Showing papers on "Proteolytic enzymes published in 1984"

Physiological Functions of Glucocorticoids in Stress and Their Relation to Pharmacological Actions

Abstract: Introduction and Background Modern glucocorticoid endocrinology is a colorful, richly varied, but formless discipline—a profusion of cellular, physiological and pharmacological effects, seemingly unrelated through any central hormonal function. A current list of glucocorticoid effects might include such disparate items as stimulation of hepatic gluconeogenesis, inhibition of glucose uptake by peripheral tissues, suppression of inflammation, enhanced excretion of a water load, induction in various cells of tryptophan oxygenase and glutamine synthetase, suppression of numerous immune reactions, inhibition of secretion of several hormones and neuropeptides, and inhibition of activity of plasminogen activator and other neutral proteinases. Judging from recent writings on glucocorticoid physiology, an item that might be low on the list or missing altogether is “increased resistance to stress”.

Fibrinogen and Fibrin

Abstract: Fibrinogen is a soluble plasma protein that is converted to polymeric fibrin in response to damage to the vascular system The clotting process is initiated when platelets aggregate at the wound site Their disruption releases biologically active amines and a proteolytic cascade follows which culminates in the conversion of fibrinogen to fibrin The fibrin polymer forms the matrix of the tangle of cellular and molecular substances called the blood clot Atomic-level details are now in hand for many of the interactions that hold fibrin units together, although some aspects have yet to be resolved Of necessity, fibrin clots need to be dismantled when they are no longer needed, an operation largely accomplished by the proteolytic enzyme plasmin Various regulatory phenomena are involved in maintaining the balance between intravascular fluidity and clots that prevent blood loss A variety of hereditary conditions, including mutant fibrinogens, can predispose individuals to either thrombosis or bleeding Key concepts: The underlying fabric of blood clots is a protein polymer called fibrin Fibrin clots are formed in response to injuries to any part of the vascular system The conversion of soluble fibrinogen molecules to insoluble fibrin depends on thrombin generated from prothrombin Keywords: fibrinogen; fibrin; clot stabilisation; fibrinolysis; blood clotting; X-ray structures

Evolution of proteolytic enzymes

Abstract: Proteolytic enzymes have many physiological functions, ranging from generalized protein digestion to more specific regulated processes such as the activation of zymogens, blood coagulation and the lysis of fibrin clots, the release of hormones and pharmacologically active peptides from precursor proteins, and the transport of secretory proteins across membranes. They are present in all forms of living organisms. Comparisons of amino acid sequences, three-dimensional structures, and enzymatic reaction mechanisms of proteases indicate that there are distinct families of these proteins. Changes in molecular structure and function have accompanied the evolution of proteolytic enzymes and their inhibitors, each having relatively simple roles in primitive organisms and more diverse and more complex functions in higher organisms.

The nucleotide and deduced amino acid sequences of the encephalomyocarditis viral polyprotein coding region

Abstract: The nucleotide sequence of 7200 bases of encephalomyocarditis (EMC) viral RNA, including the complete polyprotein-coding region, was determined. The polyprotein is encoded within a unique translational reading frame, 6870 bases in length. Protein synthesis begins with the sequence Met-Ala-Thr, and ends with the sequence Leu-Phe-Trp, 126 bases from the 3' end of the RNA. Viral capsid and noncapsid proteins were aligned with the deduced amino acid sequence of the polyprotein. The proteolytic processing map follows the standard 4-3-4 picornaviral pattern except for a short leader peptide (8 kd), which precedes the capsid proteins. Identification of the proteolytic cleavage sites showed that EMC viral protease, p22, has cleavage specificity for gln-gly or gln-ser sequences with adjacent proline residues. The cleavage specificity of the host-coded protease(s) includes both tyr-pro and gln-gly sequences.

