Showing papers on "Proteolytic enzymes published in 1977"

Binding of chloromethyl ketone substrate analogs to crystalline papain

Abstract: Papain (EC 3.4.22.2) is a proteolytic enzyme, the three-dimensional structure of which has been determined by x-ray diffraction at 2.8 A resolution (Drenth, J., Jansonius, J.N., Koekoek, R., Swen, H. M., and Wothers, B.G. (1968), Nature (London) 218, 929-932). The active site is a groove on the molecular surface in which the essential sulfhydryl group of cysteine-25 is situated next to the imidazole ring of histidine-159. The main object of this study was to determine by the difference-Fourier technique the binding mode for the substrate in the groove in order to explain the substrate specificity of the enzyme (P2 should have a hydrophobic side chain (Berger and Schechter, 1970) and to contribute to an elucidation of the catalytic mechanism. To this end, three chloromethyl ketone substrate analogues were reacted with the enzyme by covalent attachment to the sulfur atom of cysteine-25. The products crystallized isomorphously with the parent structure that is not the native, active enzyme but a mixture of oxidized papain (probably papain-SO2-) and papain with an extra cysteine attached to cysteine-25. Although this made the interpretation of the difference electron density maps less easy, it provided us with a clear picture of the way in which the acyl part of the substrate binds in the active site groove. The carbonyl oxygen of the P1 residue is near two potential hydrogen-bond donating groups, the backbone NH of cysteine-25 and the NH2 of glutamine-19. Valine residues 133 and 157 are responsible for the preference of papain in its substrate splitting. By removing the methylene group that covalently attaches the inhibitor molecules to the sulfur atom of cysteine-25 we obtained acceptable models for the acyl-enzyme structure and for the tetrahedral intermediate. The carbonyl oxygen of the P1 residue, carrying a formal negative charge in the tetrahedral intermediate, is stabilized by formation of two hydrogen bonds with the backbone NH of cysteine-25 and the NH2 group of glutamine-19. This situation resembles that suggested for the proteolytic serine enzymes (Henderson, R., Wright, C. S., Hess, G. P., and Blow, D. M. (1971), Cold Spring Harbor Symp. Quant. Biol. 36, 63-70; Robertus, J. D., Kraut, J., Alden, R. A., and Birktoft, J. J. (1972b), Biochemistry 11, 4293-4303). The nitrogen atom of the scissile peptide bond was found close to the imidazole ring of histidine-159, suggesting a role for this ring in protonating the N atom of the leaving group (Lowe, 1970). This proton transfer would be facilitated by a 30 degrees rotation of the ring around the C beta-Cgamma bond from an in-plane position with the sulfur atom to an in-plane position with the N atom. The possibility of this rotation is derived from a difference electron-density map for fully oxidizied papain vs. the parent protein.

Cleavage at glutamic acid with staphylococcal protease.

Abstract: Publisher Summary Proteolytic enzymes catalyzing the hydrolysis of peptide bonds involving exclusively the basic amino acid residues lysine and arginine have been available for many years. Trypsin is by far the best known enzyme exhibiting this high degree of specificity and, for that reason, it has played a central role in the studies of the primary structure of proteins. Recently, enzymes that specifically cleave peptide bonds at the carboxyl group of the acidic amino acid residues, aspartic acid and glutamic acid, have been discovered. One of these enzymes, staphylococcal protease, has this specificity and can be further restricted to glutamyl bonds only under certain controlled conditions. This enzyme can be used for the determination of the amino acid sequences of several proteins and proved to be another valuable tool for such studies. Staphylococcal protease shows a marked preference for certain aspartyl bonds when used in ammonium bicarbonate or acetate buffer. The staphylococcal protease is fully active in the presence of 0.2% sodium dodecyl sulfate and retains 50% of its activity in a 4 M urea solution. Digestion under these conditions could be attempted for proteins or peptides that are not readily attacked by the protease under nondenaturing conditions.

Injectable collagen for soft tissue augmentation.

Abstract: Collagen from allogenic and xenogeneic sources has been made soluble by controlled proteolytic digestion. The monomeric, telopeptide-poor collagen so prepared forms a cohesive mass when warmed to body temperature (upon injection), creating a structural basis for new soft connective tissue. We have treated 28 patients with human and/or bovine collagen injections for the correction of soft tissue contour defects, and we have followed them for 3 to 18 months. In most instances, there has been lasting and substantial correction of the defects treated. The complications have been few and transient.

