A serine protease from a detergent-soluble extract of Leishmania (Leishmania) amazonensis

Proteases mediate important crucial functions in parasitic diseases, and their characterization contributes to the understanding of host-parasite interaction. A serine protease was purified about 43-fold with a total recovery of 60% from a detergent-soluble extract of promastigotes of Leishmania amazonensis. The purification procedures included aprotinin-agarose affinity chromatography and gel filtration high performance liquid chromatography. The molecular mass of active enzyme was 110 kDa by native gel filtration HPLC and by SDS-PAGE gelatin under non-reducing conditions. Under conditions of reduction using SDS-PAGE gelatin analyses the activity of enzyme was observed in two proteins of 60 and 45 kDa, suggesting that the enzyme may be considered as a dimer. The Leishmania protease was not glycosylated, and its isoelectric point (pI) was around 4.8. The maximal protease activity was at pH 7.0 and 28 degrees C, using a-N-o-tosyl-L-arginyl-methyl ester (L-TAME) as substrate. Assays of thermal stability indicated that this enzyme was totally denatured after pre-treatment at 42 degrees C for 12 min and preserved only 20% of its activity after pre-treatment at 37 degrees C for 24 h, in the absence of substrate. Hemoglobin, bovine serum albumin (BSA), ovalbumin and gelatin were hydrolyzed by Leishmania protease. Inhibition studies indicated that the enzyme belonged to a serine protease class because of a significant impediment by serine protease inhibitors such as benzamidine, aprotinin, and antipain. The activity of the present serine protease is negatively modulated by calcium and zinc and positively modulated by manganese ions. This is the first study that reports the purification of a protease from a detergent-soluble extract of Leishmania species.     

Purification and characterization of an extracellular protease from Penicillium chrysogenum Pg222 active against meat proteins

An extracellular protease from Penicillium chrysogenum (Pg222) isolated from dry-cured ham has been purified. The purification procedure involved several steps: ammonium sulfate precipitation, ion-exchange chromatography, filtration, and separation by high-performance liquid chromatography. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and gel filtration, the purified fraction showed a molecular mass of about 35 kDa. The hydrolytic properties of the purified enzyme (EPg222) on extracted pork myofibrillar proteins under several conditions were evaluated by SDS-PAGE. EPg222 showed activity in the range of 10 to 60 degrees C in temperature, 0 to 3 M NaCl, and pH 5 to 7, with maximum activity at pH 6, 45 degrees C, and 0.25 M NaCl. Under these conditions the enzyme was most active against tropomyosin, actin, and myosin. EPg222 showed collagenolytic activity but did not hydrolyze myoglobin. EPg222 showed higher activity than other proteolytic enzymes like papain, trypsin, and Aspergillus oryzae protease. The N-terminal amino acid sequence was determined and was found to be Glu-Asn-Pro-Leu-Gln-Pro-Asn-Ala-Pro-Ser-Trp. This partial amino acid sequence revealed a 55% homology with serine proteases from Penicillium citrinum. The activity of this novel protease may be of interest in ripening and generating the flavor of dry-cured meat products.     

A metalloproteinase extracellularly released by Crithidia deanei

Actively motile cells from a cured strain of Crithidia deanei released proteins in phosphate buffer (pH 7.4). The molecular mass of the released polypeptides, which included some proteinases, ranged from 19 to 116 kDa. One of the major protein bands was purified to homogeneity by a combination of anion-exchange and gel filtration chromatographs. The apparent molecular mass of this protein was estimated to be 62 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The incorporation of gelatin into SDS-PAGE showed that the purified protein presented proteolytic activity in a position corresponding to a molecular mass of 60 kDa. The enzyme was optimally active at 37 degrees C and pH 6.0 and showed 25% of residual activity at 28 degrees C for 30 min. The proteinase was inhibited by 1,10-phenanthroline and EDTA, showing that it belonged to the metalloproteinase class. A polyclonal antibody to the leishmanial gp63 reacted strongly with the released C. deanei protease. After Triton X-114 extraction, an enzyme similar to the purified metalloproteinase was detected in aqueous and detergent-rich phases. The detection of an extracellular metalloproteinase produced by C. deanei and some other Crithidia species suggests a potential role of this released enzyme in substrate degradation that may be relevant to the survival of trypanosomatids in the host.     

