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:3.4.21.1 (chymotrypsin)
10,938 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Adenylate deaminase (EC 3.5.4.6) may help to regulate the adenine nucleotide catabolism characteristic of such disease states as myocardial ischaemia. We report analysis of the molecular, kinetic and allosteric properties of rabbit heart adenylate deaminase when extracted and purified under phosphate-free conditions (i.e., with Hepes/KOH). The enzyme's subunit molecular mass (approximately 81 kDa), pI (6.5), substrate specificity for 5'-AMP, and activation by K+ were identical in the absence or presence of phosphate. At each chromatographic step during isolation without phosphate, cardiac adenylate deaminase showed a lower apparent activity as compared with the enzyme prepared with phosphate present. Kinetic constants for the phosphate-free rabbit heart adenylate deaminase preparation (Km 0.54 mM AMP; Vmax. 1.4 mumol/min per mg of protein) were approximately 10-fold lower than those of the enzyme isolated with phosphate. The same irreversible decrease in kinetic constants could be achieved by dialysing phosphate from the phosphate-containing enzyme preparation. The relationship between enzyme activity and substrate concentration was sigmoidal in the presence of phosphate, but hyperbolic in its absence. Cardiac adenylate deaminase under phosphate-free conditions was no longer allosterically activated by ATP and ADP, yet remained inhibitable by GTP. Enzyme inhibition by the transition-state mimic coformycin was not influenced by phosphate status. The phosphate-free preparation of rabbit heart adenylate deaminase was markedly labile and extremely susceptible to proteolysis by trypsin or chymotrypsin. The inactivation kinetics and fragmentation pattern in response to controlled proteolysis depended on whether the enzyme had been isolated with or without phosphate present, suggesting a conformational difference between the two enzyme preparations. These data constitute direct evidence that the absence of phosphate irreversibly converts cardiac adenylate deaminase into a pseudo-isoenzyme with distinct kinetic, regulatory and stability properties.
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PMID:Cardiac adenylate deaminase: molecular, kinetic and regulatory properties under phosphate-free conditions. 800 40

Normal human platelets aggregated by thrombin undergo the release reaction and are not readily deaggregated by the combination of inhibitors hirudin, prostaglandin E1 (PGE1) and chymotrypsin. Released adenosine diphosphate (ADP) plays an important role in the stabilization of thrombin-induced human platelet aggregates. Since ticlopidine inhibits the platelet responses to ADP, we studied thrombin-induced aggregation and deaggregation of 14C-serotonin-labeled platelets from 12 patients with cardiovascular disease before and 7 days after the oral administration of ticlopidine, 250 mg b.i.d. Before and after ticlopidine, platelets stimulated with 1 U/ml thrombin aggregated, released about 80-90% 14C-serotonin and did not deaggregate spontaneously within 5 min from stimulation. Before ticlopidine, hirudin (5x the activity of thrombin) and PGE1 (10 mumol/l) plus chymotrypsin (10 U/ml) or plasmin (0.06 U/ml), added at the peak of platelet aggregation, caused slight or no platelet deaggregation. After ticlopidine, the extent of platelet deaggregation caused by the same inhibitors was significantly greater than before ticlopidine. The addition of ADP (10 mumol/l) to platelet suspensions 5 s after thrombin did not prevent the deaggregation of ticlopidine-treated platelets. Thus, ticlopidine facilitates the deaggregation of thrombin-induced human platelet aggregates, most probably because it inhibits the effects of ADP on platelets.
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PMID:Ticlopidine facilitates the deaggregation of human platelets aggregated by thrombin. 816 51