Nascent prehormones are intermediates in the biosynthesis of authentic bovine pituitary growth hormone and prolactin

Abstract: Major translation products of bovine pituitary RNA in a wheat germ cell-free system were identified as larger forms (prehormones) of growth hormone and prolactin containing amino-terminal extensions of 26 or 27 and 30 amino acid residues, respectively. However, translation of bovine pituitary RNA in the wheat germ cell-free system in the presence of microsomal membranes prepared from canine pancreas or bovine pituitary yielded products that were of the same size as authentic growth hormone and prolactin; by partial amino-terminal sequence analysis they were shown to contain the correct unique amino-terminal sequence of prolactin and the two correct amino termini of authentic growth hormone; moreover, they were found to be segregated within the microsomal vesicles in that they were largely inaccessible to degradation by proteolytic enzymes. When microsomal membranes were present after rather than during translation, prehormones were neither cleaved nor segregated. These results strongly suggest that the synthesis and segregation of the authentic hormone observed in the presence of membranes proceeds via a nascent prehormone rather than a completed prehormone.

Reconstitution of Semliki forest virus membrane

Abstract: The spike glycoproteins of the Semliki forest virus membrane have been incorporated into vesicular phospholipid bilayers by a detergent-dialysis method. The detergent used was beta-D-octylglucoside which is nonionic and has an exceptionally high critical micellar concentration which facilitates rapid removal by dialysis. The vesicles obtained were of varying sizes and had spikes on their surface. Two classes of vesicles were preferentially formed, small protein-rich and large lipid-rich (average lipid to protein weight ratios, 0.22 and 3.5, respectively). Both classes of vesicles retained the hemagglutinating activity of the virus. The proteins were attached to the lipid bilayer by hydrophobic peptide segments, as in the viral membrane. Most of the proteins were accessible to proteolytic digestion from the outside, suggesting an asymmetric orientation.

Maize chloroplast DNA fragment encoding the large subunit of ribulosebisphosphate carboxylase.

Abstract: In vitro linked transcription-translation of chloroplast DNA has been used to show that the large subunit of ribulose-1,5-bisphosphate carboxylase [3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39] is encoded by Zea mays chloroplast DNA. A BamHI-generated chloroplast DNA sequence cloned in Escherichia coli is shown to direct the in vitro synthesis of this protein identified as large subunit by its size, serological properties, and limited proteolytic digestion products.

Purification and characterization of a cardioexcitatory neuropeptide from the central ganglia of a bivalve mollusc.

Abstract: We have purified a cardioexcitatory substance, previously designated peak C, from ganglia of the Sunray Venus clam, Macrocallista nimbosa Low concentrations (10−9 -10−8M) of this substance not only excite the isolated clam heart, but also produce tonic contractions of the isolated radula protractor muscle of the whelk, Busycon contrarium These two muscle preparations have therefore been used as a parallel bioassay for peak C Peak C is inactivated by proteolytic enzymes, has an isoelectric point greater than pH 10 and has an ultraviolet absorption spectrum similar to that of phenylalanine On thin layer chromatograms, peak C separates into two components; one of these is probably a partially oxidized form produced during purification Both components react with ninhydrin and with the Sakaguchi reagent for guanidino groups The amino acid composition of peak C is Phe200 Met081 Arg112 N-terminal analysis, one round of Edman-dansyl degradation, and tryptic digestion are consistent with the ide

Collagenase is a component of the specific granules of human neutrophil leucocytes.

Abstract: Azurophil and specific granules were isolated from human polymorphonuclear neutrophil leucocytes. Collagenase was almost exclusively a component of the specific granules. This finding is in contrast with the distribution of other proteolytic enzymes, which are localized in the azurophil (or lysosomal) granules.

Developmental pattern of small intestinal enterokinase and disaccharidase activities in the

