Mouse submandibular gland prorenin-converting enzyme is a member of glandular kallikrein family

Mouse submandibular gland prorenin-converting enzyme (PRECE) consists of the two polypeptide chains of 17 and 10 kDa and cleaves mouse Ren-2 prorenin at a dibasic site to yield mature renin. Western blot analysis using an antiserum against this enzyme gave rise to multiple bands in mouse submandibular glands, suggesting that PRECE is a member of a protease family. Partial amino acid sequence analysis of purified PRECE and cloning and sequence analyses of its cDNA indicated that it is identical to the mGK-13 gene product, epidermal growth factor-binding protein type B, which is a member of the glandular kallikrein family and is involved in maturation of epidermal growth factor. Conditioned medium from Chinese hamster ovary cells transfected with an expression plasmid for PRECE had prorenin converting activity. These results indicate that PRECE is involved in the maturation of two bioactive polypeptides expressed in mouse submandibular glands, Ren-2 renin and epidermal growth factor.     

Demonstration of protease activity for epidermal growth factor-binding protein

The epidermal growth factor can be isolated from the male mouse submaxillary gland as part of a high molecular weight complex. The complex is composed of two molecules of epidermal growth factor and two molecules of epidermal growth-factor binding protein (J.M. Taylor, W.M. Mitchell, and S. Cohen, 1974, J. Biol. Chem.249, 3198–3203). The proteolytic activity of epidermal growth-factor binding protein was demonstrated by its self-proteolysis in moderate (3–7 m) concentrations of urea, and, its inhibition by formation of a complex with pancreatic trypsin inhibitor. This complex was characterized by its pI and by its ability to yield pancreatic trypsin inhibitor and epidermal growth factor-binding protein in sodium dodecyl sulfate-urea gel electrophoresis. The association equilibrium constant was determined to be 3.6 × 10⁷m^?1 by inhibition studies of the esteropeptidase. These results, which indicate that epidermal growth factor-binding protein is capable of autodigestion and of forming a stable complex with a macromolecular inhibitor of trypsin, lend strong support to the hypothesis that epidermal growth factor-binding protein is capable of cleaving a larger precursor by its proteolytic action.     

Characterization of the Proteinase that Initiates the Degradation of the Trypsin Inhibitor in Germinating Mung Beans (Vigna radiata)

The proteinase (proteinase F) responsible for the initial proteolysis of the mung bean (Vigna radiata) trypsin inhibitor (MBTI) during germination has been purified 1400-fold from dry beans. The enzyme acts as an endopeptidase, cleaving the native inhibitor, MBTI-F, to produce the first modified inhibitor form, MBTI-E. The cleavage of the Asp76-Lys77 peptide bond of MBTI-F occurs at a pH optimum of 4.5, with the tetrapeptide Lys-Asp-Asp-Asp being released. Proteinase F exhibited no activity against the modified inhibitor forms MBTI-E and MBTI-C. Vicilin, the major storage protein of the mung bean, does not serve as a substrate for proteinase F between pH 4 and 7. Proteinase F is inhibited by phenylmethylsulfonyl fluoride, chymostatin, p-hydroxymercuribenzoate, and p-chlorophenylsulfonate, but not by iodoacetate and CuCl₂. It is not activated by dithiothreitol, and is stable for extended periods of time (10 months, 4°C, pH 4.0) in the absence of reducing agents. An apparent molecular weight of 65,000 was found for proteinase F by gel filtration. Subcellular fractionation in glycerol suggests that greater than 85% of the proteinase F activity is found in the protein bodies of the ungerminated mung bean. The same studies indicate that at least 56% of the MBTI of the seed is also localized in the protein bodies.     

Secretion of epidermal growth factor The role of calcium in stimulus-secretion coupling and structural modification of the growth factor molecule during secretion

Secretion of epidermal growth factor by mouse submandibular salivary glands was studied in vitro to determine the second messenger involved in stimulus-secretion coupling and also to determine whether epidermal growth factor is secreted in the molecular form in which it occurs within the glandular cells. The presence of Ca²⁺ in the extracellular fluid was found to be necessary for the normal secretion of epidermal growth factor that follows activation of α-adrenoceptors. Dibutyryl cyclic AMP (1 mM) did not evoke any secretion of the growth factor. Secreted epidermal growth factor retained its ability to bind to anti-epidermal growth factor antibodies and to epidermal growth factor-binding sites on liver cell membranes. However, during secretion the 74 000-dalton, 4-subunit complex, in which epidermal growth factor occurs within the cells, dissociated. The adrenalin-stimulated submandibular salivary gland did not appear to release any material into the incubation medium which could modify the complex and produce the dissociation. It is suggested that the dissociation of the high molecular weight molecule containing epidermal growth factor results from the loss of the arginyl residue from the C-terminus of epidermal growth factor at some time during the secretion process.     

