Protease Activity Bands Research Articles

Differential inhibition of digestive proteases by tannin in two size groups of rohu (Labeo rohita,Hamilton): A biochemical and zymography study

This study was intended to evaluate tannin mediated inhibition of digestive proteases in two different size groups (fingerlings, F, 4.5 ± 0.7 cm; advanced fingerlings, AF, 18.2 ± 1.6 cm) of rohu, Labeo rohita. Graded levels (50, 100, 150 and 200 nM) of tannin (Gallotannin, 99% purity) were added to the enzyme extracts (30°C, 1 hr) prior to determination of enzyme activities. Changes in the activity of trypsin, chymotrypsin and total protease in relation to the control sets were determined through biochemical assay of enzymes and SDS-PAGE zymography. The study revealed that tannin significantly inhibited trypsin, chymotrypsin and total protease activities in a dose dependent manner as evident from the regression equations. The degree of the inhibition appeared to be significantly higher for the F in contrast to the AF (F5,66 = 282.311; p < 0.0001). Trypsin activities were reduced by 8.04 ± 0.19% to 52.68 ± 0.72% and 5.61 ± 0.22% to 39.46 ± 0.19% in F and AF, respectively. The reduction in Chymotrypsin activities ranged between 16.11 ± 0.03% to 38.02 ± 0.27% in F and 6.31 ± 0.07% to 22.80 ± 0.32% in AF. Total protease activities were reduced by 10.9 ± 0.07% to 49.60 ± 0.32% in L. rohitaF, whereas, it ranged between 5.19 ± 0.06% to 32.60 ± 0.13% in AF. On a comparative scale, the difference in tannin induced inhibition in F and AF were more prominent for trypsin and chymotrypsin than total protease. Further, nine protease activity bands (15.9–69 kD) with different electromobility were noticed in both the size groups. Subsequent densitometry analysis revealed that the average densities of the protease activity bands were gradually decreased with increasing level of tannin exposure (50–200 nM). The study might indicate adaptive tolerance to tannin in larger size groups and emphasizes the need for removal of tannin in the plant feedstuffs, especially for feeding the fingerlings of L. rohita.

Bioprocess enhancement of feather degradation using alkaliphilic microbial mixture

ABSTRACT1. The main aim of this work is to develop a robust method to generate a microbial mixture which can successfully degrade poultry feathers to overcome environmental problems.2. Four different alkaliphilic microbes were isolated and shown to degrade poultry feathers.3. Two of the isolates were phylogenetically identified as Lysinibacillus and the others were identified as Nocardiopsis and Micrococcus.4. The best microbial co-culture for white and black feather degradation was optimised for pH, temperature and relative population of the isolates to achieve almost 96% of degradation compared with a maximum of 31% when applying each isolate individually.5. The maximum activity of keratinase was estimated to be 1.5 U/ml after 3 d for white feathers and 0.6 U/ml after 4 d for black feathers in a basal medium containing feather as the main carbon source. Additionally, non-denaturing polyacrylamide gel electrophoresis showed 4 and 3 protease activity bands for white and black feather, respectively.6. This study provides a robust method to develop potential new mixtures of microorganisms that are able to degrade both white and black feathers by applying a Central Composite Design.

Water-soluble proteinase activity of gastric fluids in the European whip snake, Hierophis viridiflavus : an experimental preliminary survey

Understanding the mechanism of metabolic digestion and of food assimilation in wild snake species is of fundamental importance to compare trophic niches, their adaptive significance and evolutionary patterns of diet selection for different snake species. From two adult Hierophis viridiflavus males in fasting condition, we sampled gastric fluids using fiberoptic endoscopy. We analyzed the proteolytic activities (optimal pH and temperature, response to inhibitors) of snake samples by zymography techniques. The two major protease activity bands were always detected at every tested pH. Activity bands were also detectable over 37 °C; in particular both bands showed the highest activity ranging from 55 °C to 60 °C. Ranging from 65 °C to 85 °C, one band remained visible while the other band disappeared over 60 °C. The pattern of proteolytic enzymes activity of sampled gastric fluids highlighted a scenario composed of few active proteases, reflecting the feeding status (e.g., fasting) of studied snakes. Detection of proteolytic activity until 85 °C, as in prokaryotic organisms, supported the hypothesis about the presence of proteases of exogenous source.

