Showing papers on "Proteolytic enzymes published in 1983"

Reduction of the extent of ischemic myocardial injury by neutrophil depletion in the dog.

Abstract: Accumulation of polymorphonuclear neutrophils during the acute inflammatory response may exacerbate tissue injury through the release of activated oxygen products or proteolytic enzymes or both. To assess the role of neutrophils in acute myocardial infarction, circulating neutrophil levels in dogs were reduced by 77 +/- 2% (mean +/- SEM) by administering rabbit antiserum to dog neutrophils. Acute myocardial infarction was induced in open-chest anesthetized dogs by 90 minutes of left circumflex coronary artery occlusion followed by 6 hours of reperfusion. Dogs treated with neutrophil antiserum (n = 8) developed myocardial infarcts that were an average of 43% smaller than infarcts in dogs treated with nonimmune rabbit serum (n = 7) (27.0 +/- 4.5% vs 47.1% +/- 7.5% of the area at risk, p less than 0.05). In a saline-treated control group (n = 8), infarct size was 48.0 +/- 4.7% of the area at risk, a value not significantly different from that of the nonimmune serum group but significantly greater than that in the neutrophil antiserum dogs (p less than 0.05). There were no major hemodynamic differences between groups. Histopathologic examination revealed that infarcted myocardium from dogs given saline or treated with nonimmune serum had a substantial neutrophilic infiltrate, which was virtually absent in infarcted tissue from dogs treated with neutrophil antiserum. These observations suggest that neutrophil accumulation in response to myocardial ischemia may be responsible for a substantial portion of the irreversible myocardial injury resulting from temporary coronary artery occlusion.

Detection and activity of lactacin B, a bacteriocin produced by Lactobacillus acidophilus.

Abstract: A total of 52 strains of Lactobacillus acidophilus were examined for production of bacteriocins. A majority (63%) demonstrated inhibitory activity against all members of a four-species grouping of Lactobacillus leichmannii, Lactobacillus bulgaricus, Lactobacillus helveticus, and Lactobacillus lactis. Four L. acidophilus strains with this activity also inhibited Streptococcus faecalis and Lactobacillus fermentum, suggesting a second system of antagonism. Under conditions eliminating the effects of organic acids and hydrogen peroxide, no inhibition of other gram-positive or -negative genera was demonstrated by L. acidophilus. The agent produced by L. acidophilus N2 and responsible for inhibition of L. leichmannii, L. bulgaricus, L. helveticus, and L. lactis was investigated. Ultrafiltration studies indicated a molecular weight of approximately 100,000 for the crude inhibitor. The agent was sensitive to proteolytic enzymes and retained full activity after 60 min at 100 degrees C (pH 5). Activity against sensitive cells was bactericidal but not bacteriolytic. These characteristics identified the inhibitory agent as a bacteriocin, designated lactacin B. Examination of strains of L. acidophilus within the six homology groupings of Johnson et al. (Int. J. Syst. Bacteriol. 30:53-68, 1980) demonstrated that production of the bacteriocin lactacin B could not be used in classification of neotype L. acidophilus strains. However, the usefulness of employing sensitivity to lactacin B in classification of dairy lactobacilli is suggested. Images

Proteinases in normal bovine milk and their action on caseins.

Abstract: Native proteolytic enzymes in good quality normal bovine milk readily hydrolysed the caseins during incubation or storage, producing the gamma-caseins, proteose-peptone components 5 (PP5) and 8-fast (PP8F) and a considerable number of other unidentified fragments, many of which were also subsequently found in the proteose-peptone fraction. The rate of casein hydrolysis was greater in pasteurized than in raw milk, with beta-casein being slightly more susceptible to attack than alpha S1-casein. Measurements of gamma-casein and proteose-peptone formation have been made and it was found that PP5 was an intermediate product that was subject to further proteolysis while PP8F was a stable end-product. With the exception of component 3 (PP3), virtually all constituents of the proteose-peptone fraction increased during storage and appeared to be products of the action of proteolytic enzymes. Further evidence was obtained from the effects of various inhibitors that the principal proteinase of normal milk is plasmin, but slight differences were apparent between the protein breakdown patterns induced by storage and by added plasmin, which was consistent with the presence of more than one proteinase. Incubations in the presence of soya bean trypsin inhibitor to prevent plasmin action clearly revealed that another enzyme(s) was also involved.