Effect of alkyl alcohols on partially unfolded state of Proteinase K: Differential stability of α-helix and β-sheet rich regions of the enzyme

Proteinase K (E.C. 3.4.21.64), a serine proteinase from fungus Tritirachium album, has been used as a model system to investigate the conformational changes induced by monohydric alcohols at low pH. Proteinase K belongs to α/β class of proteins and maintains structural integrity in the range of pH 7.0–3.0. Enzyme acquires partially unfolded conformation (U_P) at pH 2.5 with lower activity, partial loss of tertiary structure and exposure of some hydrophobic patches. Proteinase K in stressed state at pH 2.5 is chosen and the conformational changes induced by alkyl alcohols (methanol/ethanol/isopropanol) are studied. At critical concentration of alcohol, conformational switch occurs in the protein structure from α/β to β-sheet driving the protein into O-state. Complete loss of tertiary contacts and proteolytic activity in O-sate emphasize the involvement of alpha regions in maintaining the active site of the enzyme. Moreover, isopropanol induced unfolding of proteinase K in U_P state occurred in two steps with the formation of β state at low alcohol concentration followed by stabilization of β state at high alcohol concentration. GuHCl and temperature induced unfolding of proteinase K in O-state (in 50% isopropanol) is non-cooperative as the transition curves are biphasic. This suggests that the structure of proteinase K in O-state has melted alpha regions and stabilized beta regions and that these differentially stabilized regions unfold sequentially. Further, the O-state of proteinase K can be attained from complete unfolded protein by the addition of 50% isopropanol. Hence the alcohol-induced O-state is different from native state or completely unfolded state and shows characteristics of the molten globule-like state. Thus, this state may be functioning as an intermediary in the folding pathway of proteinase K.     

Secretion of epidermal growth factor. The role of calcium in stimulcule during secretion.

Secretion of epidermal growth factor by mouse submandibular salivary glands was studied in vitro to determine the second messenger involved in stimulus-secretion coupling and also to determine whether epidermal growth factor is secreted in the molecular form in which it occurs within the glandular cells. The presence of Ca2+ in the extracellular fluid was found to be necessary for the normal secretion of epidermal growth factor that follows activation of alpha-adrenoceptors. Dibutyryl cyclic AMP (1 mM) did not evoke any secretion of the growth factor. Secreted epidermal growth factor retained its ability to bind to anti-epidermal growth factor antibodies and to epidermal growth factor-binding sites on liver cell membranes. However, during secretion the 74 000-dalton, 4-subunit complex, in which epidermal growth facto; occurs within the cells, dissociated. The adrenalin-stimulated submandibular salivary gland did not appear to release any material into the incubation medium which could modify the complex and produce the dissociation. It is suggested that the dissociation of the high molecule containing epidermal growth factor results from the loss of the arginyl residue from the C-terminus of epidermal growth factor at some time during the secretion process.     

Synthesis of Extracellular Proteinase by Pseudomonas fluorescens Under Conditions of Limiting Carbon, Nitrogen, and Phosphate

The influence of carbon, nitrogen, and phosphate concentrations on growth and proteinase production by Pseudomonas fluorescens 32A was examined. In mineral salts medium containing dialyzed skim milk supernatant as an inducer, maximum growth was obtained at 1.0 and 2.5 mM orthophosphate at 20 and 5°C, respectively. At both temperatures, 5 mM orthophosphate was required for maximum proteinase production, whereas significant inhibition was found at 10 mM. Orthophosphate was the only phosphate compound able to support growth. With sodium pyruvate as the carbon source, maximum enzyme synthesis was at 100 mM carbon at both temperatures. At both 20 and 5°C maximum growth and enzyme production was found with 10 mM NH₄Cl. A bioassay for available phosphate based on the growth of P. fluorescens 32A in phosphate-limited mineral salts medium showed that skim milk and skim milk supernatant contained 50 and 10 mM orthophosphate, respectively. Proteinase production in skim milk was 2.6- and 12-fold greater than that in optimal mineral salts medium at 20 and 5°C, respectively. These results suggest that proteinase production in milk does not occur as a result of nutrient limitation and may be regulated in part by milk phosphates.     

