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PubMed1989

Protease Ti from Escherichia coli requires ATP hydrolysis for protein breakdown but not for hydrolysis of small peptides.

Protease Ti, a new ATP-dependent protease in Escherichia coli, degrades proteins and ATP in a linked process, but these two hydrolytic functions are catalyzed by distinct components of the enzyme. To clarify the enzyme's specificity and the role of ATP, a variety of fluorogenic peptides were tested as possible substrates for protease Ti or its two components. Protease Ti rapidly hydrolyzed N-succinyl(Suc)-Leu-Tyr-amidomethylcoumarin (AMC) (Km = 1.3 mM) which is not degraded by protease La, the other ATP-dependent protease in E. coli. Protease Ti also hydrolyzed, but slowly, Suc-Ala-Ala-Phe-AMC and Suc-Leu-Leu-Val-Tyr-AMC. However, it showed little or no activity against basic or other hydrophobic peptides, including ones degraded rapidly by protease La. Component P, which contains the serine-active site, by itself rapidly degrades the same peptides as the intact enzyme. Addition of component A, which contains the ATP-hydrolyzing site and is necessary for protein degradation, had little or no effect on peptide hydrolysis. N-Ethylmaleimide, which inactivates the ATPase, did not inhibit peptide hydrolysis. In addition, this peptide did not stimulate the ATPase activity of component A (unlike protein substrates). Thus, although the serine-active site on component P is unable to degrade proteins, it is fully functional against small peptides in the absence of ATP. At high concentrations, Suc-Leu-Tyr-AMC caused a complete inhibition of casein breakdown, and diisopropylfluorophosphate blocked similarly the hydrolysis of both protein and peptide substrates. Thus, both substrates seem to be hydrolyzed at the same active site on component P, and ATP hydrolysis by component A either unmasks or enlarges this proteolytic site such that large proteins can gain access to it.

Adenosine Triphosphatases↗
PubMed1986

Selective cleavage of human ACTH, beta-lipotropin, and the N-terminal glycopeptide at pairs of basic residues by IRCM-serine protease 1. Subcellular localization in small and large vesicles.

We present a study of the cleavage specificity of IRCM-serine protease 1 from frozen porcine pituitary neurointermediate lobes using polypeptide substrates representing different segments of human pro-opiomelanocortin. Using 125I-labeled ACTH(11-24) and a 125I-labeled model beta-lipotropin (beta-LPH) peptide, the preference of this protease for cleavage C-terminal to the pairs of basic residues Lys-Arg and Lys-Lys was clearly seen. This study was extended to larger unlabeled natural human polypeptides including ACTH(1-39), beta-LPH(1-89), and the N-terminal glycopeptide (1-76), which are known to serve as substrates for further cleavage in vivo. In these substrates IRCM-serine protease 1 cleaved C-terminal to all pairs of basic residues known to be cleaved in vivo. In addition, the enzyme cleaved between two pairs of basic amino acids found in NT(1-76) which are also known to be cleaved in vivo. Many potential "tryptic-like" cleavage sites were not cleaved by the enzyme. However, IRCM-serine protease 1 cleaved C-terminal to Phe-Arg in the three melanocyte-stimulating hormone sequences of pro-opiomelanocortin. In order to better understand the physiological role of IRCM-serine protease 1, differential centrifugation was used to study the subcellular distribution of the enzyme from porcine pituitary anterior lobe homogenates. We present evidence that the active enzyme form, isolated from the subcellular fractions, possesses a similar molecular architecture as the enzyme isolated from frozen tissue (Mr 38,000 catalytic domain linked via disulfide bridge(s) to another polypeptide chain(s) to form an Mr 88,000 monomeric structure). The majority of IRCM-serine protease activity is found to be associated with small vesicles (150,000 X g for 5 h) of as yet undetermined nature. In addition, a latent activity was found to be associated with a 27,000 X g (15 min) pellet containing the majority of mature secretory granules. If IRCM-serine protease 1 participates in prohormone maturation in vivo, we propose a model in which this protease is present in an enzymatically active form in small vesicles, possibly within clathrin-coated structures (prosecretory granules) which are then transformed to mature secretory granules by a process which would also inactivate most of the enzyme.

Adrenocorticotropic Hormone↗
PubMed1988

Characterization of a tumor-associated serine protease.