Hormonal regulation of hormone-sensitive lipase in intact adipocytes: identification of phosphorylated sites and effects on the phosphorylation by lipolytic hormones and insulin

Abstract: In isolated adipocytes, fast-acting lipolytic hormones and insulin have been shown previously to control lipolysis by regulating the activity of hormone-sensitive lipase, the rate-limiting enzyme, through an increase or decrease, respectively, of the extent of phosphorylation of the enzyme. Here, we demonstrate that exposure to lipolytic hormones (corticotropin, noradrenaline) led to phosphorylation at two sites on the Mr 84,000 lipase subunit. One, designated "basal site," was phosphorylated also in the absence of any hormonal stimulation, its phosphorylation apparently not being influenced by hormones. The second, designated "regulatory site," was identical to that phosphorylated by cyclic AMP-dependent protein kinase on the isolated lipase. The regulatory site was not appreciably phosphorylated in the absence of hormones, but exposure of the cells to noradrenaline increased its phosphorylation extent to that of the basal site. Insulin or the beta-adrenergic antagonist propranolol decreased the extent of phosphorylation of the regulatory site to the low level before stimulation, apparently without effect on the basal site. Phosphoserine was the only phosphorylated amino acid residue at both sites. Limited proteolytic digestion indicated that the two sites were separated by less than about 170 amino acid residues. Thus, control of adipose tissue lipolysis by fast-acting lipolytic hormones and by insulin is exerted through the regulation of the phosphorylation state of a single phosphoserine residue in the hormone-sensitive lipase.

Multiple forms of hepatic cytochrome P-450. Purification, characterisation and comparison of a novel clofibrate-induced isozyme with other major forms of cytochrome P-450

Abstract: In the present studies, a novel form of highly purified cytochrome P-450 (cytochrome P-452) isolated from the hepatic microsomes of clofibrate-pretreated rats has been compared to the major isozymes isolated from the hepatic microsomes of rats pretreated with phenobarbital (cytochrome P-450) and 2-naphthoflavone (cytochrome P-447) using a number of biochemical criteria. The results show that these three isozymes exhibit marked structural differences from each other as judged by a complete lack of immunochemical cross-reactivity between the isozymes and the heterologous rabbit serum antibodies using Ouchterlony double diffusion, and non-identity between the limited proteolytic digestion maps of the three isozymes obtained in the presence of chymotrypsin, papain and Staphylococcus aureus V8 proteases. Furthermore, the three isozymes exhibited clear differences in their monomeric molecular weights determined on calibrated sodium dodecyl sulphate/polyacrylamide gel electrophoresis in gels of varying acrylamide concentration. Substantial differences were also observed in the substrate specificities of the isozymes, which were reflected in differences in the turnover rates and positional selectivities of the hemoproteins for some model substrates. In addition, the isozymes differed in their substrate binding affinities and their ability to interact with purified hepatic microsomal cytochrome b5, as judged using difference spectrophotometry. Finally, subtle differences were detected in the ultraviolet visible absorbance spectra of the hemoproteins in the ferric, ferrous, and carbonmonoxyferrous states. Taken collectively, the above data provides compelling evidence that fundamental differences exist between these cytochrome P-450 isozymes, further establishing the uniqueness of the major form of cytochrome P-450 induced by clofibrate pretreatment.

Localization of binding sites for carboxyl terminal specific anti-rhodopsin monoclonal antibodies using synthetic peptides.

