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PubMed2004

Efficient site-specific processing of fusion proteins by tobacco vein mottling virus protease in vivo and in vitro.

Affinity tags are widely used as vehicles for the production of recombinant proteins. Yet, because of concerns about their potential to interfere with the activity or structure of proteins, it is almost always desirable to remove them from the target protein. The proteases that are most often used to cleave fusion proteins are factor Xa, enterokinase, and thrombin, yet the literature is replete with reports of fusion proteins that were cleaved by these proteases at locations other than the designed site. It is becoming increasingly evident that certain viral proteases have more stringent sequence specificity. These proteases adopt a trypsin-like fold but possess an unconventional catalytic triad in which Cys replaces Ser. The tobacco etch virus (TEV) protease is the best-characterized enzyme of this type. TEV protease cleaves the sequence ENLYFQG/S between QG or QS with high specificity. The tobacco vein mottling virus (TVMV) protease is a close relative of TEV protease with a distinct sequence specificity (ETVRFQG/S). We show that, like TEV protease, TVMV protease can be used to cleave fusion proteins with high specificity in vitro and in vivo. We compared the catalytic activity of the two enzymes as a function of temperature and ionic strength, using an MBP-NusG fusion protein as a model substrate. The behavior of TVMV protease was very similar to that of TEV protease. Its catalytic activity was greatest in the absence of NaCl, but diminished only threefold with increasing salt up to 200 mM. We found that the optimum temperatures of the two enzymes are nearly the same and that they differ only two-fold in catalytic efficiency, both at room temperature and 4 degrees C. Hence, TVMV protease may be a useful alternative to TEV protease when a recombinant protein happens to contain a sequence that is similar to a TEV protease recognition site or for protein expression strategies that involve the use of more than one protease.

Amino Acid Sequence↗
PubMed2002

Purification and characterization of a putative virulence factor, serine protease, from Vibrio parahaemolyticus.

A protease (protease A) was successfully purified from the extracellular proteins of Vibrio parahaemolyticus no. 93, a clinical strain carrying neither tdh nor trh genes, using phenyl-Sepharose CL-4B hydrophobic interaction chromatography. The molecular mass of protease A was 43 kDa using gel filtration, which was in agreement with the results obtained from SDS-PAGE, suggesting that protease A was a monomeric protein. Additionally, the isoelectric point of this protein was 5.0. The optimum temperature and pH of protease A ranged from 40 degrees C to 50 degrees C and pH 8, respectively. Protease A activity was inhibited by serine protease inhibitors, such as phenylmethylsulfonyl fluoride and soybean trypsin inhibitor; moreover, the activity could be blocked by treatment with 20 mM of 1,10-phenanthroline, but could not be restored by adding metal ions. These results indicated that protease A is a serine protease that requires metal. The 12 N-terminal residues of protease A showed a high degree of identity (81%) to the sequence of Vibrio metschnikovii VapT serine protease. The purified protease had significant effects on the growth of Chinese hamster ovary, HeLa, Vero and Caco-2 cells and its cytotoxic activity was not blocked by gangliosides. Protease A lysed erythrocytes well but its hemolytic activity was unstable after heat treatment, indicating that protease A is able to cause hemolysis but is a heat-labile protein. The purified protease caused tissue hemorrhage and death in mice when injected both intraperitoneally and intravenously. In conclusion, this is the first report of a serine protease purified directly from the supernatant of V. parahaemolyticus and identifying it as a potential virulence factor.

Adenocarcinoma↗
PubMed1979

Assessment of protease (elastase) as a Pseudomonas aeruginosa virulence factor in experimental mouse burn infection.

