Low Molecular Weight Protease Inhibitor Research Articles

Proteinase Inhibitors Block Formation of Pemphigus Acantholysis in Experimental Models of Neonatal Mice and Skin Explants: Effects of Synthetic and Plasma Proteinase Inhibitors on Pemphigus Acantholysis

In organ culture experiments, the induction of pemphigus acantholysis is known to be blocked by the addition of serine proteinase inhibitors. Recently, nontoxic synthesized low molecular weight proteinase inhibitors have been clinically available for the treatment of disseminated intravascular coagulation and pancreatitis. To determine if these drugs are useful aids to treat patients with pemphigus, we examined the effect of omega-guanidino ester analogues, i.e., 1) gabexate mesilate, 2) camostat mesilate, and 3) nafamostat mesilate, on experimental pemphigus acantholysis in both organ culture and neonatal BALB/c mice. Furthermore, the effect of plasma natural proteinase inhibitors (alpha-1-proteinase inhibitor) isolated from human plasma was similarly examined. Results revealed that synthesized low molecular weight inhibitors (drugs) were able to inhibit the induction of acantholysis in organ culture system, but had little or no effect on lesion formation in the neonatal mouse system. By contrast, alpha-1-proteinase inhibitor could completely inhibit acantholysis formation in mice. These findings implied a possible new therapeutic approach using proteinase inhibitors for patients with pemphigus.

Evidence for the role of proteinases in uremic catabolism.

Enhanced muscle protein breakdown has been demonstrated in acutely uremic rats by numerous authors. In order to investigate the pathogenetic role of skeletal muscle proteinases leupeptin, a low-molecular weight proteinase inhibitor, was administered intraperitoneally to acutely uremic rats. Twenty-four hours after bilateral nephrectomy, leupeptin-treated animals displayed significantly lowered serum urea levels (-32%), as compared to untreated uremic rats. As a sign of muscle protein breakdown, plasma levels of Nt-methylhistidine, an indicator of myofibrillar protein degradation, were also decreased (-35%) in the uremic animals treated with leupeptin as compared to untreated uremic rats. Finally, leupeptin treatment resulted in a significant inhibition of the myofibrillar alkaline proteinase activity, a proteinase which has been related to various catabolic conditions. These findings suggest that the increased muscle protein breakdown in uremia is caused by enhanced activity of muscular proteinases and that anti-proteolytic agents display favourable effects on the enhanced protein degradation observed in acute uremia.

Elimination of the low-molecular weight proteinase inhibitor camostate (FOY 305) and its degradation products by the rat liver.

The elimination of the low molecular weight proteinase inhibitor camostate (FOY 305) was studied in rats after oral administration and in the the situ perfused rat liver. After feeding of camostate (400 mg/kg b.w.) only the metabolites (FOY 251, GBA) were detected in blood samples withdrawn from the portal and hepatic vein. This indicated a rapid degradation of FOY 305 after absorption from the gut lumen. The hepatic extraction of the anti-proteolytic active metabolite FOY 251 during a single liver passage was 23%. It remained almost constant over the period of 120 min. In the perfused rat liver, FOY 305 was given in concentrations comparable to the in vivo studies. It was eliminated by 20%. In these experiments, the compound was metabolized to FOY 251 and in minor amounts to guanidino-benzoate (GBA), the latter being an anti-proteolytic ineffective degradation product. In conclusion, a low hepatic extraction of FOY 305 led to pharmacologically effective concentrations of the active metabolite FOY 251 in the circulation after oral ingestion of the proteinase inhibitor.

Stimulation of pancreatic secretory process in the rat by low-molecular weight proteinase inhibitor

Oral application of a single dose of a new synthetic proteinase inhibitor Camostate (Foy-305) in male Wistar rats was carried out together with studies of in vitro amino acid incorporation followed by separation of proteins by two-dimensional gel electrophoresis. The aim of this experiment was to analyze changes produced by the inhibitor in total protein and individual enzyme biosynthesis. Administration of 100 mg/kg Foy-305 resulted in significant inhibition of total pancreatic protein synthesis, without changes in fractional rates for individual enzymes. 50 mg/kg Foy-305 induced a 10-fold elevation of cholecystokinin (CCK) levels in serum; this persisted for 3 h and led to a significant increase in the total rate of protein synthesis with peak values at 6 and 9 h (78% and 84% above control levels, respectively), returning to control by 15 h. Changes in fractional rates of synthesis occurred with a latency of 6 h and were restricted to amylase and the anionic form of trypsinogen and chymotrypsinogen. Amylase biosynthesis decreased by about 40% from control levels at 9 h to return to control levels by 15 h. Increased synthesis of trypsinogen and chymotrypsinogen was observed; this was also phasic. The results show similar enzyme-specific regulation as previously described for exogenous CCK stimulation and for the adaptation of the pancreas to diets enriched in protein. They demonstrate the effectiveness of pulsatory endogenous hormone release in the regulation of protein synthesis.

