Showing papers on "Proteolytic enzymes published in 1993"

Spontaneous assembly of a self-complementary oligopeptide to form a stable macroscopic membrane.

Abstract: A 16-residue peptide [(Ala-Glu-Ala-Glu-Ala-Lys-Ala-Lys)2] has a characteristic beta-sheet circular dichroism spectrum in water. Upon the addition of salt, the peptide spontaneously assembles to form a macroscopic membrane. The membrane does not dissolve in heat or in acidic or alkaline solutions, nor does it dissolve upon addition of guanidine hydrochloride, SDS/urea, or a variety of proteolytic enzymes. Scanning EM reveals a network of interwoven filaments approximately 10-20 nm in diameter. An important component of the stability is probably due to formation of complementary ionic bonds between glutamic and lysine side chains. This phenomenon may be a model for studying the insoluble peptides found in certain neurological disorders. It may also have implications for biomaterials and origin-of-life research.

Structure-based inhibitors of hiv-1 protease

Abstract: INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 544 HIV-l PROTEASE STRUCTURE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 545 Description of the HIV-J PR Molecule ....... .... .. . ... 546 Symmetry of the Enzyme and Its Crystallographic Manifestation . . . . . . . . . . . 549 Substrate Specificity of HIV PR . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . 554 SUBSTRATE-ANALOG INHIBITOR DESIGN ......... . 555 INHIBITOR COMPLEXES OF HIV-l PR . . . . . . . . . . . . . . . . . . . . . . . . . . . 558 Common Structural Features . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . 559 Subsites and Subsite-Inhibitor Interactions . . . . . . . . . . . . . . . . . . . . . . . . . 561 Transition-State Inhibitor Complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 566 STRUCTURE-BASED INHIBITOR DESIGN . . . . . . . . . . . . . . . . . . . . . . . . . 573 Use of Structure for New Lead Discovery . . . . . . . ... . . .. . . . ... . . ... 575 SCREENING AND SERENDIPITY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 577 SUMMARY AND CONCLUSIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 579 Is Symmetry Important in Inhibitor Design? . . . . . . . . . . . . . . . . . . . . . . . . 579 Subsitellnhibitor Contacts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 579 Conclusions .... . . .. . 580

Role of Matrix Metalloproteinases in Human Periodontal Diseases.

Abstract: Matrix metalloproteinases (MMP) are a family of proteolytic enzymes that mediate the degradation of extracellular matrix macromolecules, including interstitial and basement membrane collagens, fibronectin, laminin, and proteoglycan core protein. The enzymes are secreted or released in latent form and become activated in the pericellular environment by disruption of a Zn++-cysteine bond which blocks the reactivity of the active site. The major cell types in inflamed and healthy periodontal tissues (fibroblasts, keratinocytes, endothelial cells, and macrophages) are capable of responding to growth factors and cytokines, as well as to products released from the microbial flora by induction of transcription of 1 or more MMP genes. Cytokines that are likely to regulate expression of MMP genes in periodontal tissues include IL-1, TNF-α, and TGF-α. In addition, triggered PMN leukocytes which express only 2 MMP (PMN-CL and Mr 92K GL) release these enzymes from specific granule storage sites in response to a number of stimuli. The evidence that MMP are involved in tissue destruction in human periodontal diseases is still indirect and circumstantial. Cells isolated from normal and inflamed gingiva are capable of expressing a wide complement of MMP in culture and several MMP can be detected in cells of human gingiva in vivo. In addition, PMN-CL and Mr 92K GL are readily detected in gingival crevicular fluid from gingivitis and Periodontitis patients. Osteoclastic bone resorption does not appear to directly involve MMP, but a body of evidence suggests that bone resorption is initiated by removal of the osteoid layer by osteoblasts by means of a collagenase-dependent process. J Periodontol 1993; 64:474-484.

The DNA-binding domain of p53 contains the four conserved regions and the major mutation hot spots.

Abstract: Mutations in the p53 tumor suppressor gene are the most commonly observed genetic alterations in human cancer. The majority of these mutations occur in the conserved central portion of the gene, but there has been little information about the function of this region. Using proteolytic digestion of the 393-amino-acid human p53 protein, we have identified a 191-amino-acid protease-resistant fragment (residues 102-292) that corresponds to the central portion of p53, and we show that this core fragment is the sequence-specific DNA-binding domain of the protein. DNA binding is inhibited by metal chelating agents, and we find that the core domain contains zinc. Proteolytic digests also reveal a 53-amino-acid carboxy-terminal domain which we show to be the tetramerization domain of p53.

