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)

Appropriately fixed preparations stained by a modification of the Guard (1959) reaction for "sex chromatin" display selective staining of interphase chromatin and mitotic or meiotic chromosomes. This is a regressive staining method which seems to depend on the selective displacement of an acidic dye from less basic structures, and retention of the dye at more basic sites. The results obtained with the reaction can be controlled by the length of time that the preparations are "differentiated" in solutions containing phosphomolybdic and phosphotungstic acids (polyacids). After three- or four-hour exposures to polyacid solutions, all chromatin is stained. However, with longer differentiation, "condensed" chromatin can be stained preferentially. Of a number of fixatives investigated, only 10% formalin, ethanol-acetic acid (3:1), and Bouin's solution proved useful. Others resulted in diminished specificity or a total loss of selectivity. The most intense staining was obtained after formalin fixation. Less intense dyebinding was observed after fixation in 3:1 - probably due to extraction of some histone fractions-and the least amount of dye was bound in Bouin's-fixed chromatin - probably due to blockage of arginine residues by picric acid. The reaction was not affected by enzymatic removal of nucleic acids or the extraction of lipids. It was diminished by treatment with trypsin or weak acetylation, and it was completely prevented by strong acetylation, deamination, or extraction of basic proteins with HCl. The results presented suggest that the modified Guard (1959) procedure selectively demonstrates basic nucleoproteins. Further, by the use of regressive differentiation in polyacid solutions, the retention of dye in more condensed chromatin can be favored.
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PMID:Cytochemical evaluation of the Guard procedure a regressive staining method for demonstrating chromosomal basic proteins. I. Effects of fixation, blocking reactions, selective extractions, and polyacid "differentiation". 6 Mar 23

Various reagents were tested for the purpose of developing an improved Giemsa staining technique for the differential staining of sister chromatids in human chromosomes. Reagents like acids, bases, buffers, protein denaturants and proteolytic enzymes were all potent inducers of differential staining. The best results were obtained by brief trypsinization followed by extraction of nucleic acids by incubation in hot HCl. There was poor contrast between unifilarly and bifilarly BrdU substituted chromatids in slides from which trypsin treatment was omitted. The method of slide preparation as they affect the spreads of BrdU substituted metaphases were also evaluated. The results support the role of these reagents in the conformational changes and structural lesions of chromosomal protein leading to differential staining.
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PMID:An analysis of factors for the differential staining of sister chromatids in human chromosomes using Giemsa. 8 99

A convenient and quick method using trypsin-orcein for banding plant chromosomes (O-banding) is suggested. The technique is directly applicable to meristematic tissues (e.g. root tips) and involves the treatment of root tips with 1-2% solution of trypsin either in buffer or in 0.5 N HCl for 5-10 minutes at 37 C or for 30-60 minutes near 0 C followed by staining with 1.5% acetic orcein: 1 N HCl (19:1). Dark staining bands are reproducible and species specific. These bands possibly represent specific DNA-protein-dye interaction.
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PMID:Trypsin-orcein banding in plant chromosomes. 9 27

In patients without pancreatic disease Aminofusin L forte infused for 1 h did not stimulate gastric or pancreatic secretion. On the other hand, infusion of casein by hydrolysate led to a significant increase in the concentration and total output of HCl. 4 h intravenous infusion of Aminofusin L forte caused a transient but significant rise of the bicarbonate concentration, amylase activity and above all trypsin activity and output. The results show that N solutions used for parenteral protein nutrition influence in a different way both gastric and pancreatic secretion.
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PMID:The effect of some N solutions for parenteral nutrition on gastric and pancreatic secretion. 11 12

Earlier studies have shown that a substance(s) released from the egg jelly of the toad Bufo arenarum is required for fertilization. In this paper some properties of this diffusible factor were further examined, and a procedure was designed for its isolation from crude egg extracts. The active component is soluble in water and ethanol, and insoluble in chloroform, ether and n-butanol. The biological activity is stable to liophylization and to heat, and remains unaffected after trypsin treatment. In contrast, it is impaired after treatment with ethyl acetate, 0.1 N HCl or chloroform, and is completely destroyed after converting the diffusible factor into ash. Data are presented showing that the recovery of fertilizability of extracted eggs in the bioassay system as carried out under present conditions, cannot be ascribed to a pH alteration of the insemination medium. This lends further support to the view that diffusible factor activity is not mediated through a pH effect. The factor was purified by gel chromatography coupled with desalting and paper chromatography. The active molecule is of low molecular weight and appears associated with a high pH ninhydrin-positive fraction.
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PMID:Properties and isolation of the diffusible factor involved in Bufo arenarum fertilization. 11 83