1. ATP-sensitive K+ (K+ATP) channels are believed to make an important contribution to the increased cellular K+ efflux and shortening of the action potential duration (APD) during metabolic inhibition, hypoxia, and ischaemia in the heart. The mechanisms by which the activity of the K+ATP channel is regulated during conditions of metabolic impairment are not completely clear. Extrinsic factors such as increased [ADP]i, acidosis, and stimulation of adenosine receptors appear to decrease the K+ATP channel's sensitivity to closure by [ATP]i. The purpose of this study was to determine whether the K+ATP channel itself is intrinsically altered by the processes associated with metabolic impairment. 2. Isolated guinea-pig ventricular myocytes were metabolically inhibited in glucose-free 1.8 mM Ca2+ Tyrode solution containing 9 microM rotenone and 0.9 microM carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) while recording unitary currents through K+ATP channels in cell-attached patches. When K+ATP channel activity became maximal, the patch was excised (inside-out) into 150 mM K+ bath solution containing different ATP concentrations. The Kd for suppression by [ATP]i ([ATP]i causing half-maximal suppression of current through K+ATP channels) was markedly increased to 305 microM (n = 9) compared to patches excised from control myocytes not exposed to metabolic inhibitors (Kd = 46 microM, n = 28). 3. A [Ca2+]i-dependent process was involved in K+ATP channel modification during metabolic inhibition. Removal of extracellular Ca2+ during metabolic inhibition led to an intermediate decrease in the ATP sensitivity of the K+ATP channels (Kd = 120 microM, n = 6). In myocytes that were pretreated with 10 microM ryanodine in addition to removing extracellular Ca2+, the reduction in ATP sensitivity was completely prevented (Kd = 23 microM, n = 6). 4. In inside-out membrane patches excised from control non-metabolically inhibited myocytes, elevated free [Ca2+]i (2 microM) did not alter the sensitivity of the K+ATP channel to closure by [ATP]i, suggesting that in metabolically inhibited myocytes elevated [Ca2+]i acted indirectly. K+ATP channel run-down was found to increase the sensitivity of K+ATP channels to closure to [ATP]i (Kd = 16 microM, n = 13). 5. Inside-out membrane patches excised from control non-metabolically inhibited myocytes were also exposed to various proteases, phospholipases and other reagents that may be activated during metabolic inhibition. Trypsin and chymotrypsin treatment increased the Kd from 39 to 213 microM (n = 8) and 110 microM (n = 5), respectively. Calpain I had no apparent effect on the Kd.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:ATP-sensitive K+ channel modification by metabolic inhibition in isolated guinea-pig ventricular myocytes. 822 32

The platelet-membrane glycoprotein IIb-IIIa (GPIIb-IIIa) complex is essential for platelet aggregation and is involved in the attachment of platelets to thrombogenic surfaces. This study shows the retention of GPIIb and GPIIIa on immobilized fibrinogen after Triton X-100 (Sigma Chemical Co, St Louis, MO) lysis of adherent platelets. Glycoproteins were detected using subunit specific monoclonal antibodies in a modified enzyme-linked immunosorbent assay procedure. GPIIb-IIIa retention was judged to be specific relative to GPIb recovery, and was modulated by platelet activation. Platelet exposure to adenosine diphosphate or thrombin, but not A23187 or chymotrypsin, markedly enhanced GPIIb and GPIIIa recovery relative to that observed with unstimulated platelets, or prostaglandin E1-treated platelets. Moreover, lysis of adherent platelets in the presence of 10 mmol/L EDTA, under conditions promoting GPIIb-IIIa complex dissociation (pH 8.1, 60 minutes, 37 degrees C), had no effect on GPIIb or GPIIIa subunit recovery. Platelet activation with Zn+2 also enhanced GPIIb and GPIIIa recovery on fibrinogen-coated surfaces over that observed with unstimulated platelets, but GPIIb and IIIa retention was EDTA sensitive. This correlated with the EDTA-reversible nature of Zn+2-activated platelet adhesion to fibrinogen-coated surfaces. The data (1) show that platelet adhesion to fibrinogen is accompanied by the induction of high-affinity interactions between GPIIb-IIIa and immobilized fibrinogen that are EDTA-resistant and enhanced by platelet activation with some but not all agonists, and (2) implicate these interactions in stabilizing platelet contacts with fibrinogen-coated surfaces.
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PMID:Glycoprotein IIb and IIIa retention on fibrinogen-coated surfaces after lysis of adherent platelets. 824 6

Tetrafibricin; a nonpeptidic fibrinogen inhibitor from microbial origin, showed potent antiaggregation activities on human platelet aggregation induced by either ADP, thrombin or collagen (IC50s = 5.6, 7.6 and 11 microM, respectively) in platelet rich plasma. The ability to inhibit aggregation in platelets treated with chymotrypsin confirmed the GPIIb/IIIa blockage of tetrafibricin. Tetrafibricin blocked the release of serotonin induced by ADP but it did not block the release reaction induced by thrombin. When added to platelets formerly aggregated with ADP, tetrafibricin caused rapid and complete deaggregation. As for the selectivity among other Arg-Gly-Asp -dependent integrins, tetrafibricin seems to be more specific for glycoprotein (GP) IIb/IIIa than RGDS is. This is because it had no effect on adhesion of bovine aortic endothelial cells to RGD-containing proteins. Tetrafibricin is the first nonpeptidic fibrinogen receptor inhibitor that may be valuable for the study on platelet aggregation inhibitors.
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PMID:Tetrafibricin: a nonpeptidic fibrinogen receptor inhibitor from Streptomyces neyagawaensis. (II). Its antiplatelet activities. 830 83