Structural and immunological similarities between simian sarcoma virus gene product(s) and human platelet-derived growth factor

Abstract: The predicted amino acid sequence of the simian sarcoma virus (SSV) transforming gene product, p28SIS, closely corresponds to that of human platelet-derived growth factor (PDGF). We demonstrate that p28SIS rapidly undergoes a series of discrete processing steps including dimer formation and proteolytic digestion to yield molecules structurally and immunologically resembling biologically active PDGF.

Polymers containing enzymatically degradable bonds, 8. Degradation of oligopeptide sequences in N-(2-hydroxypropyl)methacrylamide copolymers by bovine spleen cathepsin B

Abstract: Three types of copolymers of N-(2-hydroxypropyl)methacrylamide(HPMA) were prepared which contain oligopeptide sequences: (a) HPMA copolymers containing oligopeptide side-chains terminated with p-nitroaniline; (b) soluble HPMA copolymers containing oligopeptide sequences in crosslinks connecting two poly(HPMA) chains; (c) a hydrophilic gel, i.e. a three dimensional copolymer of HPMA containing an oligopeptide sequence in the crosslinks. These polymeric substrates (suitable as drug carriers) containing potentially degradable oligopeptide sequences were incubated with an intracellular proteolytic enzyme, bovine spleen cathespin B. The degradation process of the substrates made it possible to reveal the relationship between the structure of oligopeptide sequences and their degradability. The results suggest an important role played by cathepsin B in the degradation of polymeric substrates investigated in this study under physiological conditions.

Alpha 1 antitrypsin deficiency: the clinical and physiological features of pulmonary emphysema in subjects homozygous for Pi type Z. A survey by the British Thoracic Association.

Abstract: Hereditary deficiency of alpha 1 antitrypsin, the main serum inhibitor of proteolytic enzymes is associated with pulmonary emphysema of early onset. A multicentre survey of this disorder was started in 1976 and details of 166 subjects homozygous for the Z phenotype form the main body of this report. There were 126 index cases who were identified through chest clinics and 40 non-index cases who were identified through family studies. The index cases and many of the non-index cases had severe radiological and physiological abnormalities. A history of cigarette smoking had a significant effect upon the prognosis, but sex and occupational exposure to dust or fumes did not. There was a wide variance in lung function even among those who had never smoked.

Antiestrogen-Binding Sites Distinct from the Estrogen Receptor: Subcellular Localization, Ligand Specificity, and Distribution in Tissues of the Rat

Abstract: Antiestrogens are known to interact with the estrogen receptor system of target cells. In addition, it is possible that recently identified estrogen-noncompetable, antiestrogenspecific binding sites may be of importance in mediating and/or modulating the actions of antiestrogens, and hence, we have examined their nature and properties. We find these sites to fractionate with what is likely the microsomal fraction of rat uterus with only very small amounts in high speed cytosol. Antiestrogen-specific binding sites are present in the 800 ×g × 10 min or 12,000 or 20,000 × g × 30 min supernatants, but are pelleted upon centrifugation at 100,000 or 180,000 × g × 60 min. These sites are present in the 12,000 × g supernatant fraction of almost all rat tissues examined. They are highest in concentration in the liver, uterus, esophagus, ovary, brain, and kidney; lower levels are found in lung and spleen, and negligible amounts are seen in muscle, heart, and serum. Hence, these sites are not localized exclusively o...

Chromophore structure of the physiologically active form (P(fr)) of phytochrome.

Abstract: Chromopeptides were prepared by proteolytic digestion of phytochrome (far-red absorbing form, Pfr) and of phycocyanin. The phycocyanobilin peptide, the chromophore of which is Z,Z,Z-configurated, was modified to the Z,Z,E isomeric chromophore. It has been demonstrated earlier that the Pfr chromopeptide and the Z,Z,E-configurated phycocyanin chromopeptide behave similarly with regard to spectral and chromatographic properties and reactivity. We present evidence here, obtained by high-resolution 1H NMR spectroscopy, that both the modified phycocyanobilin chromophore and the phytochrome chromophore obtained directly from Pfr are 15E-configurated.

Pseudomonas aeruginosa elastase and its role in pseudomonas infections

Abstract: Most strains of Pseudomonas aeruginosa produce three proteases with broad substrate specificities. One of these enzymes has elastolytic activity (P. aeruginosa elastase). This elastase has tissue-damaging activity and is capable of degrading various plasma proteins such as immunoglobulins, coagulation and complement factors, and alpha-proteinase inhibitor. There is evidence for a role of elastase in localized infections such as experimental pseudomonas keratitis, pneumonia, and burn infection. Once colonization and invasion has occurred and septicemia has been established, these enzymes are probably less important. Elastase is probably best classified as a virulence-enhancing factor in certain types of infections.