Abstract: The binding sites for four monoclonal antibodies, rho 1D4, rho 3C2, rho 3A6, and rho 1C5, have been localized within the C-terminal region of bovine rhodopsin: Asp18'-Glu-Ala16'-Ser-Thr-Thr-Val12'-Ser-Lys-Thr-Gl u8'-Thr-Ser-Gln-Val4'-Ala-Pr o -Ala1'. Antibody binding sites were localized by using synthetic C-terminal peptides in conjunction with solid-phase competitive inhibition assays and limited proteolytic digestion of rhodopsin in conjunction with electrophoretic immunoblotting techniques. Binding of the rho 1D4 and rho 3C2 antibodies to immobilized rhodopsin was inhibited with peptides of length 1'-8' and longer. Antibody rho 1D4 binding was not inhibited by peptides 2'-13' or 3'-18', indicating that the C-terminal alanine residue of rhodopsin was required. Similar competitive inhibition studies indicated that the antibody rho 3A6 required peptides of length 1'-12' and longer whereas rho 1C5 required peptide 1'-18'. Peptide 3'-18' was as effective as 1'-18' in inhibiting rho 3A6 binding to rhodopsin, but replacement of glutamic acid in position 8' with glutamine abolished competition. This substitution had little effect on the binding of antibody rho 1C5. Thus, Glu8' was essential for rho 3A6 binding but not for the binding of the rho 1C5 antibody. Cleavage of the seven amino acid C-terminus from rhodopsin and further cleavage to F1 (Mr 25 000) and F2 (Mr 12 000) fragments with Staphylococcus aureus V8 protease abolished binding of rho 1D4 antibody to the membrane-bound rhodopsin fragments.(ABSTRACT TRUNCATED AT 250 WORDS)

Extraction and isolation of a leukotoxin from Actinobacillus actinomycetemcomitans with polymyxin B.

Abstract: A leukotoxin from Actinobacillus actinomycetemcomitans was isolated by a procedure that includes polymyxin B extraction, ion-exchange chromatography, and gel filtration chromatography. The procedure resulted in the recovery of 48% of the toxin with a 99-fold increase in specific activity. The isolated toxin has a molecular mass of 180,000 daltons by gel filtration and 115,000 daltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It retains all the major biological characteristics previously documented for crude leukotoxin preparations, including susceptibility to heat and proteolytic enzymes and neutralization by sera from patients with juvenile periodontitis. The isolated leukotoxin destroys human but not rat or guinea pig polymorphonuclear leukocytes and has no apparent effect on human erythrocytes. The availability of the A. actinomycetemcomitans leukotoxin should facilitate studies on its chemistry and mode of action as well as its role in the pathogenesis of human periodontal disease.

Granulocyte Neutral Proteases and Pseudomonas Elastase as Possible Causes of Airway Damage in Patients with Cystic Fibrosis

Abstract: We studied the possible role of granulocyte neutral proteases as mediators of airway destruction in patients with cystic fibrosis (CF) who were infected with Pseudomonas aeruginosa. We measured the enzymatic activities of bronchial secretions on purified radioactively labeled complement component three (C3), elastin, and a granulocyte elastase-specific substrate. Bronchial secretions from 18 patients with CF who were infected with P aeruginosa had a significantly higher mean value for C3 cleaving, elastolytic, and granulocyte elastase-like activity than did two control groups. High enzymatic activities were observed in patients with CF who have advanced bronchial disease (that had been determined by a clinical scoring system). Kinetics of proteolysis of radioactively labeled C3 and inhibition profiles of the activities of the three enzymatic activities studied suggest that they are mainly derived from granulocytes. In addition, 20 of 31 strains of P aeruginosa isolated from patients with CF inactivated purified alpha 1-antiprotease in vitro. We postulate that granulocyte neutral proteases and P aeruginosa may act synergistically in the airways of patients with CF and may contribute to the destruction of elastin and inactivation of C3.

Degradation of the epidermal-dermal junction by proteolytic enzymes from human skin and human polymorphonuclear leukocytes

Abstract: The degradation of normal human skin by the human polymorphonuclear leukocyte proteinases cathepsin G and elastase, and by a human skin chymotrypsin-like proteinase that appears to be a mast cell constituent, was examined. Enzymes were incubated with fresh, split-thickness skin for up to 8 h; the tissue was examined ultrastructurally and immunohistochemically using antibodies to known basement membrane constituents. In all cases, the primary damage observed was at the epidermal-dermal junction. Elastase degraded the lamina densa leaving scattered and disorganized anchoring fibrils, dermal microfibril bundles, and normal-appearing collagen fibers. Immunohistochemically, type IV collagen, laminin, KF1 antigen, and EBA antigen were absent. The bullous pemphigoid antigen was present and localized on the basal cells. Epidermal-dermal separation produced by the chymotrypsin-like proteinases, cathepsin G, and the human skin proteinase, was confined to the lamina lucida. The lamina densa and sub-lamina densa fibrillar network remained intact. The human skin chymotrypsin-like proteinase produced extensive epidermal-dermal separation, while cathepsin G, at comparable concentrations, produced only focal separations. Immunohistochemically, all antigens were present after incubation with enzyme. The bullous pemphigoid antigen, however, was found on the epidermal side of the split, while laminin was found on the dermal side. These results show that the epidermal-dermal junction is highly susceptible to neutral serine proteinases located in mast cells and polymorphonuclear leukocytes. Although all the proteinases produce epidermal-dermal separation, the patterns and extent of degradation are different. The distinctive patterns of degradation may provide a clue to the involvement of these proteinases in skin diseases.

