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)

Elongation factor 2 (eEF-2) can interact not only with guanylic nucleotides but also with adenylic ones, as was shown by intrinsic fluorescence quenching studies [Sontag, B., Reboud, A.M., Divita, G., Di Pietro, A., Guillot, D. & Reboud, J.P. (1993) Biochemistry 32, 1976-1980]. Here we studied sites of these interactions by using photoactivable 8-azido-[gamma-32P]GTP and 8-azido-[gamma-32P]ATP. Photoincorporation of the radioactive GTP derivative into eEF-2 was prevented by the previous addition of GTP and GDP. The addition of adenylic nucleotides (ATP, ADP) and some adenylic derivatives [NAD+, NADH,poly(A)] decreased the photoincorporation by only 40% at most. However, photoincorporation of the radioactive ATP derivative was prevented by the previous addition not only of adenylic compounds [ATP, ADP, NAD+, NADH, poly(A)] but also of GTP and GDP. Photoincorporation of radioactive nucleotide derivatives was not decreased by the addition of other nucleotidic compounds [UTP, poly(U), ITP, NADP+, NADPH]. ATP and GTP acted as non-competitive inhibitors of the photoincorporation of 8-azido-[gamma-32P]GTP and 8-azido-[gamma-32P]ATP, respectively. eEF-2 photolabeled with these radioactive nucleotide derivatives was submitted to trypsin digestion under different conditions and the labeled peptidic fragments identified after HPLC purification and gel electrophoresis by N-terminal sequencing. An octapeptide, Y264FDPANGK271, was the only peptide photolabeled with 8-azido-[gamma-32P]GTP whereas a N-terminal fragment of about 7 kDa was the only one photolabeled with 8-azido-[gamma-32P]ATP. The different results support the hypothesis that guanylic and adenylic nucleotides do not interact with the same site of eEF-2.
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PMID:Photoaffinity labeling of elongation factor-2 with 8-azido derivatives of GTP and ATP. 861 59

Treatment of eIF-2B and eIF-2 with NEM abolishes nucleotide exchange and GTP-binding activities of the proteins. Incubation of eIF-2B with [14C]NEM results in strong labeling of the 82- and 55-kDa subunits and with less labeling of the other subunits. Preincubation of eIF-2B with eIF-2 interferes with [14C]NEM labeling of the 82- and 55-kDa subunits. All three (alpha, beta, and gamma) subunits of eIF-2 are labeled strongly by [14C]NEM. Limited digestion of eIF-2B with trypsin inhibits nucleotide exchange activity but does not interfere with GTP binding. Under these conditions, the 65-kDa subunit is degraded completely while the other subunits remain intact. Treatment of eIF-2 with trypsin results in the generation of eIF-2 lacking the beta-subunit (eIF-2 alpha gamma). eIF-2(alpha gamma) binds [3H]GDP equally well as intact elf-2. In the presence of elf-2B, the exchange of [3H]GDP for GTP from elf-2. [3H]GDP prepared with eIF-2(alpha gamma) is diminished considerably. [3H]GTP binding to eIF-2(alpha gamma) is also four- to five-fold less than to intact eIF-2. In addition, the association of eIF-2B with intact eIF-2, but not with eIF-2(alpha gamma), reduces by two-fold the rate and extent of removal of 32P by alkaline phosphatase from CK-2-phosphorylated 82-kDa subunit.
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PMID:The interaction of rabbit reticulocyte guanine nucleotide exchange factor eIF-2B with chain initiation factor 2: studies with N-ethylmaleimide and trypsin. 868 43

