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

The effects of methylmalonic (MMA) and propionic acid (PPA), metabolites that accumulate in methylmalonic and propionic acidemia respectively, on [3H]glutamate binding, adenylate cyclase activity and [U-14C]acetate incorporation into lipids were investigated in rat cerebral cortex. Neither acid effected [3H]glutamate binding, regardless of the presence of sodium in the incubation medium. Also, the acids had no effect on basal or GMP-PNP-stimulated adenylate cyclase activity. These results suggest that MMA and PPA do not interact with glutamate binding sites and have no effect on basal or guanine nucleotide-stimulated adenylate cyclase activity. In contrast, [U-14C]acetate incorporation into brain lipids was significantly blocked by both acids, the effects being more pronounced with PPA, indicating an inhibition of brain lipid biosynthesis caused by MMA and PPA. These results may explain at least in part the hypomyelinization and/or demyelinization characteristic of patients affected by methylmalonic acidemia and propionic acidemia.
Biochem Mol Biol Int 1997 Sep
PMID:Effects of methylmalonate and propionate on [3H]glutamate binding, adenylate cyclase activity and lipid synthesis in rat cerebral cortex. 930 32

Alcohol suppresses reproduction in humans, monkeys and small rodents by suppressing release of luteinizing hormone (LH). The major action is on the hypothalamus to decrease release of LH-releasing hormone (LHRH). The release of LHRH is controlled by nitric oxide (NO). The hypothesized pathway is via norepinephrine-induced release of NO from NOergic neurons which activates LHRH release. We have evaluated details of this process in male rats by incubating medial basal hypothalamic (MBH) explants in vitro and examining the release of NO and metabolites generated by NO which control LHRH release. Norepinephrine increased release of NO as measured by determining the content of the enzyme at the end of the experiment (30 min) by adding [14C]arginine to the homogenate and measuring its conversion to [14C]citrulline since this is formed in equimolar quantities with NO by nitric oxide synthase (NOS). Since this increase in content presumably caused by activation of the enzyme by norepinephrine was blocked by the alpha 1 receptor blocker prazosin, it appears that alpha 1 receptors activate NOS by increasing intracellular free calcium in the NOergic neuron which combines with calmodulin to activate nitric oxide synthase. The release of LHRH induced by nitroprusside (NP), a donor of NO, results in an increase in cyclic (c)GMP in the medium supporting the activation of guanylate cyclase by nitroprusside. This activation is important in releasing LHRH since addition of 8-monobutyryl cGMP also released the peptide. Ethanol had no effect on the content of NO or the increase in content induced by norepinephrine indicating that it did not act on NOS. Earlier experiments indicated that prostaglandin E2 (PGE2) was important in releasing LHRH. PGE2 is produced by activation of cyclooxygenase by NO since this could occur following addition of the NO donor nitroprusside. Not only does NP increase PGE2 release, but also the conversion of [14C]arachidonic acid to its metabolites, particularly PGE2. Ethanol acts at this step since it completely blocks the release of LHRH induced by NP and the increase in PGE2 induced by NP. Therefore, the results support the theory that norepinephrine acts to stimulate NO release from NOergic neurons. This NO diffuses to the LHRH terminals, where it activates guanylate cyclase, leading to an increase in cGMP. At the same time, it also activates cyclooxygenase. The increase in cGMP increases intracellular free calcium, required for activation of phospholipase A2. Phospholipase A2 converts membrane phospholipids into arachidonic acid, the substrate for conversion by the activated cyclooxygenase to PGE2 which then activates the release of LHRH. Since alcohol inhibits conversion of labeled arachidonic acid to PGE2, it must act either directly to inhibit cyclooxygenase or by some other mechanism which, in turn, inhibits the enzyme.
Mol Psychiatry 1997 Sep
PMID:The mechanism of action of alcohol to suppress gonadotropin secretion. 932 22

His92 of Ribonuclease T1 combines functional and structural features involving both imidazole nitrogens. To evaluate the use of Asn and Gln substitutions in dissecting the properties of histidines, we analysed the consequences of the His92Gln and His92Asn substitutions on the enzyme's structure, function, and conformational stability by protein engineering and X-ray crystallographic methods. In the X-ray structures of wild-type and His92Gln RNase T1 in complex with 2'-GMP the His92-N epsilon 2 and Gln92-N epsilon 2 atoms are isosterically equivalent. Similarly, the His92N delta 1H...OAsn99 hydrogen bond observed in wild-type is replaced by an equivalent Asn92N delta 2H...OAsn99 in the His92Asn mutant structure. Double mutant cycles at a single position were used to analyse the intermolecular and intramolecular interactions of the exchangeable proton and the individual histidine nitrogens. Urea denaturation measurements as a function of pH revealed that the exchangeable proton of His92, rather than its imidazole ring is contributing about 1 kcal/mol to the conformational stability of RNase T1. The stabilizing and the destabilizing effects of the (His-->Gln) and the (His-->Asn) mutations on urea denaturation of RNase T1 at pH 9.0 suggest that the unprotonated N delta 1 and N epsilon 2 atoms contribute in a compensating way to the conformational stability of RNase T1. A comparative study of the kinetics of all mutants suggests that the protonated His92 imidazole is a strictly co-operative catalytic device.
J Mol Biol 1998 Jan 30
PMID:Dissecting histidine interactions of ribonuclease T1 with asparagine and glutamine replacements: analysis of double mutant cycles at one position. 946 38

