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)

Streptomyces griseus trypsin has been isolated from Pronase by ion-exchange chromatography on CM-Sephadex and SE-Sephadex. The isolated enzyme was homogeneous by the criteria tested except for a low degree of contamination by an enzyme with nontryptic activity. The latter could be partially resolved by chromatography on Bio-Rex 70. The molar absorbancy at 280 nm was found to be 3.96 times 10-4 M-1/cm and the E1cm1% was found to be 17.3. The molecular weight was 22,800 plus or minus 800. The enzyme was found to be stable at 0 degrees from pH 2 to 10. At 30 degrees the enzyme was maximally stable at pH 3-4 and significantly stabilized in the neutral and alkaline range by 15 mM Ca2+. Some evidence was obtained for a reversible denaturation of the enzyme at pH 12.0 and 2.0. The K-m for N-alpha-benzoyl-L-arginine ethyl ester at pH 8.0 in 20 mM CaCl2-0.1 M KCl-10 mM Tris-HCl buffer at 30 degrees was found to be 7.7 plus or minus 1.9 times 10-6 M and the esterase activity was observed to be dependent on an ionizing group with pK-a equals 5.85. In 2H2O this pKa was increased to 6.35 and the rate of hydrolysis dicreased threefold. The rate of hydrolysis was independent of pH between 8 and 10. The inhibition of the enzyme with L-1-chloro-3-tosylamido-4-phenyl-2-butanone was shown to be associated with the alkylation of its single histidine residue. This residue is present in a homologous amino acid sequence as the active-site histidine in trypsin and chymotrypsin. Optical rotatory dispersion and circular dichroism measurements over the pH range 5.3-10.5 indicated no significant conformational change until the pH was increased above 10.1. The observation that, under the conditions tested, acetylation and carbamylation of the NH2-terminal valine were incomplete is consistent with the view that this group is buried as an ion pair and only becomes available for deprotonation and reaction upon denaturation of the enzyme at pH values greater than 10.0.
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PMID:Enzymic and physicochemical properties of Streptomyces griseus trypsin. 23 80

An improved purification procedure is described for the sigma subunit of escherichia coli DNA-dependent RNA polymerase [ribonucleoside triphosphate:RNA nucleotidyl-transferase, EC 2.7.7.6]. The method involves chromatography of purified RNA polymerase on single-stranded DNA-agarose, Bio-Rex 70, and finally Ultragel AcA44. The sigma factor obtained is electrophoretically pure with a yield of about 40%. A number of the chemical--physical properties of sigma are presented. A molecular weight of 82,000 was determined by phosphate buffered sodium dodecyl sulfate--polyacrylamide gel electrophoresis. Ultraviolet absorption spectra were used to determine an E280nm 1% of 8.4. The amino acid composition and 12-residue N-terminal sequence (Met-Glx-Glx-Asx-Pro-Glx-(Ser or Cys)-Glx-Leu-Lys-Leu-Leu) of sigma have been determined. The isoelectric focusing properties of sigma are presented. Denaturation--renaturation studies indicate that sigma is capable of an unusually rapid and complete recovery of activity after being subjected to denaturing conditions. A stable, 40,000-dalton fragment is generated from sigma by mild trypsin treatment.
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PMID:Purification and properties of the sigma subunit of Escherichia coli DNA-dependent RNA polymerase. 37 77

D was purified to homogeneity from outdated human plasma by successive chromatography on Bio Rex 70, Sephadex G-200, Bio Rex 70, and Sephadex G-75. Column fractions were monitored for D activity by a hemolytic diffusion plate assay. The overall yield was approximately 4% by activity. A m.w. of 22,900 daltons was established by sedimentation equilibrium. Amino acid analyses have been obtained and Isoleucine has been determined as the NH2-terminus. Incubation of D with purified B and CoVF in the presence of Mg++ resulted in cleavage of B, as judged by SDS-polyacrylamide gel electrophoresis and immunoelectrophoresis. D hydrolyzed certain synthetic amino acid esters of arginine, lysine, and tyrosine. Benzoyl-L-arginine methyl esters (BAME) was the most sensitive substrate for D among those tested. The substrate profile of D was dinstinct when compared to that of CIs, CIr, plasmin, urokinase, and trypsin. Both the enzymatic and hemolytic activity of D were irreversibly inhibited by treatment with 10 mM DFP as well as by reduction and alkylation.
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PMID:Human factor D of the alternative complement pathway: purification and characterization. 87 24

