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PubMedDate not supplied

[Restoration of the protease--alpha 1-protease inhibitor system after exposure of rats to cold].

The trasilol injection and warming of rats were investigated for their influence on activities of proteinase and alpha-1-proteinase inhibitor (alpha-1PT) after two-hour- and a week-long cold stress. The one-hour return to normal temperature is the most effective method of proteinase-antiproteinase correction after two-hour cold stress. Nevertheless, the return of rats to the normal temperature even for 24 hours after a week cold stress is not enough for the proteinase and alpha-1-PI balancing normalization. The trasilol injection is a more effective method in the last case.

Animals↗
PubMed1998

Analysis of protease activity in human otitis media.

Chronic otitis media is a common problem associated with a nonintact tympanic membrane frequently involving Staphylococcus aureus and Pseudomonas aeruginosa. The virulence of Pseudomonas bacteria is related to the production of two matrix metalloproteinases, elastase and alkaline protease. Serine proteases, such as neutrophil elastase, are produced by the host inflammatory response. These proteases are thought to contribute to tissue destruction and assist bacterial invasion during infection. This preliminary study was done to identify protease activity in otorrhea samples from patients with otitis media and a nonintact tympanic membrane and to examine the ability of selective protease inhibitors to decrease protease activity. Ilomostat (galardin) is a synthetic, specific inhibitor of matrix metalloproteinases including P. aeruginosa elastase and alkaline protease, whereas alpha1-antitrypsin inhibits serine proteases including neutrophil elastase. Samples were collected and cultured from 20 patients with otorrhea resulting from tympanic membrane perforations or pressure-equalization tubes. A protease assay that used azocasein as the substrate was used to quantify protease activity, with and without addition of selective protease inhibitors. Cultures revealed P. aeruginosa alone in 7 samples, P. aeruginosa plus other organisms in 10, and S. aureus alone in 3. Protease activity was detected in 15 (75%) of the samples. A statistically significant (p < 0.05) decrease in protease activity was seen with the addition of alpha1-antitrypsin or Ilomostat plus alpha1-antitrypsin, but not with Ilomostat alone. Analyzing the 10 samples with the highest protease activity, a statistically significant decrease in activity was seen with Ilomostat or alpha1-antitrypsin alone and with both Ilomostat and alpha1-antitrypsin together. Bacteriologic type, source of sample, age and gender of the subject, and duration of infection were not significantly related to protease activity. This is the first study to quantify protease activity and inhibition by selective protease inhibitors in human otorrhea. Protease inhibitors effectively decrease protease activity in most cases and in addition to standard antibiotic therapy might prove beneficial in the treatment of otitis media with a nonintact tympanic membrane. This study supports future clinical investigations into the role of proteases and inhibition of protease activity in the treatment of otitis media.

Anti-Bacterial Agents↗
PubMed1994

Neutralizing monoclonal antibodies to an extracellular Pseudomonas cepacia protease.

Pseudomonas cepacia produces at least two extracellular proteases with apparent molecular masses of 36,000 and 40,000 Da. The 36-kDa protease has high proteolytic activity and the 40-kDa protease has low proteolytic activity with hide powder azure as a substrate. Monoclonal antibodies (MAbs) were raised against the purified 36- and 40-kDa proteases. Several MAbs directed against the 36-kDa protease were found to recognize the 40-kDa protease by Western immunoblot analysis. Similarly, a MAb directed against the 40-kDa protease recognized the 36-kDa protease, suggesting that these two proteases may be immunologically related. A MAb directed against the 36-kDa protease, designated 36-6-8, and a MAb directed against the 40-kDa protease (MAb G-11) cross-reacted with other extracellular proteases, such as Pseudomonas aeruginosa elastase and alkaline protease, Pseudomonas pseudomallei protease, and the Vibrio cholerae hemagglutinin/protease. MAb 36-6-8 neutralized the P. cepacia 36-kDa protease, P. aeruginosa elastase, P. pseudomallei protease, and V. cholerae hemagglutinin/protease but did not affect P. aeruginosa alkaline protease activity. In contrast, MAb G-11 to the 40-kDa protease neutralized only the P. cepacia 36-kDa protease. This evidence suggests that the neutralizing MAb, 36-6-8, recognizes an epitope conserved among some metalloproteases. This epitope may lie at or near the active site of the P. cepacia 36-kDa protease and P. aeruginosa elastase.