We have earlier identified and characterized a tumor-associated trypsin inhibitor (TATI) in the urine of patients with gynecological cancer. Elevated levels of TATI occur in urine and serum of cancer patients (Stenman, U. H., Huhtala, M. L., Koistinen, R. & Seppälä, M. (1982) Int. J. Cancer 30, 53-57). To explain the elevation of TATI in cancer, we have postulated the existence of a tumor-associated protease reacting with TATI. Such a protease, tentatively called protease T, was found in cyst fluid from mucinous ovarian tumors. The protease occurs in complex with TATI, and its protease activity can be measured only after dissociation of the complex. This is achieved by reversed phase chromatography at low pH and elution with an isopropyl alcohol gradient. Protease T is inhibited by phenylmethanesulphonyl fluoride indicating that it is a serine protease. Its optimum activity at pH 9.1 and molecular mass of 24 kDa in gel chromatography are similar to those of trypsin but the substrate specificity is not identical and its isoelectric point (pI) is about 4.0, which is lower than the corresponding values of both cationic (pI 9) and anionic trypsin (pI 5). Protease T could be associated with the elevation of TATI seen in certain tumor patients.

Biomarkers, Tumor↗
PubMed1985

Purification and characterization of a cytosol protease from dormant cysts of the brine shrimp Artemia.

A thiol protease has been isolated and purified from the postribosomal fraction of encysted embryos of the brine shrimp Artemia using a six-step procedure. The purified enzyme has a molecular weight of 55,000 +/- 4,200 and is composed of subunits of Mr 31,500 +/- 559 and 25,867 +/- 1,087. Isoelectric focusing revealed two discrete bands, one at pH 4.6 and the other at pH 5.1. The protease appears to be a member of the thiol group of proteases based on its inhibition by leupeptin, antipain, chymostatin, Ep-475, and several other thiol protease inhibitors. The enzyme was stimulated by heavy metal chelators and thiol reagents. At pH 3.5-4.0 the thiol protease hydrolyzed a wide range of proteins including bovine serum albumin, hemoglobin, Artemia embryo soluble proteins, Artemia lipovitelline, and protamine, whereas at pH 6.0-6.5 the enzyme showed a high degree of specificity for Artemia elongation factor 2 and lipovitelline alpha 1. The total amount of protease activity in crude homogenates of Artemia embryos decreased by about 50% during the first 24 h of development, while the amount of free, active enzyme decreased proportionally for 9 h of development then remained constant during the next 26-27 h of development. These changes in protease activity appear to reflect changing levels of an endogenous protease inhibitor during development.

Animals↗
PubMedDate not supplied

[Partial purification and properties of proteases of bitoxibacillin].

The paper describes properties of proteases A and B isolated from the biological insecticide bitoxibacillin by sulphate precipitation and Sephadex G-75 gel filtration. Proteases A and B of bitoxibacillin belong to the neutral bacterial proteases. pH optimum was found to be 6.0 and 7.5 for detection of proteolytic activity of protease A and protease B, respectively. Thermal stability of proteases A and B was similar and increased by 25% upon addition of CaCl2. Both proteases were inhibited with EDTA. The molecular weight of proteases A and B was estimated to be 57,000 and 47,000, respectively.

Bacillus thuringiensis↗
PubMed1981

[Studies on degradation of cartilage proteoglycan by rheumatoid synovial tissue. Part II: On the property of acid and neutral proteases obtained from rheumatoid synovial tissue (author's transl)].

1. Proteolytic enzymes are likely to play the main role on the proteoglycan (PG)-degrading activity of rheumatoid synovium. In this paper, the presence of cathepsin D, cathepsin B, lysosomal elastase and cathepsin G in rheumatoid synovium is established by isolation, purification, and characterization of these proteases. 2. The degradation of MgCl2-extracted PG from bovine nasal cartilage was performed by using these proteases and the property of the products was studied by the viscosity, Sepharose CL-4B chromatography, Agarose/polyacrylamide-gel electrophoresis, hexosamine analysis and amino acids analysis. 3. These proteases reduced the viscosity of PG solutions and the reaction was inhibited by addition of pepstatin, antipain, elastatinal and chymostatin for each protease. 4. The size and chemical composition of the degradation products varied with the different proteases. Of the four proteases, cathepsin G produced the largest glycosaminoglycan multi-chain peptides and cathepsin B produced the smallest contained chondroitin single-chain peptide. Each protease specifically split PG core protein and the degradation products particularly indicated the characteristic structure of core peptides. 5. The results suggest that these proteases may be contributed to the breakdown of cartilage PG in rheumatoid arthritis.