Tropomyosin was found to undergo only limited digestion by trypsin at 0 degrees C and the two segments that accumulated amounted to two-thirds of the original protein. They are referred to as segments A and B. These segments were not resistant to trypsin digestion at 20 degrees C and at the latter temperature no large fragments remained as judged by disc gel electrophoresis. Segments A and B were separated from each other on the basis of solubility differences and were found to have molecular weights of 24600 and 21900 respectively. Each of the segments appeared to retain about 70-75% of the helical conformation as judged by circular dichroism at 20 degrees C. However, the segments did not show any of the inhibitory activity of the parent tropomyosin molecule when mixed with troponin in the Mg2+-actomyosin ATPase system. Amino acid analysis showed that the portion of tropomyosin that was digested by trypsin (EC 3.4.21.4) had a lower content of the helix stabilizing residues Glu and Leu and a higher content of the helix-destabilizing residues Arg and Lys. These differences indicate that the digested portion should be less stable in the helical conformation than the two trypsin-resistant segments. End group determinations along with the results of the amino acid analysis indicated that segment A was probably derived from the central one-third of tropomyosin and segment B from the C-terminal one-third. By the process of elimination the N-terminal third appears to have been more liable region that was digested by trypsin. The segments A and B were shown to differ in their stability to denaturation by guanidine-HCl and elevated temperature. All of these observations indicate that tropomyosin is not a uniform structure and is composed of regions of different stability.
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PMID:The structure and stability of trypsin-resistant segments from rabbit tropomyosin. 13 16

p-Nitrobenzyl p-toluenesulfonyl-L-arginine has been synthesized. A number of trypsin-like enzymes can catalyze the hydrolysis of this ester leading to formation of p-nitrobenzyl alcohol. After separation from the ester and p-toluenesulfonylarginine by extraction into chloroform, the p-nitrobenzyl alcohol liberated can be measured spectrophotometrically at 271 nm. Under the conditions of the assay, the hydrolysis of 1 micronmol/ml of the ester is equivalent to an absorbance change of 4.45 cm-1 at 271 nm. With 0.10 mM p-nitrobenzyl p-toluenesulfonyl-L-arginine in 0.1 M Tris-HCl at pH 8.4 and 30 degrees, the enzymatic hydrolysis is linearly proportional to time up to consumption of 60% of the ester. Product formation is proportional to enzyme concentration with 0.05 to 0.2 NIH clotting units/ml for bovine or human thrombin, 0.005 to 0.02 CTA units/ml for human plasmin, and 0.01 to 0.04 microgram/ml protein for bovine pancreatic trypsin. In 0.1 M Tris-HCl at pH 8.4 and 30 degrees, Km is 14 micrometer and Vmax is 0.037 micronmol/min/NIH unit/ml for bovine thrombin, Km is 78 micrometer and Vmax is 0.31 micronmol/min/CTA unit/ml for human plasmin, and Km is 12 micrometer and Vmax is 138 micronmol/min/mg protein/ml for bovine trypsin. With bovine thrombin, activities at pH 7.3 and at pH 9.2 were 30% lower and 40-50% higher than the rate at pH 8.4. Samples of bovine and human thrombin ranging in specific clotting activity from 59 to 2133 NIH units/mg protein showed esterase activities varying from 0.15 to 0.4 micronmol p-nitrobenzyl alcohol formed/10 min/NIH unit.
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PMID:Assay of the esterase activity of thrombin, plasmin and trypsin with a chromogenic substrate p-nitrobenzyl p-toluenesulfonyl-l-arginine. 14 54

Rates of hydrolysis of the newly developed peptide chromogenic substrates S-2160 (N-Bz-Phe-Val-Arg-pNA, HCl), S-2238 (H-D-Phe-Pip-Arg-pNA, 2HCl), S-2222 (N-Bz-Ile-Glu-Gly-Arg-pNA, HCl), and S-2251 (H-D-Val-Leu-Lys-pNA, 2HCl) from AB Kabi Peptide Research and Chromozym TH (Z-Gly-Pro-Arg-pNA, HCl) from Pentapharm Limited were tested against highly purified preparations of human plasmin, bovine trypsin, human alpha thrombin, and bovine factor Xa. S-2160, S-2238, and Chromozym TH are sensitive to thrombin, Chromozym TH and S-2238 exhibiting a substantially greater sensitivity than S-2160. All 3 substrates are insensitive to factor Xa but hydrolyzed to varying degrees by plasmin and trypsin. In contrast, S-2222 is sensitive to Xa and insensitive to thrombin. S-2251 is relatively plasmin-specific, being resistant to the clotting enzymes thrombin and Xa. S-2251 exhibits even greater sensitivity to the SK-plasmin complex than to plasmin. In addition, the substrate Chromozym PK (N-Bz-Pro-Phe-Arg-pNA, HCl) was evaluated and found to be relatively specific for plasma kallikrein. Assays for antithrombin III and heparin using S-2222 as the substrate and factor Xa as the enzyme, plasma plasminogen and plasmin inhibitors using S-2251 as the substrate, and plasma prekallikrein and kallikrein inhibitors using Chromozym PK as the substrate have been developed. Synthetic peptides mimicking amino acid sequences adjacent to proteolytic activation cleavage of plasma serine protease precursors appear to be sensitive and relatively specific tools applicable to kinetical and clinical studies of these enzymes and their inhibitors.
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PMID:Serine protease specificity for peptide chromogenic substrates. 14 72