The data presented indicate that in experimental Pseudomonas aeruginosa infection of mice, protease enhances the virulence of the organism. Anesthetized CBA/Lü mice were subjected to a 15-s flame burn and infected with a wild-type protease-producing strain and two of its protease-deficient mutants. The average bacterial cell mean lethal dose (LD50) of 3.8 +/- 0.3 standard deviation (log10) for mice infected with the protease-producing P. aeruginosa was at least 1 log lower than the LD50 of the protease-deficient mutants (0.02 greater than P greater than 0.01). The addition of purified protease to the infecting inoculum of protease-deficient strains reduced the LD50. Although the generation time in vitro was the same for all three bacterial strains used, there were consistently fewer viable bacteria in the blood of mice infected with protease-deficient strains than in those infected with the protease-producing strain. When a protease-deficient strain was mixed with the protease-producing wild-type strain, the number of protease-producing pseudomonas found in the blood remained constant, whereas the number of protease-deficient organisms increased, suggesting that protease contributed to the invasiveness of the organisms. The survival of mice infected with protease-producing pseudomonas was enhanced by antiprotease serum. Antiprotease serum had no effect in mice infected with protease-deficient mutants.

Animals↗
PubMed1996

Bcl-2 expression prevents activation of the ICE protease cascade.

The Bcl-2 family and the ICE family of cysteine proteases play important roles in regulating cell death. We show here that induction of cell death by a Ca2+ ionophore or hypoxia results in increased levels and activity of active ICE(-like) proteases and the subsequent activation of CPP32/Yama(-like) proteases, and that inhibition of these protease activities reduces the extent of cell death. Overexpression of the anti-apoptotic proteins Bcl-2 or Bcl-xL inhibits the cell death and the activation of ICE(-like) and CPP32/Yama(-like) proteases, indicating that Bcl-2 and Bcl-xL act upstream of these proteases. We also show that specific inhibition of ICE(-like) proteases in vivo prevents activation of CPP32/Yama(-like) proteases, whereas inhibition of CPP32/Yama(-like) proteases does not prevent activation of ICE(-like) proteases, suggesting the existence of a protease cascade in vivo that requires ICE(-like) proteases for activation of CPP32/Yama(-like) proteases. Induction of necrotic cell death by KCN also induces activation of ICE(-like) proteases but not of CPP32/Yama(-like) proteases, and Bcl-2 and Bcl-xL inhibit the activation and the cell death, suggesting that the functional site of Bcl-2 and Bcl-xL is also upstream of ICE(-like) proteases in at least some forms of necrosis.

Adrenal Gland Neoplasms↗
PubMed1990

Cyst fluid proteases.

The precise origin of breast cyst fluid remains obscure. Molina has presented evidence that type II cysts (high Na/K ratio) may be transudative, that is, partly derived from plasma elements which enter through gap junctions, while Type I cysts (high K/Na ratio) are primarily secretory. In transudative cysts, plasma protease inhibitors may be present, but the balance between protease and its inhibitors may fluctuate as a result of as yet undetermined circumstances. An imbalance between the protease activity of cyst fluid and its inhibitors may be involved in the pathogenesis of breast gross cystic disease. Accumulation of protein fragments with resistant bonds would produce an elevated oncotic pressure causing a shift of fluid into the cyst capsule. Albumin is a good substrate for the protease, which may account for its low concentration in cyst fluid. The major protease fraction closely corresponds to the progesterone binding protein (GCDFP-24) described by Haagensen. Affinity columns containing aprotinin or benzamidine ligands retain the protease which can then be eluted with 0.5 M NaCl. The HD1 protease and progesterone binding protein are either tightly complexed or are the same protein. Cyst fluid is a complex mixture of biomolecules. If the progesterone binding protein is a protease, many questions must be answered concerning the influence of cyst fluid steroids, lipids, anions, and cations on enzyme action. Determination of the amino acid sequence of HD1 may help elucidate the source of the enzyme and its relationship to other tissue proteases. Human plasma contains inhibitors of this protease activity. When pooled, dialyzed plasma was mixed with pooled, dialyzed cyst fluid, the ratio of plasma/cyst fluid at which all activity was inhibited was 6/1. A comparison of the rate of cleavage of three 14C-protein substrates shows that cyst fluid proteases cleave in a characteristic manner, distinct from either trypsin or calpain. A simple method for semiquantitative estimation of protease activity in cyst fluid is described which utilizes prestained Coomassie blue-albumin containing agarose gel plates. All cyst fluids tested had protease activity but showed variability in their ability to cleave 14C-albumin by a factor of 4. There is much direct and indirect evidence that proteases are involved in the cancer process. In view of the higher than normal incidence of breast cancer in women who have had gross cystic breast disease, the possibility exists that an imbalance between these proteases and their inhibitors is somehow involved.