Purification and characterization of an beta-D-xylosidase from Candida utilis IFO 0639

An intracellular beta-D-xylosidase from Candida utilis IFO 0639 was purified to homogeneity through four chromatographic steps. The molecular mass of the enzyme was estimated to be 92 kDa by SDS-PAGE. The enzyme had an isoelectric point at 5.6, and was most active at pH 6.0 and at around 40 degrees C. Ethanol at an optimal concentration (10%, v/v) stimulated the initial enzyme activity by 57%. D-Xylose, the product of the beta-D-xylosidase, has no effect on the enzyme activity at 300 mM. The beta-D-xylosidase was highly specific to the beta-D-xylopyranoside configuration. The enzyme hydrolyzed beta-1,4-linked xylo-oligosaccharides with chain lengths from 2 to 5 by releasing xylose from the non-reducing end. It showed no activity against xylan. The enzyme efficiently released monoterpenols from an aroma precursor extracted from Muscat grape juice. The fermentation of Muscat juice coupled with the enzyme addition produced a small increase in the concentration of monoterpenols.     

Leishmania (Leishmania) amazonensis: purification and characterization of a promastigote serine protease

Pathogenic protozoan proteases play crucial roles in the host-parasite interaction, and its characterization contributes to the understanding of protozoan disease mechanisms. A Leishmania amazonensis promastigote protease was purified 36-fold, using aprotinin-agarose affinity chromatography and gel filtration high performance liquid chromatography, yielding a total recovery of 49%. The molecular mass of active enzyme obtained from native gel filtration HPLC and SDS-PAGE under conditions of reduction and non-reduction was 68 kDa, suggesting that the enzyme may exist as a monomer. The protease isoelectric point (pI) was around 4.45 and, as demonstrated by deglycosylation assay, it did not have any carbohydrate content. The optimal pH and temperature of the enzyme were 8.0 and 28 degrees C, respectively, determined using alpha-N-rho-tosyl-L-arginyl-methyl ester (L-TAME) as substrate. Assays of thermal stability indicated that 50% of the enzymatic activity was preserved after 4 min of pre-treatment at 42 degrees C and after 24 h of pre-treatment at 37 degrees C, both in the absence of substrate. Hemoglobin, bovine serum albumin (BSA), ovalbumin, and both gelatin and peptide substrates containing arginine in ester bound were hydrolyzed by 68 kDa protease. The insulin beta-chain was also hydrolyzed by the protease, and four peptidic bonds (L11-V12, E13-A14, L15-Y16, and Y16-L17) were susceptible to the 68-kDa protease action. Inhibition studies suggested that the enzyme belonged to a serine protease class inhibited by calcium ions and activated by manganese ions. These findings demonstrate that the L. amazonensis 68-kDa serine protease differs from those of other protozoan parasites.     

A novel extracellular subtilisin-like protease from the hyperthermophile<Emphasis Type="Italic"> Aeropyrum pernix</Emphasis> K1: biochemical properties,cloning, and expression

A novel extracellular serine protease designated Pernisine was purified to homogeneity and characterized from the archaeon Aeropyrum pernix K1. The molecular mass, estimated by SDS-PAGE analysis and by gel filtration chromatography, was about 34 kDa suggesting that the enzyme is monomeric. Pernisine was active in a broad range of pH (5.0-12.0) and temperature (60-120 degrees C) with maximal activity at 90 degrees C and between pH 8.0 and 9.0. In the presence of 1 mM CaCl(2) the activity, as a function of the temperature, reached a maximum at 90 degrees C but at 120 degrees C the enzyme retained almost 80% of its maximal activity. Activity inhibition studies suggest that the enzyme is a serine metalloprotease and biochemical data indicate that Pernisine is a subtilisin-like enzyme. The protease gene, identified from the sequenced genome of A. pernix, was amplified from total genomic DNA by PCR technique to construct the expression plasmid pGEX-Pernisine. The Pernisine, lacking the leader sequence, was expressed in Escherichia coli BL21 strain as a fusion protein with glutathione- S-transferase. The biochemical properties of the recombinant enzyme were found to be similar to those of the native enzyme.     

Purification and characterization of an extracellular protease from the fish pathogen Yersinia ruckeri and effect of culture conditions on production.