The ubiquitous m7G cap of eukaryotic mRNAs and of precursors to the spliceosomal small nuclear RNAs (snRNAs) is the result of an essential RNA modification acquired during transcript elongation. In trypanosomes, the m7G cap is restricted to the spliced leader (SL) RNA and the precursors of U2, U3, and U4 snRNAs. mRNA capping in these organisms occurs posttranscriptionally by trans splicing, which transfers the capped SL sequence to the 5' ends of all mRNAs. The SL cap is the most elaborate cap structure known in nature and has been shown to consist of an m7G residue followed by four methylated nucleotides. Using Crithidia fasciculata, we have characterized and purified the guanylyltransferase (capping enzyme), which transfers GMP from GTP to the diphosphate end of RNA. The corresponding gene codes for a protein of 697 amino acids, with the carboxy-terminal half of the C. fasciculata guanylyltransferase containing the six signature motifs previously identified in yeast capping enzymes. The amino-terminal half contains a domain that displays no resemblance to any other domain associated with capping enzymes. Intriguingly, this region harbors a consensus sequence for a phosphate-binding loop which is found in ATP- and GTP-binding proteins. This two-domain structure is also present in the Trypanosoma brucei capping enzyme, which shows 44% overall identity with the C. fasciculata capping enzyme. Thus, this structure appears to be common to all trypanosomatid protozoa and defines a novel class of capping enzymes.
Mol Cell Biol 1998 Aug
PMID:Trypanosome capping enzymes display a novel two-domain structure. 967 71

We have examined the RNA-capping enzyme activities of bluetongue virus (BTV) minor core protein, VP4. Recombinant BTV VP4 protein was purified to homogeneity from insect cell culture infected with a baculovirus VP4 of BTV serotype 10. We demonstrate that the purified protein, and VP4 encapsidated in core-like particles, react with GTP and covalently bind GMP via a phosphoamide linkage, a characteristic feature of guanylyltransferase enzyme. VP4 also catalyses a GTP-PPi exchange reaction indicating that the protein is the guanylyltransferase of the virus. In addition, VP4 possesses an RNA 5'-triphosphatase activity which catalyses the first step in the RNA-capping sequence. Further, an inorganic pyrophosphatase activity was identified which may aid the transcription activity within the virus by removing inorganic pyrophosphate which is an inhibitor of the polymerization reaction. Finally, the direct evidence of VP4 capping activity has been obtained by demonstrating in vitro transfer of GMP to the 5' end of in vitro synthesized BTV ssRNA transcripts to form a cap structure.
J Mol Biol 1998 Jul 31
PMID:Guanylyltransferase and RNA 5'-triphosphatase activities of the purified expressed VP4 protein of bluetongue virus. 967 55

A novel function of initiation factors IF1 and IF2 in Escherichia coli translation has been identified. It is shown that these factors efficiently catalyse dissociation of peptidyl-tRNAs with polypeptides of different length from the P-site of E. coli ribosomes, and that the simultaneous presence of both factors is required for induction of drop-off. The factor-induced drop-off occurs with both sense and stop codons in the A-site and competes with peptide elongation or termination. The efficiency with which IF1 and IF2 catalyse drop-off decreases with increasing length of the nascent polypeptide, but is quite significant for hepta-peptidyl-tRNAs, the longest polypeptide chains studied. In the absence of IF1 and IF2 the rate of drop-off varies considerably for different peptidyl-tRNAs, and depends both on the length and sequence of the nascent peptide. Efficient factor-catalysed drop-off requires GTP but not GTP hydrolysis, as shown in experiments without guanine nucleotides, with GDP or with the non-cleavable analogue GMP-PNP.Simultaneous overexpression of IF1 and IF2 in vivo inhibits cell growth specifically in some peptidyl-tRNA hydrolase deficient mutants, suggesting that initiation factor-catalysed drop-off of peptidyl-tRNA can occur on a significant scale in the bacterial cell. Consequences for the bacterial physiology of this previously unknown function of IF1 and IF2 are discussed.
J Mol Biol 1998 Aug 14
PMID:Initiation factors IF1 and IF2 synergistically remove peptidyl-tRNAs with short polypeptides from the P-site of translating Escherichia coli ribosomes. 969 45