蕃茄蛋白酶抑制剂Ⅱ_a、Ⅱ_b的分离纯化及性质研究

经硫酸铵分级、CM-纤维素柱层析和Sephadex G-75柱层析,从野生型秘鲁蕃茄未成熟果实的匀浆液中分离纯化了蛋白酶抑制剂Ⅱa、Ⅱb。纯化的蛋白酶抑制剂经SDS-PAGE鉴定呈单一带,分子量均为17kD。Western blotting结果表明蛋白酶抑制剂Ⅱa、Ⅱb均与马铃薯蛋白酶抑制剂Ⅱ有血清学上的交叉反应。抑制蛋白酶活性测定显示两种蛋白酶抑制剂都表现強的抑制胰凝乳蛋白酶的活性,但对胰蛋白酶的抑制活性蛋白酶抑制剂Ⅱb強于蛋白酶抑制剂Ⅱa。     

Proteomic analysis of secretion from human transplanted submandibular gland replacing lacrimal gland with severe keratoconjunctivitis sicca

Purpose: Proteomic analysis of secretions from transplanted or non-transplanted submandibular glands in patients with severe keratoconjunctivitis sicca and tears from normal eyes. Experimental design: Secretions from submandibular glands transplanted to replace lacrimal glands and non-transplanted submandibular glands were collected at 1 year from 5 patients with severe keratoconjunctivitis sicca undergoing transplantation, and tears were collected from 3 normal subjects. 2-D electrophoresis (2-DE), then mass spectrometry was used to identify proteins. Western blot analysis was used to confirm protein expression. Results: We identified 34 and 11 distinct proteins in the saliva from transplanted submandibular glands and tears, respectively. The saliva from transplanted submandibular glands contained almost all the proteins abundant in tear fluid. The functions of identified proteins in the saliva from transplanted submandibular gland were mainly immune response and anti-bacterial. In total, 7 proteins showed differential expression between the saliva of transplanted and non-transplanted submandibular glands. The upregulation of short palate, lung and nasal epithelium carcinoma-associated protein 2 and carbonic anhydrase VI was confirmed by Western blot analysis. Conclusions: Identified proteins in saliva from transplanted submandibular glands may protect ocular structures. These findings can help in understanding the functional status of transplanted submandibular glands.     

Trypanosoma cruzi: Isolation and characterization of a proteinase

Lysates of Trypanosoma cruzi epimastigotes were able to hydrolyze casein (K_m = 2.5 mg/ml) as well as bovine and human hemoglobins (K_m = 12.2 mg/ml); there was optimum activity was around pH 7.0. The proteinase activity detected with these substrates was enhanced by sodium diaminotetraacetate (EDTA) and reducing agents (SO^2?₃, mercaptoethanol, cysteine) and was inhibited by sulfhydryl reagents, thus suggesting an SH-dependent enzyme. Purification (60×) of the proteinase was carried out as follows: (1) precipitation at ?20 C, pH 4.5, with 80% acetone, (2) gel filtration on Sephadex G-200, (3) affinity chromatography on Sepharose 4B covalently linked to p-aminophenyl mercuric acetate. Only a single component (with an estimated molecular weight of 60,000) was detected in purified preparations by polyacrylamide gel electrophoresis. However, in addition to the major component identified as a proteinase, crossed immunoelectrophoresis experiments indicated the presence of at least three other antigens that apparently were devoid of proteinase activity. Optimum pH activity of the purified preparations was around pH 6.0 for casein and pH 3.0 for hemoglobins, but these activities probably are due to the one enzyme since they were altered identically by the same agents.     

Isolation and general characterization of a heat-stable proteinase from Pseudomonas fluorescens AFT 36

An extracellular proteinase from Pseudomonas fluorescens, strain AFT 36, was isolated to homogeneity by chromatography on DEAE-cellulose and Sephadex G-150; a 230-fold increase in specific activity with a recovery of 53% was obtained. The enzyme was optimally active at pH 6.5 and 45°C; activity declined rapidly at higher temperatures but significant activity persisted down to 4°C. Activity was strongly inhibited by 10^?3 M EDTA and was partially restored by addition of Zn²⁺, Ca²⁺ or Co²⁺. The K_m values on methylated casein and sodium caseinate were 18.2 and 7.1 mg/ml, respectively. The enzyme was very labile in phosphate buffer and in a milk salts buffer at 55°C but was very stable in the latter at more than 80°C.     

