Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Saccharomyces cerevisiae mating type a cells enlarged and elongated when exposed to alpha-factor, a sex pheromone produced by mating-type alpha cells. This morphogensis required exogenous-D-glucose, nitrogen, and phosphate, and cells in exponential phase responded better than stationary-phase cells. Morphogenesis was blocked by cycloheximide and by inhibitors of cell wall biosynthesis such as 2-deoxy-D-glucose, 2-deoxy-2-fluoro-D-glucose, and 2-deoxy-2-fluoro-D-mannose, but not by polyoxin D. One to two hours after addition of pheromone, a cells became more susceptible to lysis by glucanases, a change that was dependendent on the dose of alpha-factor and was blocked by drugs that block morphogenesis. On the other hand, treatment with alpha-factor did not increase susceptibility to attack by trypsin, subtilisin, or exo-alpha-mannanase. Radioactive label, incorporated into cell wall polysaccharides during treatment with alpha-factor, was not secreted into the medium during morphogenesis. Analysis of the labeled wall polymers showed that alpha-factor-treated cells contain more glucan and less mannan than control cells, and that the mannan of treated cells contains an increased proportion of shorter side chains and unsubstituted backbone mannose units. Thin-section electron microscopy of treated cells revealed that the cell wall possesses a diffuse outer layer in the extension and is thinner at the tip.
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PMID:Morphogenic effects of alpha-factor on Saccharomyces cerevisiae a cells. 77 42

1. A trypsin and chymotrypsin inhibitor was isolated by extraction of chick-pea meal at pH8.3, followed by (NH4)2SO4 precipitation and successive column chromatography on CM-cellulose and calcium phosphate (hydroxyapatite). 2. The inhibitor was pure by polyacrylamide-gel and cellulose acetate electrophoresis and by isoelectric focusing in polyacrylamide gels. 3. The inhibitor had a molecular weight of approx. 10000 as determined by ultracentrifugation and by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. A molecular weight of 8300 was resolved from its amino acid composition. 4. The inhibitor formed complexes with trypsin and chymotrypsin at molar ratios of 1:1. 5. Limited proteolysis of the inhibitor with trypsin at pH3.75 resulted in hydrolysis of a single-Lys-X-bond and in consequent loss of 85% of the trypsin inhibitory activity and 60% of the chymotrypsin inhibitory activity. Limited proteolysis of the inhibitor with chymotrypsin at pH3.75 resulted in hydrolysis of a single-Tyr-X-bond and in consequent loss of 70% of the trypsin inhibitory activity and in complete loss of the chymotrypsin inhibitory activity. 6. Cleavage of the inhibitor with CNBr followed by pepsin and consequent separation of the products on a Bio Gel P-10 column, yielded two active fragments, A and B. Fragment A inhibited trypsin but not chymotrypsin, and fragment B inhibited chymotrypsin but not trypsin. The specific trypsin inhibitory activity, on a molar ratio, of fragment A was twice that of the native inhibitor, suggesting the unmasking of another trypsin inhibitory site as a result of the cleavage. On the other hand, the specific chymotrypsin inhibitory activity of fragment B was about one-half of that of the native inhibitor, indicating the occurrence of a possible conformational change.
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PMID:A trypsin and chymotrypsin inhibitor from chick peas (Cicer arietinum). 79 Dec 69

The effects of pyocin S2, a bacteriocin produced by Pseudomonas aeruginosa strain M47, on several processes in susceptible bacterium have been examined. Lipid synthesis, measured in terms of [32P]phosphate, [14C]acetate or [2-3H]glycerol incorporation into lipid fractions, was halted almost completely soon after pyocin S2 addition. When cell suspensions were treated with various amounts of pyocin S2, the extent of inhibition of lipid synthesis was proportional to the ratio of killed bacteria. Protein synthesis was not essential for the inhibition. Degradation of lipid due to pyocin S2 was not detected. Pyocin S2 also affected protein and nucleic acid syntheses, but these inhibitions appeared with a delay of about 10 min after the cessation of lipid synthesis. The effect of trypsin [EC 3.4.21.4] on the viability of cells which had adsorbed pyocin S2 was also investigated: the cells went through a period when the destruction of pyocin S2 by trypsin restored the colony-forming ability of the cells (stage I). Then transition to a second state in which the cells lost viability irrespective of trypsin treatment (stage II) took place. The transition from stage I to stage II depended on the energy metabolism of the cells and followed first-order kinetics with a rate proportional to the number of killing units of adsorbed pyocin S2. The residual capacity for lipid synthesis in cells which had adsorbed pyocin S2 after trypsin treatment at various times indicated that lipid synthesis was inhibited only in the cells at stage II of pyocin S2 action.
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PMID:Preferential inhibition of lipid synthesis by the bacteriocin pyocin S2. 81 53