Antibodies, Bacterial↗
PubMed1998

Substrates and inhibitors of human T-cell leukemia virus type I protease.

HTLV-I is an oncogenic retrovirus that is associated with adult T-cell leukemia. HTLV-I protease and HTLV-I protease fused to a deca-histidine containing leader peptide (His-protease) have been cloned, expressed, and purified. The refolded proteases were active and exhibited nearly identical enzymatic activities. To begin to characterize the specificity of HTLV-I, we measured protease cleavage of peptide substrates and inhibition by protease inhibitors. HTLV-I protease cleavage of a peptide representing the HTLV-I retroviral processing site P19/24 (APQVLPVMHPHG) yielded Km and kcat values of 470 microM and 0.184 s-1 while cleavage of a peptide representing the processing site P24/15 (KTKVLVVQPK) yielded Km and kcat values of 310 microM and 0.0060 s-1. When the P1' proline of P19/24 was replaced with p-nitro-phenylalanine (Nph), the ability of HTLV-I protease to cleave the substrate (APQVLNphVMHPL) was improved. Inhibition of HTLV-I protease and His-protease by a series of protease inhibitors was also tested. It was found that the Ki values for inhibition of HTLV-I protease and His-protease by a series of pepsin inhibitors ranged from 7 nM to 10 microM, while the Ki values of a series of HIV-1 protease inhibitors ranged from 6 nM to 127 microM. In comparison, the Ki values for inhibition of pepsin by the pepsin inhibitors ranged from 0.72 to 19.2 nM, and the Ki values for inhibition of HIV-1 protease by the HIV protease inhibitors ranged from 0.24 nM to 1.0 microM. The data suggested that the substrate binding site of HTLV-I protease is different from the substrate binding sites of pepsin and HIV-1 protease, and that currently employed HIV-1 protease inhibitors would not be effective for the treatment of HTLV-I infections.

Amino Acid Sequence↗
PubMedDate not supplied

Characterization of senescence-associated proteases in postharvest broccoli florets.

We characterized the senescence-associated proteases of postharvest broccoli (Brassica oleracea L. var Green King) florets, using class-specific protease inhibitors and gelatin-polyacrylamide gel electrophoresis. Different classes of senescence-associated proteases in broccoli florets were partially characterized for the first time. Protease activity of broccoli florets was depressed by all the inhibitors and showed different inhibition curves during postharvest. The hydrolytic activity of metalloprotease (EC 3.4.24. - ) and serine protease (EC 3.4.21. - ) reached a maximum, 1 day after harvest (DAH), then decreased, while the hydrolytic activity of cysteine protease (EC 3.4.22. - ) and aspartic protease (EC 3.4.23. - ) increased throughout the postharvest senescence based on the calculated inhibition percentage of protease activity. The senescence-associated proteases were separated into seven endoprotease (EP) groups by gelatin-polyacryamide gel electrophoresis and classified into EP1 (metalloprotease), EP2 (metalloprotease and cysteine protease), EP3 (serine protease and aspartic protease), EP4, EP5, EP7 (cysteine protease), and EP6 (serine protease) based on the sensitivity of class-specific protease inhibitors. The proteases EP2, EP3, and EP4 were present throughout the postharvest stages. EP3 was the major EP at all times during senescence; EP4 intensity of activity increased after 2 DAH; EP6 and EP7 clearly increased after 4 DAH. Our results suggest that serine protease activity contributes to early stage (0-1 DAH) and late stage (4-5 DAH) of senescence; metalloprotease activity was involved in the early and intermediate stages (0-3 DAH) of senescence; and cysteine protease and aspartic protease activities participated in the whole process of broccoli senescence.