Peptide linkers for improved oligonucleotide delivery

Abstract: A covalently linked conjugate of an oligonucleotide (ODN) with a peptide and a carrier or targeting ligand (ODN-peptide-carrier) includes a therapeutic oligonucleotide which is capable of selectively binding to a target sequence of DNA, RNA or protein inside a target cell. The ODN is covalently linked to a peptide which is capable of being cleaved by proteolytic enzymes inside the target cell. The peptide, in turn is covalently linked to a carrier or targeting ligand moiety which facilitates delivery of the entire ODN-peptide-carrier conjugate into the cell, and preferably into a specific target tissue type. Inside the cell, the peptide is cleaved, releasing the ODN which, by binding to the target DNA, RNA or protein sequence, brings about a beneficial result.

Structure of the cobalt-dependent methionine aminopeptidase from Escherichia coli: a new type of proteolytic enzyme.

Abstract: The X-ray structure of Escherichia coli methionine aminopeptidase (MAP) has been determined to 2.4-A resolution and refined to a crystallographic R-factor of 18.2%. The fold is novel and displays internal pseudo-2-fold symmetry which structurally relates the first and second halves of the polypeptide chain. The topology consists of a central antiparallel beta-sheet covered on one side by two pairs of alpha-helices and by a C-terminal loop. The other face of the beta-sheet, together with some irregular loops, forms the active site, which contains two cobalt ions 2.9 A apart. These metal ions are liganded by the side chains of Asp 97, Asp 108, Glu 204, Glu 235, and His 171 with approximate octahedral coordination. In terms of both the novel backbone fold and the constitution of the active site, MAP appears to represent a new class of proteolytic enzyme.

Plantaricins S and T, Two New Bacteriocins Produced by Lactobacillus plantarum LPCO10 Isolated from a Green Olive Fermentation.

Abstract: Twenty-six strains of Lactobacillus plantarum isolated from green olive fermentations were tested for cross-antagonistic activities in an agar drop diffusion test. Cell-free supernatants from four of these strains were shown to inhibit the growth of at least one of the L. plantarum indicator strains. L. plantarum LPCO10 provided the broadest spectrum of activity and was selected for further studies. The inhibitory compound from this strain was active against some gram-positive bacteria, including clostridia and propionibacteria as well as natural competitors of L. plantarum in olive fermentation brines. In contrast, no activity against gram-negative bacteria was detected. Inhibition due to the effect of organic acids, hydrogen peroxide, or bacteriophages was excluded. Since the inhibitory activity of the active supernatant was lost after treatment with various proteolytic enzymes, this substance could be classified as a bacteriocin, designated plantaricin S. Plantaricin S was also sensitive to glycolytic and lipolytic enzymes, suggesting that it was a glycolipoprotein. It exhibited a bactericidal and nonbacteriolytic mode of action against indicator cells. This bacteriocin was heat stable (60 min at 100 degrees C), active in a pH range of 3.0 to 7.0, and also stable in crude culture supernatants during storage. Ultrafiltration studies indicated that plantaricin S occurred as multimolecular aggregates and that the size of the smallest active form is between 3 and 10 kDa. In sodium dodecyl sulfate-polyacrylamide gels, plantaricin S migrated as a peptide of ca. 2.5 kDa. Maximum production of plantaricin S was obtained in a fermentor system in unregulated pH and log-phase cultures of L. plantarum LPCO10 in MRS broth plus 4% NaCl. In these culture conditions, a second bacteriocin (designated plantaricin T) was produced in late-stationary-phase cultures of L. plantarum LPCO10. On the basis of its biological activity, its sensitivity to various enzymes, and its molecular weight (lower than that of plantaricin S) as assessed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, plantaricin T appeared different from plantaricin S. Curing experiments with L. plantarum LPCO10 resulted in the appearance of variants that no longer produced either of the two bacteriocins but that were still immune to both of them.

The Enzymatic Coagulation of Milk

Abstract: After milk has been treated with chymosin or other milk coagulating enzymes, there is little apparent reaction for some time, and then the milk coagulates rapidly. This phenomenon, which is the first step of cheesemaking, results from two processes, the first being the attack on κ-casein, which stabilizes the casein micelles, by the proteolytic enzymes (chymosin, pepsin or microbial proteinases) contained in the rennet, and the second being the subsequent clotting of the micelles which have been destabilized by this enzymatic attack. These processes have been described as the primary and secondary stages of the renneting reaction. This sequential description of the reaction is, however, somewhat oversimplified, since it is not necessary for the enzymatic reaction to be complete before aggregation of partly renneted micelles can occur. Indeed, the milk may begin to clot well before the enzymatic cleavage of the κ-casein is complete.1,2,3

Human immunodeficiency virus type 1 Vpu protein is an oligomeric type I integral membrane protein.