Limited tryptic digestion of either synthase I or D forms resulted in the appearance of a new glucose 6-phosphate-dependent form which was composed of 75,000 molecular weight subunits. Early in tryptic digestion, an intermediate 78,000 subunit was also observed with both forms of the enzyme. The NH2-terminal dipeptide sequence of the 75,000 subunit of both forms was the same as that of the original 85,000 subunit (Pro-Leu-) indicating degradation at or near the COOH-terminal end without affecting the NH2-terminal end. Studies interrelating loss of organic phosphate, increase in glucose 6-phosphate dependency, and retention of the NH2-terminal sequence during limited tryptic digestion suggest that there are phosphorylation sites at or near the COOH-terminal, as well as sites within the core of the subunit, which are of importance in the synthase I to D form interconversion reaction via phosphorylation. The pathway of limited tryptic proteolysis of either synthase I or D forms was the same as judged by the molecular weights of the subunit intermediates: 85,000 leads to 78,000 leads to 75,000. A Ca2+-stimulated proteinase activity was also detected in some highly purified preparations of the synthase D form, which led to the appearance of subunits of molecular weight 78,000 and 75,000 together with phosphopeptide(s). These findings suggest that the pathway of proteolysis of the Ca2+-stimulated proteinase is similar to that of trypsin.
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PMID:Structural studies on rabbit muscle glycogen synthase. II. Limited proteolysis. 81 60

Bacterial neutral protease of Bacillus polymyxa was found to disperse mammalian tissues and cells. Primary cell cultures were obtained from several tissues after treatments with 200 to 2,000 Kunitz unit per ml of this protease in either a phosphate buffer solution, a balanced salt solution or a tissue culture medium supplemented with serum. Monolayer cultures wither in their early passage levels or of established strains were harvested by a treatment with this protease, and proliferated again in monolayer after its removal. A growing culture of strain L-929 was kept in monodisperse suspension in the presence of this protease. In contrast to trypsin, this protease was found active in the presence of serum, stable during incubation and scarcely injured cells.
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PMID:Tissue dispersion, cell harvest and fluid suspension culture by the use of bacterial neutral protease. 81 56

ATP citrate lyase was purified by two different procedures from the livers of rats first starved and then fed with a fat-deficient and high carbohydrate-glycerol diet. These enzyme preparations were judged homogeneous by sedimentation equilibrium and polyacrylamide gel electrophoresis. The molecular weight of the native enzyme was around 4.4 X 10(5) as determined by sedimentation equilibrium. On sodium dodecyl sulfate gel electrophoresis the enzyme usually showed a single protein band with an estimated molecular weight of 1.2 X 10(5). A similar value for the molecular weight of the subunit was obtained by gel filtration on 6% agarose in the presence of 6 M guanidinium chloride. The molecular weight of this polypeptide chain was estimated by sedimentation equilibrium to be around 1.1 X 10(5). These results indicated that ATP citrate lyase has a subunit structure of four polypeptides of similar size. The extinction coefficient of the dry protein and its amino acid composition are also reported. Some batches of fully active enzyme, judged to be homogeneous by sedimentation equilibrium and polyacrylamide gel electrophoresis, showed two additional major polypeptides (Mr approximately 7.1 X 10(4) and 5.5 X 10(4)) on sodium dodecyl sulfate gel electrophoresis. Studies on the polypeptides produced by proteolytic modification of the native enzyme by trypsin indicated that the additional protein bands observed on sodium dodecyl sulfate gel electrophoresis with some of the batches of enzyme could have been formed by limited proteolysis ("nicking") of the original 1.1 X 10(5) subunit. Trypsin treatment of the native enzyme did not affect the enzyme activity, whereas chymotrypsin and pronase treatment inactivated the enzyme. The trypsin-treated enzyme, which contained only the two smaller polypeptides, did not differ significantly from the untreated enzyme with respect to sedimentation behavior, phosphorylation by ATP, Km for citrate, and immunoreactivity, but it was more heat-labile than the untreated enzyme. The phosphate group on the phosphorylated "nicked" enzyme was located on the larger polypeptide fragment.
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PMID:Structure of ATP citrate lyase from rat liver. Physicochemical studies and proteolytic modification. 82 50

