Showing papers on "Proteolytic enzymes published in 1975"
TL;DR: The gonococcus and meningococcus, which infect human mucosal surfaces, elaborate a highly specific proteolytic enzyme which cleaves the immunoglobulin A1 subclass of the principal mucosal antibody, immunogloba A (IgA), which lies in a unique region of the IgA heavy chain.
Abstract: The gonococcus and meningococcus, which infect human mucosal surfaces, elaborate a highly specific proteolytic enzyme which cleaves the immunoglobulin A1 subclass of the principal mucosal antibody, immunoglobulin A (IgA). The susceptible Pro-Thr bond lies in a unique region of the IgA heavy chain; the IgA2 subclass, lacking this peptide bond, is enzyme resistant.
255 citations
TL;DR: An attempt to produce wide ranging changes in the ionic composition of the intracellular medium of neurones isolated from the snail Helix pomatia.
Abstract: IT has become possible recently to isolate single neurones from molluscan ganglia by the use of proteolytic enzymes, so that the external solution can be changed easily1,2. One advantage is that the absence of the axon makes the application of the voltage clamp technique to isolated neurones more effective3. We describe here a further development—an attempt to produce wide ranging changes in the ionic composition of the intracellular medium of neurones isolated from the snail Helix pomatia.
231 citations
TL;DR: Tests of the active fraction after digestion with proteolytic enzymes suggest that the pheromone may be a portion of a protein or a substance bound to a protein.
Abstract: The sexual development of female mice is accelerated by exposure to an adult male or to male urine. The component of the urine responsible for this effect is androgen-dependent, heat labile, nondialysable, precipitatable with ammonium sulphate, and is not extractable in ether. These results indicate that the pheromone causing accelerated sexual development is associated with a protein component of male urine. Tests of the active fraction after digestion with proteolytic enzymes suggest that the pheromone may be a portion of a protein or a substance bound to a protein.
186 citations
TL;DR: Papain digestion of closed, right side out vesicle from pig, rat and rabbit jejunum brush border induces the release of the hydrolases bound to the membrane without grossly affecting the lipid bilayer limiting the vesicles, which definitely proves that intestinal hydrolase are surface components attached to the external side of the membrane.
164 citations
TL;DR: The results suggest that the appearance of a sulfhydryl-type endopeptidase activity is a necessary prerequisite for the rapid metabolism of the reserve proteins which accompanies germination.
Abstract: The autodigestive proteolytic activity of extracts of cotyledons of mung beans (Phaseolus aureus Roxb.) increased 4- to 5-fold during germination. A similar increase was found in the ability of these extracts to digest added casein or mung bean globulins. The increase occurred after a 2-day lag during the next 2 to 3 days of germination and coincided with the period of rapid storage protein breakdown. To understand which enzyme(s) may be responsible for this increase in proteolytic activity, the hydrolytic activity of cotyledon extracts toward a number of synthetic substrates and proteins was measured. Germination was accompanied by a marked decline in leucine aminopeptidase, while carboxypeptidase increased about 50%. There were no dramatic changes in either α-mannosidase or N-acetyl-β-glucosaminidase, enzymes which may be involved in the metabolism of the carbohydrate moieties of the reserve glycoproteins. The increase in general proteolytic activity was closely paralleled by a 10-fold increase in endopeptidase activity. This activity was inhibited by sulfhydryl reagents such as N-ethylmaleimide. Studies with inhibitors of proteolytic enzymes showed that reagents which blocked sulfhydryl groups also inhibited the rise in general proteolytic activity. Our results suggest that the appearance of a sulfhydryl-type endopeptidase activity is a necessary prerequisite for the rapid metabolism of the reserve proteins which accompanies germination.
163 citations
TL;DR: The stimulation of epidermal proliferation in organ cultures of chick embryo skin is dependent upon a number of conditions, among which are the age of the embryo and the presence of dermal cells.