Frog (Rana catesbiana) rod outer segment disc membranes contain a cyclic nucleotide phosphodiesterase (EC 3.1.4.17) which is activated by light in the presence of ATP. This enzyme is firmly bound to the disc membrane, but can be eluted from the membrane with 10 mM Tris-HCl buffer, pH 7.4 and 2 mM EDTA. The eluted phosphodiesterase has reduced activity, but can be activated approximately 10-fold by polycations such as protamine and polylysine. The eluted phosphodiesterase can no longer be activated by light in the presence of ATP, that is, activation by light apparently depends on the native orientation of phosphodiesterase in relationship to other disc membrane components. The eluted phosphodiesterase was purified to homogeneity as judged by analytical polyacrylamide gel electrophoresis and polyacrylamide gel isoelectric focusing. The over-all purification from intact retina was approximately 925-fold. The purification of phosphodiesterase from the isolated rod outer segment preparation was about 185-fold with a 28% yield. Phosphodiesterase accounts for approximately 0.5% of the disc membrane protein. The eluted phosphodiesterase (inactive form) has a sedimentation coefficient of 12.4 S corresponding to an approximate molecular weight of 240,000. Sodium dodecyl sulfate polyacrylamide gel electrophoresis separates the purified phosphodiesterase into two subunits of 120,000 and 110,000 daltons. With cyclic 3':5'-GMP (cGMP) as substrate the Km for the purified phosphodiesterase is 70 muM. Protamine increases the Vmax without changing the Km for cGMP. The isoelectric point (pI) of the native dimer is 5.7. Limited exposure of the eluted phosphodiesterase (inactive form) to trypsin produces a somewhat greater activation than is obtained with 0.5 mg/ml of protamine. The trypsin-activated phosphodiesterase has a sedimentation coefficient of 7.8 S corresponding to an approximate molecular weight of 170,000. The 110,000-dalton subunit is much less sensitive to trypsin hydrolysis and the 120,000-dalton subunit is rapidly replaced by smaller fragments. On the basis of the molecular weight of the purified phosphodiesterase (240,000) and the concentrations of phosphodiesterase and rhodopsin in the rod outer segment, it is estimated that the molar ratio ophosphodiesterase to rhodopsin in the rod outer segment is approximately 1:900. Since all of the disc phosphodiesterase molecules are activated when 0.1% of the rhodopsins are bleached, we conclude that in the presence of ATP 1 molecule of bleached rhodopsin can activate 1 molecule of phosphodiesterase.
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PMID:Purification and properties of the light-activated cyclic nucleotide phosphodiesterase of rod outer segments. 16 36

Collagenase (EC 3.4.24.3) activity can be measured directly in homogenates of the involuting rat uterus. Latent forms of collagenase are activated by a brief exposure to trypsin; trypsin activity is then blocked with soybean trypsin inhibitor. Homogenizing conditions have been developed that permit 90-95% recovery of the total active and latent collagenase activity in a 6000 X g pellet, where it is presumably bound to its collagen substrate. This insoluble activity can then be extracted by heating to 60 degrees C for 4 min in 0.04 M Tris - HCl buffer, pH 7.5, containing 0.1 M CaCl2. Methods are presented for the estimation of the recovery of collagenase in the extracts; this approximates 65-70% of the total. Small amounts of activity can also be extracted from rat liver and kidney. This extraction procedure should be of use in purifying collagenase without culturing the enzyme-producing tissue and in the direct assay of tissue collagenase activity. The activity extracted from rat uterus has been proven to be collagenase by its characteristic pattern of collagen breakdown products on disc electrophoresis and by the split of tropocollagen at interband 41 as shown by electron microscopy of reconstituted fragments. The activity is inhibited by EDTA, and this inhibition is not reversed by calcium or zinc ions.
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PMID:Extraction of collagenase from the involuting rat uterus. 18 74


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