Amino Acids↗
PubMed1995

Mammalian cysteine protease inhibitors: biochemical properties and possible roles in tumor progression.

The endogenous cysteine protease inhibitors represent the final level at which cysteine protease activity can be regulated. These inhibitors are subdivided into three families (stefins, cystatins and kininogens) which belong to the protein superfamily, cystatins. Cystatins do not form a covalent bond with cysteine proteases, but instead cover the active site cleft blocking access to the active site. The most important biochemical characteristics of the cystatins are described in the first part of this review. Alterations in the balance between endogenous cysteine protease inhibitors and cysteine proteases have been postulated to contribute to malignant progression. A few studies have demonstrated the enrichment of cysteine protease inhibitor activity in the membrane fraction of tumors/cells. Evidence is accumulating that an inverse correlation exists between the level of stefin A, one of the cysteine protease inhibitors, and malignant progression. Stefin A has even been hypothesized to be a tumor suppressor. However, additional studies are necessary in order to prove functional roles for the individual cysteine protease inhibitors in tumor growth and progression.

Animals↗
PubMed1994

A new protease activity assay using fluorescence polarization.

Fluorescence polarization (FP) technology was used to develop an assay for protease activity that is more sensitive than other nonradioactive protease assays and requires no separations, precipitations or transfers of the reaction mixture. FP measures changes in the molecular volume of fluorescently labeled molecules. In the assay described in the present studies, changes in molecular volume due to the cleavage of intact fluorescein thiocarbamoyl (FTC)-casein molecules to smaller FTC peptides were measured. The sensitivity of the FP protease assay was compared with a non-FP fluorescent assay. The FP-based assay was twofold to twentyfold more sensitive than the non-FP assay, depending on the protease tested. Because measurements were taken in real time, the progress of the reaction was followed both kinetically and at a single time point. This assay provided a sensitive measure of activity for the three common protease classes: serine proteases, sulfhydryl proteases and acid proteases.

Animals↗
PubMed1993

Identification of the herpes simplex virus-1 protease cleavage sites by direct sequence analysis of autoproteolytic cleavage products.

Herpes simplex virus type-1 (HSV-1) encodes a protease responsible for proteolytic processing of the virus assembly protein, ICP35 (infected cell protein 35). The coding region of ICP35 is contained within the gene that encodes the protease, and ICP35 shares amino acid identity with the carboxyl-terminal 329 amino acids of the protease. The HSV-1 protease was expressed in Escherichia coli as a fusion protein containing a unique epitope and the protein A Fc binding domain at its carboxyl terminus. The fusion protease underwent autoproteolytic cleavage at two distinct sites. The size of the cleavage products containing the carboxyl-terminal epitope mapped one cleavage site near the carboxyl terminus of the protease corresponding to the proteolytic processing site of ICP35, and the second site proximal to the amino terminus consistent with previous data. The carboxyl-terminal autoproteolytic cleavage products were partially purified on an IgG affinity column by virtue of the protein A Fc binding domain and subjected to direct amino-terminal sequence analysis. Protein sequencing revealed that cleavage occurs between the Ala and Ser residues at amino acids 610/611 and 247/248 of the HSV-1 protease. The flanking sequences share homology with each other and are highly conserved in homologous proteases of other herpes viruses.

Amino Acid Sequence↗
PubMed1993

Structural analysis of chicken factor B-like protease and comparison with mammalian complement proteins factor B and C2.

Chicken complement factor B-like protease is a glycoprotein of 95 kDa. Activation of chicken serum complement with inulin cleaved the B-like protease into an N-terminal Ba fragment of 37 kDa and a C-terminal Bb fragment of 60 kDa. The whole protein and the two fragments were purified by affinity chromatography using mAb to chicken Ba or Bb followed by ion exchange chromatography. Amino acid sequencing showed that chicken B-like protease was cleaved at a site homologous to that cleaved in mammalian complement components B and C2 on activation. Limited tryptic digestion of the B-like protease generated fragments similar to Ba and Bb. More than 200 residues of the Ba sequence and two N-linked glycosylation sites were established by amino acid sequencing of peptides derived by digestion with four proteases. Comparison of human and mouse C2 and B sequences indicated a slower evolutionary rate for B (85% sequence identity) than for C2 (74% sequence identity). Comparison of chicken Ba to human and mouse C2b and Ba showed 42 to 45% sequence identity with respect to C2b fragments, and 46 to 49% sequence identity with respect to Ba fragments. Taking the slower evolutionary rate of factor B into account, chicken factor B-like protease seems to be equally related to mammalian complement components B and C2, and the B-like protease most likely represents the present-day descendant of a common ancestral protein for mammalian B and C2. This conclusion is in agreement with the requirement for the B-like protease in both classical and alternative activation pathways for chicken complement, and with the apparent lack of a chicken serum protein with exclusive C2 activity.

