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)

A substrate protein for botulinum C3 ADP-ribosyltransferase (C3 exoenzyme) in human platelets was purified to apparent homogeneity from the cytosol by ammonium sulfate fractionation and successive chromatography on columns of DEAE-Sepharose, hydroxylapatite, phenyl-Sepharose, and TSK phenyl-5PW. The purified protein yielded an amino acid sequence identical to that of rhoA protein. When platelet cytosol and membranes were incubated with C3 exoenzyme and [32P]NAD and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing, they gave only one [32P]ADP-ribosylated band on each electrophoresis that showed an M(r) of 22,000 and a pI of 6.0. The radioactive bands from the two fractions co-migrated with each other and with the [32P]ADP-ribosylated purified protein. When these radioactive products were partially digested with either alpha-chymotrypsin or trypsin and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the same digestion pattern was found in the three samples. These results suggest that the ADP-ribosylation substrate for C3 exoenzyme in the platelet cytosol and membrane is rhoA protein and that it is the sole substrate detectable in human platelets.
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PMID:A rho gene product in human blood platelets. I. Identification of the platelet substrate for botulinum C3 ADP-ribosyltransferase as rhoA protein. 132 15

To examine whether immature rat Sertoli cells in culture secrete a factor(s) which stimulates testosterone production by mature mouse Leydig cells, Sertoli cell-enriched cultures were prepared from 3-week-old male rats with trypsin and collagenase. Sertoli cells were plated at an initial density of 3-5 x 10(6) cells/35 mm well and cultured in 3 ml serum free media supplemented with insulin (10 micrograms/ml). Sertoli cell culture medium (SCCM) collected every 3rd day was added to Leydig cells (10(6) cells in 1 ml of MEM with 2% steroid-free FCS) prepared from 10-week-old mice by mechanical separation and incubated for 3 h at 34 degrees C. Secreted testosterone was determined by RIA. SCCM 15 times concentrated by Amicon YM10 membrane demonstrated a dose-dependent stimulation of testosterone production, whereas there was no effect on testosterone secretion when Leydig cells were maximally stimulated by LH. Leydig cell stimulating activity was retained by both a dialysis membrane with a pore size of 24 A and an ultrafiltration membrane with a molecular weight cut-off of 10 kDa. However, activity was reduced by heating at 60 degrees C for 30 min and almost lost after incubation with 0.1% trypsin for 1 h at 37 degrees C. This activity was not retained by means of a Con A-Agarose column and was demonstrated only in break-through fractions. HPLC gel filtration of a 15 times concentrated SCCM preparation on a TSK gel G3000SW revealed Leydig cell-stimulating activity at approximately 13 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:A partial characterization of a Sertoli cell-secreted protein stimulating Leydig cell testosterone production. 139 52

Three types of caseinolytic proteases (Pase-A, Pase-B, and Pase-C) were isolated and purified from culture supernatants of Porphyromonas gingivalis 381 by the combined procedures of acetone precipitation, gel filtration, solubilization with octylthioglucoside followed by affinity chromatography on arginine-Sepharose 4B, high-performance liquid chromatography (HPLC) on Biofine IEC-DEAE, and HPLC on TSK-G4000SW. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Pase-A and -B showed diffuse protein bands of 105 to 110 and 72 to 80 kDa, respectively, and Pase-C showed a clear band of about 44 kDa. Pase-B and -C hydrolyzed some synthetic substrates for trypsin, but Pase-B did not act on the carboxyl side of lysine in insulin chain B or on a synthetic substrate which trypsin and Pase-C acted on. Pase-A did not act on the synthetic substrates but cleaved the peptide bonds Glu-Ala and Ala-Leu of insulin. Leupeptin inhibition of the caseinolytic activity of both Pase-A and -B was similar to its inhibition of Pase-C. Phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate strongly inhibited Pase-A, but no significant effect on the other enzymes was observed, suggesting that only Pase-A is a serine protease. The inhibitory characteristics of Pase-B and -C were very similar. Pase-A was not thiol dependent for enzyme activity, but Pase-B was strongly dependent, i.e., even more so than Pase-C. Pase-A inactivated the inhibitory activity of plasma alpha-1-antitrypsin, but the other two did not. These results show that P. gingivalis produces different types of proteases other than the trypsinlike protease generally reported.
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PMID:Purification and characterization of three types of proteases from culture supernatants of Porphyromonas gingivalis. 187 30

