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)

Trypsin and chymotrypsin were used as probes of conformation of G-actin molecule. The pattern of fragments produced has been analyzed by sodium dodecyl sulfate gel electrophoresis. G-actin is known to be nonrefractory to proteolysis [Jacobson, G.R., and Rosenbusch, J.P. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 2742-2746]. It is really true that G-actin is cut easily into a 33-kDa fragment by trypsin or chymotrypsin, but only when free ATP is present in the medium. After the removal of free ATP from the medium, G-actin became more refractory to proteolysis. The amounts of degradation of G-actin depended on the ATP concentration in the medium with saturating at about 0.5 mM. epsilon-ADP also had the effect and its fluorescence spectrum was changed on the addition of G-actin. After the removal of free ATP, G-actin still bound 1 mol/mol of ATP. So, the present results suggest the presence of a second ATP interaction site on G-actin and that ATP interaction at this site induces conformational changes in G-actin molecule.
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PMID:Structural aspects of skeletal muscle G-actin molecule as studied by proteolytic digestion: effect of nucleotide. 319 Jul 15

Ribonucleotide reductase catalyzes the critical reaction in which the deoxyribonucleotides required for DNA replication are synthesized de novo. This enzyme consists of two non-identical protein subunits, both of which are required for enzymatic activity. These subunits consist of a non-heme iron and an effector-binding subunit. These subunits are not coordinately regulated as the cells pass from G1 to the S phase of the cell cycle. Studies carried out with the holoenzyme and the isolated subunits indicate that the effector-binding subunit is more susceptible to chymotrypsin and the sulfhydryl reagents, pCMB and NEM, than is the non-heme iron subunit. The non-heme iron subunit is more susceptible to trypsin than is the effector-binding subunit. The presence of ATP or dATP protects the effector-binding subunit from proteolysis by either trypsin or chymotrypsin. The loss of activity in the holoenzyme, as a result of proteolysis, parallels the loss of the particular subunit. These results demonstrate that the protein properties of the subunits are significantly different to account for the differential turnover. The binding of nucleotides to the effector-binding site(s), which in turn regulates ribonucleotide reductase activity, is very specific. Formycin 5'-triphosphate and etheno-ATP could not replace ATP in the CDP reductase reaction. 2',3'-DideoxyATP was 5-fold less active than dATP as a negative effector; etheno-dATP was not inhibitory. AraGTP and BuPdGTP could not replace dGTP as a positive effector of ADP reduction. BuPdGTP, but not araGTP, served as an inhibitor of CDP reduction. 2',3'-DideoxyTTP was much less active as either an activator of GDP reduction or an inhibitor of ADP reduction. These studies indicate that the binding to the allosteric sites is highly specific and suggest that the structural requirements for the binding of activators are different from the structural requirements for the binding of inhibitors.
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PMID:Protein properties of the subunits of ribonucleotide reductase and the specificity of the allosteric site(s). 354 6

A variety of proteases have been evaluated as potential structural and conformational probes of nonphosphorylated and phosphorylated phosphorylase kinase. In general, the enzyme's alpha subunit is rapidly degraded, followed in most cases by hydrolysis of the beta subunit; the gamma subunit is resistant to most proteases. Trypsin clearly distinguishes between the nonactivated and activated conformers of phosphorylase kinase, in that the beta subunit in phosphorylated enzyme, as opposed to nonphosphorylated enzyme, is markedly protected from tryptic attack. In contrast, only a small difference in the rates of proteolysis of the alpha subunit in phosphorylated and nonphosphorylated enzyme is seen, even when a protease is used that is highly selective for the alpha subunit, such as chymotrypsin or endoproteinase Arg C. Incubation of nonphosphorylated phosphorylase kinase with either Mg2+ or Ca2+, which are activating cations, also protects the beta subunit from tryptic hydrolysis, whereas Mn2+, which inhibits the kinase activity, has little effect on proteolysis. The allosteric activator ADP also causes the beta subunit to become refractory to trypsin and mimics the effects of phosphorylation. Similar effector-induced conformational changes in the beta subunit are also observed with enzyme in which the alpha subunit has previously been selectively destroyed. These data indicate that activation of phosphorylase kinase by dissimilar mechanisms is associated with a conformational change in the enzyme's beta subunit that is detectable by trypsin and confirm earlier studies from this laboratory employing a chemical cross-linker as a conformational probe for activated and nonactivated conformers of the enzyme (Fitzgerald, T. J., and Carlson, G. M. (1984) J. Biol. Chem. 259, 3266-3274).
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PMID:Phosphorylase kinase conformers. Detection by proteases. 354 30