Brassica↗
PubMed1980

Extracellular and membrane-bound proteases from Bacillus subtilis.

Bacillus subtilis YY88 synthesizes increased amounts of extracellular and membrane-bound proteases. More than 99% of the extracellular protease activity is accounted for by an alkaline serine protease and a neutral metalloprotease. An esterase having low protease activity accounts for less than 1% of the secreted protease. These enzymes were purified to homogeneity. Molecular weights of approximately 28,500 and 39,500 were determined for the alkaline and neutral proteases, respectively. The esterase had a molecular weight of approximately 35,000. Amino-terminal amino acid sequences were determined, and the actions of a number of inhibitors were examined. Membrane vesicles contained bound forms of alkaline and neutral proteases and a group of previously undetected proteases (M proteases). Membrane-bound proteases were extracted with Triton X-100. Membrane-bound alkaline and neutral proteases were indistinguishable from the extracellular enzymes by the criteria of molecular weight, immunoprecipitation, and sensitivity to inhibitors. The M protease fraction accounted for approximately 7% of the total activity in Triton X-100 extracts of membrane vesicles. The M protease fraction was partially fractionated into four species (M1 through M4) by ion-exchange chromatography. Immunoprecipitation and sensitivity to inhibitors distinguished membrane-bound alkaline and neutral proteases from M proteases. In contrast to alkaline and neutral proteases, proteases M2 and M3 exhibited exopeptidase activity.

Amino Acid Sequence↗
PubMed1989

Workshop report from the Division of Cancer Etiology, National Cancer Institute, National Institutes of Health. Protease inhibitors as cancer chemopreventive agents.

This workshop was organized to discuss the current state of research on anticarcinogenic protease inhibitors with regard to their potential use as human cancer chemopreventive agents. Previous studies have indicated that protease inhibitors can be powerful anticarcinogenic agents for animals and cells in culture and that human populations known to have high concentrations of protease inhibitors in the diet have low overall cancer mortality rates. In the workshop discussions, emphasis was placed on certain dietary protease inhibitors, such as the soybean-derived Bowman-Birk inhibitor and chymotrypsin inhibitor 1 from potatoes and some of the highly purified protease inhibitors of microbial origin provided by the Japan Society for the Promotion of Science, which have already been shown to contain anticarcinogenic activity in laboratory studies. Potential adverse side effects of dietary protease inhibitors were also considered, specifically, their possible effects on the pancreas and in causing decreased growth rates in young organisms. It was pointed out that the pancreata of a few species, notably rats and chicks, are extraordinarily sensitive to dietary protease inhibitors. Rats fed diets containing high concentrations of soybean-derived protease inhibitors (raw soy flour) had enlarged pancreata; increased pancreatic growth is thought to accelerate cancer development in the pancreas. The effect of raw soy flour on the growth of the rat pancreas has not been shown to occur in most other species tested (examples include hamsters, mice, dogs, pigs, and monkeys) and is not expected to occur in humans. There is no evidence that dietary protease inhibitors have adverse effects on the human pancreas. In fact, it has been observed that human populations with high levels of dietary protease inhibitors have decreased rates of pancreatic cancer. Dietary concentrations of protease inhibitors which have been shown to be anticarcinogenic have not produced decreased growth rates in animals or any type of pancreatic pathology. In general, there was a high level of enthusiasm at the workshop for the further development of protease inhibitors as chemopreventive agents. Recommendations for future research include: (a) research and development of sources of protease inhibitors; (b) analysis of human foods for protease inhibitor content; (c) evaluation of cancer incidence data in relation to protease inhibitor content and characteristics in the diet of human populations; (d) animal studies on the efficacy of protease inhibitors in cancer prevention; and (e) studies on the mechanism of action of anticarcinogenic protease inhibitors.

Humans↗
PubMed1996

Purification of alkaline proteases from a Bacillus strain and their possible interrelationship.

