Showing papers on "Proteolytic enzymes published in 1979"

The use of proteolytic enzymes to improve immunoglobulin staining by the PAP technique.

Abstract: Proteolytic enzymes, protease and trypsin have recently been introduced to reduce the inconsistency hitherto encountered in the unlabelled antibody-enzyme method using PAP. This study investigated factors determining the optimum conditions for use of such enzymes in order to establish which one is most suitable. Trypsin was the most effective enzyme; however, its activity decreased over 3 h, a feature paralleled immunocytochemically. Method and duration of fixation appears to influence the required time of exposure to trypsin in order that consistent immunostaining may be produced. Treatment of sections with trypsin prior to the use of the unlabelled antibody-enzyme method using PAP renders the technique reliable, provided the enzyme is used in a carefully controlled manner.

Membrane proteins specified by herpes simplex viruses. V. Identification of an Fc-binding glycoprotein.

Abstract: A glycoprotein with affinity for the Fc region of immunoglobulin was isolated from extracts of cultured cells infected with herpes simplex virus type 1, and experiments were done to characterize its properties and to investigate whether it could account for the Fc-binding activity previously demonstrated on the surfaces of intact herpes simplex virus-infected cells. The technique of affinity chromatography was used to identify and isolate the Fc-binding glycoprotein and to demonstrate the specificity of its interaction with immunoglobulin G-Fc. Although three electrophoretically distinguishable Fc-binding polypeptides were identified by affinity chromatography, these three species appear to be different forms of the same translation product, based on comparisons of proteolytic digestion products and on the kinetics of appearance of each form after a brief pulse with radioactive amino acids. The results suggest that one polypeptide, designated pE, is processed to yield gE1, which is in turn processed to yield gE2. Both gE1 and gE2 are glycosylated membrane proteins and both can be labeled by the lactoperoxidase-catalyzed radioiodination of intact infected cells, indicating the presence of these proteins in surface membranes of the cells. Increases in the amounts of gE1 and gE2 at the cell surface were found to parallel the increase in Fc-binding activity of intact infected cells.

Measuring protein quality.

Abstract: An alternative to the time-consuming and expensive PER assay for measuring food protein quality is needed by the food industry. Many biological and chemical-based assays for measuring protein quality have been described in the literature. Most of these are still too complicated, time-consuming, or too narrow in the range of foods they will test for daily quality control use. In the past five years, rapid methods have been developed that employ chemical assays for essential amino acid composition and availability or biological assays that measure protein digestibility and growth on food proteins. Most of these assays can be completed in five days or less and are applicable to a broad range of foods. These developments have brightened the prospects for the eventual development of a rapid assay that the food industry routinely can use to monitor protein quality. This paper has discussed two assays that were tested with a wide variety of foods and that take less than 72 hr to complete. The C-PER assay, uses data on the in vitro protein digestibility and EAA composition of a food protein to predict its protein quality in terms of PER. The C-PER technique is not limited by the protein, fat, additive or spice levels in the food to be tested, and is therefore applicable to a wide range of food ingredients and processed foods. The second assay is based on the growth of the protozoanTetrahymena thermophila WH14 on a proteolytic enzyme hydrolyzed food sample along with in vitro protein digestibility data to predict protein quality in terms of T-PER. Because theTetrahymena are more difficult to control on a day to day basis, the error of the T-PER estimate is greater than that for the C-PER estimate. Also, sinceTetrahymena growth is greatly affected by various food additives and spices, caution should be used when this assay is used to measure protein quality in foods where the composition is not definitely known. The T-PER assay is best suited for assaying protein quality in protein-containing food ingredients, such as meats, flours, protein concentrates and isolates, or on foods where the exact composition is known.

Scleroderma: increased biosynthesis of triple-helical type I and type III procollagens associated with unaltered expression of collagenase by skin fibroblasts in culture.