Digestive physiology and characterization of digestive cathepsin L-like proteinase from the sugarcane weevil Sphenophorus levis

Sugarcane is an important crop that has recently become subject to attacks from the weevil Sphenophorus levis, which is not efficiently controlled with chemical insecticides. This demands the development of new control devices for which digestive physiology data are needed. In the present study, ion-exchange chromatography of S. levis whole midgut homogenates, together with enzyme assays with natural and synthetic substrates and specific inhibitors, demonstrated that a cysteine proteinase is a major proteinase, trypsin is a minor one and chymotrypsin is probably negligible. Amylase, maltase and the cysteine proteinase occur in the gut contents and decrease throughout the midgut; trypsin is constant in the entire midgut, whereas a membrane-bound aminopeptidase predominates in the posterior midgut. The cysteine proteinase was purified to homogeneity through ion-exchange chromatography. The purified enzyme had a mass of 37 kDa and was able to hydrolyze Z-Phe-Arg-MCA and Z-Leu-Arg-MCA with k_cat/K_m values of 20.0 ± 1.1 μM⁻¹ s⁻¹ and 30.0 ± 0.5 μM⁻¹ s⁻¹, respectively, but not Z-Arg-Arg-MCA. The combined results suggest that protein digestion starts in the anterior midgut under the action of a cathepsin L-like proteinase and ends on the surface of posterior midgut cells. All starch digestion takes place in anterior midgut. These data will be instrumental to developing S. levis-resistant sugarcane.     

Isolation and characterization of a membrane-bound proteinase from rat liver.

The proteinase previously found in chromatin prepared from a total rat liver homogenate was purified from the rat liver mitochondrial fraction. The membrane-bound enzyme is solubilized in either 0.6% digitonin or 0.5 m phosphate buffer. After a 1330-fold purification, the enzyme appears homogeneous by acrylamide-gel electrophoresis. Sucrose density gradient centrifugation indicated a molecular weight of 22,500, a molecular weight of 23,500 ± 10% has been estimated by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme showed a high substrate specificity. Among several proteins tested, only glucagon, nonhistone chromosomal proteins, and histones are good substrates. A limited proteolysis was found for the very-lysine-rich histone H1, which was split into a high molecular weight fragment (M_r 13,000). The highly phosphorylated histone H1 isolated from regenerating rat liver 24 h after partial hepatectomy exhibited the same susceptibility to the proteinase as H1 from normal liver. Large polypeptides of a nonhistone chromosomal protein fraction were degraded more rapidly than the small ones. N-Acetyl-l-tyrosine ethyl ester was used with alcohol dehydrogenase and NAD in a coupled enzyme assay for the proteinase. The apparent Michaelis constant for the hydrolysis of N-acetyl-l-tyrosine ethyl ester is 5.0 × 10^?3m. The proteinase has catalytic properties simlar to trypsin and chymotrypsin. The pH optimum was around 8, soybean trypsin inhibitor depressed the enzymatic activity, and the serine modifying reagents diisopropyl phosphofluoridate and phenylmethanesulfonyl fluoride inactivated the enzyme. The affinity reagent for chymotrypsin-like active sites, l-1-tosylamido-2-phenylethyl chloromethyl ketone, inactivated the proteinase.     

Degradation of ribulose-1,5-bisphosphate carboxylase by proteolytic enzymes from crude extracts of wheat leaves