Abstract: The concomitant appearance of enterokinase (EK) and trypsin activities in the human intestinal mucosa is indicative of the importance of EK as an activator of trypsinogen and therefore as the key enzyme in protein digestion. Enterokinase can be detected in fetal mucosa from the 26th week of gestation on, paralleling appearance of tryptic activity in meconium. The developmental pattern of EK activity increases with age. Between 26 to 30 weeks of gestation, the EK activity is only 6% and full term babies (40 weeks) 20% of that found in older children. In contrast, lactase studies during develop­ ment show a lactase activity of only 30% in human fetuses between 26 to 34 weeks of gestation as compared to full term babies. During the same gestational period, sucrase and maltase activities reach 70% of the full term. In addition, the distributional pattern of EK differs from the disaccharidases, showing the highest activity in duodenum and the lowest in ileum, whereas disaccharidases are highest in jejunum with lower activity in duodenum and ileum. Differences in topographical distribution and time of appear­ ance of EK and disaccharidases may be attributed to differences in origin as well as subcellular localization of these enzymes. It is conceivable that the premature infant, between 26 to 30 weeks of gestation, is better equipped to deal with hydrolysis of a­ glucosides than of lactose. Recent advances in the care of premature babies in­ crease their prospects of survival. The early feeding of proteins and carbohydrates to premature babies necessi­ tates knowledge ofthe developmental pattern of enzyme activities in the fetal gastrointestinal tract. To our knowledge, no studies on the development of enteroki­ nase (EK) activity in fetal small intestine are reported. EK triggers the activation of proteolytic enzyme zymo­ gens in the gastrointestinal tract, converting trypsino­ gen into trypsin.l-4 EK, therefore, plays a key role in initiating protein digestion. This report represents a systematic study of EK activity in intestinal mucosa of human fetuses during gestation. Inasmuch as the devel­ opmental pattern of disaccharidase activities, especially lactase activity, in the last weeks of gestation is contro­ versial,5--8 we included in this report a study of disaccha­ ridase activities during the whole period of gestation.

Stability of urease in soils

Abstract: Studies with surface samples of Iowa soils selected to obtain a wide range in properties showed that the following treatments of field-moist soils had no effect on urease activity: leaching with water ; drying for 24 h at temperatures ranging from 30 to 60°C ; storage for 6 months at temperatures ranging from −20 to 40°C; incubation under aerobic or waterlogged conditions at 30 or 40°C for 6 months. No loss of urease activity could be detected when field-moist soils were air-dried and stored at 21–23°C for 2yr, but complete loss of urease activity was observed when they were dried at 105°C for 24 h or autoclaved (120°C) for 2h. Inactivation of urease in moist soils was detected at temperatures above 60°C. Treatment of field-moist soils with proteolytic enzymes which cause rapid destruction of jackbean urease did not decrease urease activity, but jackbean urease was destroyed or inactivated when added to sterilized or unsterilized soils. Although no decrease in urease activity could be detected when field-moist soils were air-dried, an appreciable (9–33%) decrease in urease activity was observed when air-dried soils were incubated under aerobic or waterlogged conditions. This decrease occurred within a few days, and prolonged incubation or repetition of the drying-incubation treatment did not lead to a further decrease in urease activity. Treatment of incubated air-dried soil with urease or glucose initially increased urease activity to a level exceeding that of the undried soil, but this activity decreased with time and eventually stabilized at the level observed for the undried soil. The work reported supports the conclusions from previous work that the native urease in Iowa soils is remarkably stable and that different soils have different levels of urease activity determined by the ability of their constituents to protect urease against microbial degradation and other processes leading to inactivation of enzymes.

Group A streptococcal antigens cross-reactive with myocardium. Purification of heart-reactive antibody and isolation and characterization of the streptococcal antigen.

Abstract: Heart-reactive antibody (HRA) appears in the sera of experimental animals inoculated with group A streptococci as well as patients with acute rheumatic fever. Adsorption of either serum with group A streptococcal membranes will remove the HRA. Blocking experiments between these two types of HRAs have demonstrated that the antibodies are directed towards different antigenic determinants on either the same or different molecules. To isolate and purify the antigen from the group A streptococcus cross-reactive with sarcolemmal sheaths of cardiac myofibers, it became necessary to purify the HRA from rheumatic fever patients' sera. Isolated gamma globulin containing all of the HRA was adsorbed onto human sarcolemmal sheaths. The specific HRA was released by using potassium iodide. Over 99 percent of the purified HRA was shown to bind the sarcolemmal sheath whereas less than 1 percent of the antibody would bind nonspecifically to other material. Preparations of group A streptococcal membrane will bind HRA purified from the sera of acute rheumatic patients at levels of 97 percent or greater. The cross-reactive antigen solubilized by nonionic detergent was purified 120-fold by column chromatography. On sodium dodecyl sulfate polyacrylamide electrophoresis, the antigen was demonstrated to be composed of four polypeptides with mol wt of 32,000, 28,000, 26,000, and 22,000 daltons, respectively. Only proteolytic enzymes could destroy the antigenic determinant whereas glycosidases and lipases had no effect. The purified antigen blocked the binding of purified HRA to normal human heart sections.

Leaf Proteolytic Activities and Senescence during Grain Development of Field-grown Corn (Zea mays L.)