We report that cultured bovine calf aorta and human adult iliac artery smooth muscle cells release a soluble factor which causes spreading and separation of cells in normally tight, cohesive epithelial colonies, similar to the morphologic changes induced by the fibroblast-derived scatter factor (SF). Smooth muscle-derived SF was heat sensitive, trypsin labile, and nondialyzable, consistent with a protein (or proteins). Its effects on epithelium were not mimicked by a variety of proteolytic enzymes, growth factors, or hormones, and were not blocked by antiproteases or by antibodies to fibronectin and basic fibroblast growth factor. Epithelial cell proliferation was unaffected or only mildly stimulated by partially purified SF at concentrations that produced cell scattering. Both smooth muscle- and MRC5 human embryo fibroblast-derived SFs could be partially purified with similar elution patterns on a number of different chromatographic columns, including DEAE-agarose, heparin-sepharose, Bio-Rex 70, concanavalin A-sepharose, and MonoQ. SF from both sources bound tightly to heparin-sepharose, requiring 1.3 to 1.4 M NaCl for elution. The morphologically obvious cell scattering effect was markedly inhibited by soluble heparin at concentrations down to 5 micrograms/ml, and this inhibition was prevented by protamine. These data suggest that vascular smooth muscle cells produce an epithelial cell scattering factor with properties similar to the fibroblast-produced factor, including a high affinity for heparin. Such factors are potentially important because they may represent a new class of proteins that primarily regulate cell mobility rather than growth and differentiation.
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PMID:Smooth muscle releases an epithelial cell scatter factor which binds to heparin. 253 11

Seryl-tRNA synthetase has been purified from the middle silk glands of Bombyx mori by successive chromatography on DEAE-Sephacel, hydroxylapatite, and Bio-Rex 70. The high abundance of seryl-tRNA synthetase in the middle silk glands may result from an adaptation of this organ for the production of the serine-rich protein, sericin. The enzyme is a dimer of Mr = 124,000 consisting of similar or identical subunits and has an oligomeric structure similar to its procaryotic and eucaryotic counterparts. Seryl-tRNA synthetase can be cleaved with trypsin to generate a fragment of Mr = 45,000 on sodium dodecyl sulfate gels; the presence of tRNASer protects the enzyme from tryptic cleavage. Conversion to the Mr = 45,000 species is accompanied by a 90% loss in aminoacyl-tRNA synthetase activity, but only a 20% loss in ATP PPi exchange activity.
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PMID:Seryl-tRNA synthetase from Bombyx mori. Purification and properties. 282 48

An attempt to identify amino groups of Naja naja siamensis neurotoxin that interact with acetylcholine receptor by a comparison of their reactivities in free and receptor bound neurotoxin. Toxicon 21, 219-229, 1983--Free Naja naja siamensis neurotoxin was acetylated with non-radioactive and acetylcholine receptor-bound neurotoxin with radioactive acetic anhydride. The toxins from the two experiments were combined and the monoacetyl derivatives isolated by chromatography on Bio-Rex 70. The yields were determined by spectrophotometry and scintillation counting. To localize the acetyl group, a radioactive monoacetyl toxin was oxidized with performic acid, digested with trypsin and a peptide with the radioactive acetyl group was isolated by gel filtration on Sephadex G-25 and high voltage paper electrophoresis. Amino acid analysis indicated from which part of the molecule the peptide was derived. In free toxin, Ac-Lys 23 and 49 account for 56% and 12%, respectively, of the monoacetyl derivatives, and in bound toxin for only 25% and 8%. Lys 49 is as reactive as Ile 1 in free toxin and 50-150% more reactive than Lys 69, 35 and 12, but it has the lowest reactivity in bound toxin, being only about half as reactive as any of these three residues. The large decrease in reactivity of Lys 23 and 49 indicates that they interact with the receptor. The proximity of the receptor makes them less accessible to acetic anhydride. The reactivities are compared to that of Lys 12, which in free toxin has the least reactive amino group. The yield of Ac-Lys 23 relative to that of Ac-Lys 12 drops from 12.4 to 1.5, or by 88%, Lys 49, 2.6 and 0.5 (81%); Ac-Ile 1, 2.6 and 1.1 (58%); Ac-Lys 69, 1.9 and 0.9 (53%); Ac-Lys 35, 1.8 and 1.0 (44%). The drop in reactivity relative to that of Lys 12 indicates a real decrease, provided that Lys 12 does not become more reactive in bound toxin. This is unlikely, since sequence homology shows that Lys 12 corresponds to Lys 15 of the neurotoxin oxiana II of Naja naja oxiana, a residue known to interact with the receptor. Sequence homology also supports the conclusion that the drop in the reactivity of Ile 1 has the same cause. The receptor-binding region of the siamensis toxin is rather large, containing the residue Lys 23 and 49, Ile 1 and probably also Lys 69 and 35.
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PMID:An attempt to identify amino groups of Naja naja siamensis neurotoxin that interact with acetylcholine receptor by a comparison of their reactivities in free and receptor-bound neurotoxin. 685 7