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J Potempa

Publications and source records attributed to J Potempa.

At least 19 recordsLinked to original sources
PubMed1992

Purification and characterization of a 50-kDa cysteine proteinase (gingipain) from Porphyromonas gingivalis.

Porphyromonas gingivalis, a Gram-negative anaerobic rod, has been closely associated with the initiation and progression of periodontal disease. This organism has been shown to produce a large number of proteolytic enzymes which can degrade a variety of tissue proteins, and these are considered to be major virulence factors. One of the proteinases produced by this organism, referred to as gingipain-1, has been purified to homogeneity from P. gingivalis culture medium by a combination of gel filtration and ion-exchange chromatography. The enzyme was found to have a molecular mass near 50 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a pH optimum in the neutral to alkaline range, and a requirement for cysteine for activation and Ca2+ for stabilization. Amino-terminal sequence analysis indicated that gingipain belongs to a new, so far unknown, subfamily of cysteine proteinases. Three unusual features of this proteinase are: (a) the stimulation of amidolytic activity by glycine-containing dipeptides; (b) a narrow specificity which is limited to peptide bonds containing arginine residues; and (c) resistance to inhibition by proteinase inhibitors in human plasma.

Adhesins, Bacterial↗
PubMed1992

Activation of complement components C3 and C5 by a cysteine proteinase (gingipain-1) from Porphyromonas (Bacteroides) gingivalis.

Complement components C3 and C5 are susceptible to limited proteolysis by an arginine-specific cysteine proteinase isolated from Porphyromonas gingivalis. This bacterium is an anaerobe commonly associated with severe periodontal disease. Infection by P. gingivalis is accompanied by an acute inflammatory response, complete with extensive neutrophil involvement. This prompted us to investigate a possible direct role for complement in periodontitis evoked by P. gingivalis. Exposure of C3 and C5 to the cysteine proteinase at molar ratios between 1:25 and 1:100 (enzyme to substrate ratios) resulted in a time-dependent, limited degradation of each component. C3 was converted in a stepwise manner to C3a-like and C3b-like fragments with evidence of extensive further degradation of the C3a-like portion of the molecule. We were unable to demonstrate C3a activity in the C3 digestion mixtures. C3 degradation appears to involve primarily the alpha-chain. Proteolysis of C5 also progresses in a stepwise manner producing an initial internal cleavage of the alpha-chain to generate 30- and 86-kDa fragments. Further digestion of the 86-kDa amino-terminal fragment of the alpha-chain leads to the release of C5a or a C5a-like fragment that is biologically active for neutrophil activation. The fact that a potent chemotactic factor, i.e. C5a, can be generated from C5 by a proteinase derived from P. gingivalis suggests a recruiting mechanism for attracting neutrophils to the gingival lesion site in periodontal disease.

Adhesins, Bacterial↗
PubMed1992

Crystal structure of cleaved equine leucocyte elastase inhibitor determined at 1.95 A resolution.

The crystal structure of active-site cleaved equine leucocyte elastase inhibitor, a member of the serpin superfamily, has been solved and refined to a crystallographic R-factor of 17.6% at 1.95 A resolution. Despite being an intracellular inhibitor with rather low sequence homology of 30% to human alpha 1-antichymotrypsin and alpha 1-proteinase inhibitor, the three-dimensional structures are very similar, with deviations only at the sites of insertions and few mobile secondary structure elements. The better resolution in comparison with the structures of other cleaved serpins allows a more precise description of the so-called R-state of the serpins.

Amino Acid Sequence↗
PubMed1992

Equine leukocyte elastase inhibitor. Primary structure and identification as a thymosin-binding protein.

The primary structure of an elastase inhibitor located in the cytoplasm of leukocytes obtained from the equine species has been determined. By sequence comparison, the protein was found to be a member of the serpin family with strong identity to plasminogen activator inhibitor-2. In contrast to other serpins this protein contained no carbohydrate and had a blocked amino terminus. Preliminary evidence indicates that the inhibitor has the additional feature of being a thymosin beta 4-binding protein, since this polypeptide was always located in purified preparations of the protein. This suggests a physiological role for cytoplasmic elastase inhibitors in the thymosin beta 4-regulated rearrangement of the cytoskeleton of leukocytes.