Tumor invasion and host extracellular matrix.

Abstract: In this review some of the major mechanistic pathways by which tumor cells are thought to invade host tissues are discussed. Tumor invasion has been conceived to be the result of pathological, close-range interactions between malignant cells and host stroma. The sequence of events that characterize invasion can be summarized as follows: (a) Tumor cell clusters break from the confinement of the primary tumor. Loss of intercellular junctions (desmosomes), alterations in the chemical composition and physical properties of the cell surface coat (loss of fibronectin and heparan sulfate; excessive amounts of hyaluronate), and loosening of cell-substrate interactions (loss of hemidesmosomes, fibronectin, and heparan sulfate), are among the most frequently listed causes of tumor cell shedding. (b) Increased proteolytic activities at the invasion front cause focal alterations in the surrounding extracellular matrix, thereby changing its physical properties. Collagenases and cathepsins, as well as elastase and other neutral proteinases are the enzymes most frequently associated with matrix destruction and invasion. In some tissues this process is effectively regulated by inhibitors of matrix-degrading, proteolytic enzymes. (c) Tumor cells migrate into the altered matrix, possibly moving as aggregates along guidance tracks provided by host structures (blood vessels, lymphatics, nerves) or matrix macromolecules (collagen and fibronectin tracks). Migration seems to be preceded by increased swelling of glycosaminoglycan (i.e., hyaluronate) in the matrix, ahead of the migrating cell population. Various host cell types (mast cells, fibroblasts, endothelial cells, macrophages, etc.) may participate in these events.

Novel renin inhibitors containing the amino acid statine

Abstract: The proteolytic enzyme renin (EC3.4.99.19) cleaves the protein substrate angiotensinogen to yield angiotensin I, the decapeptide substrate transformed by converting enzyme into the pressor substance angiotensin II1. Although the contribution of this pathway to the maintenance of normal blood pressure is unclear, it seems to be a major factor in various hypertensive states2–4. Important progress in the control of hypertension has been achieved by development of the potent inhibitors SQ-14,225 (captopril)5,6 and MK-421 (enalapril maleate)7–9 which block the generation of angiotensin II by the inhibition of angiotensin converting enzyme. An attractive alternative to the inhibition of converting enzyme would be the blockade of the preceding step in the cascade, the renin reaction. We report here new highly potent (IC50 = 10−9−10−8 M) competitive inhibitors of renin in which statine, (3S,4S)-4-amino-3-hydroxy-6-methylheptanoic acid, is incorporated into analogues of the pig renin substrate (Fig. 1).

Ganglioside‐Induced Neuritogenesis: Verification That Gangliosides Are the Active Agents, and Comparison of Molecular Species

Abstract: Gangliosides were previously reported to induce neuritogenesis in primary neuronal cultures and in some neurally derived cell lines. Because isolated gangliosides usually contain variable quantities of peptides, we investigated the possibility that neurite-stimulating activity could be caused by these contaminants. Ganglioside preparations from bovine brain and other sources were subjected to a three-step purification procedure that eliminated at least 95% of the contaminating peptides. These purified preparations retained their capacity to induce extensive neurite growth in neuro-2A murine neuroblastoma. Proteolytic digestion and a number of additional procedures were used to reduce residual contamination further without loss of activity. Several crude ganglioside samples had negative effects on neurite development until freed of their inhibitory factors, which were derived from the tissue and/or introduced during laboratory operations. This was particularly evident for bovine white matter gangliosides whose activity increased in proportion to peptide removal. When carefully purified, virtually all of 11 different gangliosides tested were highly active, with the possible exception of GM4, which demonstrated only moderate activity in a limited number of tests. All of the neutral glycolipids tested, as well as sulfatides and free sialic acid, were inactive.

Simian virus 40 and polyoma virus induce synthesis of heat shock proteins in permissive cells.