A novel protease, hydrolyzing azocasein, was identified, purified, and characterized from the culture supernatant of the fish pathogen Yersinia ruckeri. Exoprotease production was detected at the end of the exponential growth phase and was temperature dependent. Activity was detected in peptone but not in Casamino Acid medium. Its synthesis appeared to be under catabolite repression and ammonium control. The protease was purified in a simple two-step procedure involving ammonium sulfate precipitation and ion-exchange chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the purified protein indicated an estimated molecular mass of 47 kDa. The protease had characteristics of a cold-adapted protein, i.e., it was more active in the range of 25 to 42 degrees C and had an optimum activity at 37 degrees C. The activation energy for the hydrolysis of azocasein was determined to be 15.53 kcal/mol, and the enzyme showed a rapid decrease in activity at 42 degrees C. The enzyme had an optimum pH of around 8. Characterization of the protease showed that it required certain cations such as Mg(2+) or Ca(2+) for maximal activity and was inhibited by EDTA, 1,10-phenanthroline, and EGTA but not by phenylmethylsulfonyl fluoride. Two N-methyl-N-nitro-N-nitrosoguanidine mutants were isolated and analyzed; one did not show caseinolytic activity and lacked the 47-kDa protein, while the other was hyperproteolytic and produced increased amounts of the 47-kDa protein. Azocasein activity, SDS-PAGE, immunoblotting by using polyclonal anti-47-kDa-protease serum, and zymogram analyses showed that protease activity was present in 8 of 14 strains tested and that two Y. ruckeri groups could be established based on the presence or absence of the 47-kDa protease.     

Detection and characterization of protease secreted by the plant pathogen Xylella fastidiosa

Xylella fastidiosa is a pathogenic bacterium found in several plants. These bacteria secrete extracellular proteases into the culture broth as visualized in sodium-dodecyl-sulfate polyacrylamide activity gels containing gelatin as a copolymerized substrate. Three major protein bands were produced by the citrus strain with molar masses (MM) of 122, 84 and 65 kDa. Grape strain 9,713 produced two bands of approximately 84 and 64 kDa. These organisms produced zones of hydrolysis in agar plates amended with gelatin, casein and hemoglobin. Gelatin was the best substrate for these proteases. Sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE) activity gel indicated that the protease of Xylella fastidiosa from citrus and grape were completely inhibited by PMSF and partially inhibited by EDTA. The optimal temperature for protease activity was 30 degrees C with an optimal pH of 7.0. Among the proteolytic enzymes secreted by the phytopathogen, chitinase and beta-1,3-glucanase activities were also detected in cultures of Xylella fastidiosa (citrus). From these results, it is suggested that proteases produced by strains of Xylella fastidiosa from citrus and grape, belong to the serine- and metallo-protease group, respectively.     

A study of extracellular alkaline protease from Bacillus subtilis NCIM 2713

An alkaline protease was isolated from culture filtrate of B. subtilis NCIM 2713 by ammonium sulphate precipitation and was purified by gel filtration. With casein as a substrate, the proteolytic activity of the purified protease was found to be optimal at pH 8.0 and temperature 70 degrees C. The purified protease had molecular weight 20 kDa, Isoelectric point 5.2 and km 2.5 mg ml(-1). The enzyme was stable over the pH range 6.5-9.0 at 37 degrees C for 3 hr. During chromatographic separation this protease was found to be susceptible to autolytic degradation in the absence of Ca2+. Ca2+ was not only required for the enzyme activity but also for the stability of the enzyme above 50 degrees C. About 62% activity was retained after 60 min at pH 8.0 and 55 degrees C. DFP and PMSF completely inhibited the activity of this enzyme, while in the presence of EDTA only 33% activity remained. However, it was not affected either by sulfhydryl reagent, or by divalent metal cations, except SDS and Hg2+. The results indicated that this is a serine protease.     