Abstract: Publisher Summary This chapter discusses the interaction of epidermal growth factor (EGF) with cultured fibroblasts. EGF is a single-chain polypeptide containing a total of 53 amino acid residues. All the common amino acids are present except lysine, alanine, and phenylalanine. The molecule contains six half-cystine residues in disulfide bridges. EGF is antigenic and is sensitive to proteolytic digestion. It is heat stable, nondialyzable, and has an isoelectric point at pH 4.6. The chapter also discusses the complete amino acid sequence of EGF and presents the location of the three disulfide bridges. A direct stimulatory effect of EGF on epithelial cell proliferation in vitro has been demonstrated in a number of organ culture systems. These include embryonic skin, embryonic cornea, and mammary gland expiants. The effects of EGF are not species specific; expiants derived from the chick, mouse, and human are responsive. The stimulation of epidermal proliferation in organ cultures of chick embryo skin is dependent upon a number of conditions, among which are the age of the embryo and the presence of dermal cells.
142 citations
TL;DR: The enzyme, protoporphyrinogen oxidase, associated with purified mitochondria isolated from Saccharomyces cerevisiae was solubilized by sonic treatment in the presence of detergent and partially purified and was insensitive to cyanide, 2,4-dinitrophenol, and azide whereas it was inhibited in the absence of Cu-2- or Co-2+ ions, high ionic strength, heme, or hemin.
141 citations
TL;DR: It is shown that pupae of the giant silkmoth Samia cynthia have a humoral antibacterial activity, which was formed simultaneously with a selective incorporation of amino acids into eight polypeptide chains characterized by their electrophoretic behavior.
Abstract: We have previously shown that pupae of the giant silkmoth Samia cynthia have a humoral antibacterial activity, which was induced by viable, nonpathogenic gram-negative bacteria (H.G. Boman et al., 1974). We show here that this activity was formed simultaneously with a selective incorporation of amino acids into eight polypeptide chains characterized by their electrophoretic behavior. If actinomycin D or cycloheximide were given at an early time, no antibacterial activity was found. If the inhibitors were given at the time of maximum activity, there was no effect with actinomycin D but a rapid decrease of the activity in the case of cycloheximide. The results imply that the messenger ribonucleic acid was stable, but that at least one protein component was turning over. Hemolymph from immunized pupae of another giant silkmoth, Hyalophora cecropia, was fractionated by ammonium sulfate precipitation. This procedure, together with the isotope distribution after co-electrophoresis in polyarylamide gels, was used for comparing the response to injury and to different infections. Almost identical polypeptide patterns were obtained as a response to an infection with either viable Enterobacter cloacae or Bacillus subtilis. These patterns differed both qualitatively and quantitatively from the injury effect created by an injection as such. There was only a low antibacterial activity in each of the four fractions obtained by ammonium sulfate precipitation. However, a combination of three fractions restored a high killing activity. Fractionation of hemolymph from untreated pupae provided evidence for at least one preexisting factor which stimulated the killing of Escherichia coli. The osmotic pressure of the bacteria contributed to the antibacterial activity towards E. coli, but not towards B. subtitlis. The killing of E. coli was inhibited by liped A and, to a lesser extent, by an inhibitor of proteolytic enzymes. The similarities and differences with the mammalian complement system are discussed. Images
127 citations
TL;DR: Both groups of enzymes are ineffective against prothrombin whereas incubation with factor V leads to a transitory activation and the possible role of these enzymes in physiological and pathological hemostasis is discussed.
123 citations
TL;DR: It is demonstrated that the "activation" of yeast proteinases A, B and C upon incubation results from the proteolytic digestion of inhibitors rather than from activation of inactive zymogens by limited proteolysis.
119 citations
TL;DR: The chapter focuses on several cases in which proteolytic artefacts are known to have been particularly persistent, even after the existence of the problem was clearly recognized.
Abstract: Publisher Summary This chapter highlights the methods for avoiding proteolytic artefacts in studies of enzymes and other proteins from yeasts. Yeast cells contain a variety of different proteolytic enzymes. It has become increasingly apparent that these proteolytic enzymes pose a very serious problem for attempts to study other yeast proteins. The chapter refers explicitly only to Saccharomyces cerevisiae and closely related yeasts. A brief summary of the many studies is presented in which the activity of yeast proteases in vitro has presented a technical problem. The chapter focuses on several cases in which proteolytic artefacts are known to have been particularly persistent, even after the existence of the problem was clearly recognized. In the chapter, the most important properties of known yeast proteases are summarized. This information is important because a careful, thoughtful approach to the artefact problem must be conducted with the properties of the proteases in mind. The chapter also considers the virtues and limitations of the various methods that have been used, or that might be used, to cope with the protease problem.