The complete amino acid sequence of human salivary statherin, a peptide which strongly inhibits precipitation from supersaturated calcium phosphate solutions, and therefore stabilizes supersaturated saliva, has been determined. The NH2-terminal half of this Mr=5380 (43 amino acids) polypeptide was determined by automated Edman degradations (liquid phase) on native statherin. The peptide was digested separately with trypsin, chymotrypsin, and Staphylococcus aureus protease, and the resulting peptides were purified by gel filtration. Manual Edman degradations on purified peptide fragments yielded peptides that completed the amino acid sequence through the penultimate COOH-terminal residue. These analyses, together with carboxypeptidase digestion of native statherin and of peptide fragments of statherin, established the complete sequence of the molecule. The 2 serine residues (positions 2 and 3) in statherin were identified as phosphoserine. The amino acid sequence of human salivary statherin is striking in a number of ways. The NH2-terminal one-third is highly polar and includes three polar dipeptides: H2PO3-Ser-Ser-H2PO3-Arg-Arg-, and Glu-Glu-. The COOH-terminal two-thirds of the molecule is hydrophobic, containing several repeating dipeptides: four of -Gn-Pro-, three of -Tyr-Gln-, two of -Gly-Tyr-, two of-Gln-Tyr-, and two of the tetrapeptide sequence -Pro-Tyr-Gln-Pro-. Unusual cleavage sites in the statherin sequence obtained with chymotrypsin and S. aureus protease were also noted.
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PMID:Complete covalent structure of statherin, a tyrosine-rich acidic peptide which inhibits calcium phosphate precipitation from human parotid saliva. 83 35

Release of peroxidase from secretory cells of rat lacrimal gland upon cholinergic stimulation was studied in vitro with single lobules and isolated cells (lacrimocytes). Isolated lobules, kept in Eagle's medium, remain structurally intact and reaction product of peroxidase is confined to cisternae of rough endoplasmic reticulum, elements of the Golgi apparatus, and all secretory granules. Morphologically, exocytosis occurs by membrane fusion and discharge of granule content. The highest rate of peroxidase released from lobules is observed at 10(-4) M carbamylcholine. The specific activity of peroxidase released into the medium is fourfold higher as compared to the lobules. Release of peroxidase is suppressed by atropine when added before or after the addition of carbamylcholine. At 4 degrees C, no peroxidase release occurs upon cholinergic stimulation. The exocytotic release of peroxidase is dependent on energy supply, as indicated by substantial inhibition (at 37 degrees C) under anoxic conditions or in the presence of dinitrophenol, KCN, or carboxyatractyloside. Furthermore, the process is sensitive to colchicine and vinblastine. Isolated lacrimocytes, consiting of 95% secretory acinar cells, are prepared by digestion with collagenase, hyaluronidase, and trypsin. They retain the characteristic polarity of secretory cells in situ, and localization of peroxidase is the same as in lobules. Since isolated lacrimocytes respond to cholinergic stimulation in the same way as lobules, the receptors are not damaged by the isolation procedure and appear to be associated directly with the exocrine cell. Oxygen uptake by isolated lacrimocytes is about 14 nmol O2 X min-1 X 10(-6) cells; it is about doubled by uncoupling with dinitrophenol. Oxygen uptake rises by 20-30% above the resting rate upon cholinergic stimulation. This additional uptake is suppressed by atropine or by added cholinesterase, indicating that continuous receptor occupancy may be required for the energy demand by exocytosis. On the basis of the specific activity of peroxidase in the medium, the energy demand resulting from cholinergic stimulation is estimated to be 0.08 mumol ATP (or energy-rich phosphate bonds) per microgram of protein released from the lacrimocytes.
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PMID:Exocytosis in secretory cells of rat lacrimal gland. Peroxidase release from lobules and isolated cells upon cholinergic stimulation. 95 71