Purification and biochemical characterization of a protease secreted by the <Emphasis Type="Italic">Salinivibrio</Emphasis> sp. strain AF-2004 and its behavior in organic solvents

A metalloprotease secreted by the moderately halophilic bacterium Salinivibrio sp. strain AF-2004 when the culture reached the stationary growth phase. This enzyme was purified to homogeneity by acetone precipitation and subsequent Q-Sepharose anion exchange and Sephacryl S-200 gel filtration chromatography. The apparent molecular mass of the protease was 31 kDa by SDS-PAGE, whereas it was estimated as approximately 29 kDa by Sephacryl S-200 gel filtration. The purified protease had a specific activity of 116.8 mumol of tyrosine/min per mg protein on casein. The optimum temperature and salinity of the enzyme were at 55 degrees C and 0-0.5 M NaCl, although at salinities up to 4 M NaCl activity still remained. The protease was stable and had a broad pH profile (5.0-10.0) with an optimum of 8.5 for casein hydrolysis. The enzyme was strongly inhibited by phenylmethyl sulfonylfluoride (PMSF), Pefabloc SC, chymostatin and also EDTA, indicating that it belongs to the class of serine metalloproteases. The protease in solutions containing water-soluble organic solvents or alcohols was more stable than that in the absence of organic solvents. These characteristics make it an ideal choice for applications in industrial processes containing organic solvents and/or salts.     

A cysteine protease of Dieffenbachia maculata.

Plants of the genus Dieffenbachia, very popular as indoor ornamental plants, are known for their toxic as well as therapeutic properties. Their toxic manifestations have been partly attributed to their proteolytic activity. The work described in the present paper shows that stem leaves and petiole of Dieffenbachia maculata Schott, a commonly grown species, contain significant proteolytic activity, different parts showing different types of protease activities. Stem showed the highest enzyme activity and this protease was purified about 55 fold by solvent precipitation, gel filtration and ion exchange chromatography. The enzyme has a relative molecular mass of 61 kDa as determined by SDS-PAGE and has an optimum pH of 8.0 and optimum temperature of 50 degrees C. Effects of various substrates, inhibitors and activators indicate that the enzyme is a cysteine protease with leucylpeptidase activity.     

Purification and characterization of maltose phosphorylase from Bacillus sp. RK-1

Bacillus sp. RK-1 was isolated as a bacterium that produced maltose phosphorylase (MPase) in the culture supernatant. Screening was done from among about 400 isolates that could grow at 55 degrees C in a medium containing maltose as the sole carbon source. The enzyme was purified to an electrophoretically homogeneous state and some properties were investigated. The Mr of the enzyme was estimated to be 170 kDa by gel filtration and 88.5 kDa by SDS-PAGE, suggesting that it consisted of two identical subunits. The enzyme showed optimum activity around pH 6.0-7.0 and the optimum temperature was about 65 degrees C. The enzyme was stable in the range of pH 5.5-8.0 after keeping it at 4 degrees C for 24 h and retained the activity up to about 55 degrees C after keeping it for 15 min. This is the first report about an MPase that could be produced in the culture supernatant. Furthermore, these investigations showed that this MPase is one of the most thermostable ones reported so far.     

Purification and some properties of beta-N-acetyl-D-glucosaminidase from the cabbage butterfly (Pieris rapae)

A beta-N-acetyl-D-glucosaminidase (NAGase) from the cabbage butterfly (Pieris rapae) was purified. The purified enzyme was a single band on polyacrylamide gel electrophoresis and the specific activity was determined to be 8715 U/mg. The molecular weight of whole enzyme was determined to be 106 kDa by gel filtration, and the result of SDS-PAGE showed that the enzyme was a heterodimer, which contained two subunits with different mass of 59.5 and 57.2 kDa. The optimum pH and optimum temperature of the enzyme for the hydrolysis of p-nitrophenyl-N-acetyl-beta-D-glucosaminide (pNP-NAG) were investigated to be at pH 6.2 and at 42 degrees C, respectively, and the Michaelis-Menten constant (K(m)) was determined to be 0.285 mM at pH 6.2 and 37 degrees C. The stability of the enzyme was investigated and the results showed that the enzyme was stable at the pH range from 4.0 to 9.0 and at the temperature below 45 degrees C. The activation energy was 83.86 kJ/mol. The reaction of this enzyme with pNP-NAG was judged to be Ordered Bi-Bi mechanism according to the inhibitory behaviors of the products. The ionization constant, pK(e), of ionizing group at the active site of the enzyme was found to be 5.20 at 39.0 degrees C, and the standard dissociation enthalpy (DeltaH(o)) was determined to be 2.18 kcal/mol. These results showed that the ionizing group of the enzyme active center was the carboxyl group. The results of chemical modification also suggested that carboxyl group was essential to the enzyme activity. Moreover, Zn(2+), Hg(2+), Cu(2+) had strongly inhibitory effects on the enzyme activity.     