TL;DR: The three major regions of difference show how this family of proteolytic enzymes has developed from the more primitive bacterial to the relatively sophisticated pancreatic enzymes.
Abstract: Although primary structural homology between bacterial serine proteases and those from the mammalian pancreas is slight, two-thirds of the residues in the bacterial enzyme SGPB as seen at 2.8-A resolution, adopt a similar polypeptide chain conformation to that of the chymotrypsin family. The three major regions of difference show how this family of proteolytic enzymes has developed from the more primitive bacterial to the relatively sophisticated pancreatic enzymes.
TL;DR: Using an in vitro method, some factors affecting the attachment of a strain of lactobacillus to chicken crop epithelial cells have been studied and it is suggested that these concanavalin A receptors on the lactOBacillus are responsible for its attachment to crop epithel cells.
Abstract: SUMMARY: Using an in vitro method, some factors affecting the attachment of a strain of lactobacillus to chicken crop epithelial cells have been studied. Time of contact beyond 10 min, pH value, age or growth temperature of the bacterial culture, or nature of the energy source in the growth medium had little or no effect on attachment. Heating to 100 °C for 10 min, or treatment with EDTA or surface active compounds was also without effect. Treatment with sodium periodate markedly decreased adhesion, proteolytic enzymes had a smaller effect but wheat germ lipase was completely inactive. The pronounced inhibition of adhesion periodate suggested the involvement of carbohydrate. However, enzymes known to attack carbohydrate substrates were inactive in reducing adhesion. Concanavalin A, which binds specifically to certain sugar residues, reduced attachment. It is suggested that these concanavalin A receptors on the lactobacillus are responsible for its attachment to crop epithelial cells.
TL;DR: The intact lectins are much more resistant to heat, freezing and chemical treatments than the isolated A- and B-chains and are also very resistant to treatment with proteolytic enzymes, whereas the isolated chains are easily digested.
TL;DR: The apparent Km and Ki values reported in this study indicate that the preferred pyrimidine substrate is orotate, and it seems likely, therefore, that this enzyme functions in vivo as an orotated phosphoribosyltransferase.
TL;DR: Treatment of the virion with the proteolytic enzyme bromelain removes the surface projections and VPI, thus identifying this glycopolypeptide as the major structural component of the projection.
Abstract: Summary
The polypeptides of purified preparations of the coronavirus responsible for transmissible gastroenteritis of pigs have been examined by polyacrylamide gel electrophoresis. Four major polypeptides, VP1 (mol. wt. 200000), VP2 (50000), VP3 (30000) and VP4 (28500) and two minor polypeptides, VP1a (105000) and VP1b (80500) have been reproducibly demonstrated in the virion, of which VP1, VP3 and VP4 contain carbohydrate. Treatment of the virion with the proteolytic enzyme bromelain removes the surface projections and VP1, thus identifying this glycopolypeptide as the major structural component of the projection.
TL;DR: The alveolar macrophage is found to be a potential source of proteolytic enzymes in man and has a significantly greater elastase-like esterase and protease activity than those of nonsmokers.
Abstract: Proteolysis (or more specifically, elastolysis) of the lung may be involved in the pathogenesis of pulmonary emphysema. To investigate the human alveolar macrophage as a potential mediator of lung damage, elastase-like esterase and protease activity was determined in these cells as well as in alveolar lavage fluid and in peripheral blood leukocytes. Bronchoalveolar lavage was used to obtain alveolar cells and fluid in normal volunteers who were divided into two groups according to cigarette smoking history, nonsmokers and smokers. Results of these studies revealed that human alveolar macrophages possess a high activity of both elastase-like esterase and protease. Furthermore, the alveolar macrophages of cigarette smokers has a significantly greater elastase-like esterase and protease activity than those of nonsmokers. When the 4- to 5-fold increase in the number of macrophages found cigaretts smokers is taken into account there was approximately 10 times more elastase-like esterase activity and 18 times more protease activity within macrophages in the alveolar spaces of cigarette smokers' lungs. This makes the alveolar macrophage a poten potential source of proteolytic enzymes in man.