Abstract: To assess potential abnormalities in collagen metabolism in systemic scleroderma, skin fibroblast lines from patients with this disease were established and compared to control cell lines derived from healthy subjects. For studies on the biosynthesis of procollagen, the cells were incubated with [(14)C]proline in a medium supplemented with ascorbic acid and beta-aminopropionitrile, and the synthesis of nondialyzable [(14)C]hydroxyproline, in relation to DNA or cell protein, was taken as an index of procollagen formation. Five of eight scleroderma fibroblast cell lines demonstrated procollagen biosynthesis rates significantly higher than the controls, and the mean rate of procollagen synthesis by scleroderma fibroblasts was about twice that of the control cells. Control experiments demonstrated that the specific activity of the intracellular free proline was not different in scleroderma and control fibroblasts, and the mean population doubling times of the scleroderma and the control fibroblast cell lines were the same. The relative synthesis of the genetically distinct procollagens was examined by isolating type I and type III procollagens from the cell culture medium using DEAE-cellulose chromatography. The ratios of type I/III procollagens in scleroderma cell lines did not differ from the controls. The helical stability of the collagenous portion of type I and type III procollagens, estimated by the resistance of (14)C-collagen to limited proteolytic digestion with pepsin under nondenaturing conditions, was the same in both scleroderma and control cultures. The capacity of the cells to synthesize enzymatically active and immunologically reacting collagenase was also studied; no marked differences in these parameters could be observed. The results suggest that cultured skin fibroblasts from patients with scleroderma demonstrate a metabolic abnormality expressed as increased synthesis of type I and type III procollagens in a normal ratio. This abnormality may play a role in the excessive accumulation of collagen in the skin and other organs affected in scleroderma.

Demonstration of Hepatitis B e Antigen in the Core of Dane Particles

Abstract: Dane particles were isolated in a large scale from plasma of asymptomatic carriers of hepatitis B surface antigen. The core of Dane particles was exposed by treatment with NP-40 and 2-mercaptoethanol. The antigenicity of hepatitis B e antigen (HB e Ag) was not detected on the surface of Dane particles or of their cores. However, when Dane particle cores were treated with Pronase, some activity of HB e Ag was liberated from them as a small molecule. When the cores were further treated with SDS, they revealed a high activity of HB e Ag, indicating that HB e Ag existed in the core of Dane particles in a cryptic form, which can be exposed by treatment with proteolytic enzyme or SDS. Dane particles and their cores were subjected to polyacrylamide gel electrophoresis in SDS. After electrophoresis, the gel was cut into two halves, and each half was determined for polypeptide composition and for HB e Ag activity. Both of Dane particles and their cores disclosed two peaks of HB e Ag activity associated with molecules with a size of 19,000 and 45,000 daltons. These two polypeptides were the major constituents of the cores. Dane particles revealed several polypeptides in addition to the HB e Ag polypeptides, which were identified as hepatitis B surface antigen components. On the basis of the results obtained, HB e Ag is an integral component of Dane particles, the presently accepted hepatitis B virions. These results provide the basis for the close correlation between HB e Ag and Dane particles in the serum of persons infected with hepatitis B virus.

Guidelines for the Use of Thrombolytic Agents

Abstract: TWO agents that induce thrombolytic activity have recently been approved for clinical use by the Food and Drug Administration: streptokinase (marketed as Streptase by Hoechst-Roussel Pharmaceuticals, Somerville, NJ, and as Kabikinase by Kabi Group, Greenwich, CT) and urokinase (marketed as Abbokinase by Abbott Laboratories, North Chicago, IL).1 These drugs activate plasminogen and thus cause the formation of the proteolytic enzyme plasmin. (Other agents with the potential for activating the human fibrinolytic system include Thrombolysin and Actase. These drugs are mixtures of streptokinase, plasminogen, activator complex, and plasmin and have not been subjected to controlled trials in thromboembolic disease.) Clinical studies . . .

Effect of human leukocyte enzymes on tracheal mucosa and its mucociliary activity.

Abstract: Human neutrophil granulocytes contain proteolytic enzymes. In purulent bronchial and paranasal secretions these enzymes have been found extra cellulary in complex with enzyme inhibitors as well as in free form indicating saturation of the inhibiting capacity. Isolated human leukocyte enzymes, elastase and neutral protease, were found to arrest the mucociliary activity and subsequently cause superficial tissue destruction. Elastase was found to be the most potent of the enzymes. Experimental studies with elastase together with specific inhibitor indicated the importance of the enzyme inhibitors for the integrity of the mucous membrane.