Cold-active serine alkaline protease from the psychrophilic bacterium<Emphasis Type="Italic"> Pseudomonas</Emphasis> strain DY-A: enzyme purification and characterization

An extracellular protease was purified from a deep-sea psychrophilic bacterium strain DY-A which was identified as a Pseudomonas species. The optimal growth and protease-producing temperatures of the strain were all 10 degrees C, and the protease was secreted only at temperatures under 20 degrees C. The enzyme was most active at 40 degrees C and at pH 10.0. It was inhibited by phenylmethyl sulfonylfluoride and diisopropyl fluorophosphate, indicating that it is a serine protease. Chelators such as EDTA, EGTA, 1,10-phenanthroline and 2,2'-bipyridyl produced a decrease of activity. The enzyme was sensitive to denaturing agents such as SDS, urea, and guanidine HCl and resistant to thiol-containing reducing agents such as dithiotreitol. The enzyme was active towards N-succinyl-Ala-Ala-Pro-Phe- p-nitroanilide and N-succinyl-Ala-Ala-Pro-Leu- p-nitroanilide. The native molecular mass of the enzyme determined by native PAGE and SDS-PAGE was 25 kDa.     

Purification and characterization of a novel alpha-L-arabinofuranosidase from Pichia capsulata X91

An intracellular alpha-L-arabinofuranosidase from Pichia capsulata X91 was purified and characterized. The enzyme was purified to homogeneity from a cell-free extract by ammonium sulfate treatment, Concanavalin A-Sepharose, ion-exchange chromatography with DEAE Bio-Gel A agarose, arabinose-Sepharose 6B affinity chromatography, and hydroxyapatite column chromatography. The apparent molecular mass of the enzyme was estimated to be 250 kDa by native-PAGE. The enzyme molecule was suggested to be a tetramer with a subunit molecular mass of 72 kDa by SDS-PAGE. The enzyme had an isoelectric point at 5.1, and was most active at pH 6.0 and at around 50 degrees C. The alpha-L-arabinofuranosidase was active at ethanol concentrations of wine. The enzyme was inhibited by Cu2+, Hg2+, and p-chloromercuribenzoate. The enzyme hydrolyzed beet arabinan and arabinogalactan, and efficiently released monoterpenols from an aroma precursor extracted from Muscat grape juice. A considerable amount of monoterpenols was produced in the Muscat wine coupled with the enzyme addition.     

Purification and characterization of a thermostable alkaline protease from alkalophilic Thermoactinomyces sp. HS682.

Protease secreted into the culture medium by alkalophilic Thermoactinomyces sp. HS682 was purified to an electrophoretically homogeneous state through only two chromatographies using Butyl-Toyopearl 650M and SP-Toyopearl 650S columns. The purified enzyme has an apparent relative molecular mass of 25,000 according to gel filtration on a Sephadex G-75 column and SDS-PAGE and an isoelectric point above 11.0. Its proteolytic activity was inhibited by active-site inhibitors of serine protease, DFP and PMSF, and metal ions, Cu2+ and Hg2+. The enzyme was stable toward some detergents, sodium perborate, sodium triphosphate, sodium-n-dodecylbenzenesulfonate, and sodium dodecyl sulfate, at a concentration of 0.1% and pH 11.5 and 37 degrees C for 60 min. The optimum pH was pH 11.5-13.0 at 37 degrees C and the optimum temperature was 70 degrees C at pH 11.5. Calcium divalent cation raised the pH and heat stabilities of the enzyme. In the presence of 5 mM CaCl2, it showed maximum proteolytic activity at 80 degrees C and stability from pH 4-12.5 at 60 degrees C and below 75 degrees C at pH 11.5. The stabilization by Ca2+ was observed in secondary conformation deduced from the circular dichroic spectrum of the enzyme. The protease hydrolyzed the ester bond of benzoyl leucine ester well. The amino acid terminal sequence of the enzyme showed high homology with those of microbial serine protease, although alanine of the NH2-terminal amino acid was deleted.     

