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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A genomic library of Mycobacterium bovis BCG has been constructed by cloning DNA partially digested with Sau3A into the Escherichia coli expression vector pAS1. The gene coding for ornithine carbamoyl-transferase (EC.2.1.3.3; OTCase), hereafter referred to as argF, was isolated from the library by complementation of a double argF-argI mutant of E. coli and its sequence was determined. The translation initiation codon used, GTG, was identified by comparing the amino acid sequence deduced from the gene with the N-terminal sequence of the corresponding purified protein. On this basis, the M. bovis BCG OTCase monomer consists of 307 amino acid residues and displays about 44% identity with other OTCases, the most closely related homologue being the anabolic enzyme of Pseudomonas aeruginosa. The native enzyme has an estimated molecular mass of 110 kDa, suggesting a trimeric structure as is the case for most of the anabolic OTCases known from various organisms.
Mol Gen Genet 1992 Sep
PMID:Molecular cloning, characterization and purification of ornithine carbamoyltransferase from Mycobacterium bovis BCG. 140 93

Mammalian ras genes substitute for the yeast RAS gene, and their products activate adenylate cyclase in yeast cells, although the direct target protein of mammalian ras p21s remains to be identified. ras p21s undergo posttranslational processing, including prenylation, proteolysis, methylation, and palmitoylation, at their C-terminal regions. We have previously reported that the posttranslational processing of Ki-ras p21 is essential for its interaction with one of its GDP/GTP exchange proteins named smg GDS. In this investigation, we have studied whether the posttranslational processing of Ki- and Ha-ras p21s is critical for their stimulation of yeast adenylate cyclase in a cell-free system. We show that the posttranslationally fully processed Ki- and Ha-ras p21s activate yeast adenylate cyclase far more effectively than do the unprocessed proteins. The previous and present results suggest that the posttranslational processing of ras p21s is important for their interaction not only with smg GDS but also with the target protein.
Mol Cell Biol 1992 Oct
PMID:The posttranslational processing of ras p21 is critical for its stimulation of yeast adenylate cyclase. 140 40

rap1GAP is a GTPase-activating protein that specifically stimulates the GTP hydrolytic rate of p21rap1. We have defined the catalytic domain of rap1GAP by constructing a series of cDNAs coding for mutant proteins progressively deleted at the amino- and carboxy-terminal ends. Analysis of the purified mutant proteins shows that of 663 amino acid residues, only amino acids 75 to 416 are necessary for full GAP activity. Further truncation at the amino terminus resulted in complete loss of catalytic activity, whereas removal of additional carboxy-terminal residues dramatically accelerated the degradation of the protein in vivo. The catalytic domain we have defined excludes the region of rap1GAP which undergoes phosphorylation on serine residues. We have further defined this phosphoacceptor region of rap1GAP by introducing point mutations at specific serine residues and comparing the phosphopeptide maps of the mutant proteins. Two of the sites of phosphorylation by cyclic AMP (cAMP)-dependent kinase were localized to serine residues 490 and 499, and one site of phosphorylation by p34cdc2 was localized to serine 484. In vivo, rap1GAP undergoes phosphorylation at four distinct sites, two of which appear to be identical to the sites phosphorylated by cAMP-dependent kinase in vitro.
Mol Cell Biol 1992 Oct
PMID:Localization of the rap1GAP catalytic domain and sites of phosphorylation by mutational analysis. 140 53

The role of GTP hydrolysis in microtubule dynamics has been reinvestigated using an analogue of GTP, guanylyl-(alpha, beta)-methylene-diphosphonate (GMPCPP). This analogue binds to the tubulin exchangeable nucleotide binding site (E-site) with an affinity four to eightfold lower than GTP and promotes the polymerization of normal microtubules. The polymerization rate of microtubules with GMPCPP-tubulin is very similar to that of GTP-tubulin. However, in contrast to microtubules polymerized with GTP, GMPCPP-microtubules do not depolymerize rapidly after isothermal dilution. The depolymerization rate of GMPCPP-microtubules is 0.1 s-1 compared with 500 s-1 for GDP-microtubules. GMPCPP also completely suppresses dynamic instability. Contrary to previous work, we find that the beta--gamma bond of GMPCPP is hydrolyzed extremely slowly after incorporation into the microtubule lattice, with a rate constant of 4 x 10(-7) s-1. Because GMPCPP hydrolysis is negligible over the course of a polymerization experiment, it can be used to test the role of hydrolysis in microtubule dynamics. Our results provide strong new evidence for the idea that GTP hydrolysis by tubulin is not required for normal polymerization but is essential for depolymerization and thus for dynamic instability. Because GMPCPP strongly promotes spontaneous nucleation of microtubules, we propose that GTP hydrolysis by tubulin also plays the important biological role of inhibiting spontaneous microtubule nucleation.
Mol Biol Cell 1992 Oct
PMID:Role of GTP hydrolysis in microtubule dynamics: information from a slowly hydrolyzable analogue, GMPCPP. 2302 82

