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)

Heterotrimeric Go bound to the membranes of bovine brain, but Go alpha remained bound to the membranes even after activation with GTP gamma S. Furthermore, Go alpha bound to a Triton X-100-insoluble fraction of the membranes in a saturable manner. However, the 37-kDa Go alpha eliminated by trypsin at the amino-terminus could not bind to the fraction. Using a blot overlay approach of the Triton-insoluble fraction, only a 20-kDa protein was identified that interacts with Go alpha. These results indicate that Go alpha binds to a 20-kDa Triton-insoluble protein in the bovine brain membranes.
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PMID:Interaction of alpha subunit of GTP-binding protein Go with a 20-kDa Triton-insoluble membrane protein in bovine brain. 145 1

Highly purified peroxisomal membranes stripped from their peripheral membrane proteins and only minimally contaminated with other membranes, contained three GTP-binding proteins of 29, 27 and 25 kDa, respectively. Bound radioactive GTP was displaced by unlabelled GTP, GTP analogs and GDP but not by GMP or other nucleotides. GTP binding was markedly decreased by trypsin treatment of intact purified peroxisomes; it increased 2-3-fold after pretreatment of the animals with a peroxisome proliferator. We conclude that the peroxisomal membrane contains small GTP-binding proteins that are exposed to the cytosol and that are firmly anchored in the membrane. We speculate that these proteins are involved in peroxisome multiplication by fission or budding during peroxisome biogenesis and proliferation.
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PMID:Presence of small GTP-binding proteins in the peroxisomal membrane. 150 80

Cell-substrate adhesion is crucial at various stages of development and for the maintenance of normal tissues. Little is known about the regulation of these adhesive interactions. To investigate the role of GTPases in the control of cell morphology and cell-substrate adhesion we have injected guanine nucleotide analogs into Xenopus XTC fibroblasts. Injection of GTP gamma S inhibited ruffling and increased spreading, suggesting an increase in adhesion. To further investigate this, we made use of GRGDSP, a peptide which inhibits binding of integrins to vitronectin and fibronectin. XTC fibroblasts injected with non-hydrolyzable analogs of GTP took much more time to round up than mock-injected cells in response to treatment with GRGDSP, while GDP beta S-injected cells rounded up in less time than controls. Injection with GTP gamma S did not inhibit cell rounding induced by trypsin however, showing that cell contractility is not significantly affected by the activation of GTPases. These data provide evidence for the existence of a GTPase which can control cell-substrate adhesion from the cytoplasm. Treatment of XTC fibroblasts with the phorbol ester 12-o-tetradecanoylphorbol-13-acetate reduced cell spreading and accelerated cell rounding in response to GRGDSP, which is essentially opposite to the effect exerted by non-hydrolyzable GTP analogs. These results suggest the existence of at least two distinct pathways controlling cell-substrate adhesion in XTC fibroblasts, one depending on a GTPase and another one involving protein kinase C.
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PMID:A GTPase controls cell-substrate adhesion in Xenopus XTC fibroblasts. 151 94

Bovine liver glutamate dehydrogenase reacts with the bifunctional affinity label 5'-(p-(fluorosulfonyl)benzoyl)-8-azidoadenosine (5'-FSBAzA) in a two-step process: a dark reaction yielding about 0.5 mol of -SBAzA/mol of subunit by reaction through the fluorosulfonyl moiety, followed by photoactivation of the azido group whereby covalently bound -SBAzA becomes cross-linked to the enzyme [Dombrowski, K. E., & Colman, R. F. (1989) Arch. Biochem. Biophys. 275, 302-308]. We now report that the rate constant for the dark reaction is not reduced by ADP or GTP, but it is decreased 7-fold by 2 mM NADH and 40-fold by 2 mM NADH + 0.2 mM GTP, suggesting that 5'-FSBAzA reacts at the GTP-dependent NADH inhibitory site. The amino acid residues modified in each phase of the reaction have been identified. Modified enzyme was isolated after each reaction phase, carboxymethylated, and digested with trypsin, chymotrypsin, or thermolysin. The digests were fractionated by chromatography on a phenylboronate agarose column followed by HPLC. Gas-phase sequencing of the labeled peptides identified Tyr190 as the major amino acid which reacts with the fluorosulfonyl group; Lys143 was also modified but to a lesser extent. The predominant cross-link formed during photolysis is between modified Tyr190 and the peptide Leu475-Asp476-Leu477-Arg478, which is located near the C-terminus of the enzyme. Thus, 5'-FSBAzA is effective in identifying critical residues distant in the linear sequence, but close within the regulatory nucleotide site of glutamate dehydrogenase.
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PMID:Identification of amino acids modified by the bifunctional affinity label 5'-(p-(fluorosulfonyl)benzoyl)-8-azidoadenosine in the reduced coenzyme regulatory site of bovine liver glutamate dehydrogenase. 156 33

