Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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Transporters encoded in genetic loci putP, proP and proU mediate proline and/or betaine accumulation by Escherichia coli K-12. The ProP and ProU systems are osmoregulatory. Activation of ProP in response to hyperosmotic stress has been demonstrated both in vivo and in vitro. It therefore serves as a model experimental system for the analysis of osmosensory and osmoregulatory mechanisms. We developed methodologies which will facilitate the identification of proline transporter genes by functional complementation of putP proP proU bacteria. E. coli gene proP was isolated and located within a chromosomal DNA fragment. Deletion, complementation and sequence analysis revealed putative promoter and transcription termination signals flanking a 1500 base-pair open reading frame. The predicted 55 kDa ProP protein was hydrophobic. In vitro expression of proP yielded a protein whose apparent molecular mass was determined to be 42 kDa by polyacrylamide gel electrophoresis under denaturing conditions. Database searches and cluster analysis defined relationships among the ProP sequence and those of integral membrane proteins that comprise a transporter superfamily. Members of the superfamily catalyze facilitated diffusion or ion linked transport of organic solutes in prokaryotes and eukaryotes. Multiple alignment revealed particularly close correspondence among the ProP protein, citrate transporters from E. coli and Klebsiella pneumoniae and an alpha-ketoglutarate transporter from E. coli. The predicted ProP sequence differed from those closely similar sequences in possessing an extended central hydrophilic loop and a carboxyl terminal extension. Unlike other protein sequences within the transporter superfamily, the carboxyl terminal extension of ProP was strongly predicted to participate in formation of an alpha-helical coiled coil. These data suggest that the ProP protein catalyzes solute-ion cotransport. Its unusual structural features may be related to osmoregulation of its activity.
J Mol Biol 1993 Jan 05
PMID:Isolation and sequencing of Escherichia coli gene proP reveals unusual structural features of the osmoregulatory proline/betaine transporter, ProP. 842 14

The development of outbred mouse (CF1) zygotes in vitro has been studied using medium SOM in which the concentrations of NaCl (85, 105, 125 mM), glutamine (0, 1, 2 mM), and betaine (0, 1, 2 mM) were varied. The effects of the compounds were studied using a 3(3) factorial experimental arrangement. The inhibitory effect of relatively high concentrations of NaCl and the protective effect of glutamine were confirmed. Betaine, an organic osmolyte, can also protect against the deleterious effects of relatively high concentrations of NaCl. The intracellular contents of potassium and sodium have also been measured in single zygotes using X-ray electron probe spectrometry. When medium SOM contains 85 mM or 125 mM NaCl, the intracellular content of Na rises and the content of K decreases. These changes are partially reduced in the presence of 125 mM NaCl if betaine is also in the medium. Betaine has no effect on the intracellular content of K and Na if the concentration of NaCl is 85 mM. These results suggest that organic osmolytes may be required in embryo culture media to prevent excessive changes in the intracellular ionic concentration.
Mol Reprod Dev 1993 Apr
PMID:The protective action of betaine on the deleterious effects of NaCl on preimplantation mouse embryos in vitro. 847 Dec 60

Results of recent experiments indicate that the improved development of mouse embryos in medium containing a low NaCl concentration (85 mM) or the inclusion of the organic osmolyte betaine in a medium containing a high NaCl concentration (125 mM) is correlated with the maintenance of intracellular sodium concentrations that more closely approximate those found in freshly isolated embryos (Biggers et al., 1993, Mol Reprod Dev 34:380-390). We examined the effect of these different culture media on the relative rates of protein synthesis since increased levels of intracellular sodium inhibit protein synthesis; a reduced rate of protein synthesis could therefore account for the differences in development in the different media, since cell division requires protein synthesis. We observe that the ability of these media to support development and to maintain more physiological concentrations of intracellular sodium is correlated with their ability to support increased relative rates of protein synthesis. Reducing the NaCl concentration from 125 mM to 85 mM leads to a greater fraction of the embryos developing from the 2-cell stage to the 8-cell stage after 1 day of culture and a substantially improves extent of development to the morula stage after 2 days of culture. This reduction in NaCl concentration also leads to a 2.4-fold increase in the relative rate of protein synthesis in 4-cell embryos. Moreover, addition of betaine to medium containing 125 mM NaCl increases the relative rate of protein synthesis. This finding provides an explanation, at least in part, for the increase in development to the blastocyst stage exhibited by mouse embryos cultured in these media.(ABSTRACT TRUNCATED AT 250 WORDS)
Mol Reprod Dev 1993 May
PMID:Effect of sodium and betaine in culture media on development and relative rates of protein synthesis in preimplantation mouse embryos in vitro. 850 76