Abstract: The human immunodeficiency virus type 1 Vpu protein is a 16-kDa phosphoprotein which enhances the efficiency of virion production and induces rapid degradation of CD4, the cellular receptor for human immunodeficiency virus. The topology of membrane-inserted Vpu was investigated by using in vitro-synthesized Vpu cotranslationally inserted into canine microsomal membranes. Proteolytic digestion and immunoprecipitation studies revealed that Vpu was a type I integral membrane protein, with the hydrophilic domain projecting from the cytoplasmic membrane face. In addition, several high-molecular-weight proteins containing Vpu were identified by chemical cross-linking. Such complexes also formed when wild-type Vpu and a Tat-Vpu fusion protein were coexpressed. Subsequent analysis by one- and two-dimensional electrophoresis revealed that these high-molecular-weight complexes consisted of homo-oligomers of Vpu. These findings indicate that Vpu is a type I integral membrane protein capable of multimerization.

Fibroblast response to metallic debris in vitro. Enzyme induction cell proliferation, and toxicity.

Abstract: Bovine synovial fibroblasts in primary monolayer culture were exposed to particulate metallic debris. The effects of the metallic particles on the synthesis and secretion of proteolytic enzymes and on cell proliferation and viability were examined. Uniform suspensions of titanium, titanium-aluminum, cobalt, and chromium particles, ranging in size from approximately 0.1 to ten micrometers (average, one to three micrometers), were prepared; the particle concentrations (the volume of particles divided by the total volume of the suspension) ranged from 0.0005 to 5 per cent. Aliquots of the particle suspensions were added to the synovial fibroblast cultures. The final particle concentrations in the media ranged from 0.0000083 to 0.83 per cent. After seventy-two hours of exposure, each medium was harvested and was assayed for proteolytic and collagenolytic activity and for hexosaminidase levels. Neutral metalloproteases, quantified by collagenolytic and caseinolytic (proteolytic) activity, represent enzymes, secreted by cells, that are capable of degrading extracellular matrix. Hexosaminidase is a marker for lysosomal enzyme activity that can include more than thirty enzymes, such as proteases, lipases, nucleases, and phosphatases. Cell proliferation was quantified by uptake of 3H-thymidine. Cell morphology was examined by scanning electron microscopy. Titanium, titanium-aluminum, and chromium significantly stimulated 3H-thymidine uptake at low particle concentrations (p < 0.01, p < 0.002, and p < 0.002, respectively). Exposure to cobalt, even at the lowest particle concentration, resulted in a significant decrease in thymidine uptake (p = 0.027). At the highest particle concentrations, all particles were toxic, as evidenced by the absence of thymidine uptake. At high particle concentrations, all of the metals caused a decrease in caseinolytic (proteolytic) and collagenolytic activity in the culture media. Titanium elevated the lysosomal enzyme marker, hexosaminidase, except at high concentrations. Chromium and titanium-aluminum had no significant effect on hexosaminidase at any particle concentration, while cobalt decreased all enzyme markers at mid-particle to high-particle concentrations. Scanning electron microscopy demonstrated that the morphological response of fibroblasts to titanium included membrane-ruffling and extension of filopodia, typical of active fibroblasts. In contrast, exposure to cobalt at the same concentration resulted in cell crenation, indicative of cell death.

Inhibition of osteoclastic bone resorption in vivo by echistatin, an "arginyl-glycyl-aspartyl" (RGD)-containing protein.

Abstract: Osteoclastic bone resorption requires the formation of a tightly sealed compartment between the osteoclast and the mineralized bone matrix. This compartment functions as an extracellular "lysosome" which contains proteolytic enzymes and acids. Vitronectin receptors (VnR, integrin alpha v beta 3) displayed on the osteoclast cell surface may play a role in the attachment of osteoclasts to the resorption surface. VnR are known to bind to arginyl-glycyl-aspartyl (RGD)-containing matrix proteins and it has recently been reported that soluble peptides containing RGD sequences can block osteoclast attachment to bone and inhibit bone resorption in vitro. In this study echistatin, a naturally-occurring protein containing an RGD-sequence motif, was shown to completely inhibit osteoclast-mediated bone resorption in vivo. Echistatin or smaller derivative peptides may prove useful in the treatment of disorders characterized by excess bone resorption, such as osteoporosis and metastatic bone disease.