The inside-out configuration of the patch-clamp method was used to study the effects of trypsin on the activity of ATP-sensitive potassium (K-ATP) channels from isolated mouse pancreatic beta-cells. Trypsin (20 micrograms/ml) irreversibly enhanced channel activity around twofold by reducing the interburst intervals without altering the burst kinetics. No effect on the single channel conductance or the inward rectification produced by internal Mg2+ was observed: however, the protease did reduce the inhibitory effect of Mg2+ on channel activity. Trypsin both prevented rundown of K-ATP channel activity and reactivated the channels after complete rundown. These effects of trypsin were absent in the presence of trypsin inhibitor. The protease also reduced the inhibitory effect of ATP on channel activity, increasing the dissociation constant from 7 to 49 microM. Trypsin removed the activating effect of ADP (0.1 mmol/l) on channel activity and reduced the inhibitory effect of tolbutamide (0.5 mmol/l). Carboxypeptidase A did not activate K-ATP channels in excised patches, although it was able to slightly reactivate channels after complete rundown, whereas chymotrypsin increased K-ATP channel activity but it did not produce reactivation. The effects of papain were similar to those of trypsin.
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PMID:Modification of K-ATP channels in pancreatic beta-cells by trypsin. 835 Dec 6

Photoaffinity labeling with [alpha-32P]8N3GTP and [gamma-32P]8N3GTP was used to identify the guanine binding domain of the GTP regulatory site within glutamate dehydrogenase (GDH). Without photolysis, 8N3GTP mimicked the regulatory properties of GTP on GDH activity with 8N3GTP exhibiting a Ki of 5 microM while the Ki for GTP was about 0.6 microM. Under optimal photolabeling conditions saturation of photoinsertion with 1 microgram of GDH revealed an apparent Kd of 9 +/- 4 microM for [gamma-32P]8N3GTP. Photolabeling with this analog could be competitively inhibited with GTP with an apparent Kd of 12 +/- 2 microM. Other nucleotides such as ATP and NAD(P)H could not reduce the amount of photoinsertion as effectively as GTP. ADP could decrease photoinsertion, but only at much higher concentrations. NAD(P)+, GDP, AMP, and GMP had little effect on photoinsertion. Divalent cations Mg2+ and Ca2+ also reduced photoinsertion significantly while the monovalent K+ and Na+ ions had no effect. Aluminum(III)-chelate or iron(III)-chelate affinity chromatography and reversed-phase HPLC were used to purify photolabel-containing peptides generated with either trypsin or chymotrypsin. This identified a portion of the guanine binding domain within the GTP regulatory site as the region containing the sequence Ile439 to Tyr454. Photolabeling of this peptide was prevented 91% by the presence of 300 microM GTP during photolysis. Lys445 was not identified in sequence analyses of the photolabeled peptides. Also, trypsin was unable to cleave the photolabeled peptide at this site. These results suggest that Lys445 may be the residue modified by [alpha-32P]8N3GTP.
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PMID:Identification of a guanine binding domain peptide of the GTP binding site of glutamate dehydrogenase: isolation with metal-chelate affinity chromatography. 843 45

The limited proteolytic pattern of transducin, Gt, and its purified subunits with chymotrypsin were analyzed and the cleavage sites on the alpha t subunit were identified. The alpha t subunit in the GTP gamma S bound form was cleaved into a major 38 kD fragment, whereas alpha t-GDP was progressively digested into 38, 23, 21, and 15 kD fragments. The beta gamma t subunit was not very sensitive to proteolytic digestion with chymotrypsin. The gamma t subunit was not cleaved and only a small portion of beta t was digested into several fragments. In order to determine which proteolytic fragment of alpha t still contained the carboxyl terminal region, chymotrypsinization was carried out using Gt previously 32P-labeled at Cys347 by pertussis toxin-catalyzed ADP-ribosylation. The 32P-label was mainly associated with the alpha t subunit and a 15 kD fragment. The 23 and 21 kD fragments were not 32P-labeled. Analysis of amino terminal sequences of 38, 21, and 15 kD proteolytic bands allowed the identification of the major cleavage sites. Chymotrypsin had two cleavage sites in the amino terminal region of alpha t, at Leu15 and Leu19. Chymotrypsin removed 15-19 amino acid residues from the amino terminus of alpha t, generating two peptides (38 kD) which comigrates in gel electrophoresis. Chymotrypsin also cleaved at Trp207 in a conformation-dependent manner. Trp207 of alpha t-GTP gamma S was resistant to proteolysis but alpha t-GDP and the 38 kD fragments of alpha t-GDP produced the 23 and 21 kD fragments, respectively, and a 15 kD fragment containing the carboxyl terminus.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Tryptophan207 is involved in the GTP-dependent conformational switch in the alpha subunit of the G protein transducin: chymotryptic digestion patterns of the GTP gamma S and GDP-bound forms. 848 7