Amino Acid Sequence↗
PubMed1993

Secreted LasA of Pseudomonas aeruginosa is a staphylolytic protease.

Full expression of the elastolytic phenotype of Pseudomonas aeruginosa depends on LasA, an extracellular protease with restricted specificity whose mode of action on elastin and biological role is not understood. LasA exhibits amino acid sequence homology to some bacteriolytic proteases and shares several physicochemical properties with the staphylolytic protease of P. aeruginosa. This led us to examine whether the two proteases are the same. Production of LasA and staphylolytic protease by prototrophic and lasA mutant strains of P. aeruginosa was investigated. The two prototrophic strains examined, PAO1 and FRD2, exhibited extracellular staphylolytic activity and secreted LasA. LasA mutants, PAO-E64 lasA1 (Ts), FRD2128 delta lasA, and FRD244 lasA::mTn10, did not exhibit staphylolytic activity. A low level of the LasA protein was detected in the culture filtrate of the temperature-sensitive lasA mutant PAO-E64, but none was detectable in those of the deletion and insertion mutants, FRD2128, and FRD244, respectively. The staphylolytic protease was purified from the culture filtrate of P. aeruginosa strain FRD2 by DEAE-cellulose chromatography. The purified enzyme hydrolyzed pentaglycine into the respective di- and tripeptides and reacted specifically with antibodies against a synthetic peptide identical in sequence to positions 77-98 in LasA. The amino-terminal sequence of the first 15 amino acid residues of the staphylolytic protease was found to be identical with that of the secreted LasA. These results clearly indicate that LasA is a staphylolytic protease. In addition to lysing staphylococci, it may enhance elastolysis by cleaving Gly-Gly bonds, which are abundant in elastin.

Amino Acid Sequence↗
PubMed1996

Bcl-2 expression in neural cells blocks activation of ICE/CED-3 family proteases during apoptosis.

The ICE/CED-3 family of proteases has been implicated in playing a fundamental role in programmed cell death. Bcl-2 protein represses a number of apoptotic death programs, but the biochemical mechanism of its action is not known. We investigated the activation of ICE/CED-3 proteases induced by three apoptotic stimuli (staurosporine, ceramide, and serum withdrawal) in the neuronal cell line GT1-7 and in cells overexpressing Bcl-2. Rapid activation of a 17 kDa subunit of an activated member of the ICE/CED-3 family is demonstrated by affinity-labeling GT1-7 extracts from apoptotic controls cells with a biotinylated ICE/CED-3 inhibitor. This activation corresponds to an increased ICE/CED-3-like protease activity in extracts measured by a fluorogenic substrate assay. In a cell-free system, these extracts induce apoptotic morphological changes in intact nuclei. All three activities are readily inhibited by treatment of control extracts with ICE/CED-3-like protease inhibitors. Overexpressed Bcl-2 inhibits the activation of the 17 kDa protein, the ICE/CED-3-like protease activity in the fluorogenic assay, and the induction of apoptotic morphological changes in HeLa nuclei in the cell-free system, similar to results obtained with ICE/CED-3 protease inhibitors. At the mRNA level, overexpression of Bcl-2 did not alter expression of five members of the ICE/CED-3 family: CPP32, ICE, Mch 2, Nedd 2, and TX. Overexpression of Bcl-2 prevented the apoptosis-induced processing of pro-Nedd 2 to the cleaved form. These data suggest that Bcl-2 participates upstream from the function of ICE/CED-3 proteases and may inhibit apoptosis by preventing the post-translational activation of ICE/CED-3 proteases.

Animals↗
PubMed1996

A novel protease homolog differentially expressed in breast and ovarian cancer.