Semiquantitative determination of low molecular weight proteinase inhibitors in the presence of high molecular weight inhibitors by a modified radial diffusion assay

The radial diffusion assay is very suitable for the determination of proteinase inhibitors in biological fluids. By combining radial diffusion and ultrafiltration, it has become possible to directly determine low molecular weight proteinase inhibitors in mixtures with inhibitors of higher molecular weight. By this modification the inhibitor solutions to be investigated are not pipetted into wells as usually, but are applied on small pieces of dialysis membranes lying on the gel. The exclusion limit of the membrane must be of a magnitude that the inhibitors of higher molecular weight are retained, whereas the inhibitors of lower molecular weight can diffuse into the gel. The modified method can be used for the direct determination of e.g. aprotinin ( M r 6500) in the presence of α 1-proteinase inhibitor ( M r 54 000), ovoinhibitor ( M r 50 000) and ovomucoid ( M r 27 000), respectively. The modified method is suitable for the direct determination of low molecular weight inhibitors of trypsin and papain in serum, synovial fluid and saliva. Tissue extracts containing 4 M guanidine hydrochloride or 6 M urea can be investigated directly, too.

Stimulation of pancreatic secretory process in the rat by low-molecular weight proteinase inhibitor

Application of a single dose of a new type of proteinase inhibitor camostate (FOY-305) via orogastric tube was used in rats to study the dose-response relationship of resulting pancreatic stimulation. Doses up to 10 mg/kg failed to elicit any response, while significant decrease in enzyme content and increase in serum CCK-levels were observed with doses ranging from 25 to 400 mg/kg. A single dose of 100 mg/kg was selected for a time-sequence analysis, which revealed a 60 to 70% depletion of enzyme stores persisting over 6 h and reverting to control levels by 12 h. Peak increases in serum CCK-levels (15-fold above the elevation observed after regular food intake) were found after 30 min and persisted as an 8- to 10-fold elevation for at least 3 h, then declined to control levels by 9 h. This prolonged endogenous hormone release and resulting pancreatic stimulation were also verified in a separate group of animals in which volume, protein, and enzyme output were measured after cannulation of the pancreatic duct. While volume secretion was not altered by feeding a single dose of 100 mg/kg FOY-305, protein and enzyme output increased 2- to 3-fold over a period of 7 h. Fine-structural analysis of the pancreas demonstrated efficient depletion of zymogen granules from acinar cells with all doses between 50 and 400 mg/kg, accompanied by the appearance of membrane material in the acinar lumina at 3 and 6 h. The same transient increase in the number of lysosomal bodies predominantly containing mitochondria with all doses above 50 mg/kg was interpreted as increased organelle turnover due to persisting hormonal stimulation.

Proteases and their inhibitors in chronic inflammatory periodontal disease

This article reviews the current knowledge of the sources, function and interactions of proteolytic enzymes and their inhibitors in chronic inflammatory periodontal disease. Proteolytic tissue degradation is a typical phenomenon in chronic inflammatory periodontal disease. The proteolytic enzymes can be both host- and bacteria-derived. The proteases of the inflammatory cells are aimed for digestion of bacteria, enhanced locomotion through connective tissue, demarcation of the site of infection and tissue remodeling. Uncontrolled release of proteases in inflammation causes self-digestion and tissue destruction. The potential of the bacterial proteases in degradation of connective tissue is not yet known. Biochemical and immunologic mediators of inflammation are released by proteolytic reactions. Immunoglobulin-cleaving proteases present a specific mechanism in perturbation of host defenses. The 2 main protease inhibitors in serum, alpha-1-antitrypsin and alpha-2-macroglobulin, are also present in the gingival tissue fluid guarding the function of proteases. It has been suggested, although not confirmed, that deficiency in serum protease inhibiting capacity could be correlated with susceptibility to periodontal disease. Mucous secretions contain local low molecular weight protease inhibitors, but their possible rôle in saliva is not known. Bacteria-derived, antiproteolytic short peptides may prove to be useful in pharmacological control of tissue destruction at inflammatory sites.