The tryptic cleavage pattern of transducin (Gt) in solution was compared with that in the presence of phospholipid vesicles, rod outer segment (ROS) membranes kept in the dark, or ROS membranes containing light-activated rhodopsin, metarhodopsin II (Rh*). When Gt was in the high affinity complex with Rh*, the alphat subunit was almost completely protected from proteolysis. The protection of alphat at Arg310 was complete, while Arg204 was substantially protected. The cleavage of alphat at Lys18 was protected in the presence of phospholipid vesicles, ROS membranes kept in the dark, or ROS membranes containing Rh*. The cleavage of betat was slower in the presence of ROS membranes or phospholipid vesicles. When the Rh*. Gt complex was incubated with guanyl-5'-yl thiophosphate, a guanine nucleotide analog known to release the high affinity interaction between Gt and Rh*, the protection at Arg310 and Arg204 was diminished. From our results, we propose that Rh* either physically blocks access of trypsin to Arg204 and Arg310 or maintains the heterotrimer in such a conformation that these cleavage sites are not available. Since Arg204 is involved in the switch interface with betagammat (Lambright, D. G., Sondek, J., Bohm, A., Skiba, N. P., Hamm, H. E., and Sigler, P. B. (1996) Nature 379, 311-319), it may be that betagammat is implicated in protecting this cleavage site in the receptor-bound, stabilized heterotrimer. Arg310 is not near the betagammat subunit, thus we believe that the high affinity binding of Gt to Rh* physically or sterically blocks access of trypsin to this site. Thus, Arg310, only a few angstroms away from the carboxyl terminus of alphat, which is known to directly bind to Rh*, is likely to also be a part of the Rh* binding site. This is in agreement with other studies and has implications for the mechanism by which receptors catalyze GDP release from G proteins. The protection of Lys18 in the presence of phospholipid vesicles suggests that the amino-terminal region is in contact with the membrane, consistent with the crystal structure of the heterotrimer (Lambright, D. G., Sondek, J., Bohm, A., Skiba, N. P., Hamm, H. E., and Sigler, P. B. (1996) Nature 379, 311-319).
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PMID:Interaction of transducin with light-activated rhodopsin protects It from proteolytic digestion by trypsin. 893 50

We have compared the abilities of mammalian ADP-ribosylation factors (ARFs) 1, 5, and 6 and Saccharomyces cerevisiae ARF2 to serve as substrates for the rat liver Golgi membrane guanine nucleotide exchange factor and to initiate the formation of clathrin- and coatomer protein (COP) I-coated vesicles on these membranes. While Golgi membranes stimulated the exchange of GTPgammaS for GDP on all of the ARFs tested, mammalian ARF1 was the best substrate, with an apparent Km of 5 microM. In all cases myristoylation of ARF was required for stimulation. Agents that inhibit the Golgi membrane guanine nucleotide exchange factor (the fungal metabolite brefeldin A and trypsin treatment) selectively inhibited the guanine nucleotide exchange on mammalian ARF1. Taken together, these data indicate that of the ARFs tested, only mammalian ARF1 is activated efficiently by the Golgi guanine nucleotide exchange factor. The other ARFs are activated mainly by another mechanism, possibly phospholipid-mediated. Once activated, all of the membrane-associated, myristoylated ARFs promoted the recruitment of coatomer to about the same extent. Mammalian ARFs 1 and 5 were the most effective in promoting the recruitment of the AP-1 adaptor complex, whereas yeast ARF2 was the least active. These data indicate that the specificity for ARF action on the Golgi membranes is primarily determined by the Golgi guanine nucleotide exchange factor, which has a strong preference for myristoylated mammalian ARF1.
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PMID:Comparative activity of ADP-ribosylation factor family members in the early steps of coated vesicle formation on rat liver Golgi membranes. 902 Jan 26

A cDNA encoding the alpha-subunit of the heterotrimeric G-protein in rice (RGA1) was overexpressed in Escherichia coli and then isolated by Ni2+-nitrilotriacetic acid affinity chromatography. The molecular mass of RGA1 bearing a His tag was approx. 49 kDa. Immunoblot analysis using anti-RGA1 revealed that the RGA1 protein is most abundant in seedling leaves and least abundant in mature roots. It exists at particularly high levels in the immature embryo after pellicle extrusion. In addition, the RGA1 antiserum exhibited a difference in binding affinity for Galpha proteins from monocots (maize and rice) and dicots (Arabidopsis, pea, soya bean and tomato); whereas it cross-reacted with Galpha proteins of monocots, it did not with those of dicot plants. When bound to guanosine 5'-(gamma-thio)triphosphate (GTP[S]), the RGA1 protein was partially protected from tryptic proteolysis. In the presence of GTP[S], trypsin cleaved the RGA1 protein into four fragments 24, 14, 11 and 5 kDa in size. When RGA1 was bound to GDP, only the 5 kDa polypeptide was seen on SDS/PAGE after trypsin digestion. Photoaffinity labelling with [alpha-32P]GTP and a GTP[S]-binding assay revealed that RGA1 incorporated 32P and showed specific binding to a guanine nucleotide. Guanidine binding of RGA1 was affected by the concentration of MgCl2 (maximum at 2 mM). The rate of guanine nucleotide binding of RGA1 (kon,GTP[S]=0.0141+/-0.0014 min-1) and, at steady state, the kcat value for GTP hydrolysis (0.0075+/-0.0001 min-1) were very low even at 2 mM MgCl2. The binding affinity for the nucleotides examined was in the order GTP-S- >/= GTP > GDP > CTP > ATP >/= dTTP.
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PMID:Biochemical characteristics of a rice (Oryza sativa L., IR36) G-protein alpha-subunit expressed in Escherichia coli. 916 67