Differentially Secreted Proteins of Antarctic and Mesophilic Strains of Synechocystis Salina and Chlorella Vulgaris after UV-B and Temperature Stress Treatment

ABSTRACTExponential-phase cell cultures from Antarctic and mesophilic strains of the cyanobacterium Synechocystis salina and the unicellular green alga Chlorella vulgaris were subjected to UV-B and temperature stress and investigated for alterations in the spectrum of secreted proteins. In addition to constitutively secreted proteins, strain-specific stress proteins were detected on silver-stained gels. Extracellular protein patterns within a given strain, as well as between strains after exposure to each UV-B and temperature stress were notably different. UV-B exposure induced only moderate changes of the secreted proteins in both Antarctic and mesophilic Synechocystis salina strains. However, a 21 kDa protein band present in Antarctic Synechocystis salina only was identified using LC-MS/MS analysis, to contain two proteins—a peroxiredoxin and Fe-superoxide dismutase (Fe-SOD). Their presence might modulate the level of reactive oxygen species generated in membranes during UV-B stress and, thus, contribute to the higher survival rate of Antarctic Synechocystis salina. In addition, proteolytic activity in the extracellular protein samples of both species was detected on 1D zymography in a polyacrylamide gel containing gelatin as substrate. Antarctic Synechocystis salina secreted cysteine-, serine-, and metalloproteases, while mesophilic Synechocystis salina secreted mainly cysteine proteases. Subtle changes of the protease activity bands in Antarctic Synechocystis salina and Chlorella vulgaris were observed after UV-B and temperature stress. In contrast, mesophilic Synechocystis salina responded with a considerable increase in protease activity and number of activity bands after 30 min and 60 min exposure to UV-B, as well as when grown at 35°C and 40°C. Peptidases are key players in structuring and adapting cells under various stressful conditions. The varying pattern of secreted proteases among the Antarctic and mesophilic strains of Synechocystis salina and Chlorella vulgaris suggests different mechanisms for coping with UV-B and temperature stress. Characterization of extracellular proteins discovered in the present study will provide further understanding of the rich defense arsenal and mechanisms of survival of cyanobacteria and microalgae.

Profile of the body surface proteolytic systém in Apis mellifera quee

The proteolytic system on the body surface of the honey bee has been insufficiently researched. In this study the body surface proteolytic activity was examined in queens at various developmental stages (eggs, larvae, pupae and imagines) in different seasons (spring, summer, autumn, winter). Extracts of the body surface material with water and detergent were used for an in vitro analysis of the proteolytic activity and protease inhibitor level assaying, as well as for an electrophoretic separation of the extracts in polyacrylamide gels. The following methods were used: protein content testing by the Lowry method (modified by Schacterle-Pollack), protease activity testing by the Anson method and protease inhibitor activity testing by the Lee and Lin method. Our studies revealed a high protease activity in an acidic environment (pH = 2.4; the material rinsed with detergent), as well as in neutral (pH = 7) and alkaline (pH = 11.2) environments (the material rinsed with water in both cases). The highest protein concentration values were observed in the imagines from summer. The lowest activities of the proteases and protease inhibitors were determined in the eggs from summer. The highest activities of the acidic, neutral and alkaline proteases were observed in the pupae from spring. The highest number of protease activity bands in PAGE zymography was obtained for the neutral and alkaline activities in the queens for all the seasons. In the queens all the catalytic protease types were present: asparagine and cysteine proteases at pH = 2.4; cysteine proteases and metalloproteases at pH = 7 and serine proteases at pH = 11.2. These results were crucial for the analysis of immunity mechanisms on the body surface of the honey bee.

A preliminary study of the protease activities in germinating brown rice (Oryza sativa L.)