Exudates and Transudates↗
PubMed1987

Novel alkaline- and heat-stable serine proteases from alkalophilic Bacillus sp. strain GX6638.

An alkalophilic Bacillus sp., strain GX6638 (ATCC 53278), was isolated from soil and shown to produce a minimum of three alkaline proteases. The proteases were purified by ion-exchange chromatography and were distinguishable by their isoelectric point, molecular weight, and electrophoretic mobility. Two of the proteases, AS and HS, which exhibited the greatest alkaline and thermal stability, were characterized further. Protease HS had an apparent molecular weight of 36,000 and an isoelectric point of approximately 4.2, whereas protease AS had a molecular weight of 27,500 and an isoelectric point of 5.2. Both enzymes had optimal proteolytic activities over a broad pH range (pH 8 to 12) and exhibited temperature optima of 65 degrees C. Proteases HS and AS were further distinguished by their proteolytic activities, esterolytic activities, sensitivity to inhibitors, and their alkaline and thermal stability properties. Protease AS was extremely alkali stable, retaining 88% of initial activity at pH 12 over a 24-h incubation period at 25 degrees C; protease HS exhibited similar alkaline stability properties to pH 11. In addition, protease HS had exceptional thermal stability properties. At pH 9.5 (0.1 M CAPS buffer, 5 mM EDTA), the enzyme had a half-life of more than 200 min at 50 degrees C and 25 min at 60 degrees C. At pH above 9.5, protease HS readily lost enzymatic activity even in the presence of exogenously supplied Ca2+. In contrast, protease AS was more stable at pH above 9.5, and Ca2+ addition extended the half-life of the enzyme 10-fold at 60 degrees C. In contrast, protease AS was more stable at pH above 9.5, and Ca2+ addition extended the half-life of the enzyme 10-fold at 60 degrees C. The data presented here clearly indicate that these two alkaline proteases from Bacillus sp. strain GX6638 represent novel proteases that differ fundamentally from the proteases previously described for members of the genus Bacillus.

Bacillus↗
PubMed1987

Evolution in the structure and function of aspartic proteases.

Aspartic proteases (EC3.4.23) are a group of proteolytic enzymes of the pepsin family that share the same catalytic apparatus and usually function in acid solutions. This latter aspect limits the function of aspartic proteases to some specific locations in different organisms; thus the occurrence of aspartic proteases is less abundant than other groups of proteases, such as serine proteases. The best known sources of aspartic proteases are stomach (for pepsin, gastricsin, and chymosin), lysosomes (for cathepsins D and E), kidney (for renin), yeast granules, and fungi (for secreted proteases such as rhizopuspepsin, penicillopepsin, and endothiapepsin). These aspartic proteases have been extensively studied for their structure and function relationships and have been the topics of several reviews or monographs (Tang: Acid Proteases, Structure, Function and Biology. New York: Plenum Press, 1977; Tang: J Mol Cell Biochem 26:93-109, 1979; Kostka: Aspartic Proteinases and Their Inhibitors. Berlin: Walter de Gruyter, 1985). All mammalian aspartic proteases are synthesized as zymogens and are subsequently activated to active proteases. Although a zymogen for a fungal aspartic protease has not been found, the cDNA structure of rhizopuspepsin suggests the presence of a "pro" enzyme (Wong et al: Fed Proc 44:2725, 1985). It is probable that other fungal aspartic proteases are also synthesized as zymogens. It is the aim of this article to summarize the major models of structure-function relationships of aspartic proteases and their zymogens with emphasis on more recent findings. Attempts will also be made to relate these models to other aspartic proteases.

Amino Acid Sequence↗
PubMed2004

The S8 serine, C1A cysteine and A1 aspartic protease families in Arabidopsis.