Tissue transglutaminase (tTG) catalyzes a Ca(2+)-dependent transglutaminase (TGase) activity that stabilizes tissues and a GTP hydrolysis activity that regulates cell receptor signaling. The purpose of this study was to examine the true substrates for nucleotide hydrolysis and the effects of these substrates on modulating the dual enzymatic activities of tTG. We found that Mg-GTP and Mg-ATP are the true substrates of the hydrolysis reaction. tTG hydrolyzed Mg-GTP and Mg-ATP at similar rates and interacted with Mg-ATP (Km = 38 +/- 10 microM) at a 3-fold greater steady-state affinity than with Mg-GTP (Km = 130 +/- 35 microM). In addition, Mg-ATP inhibited GTP hydrolysis (IC50 = 24 microM), whereas 1 mM Mg-GTP reduced ATP hydrolysis by only 20%. Furthermore, the TGase activity of tTG was inhibited by Mg-GTP, Mg-GDP, and Mg-GMP, with IC50 values of 9, 9, and 400 microM, respectively, whereas the Mg-adenine nucleotides were ineffective. Kinetic analysis of the hydrolysis reaction demonstrates the presence of separate binding sites for Mg-GTP and Mg-ATP. Finally, we found that Mg-GTP protected tTG from proteolytic degradation by trypsin, whereas Mg-ATP was ineffective. In conclusion, we report that Mg-GTP and Mg-ATP can bind to distinct sites and serve as substrates for nucleotide hydrolysis. Furthermore, binding of Mg-GTP causes a conformational change and the inhibition of TGase activity, whereas Mg-ATP is ineffective. The implication of these findings in regulating the intracellular and extracellular function of tTG is discussed.
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PMID:Regulation of human tissue transglutaminase function by magnesium-nucleotide complexes. Identification of distinct binding sites for Mg-GTP and Mg-ATP. 943 Jul 26

Cyclic GMP phosphodiesterase, a key enzyme in phototransduction, is composed of P alpha beta and two P gamma subunits. Interaction of P gamma with P alpha beta or with the alpha subunit (T alpha) of transducin is crucial for the regulation of cGMP phosphodiesterase in retinal photoreceptors. Here we have investigated phosphorylation of P gamma by cAMP-dependent protein kinase and its functional effect on the P gamma interaction with P alpha beta or T alpha in vitro. P gamma, but not P gamma complexed with T alpha (both GTP and GDP forms), is phosphorylated. Measurement of 32P radioactivity in phosphorylated P gamma, analysis of phosphorylated P gamma by laser mass spectrometry, identification of phosphoamino acid, and phosphorylation of mutant forms of P gamma indicate that only threonine 35 in P gamma is phosphorylated. Phosphorylation of P gamma mutants also reveals that the C and N terminals of P gamma which are required for the regulation of P alpha beta functions are not involved in the P gamma phosphorylation but that arginine 33, which is ADP-ribosylated by an endogenous ADP-ribosyltransferase, is required for the phosphorylation. Phosphorylated P gamma has a higher inhibitory activity for trypsin-activated cGMP phosphodiesterase than nonphosphorylated P gamma, indicating that the P gamma-P alpha beta interaction is affected by P gamma phosphorylation. Nonphosphorylated P gamma inhibits both the GTPase activity of T alpha and the binding of a hydrolysis-resistant GTP analogue to T alpha, while P gamma phosphorylation reduces these inhibitory activities. These observations suggest that a P gamma domain containing threonine 35 is involved in the P gamma-T alpha interaction, and P gamma phosphorylation regulates the P gamma-T alpha interaction. Our observation suggests that P gamma phosphorylation by cAMP-dependent protein kinase may function for the regulation of phototransduction in vertebrate rod photoreceptors.
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PMID:Phosphorylation of the gamma subunit of the retinal photoreceptor cGMP phosphodiesterase by the cAMP-dependent protein kinase and its effect on the gamma subunit interaction with other proteins. 955 60