Proteases hydrolyse storage proteins to provide precursors for perpetuating species. The aim of this study was to investigate and characterise different proteases in germinating brown rice. The protease activity of brown rice increased sevenfold during 7 days of germination. It was highest on day 6 when determined at pH 3.5. With casein as substrate the proteases showed two catalytic groups: acidic proteases with an optimal pH of 3.5 and alkaline proteases with an optimal pH of 8.0. The acidic protease activity was inhibited by Ba(2+) and Pb(2+) but stimulated by Zn(2+) , while the alkaline protease activity was inhibited by Ca(2+) and Pb(2+) but stimulated by Mg(2+) and Zn(2+) . SDS-gelatin-PAGE assay showed two protease activity bands at pH 3.5, while two different bands with higher molecular weights were observed at pH 8.0. Inhibition assay revealed that pepstatin A and E-64 inhibited 67.63 and 38.26% respectively of the protease activity at pH 3.5, indicating the presence of aspartic and cysteine proteases. Metalloproteases played a major role under alkaline conditions (88.37% inhibition with EDTA). Germinated brown rice proteases fall into different classes with different properties. This study is helpful for their further purification.

Ontogenic changes in the digestive enzyme patterns and characterization of proteases in Indian major carp Cirrhinus mrigala

Digestive enzymes of Cirrhinus mrigala (Ham.) were studied during ontogenic development. Specific amylase activity was detected in first feeding fish. The enzyme activity decreased up to day-18 and then it increased with the age of fish to reach the highest level on day-34. Protease activity was 28.61 ± 8.90 mU mg protein−1 min−1 on day-4 and increased with the age throughout the study period. Trypsin activity was 31.86 ± 1.12 mU mg protein−1 min−1 on day-4. The activity decreased up to day-10 and from day-12 onwards increased up to day-26. Chymotrypsin activity was 14.56 ± 2.74 mU mg protein−1 min−1on day-4 and constantly increased up to day-26. A significant increase in lipase activity was observed between days-24 and 34. SDS-PAGE and substrate SDS-PAGE showed the diversity of protein (17.4–127.8 kDa) and protease activity bands (16.6–88.8 kDa) during ontogenesis. Soybean trypsin inhibitor, phenyl methyl sulphonyl fluoride, N-α-p-tosyl-l-lysine chloromethylketone and N-tosyl-l-phenylalanine chloromethylketone inhibited the protease activity up to 79.72–97.21, 65.55–94.83, 45.41–75.31 and 40.78–64.72%, respectively. Inhibition study in substrate SDS-PAGE revealed the abundance of serine proteases and the presence of isoforms of trypsin and chymotrypsin. Ethylenediamine-tetraacetate showed 5.56–22.78% inhibition of metal ion-specific enzyme activity.

Digestive proteases of three carps Catla catla, Labeo rohita and Hypophthalmichthys molitrix: partial characterization and protein hydrolysis efficiency

Characteristics and functional efficacy of digestive proteases of Catla catla, catla, Labeo rohita, rohu and Hypophthalmichthys molitrix, silver carp were studied. Total protease activity was significantly (P < 0.05) higher in rohu (1.219 ± 0.059 U mg protein )1 min )1 ) followed by silver carp (1.084 ± 0.061 U mg protein )1 min )1 ), and catla (0.193 ± 0.006 U mg protein )1 min )1 ). Trypsin activity of silver carp and rohu was 89–91% higher than catla. Chymotrypsin activity was significantly (P < 0.05) higher in silver carp compared with rohu and catla. The protease activity of rohu and silver carp displayed bell-shaped curves with maximum activity at pH 9; whereas in catla, maximum activity was found between pH 8 and 11. Inhibition of protease activity with soybean trypsin inhibitor (SBTI), phenylmethylsulfonyl fluoride (PMSF) revealed the presence of serine proteases and inhibition of activity with N-a-p-tosyl-L-lysine-chloromethyl ketone (TLCK) and N-tosyl-L-phenylalanychloromethane (TPCK) indicated the presence of trypsin-like and chymotrypsin-like enzymes in all these three carps. SDS-PAGE showed the presence of several protein bands ranging from 15.3 to 121.9 kDa in enzyme extracts of catla, rohu and silver carp. The substrate SDS-PAGE evidenced the presence of various protease activity bands ranging from 21.6–93.7, 21.6–63.8 and 26.7–98.5 kDa for catla, rohu and silver carp respectively. In pH-stat hydrolysis of Chilean fishmeal showed significantly (P < 0.05) higher degree of hydrolysis compared with soybean meal, silver cup (a commercial fish feed of Mexico) and wheat flour, with enzyme preparations of three fishes. The rate of hydrolysis was significantly (P < 0.05) higher in silver carp compared with others.