Abstract: During the lytic infection of monkey and mouse cells with simian virus 40 and polyoma virus, respectively, the preferentially increased synthesis of two host proteins of 92,000 and 72,000 Mr was observed by 15 to 20 h after infection besides the general stimulation of most cellular proteins. The incubation of uninfected monkey and mouse cell cultures for 30 to 60 min at 43.5 degrees C induced the enhanced synthesis of at least three proteins of 92,000, 72,000 and 70,000 Mr, the last one being the major heat shock protein of mammalian cells. Two-dimensional gel electrophoresis and partial proteolytic digestion confirmed that the same 92,000- and 72,000-Mr proteins are stimulated by virus infection and thermal treatment. In simian virus 40-infected CV-1 cells, we also observed the weak stimulation of a 70,000-Mr protein comigrating in gel electrophoresis with the major heat shock protein. The 92,000-, 72,000- and 70,000-Mr proteins of monkey cells are structurally very similar to the corresponding proteins of mouse cells. In immunoprecipitations, no specific association of these proteins to simian virus 40 T antigens was noticed.

Structural diversity and domain composition of a unique collagenous fragment (intima collagen) obtained from human placenta

Abstract: Intima collagen was obtained from pepsin digests of human placenta in two forms, which differ to some extent in the size of their constituent polypeptide chains (Mr 50 000-70 000). These chains are connected by disulphide bonds to large aggregates. The aggregates are arranged in a triple-helical conformation with a remarkably high thermal stability (Tm 41-62 degrees C) and are resistant to further proteolytic digestion. Reduction of as little as 5% of the disulphide bonds produces mainly monomeric triple helices (Mr about 160 000) with Tm 32 degrees C. Partially reduced material can be separated into triple-helical and non-collagenous domains by proteolysis. Pepsin releases a collagenous component with chains of Mr 38 000. Bacterial collagenase liberates two non-collagenous segments (Mr 15 000-30 000) rich in cystine. Treatment with collagenase before reduction separates intima collagen into a large fragment composed of collagenous (Tm 41 degrees C) and non-collagenous structures and a single non-collagenous segment. The data support the electron-microscopical model of intima collagen [Furthmayr, Wiedemann, Timpl, Odermatt & Engel (1983) Biochem. J. 211, 303-311], indicating that the basic unit of the fragment consists of a continuous triple helix joining two globular domains.

Characterization of Candida albicans adherence to human vaginal epithelial cells in vitro.

Abstract: Certain environmental, physical, and biochemical aspects of Candida albicans adherence to human vaginal epithelial cells were characterized by using an in vitro radiometric adherence assay. Blastospores harvested from cultures grown at 25 degrees C adhered to vaginal epithelial cells in significantly greater numbers than did blastospores isolated from cultures grown at 37 degrees C. C. albicans viability was not essential for adherence, but severe methods used to kill the blastospores did reduce their attachment. The addition of sodium chloride, divalent cations, sugars, mannan, or mannoprotein to the assay had no effect on attachment. Pretreatment of the blastospores with detergents, salts, urea, glycosidases, lipase, or pepsin did not affect adherence, but treatment with reducing agents or five proteolytic enzymes did render C. albicans nonadherent. Cell wall fragments prepared from C. albicans, but not from Candida krusei, adhered to vaginal epithelial cells. Loss of adherence after the cell walls were treated with alpha-mannosidase or papain suggests that cell wall mannoprotein is an essential component of the C. albicans adhesin.

Ultrastructure of Reichert's membrane, a multilayered basement membrane in the parietal wall of the rat yolk sac.

Abstract: The ultrastructure of Reichert's membrane, a thick basement membrane in the parietal wall of the yolk sac, has been examined in 13-14-d pregnant rats. This membrane is composed of more or less distinct parallel layers, each one of which resembles a common basement membrane. After routine fixation in glutaraldehyde followed by osmium tetroxide, the layers appear to be mainly composed of 3-8-nm thick cords arranged in a three-dimensional network. Loosely scattered among the cords are unbranched, straight tubular structures with a diameter of 7-10 nm, which mainly run parallel to the surface and to one another; they are referred to as basotubules. Permanganate fixation emphasizes the presence of a thick feltwork of irregular material around basotubules. Finally, minute dot-like structures measuring 3.5 nm and referred to as double pegs are present within the meshes of the cord network. Reichert's membranes have been treated for 2-48 h at 25 degrees C with plasmin, a proteolytic enzyme known to rapidly digest laminin and fibronectin. After a 2-h treatment, most of the substance of the cords is digested away leaving a three-dimensional network of 1.5-2.0-nm thick filaments. The interpretation is that the cords are formed of a plasmin-resistant core filament and a plasmin-extractable sheath. When plasmin treatment is prolonged for 15 h or longer, the filaments are dissociated and disappear, while basotubules are maintained. Plasmin digestion also reveals that basotubules are composed of two parts: a ribbon-like helical wrapping and tubule proper. Further changes in the tubule under plasmin influence are interpreted as a dissociation into pentagonal units suggestive of the presence of the amyloid P component. After 48 h of plasmin treatment, basotubules are further disaggregated and dispersed, leaving only linearly arranged double pegs. Reichert's membranes with or without a 2-hr plasmin treatment have been immunostained by exposure to antibodies against either laminin or type IV collagen with the help of peroxidase markers. The results indicate that the sheath of the cords contains laminin antigenicity, while the core filament contains type IV collagen antigenicity. It is proposed that Reichert's membrane consists mainly of a three-dimensional network of cords composed of a type IV collagen filament enclosed within a laminin-containing sheath. Also present are basotubules--which may contain the amyloid P component--and double pegs whose nature is unknown.