The N-terminal sequence of antileukoprotease isolated from bronchial secretion

Antileukoprotease (ALP) was isolated from bronchial secretion. The amino acid composition as well as the N-terminal sequence were determined. No homology with other low molecular weight proteinase inhibitors was found.

Role of intramuscular enzymatic changes in the development of muscular weakness in rats with experimental allergic neuritis

We investigated the role of intramuscular enzymatic changes in the development of muscular weakness in rats suffering from experimental allergic neuritis. At an initial stage without apparent clinical symptoms, enzymatic changes of similar types occurred in the muscles of the forelimbs and hind limbs. At a later stage when the weakness appeared in the hind limb but not in the forelimb, dissociation of the pattern of the enzymatic changes occurred between the two limbs. Comparison of the intramuscular enzymatic changes between the two stages and between the two limbs suggested that the increased activities of aminopeptidases and endopeptidases play some important roles in the development of muscular weakness in this experimental model. Low molecular weight protease inhibitors may thus be worthy of a trial in this disease condition.

Effects of thiol protease inhibitors on intracellular degradation of exogenous beta-galactosidase in cultured human skin fibroblasts.

The effects of low molecular weight (LMW) protease inhibitors of microbial origin were evaluated on the intracellular degradation of beta-galactosidase purified from Aspergillus oryzae and taken up by cultured human skin fibroblasts with beta-galactosidase deficiency. Only thiol protease inhibitors showed an effect to increase the enzyme activity. E-64, a specific inhibitor of thiol proteases, prolonged 3-fold a half life of the exogenous beta-galactosidase and when the enzyme was supplied as liposomes, the half life was prolonged 9-fold in these cells. The role of thiol proteases in the degradation of enzyme molecules was discussed.

Variation in the Low Molecular Weight Proteinase Inhibitors of Soybeans1

Two types of proteinase inhibitors exist in soybean [Glycine max (L.) Merr.] seed. The first type is called the Kunitz trypsin inhibitor and the second is a group generally termed the Bowman‐Birk type inhibitors. The objectives of this study were to attempt to separate and purify the Bowman‐Birk type inhibitors in soybean seeds, and to screen for variants in the USDA Named Cultivar and Type Collections. Five Bowman‐Birk type inhibitors were separated from ‘Amsoy 71’ by anion exchange column chromatography. Soybean accessions were screened electrophoretically for variants of the inhibitors. Three variants were identified: 1) lines which appear to be missing fraction III electrophoretic band (F phenotype); 2) lines which appear to be missing fraction V electrophoretic band (S phenotype); and 3) lines in which both electrophoretic bands are present (SF phenotype). Most lines examined (71%) have the SF banding pattern. The S or F banding patterns are less common with only 26 and 3%, respectively, of the lines screened.

Ultrastructural localization of the low molecular weight protease inhibitor in human bronchial glands.

Light microscopically the low molecular weight protease inhibitor (LMI) and lysozyme have both been demonstrated in the cytoplasm of serous cells of bronchial glands. By immunoelectron microscopy LMI was demonstrated in secretory granules of these serous cells. Many granules showed a peripheral or bean-shaped staining pattern, other granules were uniformly stained or not stained at all. In contrast to this latter finding, virtually all granules were found positive for the presence of lysozyme, suggesting that there is no association of LMI with lysozyme at the ultrastructural level.

Purification and properties of a progesterone-induced plasmin/trypsin inhibitor from uterine secretions of pigs and its immunocytochemical localization in the pregnant uterus.