The Arabidopsis thaliana genome has over 550 protease sequences representing all five catalytic types: serine, cysteine, aspartic acid, metallo and threonine (MEROPS peptidase database, http://merops.sanger.ac.uk/), which probably reflect a wide variety of as yet unidentified functions performed by plant proteases. Recent indications that the 26S proteasome, a T1 family-threonine protease, is a regulator of light and hormone responsive signal transduction highlight the potential of proteases to participate in many aspects of plant growth and development. Recent discoveries that proteases are required for stomatal distribution, embryo development and disease resistance point to wider roles for four additional multigene families that include some of the most frequently studied (yet poorly understood) plant proteases: the subtilisin-like, serine proteases (family S8), the papain-like, cysteine proteases (family C1A), the pepsin-like, aspartic proteases (family A1) and the plant matrixin, metalloproteases (family M10A). In this report, 54 subtilisin-like, 30 papain-like and 59 pepsin-like proteases from Arabidopsis, are compared with S8, C1A and A1 proteases known from other plant species at the functional, phylogenetic and gene structure levels. Examples of structural conservation between S8, C1A and A1 genes from rice, barley, tomato and soybean and those from Arabidopsis are noted, indicating that some common, essential plant protease roles were established before the divergence of monocots and eudicots. Numerous examples of tandem duplications of protease genes and evidence for a variety of restricted expression patterns suggest that a high degree of specialization exists among proteases within each family. We propose that comprehensive analysis of the functions of these genes in Arabidopsis will firmly establish serine, cysteine and aspartic proteases as regulators and effectors of a wide range of plant processes.

Amino Acid Sequence↗
PubMed2001

Purification and characterization of two extracellular alkaline proteases from a newly isolated obligate alkalophilic Bacillus sphaericus.

Two novel extracellular serine proteases were purified to homogeneity from the cell-free culture filtrate of an obligate alkalophilic Bacillus sphaericus by a combination of ultrafiltration, ammonium sulfate precipitation and chromatographic methods. The enzymes showed similar substrate specificities, but differed in hydrophobicity and molecular mass. Protease A was a monomeric protease with a relative molecular mass (M(r)) of 28.7 kDa, whereas protease B, with a M(r) of 68.0 kDa, apparently consisted of smaller subunits. The purified protease A had a specific activity on hemoglobin of 5.1 U/mg protein compared to 40.9 U/mg protein in the case of protease B. Both proteases were most active on SAAPF-pNa, a substrate for chymotrypsin-like serine proteases. However, the K(m) values of these two proteases on SAAPF-pNa were higher than that for alpha-chymotrypsin, indicating a lower affinity of proteases A and B for this substrate compared to chymotrypsin. Unlike other Bacillus serine proteases, neither protease A nor B stained with Coomasie blue R-250, even with loading of a large amount of protein, and they stained poorly with the silver staining method. However, NH(2)-terminal amino acid sequencing of protease B revealed a high similarity with subtilisin Carlsberg (67% homology). Almost total inhibition of both proteases by PMSF, but very little/no inhibition by trypsin and chymotrypsin inhibitors (TPCK and TLCK) or thiol reagents (PCMB and iodoacetic acid), further supported the view that the enzyme belonged to the serine protease family.

Amino Acid Sequence↗
PubMed2003

Improvement of the nutritive value of soybean meal by protease and alpha-galactosidase treatment in broiler cockerels and broiler chicks.