Abstract: Some proteolytic enzymes occurring in the leaves of field-grown corn (Zea mays) (B73) were identified and partially characterized. Changes in activities of several proteolytic enzymes and in concentrations of protein and chlorophyll as a function of intraleaf segments (tip to base), leaf position, and leaf senescence during grain development and maturation were followed in crude leaf extracts. The aminopeptidase (not affected by sulfhydryl or fluoride reagents) was most active at pH 7, while the carboxypeptidase(s) (sensitive to fluoride, but insensitive to sulfhydryl reagents) was most active in the acid range, pH 3 to 6. The presence of two or more endopeptidases is indicated. Endopeptidase (caseolytic) activity at pH 5.4 appeared to be stimulated by sulfhydryl groups or EDTA, while caseolytic activity at pH 7.5 was not. Visually, individual leaf senescence starts at the leaf tip and the necrotic (brown) V-shaped area enlarges progressively toward the leaf base. Canopy senescence occurs in two phases. Foliar symptoms are first observed on the bottom leaf and then in sequential order up the plant. Subsequently, senescence occurs on the top leaf and moves downward. These foliar senescence symptoms are paralleled by decreases in exopeptidase activities, protein, and chlorophyll concentrations and by increases in endopeptidase activities. During development and maturation of the grain, both aminopeptidase and carboxypeptidase activity of the middle half of the ear leaf increased (2- to 3-fold) during the onset of the visual reproductive phase (tassel and car emergence). However, during grain development and plant senescence, both activities decreased rapidly and concurrently with the loss of protein and chlorophyll from this leaf section. In contrast, caseolytic activity at both pH 5.4 and 7.5 increased gradually during the early reproductive phase and rapidly with leaf senescence. The fastest rate of increase in caseolytic activities was concurrent with the most rapid loss of protein from the leaves. The coincidence of these events suggests a major role for the caseolytic enzymes in initiating the rapid hydrolysis of leaf protein.

Factors affecting genetic transformation of Neisseria gonorrhoeae.

Abstract: Piliated gonococci were competent in genetic transformation in all stages of growth in minimal and enriched media, but nonpiliated cells were almost totally incompetent. Uptake of deoxyribonucleic acid into a deoxyribonuclease-insensitive state was observed only in competent piliated cells. Competence was not affected by washing of competent cells or treatment of competent cells with proteolytic enzymes. Expression of competence required presence of any of several different monovalent or divalent cations, as well as a utilizable source of energy. Efforts to produce genotypically or phenotypically competent derivatives of nonpiliated cells were unsuccessful. These experiments are consistent with the idea that pili may play a role in the irreversible uptake of transforming deoxyribonucleic acid by the gonococcus, but fail to provide evidence for other types of competence factors.

THE MODE OF BINDING OF β-GLUCANS AND PENTOSANS IN BARLEY ENDOSPERM CELL WALLS

Abstract: β-Glucans in barley endosperm cell walls exist as polymers of very high molecular weight (about 4 × 107 daltons) containing firmly linked peptide sequences. This peptidic material is an essential part of the structure of the β-glucan complex as it exists in the cell wall. Rupture of peptide bonds by hydrazinolysis or with the proteolytic enzyme thermolysin gives β-glucans similar in size to those from short-grown green malts (about 106 daltons). This suggests that proteolysis is the first step in β-glucan degradation. Large β-glucans are not all precipitated in 30% (w/v) ammonium sulphate; only 34% of the β-glucan in a hot aqueous extract of cell walls is precipitated. The amount is increased to 63% if the cell walls have been previously dehydrated. Prolonged incubation of cell wall β-glucan at 40°C, mechanical stress, chromatography lasting 8–10 h at or above 65°C, or chromatography in M sodium chloride causes some disassociation of high molecular weight β-glucan to a size of about 107 daltons. Heating a solution for 1 h at 100°C does not disassociate the β-glucan. Pentosans isolated from cell walls are not covalently linked to the β-glucans and can be separated from them by molecular sieve chromatography. They have a higher xylose/arabinose ratio than previously reported for barley pentosans. The pentosan molecules extracted by water are smaller (106 daltons) than those extracted by alkali (5 × 106 daltons). Little difference was observed in the chemical or physical properties of cell wall materials of barley cultivars of different malting qualities.

Studies on the role of exogenous proteolytic enzymes in digestion processes in fish

Abstract: Studies were carried out on the proteolytic activity of the fry of common carp, rainbow trout, grass carp, and whitefish, as well as on the activity of digestive organs of adult common carp and rainbow trout. Activity of exogenous enzymes in relation to endogenous ones was assessed on the basis of the proteolytic value of fish food and the activity of digestive organs. It was found that the share of proteolytic enzymes of natural food in the digestion process in fish was high. Beginning from a weight of 50–100 g for common carp and 10 g for rainbow trout, the relation between the daily enzymatic ration and the weight of fish indicates the cooperation of an approximately constant amount of exogenous enzymes.