BACKGROUND: Using differential display (DD), we discovered a new member of the serine protease family of protein-cleaving enzymes, named protease M. The gene is most closely related by sequence to the kallikreins, to prostate-specific antigen (PSA), and to trypsin. The diagnostic use of PSA in prostate cancer suggested that a related molecule might be a predictor for breast or ovarian cancer. This, in turn, led to studies designed to characterize the protein and to screen for its expression in cancer. MATERIALS AND METHODS: The isolation of protease M by DD, the cloning and sequencing of the cDNA, and the comparison of the predicted protein structure with related proteins are described, as are methods to produce recombinant proteins and polyclonal antibody preparations. Protease M expression was examined in mammary, prostate, and ovarian cancer, as well as normal, cells and tissues. Stable transfectants expressing the protease M gene were produced in mammary carcinoma cells. RESULTS: Protease M was localized by fluorescent in situ hybridization analysis to chromosome 19q13.3, in a region to which other kallikreins and PSA also map. The gene is expressed in the primary mammary carcinoma lines tested but not in the corresponding cell lines of metastatic origin. It is strongly expressed in ovarian cancer tissues and cell lines. The enzyme activity could not be established, because of difficulties in producing sufficient recombinant protein, a common problem with proteases. Transfectants were selected that overexpress the mRNA, but the protein levels remained very low. CONCLUSIONS: Protease M expression (mRNA) may be a useful marker in the detection of primary mammary carcinomas, as well as primary ovarian cancers. Other medical applications are also likely, based on sequence relatedness to trypsin and PSA.

Amino Acid Sequence↗
PubMed1997

Characterization of a novel, membrane-bound, 80-kDa matrix-degrading protease from human breast cancer cells. Monoclonal antibody production, isolation, and localization.

A major, apparently novel extracellular matrix-degrading protease was previously identified and partially isolated from hormone-dependent but not from hormone-independent human breast cancer cells (Shi, Y. E., Torri, J., Yieh, L., Wellstein, A., Lippman, M. E., and Dickson, R. B. (1993) Cancer Res. 53, 1409-1415). Although initially the 80-kDa protease was identified from breast cancer cell-conditioned medium, immunofluorescence staining of breast cancer cells with anti-80-kDa protease monoclonal antibody 21-9 showed that in addition to its detection in intracellular compartments, the protease was uniformly localized around periphery of the cells with more intensive staining on the pseudopodia and membrane ruffles. A surface biotinylation technique confirmed the plasma membrane localization of the protease. In addition, the 80-kDa protease could not be washed from the membrane fraction of homogenized breast cancer cells with high concentrations of salts or with EDTA. The 80-kDa protease may noncovalently associate with other protein(s) to form complexes, the 95- and 110-kDa proteases. Both complexes showed gelatinolytic activity and bore the epitopes recognized by monoclonal antibody 21-9. Furthermore, both complexes could be converted to 80-kDa forms by boiling in SDS in the absence of reducing agents. Expression of this novel, integral membrane gelatinase could allow breast cancer cells an alternative to other previously described matrix-degrading enzymes for degradation of the extracellular matrix in close proximity to their surfaces.

Antibodies, Monoclonal↗
PubMed1997

Protease activation is required for glucocorticoid-induced apoptosis in chronic lymphocytic leukemic lymphocytes.

Recent work has demonstrated that glucocorticoids, nucleoside analogues, and other cancer chemotherapeutics induce apoptosis in chronic lymphocytic leukemia (CLL) cells. In this study, we investigated the involvement of protease activation in these responses using selective peptide inhibitors of the interleukin-1beta converting enzyme (ICE)/caspase family and a Ca2+-activated protease we recently implicated in thymocyte apoptosis. Apoptosis was associated with proteolytic cleavage of poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) and increased caspase protease activity, and cell-permeant caspase antagonists [zVAD(OMe)fmk and Boc-D(OBzl)cmk] blocked apoptosis in response to the glucocorticoid methylprednisolone or the nucleoside analogue fludarabine, indicating that caspase activation was required for these responses. However, a peptide-based inhibitor of the Ca2+-dependent lamin protease (zAPFcmk) also completely suppressed DNA fragmentation and the cleavage of lamin B1 . Strikingly, treatment of cells with zAPFcmk alone led to characteristic PARP cleavage, depletion of the precursor forms of two ICE family proteases (CPP32 and ICH-1), and phosphatidylserine exposure, suggesting that blockade of the lamin protease led to activation of the ICE family. Our results implicate the lamin protease as a target for Ca2+ during chemotherapy-induced apoptosis in CLL lymphocytes, and they identify a novel functional interaction between the protease and members of the ICE family.