Alkalophilic Bacillus sp. KSM-K16 produced three alkaline proteases, as detected by polyacrylamide gel electrophoresis (PAGE). The major protease, designated M protease, was recently purified to homogeneity and its properties were characterized. In the present study, two minor proteases, designated H protease and N protease, were purified to homogeneity from cultures of this organism. H protease had a molecular mass of 28 kDa, as estimated by sodium dodecyl sulfate/PAGE (SDS-PAGE) and its maximum activity against casein was observed at pH 11.0 and at 55 degrees C. N protease consisted of two polypeptide chains with molecular masses of 12.5 kDa and 14.5 kDa, as estimated by SDS-PAGE, although it migrated as a single protein band during non-denaturing PAGE. Its maximum activity was observed at pH 11.0 and at 60 degrees C. The amino-terminal sequences of H protease and of the 14.5-kDa polypeptide of N protease were identical to that of M protease. The electrophoretic relationship between the three enzymes was examined after they had been stored at different pH values and at 5 degrees C. M protease was converted to H protease more rapidly at pH 11 than at pH 8 or below, and H protease was converted to M protease at pH 8 or below but not at pH 11. N protease appeared to be the autolytic product of the M and H proteases.

Amino Acid Sequence↗
PubMed1992

Evidence for controlled autoproteolysis of alkaline protease. A mechanism for physiological regulation of conidial discharge in Conidiobolus coronatus.

The alkaline serine protease of Conidiobolus coronatus was shown to be involved in its conidial discharge [Phadatare, S., Srinivasan, M. C., Deshpande, M. (1989) Arch. Microbiol. 153, 47-49]. To understand the regulation of conidial discharge, the mechanism of control of protease activity was investigated, which revealed the presence of two electrophoretically separable intracellular proteases (protease I and protease II). The formation of smaller and less-active protease II coincided with the decrease in conidial discharge. In order to trace the origin of protease II, the corresponding purified extracellular enzymes were compared with respect to their biochemical, physiochemical and immunological properties. The biochemical properties, such as optimum pH and temperature, stability, sensitivity to metal ions and substrate specificity were closely similar for both proteases. Amino acid analysis revealed that protease II is completely similar to protease I, though protease I contains an additional portion which is not contained in protease II. Western-blot ELISA, immunotitration and determination of antigenic valencies also revealed the structural similarity between the two proteases. Purified protease I showed partial degradation to protease II in vitro, the process being sensitive to phenylmethylsulfonyl fluoride, indicating its proteolytic nature. These results suggest that the formation of a less-active protease by autoproteolysis represents a novel means of physiological regulation of protease activity, which in turn regulates the conidial discharge in C. coronatus.

Amino Acids↗
PubMed1986

Gastric proteases of the Greenland cod Gadus ogac. II. Structural properties.

Three gastric proteases were isolated from the stomach mucosa of the Greenland cod (Gadus ogac). The cod proteases were all less stable to heating and protease 1 retained less activity at 5 degrees C when the pH was greater than 5 in comparison with porcine pepsin. The activities of cod proteases 1 and 2, with hemoglobin as the substrate, were doubled in the presence of 25 mM NaCl, while cod protease 3 and porcine pepsin were not stimulated by the salt. The cod proteases did not cross-react with antibodies raised against porcine pepsin. However, some cross-reactivity was noted with antibodies raised against proteases from psychotrophic pseudomonads. The molecular weights of all the cod proteases were in the range of 36,000-38,000. The amino acid compositions of the cod proteases as compared by the Metzger difference index differed from the mammalian gastric proteases by about the same extent that pepsin, gastricsin, and chymosin differ from each other. Of the cod enzymes, protease 1 differed from mammalian gastric proteases, while cod proteases 3 was more like chymosin with respect to amino acid composition. Cod protease 1 had the lowest hydrophobicity index and chymosin had the highest. The hydrophobicity indices of cod proteases 2 and 3 were intermediate between that of porcine pepsin and bovine chymosin. It is suggested that the Greenland cod proteases represent less differentiated forms of gastric proteases than the mammalian pepsins, gastricsins, and chymosins.

Amino Acids↗
PubMed1998

Pseudomonas aeruginosa protease IV produces corneal damage and contributes to bacterial virulence.