We purified a large amount of dynamin with high enzymatical activity from rat brain tissue by a new procedure. Dynamin 0.48 mg was obtained from 20 g of rat brain. The purity of dynamin was almost 98%. Dynamin plays a role of GTPase rather than ATPase. In the absence of microtubules, Michaelis constant (Km) and maximum velocity (Vmax) for dynamin GTPase were 370 microM and 0.25 min-1, respectively, and in their presence, both were significantly accelerated up to 25 microM and 5.5 min-1. On the other hand, the ATPase activity was very low in the absence of microtubules, and even in their presence, Km and Vmax for dynamin ATPase were 0.2 mM and 0.91 min-1. Despite slow GTPase turnover rate in the absence of microtubules, binding of GTP and its nonhydrolizing analogues was very fast, indicating that GTP binding step is not rate limiting. Dynamin did not cause a one-directional consistent microtubule sliding movement just like kinesin or dynein in the presence of 2 mM ATP or 2 mM GTP. We observed the molecular structure of dynamin with low-angle rotary shadowing technique and revealed that the dynamin molecule is globular in shape. Gel filtration assay revealed that these globules were the oligomers of 100-kDa dynamin polypeptide. Dynamin bound to microtubules with a 1:1 approximately 1.2 molar ratio in the absence of GTP. Quick-freeze deep-etch electron microscopy of the dynamin-microtubule complex showed that dynamin decorates the surface of microtubules helically, like a screw bolt, very orderly and tightly with 11.4 +/- 0.9 (SD)nm period. Contrary to the previous report, microtubules make bundles by the attachment of the dynamin helixes around each adjacent microtubule, and no cross-bridge formation was observed.
Mol Biol Cell 1992 Oct
PMID:Interaction of dynamin with microtubules: its structure and GTPase activity investigated by using highly purified dynamin. 142 74

Epidermal growth factor (EGF) can stimulate inositol lipid hydrolysis in rat hepatocytes and can accelerate GTP/GDP exchange in hepatic membranes. Both of these responses can be abolished by pretreatment with pertussis toxin, suggesting that EGF may regulate phospholipase C (PLC) activity via a guanine nucleotide-binding regulatory protein (G protein) in liver cells. In contrast, in A431 human epidermoid carcinoma cells EGF can induce a rapid phosphorylation of PLC-gamma on tyrosine residues that increases the activity of immunoprecipitated PLC-gamma, suggesting that tyrosine phosphorylation of PLC-gamma may be the mechanism for EGF-stimulated inositol trisphosphate production in these cells. To determine the importance of the phosphorylation of PLC-gamma on tyrosine residues in a system where the EGF receptor apparently couples to a G protein, the effect of EGF on tyrosine phosphorylation of PLC-gamma was examined in rat hepatocytes. PLC-gamma was immunoprecipitated from cell lysates with a PLC-gamma antiserum and its tyrosine phosphorylation state was determined using both Western blot analysis with phosphotyrosine antibodies and direct measurement of phosphorylated amino acids. The results were compared with analogous experiments performed with A431 cells and another cultured cell line expressing high levels of human EGF receptors, Rat1hER fibroblasts. Although the amount of PLC-gamma in rat hepatocytes is similar to that in A431 cells and slightly higher than that in Rat1hER cells, EGF causes a barely detectable increase in the phosphorylation of PLC-gamma on tyrosine in hepatocytes, whereas it stimulates a significant degree of phosphorylation of PLC-gamma on tyrosine in Rat1hER or A431 cells. Pretreatment of hepatocytes with pertussis toxin abolishes the ability of EGF to activate PLC, as determined by an increase in intracellular Ca2+, but has no effect on the small amount of phosphate incorporated into tyrosine residues on the PLC-gamma protein, demonstrating that this low level of PLC-gamma phosphorylation does not correlate with changes in PLC activity. The data suggest that phosphorylation of PLC-gamma on tyrosine is not important for EGF-enhanced PLC activity in hepatocytes. This conclusion implies that EGF may use a mechanism to regulate PLC activity in hepatocytes that is different from that used in cultured cells expressing high levels of EGF receptors.
Mol Pharmacol 1992 Nov
PMID:Epidermal growth factor activates phospholipase C in rat hepatocytes via a different mechanism from that in A431 or rat1hER cells. 143 49