TL;DR: Cell suspensions from monolayer cultures of 3T3, SV-3T 3, BHK, BS-C-1, or macrophages were prepared by brief exposure of the cultures to the amide anesthetic lidocaine by applicable to physiological, biochemical, or immunological studies in which exposure of cells to proteolytic enzymes is to be avoided.
Abstract: Cell suspensions from monolayer cultures of 3T3, SV-3T3, BHK, BS-C-1, or macrophages, were prepared by brief exposure of the cultures to the amide anesthetic lidocaine. Cells were subcultivated six times without marked impairment of plating efficiency. The method should be applicable to physiological, biochemical, or immunological studies in which exposure of cells to proteolytic enzymes is to be avoided.
TL;DR: Uukuniemi virus, grown in chicken embryo fibroblasts, has been studied by electron microscopy using negative staining, thin sectioning, and freeze-etching techniques to suggest that these units are penton-hexon clusters arranged in a T = 12, P = 3, icosahedral surface lattice.
Abstract: Uukuniemi virus, grown in chicken embryo fibroblasts, has been studied by electron microscopy using negative staining, thin sectioning, and freeze-etching techniques. The spherical virus particle measures about 95 nm in diameter. Its envelope consists of a 5-nm thick membrane covered by 8- to 10-nm long surface projections. These are composed of two polypeptides species of about the same size. Both of them can be removed by digestion with the proteolytic enzyme thermolysin except for a small fragment. The enzyme-treated particles are smooth surfaced and extremely deformable. The glycopolypeptides are clustered to form hollow cylindrical morphological units, 10 to 12 nm in diameter, with a 5-nm central cavity. Both negative staining and freeze-etching suggest that these units are penton-hexon clusters arranged in a T = 12, P = 3, icosahedral surface lattice. The membrane to which the surface subunits are attached is probably a lipid bilayer as evidenced by its double-track appearance in thin sections and the tendency of the freeze fracturing to occur within it. The strand-like nucleoprotein appears from thin-sectioning results to be to a large part located in a zone underneath the membrane.
TL;DR: Early evidence regarding the chemical properties of the modified enzyme suggests that tyrosine, rather than lysine, histidine, or arginine, may be the residue modified, and the functional consequences of successive modification with different reagents indicate that each of them reacts with the same protein residue.
Abstract: Synthesis of a series of active N-hydroxysuccinimide esters of aliphatic and aromatic amino acids has yielded a new class of reagents for the covalent modification of proteolytic enzymes such as thermolysin. The activities of aliphatic acyl amino acid thermolysins are from 1.7 to 3.6 times greater than that of the native enzyme when hydrolyzing durylacryloyl-Gly-Leu-NH2, the substrate employed most widely. By comparison, the aromatic acylamino acid derivatives are "superactive," their activities being as much as 70-fold greater. Apparently, the aromatic character of the amino acid introduced is a critical variable in the determination of the functional response. The increased activity is completely restored to that of the native enzyme by deacylation with nucleophiles, such as hydroxylamine, and the rate of restoration of native activity is a function of the particular acyl group incorporated. Preliminary evidence regarding the chemical properties of the modified enzyme suggests that tyrosine, rather than lysine, histidine, or arginine, may be the residue modified. The functional consequences of successive modification with different reagents, moreover, indicate that each of them reacts with the same protein residue. The competitive inhibitors beta-phenyl-propionyl-Phe and Zn-2+ do not prevent modification with these active esters. Hence, the site(s) of their inhibitory action differ(s) from that at which modification occurs. The structure of the substrate is also a significant variable which determines the rate at which each acyl amino acid thermolysin hydrolyzes peptides. Depending on the particular substrate, the activity of aromatic derivatives can be as much as 400-fold greater than that of the native enzyme, and the resultant activity patterns can be ordered in a series characteristic for each enzyme derivative.
TL;DR: The results confirm and extend previous observations by Hirst that influenza C receptors differ from receptors for myxoviruses and suggest they do not contain sialic acid, and conclude that “influenza C” lacks an α-neuraminidase.