Mechanisms of rhabdomyolysis.

Abstract: Rhabdomyolysis is a common disorder that occurs as a primary disease or as a complication of a broad spectrum of other diseases. Although some cases are caused by hereditary metabolic or structural abnormalities of the skeletal muscle cell, the majority of cases occur in healthy persons as a result of exhaustive exercise, infections, intoxications, deficiency states, or trauma. Although the causes of rhabdomyolysis are diverse, current evidence suggests that there may be a common final pathway that mediates cellular injury. Thus some noxious factor, perhaps a drug that injures the plasma membrane of the cell, a toxin that activates a cytolytic enzyme, a factor that interferes with metabolism and disrupts the integrity of the skeletal muscle cell, a cytokine such as tumor necrosis factor, or simple hypoxia that reduces energy production by the cell, serves to increase cellular permeability to sodium ions. When sodium ions accumulate in the cytoplasm of the cell, an increase of cytosolic or mitochondrial calcium follows. Calcium activates a variety of proteolytic enzymes that injure the cell membrane, allowing efflux of cellular components into the circulation. The ability to identify some of these components, such as myoglobin or creatine kinase, facilitates clinical recognition of rhabdomyolysis. The cytosolic components released into the circulation, under appropriate conditions, may be life threatening, eg, release of potassium causes hyperkalemic cardiotoxicity. In this review, I attempt to describe a variety of factors that are known to be injurious to skeletal muscle cells and, when possible, describe the apparent mechanism whereby these factors result in injury and disruption of the muscle cell.

Oligomerization of a membrane protein correlates with its retention in the Golgi complex

Abstract: The first membrane-spanning domain (m1) of the M glycoprotein of avian coronavirus (formerly called E1) is sufficient to retain this protein in the cis-Golgi. When the membrane-spanning domain of a protein which is efficiently delivered to the plasma membrane (VSV G protein) is replaced with m1, the resulting chimera (Gm1) is retained in the Golgi (Swift, A. M., and C. E. Machamer. 1991. J. Cell Biol. 115:19-30). When assayed in sucrose gradients, we observed that Gm1 formed a large oligomer, and that much of this oligomer was SDS resistant and stayed near the top of the stacking gel of an SDS-polyacrylamide gel. The unusual stability of the oligomer allowed it to be detected easily. Gm1 mutants with single amino acid substitutions in the m1 domain that were retained in the Golgi complex formed SDS-resistant oligomers, whereas mutants that were rapidly released to the plasma membrane did not. Oligomerization was not detected immediately after synthesis of Gm1, but occurred gradually with a lag of approximately 10 min, suggesting that it is not merely aggregation of misfolded proteins. Furthermore, oligomerization did not occur under several conditions that block ER to Golgi transport. The lumenal domain was not required for oligomerization since another chimera (alpha m1G), where the lumenal domain of Gm1 was replaced by the alpha subunit of human chorionic gonadotropin, also formed an SDS-resistant oligomer, and was able to form hetero-oligomers with Gm1 as revealed by coprecipitation experiments. SDS resistance was conferred by the cytoplasmic tail of VSV G, because proteolytic digestion of the tail in microsomes containing Gm1 oligomers resulted in loss of SDS resistance, although the protease-treated material continued to migrate as a large oligomer on sucrose gradients. Interestingly, treatment of cells with cytochalasin D blocked formation of SDS-resistant (but not SDS-sensitive) oligomers. Our data suggest that SDS-resistant oligomers form as newly synthesized molecules of Gm1 arrive at the Golgi complex and may interact (directly or indirectly) with an actin-based cytoskeletal matrix. The oligomerization of Gm1 and other resident proteins could serve as a mechanism for their retention in the Golgi complex.

Myocardial oedema: a preventable cause of reperfusion injury?