In crude extracts from the primary leaf of wheat seedlings, Triticum aestivum L., cv. Olympic, maximum proteinase activity, as determined by measuring the rate of release of amino nitrogen from ribulose-bisphosphate carboxylase (RuBPCase), was found to be obtained only when EDTA and L-cysteine were included in the extraction buffer. Highest proteinase activity was obtained by grinding at pH 6.8, although the level of activity was similar in the pH range 5.6 to 8.0; this range also coincided with maximum extractability of protein. The lower amount of RuBPCase degrading proteinase extracted at low pH was not due to an effect of pH on enzyme stability. The optimum temperature of reaction was 50° C and reaction rates were linear for at least 120 min at this temperature. In the absence of substrate the proteinase was found to be very sensitive to temperatures above 30° C, with even short exposures causing rapid loss of activity. The relation between assay pH and RuBPCase degradation indicated that degradation was restricted to the acid proteinase group of enzymes, with a pH optimum of 4.8, and no detectable activity at a pH greater than 6.4. The levels of extractable RuBPCase proteinase exhibited a distinct diurnal variation, with activity increasing during the latter part of the light period and then declining once the lights were turned off. The effect of leaf age on the level of RuBPCase, RuBPCase proteinase and total soluble protein was investigated. Maximum RuBPCase activity occurred 9 days after sowing as did soluble protein. After the maximum level was obtained, the pattern of total soluble protein was shown to be characterised by three distinct periods of protein loss: I (day 9–13) 125 ng leaf^-1 day^-1; II (day 15–27) 11 ng leaf^-1 day^-1; III (day 29–49) 22 ng leaf^-1 day^-1. Comparison of the pattern of RuBPCase activity and total protein suggest that the loss of RuBPCase may be largely responsible for the high rate of protein loss during period I. Proteinase activity increased sharply during the period of most rapid loss of RuBPCase activity, and because the specific activity of RuBPCase also declined, we concluded that RuBPCase was being degraded more rapidly than the other proteins. Once the majority of the RuBPCase was lost, there did not appear to be a direct relation between RuBPCase proteinase activity and rate of total soluble protein loss, since the proteinase exhibited maximum activity during the slowest period of protein loss (II), and was declining in activity while the rate of protein loss remained stable during the third and final period of total protein loss.Abbreviations RuBPCase ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39) - TCA trichloroacetic acid Supported by the Wheat Industry Research Council of Australia and the Australian Research Grants Committee D2 74/15052     

In-silico comparative study of inhibitory mechanism of Plant Serine Proteinase Inhibitors

The nematodes like root-knot and cyst are plant-parasitic pest found in horticultural and agricultural crops. They do damages in the roots of plants as a result losses million tons of production. High cost of nematicides and environment safety concern has necessitated finding of some alternative methods. Under Integrated Pest Management (IPM) such problems are solving significantly by means of target gene inhibition, agrobacterium mediated transformation etc. One of this strategy use Plant Proteinase Inhibitors (PIs) gene which are used to control the proteolysis mechanism of Pest by inhibiting gut Serine Proteinase (SP). Present work investigates the utility of computer aided methods to study the mechanism of Protein-Protein interactions and thereby inhibition of Serine Proteinase by PIs. Hence 3D models of Serine Proteinase as well as Serine Proteinase Inhibitors (SPIs) generated using homology modeling. Validations of constructed models have been done by PROCHECK, VERIFY3D, ERRAT and PROSA. Prediction of Protein interacting surface patches and site specific protein docking was performed by using ZDOCK Server. Backbone refinement of output protein complexes was executed in Fiber Dock server. Interaction study between SP and SPIs complexes shows their comparative inhibition efficacy, measured in terms of number of hydrogen bonds, Van dar wall attraction and docking energy. This work reported that Vigna marina and Phaseolus oligospermus are having better inhibition efficiency in comparison to other inhibitors.     

Purification and properties of a fibrinogenase from the venom of Naja nigricollis

A fibrinogenolytic proteinase from the venom of Naja nigricollis was purified by chromatography on Bio-Rex 70 and Phenyl-Sepharose. The purified enzyme, designated proteinase F1, was homogeneous by the criterion of SDS-polyacrylamide gel electrophoresis, and consisted of a single chain with a molecular weight of 58 000. Purified proteinase F1 had approximately 15-fold more proteinase activity than the crude venom, based on its ability to inactive α₂-macroglobulin. The enzyme acted on only the Aα-chain of fibrinogen and left the Bβ- and γ-chains intact. The pH optimum for this fibrinogenolytic activity was in the range of pH 8 to 10. In addition to its activity on fibrinogen, proteinase F1 was active on α₂-macroglobulin and fibronectin, but did not degrade casein, hemoglobin or bovine serum albumin. The enzyme was not inhibited by inhibitors of serine proteinases, cysteine proteinases or acid proteinases, but only by the metalloproteinase inhibitor, EDTA. The inhibition by EDTA could be prevented by Zn²⁺, but not by Ca²⁺ or Mg²⁺.     

