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:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The influence of various proteinases on GTP hydrolysis was studied in membranes of human platelets. Of the proteinases examined, trypsin, acrosin and a recently described trypsin-like proteinase from bovine sperm, but not chymotrypsin, increased GTP hydrolysis. Similar to what was described previously for hormone-like agents, the stimulation of GTP hydrolysis by the proteinases was only observed at low GTP concentrations, with apparent Km values of 0.2-0.3 microM-GTP. Stimulation of the high-affinity GTPase by the proteinases occurred without apparent lag phase and was constant over a long period of incubation. The proteinase inhibitors leupeptin and soya-bean trypsin inhibitor blocked the stimulation of GTP hydrolysis, but did not reverse the effect of the proteinases. Treatment of platelet membranes with N-ethylmaleimide, which eliminates Gi-protein (inhibitory guanine-nucleotide-binding protein)-related GTPase stimulation by adrenaline, decreased stimulation of GTP hydrolysis by the proteinases only partially. Activation of GTP hydrolysis by the proteinases was partially additive with that caused by adrenaline, whereas thrombin stimulation was not increased further. The data indicate that, similarly to the proteinase thrombin, trypsin and trypsin-like proteinases can activate GTP-hydrolysing protein(s) that exhibit high affinity for GTP in platelet membranes. It is suggested that the proteinases interact in platelet membranes with a receptor site similar to that used by thrombin and that the observed GTPase stimulation is a reflection of a proteinase-receptor interaction with a guanine-nucleotide-binding regulatory protein.
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PMID:Stimulation of high-affinity GTPase by trypsin and trypsin-like proteinases in membranes of human platelets. 283 22

The guanine nucleotide-binding proteins (G proteins), which transduce hormonal and light signals across the plasma membrane, are heterotrimers composed of alpha, beta, and gamma subunits. Activation of G proteins by guanine nucleotides is accompanied by dissociation of the heterotrimer: G + alpha.beta.gamma in equilibrium alpha G + beta.gamma. Brain contains several G proteins of which the most abundant are alpha 39.beta.gamma and alpha 41.beta.gamma. We have used proteolysis by trypsin to study the functional domains of the alpha subunits. In the presence of guanosine 5'-(3-O-thio)triphosphate, trypsin removes a 2-kDa peptide from the amino terminus of these proteins (Hurley, J. B., Simon, M. I., Teplow, D. B., Robishaw, J. D., and Gilman, A. G. (1984) Science 226, 860-862; Winslow, J. W., Van Amsterdam, J. R., and Neer, E. J. (1986) J. Biol. Chem. 261, 7571-7579). Tryptic cleavage does not affect the GTPase activity of the truncated molecule nor the apparent Km for GTP. However, removal of the 2-kDa amino-terminal peptide prevents association of the alpha subunits with beta.gamma. Since the apparent substrate for pertussis toxin-catalyzed ADP-ribosylation is the alpha.beta.gamma heterotrimer, the trypsin-cleaved alpha subunit is not a substrate for the toxin. Digestion of the carboxyl terminus of alpha 39 with carboxypeptidase A prevents ADP-ribosylation by pertussis toxin but does not interfere with the formation of alpha 39.beta.gamma heterotrimers. We do not yet know whether the amino-terminal region of alpha 39 interacts with beta gamma directly or whether it is necessary to maintain a conformation of alpha 39 which is required for heterotrimer formation. Further studies are needed to define the nature of the contracts between alpha and beta gamma subunits since understanding the structural basis for their reversible interaction is fundamental to understanding their function.
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PMID:The amino terminus of G protein alpha subunits is required for interaction with beta gamma. 313 54