Fibrinogen-platelet interaction was studied in suspensions of platelets obtained from patients with uncontrolled diabetes mellitus of long duration and from control individuals. Fibrinogen binding sites were exposed by stimulating platelets with ADP or with chymotrypsin. There was no significant difference in fibrinogen mediated aggregation between ADP-stimulated platelets of 80 control and 47 diabetic subjects. The Km values for fibrinogen mediated aggregation of ADP-stimulated platelets obtained from control and diabetic donors were 1.39 +/- 0.13 X 10(-7)M and 1.44 +/- 0.13 X 10(-7)M; the Vmax values (expressed in arbitrary light transmission units) were 87.8 +/- 3.14 and 92.8 +/- 4.5 (mean +/- S.E.M.). The analysis of variance showed no significant relationship between Km, Vmax, age and sex in control group; in patient group there was no significant relationship between Km, Vmax, age, sex, type of diabetes, presence of vascular complications and type of treatment (insulin and/or oral hypoglycemic agents). Fibrinogen mediated aggregation of chymotrypsin-treated platelets showed similar pattern in 25 control and in 25 diabetic donors. In 24 normal individuals and in 24 diabetic patients Scatchard analysis revealed 48,820 +/- 5350 fibrinogen binding sites per one normal platelet (Kd = 4.7 X 10(-7)M) and 45,350 +/- 4663 sites per one diabetic platelet (Kd = 3.5 X 10(-7)M). Our data suggest a normal pattern of interaction between fibrinogen and fully activated platelets of diabetic subjects.
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PMID:Fibrinogen interaction with platelets of diabetic subjects. 360 36

The following lines of evidence suggest that MP100, a putative ADP receptor, and GPIIIa are distinct proteins. [3H]FSBA incorporated equally into normal and thrombasthenic platelets (less than 5% GPIIIa), quantitatively as well as qualitatively. The dose-dependent inhibition of ADP-induced platelet shape change by FSBA is identical for normal and thrombasthenic platelets. Polyclonal rabbit antibodies precipitate MP100 and GPIIIa, but monoclonal antibodies directed against GPIIb/GPIIIa complex GPIIIa and P1A1 fail to precipitate the ADP receptor protein. A monoclonal antibody which inhibits ADP-induced platelet aggregation and fibrinogen binding fails to inhibit ADP-induced shape change. Thus, both functional and immunochemical evidence clearly indicates the distinct character of the ADP and fibrinogen receptors. We hypothesize that conformational changes in an ADP receptor on binding ADP or proteolytic changes as with chymotrypsin digestion may be responsible for exposure of the normally latent fibrinogen receptor (GPIIb/GPIIIa complex).
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PMID:Separation of the 100-kDa membrane protein mediating ADP-induced platelet shape change and activation from glycoprotein IIIa. 360 28

Close platelet-to-platelet contact induced by weak agonists in a medium with a low concentration of Ca2+ leads to thromboxane A2 (TXA2) formation, release of granule contents, and secondary aggregation. These responses do not occur in a medium containing Ca2+ in the physiological range (1 to 2 mmol/L). Experiments were done to determine whether feedback amplification is required to generate amounts of TXA2 that are sufficient to cause secondary aggregation and the reactions associated with it, or whether close platelet-to-platelet contact alone is sufficient to generate enough TXA2 to produce these responses. Platelets were washed and resuspended in a modified Tyrode solution to which no calcium salt was added that contained 0.35% albumin and apyrase. This medium contains 20 mumol/L Ca2+ and 1 mmol/L Mg2+. Platelets were aggregated with adenosine diphosphate (ADP) in the presence of fibrinogen, agglutinated with polylysine, or after pretreatment with chymotrypsin, aggregated with fibrinogen. In the low-Ca2+ medium, all these agonists caused platelets to adhere to each other, followed by secondary aggregation with TXA2 formation and release of granule contents. When Ca2+ (1 to 2 mmol/L), aspirin, or the thromboxane receptor blocker BM 13.177 was present, the secondary responses did not occur; dazoxiben decreased thromboxane formation, but did not prevent secondary aggregation or release. Aspirin-treated platelets were less responsive to ADP, U46619, or TXA2 in the low-Ca2+ medium, which indicated that the secondary responses of untreated platelets were not caused by a generalized increase in sensitivity. The reactions that result from close platelet-to-platelet contact in a low-Ca2+ medium can be caused by a wide variety of weak agonists; the secondary aggregation response and release of granule contents are dependent on TXA2 formation and on feedback amplification by TXA2 or the prostaglandin endoperoxides. The secondary responses caused by weak agonists in citrated platelet-rich plasma (which has a concentration of Ca2+ similar to the low-Ca2+ medium used in the present studies) do not occur at the concentration of Ca2+ in circulating blood and thus may have little biologic relevance.
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PMID:Thromboxane A2 causes feedback amplification involving extensive thromboxane A2 formation on close contact of human platelets in media with a low concentration of ionized calcium. 362 Jun 98