PURPOSE: A Pseudomonas mutant deficient in protease IV has significantly reduced virulence in experimental keratitis. In the present study, the corneal toxicity of purified protease IV and its ability to augment the virulence of protease-IV-deficient bacteria were analyzed. METHODS: The toxicity of purified protease IV was determined by intrastromally injecting the exoenzyme (20-200 ng) into the cornea. The effects of protease IV on the corneal virulence of the protease-IV-deficient strain, PA103-29::Tn9, were determined by injecting eyes with 1000 CFU of log phase bacteria plus either 200 ng active purified protease IV or 200 ng heat-inactivated protease IV. Changes in ocular disease, determined by slit-lamp examination, were measured at 3, 16, 22, and 27 hours after infection. Colony-forming units per cornea were quantified at 27 hours after infection. RESULTS: Purified protease IV at doses from 50 to 200 ng induced epithelial defects within 3 hours of injection. Injection of 20 ng active protease IV or heat-inactivated protease IV (200 ng) had no effect on ocular tissue. Corneal virulence of the protease-IV-deficient strain was augmented by intrastromal injection with purified protease IV but not with heat-inactivated protease IV (P < or = 0.0001). Neither active nor heat-inactivated protease IV altered the growth of bacteria in the cornea (6 log units; P = 0.81). CONCLUSIONS: The important role of protease IV in corneal virulence was demonstrated by direct toxicity and by its ability to significantly augment the virulence of protease-IV-deficient Pseudomonas.

Animals↗
PubMed1996

Characterization and determination of the relative abundance of two types of insulin-like growth factor binding protein-5 proteases that are secreted by human fibroblasts.

We previously reported that human fibroblasts secrete a protease into their conditioned medium that cleaves insulin-like growth factor binding protein-5 (IGFBP-5) into non-IGF-I binding fragments. Because the protease activity in the fibroblast medium has characteristics of both serine and metalloproteases, the activity was purified and analyzed to determine whether it retained serine or metalloprotease properties. The protease was purified by heparin Sepharose affinity chromatography followed by alpha1 antichymotrypsin affinity or gelatin agarose chromatography. The heparin Sepharose purified material degraded IGFBP-5 into 22-, 17-, and 16-kDa fragments. Amino acid sequencing showed that the 22-kDa fragment contained the amino-terminus of the protein. The protease activity in the fibroblast conditioned medium that was purified by heparin Sepharose was inhibited by both serine and metalloprotease inhibitors. To attempt to separate these activities, the heparin Sepharose purified activity was further purified by gelatin agarose chromatography. The IGFBP-5 protease activity that did not bind to gelatin agarose was inhibited by serine protease inhibitors, such as 3,4 dichloroisocoumain (3,4 DCI), whereas tissue inhibitor metalloprotease-1 (TIMP-1) had minimal activity. When this same pool of protease activity that had been eluted from heparin Sepharose was applied to an alpha1 antichymotrypsin peptide affinity column, the protease activity that bound to the column was inhibited by 3,4 dichloroisocoumain, but was not inhibited by TIMP-1. In contrast, the activity that did not adhere to this column was inhibited by TIMP-1. IGFBP-5 zymography showed that the Mr estimate of the protease that was inhibited by serine protease inhibitors was 92 kDa, whereas gelatin zymography showed that the metalloproteases had Mr estimates of 72, 69, and 55 kDa. When the protease activity in the crude conditioned medium was analyzed by zymography, almost all of the detectable protease had an Mr estimate of 92 kDa, suggesting that the metalloproteases that were detected in the partially purified fractions were inactive in the medium. In summary, fibroblasts secrete a 92-kDa protease that cleaves IGFBP-5 into 22-, 17-, and 16-kDa fragments. The protease inhibitor specificity results, chromatographic characteristics, and zymographic analyses suggest that this is a serine protease. Although metalloproteases are secreted by these cells, the 92-kDa serine protease is the predominate form of activity in the conditioned medium that cleaves IGFBP-5.

Cells, Cultured↗