Nicotinamide 1,N6-ethenoadenine dinucleotide (etheno-NAD, epsilon-NAD), a fluorescent analogue of NAD, was able to serve as a substrate for the bacterial toxin-catalyzed epsilon-ADP ribosylation of signal-transducing G-proteins. Pertussis toxin and transducin were used as a model system to characterize this reaction. Similar to ADP ribosylation using NAD as substrate, the epsilon-ADP ribosylation occurs at the carboxyl-terminal 5-kDa tryptic fragment of the T alpha subunit of transducin with the same labeling stoichiometry; however, the rate of labeling is slightly slower. epsilon-NAD competes with NAD as a substrate which suggests that the epsilon-ADP ribosylation occurs at Cys-347 of the T alpha subunit. The biochemical effects of epsilon-ADP ribosylation on transducin are similar to those of ADP ribosylation and include inhibition of the GTPase and [3H]Gpp(NH)p-binding activities. The epsilon-ADP-ribosylated transducin exhibits a fluorescent spectrum which resembles that of epsilon-ADP with an excitation maximum at 292 nm and an emission maximum of 413 nm. Removal of the amino-terminal peptide of epsilon-ADP-ribosylated T alpha with either Staphylococcus aureus V8 protease or trypsin results in a decrease in the emission intensity. This result suggests that the amino- and carboxyl-terminal peptides of the T alpha molecule may interact with each other as suggested previously (Hingorani, V. N., and Ho, Y.-K. (1987) FEBS Lett. 220, 15-22). epsilon-NAD should prove to be a useful fluorescent substrate for future studies of the ADP ribosylation reaction in biological systems.
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PMID:Fluorescent labeling of signal-transducing G-proteins. Pertussis toxin-catalyzed etheno-ADP ribosylation of transducin. 314 31

The structure of the guanosine nucleotide binding site of EF-2 was studied by affinity labelling with the GTP analogue, oxidized GTP (oGTP), and by amino acid sequencing of polypeptides generated after partial degradation with trypsin and N-chlorosuccinimide. Native EF-2 contains two exposed trypsin-sensitive cleavage sites. One site is at Arg66 with a second site at Lys571/Lys572. oGTP was covalently bound to the factor between Arg66 and Lys571. After further cleavage of this fragment with the tryptophan-specific cleavage reagent N-chlorosuccinimide, oGTP was found associated with a polypeptide fragment originating from a cleavage at Trp261 and Trp343. The covalent oGTP . EF-2 complex was capable of forming a high-affinity complex with ribosomes, indicating that oGTP, in this respect, induced a conformation in EF-2 indistinguishable from that produced by GTP. Although GTP could be substituted by non-covalently linked oGTP in the factor and ribosome-dependent GTPase reaction, the factor was unable to utilize the covalently bound oGTP as a substrate. This indicates that the conformational flexibility in EF-2 required for the ribosomal activation of the GTPase was inhibited by the covalent attachment of the nucleotide to the factor. EF-2 cleaved at Arg66 were unable to form the high-affinity complex with ribosomes while retaining the ability to form the low-affinity complex and to hydrolyse GTP. The second cleavage at Lys571/Lys572 was accompanied by a total loss of both the low-affinity binding and the GTPase activity.
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PMID:Structural and functional studies of the interaction of the eukaryotic elongation factor EF-2 with GTP and ribosomes. 333 67

Cyclic guanosine 3',5'-monophosphate phosphodiesterase in crude extracts from bovine rod outer segments can be activated by the addition of bleached rod outer segment membranes and GTP. In the absence of rhodopsin-containing membranes, the phosphodiesterase specific activity decreases with increasing concentration. A trypsin-sensitive inhibitor believed to be responsible for this phenomenon can be separated from the phosphodiesterase by DEAE-cellulose chromatography of the crude extract. Phosphodiesterase eluted from the DEAE-cellulose column shows considerably less concentration-dependence than in the crude extract. This partially purified phosphodiesterase was used as the substrate to assay for inhibitor. A GTPase which is active only in the presence of bleached rod outer segment membranes coelutes with the phosphodiesterase and is distinct from the phosphodiesterase inhibitor we have isolated.
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PMID:Isolation of an inhibitory protein for the cyclic guanosine 3','5'-monophosphate phosphodiesterase of bovine rod outer segments. 611 78