Pertussis toxin (PT)-catalyzed ADP-ribosylation of transducin (Gt) is stimulated by ATP. In the absence of ATP, PT exhibited an approximately 20-fold lower linear velocity than the recombinant S1 subunit (rS1) in catalyzing the ADP-ribosylation of Gt. In the presence of 0.1 mM ATP, the linear velocities of rS1 and PT were essentially identical. ATP increased the kcat of PT-catalyzed ADP-ribosylation of Gt without altering the Kmapp for either Gt or NAD. Further, in the presence of ATP, PT exhibited similar kinetic constants under conditions of variable Gt and variable NAD as rS1 in catalyzing the ADP-ribosylation of Gt. The S1 subunit of PT was cleaved by chymotrypsin to a single immunoreactive peptide in the absence of ATP, while three immunoreactive peptides were generated in the presence of ATP. The S1 subunit of PT was not cleaved by trypsin in the absence of ATP, at the concentrations of trypsin used, while two immunoreactive peptides were produced in the presence of ATP. The immunoreactive peptides produced either by chymotrypsin or trypsin cleavage of the S1 subunit of PT in the presence of ATP were indistinguishable from those produced by cleavage of rS1 with the same protease. Chymotryptic and tryptic cleavage of rS1 was not altered by ATP. When PT was incubated with ATP prior to Bio-Gel P-100 gel filtration, approximately 8% of the S1 subunit dissociated from the B oligomer, as determined by ADP-ribosyltransferase assays of the column eluant. This increased to 20% when ATP was included in the column buffer. The presence of dithiothreitol and NAD in addition to ATP did not affect the amount of dissociated S1 subunit. Our data further indicated that activation of PT by ATP was a reversible process. Together, these data showed that ATP quantitatively converted the S1 subunit of PT to a form which was kinetically and conformationally identical with rS1, while only a fraction of the S1 subunit was dissociated from the B oligomer. These results indicate that both S1 subunit which is bound to the B oligomer as well as dissociated S1 subunit are capable of catalyzing the ADP-ribosylation of Gt.
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PMID:Molecular characterization of the in vitro activation of pertussis toxin by ATP. 850 98

In order to further characterize low density lipoprotein (LDL)-platelet interaction, we investigated the effect of protease pretreatment of human platelets on the subsequent binding of iodinated LDL (125I-LDL). Our results showed that the platelet LDL receptor had a proteolytic susceptibility different from that of both classical LDL receptors and the fibrinogen receptor. Platelet pretreatment with chymotrypsin, trypsin, and pronase (at 50 micrograms/mL) had no effect on 125I-LDL binding, whereas fibroblast 125I-LDL binding was markedly reduced. Mild proteolytic digestion, however (up to 1 mg/mL), was helpful in characterizing the platelet LDL receptor. Scatchard analysis showed that chymotrypsin did not modify LDL binding characteristics, whereas trypsin and pronase altered maximal number of binding sites (Bmax) without variation in dissociation constant. Trypsin increased Bmax approximately twofold (2156 +/- 327 binding sites on control platelets vs. 5246 +/- 296 on treated platelets, P < 0.001, mean +/- SEM, n = 5), but pronase decreased Bmax about 50% (2017 +/- 275 control vs. 1153 +/- 195 treated, P < 0.001). A minimum of 30 min preincubation was required to detect significant effects, and apparent equilibrium was reached by 60 min. Maximal increase in platelet LDL binding sites induced by trypsin was observed at a protein concentration of 1 mg/mL at 37 degrees C, whereas at 4 degrees C no effect was found. In contrast, maximal pronase-inhibitory effect also was observed at 37 degrees C but at higher protein concentration (10 mg/mL). Aprotinin, phenylmethylsulfonylfluoride, and soybean trypsin inhibitor were capable of fully blocking both the stimulation and the inhibition of platelet LDL binding induced by trypsin and pronase, respectively. Platelet pretreatment with both chymotrypsin and pronase (0.5 mg/mL) activated fibrinogen binding sites to a similar extent as ADP (100 microM). Furthermore, LDL (at a protein concentration of 0.3 mg/mL) increased by 81 +/- 6% the binding of fibrinogen to both protease- and ADP-stimulated platelets, but was unable to activate fibrinogen binding sites in unstimulated platelets. Overall, the results suggest that platelet LDL receptor presents a different proteolytic susceptibility in comparison with both "classical" LDL receptor and fibrinogen receptor.
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PMID:Proteolytic susceptibility of platelet low density lipoprotein receptor. 853 80


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