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)

Cytoplasmic elongation factor 1 alpha (EF-1 alpha) was purified to homogeneity from the yeast Saccharomyces cerevisiae using a large-scale procedure. The three steps of purification used were batch adsorption on phosphocellulose, phosphocellulose chromatography and, as the last step, GDP-Sepharose or Biorex column chromatography. The protein is very basic (pI = 9.2) and has an apparent molecular mass of 49 kDa, as determined by polyacrylamide gel electrophoresis using denaturing conditions. It is one of the most abundant proteins in yeast (about 5% of total soluble protein), as shown by two-dimensional gel electrophoresis and by immunological titration. A strong immunological and structural homology was found between yeast EF-1 alpha and elongation factors from other sources. Common immunological features were found between yeast and wheat germ EF-1 alpha. Tryptic hydrolysis of yeast EF-1 alpha in the presence of 25% glycerol generated a large trypsin-resistant polypeptide (Mr = 43,000) which had the same NH2-terminal sequence as the proteolyzed product from rabbit reticulocyte, Artemia salina EF-1 alpha and Escherichia coli EF-Tu. Completed DNA sequence determination of one structural gene for yeast EF-1 alpha confirmed a remarkable conservation of several protein sequence domains in yeast and animal EF-1 alpha (Cottrelle, P., Thiele, D., Price, V., Memet, S., Micouin, J.Y., Marck, C., Buhler, J.M. Sentenac, A., and Fromageot, P. (1985) J. Biol. Chem. 260, 3090-3096).
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PMID:Elongation factor 1 alpha from Saccharomyces cerevisiae. Rapid large-scale purification and molecular characterization. 388 5

A 2.7 angstrom resolution x-ray diffraction analysis of a trypsin-modified form of the Escherichia coli elongation factor Tu reveals that the GDP-binding domain has a structure similar to that of other nucleotide-binding proteins. The GDP ligand is located at the COOH-terminal end of the beta sheet and is linked to the protein via a Mg2+ ion salt bridge. The location of the guanine ring is unusual; the purine ring is located on the outer edge of the domain, not deep within a hydrophobic pocket. The amino acids from Pro10 to Arg44 and from Gly59 to Glu190 have been assigned to the electron density with computer graphic techniques, and the resulting model is consistent with all known biochemical data. An analysis of the structure reveals that four regions of the amino acid sequence that are homologous with the family of ras oncogene proteins, termed p21, are located in the vicinity of the GDP-binding site, and most of the invariant amino acids shared by the proteins interact directly with the GDP ligand.
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PMID:Structure of the GDP domain of EF-Tu and location of the amino acids homologous to ras oncogene proteins. 389 65

A simple procedure for the preparation of 10-500 mg of the Escherichia coli elongation Tu-Ts complex is described. The protocol is based on the separate purification and quantitation of EF-Tu-GDP and EF-Ts, followed by mixing of equimolar amounts of each protein and removal of the displaced GDP by dialysis. Single crystals grown from the final product have been analyzed by X-ray diffraction techniques. The procedure is also applicable to the bulk preparation and crystallization of the trypsin-modified Tu-Ts complex. Quantitation of the elongation factors by three methods is presented.
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PMID:Bulk preparation and crystallization of the Escherichia coli elongation factor Tu-Ts complex. 390 45

Ornithine transcarbamoylase (OTC) is inactivated by liver lysosomes. Carbamoyl phosphate prevents the inactivation of OTC by lysosomes, while ATP, ADP, GTP, GDP 1,N6-ethenoadenosine 5'-triphosphate and particularly epsilon-ATP stimulate it. Both stimulation and protection occur at concentrations within the physiological range of ATP and carbamoyl phosphate. Inactivation of OTC is followed by extensive proteolysis. Since the inactivation is prevented by leupeptin, antipain and L-(tosylamido-2-phenyl)ethylchloromethyl ketone, the proteolytic susceptibility of OTC to lysosomes could be due to thiol endopeptidase(s). 1,N6-Ethenoadenosine 5'-triphosphate also markedly increases OTC susceptibility to trypsin and elastase. ATP analogs had no stimulatory effect on OTC inactivation by lysosomes; none of the inhibitors of ATPases tested inhibited the ATP effect. The ATP stimulation does not require Mg2+. These findings indicate a new role for ATP, GTP and related nucleotides in protein breakdown. The ATP, ADP, GTP, GDP stimulation, together with the carbamoyl protection of OTC, agree well with the molecular plasticity hypothesis model.
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PMID:Purine nucleotides stimulate while carbamoyl phosphate protects inactivation of ornithine transcarbamoylase by disrupted lysosomes. 399 99