The first stage of amplification in the cyclic GMP cascade in bovine retinal rod is carried out by transducin, a guanine nucleotide regulatory protein consisting of two functional subunits, T alpha (Mr approximately 39,000) and T beta gamma (Mr approximately 36,000 and approximately 10,000). Limited trypsin digestion of the T beta gamma subunit converted the beta polypeptide to two stable fragments (Mr approximately 26,000 and approximately 14,000). The GTPase and Gpp(NH)p binding activities were not significantly affected by the cleavage. Trypsin digestion of the T alpha subunit initially removed a small segment from the polypeptide terminus and resulted in the formation of a single 38,000-Da fragment. When this fragment was recombined with the intact T beta gamma subunit in the presence of membranes containing photolyzed rhodopsin, the reconstituted transducin exhibited greatly reduced GTPase and Gpp(NH)p binding activities. The loss in activities was due to the inability of the cleaved T alpha to bind to the photolyzed rhodopsin. Prolonged digestion converted the 38,000-Da fragment to a transient 32,000-Da fragment and then to two stable 23,000-Da and 12,000-Da fragments. The cleavage of the 32,000-Da fragment, however, can be blocked by bound Gpp(NH)p. The 32,000-Da fragment contains the Gpp(NH)p binding site and retains the ability to activate phosphodiesterase. These results indicate that the guanine nucleotide binding and rhodopsin binding sites are located in topologically distinct regions of the T alpha subunit and proved evidence that a large conformational transition of the molecule occurs upon the conversion of the bound GDP to GTP.
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PMID:Characterization of transducin from bovine retinal rod outer segments. II. Evidence for distinct binding sites and conformational changes revealed by limited proteolysis with trypsin. 613 10

When EF-Tu was photooxidized for 20 min at 0 degrees C in the presence of 10 microM GDP and 5 microM rose bengal, the activity to promote the binding of [14C]Phe-tRNA to ribosomes was rapidly lost, while the activity to bind [3H]GDP remained intact. The activity of EF-Tu to interact with Phe-tRNA and ribosomes, as assessed by protection of [14C]Phe-tRNA against RNase A digestion and by methanol-induced uncoupled GTPase activity, respectively, was also inactivated under the above conditions. It was found, however, that these activities were fully protected in the presence of aminoacyl-tRNA and GTP, indicating that the active site(s) of EF-Tu for interaction with aminoacyl-tRNA and ribosomes could be protected against photooxidation in the ternary aminoacyl-tRNA . EF-Tu . GTP complex. Comparison of the amino acid composition of EF-Tu photooxidized in the form of EF-Tu . GDP with that of the intact EF-Tu revealed that only 1.4 residues of histidine were damaged. On the other hand, no histidine residue was lost when EF-Tu was oxidized in the presence of both aminoacyl-tRNA and GTP. The photooxidized EF-Tu . GDP was then partially degraded with trypsin and each of the resulting tryptic fragments, D, B, and C (Arai, Nakamura, Arai, Kawakita, and Kaziro (1976) J. Biochem. 79, 69-83), was analyzed for histidine content. The results indicated that fragments B, C, and D had lost 0.7, 0.5, and 0.2 residues of histidine, respectively. Since fragment B contains the cysteine residue which is essential for interaction with aminoacyl-tRNA and ribosomes, the above results suggest that a histidine residue in fragment B may also play an essential role in the interaction with aminoacyl-tRNA and ribosomes.
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PMID:Selective photooxidation of histidine residues in polypeptide chain elongation factor Tu from E. coli. 703 Oct 46