Eel liver glutamate dehydrogenase (GDH) [EC 1.4.1.3] was eightfold activated by trypsin and the molecular weight of the subunit of the native GDH decreased from 54,000 to 50,000. The C-terminal amino acid of both subunits was Thr. One peptide was released after proteolysis of the native GDH by trypsin and purified by anhydrotrypsin agarose and reversed-phase HPLC. The isolated peptide consisted of 39 amino acids and its amino acid sequence was as follows: H2NS-E-A-V-E-K-E-D-D-P-N-F-F-K-M-V-E-G-F-F-D-K-G-A-A-I- V-E-N-K-L-V-E-E-D-L-K-T-R-COOH. The peptide contained the N-terminal of the native GDH and its molecular weight was calculated to be 4,413. We concluded that the trypsin-catalyzed activation was caused by release of this peptide from the native GDH. p-Chloromercuribenzoic acid inhibited the activity of the trypsin-treated GDH, but stimulated that of the native GDH. The response of trypsin-treated GDH to ADP and GTP was decreased compared with that of the native GDH.
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PMID:The trypsin-catalyzed activation of glutamate dehydrogenase purified from eel liver. 163 63

Atrial natriuretic factor (ANF-R1) receptor is a 130-kDa protein that contains a cytoplasmic guanylate cyclase domain. We report that ATP interacts in an allosteric manner with the ANF-R1 receptor, resulting in reduced ANF binding and enhanced ANF-stimulated guanylate cyclase activity. The modulatory properties of various nucleotides indicate a preference for the adenine family with a rank order of potency of ATP greater than App(NH)p greater than or equal to ADP greater than or equal to AMP while cyclic and guanine nucleotides except GTP are inactive. The negative modulation by ATP of ANF binding is specific for the ANF-R1 receptor subtype since the amount of ANF bound by the guanylate cyclase uncoupled ANF-R2 subtype is increased in the presence of ATP. Furthermore, the effects of ATP on ANF-R1 receptor binding function are still observed with the affinity-purified ANF-R1 receptor, suggesting an allosteric binding site for ATP on the ANF-R1 receptor. In intact membranes, limited proteolysis of the ANF-R1 receptor with trypsin dose-dependently prevents the ATP-induced decrease in ANF binding concomitantly with the formation of a membrane-associated ANF-binding fragment of 70 kDa. These results confirm the direct modulatory role of ATP on hormone binding activity of ANF-R1 receptor and suggest that the nucleotide regulatory binding site is located in the intracellular domain vicinal to the protease-sensitive region.
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PMID:Allosteric modulation by ATP of the bovine adrenal natriuretic factor R1 receptor functions. 165 83

Pharmacological and biochemical characteristics of the partially purified gamma-aminobutyric acid (GABA)B receptor using baclofen affinity column chromatography have been examined. The Scatchard analysis of [3H]GABA binding to the purified GABAB receptor showed a linear relationship and the KD and Bmax values were 60 nM and 118 pmol/mg of protein, respectively. Although GTP and Mg2+ did not affect on the GABAB receptor binding, Ca2+ significantly increased [3H]GABA binding to the purified GABAB receptor in a dose-dependent manner and showed its maximum effect at 2 mM. The enhancement of the binding by Ca2+ was found to be due to the increase of Bmax by the Scatchard analysis. The treatments with pronase and trypsin significantly decreased the binding of [3H]GABA, but phospholipase A2 had no significant effect on the binding. In addition, treatment with glycosidases such as glycopeptidase A and beta-galactosidase significantly decreased the binding of [3H]GABA to the purified GABAB receptor. These results suggest that purification of the solubilized GABAB receptor by the affinity column chromatography may result in the functional uncoupling of GABAB receptor with GTP-binding protein. Furthermore, the present results suggest that cerebral GABAB receptor may be a glycoprotein and membrane phospholipids susceptible to phospholipase A2 treatment may not be involved in the exhibition of the binding activity.
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PMID:Pharmacological and biochemical characteristics of partially purified GABAB receptor. 166 62