Anaphylactic- and calcium-dependent generation of prostaglandin D2 (PGD2), thromboxane B2, and other cyclooxygenase products of arachidonic acid by dispersed human lung cells and relationship to histamine release.

Abstract: Proteolytic digestion of human lung tissue dispersed a population of cells (HDLC) containing 1 to 8% mast cells but which were free from bronchial and vascular smooth muscle. Incubation of HDLC with anti-human IgE, which released a net 24.8 + 4.3% of mast cell-derived histamine, stimulated a 14-fold increase in the generation of PGD2, a seven-fold increase in TXB2, and less than a twofold increase in PGF2 alpha, immunoreactive PGE, (i-PGE) and 6-keto-PGF1 alpha. A similar profile of prostanoid release was observed when cells were challenged with epsilon-specific anti-IgE, indicating that the response was specific to the coupling of IgE Fc receptors. The calcium ionophore A23187 also released prostanoids from HDLC in approximately the same proportions as anti-IgE. This stimulus, however, released only 50% as much PGD2 per nanogram histamine than did IgE-dependent activation, thereby showing a fundamental difference in the mechanisms by which the two agents activate mast cells and liberate arachidonic acid for oxidative metabolism. In concentration-response and time course experiments, both secretory stimuli released prostanoids and histamine in parallel. After separation of lung cells by isopyknic centrifugation, challenge with anti-IgE or A23187 released PGD2 only from those fractions containing mast cells, the amount released corresponding closely to both the mast cell concentration and net histamine release. On pooling data from all experiments, the closest correlation was found between release of PGD2 and histamine when cells were stimulated with either anti-IgE (r = 0.813, p less than 0.001) or A23187 (r = 0.763, p less than 0.001), supporting a mast cell origin for PGD2. The release of other prostanoids in fractions not containing mast cells demonstrates that macrophages, monocytes, and lymphocytes have the capacity to generate TXB2, PGF2 alpha, and i-PGE both in the absence and presence of mast cells. Thus, although mast cells are likely to be the major source of PGD2 generated upon IgE-dependent stimulation of HDLC, other cells dispersed from lung tissue have the capacity to generate prostanoids directly after activation of their IgE-Fc receptors and, indirectly after the secretion of mast cell mediators.

Monoclonal antibodies against protease-sensitive pneumococcal antigens can protect mice from fatal infection with Streptococcus pneumoniae.

Abstract: Monoclonal antibodies were raised against surface determinants of Streptococcus pneumoniae by hyperimmunizing X-linked immunodeficient (xid) CBA/N mice with the heat-killed rough strain R36A. 17 hybridomas produced antibody that bound intact R36A and did not cross-react with phosphocholine, an antigen common in the cell wall of all S. pneumoniae. The antibody produced by at least two of these hybridomas, Xi64 (IgM) and Xi126 (IgG2b), could protect mice from a lethal intravenous challenge of type 3 S. pneumoniae strains WU2 and A66 and of the type 2 strain D39. The minimum amount of antibody required to protect xid mice from 100 WU2 was 4.5 micrograms/mouse for Xi64 and 2.6 micrograms/mouse for Xi126,. Free phosphocholine, C-polysaccharide, and type 3 capsular polysaccharide all failed to inhibit the binding of Xi64 or Xi126 to R36A. These antibodies appeared to bind surface polypeptides, since treatment of R36A with either pepsin or trypsin, or of R36A lysate with trypsin, effectively eliminated the ability of Xi64 and Xi126 to bind antigens in these preparations. Binding studies indicated that these two antibodies recognized different epitopes that were expressed on several but not all serotypes of pneumococci.