1. Tube-fed broiler cockerels were used in three experiments to measure the effect of different enzyme treatments on true metabolisable energy (TME) and true nitrogen digestibility (TND) of commercial solvent-extracted, heat-treated soybean meal (SBM). 2. In experiment 1, proteases P2 and P3 (from Aspergillus niger) and alpha-galactosidase (from A. oryzae) improved TME and TND while protease P1 (from Bacillus subtilis) had little effect. The effects of enzyme treatment were similar whether treatment was applied by pre-incubation of enzymes (proteases P1, P2 and P3, 1.0 g/kg; alpha-galactosidase, 0.1 g/kg) with SBM for 2 h at 50 degrees C or by simple mixing of enzymes (proteases P1, P2 and P3, 0.25 g/kg; alpha-galactosidase, 0.1 g/kg) with SBM prior to feeding. 3. In experiment 2, the effects of mixing SBM with each of protease P1 (0 or 0.25 g/kg), protease P3 (0 or 0.25 g/kg) or alpha-galactosidase (0 or 0.1 g/kg) alone or in all possible combinations were studied. Effects of protease P1 were limited, but protease P3 and alpha-galactosidase improved TME and TND. There were significant interactions between protease P3 and alpha-galactosidase for both TME and TND; the response when enzymes were applied together was less than that expected from application of the enzyme preparations individually. 4. In experiment 3, the effect of varying the concentration of protease P3 (0, 0.1, 0.25 and 1.0 g/kg SBM dry matter) and alpha-galactosidase (0, 0.025, 0.0625 and 0.25 g/kg SBM dry matter) mixed with SBM alone or in all possible combinations of protease P3 and alpha-galactosidase were assessed. Increases in TME and TND for both enzymes were non-linear with the greatest response occurring at the lowest concentration of each enzyme. There were also interactions between the two enzyme preparations. 5. Finally, either protease P3 (0, 0.1 and 1.0 g/kg SBM dry matter) or alpha-galactosidase (0, 0.025 and 0.25 g/kg SBM dry matter) were mixed with SBM alone or in all possible combinations and treated SBMs incorporated into semi-purified diets containing 450 g SBM/kg as the sole source of dietary N and offered to growing broiler chicks for 21 d. Similar patterns of increases in chick growth rate and diet digestibility to those recorded in experiment 3 were obtained when protease P3 and alpha-galactosidase where included in the diets. 6. It is concluded that responses measured by tube-feeding SBM treated with protease P3 and alpha-galactosidase were similar to responses obtained with growing broiler chicks. Further, these responses were obtained by simple addition of enzymes to diets and nutritive value of SBM was improved by both protease and alpha-galactosidase treatment.

Animal Feed↗
PubMed1988

Characterization of 73 kDa thiol protease from Serratia marcescens and its effect on plasma proteins.

The 73-kDa protease (73K protease) was purified from a clinical isolate of Serratia marcescens kums 3958. The purified protease appeared homogeneous by sodium dodecyl sulfate polyacrylamide gel electrophoresis in the presence or absence of 2-mercaptoethanol. The protease is active in a broad pH range with maximum activity at pH 7.5-8.0. The protease appeared to be a thiol protease, since it was inhibited by sulfhydryl reactive compounds such as p-chloromercuribenzoic acid, fluorescein mercuric acetate (FMA), iodoacetamide, and N-ethylmaleimide, and the protease activity was enhanced by various reducing agents such as cysteine, glutathione, 2-mercaptoethanol, and dithiothreitol. The protease contained 2 mol of free sulfhydryl residues per mol of protease. When the protease was reacted with FMA, a maximum of 2 mol of FMA per mol of enzyme was found reacted, based on fluorescence quenching in which the enzyme inactivation was paralleled linearly with the loss of both SH groups. This indicates possible equal involvement of the two thiol groups for the enzyme activity. The inactivation of the protease by FMA was partially restored by a dialysis in the presence of cysteine or dithiothreitol. The protease was not inhibited by high molecular weight kininogen but was inhibited by alpha 2-macroglobulin. The protease bound stoichiometrically to alpha 2-macroglobulin with 1:1 molar ratio and 25% activity remained constant even after the addition of 4 molar excess of alpha 2-macroglobulin. The protease extensively degraded IgG, IgA, fibronectin, fibrinogen, and alpha 1-protease inhibitor.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1998

Molecular and biotechnological aspects of microbial proteases.