In this report, effects of alpha 1-adrenergic stimulation on phosphatidylcholine (PC) hydrolysis and the subsequent generation of water-soluble choline metabolites were investigated after preincubation of isolated cardiac myocytes of adult rats with [methyl-3H]choline. Choline uptake into cardiac myocytes was apparently mediated by a choline carrier which could be inhibited by hemicholinium-3. Analysis of the intracellular choline metabolites was performed by HPLC. Adrenergic stimulation of cardiac myocytes by (-)-phenylephrine, which is also known to activate the phosphoinositide signaling system, induced the generation of betaine as a selective signal transduction response. Agonist-induced generation of betaine in cardiac myocytes was maximal at 10 min after stimulation, and was optimal at physiologically relevant (-)-phenylephrine concentrations (1-10 microM). Betaine accumulation was transient, and no betaine remained detectable after 15 min. CDP-choline, however, was still elevated after 15 min which is indicative of continued PC resynthesis after adrenergic stimulation. The source of betaine in cellular signalling appeared to be hydrolysis of membrane PC to phosphatidic acid and choline by phospholipase D with subsequent oxidation of choline to betaine. This is based on the observation that radioactivity in unstimulated cells is present only in the lipid phase (presumably as PC) or as phosphocholine in the aqueous phase of the cells. The latter finding suggests that choline is rapidly phosphorylated after uptake into cardiac myocytes. Collectively, these results suggest a hypothetical role of betaine in the cellular signal transduction response to alpha 1-adrenergic stimulation in cardiac myocytes.
J Mol Cell Cardiol 1996 May
PMID:Betaine generation in cardiac myocytes after adrenergic activation of phosphatidylcholine hydrolysis. 876 47

We have investigated the disposition of potentially endotoxic homocysteine (Hcy) and its transsulfuration metabolite cysteine (Cys) in 98 individuals (age range 20-66 years). Our study reports on the relationship between Hcy and two important dietary factors likely to influence plasma levels of this thiol: dietary folate and dietary methionine. chi2 analysis shows a low frequency of elevated plasma Hcy at high folate intake. This frequency for Hcy >10 micromol/liter with a folate intake >350 microg/day is significant (P < 0.02). The data reflect a tendency for elevated Hcy values to be associated with low dietary folate, although many subjects with a low dietary folate also had a low plasma Hcy. Intake of dietary methionine was found to be significantly higher in males than in females (P < .0001). This may account for the looser relationship between Hcy and its transsulfuration product, Cys, in females (R2 = 0.30) compared to males (R2 = 0.73), since conversion of methionine to SAM in males would activate cystathionine beta synthase and commit excess Hcy to transsulfuration. The generally lower methionine intake of females means that more Hcy is utilized in the remethylation cycle in which methionine is produced from the de novo methyl group of 5-methyltetrahydrofolate or from the preformed methyl group of betaine. Clearly a Hcy moiety locked up in remethylation would be further removed from Cys, the end product of transsulfuration. An increasing number of studies are clarifying the relationship between Hcy, folate, and other B vitamins. However, less attention seems to be given to the influence of dietary methionine on the disposition of Hcy. The present study supports biochemical theory and indicates that more focus should be given to the effect of dietary methionine on Hcy. These findings have particular significance since even moderate increases in plasma Hcy are associated with a toxic vascular effect. Consequently the relationship between dietary folate and Hcy levels should be a factor in evaluating recommended dietary allowances for this vitamin. The simplicity of our dietary folate questionnaire also raises the possibility of a screening test in which individuals can ascertain whether their folate intake is adequate to reduce Hcy levels to a benign value.
Biochem Mol Med 1996 Dec
PMID:The influence of dietary folate and methionine on the metabolic disposition of endotoxic homocysteine. 898 31