Journal Article•
TL;DR: The requirement for macrophage-lymphocyte interaction in NaIO4 or NG-induced lymphocyte proliferation is indicative of a central role for the macrophages in the initiation of T lymphocytes proliferation.
Abstract: Guinea pig T lymphocyte proliferation induced by sodium periodate (NaIO4) or neuraminidase-galactose oxidase (NG) occurs when lymphocytes and macrophages are cultured together after treatment of either purified T lymphocytes or macrophages with these agents Regardless of which cell initially bears the modified surface carbohydrate, lymphocyte proliferation requires the presence of viable homologous macrophages and fails to occur when they are replaced with fibroblasts, erythrocytes, L2C leukemia cells, thymocytes, PMN, line I hepatoma cells, or murine macrophages Lymphocyte proliferation resulting from NaIO4 or NG treatment of lymphocytes is diminished when these cells are treated with proteolytic enzymes or aged in in vitro culture for 48 hr By contrast, proteolytic enzyme treatment or in vitro aging has no effect on the ability of NaIO4 or NG-treated macrophages to induce lymphocyte proliferation The requirement for macrophage-lymphocyte interaction in NaIO4 or NG-induced lymphocyte proliferation is indicative of a central role for the macrophage in the initiation of T lymphocyte proliferation
TL;DR: The finding that radioactivity in the solubilized [3H]etorphine-macromolecular complex was virtually eliminated when binding had occurred in the presence of excess levorphanol, an active narcotic analgesic, while it was unaffected by its inactive enantiomorph dextrorphan was demonstrated.
Abstract: A [3H]etorphine-macromolecular complex has been solubilized from rat brain synaptosomal fraction by extraction with the nonionic detergent Brij 36T. Stereospecificity of binding to this solubilized complex was demonstrated by the finding that radioactivity in the complex was virtually eliminated when binding had occurred in the presence of excess levorphanol, an active narcotic analgesic, while it was unaffected by its inactive enantiomorph dextrorphan. Bound radioactivity was dissociated by proteolytic enzymes, sulfhydryl reagents, and heat, suggesting the presence of protein. The bound solubilized macromolecular moiety may be the opiate receptor.
TL;DR: Results suggest that the VS viral glycoprotein is an amphipathic molecule, the hydrophilic portion of which contains all the carbohydrate and a lipophilic tail segment which forms lipid or detergent micelles, thus rendering it resistant to proteolysis.
Abstract: The proteolytic enzyme, thermolysin, degraded the external segment of the membrane glycoprotein of intact vesicular stomatitis (VS) virions but left behind a small nonglycosylated fragment, presumably embedded in the virion membrane Other proteases generated membrane-associated glycoprotein fragments differing somewhat in molecular weight The thermolysin-resistant, virion-associated fragment, which can be selectively solubilized by either Triton X-100 or chloroform/methanol, has a molecular weight of 5,200 Amino acid analysis of the glycoprotein fragment reveals a preponderance of hydrophobic amino acids (64% of the residues); the amino-terminal amino acid is alanine as determined by dansylation Cyanogen bromide digestion of the tail fragment generated two peptides, confirming the presence of one methionine residue per thermolysin-resistant glycoprotein fragment The secondary structure of this glycoprotein tail peptide is maintained by at least one disulfide bridge Thermolysin treatment is isolated VS viral glycoprotein in the presence of Triton X-100 also generated a hydrophobic peptide fragment which is very similar to the virion-associated glycoprotein fragment The amino acid terminus of intact glycoprotein was also found to be alanine as was its dansylated Triton-micellar fragment that resisted thermolytic degradation; this finding suggests that the amino-terminal end of the VS viral glycoprotein is embedded in the virion membrane These results suggest that the VS viral glycoprotein is an amphipathic molecule, the hydrophilic portion of which contains all the carbohydrate and a lipophilic tail segment which forms lipid or detergent micelles, thus rendering it resistant to proteolysis
TL;DR: It is reported here that mouse PCs in culture develop a plaque-forming activity against isologous BrMRBC as well as against SRBC, and it is shown that these two types of erythrocytes share some common antigen.