Mechanism of Proteinase Release from Lactococcus lactis subsp. cremoris Wg2

The procedure generally used for the isolation of extracellular, cell-associated proteinases of Lactococcus lactis species is based on the release of the proteinases by repeated incubation and washing of the cells in a Ca²⁺-free buffer. For L. lactis subsp. cremoris Wg2, as many as five incubations for 30 min at 29°C are needed in order to liberate 95% of the proteinase. Proteinase release was not affected by chloramphenicol, which indicates that release is not the result of protein synthesis during the incubations. Ca²⁺ inhibited, while ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) stimulated, proteinase release from the cells. The pH optimum for proteinase release ranged between 6.5 and 7.5, which was higher than the optimum pH of the proteinase measured for casein hydrolysis (i.e., 6.4). Treatment of cells with the serine proteinase inhibitor phenylmethylsulfonyl fluoride prior to the incubations in Ca²⁺-free buffer reduced the release of the proteinase by 70 to 80%. The residual proteinase remained cell associated but could be removed by the addition of active L. lactis subsp. cremoris Wg2 proteinase. This suggests that proteinase release from cells of L. lactis subsp. cremoris Wg2 is the result of autoproteolytic activity. From a comparison of the N-terminal amino acid sequence of the released proteinase with the complete amino acid sequence determined from the nucleotide sequence of the proteinase gene, a protein of 180 kilodaltons would be expected. However, a proteinase with a molecular weight of 165,000 was found, which indicated that further hydrolysis had occurred at the C terminus.     

Purification and properties of a proteinase from Vipera lebetina (snake) venom

A proteinase from the venom of Vipera lebetima was purified by chromatography on Sephadex G-100 and CM-cellulose. The purified proteinase was homogeneous on SDS-polyacrylamide gel electrophoresis and consisted of a single chain with molecular weight of 37 000±1500. The isoelectric point of the proteinase was over 10. The enzyme was active on casein but not on esters and amides of arginine. It split the oxidized insulin B-chain at the peptide bonds of Tyr¹⁶-Leu¹⁷, Phe²⁴-Phe²⁵ and Phe²⁵-Tyr²⁶, and glucagon at the bonds Tyr¹⁰-Ser¹¹, Leu¹⁴-Asp¹⁵ and Leu²⁶-Met²⁷. The enzyme was inhibited by DFP and PMSF, and partially by soybean trypsin inhibitor, but not with EDTA.     

PURIFICATION AND CHARACTERIZATION OF A PROTEINASE FROM THE PROBIOTIC Lactobacillus rhamnosus OXY

A proteinase produced by the human gastrointestinal isolate Lactobacillus rhamnosus strain OXY was identified and characterized. The prtR2 gene coding for proteinase activity was detected in the examined strain. The PCR primers used were constructed on the basis of the sequence of the prtR2 proteinase gene from Lactobacillus rhamnosus GG. The enzyme was purified by fast protein liquid chromatography (FPLC) using CM-Sepharose Fast Flow and Sephacryl S-300 columns. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the enzyme had a relatively low molecular mass of 60 kD. Protease activity was observed at a pH range from 6.5 to 7.5 with optimum k _cat/K _m values at pH 7.0 and 40°C. Maximum proteolytic activity (59 U mL^?1) was achieved after 48 hr of cultivation. The activity of the enzyme was inhibited only by irreversible inhibitors specific for serine proteinases (PMSF and 3,4-dichloro-isocumarine), suggesting that the enzyme was a serine proteinase. Proteinase activity was increased by Ca²⁺ and Mg²⁺, and inhibited by Cu²⁺, Zn²⁺, Cd²⁺, and Fe^2+.     

Extracellular proteinase fromLactobacillus murinus

Lactobacillus murinus CNRZ 313 produced an extracellular proteinase irrespective of the Ca²⁺ content in the culture medium. Proteinase activity was optimal at 37 °C and pH 7.5 in phosphate buffer (0.2 mol/L). It was stimulated by Mg²⁺ and Mn²⁺ and was inhibited by Zn²⁺. Ca²⁺ did not affect the enzymic activity but the proteinase liberated in the presence of this ion is more stable. The enzyme was purified to homogeneity from cell-free culture medium.