Pertussis toxin (PTX) ADP-ribosylates alpha subunits of GTP-binding proteins (G proteins) when they are in association with beta gamma dimers, and free alpha subunits are thought not to be substrates under standard assay conditions. We now report the rather unexpected discovery that synthetic peptides encompassing the last 10-20 amino acids of alpha subunits of PTX-sensitive G proteins are substrates for PTX by themselves and in the absence of beta gamma dimers. As determined for G13, the Km of PTX for the 20-amino acid carboxyl-terminal peptide is 10-fold higher than that for the trimeric G protein. Interestingly, PTX ADP-ribosylates the free full length alpha 13 subunit with a Km not different from that of the trimer but with a Vmax that is only 1% of that with which it ADP-ribosylates the trimer. It follows that the primary role of beta gamma dimers in ADP-ribosylation of G proteins is one of increasing the Vmax of the reaction without affecting the Km of the substrate for the toxin. Mutant peptides lacking the ADP-ribose acceptor site act as competitive inhibitors.
Mol Pharmacol 1992 Nov
PMID:Peptide inhibitors of ADP-ribosylation by pertussis toxin are substrates with affinities comparable to those of the trimeric GTP-binding proteins. 143 50

Guanine nucleotides such as guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) have been found to increase the binding of antagonists to adenosine A1 receptors. This response can be attributed either to a direct effect of GTP on receptors to increase antagonist affinity or to an indirect effect to decrease the affinity of receptors for a pool of endogenous adenosine that cannot be readily removed from membranes. In this study, adenosine content was measured in preparations of membranes and 3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonate (CHAPS)-solubilized receptors by a sensitive radioimmunoassay. In both preparations, pools of adenosine (2.5-10 pmol/mg of protein) were detected that were resistant to deamination by added adenosine deaminase (0.5-3 units/ml) unless membrane lipids were first dissolved in acetone. Electron microscopic examination of crude CHAPS-solubilized receptors revealed the existence of small vesicles (< 1 microns in diameter). Furthermore, most "solubilized" receptors were retained by a 0.1-microns filter. The effects of GTP gamma S were evaluated on the binding of an antagonist, 3-(4-amino-3-125I-phenethyl)-1-propyl-8-cyclopentylxanthine (125I-BW-A844U), to A1 receptors of bovine brain membranes, receptors solubilized in CHAPS (crude solubilized), or receptors partially co-purified with G proteins by agonist affinity chromatography (partially purified). GTP gamma S (10 microM) increased antagonist binding to membranes (20-50%) and crude CHAPS-solubilized receptors (> 200%) but increased binding to partially purified receptors by only 10-15%. GTP gamma S decreased agonist (125I-N6-aminobenzyladenosine) binding and increased antagonist Bmax, but did not significantly decrease (5%) the dissociation rate of the antagonist. Omission of Mg2+ mimicked the effects of GTP gamma S on agonist and antagonist binding and increased both the association and dissociation rates of 125I-BW-A844U. These data suggest that a Mg(2+)-dependent GTP gamma S-induced increase in antagonist binding to membranes and solubilized receptors is primarily due to unmasking of cryptic binding sites occupied by contaminating vesicular adenosine. These findings are consistent with the observation that adenosine receptor antagonists have been found to have little or no inverse agonist physiological effects in well oxygenated tissues.
Mol Pharmacol 1992 Nov
PMID:Indirect effect of guanine nucleotides on antagonist binding to A1 adenosine receptors: occupation of cryptic binding sites by endogenous vesicular adenosine. 143 51

Affinity labeling of 40S subunits from human placenta with 4-(N-2-chloroethyl-N-methylamino)benzylmethyl-[32P]phosphoamide s of oligoribonucleotides pAUG and pAUGU3 was studied. Covalent attachment of these derivatives to 40S subunits within the complexes with 40S subunits, formed in the presence of Met-tRNAf.eIF-2.GTP, was detected. Both rRNA and ribosomal proteins were modified. Fragments of 18S rRNA, containing sites of the reagent attachment were identified: 1058-1164 for pAUG derivative and 976-1057--for pAUG and pAUGU3 ones. The data obtained allowed to conclude that the presence of the neighbouring codon at the A-site, regardless of the presence of the tRNA in it, affects significantly the arrangement of the trinucleotide template in the codon-anticodon interaction region. The large subunit does not cause significant alterations in the structural organization of the codon-anticodon interaction region.
Mol Biol (Mosk)
PMID:[Affinity modification of the 40S subparticle from human placenta with derivatives of pAUG and pAUGU3]. 143 86

We describe the sequence and characterization of the Bacillus subtilis flhF gene. flhF encodes a basic polypeptide of 41 kDa that contains a putative GTP-binding motif. The sequence of FlhF reveals a structural relationship to two Escherichia coli proteins, Ffh and FtsY, as well as to other members of the SRP54 family, in a domain presumed to bind GTP. flhF is located in a large operon consisting of chemotaxis and flagellar genes. Cells deficient in flhF are nonmotile. Through the use of anti-flagellar antibodies we have established that flhF is a flagellar (fla) gene. Thus, flhF is a unique flagellar gene in that it encodes a GTP-binding protein with similarities to members of the SRP54 family of proteins. These data suggest that flagellar biosynthesis in B. subtilis requires GTP.
Mol Microbiol 1992 Sep
PMID:flhF, a Bacillus subtilis flagellar gene that encodes a putative GTP-binding protein. 144 78


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