Abstract: WE have reported that peritoneal cells (PCs) from unstimulated mice can, after 2 h of incubation in the appropriate medium, start to secrete anti-sheep erythrocyte (SRBC) antibodies1,2. We extended this observation by showing that PCs cultured in standard conditions for 4–6 d in the absence of SRBC, can form numerous haemolytic plaques, demonstrable by the ordinary techniques of local haemolysis (in Agarose, in liquid layer or in cellulose gum3). The immunological nature of this plaque-forming activity was established by the following criteria: complement dependency, inhibition by anti-mouse IgM serum and immunological specificity. Thus, we concluded3,4 that PCs of normal adult mice had been stimulated previously by an unknown immunogen sharing common or cross-reacting determinants with SRBC, and that tissue culture conditions would derepress the built-in capacity of these cells to produce antibodies. Treatment of autologous erythrocytes by the proteolytic enzyme bromelain revealed that normal mouse spleen cells regularly produce antibodies against their own erythrocytes5,6. This led us to investigate the behaviour of normal PCs towards SRBC or mouse red blood cells (MRBCs) treated by bromelain (BrMRBCs). We report here that mouse PCs in culture develop a plaque-forming activity against isologous BrMRBC as well as against SRBC, and we show that these two types of erythrocytes share some common antigen.
TL;DR: The decrease of hydrolytic activity after 10 days of age seems to parallel a decrease in the rates of protein breakdown in vivo, showing parallel behavior with decreasing protein turnover.
TL;DR: These studies confirm the release of pancreatic enzymes during bile-induced pancreatitis, and quantify an important protective role for plasma protease inhibitors in this situation.
Journal Article•
TL;DR: It was found that not only NZB mice, a known autoimmune strain, but also several normal strains of mice contain cells in small numbers in their spleens and in largerNumbers in their peritoneal cavities which will form plaques against bromelain-treated MRBC.
Abstract: Treatment of mouse erythrocytes with the proteolytic enzymes, bromelain, reveals antigenic determinants not normally exposed on the erythrocyte surface. It was found that not only NZB mice, a known autoimmune strain, but also several normal strains of mice contain cells in small numbers in their spleens and in larger numbers in their peritoneal cavities which will form plaques against bromelain-treated MRBC. During in vitro culture the number of anti-BR-MRBC PFC increases slightly in the spleen cell populations whereas the number of these PFC in peritoneal cells increases dramatically to as many as 100,000 PFC/10(6) cells. The plaques detected in this assay contain a central lymphoid cell and their development, which requires the presence of complement and protein synthesis, is inhibited by anti-mouse immunoglobulin.
TL;DR: The fixation of the third component of complement (C3) results in many important biological phenomenon, among which are immune adherence, enhancement of phagocytosis, and the release of an anaphylatoxin which is a potent releaser of histamine.
Abstract: The fixation of the third component of complement (C3) results in many important biological phenomenon, among which are (a) immune adherence (1), (b) enhancement of phagocytosis (2,3), (c) the release of an anaphylatoxin which is a potent releaser of histamine (4), and (d) the feedback activation of the alternative pathway (5,6). The physiological mechanisms involving C3 fixation require the generation of a C3 convertase which may occur by two separate pathways. C3 convertase can be generated, in the form of C42, by the so-called classical pathway of activation or in the form C3b,B by the alternative or properdin pathway (7). In both cases, C3 is converted to C3b by cleavage of a small peptide, C3a. Normal human serum contains an inactivator of activated C3b. This C2b inactivator or conglutinogen-activating factor (KAF) has been shown to inhibit both immune hemolysis and the immune adherence properties of C3b and to cause cleavage of C3b in the fixed and fluid- phase stages (8-11). Although it is known that the C3b inactivator is not depleted during its reaction with C3b and that C3b treated with the C3b inactivator becomes extremely sensitive to proteolytic digestion by trypsin and “trypsin-like” enzymes (9), the exact molecular nature of the action of the C3b inactivator on C3b has not been studied. In an effort to delineate the products of this interaction, purified C3b and C3b inactivator were allowed to react for various specific lengths of time and the products of these reactions were then analyzed.
TL;DR: It is concluded that the faecal enzymes of the fungus-growing ants are derived from the mycelial fluid upon which they feed, and the basis for the symbiosis between the attine ants and the fungi which they cultivate in their nests is reinterpreted in the context of this finding.