The action of proteolytic enzymes on the glycoprotein from pig gastric mucus.

Abstract: A glycoprotein of mol.wt. 2x10(6) was isolated in homogeneous form from pig gastric mucus by isopycnic centrifugation in CsCl but without enzymic digestion or reductive cleavage of disulphide bonds. Digestion of the purified glycoprotein with trypsin, pepsin or Pronase resulted in the formation of glycoprotein subunits, of mol.wt. 5.2x10(5)-5.8x10(5), one-quarter that of the undigested glycoprotein. The glycoprotein subunits were isolated by gel filtration and shown to contain all the carbohydrate present in the undigested glycoprotein, but 18.6-25.6% of the total amino acids originally present were lost on digestion. The relative amount of threonine, serine and proline had increased from 41% (w/w) in the undigested glycoprotein to 61-67% of the total amino acids in the glycoprotein subunits after digestion. The results support the previously proposed structure for the glycoprotein, namely that of four subunits joined by disulphide bridges. These results show the presence of two distinct regions in the glycoprotein molecule, one rich in threonine, serine and proline, which is glycosylated and resistant to proteolyis, whereas the other, with an amino acid composition more characteristic of a globular protein, is not glycosylated and is susceptible to proteolysis. In addition, the region that is susceptible to proteolysis contains the disulphide bridges which join the glycoprotein subunits together to form the gastric glycoprotein.

Gingival Fluid and Serum in Periodontal Diseases: II. Evidence for Cleavage of Complement Components C3, C3 Proactivator (Factor B) and C4 in Gingival Fluid

Abstract: 1. C3 activation products C3c and C3d can be demonstrated in gingival pocket fluid from patients with severe periodontitis. 2. Quantitatively, C3d levels in GF are comparable to levels attained consequent to immune-complex and tryptic activation of human serum. 3. The alternate pathway of complement activation occurs in the periodontal pocket, as demonstrated by conversion of C3 proactivator (Factor B) to C3-activator Bb. 4. C4 is present in an altered form in some, but not all, GF's. This form is electrophoretically similar to that generated from serum by C1s and several proteolytic enzymes.

Mechanisms of zymogen activation.

Abstract: It is now well recognized that proteolytic enzymes play key roles in the regulation of or control over the action of other proteins (1-3). Such enzymes can be found in all species from bacteria to man and in control of diverse systems, which include hormone production, bacteriophage assembly (4), development, fertilization (5), digestion, defense against invading organisms (6), and tissue repair (7, 8). In most cases the proteolytic enzymes are known to be synthesized as inactive precursor proenzymes, or zymogens (9). They are activated by proteolytic cleavage of a single peptide bond in the proenzyme and so become catalytically active. Further control over the degree of specificity for a target molecule or molecules is determined by the degree of specificity inherent to the enzyme. Further control over the time and location of action is often carried out by protein inhibitors of the requisite specificity. There are several different classes of proteolytic enzymes, which were classified first according to their susceptibility toward different inhibitors (10) and later ac­ cording to their homologous amino acid sequences or structures within each class (II). There are many excellent papers and reviews that deal with all aspects of the collected information of the proenzymes of proteases (e.g. 1-3, 9, 11). Therefore, in this review we consider the single question of why the zymogens of the serine proteases-the enzyme class about which most is known-are relatively inactive. We consider this in the light of most recent information about the known structures of two proenzymes, chymotrypsinogen (12) and trypsinogen (13), and their ac­ tivated counterparts, a -chymotrypsin (14) and trypsin (15).

Properties of a Protein Activator of NAD Kinase from Plants

Abstract: Purification of pea ( Pisum sativum ) seedling NAD kinase by DEAE-cellulose column chromatography resulted in loss of activity, due to dissociation of an activator from the enzyme. The purified enzyme preparation, which was almost completely inactive, regained the activity when the activator was added back. The activator was purified 320-fold by ion exchange chromatographies. The activator was susceptible to proteolytic enzymes, but not to ribonuclease, glucoamylase or pectinase, indicating that it is of a protein nature. This protein was relatively stable in boiling water, but susceptible to acid or alkali, especially under high temperatures. Restoration of catalytic activity of inactive enzyme was proportional to amounts of the activator added. Gel filtration indicated that molecular weight of the activator was 28,000. The activator was found in extracts from various plants.