The porcine uterus secretes a group of basic, low molecular weight protease inhibitors under the influence of progesterone, but not estrogen. One of these inhibitors (Mr approximately 14,500) which inhibits trypsin, plasmin, and chymotrypsin, but not other proteases tested, has been purified 10- to 15-fold from uterine secretions of pseudopregnant pigs using Sephadex G-100 chromatography, CM-cellulose ion exchange chromatography, and Sephadex G-50 or Bio-Gel P-10 chromatography. The inhibitor which is relatively heat- and pH-stable forms a 1:1 molar complex with trypsin which is not dissociated in sodium dodecyl sulfate except by boiling. Chymotrypsin appears to bind at the same site on the inhibitor as trypsin. The inhibitor is high in half-cysteine residues and basic amino acids, and appears not to be a glycoprotein. Antiserum has been raised against the purified inhibitor in rabbits and used to test its distribution in pigs using the immunoperoxidase-staining technique on tissue sections. The inhibitor is associated only with the glandular and surface epithelium of the uterus. Endometrial explants from pseudopregnant animals, cultured in presence of L-[3H]leucine, release the inhibitor in radioactive form indicating that it is a uterine product. The antiserum against the inhibitor cross-reacts with at least three other, basic, low molecular weights plasmin/trypsin inhibitors in porcine uterine secretions, suggesting that a family of isoinhibitors exists which may constitute up to 15% of the protein in porcine uterine secretions. The inhibitor(s) appears to coat and to be taken up by the trophoectoderm cells of the elongating blastocyst during pregnancy. It is suggested that the inhibitors may serve to protect the uterus from proteases released by the porcine trophoblast or to prevent degradation of essential macromolecules, such as uteroferrin, which have to be taken up by the conceptus.

Localization of low molecular weight protease inhibitor in serous secretory cells of the respiratory tract.

We prepared in rabbits an antiserum against low molecular weight protease inhibitor (LMI) purified from the sputum of patients with purulent bronchitis. Using this antiserum in an immunoperoxidase staining method we found that this inhibitor was located exclusively in the serous cells of the submucosal glands of human upper and lower airways. The inhibitor was localized also in serous cells of the sublingual and submandibular glands. In contrast, LMI could not be demonstrated in the serous cells of the parotid gland. In the tissues investigated a strong association between the localization of the protease inhibitor and lysozyme was observed. Our observations indicate that the inhibitor may be present together with lysozyme as a secretory product in the serous cell granules. The possible consequences of the coexistence of these two proteins in the defense mechanism of the respiratory tract is discussed.

Human seminal plasma inhibition of antibody complement-mediated killing and opsonization of Neisseria gonorrhoeae and other gram-negative organisms.

Seminal plasma diluted 1:5-1:1,000 gave marked inhibition of serum antibody complement-mediated bactericidal and opsonic effects against Neisseria gonorrhoeae and other gram-negative organisms. Serum that was bactericidal at a dilution of 1:5,120 was not bactericidal at a dilution of 1:10 when seminal plasma was added. Bactericidal action of immune human or rabbit sera, or purified immunoglobulin (Ig)G or IgM plus complement for six strains of N. gonorrhoeae, serogroups A, B, C, and Y of Neisseria meningitidis, Escherichia coli and other gram-negative rods was inhibited by seminal plasma. Using C8- or C7-deficient sera as antibody and complement sources, opsonization, phagocytosis, and killing of N. gonorrhoeae and E. coli 014-K7 were inhibited by seminal plasma. Opsonization, phagocytosis, and killing of Staphylococcus aureus 502A was not inhibited. For the gram-negative organisms, the early phase of the opsonization process, probably complement activation, appeared to be inhibited rather than the ingestion or polymorphonuclear leukocyte killing steps; addition of seminal plasma yielded a significant reduction in the percentage of polymorphonuclear cells with associated bacteria. Seminal plasma did not prevent attachment of IgG, IgM, or IgA antibodies to gonococci. It reduced serum hemolytic whole complement activity by 25%. The seminal plasma inhibitor was of low molecular weight and was stable at 56 degrees C for 30 min, but inhibitory activity was lost after heating to 100 degrees C for 10 min. It is likely that the inhibitory factor(s) is a low-molecular weight protease or protease inhibitor. Seminal plasma probably has an important role in inhibition of complement and antibody functions in the genital tract. It may enhance pathogenesis of agents of sexually transmitted diseases.

Localization and quantitation of a low molecular weight proteinase inhibitor, antileukoprotease, in the human uterus.

Antileukoprotease was demonstrated in the cervical mucosa, but was absent in the endometrium. It was shown immunohistologically to predominate in the epithelial lining of the cervical crypts. High concentrations were found in the cervical mucus. Although antileukoprotease was not present in the endometrium, low concentrations were found in the uterine fluid, probably secondary to diffusion from the cervical mucus. The concentration of antileukoprotease in cervical mucus varied greatly during the menstrual cycle, but no midcycle minimum was found. Increased concentrations were found in samples obtained during the week preceding onset of the menstruation in most women. Antileukoprotease was present in uterine fluid from intra uterine device (IUD) non-users in its free form. In fluid from IUD users it was partly in its free form, and partly in a complexed form. The major part of the non-complexed portion was biologically active. The complex consisted, at least partly, of elastase bound to antileukoprotease. Antileukoprotease may thus contribute to the neutralization of granulocyte elastase in the uterine fluid of IUD users.