The RNA-DNA helicase activity of Escherichia coli transcription termination factor rho can be significantly enhanced at lower potassium chloride and magnesium acetate concentrations than previously used. Decreasing the potassium chloride concentration from 150 to 50 mM increases the rate of release at least 4-fold, while at lower magnesium concentrations less ATP is required for maximal duplex disruption. For all concentrations tested (between 0.1 and 5 mM), the optimal magnesium and ATP concentrations are interdependent; a roughly equimolar ratio gives the maximal rate of RNA release, although peak height and breadth vary. Surprisingly, rho behaves differently with an RNA-RNA duplex, which cannot be efficiently disrupted at magnesium concentrations below 1 mM. Above 2.0 mM, release does occur efficiently suggesting that Mg2+ promotes some structural transition in the RNA-RNA helix to a rho-susceptible conformation. In addition to Mg2+, helicase activity requires hydrolysis of nucleoside triphosphates, but for all four standard NTPs the rates of NTP hydrolysis do not correlate uniformly with the rates of RNA release. Based on the ratio of the rate of RNA release to the rate of NTP hydrolysis, rho utilizes ATP most efficiently. The 2-4-fold weaker coupling of hydrolysis to duplex disruption for the other three NTPs demonstrates that NTP utilization is not, on its own, sufficient for efficient helicase activity. The less efficient coupling with GTP, CTP, and UTP correlates with conformational differences in the protein complex as probed by mild trypsin digestion. The implications of our findings for substrate specificity and energy coupling in the helicase reaction are discussed.
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PMID:Specificity and efficiency of rho-factor helicase activity depends on magnesium concentration and energy coupling to NTP hydrolysis. 169 Jul 11

Monoclonal antibody 4A (mAb 4A) against the T alpha subunit of transducin has been widely used to study the structure and function of signal transducing GTP-binding proteins involved in the regulation of visual excitation, hormonal regulation of adenylyl cyclase and ionic channels. Results of mapping the epitope-binding site of mAb 4A on T alpha have been controversial. Hamm and co-workers (Deretic, D., and Hamm, H. E. (1987) J. Biol. Chem. 262, 10839-10847) reported that mAb 4A interacts with T alpha at the carboxyl-terminal peptide, whereas Fung and co-workers (Navon, S. E., and Fung, B. K.-k. (1988) J. Biol. Chem. 263, 489-498) showed that mAb 4A binds mainly at the amino-terminal peptide. In this report, we examine the epitope-binding site of mAb 4A by Western immunoblotting of the proteolytic fragments of T alpha generated by submaxillary Arg-C protease digestion. Submaxillary Arg-C protease cleaved T alpha at two sites, Arg-204 and Arg-310, generating two major fragments of apparent size 35 (T alpha'SM-35) and 23 kDa (T alpha'SM-23). Both fragments contain the amino-terminal peptide of T alpha but lack the carboxyl-terminal peptide. Western immunoblotting showed that mAb 4A cross-reacted with both peptides. Treatment of T alpha'SM-35 and T alpha'SM-25 with L-1-(tosylamido)-2-phenyethyl chloromethyl ketone-trypsin removed the amino-terminal 2-kDa peptide with concomitant loss of mAb 4A reactivity. This observation unequivocally confirms the result of Fung and co-workers that the epitope for mAb 4A is located on the amino-terminal 2-kDa peptide of T alpha. This conclusion should provide a more accurate interpretation of results in the literature as well as of future studies in which mAb 4A is used.
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PMID:Identification of monoclonal antibody 4A-binding site on the transducin alpha subunit. Immunoblotting of submaxillary Arg-C protease fragments of transducin. 170 Sep 81

We have initiated the characterization of the DNA helicases from HeLa cells, and we have observed at least 4 molecular species as judged by their different fractionation properties. One of these only, DNA helicase I, has been purified to homogeneity and characterized. Helicase activity was measured by assaying the unwinding of a radioactively labelled oligodeoxynucleotide (17 mer) annealed to M13 DNA. The apparent molecular weight of helicase I on SDS polyacrylamide gel electrophoresis is 65 kDa. Helicase I reaction requires a divalent cation for activity (Mg2+ greater than Mn2+ greater than Ca2+) and is dependent on hydrolysis of ATP or dATP. CTP, GTP, UTP, dCTP, dGTP, dTTP, ADP, AMP and non-hydrolyzable ATP analogues such as ATP gamma S are unable to sustain helicase activity. The helicase activity has an optimal pH range between pH8.0 to pH9.0, is stimulated by KCl or NaCl up to 200mM, is inhibited by potassium phosphate (100mM) and by EDTA (5mM), and is abolished by trypsin. The unwinding is also inhibited competitively by the coaddition of single stranded DNA. The purified fraction was free of DNA topoisomerase, DNA ligase and nuclease activities. The direction of unwinding reaction is 3' to 5' with respect to the strand of DNA on which the enzyme is bound. The enzyme also catalyses the ATP-dependent unwinding of a DNA:RNA hybrid consisting of a radioactively labelled single stranded oligodeoxynucleotide (18 mer) annealed on a longer RNA strand. The enzyme does not require a single stranded DNA tail on the displaced strand at the border of duplex regions; i.e. a replication fork-like structure is not required to perform DNA unwinding. The purification of the other helicases is in progress.
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PMID:A DNA helicase from human cells. 170 1


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