Identification, purification, and characterization of a secretory serine protease in an Indian strain of Leishmania donovani

An aprotinin sensitive serine protease was identified in the culture supernatant of the Indian strain of Leishmania donovani (MHOM/IN/1983/AG83). The protease was subsequently purified and characterized. The apparent molecular mass of the enzyme was 115 kDa in SDS-PAGE under non-reducing condition, while on reduction it showed a 56 kDa protein band indicating that the protease is a dimeric protein. The purified enzyme was optimally active at the pH and temperature of 7.5 and 28 degrees C, respectively. Assays of thermal stability indicated that the enzyme preserved 59% of activity even after pretreatment at 42 degrees C for 1 h. The purified protease was not glycosylated and its isoelectric pI was 5.0. N-alpha-p-tosyl-L-arginine methylester (TAME) appeared to be relatively better substrate among the commonly used synthetic substrates. The enzyme was inhibited by Ca(2+) and Mn(2+), but activated by Zn(2+). The protease could play important role(s) in the pathogenesis of visceral leishmaniasis or kala-azar.     

A thermostable alpha-arabinofuranosidase from xylanolytic Bacillus pumilus: purification and characterisation

Bacillus pumilus PS213 secretes an alpha-L-arabinofuranosidase (AF) when grown in the presence of arabinogalactan or oat meal. The enzyme has been purified to homogeneity and characterised. Its molecular mass, as determined by gel filtration, is 220 kDa, while sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) showed a single band of approximately 60 kDa. According to the result of the mass spectrometry analysis showing a molecular mass of 56 kDa, the enzyme should be a homotetramer. The isoelectric point was found to be 5.2, the enzyme activity was optimal at 55 degrees C and pH 7.0. The enzyme retained 80% of its activity after 2 h at 65 degrees C and lost 50% of activity at 75 degrees C after 135 min. The Michaelis constant K(m) and V(max) for p-nitrophenylarabinofuranoside at 37 degrees C were 1.7 mM and 52.9 U mg(-1), respectively. N-terminal sequence analysis and internal peptide fragments showed homology with glycosyl hydrolases of family 51.     

Purification and properties of invertase from the flowers of Woodfordia fruticosa

Invertase (beta-fructofuranosidase, EC 3.2.1.26) was purified from the flowers of Woodfordia fruticosa, which is used to prepare certain fermented Ayurvedic drugs. The enzyme was purified to near homogeneity as judged by native PAGE with a yield of 10.7%, using (NH4)2SO4 fractionation, followed by gel filtration through Sepharose 4B and DEAE cellulose chromatography at pH 6.8 and 4.42. The molecular mass of the purified enzyme as determined by elution through Sepharose 4B gel column was found to be approximately 280 kDa. SDS-PAGE of the purified enzyme showed that the enzyme is composed of three subunits with molecular mass of 66, 43 and 40 kDa. The enzyme showed a broad pH optimum between 4.0-7.0. Optimum assay temperature was 37 degrees C and above 45 degrees C, the enzyme activity slowly declined and inactivated around 80 degrees C. The apparent Km value of the enzyme for sucrose was 160 mM.     

Extracellular proteinase fromEnterococcus faecalis subsp.liquefaciens

An extracellular proteinase from Enterococcus faecalis subsp. liquefaciens has been purified 780-fold by a method including gel filtration on Sephadex G-50 and affinity chromatography with gramicidin J as ligand. Approximately 15% of the original enzyme activity was recovered. A purification of 14,800-fold, with 11.4% yield, may be reached using chromatofocusing as final step in the purification procedure. The molar mass of the enzyme has been estimated to be approximately 30 kDa by Sephadex gel filtration and approximately 26 kDa by SDS-PAGE. The isoelectric point has been found to be 4.6. Maximum enzyme activity of the proteinase has been observed at pH 7.5 and 45 degrees C. The enzyme hydrolyzed bovine serum albumin, alpha-lactoalbumin, beta-lactoglobulin, casein and pork myofibrillar and sarcoplasmic proteins. The extracellular proteinase was very stable; the enzyme maintained its activity in cell-free extracts over a very wide range of temperatures (-25 to 37 degrees C) for at least 2 months. At 12 degrees C, it was stable in the pH range of 5.5 to 8.0.