Trigramin, a highly specific inhibitor of fibrinogen binding to platelet receptors, was purified to homogeneity from Trimeresurus gramineus snake venom. Trigramin is a single chain (approximately 9 kDa) cysteine-rich peptide with the Glu-Ala-Gly-Glu-Asp-Cys-Asp-Cys-Gly-Ser-Pro-Ala NH2-terminal sequence. Chymotryptic fragmentation showed the Arg-Gly-Asp sequence in trigramin. Trigramin inhibited fibrinogen-induced aggregation of platelets stimulated by ADP (IC50 = 1.3 X 10(-7)M) and aggregation of chymotrypsin-treated platelets. It did not affect the platelet secretion. Trigramin was a competitive inhibitor of the 125I-fibrinogen binding to ADP-stimulated platelets (Ki = 2 X 10(-8) M). 125I-Trigramin bound to resting platelets (Kd = 1.7 X 10(-7) M; n = 16,500), to ADP-stimulated platelets (Kd = 2.1 X 10(-8) M; n = 17,600), and to chymotrypsin-treated platelets (Kd = 8.8 X 10(-8) M; n = 13,800) in a saturable manner. The number of 125I-trigramin binding sites on thrombasthenic platelets amounted to 2.7-5.4% of control values obtained for normal platelets and correlated with the reduced number of GPIIb-GPIIIa molecules on the platelet surface. EDTA, monoclonal antibodies directed against the GPIIb-GPIIIa complex, and synthetic peptides (Arg-Gly-Asp-Ser and Tyr-Gly-Gln-Gln-His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val) blocked both 125I-fibrinogen binding and 125I-trigramin binding to platelets. Fibrinogen binding was more readily inhibited by these compounds than was trigramin binding. Monoclonal antibodies directed either against GPIIb or GPIIIa molecules did not block the interaction of either ligand with platelets. Reduced, S-pyridylethyl, trigramin did not inhibit platelet aggregation and fibrinogen binding to platelets and it did not bind to platelets, suggesting that the secondary structure of this molecule is critical for expression of its biological activity.
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PMID:Trigramin. A low molecular weight peptide inhibiting fibrinogen interaction with platelet receptors expressed on glycoprotein IIb-IIIa complex. 368 Feb 47

The Baumgartner perfusion apparatus has been used for quantitative comparison of the interaction of platelets with subendothelium in the presence of microvesicles derived from SKNMC (human neuroblastoma) cells, which aggregate platelets by an adenosine diphosphate (ADP)-dependent mechanism, and U87MG (human glioblastoma) cells, which function by a thrombin-dependent mechanism. The derived microvesicles from each line were as effective as the intact cells in inducing thrombogenesis on both undigested and alpha-chymotrypsin-digested subendothelium. Thrombus size on digested vessels was greater than on undigested vessels by fivefold for SKNMC cells and microvesicles and by 20-fold for U87MG cells and sevenfold for U87MG microvesicles. The results show that microvesicles from both cell lines initiate interactions between platelets and subendothelium identical to those caused by intact tumor cells. The results also demonstrate that intact tumor cells in the circulation may not be necessary for the thromboembolic complications of malignancy.
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PMID:Morphometric evaluation of thrombogenesis by microvesicles from human tumor cell lines with thrombin-dependent (U87MG) and adenosine diphosphate-dependent (SKNMC) platelet-activating mechanisms. 378 31

Ajoene, the major antiplatelet compound derived from garlic inhibits the fibrinogen-supported aggregation of washed human platelets (ID50 = 13 microM) and, inhibits binding of 125I-fibrinogen to ADP-stimulated platelets (ID50 = 0.8 microM). In both cases, the inhibition is of the mixed non-competitive type. Furthermore, fibrinogen-induced aggregation of chymotrypsin-treated platelets is also inhibited by ajoene in a dose-dependent manner (ID50 = 2.3 microM). Other membrane receptors such as ADP or epinephrine receptors are not affected by ajoene. Ajoene strongly quenches the intrinsic fluorescence emission of purified glycoproteins IIb-IIIa (ID50 = 10 microM). These results indicate that the antiaggregatory effect of ajoene is causally related to its direct interaction with the putative fibrinogen receptor.
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PMID:The molecular basis of the antiplatelet action of ajoene: direct interaction with the fibrinogen receptor. 380 Sep 91

Washed human platelets are damaged by two neutral proteases from human leukocytes (elastase-like protease, ELP and chymotrypsin-like protease, CLP). The damage is manifested as inhibited aggregation by ristocetin and collagen, and enhanced aggregation by ADP in the presence of fibrinogen. Similarly to alpha-chymotrypsin (alpha-CT), CLP also increases binding of 125I-fibrinogen to platelets and renders platelets aggregable by human fibrinogen. ELP is less effective in this respect possibly due to damage to platelet receptors for fibrinogen. In the plasma medium platelets are not sensitive to leukocytic proteases added at concentrations that provoke some prolongation of the time of plasma clotting by thrombin.
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PMID:Alterations of blood platelet function induced by neutral proteases from human leukocytes. 389 61


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