Effect of Stage of Lactation and Parity on Mammary Gland Cell Renewal

Milk production is a function of the number and activity of mammary epithelial cells, regardless of stage of lactation. Milk yield is generally higher in multiparous cows than in primiparous cows, but persistency is usually greater in the latter group. We compared several measures related to metabolic activity, apoptosis, and endocrine control of mammary cell growth in 8 primiparous and 9 multiparous cows throughout lactation. Mammary gland biopsies were taken in early [10 d in milk (DIM)], peak (50 DIM), and late (250 DIM) lactation to evaluate gene expression and determine DNA and fatty acid synthase (FAS) content. Milk samples taken the day before the biopsies were used to detect protease activities and to determine stanniocalcin-1 (STC) concentrations. Blood samples served to measure insulin-like growth factor-1, prolactin, and STC concentrations. Milk yield was higher in multiparous cows than in primiparous cows at the 10 DIM (32.8±1.3 and 25.2±0.8 kg/d) and 50 DIM (38.0±1.2 and 29.8±1.1 kg/d), but it was the same for both groups at 250 DIM (23.9±1.5 and 23.8±1.1 kg/d). Except for stearoyl-coenzyme A desaturase, expression of genes related to milk synthesis was not affected by stage of lactation. However, gene expression of acetyl-coenzyme A carboxylase, β-casein, and FAS was lower in early lactation in primiparous cows. Expression of both proapoptotic bax and antiapoptotic bcl-2 genes was higher in primiparous cows, whereas the bax-to-bcl-2 ratio was not changed. Mammary DNA concentration was higher in multiparous cows, as was the amount of FAS protein in early lactation. Two bands of protease activity were found in milk samples, and one of the bands had an apparent molecular weight similar to gelatinase A and was dependent on the stage of lactation. Serum insulin-like growth factor-1 increased with day of lactation and was higher in primiparous cows. Serum prolactin decreased in late lactation, but peak values were observed in early lactation for primiparous cows and peak lactation for multiparous cows. Milk STC content increased with advancing lactation. The results are consistent with a lower degree of differentiation and a greater capacity for cell renewal in the mammary gland of primiparous cows.

A Conserved Subtilisin Protease Identified in Babesia divergens Merozoites

Invasion of erythrocytes is an integral part of the Babesia divergens life cycle. Serine proteases have been shown to play an important role in invasion by related Apicomplexan parasites such as the malaria parasite Plasmodium falciparum. Here we demonstrate the presence of two dominant serine proteases in asexual B. divergens using a biotinylated fluorophosphonate probe. One of these active serine proteases (p48) and its precursors were recognized by anti-PfSUB1 antibodies. These antibodies were used to clone the gene encoding a serine protease using a B. divergens cDNA library. BdSub-1 is a single copy gene with no introns. The deduced gene product (BdSUB-1) clearly belongs to the subtilisin superfamily and shows significant homology to Plasmodium subtilisins, with the highest degree of sequence identity around the four catalytic residues. Like subtilisin proteases in other Apicomplexan parasites, BdSUB-1 undergoes two steps of processing during activation in the secretory pathway being finally converted to an active form (p48). The mature protease is concentrated in merozoite dense granules, apical secretory organelles involved in erythrocyte invasion. Anti-PfSUB1 antibodies have a potent inhibitory effect on erythrocyte invasion by B. divergens merozoites in vitro. This report demonstrates conservation of the molecular machinery involved in erythrocyte invasion by these two Apicomplexan parasites and paves the way for a comparative analysis of other molecules that participate in this process in the two parasites.