Sea urchin sperm were demembranated and reactivated with a solution containing 0.04% Triton X-100 and 0.03 mM ATP. The ATP concentration was then lowered abruptly by diluting the sperm suspension 50-fold into reactivating solution containing no ATP. The flagella of the sperm in the diluted suspension were not motile, but they were bent into a variety of stationary rigor wave forms closely resembling the wave forms occurring at different stages of the flagellar bending cycle during normal movement. The form of these rigor waves was unchanged upon storage for several hours in the presence of dithiothreitol and EDTA. Addition of 1 microM ATP induced slow relaxation of the waves, with most of the sperm becoming partially straightened over a period of about 30 min; somewhat higher concentrations gave a more rapid and complete relaxation. Concentrations of ATP above 10 microM induced resumption of normal beating movements. Addition of ITP, GTP, or GDP (up to 1 mM) produced no relaxation of the rigor waves. Digestion with trypsin to an extent sufficient to disrupt the radial spokes and the nexin links caused no change in the rigor wave forms, suggesting that these wave forms could be maintained by the dynein cross-bridges between the outer doublet tubules of the flagellar axoneme. Study of the effects of viscous shear on the rigor wave axonemes has shown that they are resistant to distortion by bending, although they can be twisted relatively easily.
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PMID:Properties of flagellar "rigor waves" formed by abrupt removal of adenosine triphosphate from actively swimming sea urchin sperm. 421 20

It has been previously shown that trypsin treatment of rat liver plasma membranes causes the solubilization of a guanylate cyclase of Mr = 140,000 (Lacombe, M. L., Haguenauer-Tsapis, R., Stengel, D., Ben Salah, A., and Hanoune, J. (1980) FEBS Lett. 116, 79-84). In this study, we observed that addition of Mn-GTP during this step greatly protected the enzyme from proteolytic degradation. This effect was specific for guanine nucleotides, being weaker for other nucleotides triphosphate and GDP, and absent for cyclic GMP and GMP. Metal-GTP complex was required with a strict specificity for Mn2+. In addition to the Mr = 140,000 enzyme, trypsin solubilization in the presence of Mn-GTP led to the formation of a small and active form of guanylate cyclase. Based on its behavior on Ultrogel AcA 34 and sucrose gradients, its apparent Mr was calculated to be 68,000. Both forms could be well separated by high performance liquid chromatography and were shown to be sequentially solubilized (the larger appearing before the smaller species). Mr = 140,000 species, but not the cytosolic enzyme, was able to generate the Mr = 68,000 enzyme upon tryptic treatment in the presence of Mn-GTP. The Mr = 140,000 and 68,000 enzymes exhibited Michaelis-Menten kinetics (Hill coefficient = 1) with Km for Mn-GTP of 130 and 70 microM, respectively. The proteolytically solubilized enzymes were strickingly heat labile and highly protected by Mn-GTP. These results support the hypothesis that the rat liver membrane-bound guanylate cyclase has a dimeric structure similar to that of the cytosolic enzyme. They also suggest a possible role for GTP in limiting the degradation rate of membrane guanylate cyclase in vivo and, thus, in regulating the active enzyme concentration.
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PMID:Guanine nucleotides allow the trypsin solubilization of an active Mr = 68,000 guanylate cyclase. 613 89

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

A remarkable and immediate decrease in GDP-mannose:retinyl phosphate mannosyltransferase activity was found on pre-incubation of rat liver postnuclear membranes with phospholipase A2 or phospholipase C. Under the same conditions of pre-incubation (1 min at 37 degrees C) trypsin did not affect the enzyme activity, whereas pre-incubation for 30 min with trypsin and Pronase abolished enzyme activity. The lipid extract of untreated rat liver membranes partially restored enzyme activity after phospholipase treatment. Sphingomyelin was as active as the endogenous lipids. Other phospholipids were less active in the following order: phosphatidylcholine greater than phosphatidylethanolamine greater than phosphatidylinositol = phosphatidylserine. Dolichyl phosphate mannose synthesis was inhibited less (33%) by phospholipase C than was Ret-P-Man synthesis (98.5%) under identical conditions of incubation, which included 0.025% Triton. However, retinyl phosphate mannose synthesis by purified endoplasmic reticulum was found to be resistant to phospholipase C. Mixing experiments failed to demonstrate an inhibitory effect of the phospholipase-treated postnuclear membrane fraction on the synthetic activity of the endoplasmic reticulum, thus excluding the release of an inhibitory factor from the postnuclear membranes.
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PMID:Retinyl phosphate mannose synthesis in rat liver membranes. Phospholipase sensitivity and phospholipid requirement. 619 16