Differences in A and B forms of monoamine oxidase revealed by limited proteolysis and peptide mapping

Abstract: Monoamine oxidase (MAO, EC 1.4.3.4) is a mitochondrial outer membrane enzyme which deaminates amine transmitters in the nervous system and biogenic amines throughout the body. At least two types of MAO activity have been identified which differ in their specificities for substrates and sensitivities to inhibitors1–3. Type A preferentially deaminates 5-hydroxy-tryptamine and noradrenaline and is more sensitive to inhibition by clorgyline. Type B deaminates phenylethylamine and benzylamine and is more sensitive to inhibition by deprenyl. Tissues from several species express MAO A alone, MAO B alone, or both types of MAO. Furthermore, the two types of activity follow different developmental sequences in certain tissues. Models to explain the molecular and genetic regulation of this enzyme4 raise two questions: (1) are there different enzyme molecules associated with A and B types of activity, and (2) if there is only one enzyme molecule, how do differences in the microenvironment of this enzyme within the outer mitochondrial membrane produce two types of activity? Attempts to separate and purify the different types of activity have involved solubilisation and lipid extraction procedures that alter the kinetics of the enzyme and differentially affect the A and B types of activity5–8. Immunological studies9,10 have produced equivocal results. None of these observations has elucidated the molecular basis of the differences between the types of MAO activity. Here, we have used the technique of proteolytic digestion and peptide mapping to demonstrate that there are distinct enzyme molecules associated with the A and B types of MAO activity. This finding implies that the A and B forms of MAO may be coded for by separate gene loci and represent evolutionarily related proteins which have diverged in function.

Membrane biogenesis: cotranslational integration of the bacteriophage f1 coat protein into an Escherichia coli membrane fraction

Abstract: The coat protein (CP) of bacteriophage f1 is integrated into an Escherichia coli plasma membrane fraction consisting of inverted vesicles when it is synthesized in a cell-free, coupled transcription--translation system supplemented with the inverted vesicles. By using proteolytic enzymes as probes, we found by subsequent peptide mapping and determination of the sequence of the proteolytic products that CP was inserted into the inverted vesicles in an orientation indistinguishable from that in inverted vesicles prepared from infected E. coli: only a COOH-terminal portion of approximately 10 residues was accessible to proteolysis, whereas the remainder of CP (CP') was entirely protected. Protection of CP' was dependent on the integrity of the vesicle membrane, because it was abolished when proteolysis was done in the presence of nonionic detergents. Insertion was observed when the inverted vesicles were present during translation in the cell-free system, not when they were added after translation. Thus, the asymmetric insertion of this type of integral membrane protein is strictly coupled to translation. These findings are discussed with respect to prokaryotic membrane biogenesis and are related to bacteriophage f1 assembly and infection.

Cell-Free Transcription and Translation of Total Spinach Chloroplast DNA

Abstract: A highly efficient cell-free Escherichia coli system for the coupled transcription/translation of chloroplast DNA is described. Evidence for the synthesis of more than 20 discrete size classes of [35S]-methionine-labelled polypeptides has been obtained by dodecylsulphate/polyacrylamide gel electrophoresis of the products formed in a reaction programmed with spinach chloroplast DNA. One of the major radioactive products co-electrophoresed with the large subunit (Mr 55000) of ribulosebisphosphate carboxylase (fraction 1 protein). Its identity as the large subunit was established by comparing its partial proteolytic digestion products with those of purified large subunit protein. Because the level of radioactivity in many of the other polypeptides synthesized in vitro is so high, their identification likewise should be possible by comparing them with known chloroplast proteins. When spinach chloroplast DNA that had been digested with certain restriction endonucleases was used to programme the transcription/translation system, some of the polypeptides, which were observed using undigested DNA, were not detectable among the products. These results are discussed with regard to the distribution of restriction enzyme sites in the operons for some of the polypeptides, and with regard to the information they can provide on the location of genes on a restriction enzyme cleavage map of chloroplast DNA.

Membrane biogenesis. In vitro cleavage, core glycosylation, and integration into microsomal membranes of sindbis virus glycoproteins.