Neuropeptide Y receptor in the rat brain.

Abstract: The specific binding of the chloramine-T iodinated neuropeptide Y (125I-NPY) to membranes from rat cerebral cortex was investigated using equilibrium binding and kinetic methods. The equilibrium binding of 125I-NPY at 37°C was characterized by a Kd value of 0.38 nM. The receptor densities in the cerebral cortex, hypothalamus and cerebellum were 0.45 pmol/mg, 0.47 pmol/mg and 0.04 pmol/mg protein respectively. The binding site for 125I-NPY was sensitive to treatment with proteolytic enzymes and thiol reagents. The binding showed a sharp optimum at pH 7 - 7.7 and was inhibited by increasing concentrations of Mg2+

Proteases and oxidants in experimental pulmonary inflammatory injury.

Abstract: We have examined various biochemical parameters of pulmonary inflammation in experimental animals. Intrabronchial instillation of glucose oxidase-glucose (GO/G) to produce oxidants or formylated norleu-leu-phe (FNLP) or phorbol myristate acetate (PMA) as leukocytic stimuli induced severe acute pulmonary injury in New Zealand white rabbits. PMA also induced inflammation when administered intravenously. Each stimulus induced transudation of protein from the vascular space into the pulmonary tissues, and an influx of leukocytes during the 4-6 h period of the experiment. Pathophysiologic changes were measured by edema formation (transudation of 125I-bovine serum albumin), and histologic examination. Biochemical analysis was performed by measuring concentrations of potentially injurious agents in bronchoalveolar lavage (BAL) fluid. Increased acid protease and myeloperoxidase levels were found in the BAL fluid after administration of either of the stimuli. Evidence of oxidant generation in vivo was obtained in two different ways. In the first, specific activities for catalase were measured in the BAL fluid in the presence or absence of 3-amino, 1,2,4 triazole (AT), injected at intervals before obtaining BAL fluid. In the presence of AT, specific activities for catalase dropped to 0.22 after a double instillation of FNLP and to 0.15 in the presence of GO/G. In neutrophil-depleted FNLP animals, catalase was not greatly inhibited by AT (sp act 0.90). In the second, intracellular levels of total glutathione (GSH + GSSG) in whole lung tissue and alveolar macrophages decreased when stimuli of neutrophils were administered. Intrabronchially instilled PMA, e.g., caused a drop of glutathione in whole lung tissue from the control value of 2.3 mumol GSH equivalent/100 mg dry wt to 0.54 mumol GSH equivalent/100 mg dry wt at 4 h. Neutrophil depletion and superoxide dismutase protected from this effect. From these results, we conclude that O-2 or its metabolites can initiate severe pulmonary injury as shown by the effect of GO/G and that, during development of pulmonary injury, stimulated neutrophils generate oxidants and release proteolytic enzymes into the surrounding tissues.

Physiology and Pathophysiology of Neutral Proteinases of Human Granulocytes

Abstract: The occurence of proteolytic enzymes in polymorphonuclear granulocytes was first demonstrated in 1888 by the famous clinician and biochemist Friedrich von Muller1 who showed that a glycerine extract of fresh pus digests fibrin or coagulated protein at a neutral or weakly alkaline pH. Later on at the end of the last and the beginning of this century further characterization of the enzymes including their serum antiproteases was achieved by German and American scientists2,3,4. However, the neutral proteases then became largely forgotten as the result of the attention paid to the acid-cathepsins of the rabbit leukocyte, a convenient but somewhat misleading cell.