1. High-affinity estrogen-binding sites can be solubilized from the liver chromatin of estrogenized chickens by treatment of the chromatin with 2 M KCL/5 M urea and fractionation on hydroxylapatite. Two estrogen-binding proteins are eluted from hydroxylapatite columns by 20mM phosphate (binding protein I) and 200mMphosphate (binding protein II), respectively. 2. The binding protein I is part of a non-histone protein fraction containing acid-soluble and insoluble proteins, whereas the binding protein II elutes together with high molecular weight nonhistone proteins containing acid insoluble proteins only. Both binding proteins exhibit the smae affinity for estradiol (Kd approximately 10(-9) M). 3. From chromatin of untreated chickens very small amounts of binding protein I (0.1 pmol/mg protein compared to 1.9 pmol/mg protein from estrogenized chickens) with the smae affinity for estradiol as that from estrogenized animals can be solubilized. Binding protein II is not detectable. 4. The "soluble nuclear estrogen receptor" extracted from crude liver nucleir of estrogenized chickens by 0.5 M KCL behaves on hydroxylapatite very similarly to salt/urea-dissociated chromatin with respect to the binding protein I. No binding protein II, however, can be demonstrated. 5. Chromatography of various preparations on Bio-Gel A-1.5 m indicates that the binding protein II is a residual chromatin fragment containing an unseparated binding protein-DNA complex, whereas the binding protein I represents the solubilized nucleic-acid-free chromosomal estrogen receptor. The "soluble nuclear receptor" and the binding protein I, however, are not identical with respect to their chromatographic behaviour on Bio-Gel A-1.5m, even though their estrogen binding entity remaining after trypsin treatment seems to be very similar.
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PMID:Solubilization of the chromatin-bound estrogen receptor from chicken liver and fractionation on hydroxylapatite. 96 52

Two peptide bonds of staphylococcal enterotoxin C, were hydrolyzed concurrently at quite different rates during limited digestion with trypsin. A Lys-Val at about position 92 in the disulfide loop was the first bond cleaved, followed by a Lys-Asx at about position 57 on the NH2-terminal side of the loop. Preparations of singly cleaved material (enterotoxin C1-T1) contained about 93% of the cleaved protein and 7% unreacted enterotoxin. Preparations of the doubly cleaved material (enterotoxin C1-T2) consisted of 98% enterotoxin C1-T2 and 2% enterotoxin C1-T1. In the absence of denaturant, enterotoxin C1-T2 behaved as a single particle. It gave a single peak on Sephadex G-75 with a sedimentation coefficient of 2.85 S and a molecular weight of 29,100 by sedimentation equilibrium. Circular dichroic spectra indicated only minor conformational differences between enterotoxins C1-T2 and C1. However conformational stability was significantly affected with the unfolding of enterotoxin C1-T2 in 4 M guanidine hydrochloride proceeding at about twice the rate of native enterotoxin. Enterotoxin C1-T2 was separated into 6,500 and 22,000 molecular weight polypeptides by gel filtration on Sepharose 6B in 6 M guanidine hydrochloride. Complementation (as measured by CD spectra, serologic activity and mitogenicity) of the two polypeptides was readily achieved from solution in 6 M guanidine hydrochloride by dialysis against phosphate buffer. The 22,000 molecular weight polypeptide was further separated into two peptides (Mr = 4,000 and 19,000 after alkylation of the reduced disulfide bridge. Summation of the amino acid composition of the constituent peptides of enterotoxin C1-T2 agreed well with the composition of enterotoxin C1. A comparison of the 6,500 and 4,000 molecular weight polypeptides from enterotoxin C1-T2 with structurally equivalent segments of enterotoxin B suggested structural homology between the two antigenic variants. Enterotoxins C1, C1-T1, and C1-T2 gave reactions of complete identity in Ouchterlony immunodiffusion and were indistinguishable in the quantitative precipitin reaction. Enterotoxins C1-T1 and C1-T2 were highly mitogenic but were slightly less potent than the native enterotoxin. Enterotoxin C1-T2 had equivalent emetic activity to enterotoxin C1 in rhesus monkeys. It is suggested that the exceptional lability to limited enzymic hydrolysis exemplified by enterotoxin C1 is associated with beta turn structures at protein surfaces.
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PMID:Effect of single and double peptide bond scission by trypsin on the structure and activity of staphylococcal enterotoxin C. 96 78


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