Protease inhibitors in middle ear effusions.

As the presence of granulocytes in middle ear fluid in serous otitis media implicates a potential risk of extracellular release of granulocyte proteases, the main protease inhibitors in serum, alpha 1-antitrypsin, alpha 1-antichymotrypsin and alpha 2-macroglobulin, were immunochemically quantitated in such effusions. The mean concentration of alpha 1-antitrypsin was found to be higher in middle ear effusion and the mean concentration of alpha 1-antichymotrypsin slightly lower than in plasma. On comparison with albumin, however, lower values than expected were found in the middle ear fluid for the two inhibitors. The high molecular weight alpha 2-macroglobulin was significantly lower than in plasma. Furthermore a low molecular weight protease inhibitor, which is thought to be locally produced in the respiratory epithelium, was demonstrated in the effusion.

Heat-induced fragmentation of human alpha 2-macroglobulin.

Previous studies have demonstrated that human plasma alpha 2-macroglobulin (alpha 2 M) possesses a single subunit chain (Mr approximately 185,000) when incubated with dodecyl sulfate and dithiothreitol at 37 degrees C and analyzed by dodecyl sulfate-gel electrophoresis. The present study details the observation that heating alpha 2 M to 90 degrees C under identical conditions produces at least two additional polypeptide chains, termed bands II and III, with apparent molecular weights of 125,00 and 62,000. The generation of these fragments is enhanced by increasing the time of incubation. The appearance of band II composition of the buffer, dodecyl sulfate concentrations, or alpha 2 M protein concentration in the incubation mixture. The electrophoretic bands II and III of alpha 2 M have dissimilar 125I-labeled tryptic peptide digests and also differ in their amino acid composition. The heat-induced fragmentation of alpha 2M is not affected by the inclusion of a variety of low molecular weight protease inhibitors, suggesting that the appearance of bands II and III is not due to enzyme-catalyzed hydrolysis. When the subunit chain of alpha 2M is first cleaved by trypsin into the previously described Mr = 85,000 derivative, neither band II nor III material, nor other lower molecular weight products are generated by heat treatment. Furthermore, preincubation of alpha 2M with methylamine prevents fragmentation of the subunit chain. These results indicate that these fragments are neither pre-existing subunits of alpha 2M nor derivatives formed prior to treatment for gel analysis. These data provide evidence that a covalent bond in the alpha 2M molecule is unusually susceptible to heat-induced cleavage.

Purification, partial characterization, and immunological relationships of multiple low molecular weight protease inhibitors of soybean

Five protease inhibitors, I–V, in the molecular weight range 7000–8000 were purified from Tracy soybeans by ammonium sulfate precipitation, gel filtration on Sephadex G-100 and G-75, and column chromatography on DEAE-cellulose. In common with previously described trypsin inhibitors from legumes, I–V have a high content of half-cystine and lack tryptophan. By contrast with other legume inhibitors, inhibitor II contains 3 methionine residues. Isoelectric points range from 6.2 to 4.2 in order from inhibitor I to V. Molar ratios (inhibitor/enzyme) for 50% trypsin inhibition are I = 4.76, II = 1.32, III = 3.22, IV = 2.17, V = 0.97. Only V inhibits chymotrypsin significantly (molar ratio = 1.33 for 50% inhibition). The sequence of the first 16 N-terminal amino acid residues of inhibitor V is identical to that of the Bowman-Birk inhibitor; all other observations also indicate that inhibitor V and Bowman-Birk are identical. The first 20 N-terminal amino acid residues of inhibitor II show high homology to those of Bowman-Birk inhibitor, differing by 1 deletion and 5 substitutions. Immunological tests show that inhibitors I through IV are fully cross-reactive with each other but are distinct from inhibitor V.

The interactions between a low molecular weight protease inhibitor of bronchial mucus and chymotrypsin-like cationic proteins of granulocytes.

The proteolytic activity of the chymotrypsin-like cationic proteins of human granulocytes is inhibited by complex formation with the acid-stable low molecular weight protease inhibitor of human bronchial mucus (Ki = 1.0 x 10(7)M). The molar combining ratio is 1:1.