Isolation and partial purification of a clonidine-displacing endogenous brain substance

Abstract: A new compound, designated clonidine-displacing substance (CDS), has been isolated from calf brain by ion-exchange chromatography, zone electrophoresis and high-performance liquid chromatography. CDS binds specifically to α2-adrenergic receptors in rat brain and human platelet membranes, as measured in direct binding experiments using [3H]clonidine and [3H]yohimbine respectively. Unlike clonidine or other α2-agonists, CDS does not affect basal levels of adenylate cyclase in human platelets at the highest concentrations obtainable. The apparent molecular mass of the compound is estimated to be 500 ± 50 Da, as determined by gel-filtration chromatography on Sephadex G-15. The new compound is thermostable, not affected by proteolytic enzymes, such as trypsin. chymotrypsin, pronase, papain and pyroglutamase, or by boiling in 0.2 M HCl for 5min. It does not bind to α1receptors in rat brain or to β-adrenergic receptors in turkey erythrocytes, since it is unable to displace [3H]prazosin and [125I]cyanopindolol from α1 and β-receptors respectively.

Compositions containing odor purified proteolytic enzymes and perfumes

Abstract: Compositions containing proteolytic enzymes having no detectable odor at a concentration of less than about 0.002 Anson units per gram of distilled water, and selected perfume materials for improved odor. Heavy-duty liquid detergents are preferred.

Pathogenesis of Serratial Infection: Activation of the Hageman Factor-Prekallikrein Cascade by Serratial Protease

Abstract: A serratial protease with an apparent molecular weight of 56,000 (56K protease), which had been purified from the culture supernatant of a strain of Serratia marcescens isolated from a corneal lesion of a human eye [Matsumoto, K. et al. (1984) J. Bacteriol. 157, 225-232], greatly enhanced vascular permeability when injected into guinea pig skin. The 56K protease, which requires zinc ion for activity, was found to possess plasma kallikrein-like properties in vitro as judged by (i) preferential amidolysis of carbobenzoxy-Phe-Arg-4-methylcoumaryl-7-amide and Pro-Phe-Arg-4-methylcoumaryl-7-amide, which are known substrates for plasma kallikrein; (ii) release of kinin from high-molecular-weight kininogen; and (iii) prompt activation of Hageman factor followed by generation of kallikrein from plasma prekallikrein. These results suggest that the 56K protease enhances vascular permeability through activation of a Hageman factor-kallikrein-kinin pathway in vivo, and this molecular process appears to be a rational mechanism of enhancement of permeability and serratial pathogenesis.

Liquid detergents containing boric acid and formate to stabilize enzymes

Abstract: Heavy-duty liquid detergents containing anionic surfactant, fatty acid, builder, proteolytic enzyme, boric acid or a boron compound capable of forming boric acid in the composition, formate, and calcium ion are disclosed. The combination of boric acid and formate provides improved protease stability in the compositions.

Intestinal transmission of macromolecules (BSA and FITC-labelled dextrans) in the neonatal pig. Influence of age of piglet and molecular weight of markers.

Abstract: The intestinal transmission of two macromolecular markers, of similar molecular weight but different susceptibility to proteolytic digestion, was investigated in the neonatal pig. Piglets of varying age (0 h-7 days old) were given a mixture of bovine serum albumin (BSA) and fluorescein-isothiocyanate labelled dextran 70,000 (FITC-D 70) by stomach tube, and the serum concentrations were determined 2 h after feeding. A high correlation between the patterns of transmission were obtained for the two marker substances (r=0.91, n=39). Furthermore, a rapid decrease in the transmission of the markers was observed during the first day of life in suckled piglets, and intestinal macromolecular closure was well developed in the piglets after 18-36 h of life. These findings indicate that 'closure' is unrelated to changes in intestinal proteolytic activity. After closure, only small amounts of the markers were transmitted to the serum. During the first day of life, great individual differences in the transmission were found between piglets. As shown by feeding different-sized FITC-D (MW = 3,000-70,000 dalton) and unconjugated FITC (MW = 389 dalton), molecules having a molecular weight greater than 3,000 daltons were excluded upon macromolecular closure. On the other hand, smaller molecules like FITC were transmitted across the intestinal barriers independent of closure.