Abstract: Sindbis virus 26S RNA has been translated in a cell-free protein-synthesizing system from rabbit reticulocytes. When the system was supplemented with EDTA-stripped dog pancreas microsomal membranes, the following results were obtained: (a) Complete translation of 26S RNA, resulting in the production, by endoproteolytic cleavage, of three polypeptides that are apparently identical to those forms of C, PE2, and E1 that are synthesized in vivo by infected host cells during a 3-min pulse with [35S]methionine. (b) Correct topological deposition of the three viral polypeptides--in vitro-synthesized PE2 and E1 forms are inserted into dog pancreas microsomal membranes in a orientation which, by the criterion of their limited (or total) inaccessibility to proteolytic probes, is indistinguishable from that of their counterparts in the rough endoplasmic recticulum of infected host cells; in vitro-synthesized C is not inserted into membranes and therefore is accessible to proteolytic enzymes, like its in vivo-synthesized counterpart. (c) Core glycosylation of in vitro-synthesized PE2 and E1 forms, as indicated by binding to concanavalin A Sepharose and subsequent elution by alpha-methylmannoside.

The nature of the hydroxyapatite-binding site in salivary acidic proline-rich proteins

Abstract: Protein A and C, which are major components of the acidic proline-rich proteins in human saliva, were digested, before or after adsorption to hydroxyapatite, with alkaline phosphatase, trypsin, thermolysin and a proteinase preparation from salivary sediment. The results demonstrate that the binding site is located in the proline-poor N-terminal part of the protein, possibly between residues 3 and 25. Phosphoserine is necessary for maximal adsorption of the proteins to hydroxyapatite. When proteins A and C are adsorbed to hydroxyapatite before proteolytic digestion there is a protection of some of the susceptible bonds in the N-terminal part of the proteins and a gradual removal of the proline-rich C-terminal part. Thermolysin can cleave susceptible bonds in the part of the protein that remains bound to hydroxyapatite, but at least some of the resulting peptides are retained on the mineral. Since the ability of the proteins to inhibit hydroxyapatite formation and to bind calcium is located in the N-terminal proline-poor part, it is possible that these activities are retained after proteolytic digestion of the adsorbed proteins.

Proteolytic Enzyme in Porcine Immature Enamel

Abstract: A proteolytic enzyme cleaving the main component of enamel proteins obtained from immature enamel has been purified from a soluble extract of porcine immature enamel. It is optimally active around pH 6 against enamel protein. It is completely inhibited by phenylmethylsulfonyl fluoride and diisopropyl phosphofluoridate, and partially by benzamidine. EDTA does not affect its activity. The enzyme seems to sever initially enamel protein into two segments, one containing lysine, arginine and tyrosine and the other being free from these amino acids.

Higher molecular weight forms of immunoreactive somatostatin in mouse hypothalamic extracts: evidence of processing in vitro.

Abstract: Extracts of mouse hypothalamus made in acid/urea containing protease inhibitors were analyzed for somatostatin immunoreactivity after molecular sieve filtration on Sephadex G-50. Higher molecular weight (higher-Mr) somatostatin-like forms with apparent molecular weights of 15,000, 10,000, and 6000 could be identified, besides the molecular weight 1600 somatostatin. Immunological identities with somatostatin were unambiguously demonstrated by the analysis of the displacement curves in the radioimmunoassay. The Mr 15,000, 6000, and 1600 species were purified by affinity chromatography on an anti-somatostatin immune serum covalent conjugate with Sepharose used as immunoadsorbant. After disulfide reduction by dithiothreitol, the size of the Mr 15,000 and 6000 somatostatin-like species was assessed either by molecular sieve filtration or by polyacrylamide gel electrophoresis. The results indicated that the higher-Mr somatostatin-like species isolated from the hypothalamus did not result from hormone polymerization by means of disulfide interchange. The processing in vitro of the 15,000 higher-Mr form of somatostatin was achieved by proteolytic enzymes coeluted with this species during the fractionation of hypothalamic extracts. Under neutral pH conditions the intermediary higher-Mr forms were generated together with the Mr 1600 somatostatin-like species. This processing activity could be either strongly inhibited at acidic pH or in acid/urea medium or else eliminated by selective immunoadsorption of the 15,000 higher-Mr form. Neither trypsin nor the γ subunit of 7S nerve growth factor was able to produce this processing, suggesting that enzymes with other kinds of specificity may be involved. It is concluded that somatostatin biosynthesis in the mouse hypothalamus may occur via a high-Mr precursor that is processed into intermediary forms leading to the tetradecapeptide hormone.

