHN2 to label cysteine proteinases in a variety of human tissues was investigated. The inhibitor bound only to cathepsin B in tissues homogenized at pH 5.0. When liver was autolysed at pH 4.0 for up to 4 h, the inhibitor also bound to a protein of Mr 25,000. This was identified immunologically and chromatographically as cathepsin L. Both cathepsins B and L were found primarily in kidney, liver and spleen. In spleen, an additional protein of Mr 25,000 was also labelled. This protein could not be precipitated by antibodies to any of cathepsins B, H and L. This protein has tentatively been identified as human cathepsin S by its tissue distribution, chromatographic properties and molecular size. This work clearly shows that peptidyldiazomethanes are specific probes for cysteine proteinases, and that benzyloxycarbonyl-(125I)Tyr-Ala-CHN2 binds to three such enzymes in human tissues.     

Schistosoma mansoni: differential expression of cathepsins L1 and L2 suggests discrete biological functions for each enzyme

Schistosoma mansoni cathepsins L1 (SmCL1) and L2 (SmCL2) were expressed as active recombinant proteinases in Saccharomyces cerevisiae. The recombinant enzymes exhibited substrate preferences characteristic of cathepsin-L-like cysteine proteinases. However, the enzymes differed in their substrate specificities; SmCL1 cleaved Boc-Val-Leu-Lys-NHMec with a higher efficiency than it cleaved Z-Phe-Arg-NHMec, whereas the opposite was true for SmCL2. The enzymes also differed in their pH profiles of activity; SmCL1 exhibited a broad pH profile with an optimum of pH 6. 5, while SmCL2 was active only in the acidic pH range with an optimum of 5.35. Immunoblot and RT-PCR analyses revealed that the native forms of both SmCL1 and SmCL2 are expressed in male and female worms, but at higher levels in adult female compared to male schistosomes. Additionally, both enzymes were observed in the excretory/secretory products of adult worms. The RT-PCR analysis indicated that neither enzyme is expressed in S. mansoni eggs or in miracidia, suggesting that the cathepsin-L-like activity that has been previously reported to be expressed in these stages may be the product of another gene(s). Cercariae do not express SmCL2, but appear to express SmCL1 in its inactive precursor form. Together with the findings of previous immunolocalization and phylogenetic analyses, the results reported here demonstrate that SmCL1 and SmCL2 are distinct cathepsin cysteine proteinases and strongly suggest that they play discrete biological roles.     

Cathepsins J and K: high molecular weight cysteine proteinases from human tissues

Human tissue extracts contained two high Mr proteinases active in hydrolyzing the fluorogenic substrate Cbz-phe-arg-aminomethylcoumarin. By gel filtration chromatography, cathepsins J and K had apparent molecular weights of 230,000 and 650,000, respectively. Both enzymes were cysteine proteinases with optimum activity at pH 6.2-6.8; neither had aminopeptidase activity. Human kidney, lung and spleen were rich sources of these enzymes, while liver contained moderate amounts. Cathepsins J and K were partially characterized and appeared to differ from the mammalian high Mr cysteine proteinases described in the literature. In rat liver and kidney and in mouse liver, cathepsin J was localized in the particulate fraction, whereas cathepsin K was not detected in these tissues.