Proteases represent the class of enzymes which occupy a pivotal position with respect to their physiological roles as well as their commercial applications. They perform both degradative and synthetic functions. Since they are physiologically necessary for living organisms, proteases occur ubiquitously in a wide diversity of sources such as plants, animals, and microorganisms. Microbes are an attractive source of proteases owing to the limited space required for their cultivation and their ready susceptibility to genetic manipulation. Proteases are divided into exo- and endopeptidases based on their action at or away from the termini, respectively. They are also classified as serine proteases, aspartic proteases, cysteine proteases, and metalloproteases depending on the nature of the functional group at the active site. Proteases play a critical role in many physiological and pathophysiological processes. Based on their classification, four different types of catalytic mechanisms are operative. Proteases find extensive applications in the food and dairy industries. Alkaline proteases hold a great potential for application in the detergent and leather industries due to the increasing trend to develop environmentally friendly technologies. There is a renaissance of interest in using proteolytic enzymes as targets for developing therapeutic agents. Protease genes from several bacteria, fungi, and viruses have been cloned and sequenced with the prime aims of (i) overproduction of the enzyme by gene amplification, (ii) delineation of the role of the enzyme in pathogenecity, and (iii) alteration in enzyme properties to suit its commercial application. Protein engineering techniques have been exploited to obtain proteases which show unique specificity and/or enhanced stability at high temperature or pH or in the presence of detergents and to understand the structure-function relationships of the enzyme. Protein sequences of acidic, alkaline, and neutral proteases from diverse origins have been analyzed with the aim of studying their evolutionary relationships. Despite the extensive research on several aspects of proteases, there is a paucity of knowledge about the roles that govern the diverse specificity of these enzymes. Deciphering these secrets would enable us to exploit proteases for their applications in biotechnology.

Amino Acid Sequence↗
PubMed1980

Purification and characterization of exocellular proteases produced by a clinical isolate and a laboratory strain of Pseudomonas aeruginosa.

Exocellular protease production was examined in two separate strains of Pseudomonas aeruginosa, one a clinical isolate and the other a laboratory strain. Both strains produced two separate proteases (proteases 1 and 2) which were indistinguishable from one strain to the other. The two proteases were purified by a two-step procedure of gel filtration chromatography followed by ion-exchange chromatography. Proteases 1 and 2 were shown to be distinct serologically and unrelated by physiochemical parameters examined. Protease 1 was the major exocellular protein produced and contributed about 95% of the total protease activity of the culture. It was etimated to have a molecular weight of 34850 and was also shown to contain 10% glucosamine by weight. Protease 2, in contrast, had an estimated molecular weight of 52750 and contained no detectable carbohydrate. Proteases 1 and 2 were both stimulated by Ca2+, and Mg2+ and inhibited by Co2+Zn2+, and 1,10-o-phenanthroline. Protease 1 was also inhibited by EDTA. In addition to protease activity, both proteases 1 and 2 demonstrated elastase activity as well as a limited collagenase activity. Specificity of the two protease against synthetic peptides was, however, quite different. Protease 1, but not protease 2, showed a preference for peptide bonds in which the amino group was contributed by an amino acid with a hydrophovic R group.

Humans↗
PubMed1990

Clipsin, a chymotrypsin-like protease in rat brain which is irreversibly inhibited by alpha-1-antichymotrypsin.

The protease inhibitor alpha-1-antichymotrypsin, which binds to chymotrypsin-like enzymes in a sodium dodecyl sulfate-resistant manner, has been shown recently to be both a normal constituent of brain and an integral component of the neuritic plaques that form in Down's syndrome and Alzheimer's disease. We have now identified in rat brain a Mr 25,000 alpha-1-antichymotrypsin-binding protein classified as a chymotrypsin-like protease by its inhibitor profile and substrate specificity. Release of 125I-labeled breakdown products from bands containing the protease in substrate-linked polyacrylamide gels was examined in parallel with hydrolysis of tetrapeptide chromogenic substrates in vitro to establish conditions under which the Mr 25,000 protease was the only activity being measured in vitro. The protease was completely membrane associated but was extractable using 1 M MgCl2; prior extraction of detergent- and low ionic strength-soluble proteins from membranes was used to increase its specific activity. The formation of sodium dodecyl sulfate-resistant bonds between human alpha-1-antichymotrypsin and the protease (kassoc = 2.9 X 10(6) M-1 s-1) was used to titrate the concentration of free protease solubilized from membranes. The protease cleaved both succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, and methoxy-succinyl-Ala-Ala-Pro-Met-p-nitroanilide, the latter being of interest because cleavage after a methionine residue is predicted to generate the amino terminus of the neuritic plaque component beta-amyloid from its precursor protein. In fact, the solubilized protease degraded 90% of membrane-associated beta-amyloid precursor protein detected by Western blot analysis. The protease was kinetically distinct from both chymotrypsin and cathepsin G in direct comparisons and did not match kinetic values published for the rat mast cell proteases against comparable substrates; we therefore refer to the protease with the descriptive acronym clipsin (for chymotrypsin-like protease). Proteases similar to and potentially identical to clipsin were detected by enzymography in other organs from rat (most notably spleen and adult lung). The enzyme in brain was distinguished by a narrow window of elevated activity surrounding postnatal day 5, which was 12-14-fold higher than levels in day 1 or adult brain. Because independent lines of evidence suggest that a brain chymotrypsin-like protease may be involved in the etiology of Down's syndrome and Alzheimer's disease, clipsin is discussed as a candidate for such a role.