Study of digestive enzyme activities and partial characterization of digestive proteases in a freshwater teleost, Labeo rohita, during early ontogeny

Digestive enzymes of freshwater fish Labeo rohita (family Cyprinidae; class Actinopterygii; infraclass Teleostei; order Cypriniformes) were studied during ontogenic development. Amylase, protease and lipase activities showed a polynomial relationship with age of rohu, whereas trypsin, chymotrypsin and lipase activities exhibited exponential trends with the age of rohu larvae. SDS-PAGE of crude enzyme extract revealed that the proteases of higher molecular weight (MW) appeared during early ontogeny, whereas low MW proteases were observed at a later stage. Substrate SDS-PAGE supported the quantitative study of protease activities as evidenced with the increasing number and intensity of activity bands with the age of fish. The number of protease activity bands observed in 4, 10, 12 and 24 DAH (days after hatching) larvae were 5, 7, 8 and 9, respectively. Inhibition of protease activities with soybean trypsin inhibitor (SBTI) (58.6–81.2%), phenyl methyl sulphonyl fluoride (PMSF) (55.6–70%), N-α-p-tosyl-l-lysine chloromethyl ketone (TLCK) (41.1–52.3%) and N-tosyl-l-phenylalanine chloromethyl ketone (TPCK) (27.9–44.5%) indicated the presence of serine proteases, trypsin- and chymotrypsin-like enzymes in rohu larvae. Inhibition with SBTI and TPCK showed power, TLCK and ethylenediaminetetraacetic acid showed exponential, whereas PMSF showed polynomial relationships during the study period.

Functional changes in digestive enzymes and characterization of proteases of silver carp (♂) and bighead carp (♀) hybrid, during early ontogeny

Study of digestive enzyme and partial characterization of proteases of silver carp (♂) and bighead carp (♀) hybrid during early ontogeny was performed. Specific amylase activity was observed in 4 DAH (0.07 ± 0.01 mg maltose mg protein − 1 min − 1 ) carp hybrid. Specific amylase activity showed polynomial relationship with the age of fish. Total protease, trypsin and chymotrypsin activities were 14.37 ± 2.21, 11.38 ± 1.67 and 2.83 ± 0.50 mUnits mg protein − 1 min − 1 in 4 DAH fish, respectively. Total protease activity showed exponential trend, whereas trypsin and chymotrypsin activities showed polynomial relationships with the increasing age of the fish. Lipase activity was 2.33 ± 0.18 mUnits in 4 DAH carp hybrid. Lipase activity showed polynomial trend with the increasing age of fish. In inhibition study, SBTI, PMSF, TLCK and TPCK inhibited the protease activity by 83.0% to 92.0%, 71.0% to 81.0%, 45.1% to 55.5% and 35.8% to 48.2%, respectively. SDS-PAGE showed the presence of various protein bands (20.9–69.4 kDa) in carp hybrid during ontogenic development. Substrate SDS-PAGE revealed the presence of several protease activity bands (19.1–73.7 kDa) in digestive tissue extract of fish during ontogenesis. Inhibition of protease activity bands in substrate SDS-PAGE with SBTI and PMSF revealed the abundance of serine proteases and inhibition of activity bands with TLCK and TPCK evidenced the presence of more than one isoform of trypsin and chymotrypsin in the digestive tissue extract of hybrid carp.

Digestive enzyme patterns and evaluation of protease classes in Catla catla (Family: Cyprinidae) during early developmental stages

Digestive enzymes of Catla catla were studied during ontogenic development. Specific amylase activity was 0.12 ± 0.01 mg maltose mg protein − 1 h − 1 in fish 4 days after hatching (DAH) and reached a maximum on (0.41 ± 0.12 mg maltose mg protein − 1 h − 1 ) 34 DAH. Total protease activity was minimum (123.2 ± 16.5 mU mg protein − 1 min − 1 ) on day-8 and reached its highest level (2713 ± 147.2 mU mg protein − 1 min − 1 ) on day-32. Trypsin activity showed constant increasing trend from day-16 onwards and was maximum on day-34 (118.1 ± 7.09 mU mg protein − 1 min − 1 ). Highest chymotrypsin activity was found on day-32 (1789.0 ± 111.7 mU mg protein − 1 min − 1 ). Lipase activity was detected in 4 DAH catla. Lipase activity increased steadily from day-22 onwards. SDS-PAGE of crude enzyme extracts showed that high molecular mass bands (41.8–127.8 kDa) appeared during the early stages followed by low molecular mass bands (17.8–37.2 kDa). The number of protease activity bands in substrate SDS-PAGE increased with age of fish. During ontogenesis of carp, soybean trypsin inhibitor (SBTI), PMSF and TLCK inhibited 75.5 ± 1.19% to 92.8 ± 0.85%, 53.3 ± 9.47% to 90.5 ± 2.6% and 39.8 ± 3.8% to 84.7 ± 1.54% of total protease activity, respectively. There was only 2.58 ± 0.66% to 10.21 ± 0.09% inhibition of protease activity with EDTA. SBTI and PMSF inhibited 8 and 4 activity bands, respectively. TLCK, a specific trypsin inhibitor, inhibited four trypsin-like enzymes in carp during ontogenesis.