Abstract: Myocardial ischaemia increases cellular and extracellular osmolarity, alters membrane permeability to ions and causes moderate cell swelling and interstitial oedema. Ischaemia also reduces the mechanical resistance of the sarcolemma of myocytes, probably as a result of proteolytic digestion of the connections between cell membrane and the cellular scaffold. During reperfusion, the abrupt normalisation of extracellular osmotic pressure results in marked osmotic cell swelling. In the clinical situation, the ability of mechanical stress imposed by cell swelling to disrupt the weakened sarcolemma of viable myocytes during reperfusion has not been definitively established. Observations demonstrating the important role of mechanical stress caused by contraction and cell to cell interaction in myocyte necrosis support the hypothesis that osmotic cell swelling may actually produce lethal reperfusion injury. This hypothesis has been investigated by analysing the effect of hyperosmotic reperfusion with mannitol on final infarct size after coronary occlusion. Studies using highly hyperosmotic reperfusion after relatively short periods of ischaemia have yielded positive results, while studies using intravenous mannitol at the time of reperfusion, and more closely resembling clinical situations, have failed to detect any beneficial effect. Myocardial oedema could also contribute to postischaemic functional derangements, such as reperfusion arrhythmias and stunning, and could modify the passive mechanical properties of the infarcts and alter ventricular remodelling. Interventions aimed to limit myocardial oedema will probably not play a role as a co-adjuvant therapy in patients with acute myocardial infarction receiving thrombolytic treatment. However, they should probably form part of controlled reperfusion strategies to be evaluated in patients with acute myocardial infarction in whom reperfusion is accomplished by percutaneous transcoronary angioplasty or surgery.

New perspectives on basic mechanisms in lung disease. 6. Proteinase imbalance: its role in lung disease.

Abstract: The hypothesis, some 30 years ago, that NE was the sole proteolytic agent responsible for the development of emphysema seems naive in retrospect. The availability of technology to measure NE facilitated the early research into the relationship between NE and lung disease. Despite an abundance of information on the activity of NE in the lung, it will probably require prospective studies in man with specific NE inhibitors or control at the gene level to establish a causal relationship between NE and lung disease. Parallel research has resulted in the isolation and characterisation of NE inhibitors other than PI and, indeed, alternative proteolytic enzymes that might contribute to lung disease. It is perhaps impossible now to think that a single proteinase, however omnipotent it may be, causes lung diseases as diverse as emphysema and fibrosis. An important aspect that is emerging is the interrelationship between proteolytic enzymes produced by different, or sometimes the same, cells that could potentiate tissue proteolysis. The evidence suggests that there is likely to be coordinated action between neutrophils, macrophages, and possibly mesenchymal proteinases which can activate or inactivate each other. In addition, one class of proteinases often appears able to proteolytically inactivate inhibitors of the opposite class, which presumably could amplify proteolysis if it occurred in vivo. Although the work on this aspect of proteinase activity is in its infancy, one suspects that part of the normal regulation of proteinase activity might include compartmentalisation. For example, the neutrophil stores proteinases before appropriate release and can inactivate PI to enable proteolytic action pericellularly, whereas degradation of extracellular matrix by macrophages requires interaction between the cell and matrix which is facilitated by cell receptor bound uPA. Disintegration of these "compartments" due to oedema, proteolysis, or for mechanical reasons could, firstly, expose further extracellular matrix substrates to inflammatory and damaged cell proteinases but, secondly, might enhance proteinase potential by the cooperative action of these enzymes. It seems increasingly likely that, where proteinases play a part, there is a cocktail of proteinases that is characteristic of the injury that develops (fig). What remains unclear is why only a proportion of those susceptible, such as smokers or those with acute lung injury, develop irreversible lung disease. This suggests that there are other factors acquired or inherited that need to be considered.

Purification, partial characterization and plasmid‐linkage of pediocin SJ‐1, a bacteriocin produced by Pediococcus acidilactici

Abstract: Pediococcus acidilactici SJ-1, isolated from a naturally-fermented meat product, produced an antibacterial agent active against selected strains of Lactobacillus spp., Clostridium perfringens and Listeria monocytogenes. The agent was bactericidal against sensitive indicators, and sensitive to proteolytic enzymes; it was identified as a bacteriocin, and was designated as pediocin SJ-1. It was stable over a wide pH range (3-9), and apparently most stable in the lower part of that range. At pH 3.6, pediocin SJ-1 was stable at heat-processing temperatures within the range 65-121 degrees C; its activity decreased significantly, however, when it was heated at pH 7.0. The activity of pediocin SJ-1 on sensitive indicator cells was lost in the presence of alpha-amylase, suggesting that it contains a glyco moiety, necessary for its antibacterial action. Native pediocin SJ-1 exists in the form of monomers and aggregates (with molecular weights in the range 80-150 kDa). Pediocin SJ-1 was purified 262-fold by direct application of cell-free supernatant fluids to a cation-exchange chromatography column, and was resolved by SDS-PAGE as a single peptide band with a MW of ca 4 kDa. The original pediocin SJ-1-producing strain (bac+) harbours three plasmids of 4.6, 23.5, and 45.7 MDa. Production of pediocin SJ-1, but not immunity to SJ-1, is associated with the 4.6 MDa plasmid.