Amino Acid Sequence↗
PubMed1992

Cleavage of human immunoglobulins by serine proteinase from Staphylococcus aureus.

The serine proteinase (SP) released into the environment by most strains of S. aureus cleaves human IgG, IgM and IgA of both subclasses--IgA 1 and IgA 2. SP cleaves H chains of all immunoglobulin classes and the SC of S-IgA, the L chains are degraded partially. The SP-induced cleavage results in a large spectrum of fragments under reducing conditions within a broad range of Mr (approx. 41,000 to less than 12,400). This indicates that the enzyme does not affect the Ig molecule in the hinge region only. The degree of cleavage depends on the enzyme:substrate ratio and on the duration of incubation. The generation of small fragments is associated with the loss of antigenic determinants that results from the decreased binding of the cleaved material in the ELISA method. Partial cleavage of L chains suggests that the enzyme alters part of the molecule that is involved in antigen binding. Even if the ability of antigen binding remains preserved after cleaving Ig with SP, the antibody function is disturbed by splitting off the Fc region or by its degradation into small fragments. SP has to be considered as one of the virulence factors of S. aureus that may protect bacteria against the defence mechanisms of the host.

Electrophoresis, Polyacrylamide Gel↗
PubMed1991

Proteolytic inactivation of alpha-1-anti-chymotrypsin. Sites of cleavage and generation of chemotactic activity.

The effect of several microbial and mammalian proteinases on the inhibitory activity of human plasma alpha-1-anti-chymotrypsin (alpha-1-Achy) has been tested. Most of these enzymes caused rapid inactivation of the inhibitor by cleavage at single sites within the reactive-site loop between P5 Lys and P3' Leu, with additional cleavages also being detected in some cases near the NH2 terminus of the native protein. In contrast, two of the enzymes tested failed to inactivate alpha-1-Achy, although they could cause removal of peptides near the NH2 terminus. Studies of neutrophil chemotaxis revealed that native or NH2-terminally truncated alpha-1-Achy was not stimulatory. However, testing of two enzymatically inactivated forms of the inhibitor (alpha-1-Achy), cleaved at widely different positions within the reactive-site loop, indicated that they had become potent chemoattractants at concentrations within the nanomolar range. Competition studies using proteolytically inactivated alpha-1-proteinase inhibitor suggested that the chemotactic activity of the two inactivated serpins was probably mediated by the same mechanism. The physiological relevance of this chemotactic activity is underscored by the results of plasma elimination studies which indicate that alpha-1-Achy is eliminated at approximately the same rate as native alpha-1-Achy, thus prolonging chemotactic stimuli within the tissues.

Amino Acid Sequence↗
PubMed1991

The fluorescence quenching resolved spectra and red-edge excitation fluorescence measurements of human alpha 1-proteinase inhibitor.

The human alpha 1-proteinase inhibitor (alpha 1-PI) and its reactive site modified form (alpha 1-PI*) have been examined using the fluorescence quenching resolved spectra method. The red-edge excitation measurements were applied for the study of structural differences between these forms. The crystallographic data of alpha 1-PI* structure have shown that its polypeptide chain includes only two tryptophan residues. The fluorescence quenching data have indicated that the conversion of the intact inhibitor molecule into its nicked form is accompanied by changes in the tryptophan environments. The red-edge excitation measurements have proved that the dipolar relaxation process around the Trp-194 residue is much bigger in alpha 1-PI* form than in the nicked one.

Fluorescence↗
PubMed1991

Comparative properties of three functionally different but structurally related serpin variants from horse plasma.

Three structurally related but functionally different serpins from horse plasma were isolated and characterized. In spite of their identical N-terminal sequences, which show some similarity to that of human alpha 1-proteinase inhibitor, the reactive-centre loops of each of these proteins show extensive variation. Only inhibitor I, with a P1 methionine residue, resembles human alpha 1-PI with regard to (a) similarity of amino acid sequence in the vicinity of the reactive-site peptide bond, (b) broad inhibitory specificity, (c) sensitivity to oxidative inactivation and (d) high rate of reactivity with neutrophil elastase(s). Inhibitor II, with a P1 arginine residue, is an exclusive trypsin inhibitor, and inhibitor III is an oxidation-resistant slow-reacting elastase inhibitor with a P1 alanine residue. Comparison of association rate constants for the inhibition of horse neutrophil elastases by the three inhibitors indicates that only inhibitor I is likely to be physiologically important in the regulation of these enzymes.