Apoptosis↗
PubMed1997

Role of proteases in tumor invasion and metastasis.

Cancer remains a major cause of worldwide deaths due to ability of cancer cells to form secondary tumors at other sites by multistep process called metastasis. In order to migrate from their original site, tumor cells have to cross several barriers like basement membranes, interstitial tissues and extracellular matrices, which are composed primarily of collagen, proteoglycans, elastin, laminin and other glycoproteins. Tumor cells over express and secrete proteases which are capable of degrading the components of these barriers and thus facilitate their migration. The classes of proteases which have been implicated in the process of tumor invasion and metastasis include metalloproteases, serine proteases and cathepsins. Cancer cells in general have elevated levels of proteases belonging to more than one class. In some studies, process of invasion has been inhibited by using specific inhibitors of these proteases. Expression of some proteases has been observed only in some specific tumors. These proteases have been proposed to be of diagnostic/prognostic value. However a better understanding of the process of metastasis and tumor invasion is required before proteases can be used as therapeutic targets for blocking the spread of cancer.

Animals↗
PubMed1997

Identification of a cysteine protease responsible for degradation of sperm histones during male pronucleus remodeling in sea urchins.

We have identified a 60-kDa cysteine protease that is associated with chromatin in sea urchin zygotes. This enzyme was found to be present as a proenzyme in unfertilized eggs and was activated shortly after fertilization. At a pH of 7.8-8.0, found after fertilization, the enzyme degraded the five sperm-specific histones (SpH), while the native cleavage-stage (CS) histone variants remained unaffected. Based on its requirements for reducing agents, its inhibition by sulfhydryl blocking compounds and its sensitivity to the cysteine-type protease inhibitors (2S,3S)-trans-epoxysuccinyl-L-leucyl-amido-3-methylbutane-ethyl-es ter (E-64 d), cystatin and leupeptin, this protease can be defined as a cysteine protease. Consistently, this protease was not affected by serine-type protease inhibitors phenylmethylsulfonyl fluoride (PMSF) and pepstatin. The substrate selectivity and pH modulation of the protease activity strongly suggest its role in the removal of sperm-specific histones, which determines sperm chromatin remodeling after fertilization. This suggestion was further substantiated by the inhibition of sperm histones degradation in vivo by E-64 d. Based on these three lines of evidence, we postulate that this cysteine protease is responsible for the degradation of sperm-specific histones which occurs during male pronucleus formation.

Animals↗
PubMed1998

Purification and characterization of a protease from Actinobacillus pleuropneumoniae serotype 1, an antigen common to all the serotypes.

A high molecular-mass proteolytic enzyme of Actinobacillus pleuropneumoniae serotype 1, was purified from culture supernatants (CSN) by using DEAE-cellulose and sepharose-4B-gelatin chromatography. In 10% SDS-polyacrylamide gels copolymerized with porcine gelatin, the protease showed a single band of activity of > 200 kDa. However, minor molecular-mass proteolytic bands were observed when the protease was electrophoresed in the presence of either 5% beta-mercaptoethanol, 50 mM dithiothreitol, or 0.25 M urea. Furthermore, when the > 200-kDa purified protein was passed through a sucrose gradient, several bands with proteolytic activity were found: 62, 90, 190, and 540 kDa. The proteolytic activity was increased in the presence of calcium or zinc and was not affected after being heated at 90 degrees C for 5 min. Proteolytic activities were also observed in CSN from all A. pleuropneumoniae serotypes and biotypes. The purified protease hydrolyzed porcine IgA and IgG in vitro. In addition, by immunoblot the protease was recognized by serum of naturally infected pigs with serotypes 1 and 5, and by serum of pigs experimentally infected with serotypes 1, 2, 8, or 9. Serum of a pig vaccinated with CSN of a serotype 3 strain also recognized the protease, but not sera of pigs vaccinated with a bacterin (serotype 1). Proteins from CSN of all the serotypes, which were precipitated with 70% (NH4)2SO4, were recognized by a polyclonal antibody raised against the purified protease. Taken together these results indicate that an antigenic protease is produced in vivo by all the serotypes of A. pleuropneumoniae. The results indicate that proteases could have a role in the disease and in the immune response of pigs infected with A. pleuropneumoniae.

Actinobacillus pleuropneumoniae↗