Autoimmunity to cytoskeletal protein tropomyosin. A clue to the pathogenetic mechanism for ulcerative colitis.

Abstract: Autoimmunity has been emphasized in the pathogenesis of ulcerative colitis and various anticolon antibodies have been described. Nevertheless, the nature of the autoantigens has been elusive. Using ulcerative colitis colon-eluted IgG antibody, we earlier detected an M(r) 40,000 protein, P40, in colon extract. In our study, we purified P40 from human colon to apparent homogeneity, sequenced two peptides after proteolytic digestion, and demonstrated that the P40 belongs to the tropomyosin family. Furthermore, we demonstrated that blood serum from 95% of patients with ulcerative colitis contains antibodies that are reactive to tropomyosin.

Effect of Pseudomonas aeruginosa elastase, alkaline protease, and exotoxin A on corneal proteinases and proteins.

Abstract: Purpose To determine the effects of exoproducts from the corneal pathogen Pseudomonas aeruginosa on corneal proteinases and proteins. Methods Whole rabbit corneas were cultured in the presence or absence of broths conditioned with Pseudomonas aeruginosa, elastase, alkaline protease, and exotoxin A. Protein synthesis was assayed by adding 35S-methionine during the last 6 hours of culture. Caseinolytic assays and zymography on sodium dodecyl sulfate polyacrylamide gels containing casein and gelatin were used in the presence and absence of inhibitors to quantify and identify corneal proteinases. Results The major proteinases released by the corneas were 92/89 kD (MMP9) and 65 kD (72 kD gelatinase, MMP2) gelatinases and a 97 kD caseinase. Minor proteinases observed included 184, 166, 156, 153, 126, 111, 102, 60, 57, and 43 kD gelatinases and 170, 136, 85, and 54 kD caseinases. P. aeruginosa elastase at 1 microgram/ml cleaved the 92 kD gelatinase to yield a 77 kD active form and cleaved the 65 kD gelatinase to yield a 57 kD active form. At 25 micrograms/ml elastase, the gelatinases were degraded. P. aeruginosa alkaline protease had no effect on the 92 or 65 kD gelatinases. Both elastase and alkaline protease degraded the 97 kD caseinase. Proteinases other than elastase and alkaline protease in P. aeruginosa103- and P. aeruginosa01-conditioned broths also activated and/or degraded corneal proteinases. Exotoxin A inhibited the synthesis of the 92 kD gelatinase and most other proteins. The 72 kD gelatinase and the 97 kD caseinase were released in the presence of exotoxin A. Conclusions Pseudomonas aeruginosa exoproducts can contribute directly to keratitis caused by Pseudomonas organisms through toxic effects on corneal cells and degradation of corneal proteins and indirectly through the activation of corneal proteinases.

Myocardial matrix metalloproteinase(s): localization and activation.

Abstract: Matrix metalloproteinases (MMPs) and neutrophil elastate (NE) may each contribute to fibrillar collagen degradation in various disease states. Little, however, is known about the activation and localization of MMP in the heart. Accordingly, we extracted MMP and examined mechanisms of proMMP activation in whole tissue extracts of the adult rat myocardium. Incubation of extracts with serine proteases (i.e., trypsin or neutrophil elastase) at 37°C resulted in a time-dependent activation of proMMPs. Based on immunoblot and measurements of MMP activity by zymography, the molecular weight of active MMP was deduced to be 52 kDa. The second-order rate constant for activation of proMMP by serine protease was 5.5±0.2×105 M−1min−1 and for oxidized glutathione (GSSG) 1.5±0.1 M−1min−1. Incubation of the extract with both serine protease and GSSG increased the rate of activation 30-fold. Based on reverse zymographic analysis of collagenase inhibition, tissue inhibitors of metalloproteinases were identified. Indirect immunofluorescence localized proMMPs/MMPs to the endothelium and subendothelial space of the endocardium and throughout the interstitial space found between groups of muscle fibers. These results suggest that the mechanism of activation of MMPs by either a serine protease and by oxidizing, thiol-modifying reagents are mechanistically different and the presence of either a serine protease or GSSG synergistically increase the rate of activation of proMMPs. Our results also suggest that MMPs may be regulated by its own endogenous inhibitors. The contribution of this proteolytic enzyme to tissue remodeling and wound healing responses that occur in various diseases states remains to be established.