In a cell-free system from neutrophil cytosol GTP(&ggr ;)S can induce an increase in the number of free filament barbed ends and massive actin polymerisation and cross-linking. GTP(&ggr ;)S stimulation was susceptible to an excess of GDP, but not Bordetella pertussis toxin and could not be mimicked by aluminium fluoride, myristoylated GTPgammaS.Gialpha2 or Gbeta1gamma2 subunits of trimeric G proteins. In contrast, RhoGDI and Clostridium difficile toxin B (inactivating Rho family proteins) completely abrogated the effect of GTPgammaS. When recombinant, constitutively activated and GTPgammaS-loaded Rac1, RhoA, or Cdc42 proteins alone or in combination were probed at concentrations >100 times the endogenous, however, they were ineffective. Purified Cdc42/Rac-interactive binding (CRIB) domain of WASP or C3 transferase did not prevent actin polymerisation by GTPgammaS. The action of GTPgammaS was blocked by mM [Mg2+], unless a heat- and trypsin-sensitive component present in neutrophil plasma membrane was added. Liberation of barbed ends seems therefore to be mediated by a toxin B-sensitive cytosolic Rho-family protein, requiring a membrane-associated guanine nucleotide exchange factor (GEF) for its activation by GTPgammaS under physiologic conditions. The inefficiency of various protein kinase and phosphatase inhibitors (staurosporine, genistein, wortmannin, okadaic acid and vanadate) and removal of ATP by apyrase, suggests that phosphate transfer reactions are not required for the downstream propagation of the GTPgammaS signal. Moreover, exogenously added phosphoinositides failed to induce actin polymerisation and a PtdIns(4,5)P2-binding peptide did not interfere with the response to GTPgammaS. The speed and simplicity of the presented assay applicable to protein purification techniques will facilitate the further elucidation of the molecular partners involved in actin polymerisation.
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PMID:GTPgammaS-induced actin polymerisation in vitro: ATP- and phosphoinositide-independent signalling via Rho-family proteins and a plasma membrane-associated guanine nucleotide exchange factor. 958 May 66

Purified membrane fractions have been widely used for the study of the factors regulating the functions of Rho small GTP-binding proteins. Using brush border membranes from the rat kidney as a model, we observed that in vitro incubation of these membranes resulted in time- and temperature-dependent proteolytic degradation of Cdc42 and RhoA. Treatment of kidney brush border membranes with various nucleotides showed that GDP and GTP weakly protected Cdc42 but not RhoA and that their nonhydrolyzable counterparts, guanosine 5'-O-[beta-thio] diphosphate (GDP beta S) and guanosine 5'-O-[gamma-thio]triphosphate (GTP gamma S), were highly efficient in protecting both proteins from endogenous proteolytic activity whereas ADP and ATP were without effect. GTP gamma S also protected Cdc42 and RhoA from proteolytic degradation in crude cell membranes from several rat tissues including intestine, kidney, liver, and testis. In addition, Cdc42 and RhoA associated with brush border membranes were largely resistant to increased proteolytic degradation induced by membrane treatment with the denaturing reagent urea as well as to added trypsin when incubated in the presence of GTP gamma S. In brush border membranes, the resistance to endo- and exo-genous proteolytic activity conferred by GTP gamma S was usually lower for RhoA than for Cdc42. GTP gamma S also protected recombinant Cdc42 and RhoA from the action of proteases associated with brush border membranes. The only protease inhibitor protecting Cdc42 but not RhoA from proteolytic degradation in brush border membranes was the synthetic peptide acetyl-Tyr-Val-Ala-Asp-aldehyde, a selective inhibitor of interleukin-1 beta-converting enzyme. This latter result showed that different proteases cleaved the two Rho proteins. Taken together, these results suggest that the GTP gamma S-bound forms of Cdc42 and RhoA are maintained in a conformation that protects them from proteases found in many cell membranes.
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PMID:Guanine nucleotides protect Rho proteins from endogenous proteolytic degradation in renal membranes. 966 7

We investigated the interaction of ADP-ribosylation factor (Arf) with the secretory granules in rat parotid acinar cells. The 20. 5-kDa small-molecular-mass GTP-binding protein in the cytosolic fraction of rat parotid acinar cells was identified as ADP-ribosylation factor1 by using a pan-Arf monoclonal antibody and isotype-specific polyclonal antibodies for Arf proteins 1, 3, 5, and 6. Incubation of the cytosolic fraction with isolated secretory granule membranes in the presence of GTPgammaS resulted in the translocation of Arf1 from the cytosolic fraction to the secretory granule membranes. The translocation was not observed in the presence of GDPbetaS in place of GTPgammaS, indicating that the process is GTP-dependent. The immunoelectron microscopy experiment confirmed Arf1 is translocated to the secretory granules. A prior treatment of the granule membranes with trypsin inhibited the translocation of Arf1 at 2 mM Mg2+, but had no effect in the absence of Mg2+ (condition of spontaneous conversion of Arf-GDP to Arf-GTP). Thus, the trypsin-sensitive nucleotide exchange activity for Arf1 is probably associated with the secretory granule membranes. These results demonstrate Arf1 translocates to the secretory granules in rat parotid acinar cells.
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PMID:Translocation of Arf1 to the secretory granules in rat parotid acinar cells. 972 Nov 94


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