Amino Acid Sequence↗
PubMed1991

Effect of metalloproteinase from Staphylococcus aureus on in vitro stimulation of human lymphocytes.

Metalloproteinase (MP) produced by the majority of Staphylococcus aureus strains exerts, in a wide concentration range (0.1-100 micrograms/ml), no cytotoxic action on mononuclear leukocytes of human peripheral blood. The enzyme itself does not appreciably stimulate proliferation and differentiation of lymphocytes in culture, but affects the stimulation of both T and B lymphocytes by polyclonal activators. The action is dose-dependent. High doses of MP (100 micrograms/ml) lower the blastic transformation after stimulation with Con A, SpA, NDCM, S. aureus strain Wood 46 and with suboptimal doses of PWM. Optimal concentrations of the enzyme potentiate the stimulation of lymphocytes by PWM, PHA, S. aureus strains Cowan 1 and Wood 46, and by NDCM. The same potentiation effect was achieved whether the enzyme was added concurrently with the mitogen or 18 h later. This implies that the beginning of cell activation is not affected. A high MP concentration decreases the production of Ig in culture after stimulation with PWM whereas lower concentrations of MP enhance this production. Production of Ig after stimulation with NDCM and Wood 46 is decreased by MP. The possible action of exoproteinase from S. Aureus on the immune response during infection is discussed.

Cells, Cultured↗
PubMed1991

Antithrombin III activity in horses with colic: an analysis of 46 cases.

Antithrombin-III (AT-III) activity was measured at the time of admission in 46 horses referred with colic. Differences in AT-III activities between animals treated medically or surgically and survivors or non-survivors were compared. The mean AT-III values for the horses treated medically (76.2 per cent), surgical survivors (69.5 per cent) and surgical non-survivors (55.9 per cent) were significantly different from the reference value for healthy adults (92 to 108 per cent). The mean AT-III activity of the survivors was significantly (P less than 0.01) greater than that of the nonsurvivors. The majority of the survivors (11 to 13 horses) had AT-III activities exceeding 60 per cent of normal, whereas that of 10 of the 14 non-survivors was less than 60 per cent of normal. There was an apparent distinction between the survivors and non-survivors at approximately 60 per cent of normal AT-III activity.

Animals↗
PubMed1991

Stimulation of proteinase and amidase activities in Porphyromonas (Bacteroides) gingivalis by amino acids and dipeptides.

Proteolytic enzymes from the organism Porphyromonas gingivalis are believed to be involved in the development of periodontitis. Studies on both crude extracts and purified trypsinlike enzymes from this organism indicate that substantial stimulation of both amidase and proteinase activities can be obtained during incubation with glycine-containing compounds. We postulate that P. gingivalis may have developed this unusual property to take advantage of the glycine-rich environment which occurs during the periodontitis-associated degradation of gingival collagen. The finding of such a stimulation in crevicular fluids from discrete periodontal sites has been correlated with the presence of P. gingivalis and could be utilized for the early detection of infection by this organism during the onset of periodontitis.

Amidohydrolases↗
PubMed1991

Elastase inhibitor from mammalian neutrophil cytosol.

Bovine, pig and horse leukocyte elastase inhibitors represent the first example of functionally active intracellular serpin. Sequence analysis yields data indicating that the reactive site encompasses P1-P'1 Ala-Met sequence. Oxidation of swine (as well as equine) inhibitor reduced its association rate constants kon with the tested enzymes (by at least 1 order of magnitude). However in case of bovine inhibitor, only a slight reduction has been observed for human neutrophil elastase but not for chymotrypsin or pancreatic elastase.

Amino Acid Sequence↗
PubMed1990

Comparative properties of human alpha-1-proteinase inhibitor glycosylation variants.