Characterization of a serine protease activity in Sarcocystis neurona merozoites.

Sarcocystis neurona merozoites were examined for their ability to invade and divide in bovine turbinate (BT) cell cultures after treatment with cysteine (iodoacetamide), aspartic (pepstatin A), metallo-(1,10-phenanthroline and ethylene glycol-bis(aminoethylether)-tetraacetic acid [EGTA]), or serine (4-[2-aminoethyl]-benzenesulfonyl fluoride hydrochloride [AEBSF], phenylmethane sulphonyl fluoride [PMSF], and tosyl lysyl chloramethyl ketone [TLCK]) protease inhibitors. Significant (P < 0.01) inhibition of serine protease activity by PMSF and TLCK led to a reduction of 86 and 78% in merozoites produced in BT cell cultures, respectively, whereas AEBSF (1 mM) led to a 68% reduction in merozoites produced in BT cell cultures and a reduction of 84 and 92% at higher AEBSF concentrations (2 and 3 mM, respectively). Pepstatin A and iodoacetamide failed to cause any inhibition in merozoite production, whereas 1,10-phenanthroline and EGTA caused slight, but not significant, inhibition at 6 and 17%, respectively. In zymograms, 2 bands of protease activity between 65- and 70-kDa molecular weight were seen. The protease activity was inhibited by AEBSF but not by E-64 (cysteine protease inhibitor), EGTA, iodoacetamide, or pepstatin A. In native zymograms, the protease activity was highest between a pH range of 8 and 10. These data suggest that merozoites of S. neurona have serine protease activity with a relative molecular weight range between 65 and 70 kDa and optimal pH range between 8 and 10, which is essential for host cell entry at least in vitro. The protease activity described here could be a potential target for chemotherapy development.

Helicobacter pylori and proteolytic activity.

Protease activity of 10 different H. pylori strains, purified marker proteases and protease-positive reference bacteria (Klebsiella ozaenae, Serratia marcescens) were tested against bovine haemoglobin, porcine mucin, bovine serum albumin, gelatin and casein as substrates. After incubation in development buffer and subsequent staining with Coomassie blue, protease activity bands were demonstrated as transparent spots after polyacrylamide gel electrophoresis (PAGE) on gels with incorporated substrate. Presence of protease activity was investigated in a wide pH range (pH 2.0-9.0). Although marker proteases (0.15-0.2 microgram per slot) as well as protease-positive bacteria (2-30 micrograms per slot) clearly showed proteolytic activity in gels containing 0.1-0.2% protein mL-1, no proteolytic activity was demonstrated in any of the H. pylori strains tested. This finding indicates that H. pylori does not possess significant protease activity, as this would have been detected by this sensitive method.

Physiological and nutritional factors affecting synthesis of extracellular metalloproteases by Clostridium bifermentans NCTC 2914.

Extracellular protease production by Clostridium bifermentans NCTC 2914 occurred throughout the growth phase in batch culture. In both glucose-excess and -limited chemostats, protease formation was inversely related to the dilution rate, over the range D = 0.03 to 0.70 h-1. At high dilution rates (D greater than 0.25 h-1), protease activities were greatest under excess glucose conditions. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of chemostat culture effluents showed the presence of up to 18 bands of protease activity at low dilution rates, with apparent molecular masses ranging from about 36 to 125 kDa. High-performance liquid chromatography gel filtration of culture supernatants gave four peaks of activity at 34, 42, 60, and 102 kDa. Glucose, peptone, and phosphate stimulated protease formation, but ammonia concentrations up to 10 g liter-1 had little effect on the process. Culture pH in glucose-excess chemostats strongly influenced protease synthesis, which was maximal during growth at pH 6.4. The optimal pH of protease activity was 7.0. Although a wide variety of proteins were hydrolyzed by C. bifermentans proteases, none of the enzymes were collagenolytic. Of 21 different p-nitroanilide, beta-naphthylamide, and N-carbobenzoyl substrates tested, none were hydrolyzed. With the exception of Ca2+, divalent metal ions inhibited proteolysis. Experiments with protease inhibitors demonstrated that 1 mM EDTA inhibited protease activities in culture supernatants by over 90%, indicating that the enzymes were principally of the metalloprotease type.