Previously we have shown that the measurable soluble sialyltransferase (STase) activity released into the medium during the incubation of rat jejunal slices was dependent upon the presence of a heparin-binding fraction (HBF) from heat-inactivated serum or a trypsin-binding protein (TBP) isolated from HBF. Both HBF and TBP were able to inhibit trypsin and plasmin. The measurement of galactosyltransferase (GTase) activity which was also released in incubations was not dependent on HBF or TBP. The present study is directed towards further exploring the relationship between STase activity and protease inhibitory activity. Heat-inactivated serum from turpentine-treated rats (HTS), had higher plasmin-trypsin-inhibitory (HTS) activities compared to heat-inactivated serum from control rats (HCS). When HTS was used to supplement jejunal incubations, there was a 25-40% increase in the measurable STase activity in the incubation medium compared to similar incubations carried out in buffer alone. In contrast, with HCS the increase was 10-15%. During incubations with hepatocytes, STase activity detected in the incubation medium was increased with the incubation buffer was supplemented with HTS compared to incubations supplemented with HCS. Serum antiproteolytic activity was higher in turpentine rats compared to controls. Incubation of serum at 37 degrees C led to a progressive decrease in plasmin-trypsin-inhibitory and STase activities. TBP a plasmin and trypsin inhibitor was able to prevent the decrease in STase activity. Overall, serum STase activity was higher in the turpentine treated rats. In contrast, GTPase activity in serum as well as that detected in the medium during jejunal and hepatocyte incubations was not dependent on protease inhibitory activity.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Relationship between plasmin-trypsin-inhibitory and sialyltransferase activities. 755 56

Biochemical, structural, and functional properties of Rab5 wild-type (WT) protein were compared with those of Q79L and N133I mutants. The detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate increased guanine nucleotide binding to Rab5 WT approximately 10-fold. The single-step catalytic rate of Rab5 WT exceeded that of Q79L 12.2-fold, but the steady-state GTPase rate was only 2.8-fold greater because GDP dissociation was rate-limiting and GDP dissociation was 3.6-fold slower than for Q79L. In contrast, dissociation rates of GTP were indistinguishable. Binding to Rab5 N133I was not detectable. GTP protected Rab5 WT and Q79L from any apparent proteolysis by trypsin. A 20-kDa fragment was the major product of digestion in the presence of GDP, and 12- and 8-kDa fragments were the major products in the absence of added guanine nucleotides. Rab5 N133I underwent no apparent proteolysis with 10 mM GTP or GDP, suggesting a "triphosphate" conformation may be induced in Rab5 N133I by either GTP or GDP. Partially geranylgeranylated Rab5 WT stimulated endosome fusion in vitro, whereas unmodified Rab5 WT did not. Processed Rab5 Q79L failed to inhibit endosome fusion, and Rab5 N133I could not be geranylgeranylated. These findings identify biochemical and structural features of Rab5 proteins, providing data for the interpretation of functional assays.
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PMID:Biochemical and functional characterization of a recombinant GTPase, Rab5, and two of its mutants. 789 Jun 12

Tubulin exhibits a number of characteristic functions that can be used to identify it. They include the ability to polymerize to microtubules, GTPase activity, and the binding of numerous antimitotic drugs and fluorophores. These functions can be differentially modified by low (0.1-1.0M) urea concentrations, and such urea-induced modifications are stable over time periods of minutes to hours. These intermediate states suggest the existence of restricted regions in the protein each of which is associated with a function and its own urea sensitivity. In order of decreasing sensitivity to urea these effects are decreased rate of polymerization of tubulin to microtubules > decreased extent of polymerization approximately decreased GTPase activity > enhanced fluorescence of a rapidly binding analogue of colchicine-MTPT [2-methoxy-5-(2',3',4'-trimethoxyphenyl)tropone] approximately decreased proteolysis by trypsin (after alpha Arg339) and by chymotrypsin (after beta Tyr281) > enhanced fluorescence of 1-anilino-8-naphthalenesulfonic acid (ANS). Additional evidence for the independent behavior of the restricted regions stems from the markedly different time dependence of the response to urea. These low urea concentrations do not induce significant changes in tryptophan fluorescence, suggesting that the observed effects are due to local unfolding. At higher urea concentrations (2-4 M), the enhanced fluorescence of the ligands is abolished; MTPT fluorescence decreases at lower urea concentrations than ANS fluorescence. Moreover, tubulin becomes highly susceptible to proteolysis at multiple sites, and tryptophan emission shows a red-shift, as expected. Multistep unfolding in response to denaturants has been reported for some other proteins. Tubulin appears to be an extreme example of such local responses that proceed under milder conditions than the global transition to the unfolded state.
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PMID:Local unfolding and the stepwise loss of the functional properties of tubulin. 794 93


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