Pyruvate dehydrogenase was separated from pig heart pyruvate dehydrogenase complex by gel-permeation chromatography on a TSK G4000SWG column in the presence of 4 M urea, followed by chromatography on a calcium phosphate gel-cellulose column. The pyruvate dehydrogenase was further separated into two nonidentical subunits, alpha and beta, by high-performance liquid chromatography on a Synchropak CM-300 column in the presence of 8 M urea. The complete amino acid sequences of two subunits of pyruvate dehydrogenase were determined. The peptide fragments of S-carboxymethylated subunits were generated by treatment with endoproteinase Lys-C, endoproteinase Asp-N, trypsin, and cyanogen bromide. The subunits alpha and beta contain 361 amino acid residues (Mr 40,294) and 329 residues (Mr 35,787), respectively. The amino acid sequences of subunits alpha and beta in the pig were, respectively, 98 and 96% identical to those in humans. Hydropathy analysis and prediction of the secondary structure of two subunits suggest that the subunit alpha contains the thiamin pyrophosphate-binding domain and that the subunit beta contains segments with a high hydrophobicity.
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PMID:Novel separation and amino acid sequences of alpha and beta subunits of pig heart pyruvate dehydrogenase. 191 11

An improved method for the purification of human placental transferrin receptor (Tf-R) was developed. Fresh human placenta was homogenized in cold acetone and the acetone powder was prepared. After the acetone powder had been washed with HEPES buffer, the insoluble proteins containing Tf-R were separated by centrifugation and dissolved in Emulgen 109P-containing buffer. Tf-R was collected by affinity binding to Tf-Sepharose and extracted by consecutive treatment with 4 different kinds of buffers. Tf-R was eluted by buffer C (2 M KCl) and buffer D (0.5 M NaSCN). Tf-R was characterized as a 90-kDa monomer on gradient SDS-PAGE (4-20%) in the presence of 2-mercaptoethanol. Though there were several minor bands of 180- and above 205-kDa, all these bands were confirmed as Tf-R by Western blotting using an anti-Tf-R monoclonal antibody (OKT 9). The apparent molecular weights, measured by HPLC using a TSK-G 3,000 SW column, demonstrated that Tf-Rs eluted with buffer C were approximately 370-, 500- and above 500-kDa, but only a peak of above 500-kDa was found in Tf-R eluted with buffer D. Although the polymers of Tf-R with molecular weight of above 500-kDa were resistant to trypsin digestion, the Tf-R of 370-kDa was resistant to the enzyme only when it conjugated to the diferric Tf. The stability of the polymers of above 500-kDa to trypsin digestion suggested an advantage for the repeated use of Tf-R in the endocytosis of diferric Tf, which was performed by the translocation of Tf-R between cell surface and intracellular vesicles.
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PMID:[An improved purification of human placental transferrin receptor and biochemical properties of the receptor]. 206 90

Parathyroid hypertensive factor (PHF) is a newly described hypertensive factor that may be related to elevation of blood pressure in 30-40% of North American essential hypertensive patients. PHF is also found in several animal models of hypertension, including spontaneously hypertensive rats, and deoxycorticosterone acetate salt hypertensive rats. Plasma collected from spontaneously hypertensive rats (SHR) was used in the present study for purification of PHF. Plasma was dialyzed at a molecular mass cutoff of 1 kDa, and then ultrafiltered at a molecular mass cutoff of 5 kDa. PHF activity, as determined by bioassay (characteristic delayed hypertensive response in normotensive rat) was retained in the fraction that was greater than 1 kDa and less than 5 kDa. Dialyzed and ultrafiltered SHR plasma was fractionated by molecular-exclusion chromatography, either with Bio-Gel P-6 liquid chromatography, or TSK 2000 SW HPLC. The biological activity was detected in a discrete region corresponding to a molecular mass of 2.5-3 kDa. When the molecular-exclusion fraction was subsequently fractionated by reverse-phase HPLC, biological activity was located in a single discrete peak, which did not occur in plasma from normotensive rats prepared in a similar manner. The biologically active fraction of PHF was inactivated by trypsin; this and its UV spectrum indicate the presence of a peptide structure.
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PMID:Purification of parathyroid hypertensive factor from plasma of spontaneously hypertensive rats. 206 15

Purification of an A1 adenosine receptor of rat testes was performed using a newly developed affinity chromatography system (Nakata, H. (1989) Mol. Pharmacol. 35, 780-786). The A1 adenosine receptor was solubilized with digitonin from rat testicular membranes and then purified more than 25,000-fold by sequential use of affinity chromatography on xanthine amine congener-immobilized agarose, hydroxylapatite chromatography, re-affinity chromatography on xanthine amine congener-agarose, and finally gel permeation chromatography on TSK-3000SW. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the final preparation showed a single broad band of Mr 41,000 by autoradiography after radioiodination. This Mr 41,000 peptide was also specifically labeled with an A1 adenosine receptor affinity labeling reagent. A high affinity A1 adenosine receptor antagonist, 8-cyclopentyl-1,3-[3H]dipropylxanthine, bound saturably to the purified receptor with a KD of approximately 1.4 nM. The purified receptor also showed essentially the same specificity for adenosine agonists and antagonists as the unpurified receptor preparations, although the affinities of the purified adenosine receptor for agonists were significantly low compared to those of unpurified receptor preparations indicating that the purified A1 adenosine receptor exists as a low agonist-high antagonist affinity state. Deglycosylation of the purified testis adenosine A1 receptors with endoglycosidase F produced an increase in the mobility of the receptor protein to an apparent Mr 30,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, similar to that of deglycosylated A1 adenosine receptors of rat brain membranes. Peptide maps of the purified testis and brain A1 adenosine receptors using trypsin and V8 protease suggest that these receptors show some structural homologies.
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PMID:A1 adenosine receptor of rat testis membranes. Purification and partial characterization. 210 29