Amino Acid Sequence↗
PubMed1984

A high-molecular-weight trypsinlike protease in the skin sites of delayed hypersensitivity in guinea pigs.

A trypsinlike protease was extracted from the delayed hypersensitivity skin sites in guinea pigs. Extractable amounts of the enzyme were chronologically paralleled with the gross appearance of the inflammation, and the maximum activity from the inflamed sites at 24-36 hours was about 20 times stronger than that from normal skin, suggesting a potential role in the pathogenesis of the delayed hypersensitivity reaction. The enzyme, which suitably hydrolyzed t-butyloxycarbonyl-phenylalanyl-seryl-arginine 4-methylcoumaryl-7-amide, was partially purified by isoelectric focusing or by gel filtration. The enzyme demonstrated a single peak of activity on the former column with an apparent isoelectric point of 4.2, and in the latter it showed an apparent molecular weight of 600,000 (600K-protease). When incubated with 3H-diisopropylfluorophosphate, the enzyme lost all amidolytic activity and yielded a single band of radioactivity in polyacrylamide disk gel electrophoresis in the presence of sodium dodecyl sulfate, and a single peak of radioactivity in gel filtration, both having an apparent molecular weight of 31,000-33,000 (31K-protease). That the 600K-protease might be a complex with alpha 2-macroglobulin was ruled out. The 31K-protease was separated from the 600K-protease by gel filtration in the presence of 6 M guanidine hydrochloride, and was renatured to an active form. An apparent isoelectric point of the 31K-protease observed was 9.4, suggesting that the 600K-protease may be a complex of 31K-protease with an acidic carrier molecule(s). Both proteases, 31K- and 600K-protease, had identical substrate specificity, a pH profile of amidolytic activity, and susceptibility to exogenous protease inhibitors. However, when sensitivities to intrinsic protease inhibitors in guinea pig plasma, two kinds of trypsin inhibitor, and alpha 2-macroglobulin were compared, the 600K-protease was at least 100 times more resistant than the 31K-protease. It was supposed that one of the pathophysiologically significant functions of the complex formation might be to maintain the enzyme activity longer in vivo.

Amidohydrolases↗
PubMed1996

Pseudomonas keratitis. The role of an uncharacterized exoprotein, protease IV, in corneal virulence.