Variant forms of human alpha-1-proteinase inhibitor (alpha-1-PI), obtained by the treatment of human Hep G2 cells with specific inhibitors of glycosylation were tested for both inhibitory activity and heat stability. All were found to have the same second-order association rate with human neutrophil elastase, indicating a lack of importance of the carbohydrate moiety. In contrast, incompletely glycosylated forms of alpha-1-PI were found to be heat sensitive relative to the mature protein, suggesting a role for carbohydrate in protein stabilization.

1-Deoxynojirimycin↗
PubMed1990

Isolation of nine human plasma proteinase inhibitors by sequential affinity chromatography.

Purification of nine plasma proteinase inhibitors and one zymogen from a single batch of human plasma, using affinity chromatography has been accomplished. Those isolated were plasminogen (lysine-Sepharose), alpha-2-antiplasmin (plasminogen-Sepharose), high and low molecular weight kininogens (CM-papain-Sepharose), alpha-2-macroglobulin (Zn++ chelate-Sepharose), alpha-1-proteinase inhibitor, alpha-1-antichymotrypsin, Cl-inhibitor, inter-alpha-trypsin inhibitor (Blue-Sepharose) and antithrombin III (heparin-Sepharose). Alpha-2-macroglobulin and alpha-1-proteinase inhibitor required gel filtration as additional purification steps. Each protein was recovered in both high yield and purity.

Alpha-Globulins↗
PubMed1990

Serpins: structure and mechanism of action.

The Serpins are a major family of proteins, most of which are involved in the regulation of proteinase activity. Current data indicate that inhibitor function is dependent on formation of tight, but reversible binary complexes, with carbohydrate being unimportant for this function. The reaction takes place in a reactive site loop common to all Serpins, with the key residue for complex formation being in the P1 reactive site position. However, other contact residues are also involved as shown by the variation in specificity in a number of animal proteins with the same P1 residue. The role of other amino acid residues in Serpins which aid in conferring specificity has not yet been established. However, the availability of methods for obtaining site specific mutations should soon make it possible to determine other contact points required for Serpin function, thus allowing for the design of inhibitors which are singularly targeted with a high reaction rate towards a given proteinase.

Animals↗
PubMed1989

Stabilization vs. degradation of Staphylococcus aureus metalloproteinase.

Purified Staphylococcus aureus metalloproteinase contains trace amounts of a serine proteinase which rapidly degrades the metalloproteinase when EDTA is present. However, no degradation occurs when Ca2+ is added or if the serine proteinase is removed by immunoaffinity chromatography. Selective chelation of Zn2+ by o-phenanthroline, which reversibly inactivates the metalloproteinase, does not result in the degradation of the apometalloproteinase, even with excess of serine proteinase. These data are interpreted as follows: EDTA chelates enzyme-bound Ca2+ and Zn2+, causing irreversible inactivation as well as a conformational change in the metal-free protein. This allows proteolysis by the contaminating serine proteinase and explains why the metalloproteinase purified from serine proteinase-deficient strains of S. aureus was previously thought to be stable to autolysis.

Calcium↗
PubMed1989

Inter-alpha-trypsin inhibitor. Inhibition spectrum of native and derived forms.

The conversion of inter-alpha-trypsin inhibitor (I alpha I) into active, acid-stable derivatives by proteolytic degradation has been tested with 10 different proteinases. Of these, only plasma kallikrein, cathepsin G, neutrophil elastase, and the Staphylococcus aureus V-8 proteinase were found to be effective, each releasing more than 50% of this activity. However, a strong correlation between inhibitor degradation and significant release of acid-stable activity could only be found with the V-8 enzyme. Inhibition kinetics for the interaction of native I alpha I, the inhibitory fragment released by digestion with S. aureus V-8 proteinase, or the related urinary trypsin inhibitor, with seven different proteinases indicated that all had essentially identical Ki values with an individual enzyme and, where measurements were possible, nearly identical second order association rate constants. Significantly, none of the five human proteinases tested, including trypsin, chymotrypsin, plasmin, neutrophil elastase, and cathepsin G, would appear to have low enough Ki values to be physiologically relevant. Thus, the role of native I alpha I or its degradation products in controlling a specific proteolytic activity is still unknown.

Alpha-Globulins↗