Separation by polyacrylamide gel electrophoresis of multiple proteases in rat and bovine enamel

At least seven protein bands of protease activity were found in rat secretory enamel and six bands in bovine secretory enamel. The bovine proteases had molecular weights similar to those found in rat enamel. Two bands were found to be active in gels containing enamel proteins. All proteases were acid-resistant and calcium-dependent. In vitro digestion of secretory enamel resulted in a changing profile of proteases. Rat maturation enamel contained a 23,000 and a 33,000 molecular-weight protease which were not present in the secretory enamel. The 33,000 molecular weight protease was also active against enamel proteins. Thus multiple proteases are present in rat and bovine secretory enamel and in rat maturation enamel; some of these may be important in hydrolysis of enamel proteins.

Detection and characterization of extracellular proteases in Butyrivibrio fibrisolvens H17c

The detection and approximate molecular weights of extracellular serine protease isoenzymes produced by Butyrivibrio fibrisolvens H17c were determined by gelatin-PAGE. Nine bands of protease activity with apparent molecular weights of approximately 101000, 95000, 87000, 80000, 76000, 68000, 63000, 54000 and 42000 were detected after gelatin-PAGE of supernatants from exponential phase cultures. A tenth serine protease band with an apparent molecular weight of approximately 32000 was detected in stationary phase cells. The activities of all ten protease bands were inhibited by a serine protease inhibitor but their activities were not affected by inhibitors of trypsin-like enzymes or metallo-, sulphydryl-and carboxylproteases. The activity of all ten exoprotease bands was optimal between pH 6.0 and 7.5. The ten exoprotease bands were only detected in media containing trypticase or casamino acids as nitrogen sources. Production of the ten protease bands was not affected by the carbohydrate source.

Characterization of extracellular alkaline proteases and collagenase induction in Vibrio alginolyticus.

The number and approximate molecular weights of extracellular alkaline proteases produced by Vibrio alginolyticus were determined by gelatin-PAGE. Three major bands of protease activity with apparent molecular weights of approximately 28 000, 22 500 and 19 500 (proteases 1, 2 and 3, respectively) and two minor bands of protease activity with apparent molecular weights of approximately 15 500 and 14 500 (proteases 4 and 5, respectively) were obtained after gelatin-PAGE. The activities of the five proteases were inhibited by serine protease inhibitors but their activities were not affected by inhibitors of trypsin-like enzymes. Histidine, which inhibited V. alginolyticus collagenase, did not inhibit the activities of the alkaline serine proteases. The production of protease 1, however, was enhanced by histidine. Protease 1 production was also affected by temperature and production was depressed at 37 degrees C. Gelatin-PAGE of a commercial V. alginolyticus collagenase preparation revealed four bands of activity which were identified as collagenases with apparent molecular weights of approximately 45 000, 38 500, 33 500 and 31 000. The collagenase preparation was contaminated with two serine proteases. The release of [3H]proline from collagen matrices produced by smooth muscle cells was shown to be a sensitive assay for bacterial collagenases and was used to show that V. alginolyticus produced a basal constitutive level of extracellular collagenase. The constitutive levels of collagenase were affected by aeration.

Cytochrome c degrading activity in rat liver mitochondria

By means of a suitable zymographic technique we revealed a cytochrome c degrading system in rat liver mitochondria. After separation of the mitochondrial proteins by polyacrylamide gel electrophoresis and incubation of the gels in the presence of cytochrome c , three bands of protease activity were detected. Distinct zymogram patterns were obtained in parallel assays using lysosomes or cytosol instead of mitochondria. The soluble and the membrane components of mitochondria were analyzed by the same method to assess the intra-mitochondrial localization of these proteases.