Structures of free, substrate-bound and product-bound forms of Escherichia coli xanthine-guanine phosphoribosyltransferase (XGPRT) have been determined by X-ray crystallography. These are compared with the previously determined structure of magnesium and sulphate-bound XPRT. The structure of free XGPRT at 2.25 A resolution confirms the flexibility of residues in and around a mobile loop identified in other PRTases and shows that the cis-peptide conformation of Arg37 at the active site is maintained in the absence of bound ligands. The structures of XGPRT complexed with the purine base substrates guanine or xanthine in combination with cPRib-PP, an analog of the second substrate PRib-PP, have been solved to 2.0 A resolution. In these two structures the disordered phosphate-binding loop of uncomplexed XGPRT becomes ordered through interactions with the 5'-phosphate group of cPRib-PP. The cyclopentane ring of cPRib-PP has the C3 exo pucker conformation, stabilised by the cPRib-PP-bound Mg2+. The purine base specificity of XGPRT appears to be due to water-mediated interactions between the 2-exocyclic groups of guanine or xanthine and side-chains of Glu136 and Asp140, as well as the main-chain oxygen atom of Ile135. Asp92, together with Lys115, could help stabilise the N7-protonated tautomer of the incoming base and could act as a general base to remove the proton from N7 when the nucleotide product is formed. The 2.6 A resolution structure of XGPRT complexed with product GMP is similar to the substrate-bound complexes. However, the ribose ring of GMP is rotated by approximately 24 degrees compared with the equivalent ring in cPRib-PP. This rotation results in the loss of all interactions between the ribosyl group and the enzyme in the product complex.
J Mol Biol 1998 Oct 02
PMID:Structures of free and complexed forms of Escherichia coli xanthine-guanine phosphoribosyltransferase. 974 33

Kinetics of a self-capping RNA, Iso6, have been investigated to constrain the catalytic mechanism. The role of phosphates has been examined by varying the number of phosphates on the nucleophilic attacking group or on the RNA. While the number of phosphates in the nucleophile affects capping kinetics, only KM but not kcat is altered. The KM values for GMP, GDP, GTP and ppppG are 200, 11, 13 and 31 microM, respectively. A reaction product, pyrophosphate, is also found to strongly inhibit RNA activities through a competitive exchange mechanism with an apparent Ki of 200 nM. Uniquely strong binding of pyrophosphate supports the idea that capping originated by utilization of the initial pyrophosphate leaving group site for capping nucleophiles. In contrast to the nucleophile phosphate, change of 5' RNA terminus from triphosphate to tetraphosphate enhances the overall rate and kcat by 40%, with little effect on KM. Thus, only the leaving group appears to affect the rate of the chemical transformation. We propose two possible mechanisms that explain this apparent rate-limiting chemical step, either dissociation of pyrophosphate to form a metaphosphate monoester intermediate or formation of a circular phosphoramidate intermediate, using an internal RNA nitrogenous group. A single essential Ca ion is required for all activities.
J Mol Biol 1998 Nov 27
PMID:Kinetics at a multifunctional RNA active site. 981 16

COPII proteins are required to create transport vesicles and to select cargo molecules for transit from the ER. A reconstituted liposome budding reaction was used to detect the capture and concentration of membrane-associated v-SNARE molecules into synthetic COPII vesicles. A novel glutathione-phosphatidyl-ethanolamine conjugate (Glut-PE) was synthesized and incorporated into chemically defined liposomes to provide binding sites for GST hybrid proteins. Large liposomes containing bound cytoplasmic domains of the v-SNAREs, Sec22p or Bos1p, or of the ER resident proteins, Sec12p and Ufe1p, were exposed to COPII proteins and GMP-PNP. v-SNAREs but not resident proteins were concentrated in synthetic COPII vesicles generated from donor liposomes. We conclude that COPII proteins are necessary and sufficient for cargo selection and vesicle morphogenesis.
Mol Cell 1998 Nov
PMID:Coat assembly directs v-SNARE concentration into synthetic COPII vesicles. 984 42

Capping is targeted to pre-mRNAs through binding of the guanylyltransferase component of the capping apparatus to the phosphorylated CTD of RNA polymerase II. We report that mammalian guanylyltransferase binds synthetic CTD peptides containing phosphoserine at either position 2 or 5 of the YSPTSPS heptad repeat. CTD peptides containing Ser-5-PO4 stimulate guanylyltransferase activity by enhancing enzyme affinity for GTP and increasing the yield of the enzyme-GMP intermediate. A CTD peptide containing Ser-2-PO4 has no effect on guanylyltransferase activity. This implies an allosteric change in guanylyltransferase conformation that is specified by the position of phosphoserine in the CTD. Stimulation of guanylyltransferase increases with the number of Ser-5-phosphorylated heptads. Our results underscore how mRNA production may be regulated by the display of different CTD phosphorylation arrays during transcription elongation.
Mol Cell 1999 Mar
PMID:Distinct roles for CTD Ser-2 and Ser-5 phosphorylation in the recruitment and allosteric activation of mammalian mRNA capping enzyme. 1019 43


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