Carbohydrate-free caseinomacropeptide (CMP) was purified from rennet-hydrolysed caseinate by trichloroacetic acid precipitation and DEAE-TSK Fractogel-650 ion-exchange chromatography. To study the liberation of 106-112, 106-116 and 113-116 fragments from carbohydrate-free CMP involved in platelet function, a quantitative study was made on the rate of hydrolysis of the three peptidic bonds that are susceptible to the action of trypsin. Data were obtained from reverse-phase (Ultrabase column) and cationic-exchange (Mono S column) h.p.l.c. On the basis of the disappearance of substrate, kcat. and Km were respectively 3.95 s-1 and 0.2 mM. The two 111-112 and 112-113 bonds were split according to similar kinetic parameters (kcat. = 1.97 s-1, Km = 0.2 mM) and much faster than the 116-117 bond. The difference in susceptibility of the bonds can probably be attributed to the nature of residues flanking the primary proteolytic sites rather than to their accessibility to the proteinase. On the basis of our results the 106-116 fragment cannot be formed.
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PMID:Liberation of tryptic fragments from caseinomacropeptide of bovine kappa-casein involved in platelet function. Kinetic study. 222 16

A proteinase was purified from resting seeds of Cucurbita ficifolia by ammonium sulfate fractionation and successive chromatography on CM-cellulose, Sephacryl S-300 and TSK DEAE-2SW (HPLC) columns. Inhibition by DFP and PMSF suggests that the enzyme is a serine proteinase. The apparent molecular mass of this enzyme is ca. 77 kDa. The optimum activity for hydrolysis of casein and Suc-Ala-Ala-Pro-Phe-pNA is around pH 10.5. The following peptide bonds in the oxidized insulin B-chain were hydrolysed by the proteinase: Phe1-Val2, Asn3-Gln4, Gln4-His5, Cya7-Gly8, Glu13-Ala14, Ala14-Leu15, Cya19-Gly20, Pro28-Lys29 and Lys29-Ala30. The proteinase is more selective towards the native squash seed trypsin inhibitor (CMTI I) and primarily cuts off only its N-terminal arginine. The inhibitor devoided of the N-terminal arginine residue is still active against trypsin.
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PMID:Serine proteinase from Cucurbita ficifolia seed; purification, properties, substrate specificity and action on native squash trypsin inhibitor (CMTI I). 229 13

Chlamydomonas flagellar sexual agglutinins are responsible for the adhesion of opposite mating-type (plus and minus) gametes during the first stages of mating. Purification and partial characterization of the plus agglutinin was previously reported (Adair, W. S., C. J. Hwang, and U. W. Goodenough, 1983, Cell, 33:183-193). Here we characterize the purified minus molecule. We show it to be a high molecular weight, hydroxyproline-rich glycoprotein that migrates in the 3% stacking region of an SDS-polyacrylamide gel and is absent from two nonagglutinating minus mutants. Plus and minus agglutinins are remarkably similar, although nonidentical, in amino acid composition, molecular morphology, and reactivity in vivo and in vitro with monoclonal antibodies raised against the plus agglutinin. Moreover, the adhesiveness of both plus and minus agglutinins, when coupled to agarose beads, is abolished by thermolysin, trypsin, periodate, alkaline borohydride, reducing agents, or heat, but unaffected by exo- or endoglycosidases. The minus agglutinin, however, migrates just ahead of the plus molecule on SDS PAGE, is excluded from an anion-exchange (Mono Q) column, elutes earlier during hydrophobic interaction (Bio-gel TSK Phenyl 5PW) chromatography, and is sensitive to chymotrypsin digestion (unlike the plus agglutinin); therefore, it differs from the plus agglutinin in apparent molecular weight, net charge, relative hydrophobicity and proteolytic susceptibility. Nevertheless, our results generally demonstrate a high degree of homology between these complementary cell-cell recognition/adhesion molecules, which suggests that they are specified by genes that have a common evolutionary origin.
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PMID:Characterization of the purified Chlamydomonas minus agglutinin. 241 36


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