Upon incubation with trypsin, the adenosine-5'-triphosphatase (ATPase) activity of the nucleotide-depleted F1 is first rapidly and slightly activated and then slowly inactivated. The first phase is simultaneous with the conversion of the alpha subunit into an alpha' fragment which migrates between alpha and beta on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The second phase is related to the proteolysis of the three main subunits, alpha', beta, and gamma. Preincubation of the enzyme with low concentrations of adenosine 5'-diphosphate (ADP) or adenosine 5'-triphosphate (ATP) does not modify the slight increase of activity but efficiently prevents the inactivation induced by trypsin. The alpha leads to alpha' conversion is not affected whereas the further proteolysis of alpha', beta, and gamma does not occur. On the contrary, even high concentrations of GDP only slightly lower the trypsin-induced inactivation. The presence of endogenous tightly bound nucleotides also partially lowers the sensitivity to trypsin since F1 is less rapidly inactivated and proteolyzed than the nucleotide-depleted F1. Phosphate, at high concentrations, both slows down the first phase of activation and simultaneous alpha leads to alpha' conversion and prevents the second phase of inactivation and proteolysis of the main subunits. Pretreatment of the nucleotide-depleted F1 with trypsin under conditions where the ATPase activity is largely inhibited only slightly modifies, however, the hysteretic behavior of the enzyme: the ADP binding and the concomitant hysteretic inhibition of the residual activity are not markedly diminished. The purified ATPase-ATP synthase complex binds very few ADP's and is not hysteretically inhibited. Its ATPase activity is rapidly activated but not further inhibited by trypsin. Preincubation of the complex with ADP does not modify the effects of trypsin.
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PMID:Use of trypsin to monitor conformational changes of mitochondrial adenosinetriphosphatase induced by nucleotides and phosphate. 622 Jul 37

1. GTP, but not p[NH]ppG (guanosine 5'-[betagamma-imido]triphosphate), abolishes the sensitivity of glucagon-stimulated adenylate cyclase to the lipid-phase separations occurring in the outer half of the bilayer in liver plasma membranes from rat. 2. When either GTP or p[NH]ppG alone stimulate adenylate cyclase, the enzyme senses only those lipid-phase separations occurring in the inner half of the bilayer. 3. Trypsin treatment of intact hepatocytes has no effect on the basal, fluoride-, GTP- or p[NH]ppG-stimulated adenylate cyclase activity. However, (125)I-labelled-glucagon specific binding decays with a half-life matching that of the decay of glucagon-stimulated adenylate cyclase activity. 4. When GTP or p[NH]ppG are added to assays of glucagon-stimulated activity, the half-life of the trypsin-mediated decay of activity is substantially increased and the decay plots are no longer first-order. 5. Trypsin treatment of purified rat liver plasma membranes abolishes basal and all ligand-stimulated adenylate cyclase activity, and (125)I-labelled-glucagon specific binding. 6. Benzyl alcohol activates the GTP- and p[NH]ppG-stimulated activities in an identical fashion, whereas these activities are affected differently when glucagon is present in the assays. 7. We suggest that guanine nucleotides alter the mode of coupling between the receptor and catalytic unit. In the presence of glucagon and GTP, a complex of receptor, catalytic unit and nucleotide regulatory protein occurs as a transient intermediate, releasing a free unstable active catalytic unit. In the presence of p[NH]ppG and glucagon, the transient complex yields a relatively stable complex of the catalytic unit associated with a p[NH]ppG-bound nucleotide-regulatory protein.
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PMID:Guanosine 5'-triphosphate and guanosine 5'-[beta gamma-imido]triphosphate effect a collision coupling mechanism between the glucagon receptor and catalytic unit of adenylate cyclase. 624 58


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