Induction of lysosomal enzyme secretion by alveolar macrophages in response to the purified complement fragments C5a and C5a des-arg.

Abstract: Purified C5a and its “inactive” form, C5a des-arg, were shown to induce secretion of acid hydrolases from rabbit alveolar macrophages (AM) in a concentration-dependent manner. Secretion increased with time to 5 times above controls by 72 hr. Concentrations of these enzymes in the cell lysates did not decrease during the incubation, suggesting that synthesis of new enzyme was occurring. The lysosomal enzyme secretion was accompanied by increased pinocytosis and release of proteolytic enzymes from the macrophages. At no time was significant lactic dehydrogenase liberated, indicating that secretion was selective and not due to cell death. Data presented also suggest that C5a des-arg induced secretion from the macrophages of a chemotactic factor for neutrophils. It was concluded that C5a and C5a desarg may play a role in lung injury by interactions with AM, inducing the secretion of acid hydrolases and proteolytic enzymes that can cause tissue damage, and by regulating the influx of other inflammatory cells into the interstitium and air spaces.

Ca2+ and cyclic AMP regulate phosphorylation of same two membrane-associated proteins specific to nerve tissue

Abstract: It was shown previously that addition of cyclic AMP (cAMP) to a synaptic membrane fraction incubated with [gamma-32P]ATP stimulated the phosphorylation of two proteins, designated proteins Ia and Ib, found only in nerve tissue. Addition of Ca2+ plus veratridine to synaptosomes preincubated with 32Pi stimulated the phosphorylation of two proteins with similar apparent molecular weights. Various techniques have now been used to determine whether the two proteins phosphorylated in synaptosomes in the presence of Ca2+ plus veratridine are the same as proteins Ia and Ib phosphorylated in synaptic membranes in the presence of cAMP. The proteins phosphorylated by the two procedures were extracted under similar conditions, had similar apparent molecular weights and charges, and were digested by collagenase at similar rates and to the same radioactive intermediates and end products. Furthermore, the two sets of proteins were digested by three other proteolytic enzymes to phosphopeptides with similar molecular weights. The results indicate that Ca2+ and cAMP are each capable of regulating the phosphorylation of proteins Ia and Ib.

Ethidium fluorescence assay. Part II. Enzymatic studies and DNA-protein interactions

Abstract: Almost all DNA and RNA metabolizing enzymes can be assayed rapidly and very sensitively by exploiting the enhanced fluorescence of ethidium intercalated into duplex DNA or RNA. Denatured DNA and natural RNAs contain duplex regions due to intramolecular hydrogen-bonding and can also be sensitively measured. Where the product is truly single-stranded (e.g. dTn) it can be assayed by adding the appropriate complementary strand (e.g. dAn or rAn). Some of the assays described provide information not readily obtained by other assay procedures. Among the enzymes readily assayed are DNA and RNA polymerases, terminal deoxynucleotidyl transferases, nucleases of all varieties (e.g. single-strand specific, endonucleases including for example AP endonucleases, exonucleases, RNase H, etc.), ligases, topoisomerases including gyrases, and indirectly enzymes such as proteases and superoxide dismutase. DNA binding proteins such as histones and helix destablizing proteins can also be quantitatively assayed.

Why do skin grafts fail

Abstract: Fibrin is shown to be the agent responsible for the adherence of biological dressings and of autografts to wounds. Its presence is associated with graft success, and its absence with graft failure. The results suggest that the deposition of fibrin provides the basis for the anti-bacterial actions of biological dressings and for the sterilization of the wound under adherent autografts. The total number of bacteria per gram of tissue in the wound, though important, is not critical to the result of skin grafting. The mechanism by which different organisms cause grafts to fail is by the production of plasmin and proteolytic enzymes which dissolve the important fibrin scaffold--thus ensuring their own survival. Thus, it is the levels of these (and the numbers of organisms efficient in producing them) which cause success or failure of applied skin grafts.