A receptor on soybean membranes for a fungal elicitor of phytoalexin accumulation.

Abstract: Soybean membrane preparations specifically bound [14C]mycolaminaran, a branched β-1,3-glucan produced by Phytophthora sp. which elicits production of the phytoalexin glyceollin in soybean tissues. A Scatchard plot of the binding data disclosed the presence of a single affinity class of binding sites with a Kd value of 11.5 micromolar for the glucan. To assess the physiologic importance of mycolaminaran binding in phytoalexin elicitation, several derivatives of mycolaminaran were prepared. Reduced mycolaminaran had slightly greater elicitor activity and binding affinity than the native substance, while periodinated mycolaminaran was virtually devoid of either elicitor activity orbinding capability. Phosphorylated mycolaminaran, on the other hand, gave values for both elicitor activity and membrane binding which were intermediate between the native and periodinated preparations. No other tested carbohydrates competed with the binding of [14C]mycolaminaran. Soybean membrane preparations contained β-1,3-endoglucanase activity that degraded mycolaminaran and reduced both its efficiency as a phytoalexin elicitor and its membrane binding at temperatures above 0°C. Once [14C]mycolaminaran bound to membranes, however, it was not appreciably susceptible to glucanase attack and could not be displaced with excess unlabeled ligand. Taken collectively, the observations suggest that the membrane binding sites are mycolaminaran-specific receptors which are physiologically involved in the initiation of phytoalexin production in soybean cotyledons. Because the binding of mycolaminaran to membranes was abolished by heat and proteolytic enzymes, the receptor is probably a protein(s) or glycoprotein(s).

Effect of in Vitro Action of Serum Proteases or Exposure to Acid on Measurable Immunoreactive Somatomedin-C in Serum*

Abstract: The proportion of immunoreactive somatomedin-C (IR-Sm-C) in blood that is available for measurement in the RIA is influenced by whether the sample is processed as serum or plasma, by how promptly the sample is chilled and frozen, by whether the reaction is carried out in glass or polystyrene tubes and by whether the incubation mixture contains protamine or heparin. Although protamine buffers and polystyrene tubes increase the availability of purified somatomedin-C (Sm-C), they decrease the detectability of Sm-C in serum. By incubating serum at neutral or acid pH, this IR-Sm-C can be made available for measurement, suggesting that incubation alters the nature of the linkage between Sm-C and its binding proteins or causes a conformational change in the binding protein, resulting in greater exposure of IR-Sm-C. The increment in measurable IR-Sm-C that occurs at neutral pH appears to be due to the action of proteolytic enzymes since it is time, temperature, and pH dependent and is inhibited by a variety of pr...

Liberation of immunoreactive somatomedin-C from its binding proteins by proteolytic enzymes and heparin.

Abstract: Most of the somatomedin in serum exists as a high molecular weight complex (≈140,000) composed of binding protein subunits and small molecular weight somatomedin. These binding proteins may interfere with measurements of the somatomedins by various radioligand assays. In a companion paper, we reported that when serum is incubated at neutral pH the detectability of the somatomedin-C (Sm-C) is increased. Using gel chromatography, however, it was not possible to demonstrate release of free Sm-C from the macromolecular complex. In the studies reported here, incubation of heparinized plasma at pH 7.4 followed by gel chromatography in a heparincontaining buffer caused 70-80% of the immunoreactive Sm-C to shift from the γ-globulin region to a molecular weight which approximates that of free Sm-C. This conversion is a timedependent process which is inhibited by the proteolytic enzyme inhibitor antipain. Similar changes in the elution profile of Sm-C were observed when heparinized plasma was acidified and chromato...