PURPOSE: The role of exoproteins in the pathogenesis of Pseudomonas aeruginosa keratitis was investigated in three animal models by assessing the relationship between corneal virulence and the activities of exotoxin A, elastase, alkaline protease, and an uncharacterized protease, protease IV. METHODS: The four Pseudomonal strains tested included a prototype strain (ATCC 27853) producing exotoxin A, elastase, and alkaline protease; a parent strain (PA103) producing only exotoxin A and protease IV; a mutant (PA103-29) producing only protease IV; and a mutant (PA103-AP1) producing exotoxin A and having only approximately 5% of the protease IV activity of its parent. Corneal virulence was evaluated in the mouse scratch, rabbit scratch, and rabbit intrastromal models in terms of clinical signs (slit lamp examination, slit lamp examination), and viable bacteria. RESULTS: Protease IV, the only protease produced by PA103 and PA103-29, was found to produce a unique band on zymograms (120 kDa) and to react distinctively with a synthetic substrate. Evidence for the role of protease IV in corneal virulence included two findings: PA103-29,which produced protease IV but not the other exoproteins, caused infections that were as severe as those caused by the prototype strain (ATCC 27853) in all three models (P>0.24); and PA103-AP1, the strain deficient in 95% of the parent protease IV activity, mediated infections characterized by slit lamp examination scores significantly lower than those of infections caused by the parent (PA103) or the prototype strain (ATCC 27853) in the rabbit and mouse scratch models (P<0.02). CONCLUSIONS: Protease IV was found to be a novel Pseudomonas protease contributing to corneal virulence in rabbits and mice when infections were initiated at the corneal surface. Furthermore, production of protease IV in low quantities was sufficient for virulence when the topical stages of keratitis were bypassed by an intrastromal injection of Pseudomonas.

ADP Ribose Transferases↗
PubMed2004

Narrow substrate specificity and sensitivity toward ligand-binding site mutations of human T-cell Leukemia virus type 1 protease.

Human T-cell leukemia virus type 1 (HTLV-1) is associated with a number of human diseases; therefore, its protease is a potential target for chemotherapy. To compare the specificity of HTLV-1 protease with that of human immunodeficiency virus type 1 (HIV-1) protease, oligopeptides representing naturally occurring cleavage sites in various retroviruses were tested. The number of hydrolyzed peptides as well as the specificity constants suggested a substantially broader specificity of the HIV protease. Amino acid residues of HTLV-1 protease substrate-binding sites were replaced by equivalent ones of HIV-1 protease. Most of the single and multiple mutants had altered specificity and a dramatically reduced folding and catalytic capability, suggesting that mutations are not well tolerated in HTLV-1 protease. The catalytically most efficient mutant was that with the flap residues of HIV-1 protease. The inhibition profile of the mutants was also determined for five inhibitors used in clinical practice and inhibitor analogs of HTLV-1 cleavage sites. Except for indinavir, the HIV-1 protease inhibitors did not inhibit wild type and most of the mutant HTLV-1 proteases. The wild type HTLV-1 protease was inhibited by the reduced peptide bond-containing substrate analogs, whereas the mutants showed various degrees of weakened binding capability. Most interesting, the enzyme with HIV-1-like residues in the flap region was the most sensitive to the HIV-1 protease inhibitors and least sensitive to the HTLV-1 protease inhibitors, indicating that the flap plays an important role in defining the specificity differences of retroviral proteases.

Amino Acid Sequence↗
PubMed1987

Comparison of proteases from chloroquine-sensitive and chloroquine-resistant strains of Plasmodium falciparum.

An aminopeptidase and four hemoglobin-degrading acid proteases have been isolated from cloned strains of chloroquine-sensitive and chloroquine-resistant Plasmodium falciparum. Amino-peptidases from both strains showed similar properties including molecular weights of 63,000 and non-competitive inhibition by chloroquine; Ki = 535 and 410 microM for enzymes from the sensitive and resistant strains respectively. The acid proteases from the chloroquine-sensitive strain included a low molecular weight enzyme in the soluble fraction (protease S), an enzyme weakly associated with membrane (protease M2), and two enzymes strongly associated with membrane (proteases M3 and M4). The acid proteases from the chloroquine-resistant strain included protease S, protease M2, a second enzyme weakly associated with membrane (protease M1), and protease M3. All of the acid proteases were inhibited by ferriprotoporphyrin IX and by the chloroquine-ferriprotoporphyrin IX complex, I50 = 5-25 microM. The data were consistent with a model for chloroquine action wherein chloroquine acts to divert ferriprotoporphyrin IX from sequestration into malarial pigment, leaving ferriprotoporphyrin IX (or its chloroquine complex) to interfere with digestion of host cytosol by inhibiting hemoglobin-degrading proteases. However, the similarities among the proteases from chloroquine-sensitive and chloroquine-resistant strains of parasites suggest that chloroquine resistance does not result from changes in parasite proteases.

Aminopeptidases↗