Production of the two phospholipases C (PLCs) in Pseudomonas aeruginosa PAO1 is induced under conditions of phosphate limitation, or by the osmoprotectants choline or glycine betaine. Tn5 mutagenesis was performed on strain PAO1 to isolate mutants deficient in choline-dependent induction of PLC. Two mutants, Tn5T1 and Tn5G19, were identified which produce decreased levels of PLC in phosphate-replete media supplemented with choline. A total of 136 and 496 bp of flanking DNA from Tn5G19 and Tn5T1 was cloned by an inverse polymerase chain reaction (PCR) and sequenced. The DNA flanking the Tn5T1 insertion contains an open reading frame predicted to encode a peptide that is approx. 60% identical to the N-terminus of a previously identified protein (P35) of unknown function from Escherichia coli. The P35 gene, which is located in the nusA-infB operon in E. coll, was designated orp (osmoprotectant regulator of PLC). Haemolytic titres, total PlcH protein and beta-galactosidase activity expressed from a chromosomally inserted plcH-lacZ operon fusion were reduced in strain Tn5T1 in comparison with the parental strain (PAO1) carrying the same fusion. However, this mutant expressed several-fold higher levels of plcH message than strain PAO1 in the presence of choline, while the phosphate-starvation-dependent transcript of plcH could not be detected in this mutant. The defects in Tn5T1 are complemented by a DNA fragment, isolated from a genomic library of PAO1, that carries the orp gene. The deduced amino acid sequence of the DNA fragment cloned from Tn5G19 exhibits 84% identity with the betB gene product of E. coli that has betaine aldehyde dehydrogenase activity. This enzyme catalyses the conversion of betaine aldehyde to glycine-betaine. Unlike the parental strain, the Tn5G19 mutant could not utilize choline as a sole carbon, nitrogen and energy source, and it was deficient in betaine aldehyde dehydrogenase activity. Also, consistent with a disruption of betB in Tn5G19, choline inhibited growth of this strain in media containing 0.7 M NaCl, while glycine-betaine restores growth to wild-type levels. The defects in Tn5G19 are complemented by a DNA fragment from PAO1 that carries the betB gene. The orp gene is located between 0.6 to 6.6 min while betB is located between 10.5 to 12.5 min on the chromosome of PAO1.
Mol Microbiol 1997 Jan
PMID:Molecular characterization of mutants affected in the osmoprotectant-dependent induction of phospholipase C in Pseudomonas aeruginosa PAO1. 900 19

In order to elucidate the biochemical roles of imidazol-containing dipeptides, we have studied quenching of singlet molecular oxygen (1O2) by carnosine (beta-alanyl-L-histidine), its structural components (L-histidine, imidazole, and beta-alanine), and related natural free-radical scavengers-L-anserine (beta-alanyl-1-methyl-histidine), ergothioneine (2-thiol-L-histidine-betaine), and taurine (2-aminoethanesulfonic acid) in aqueous (D2O, pD 7) solutions by using monitoring of 1O2-phosphorescence (1270-nm). The rate constants of 1O2 quenching (Kq) by carnosine, anserine, and ergothioneine were shown to be similar [(3 +/- 1) x 10(7) M-1s-1]. Their values resembled those of free-L-histidine [Kq = (4 +/- 1) x 10(7) M-1s-1] and imidazole [Kq = (2 +/- 1) x 10(7) M-1s-1]. Non-aromatic amino acids-taurine and beta-alanine-showed very low quenching activities (Kq < 3 x 10(3) M-1c-1). The Kq values did not correlate with the literature data on abilities of the tested compounds to stimulate muscle working capacities and inhibit myeloperoxidase-catalyzed oxygenation. Thus, the dipeptides can be used as potent water-soluble protectors against 1O2 attack whereas their natural biochemical functions are most probably determined by the processes of different nature.
Biochem Mol Biol Int 1997 Apr
PMID:Quenching of singlet molecular oxygen by carnosine and related antioxidants. Monitoring 1270-nm phosphorescence in aqueous media. 911 30

The proP gene, encoding a transporter of the osmoprotecting compounds proline and glycine betaine, is expressed from two promoters. Transcription of the P2 promoter occurs at a transient period in late exponential phase and is dependent upon Fis and the RpoS (sigma38) sigma factor. Here we characterize Fis-mediated activation of the P2 promoter in vitro. We find that this promoter displays unusually high specificity for sigma38. Fis strongly activates P2 when bound to site I centered at -41 within the promoter region. There is a complex relationship involving DNA supercoiling and potassium glutamate concentration on Fis activation, but most efficient transcription occurs under high salt conditions when the superhelical density is above -0.03. The major stimulatory effect of DNA supercoiling occurs between superhelical densities of 0 to -0.02 suggesting that, while supercoiling is mechanistically important, it may not be a physiologically relevant controlling factor. However, the stimulation of transcription by high potassium glutamate concentrations may contribute to the osmotic inducibility of the P2 promoter. We show that Fis and E sigma38 bind cooperatively on supercoiled DNA to form a stable complex at P2 that involves promoter melting. Fis also binds to a second site within the proP regulatory region. While binding to this site appears to play no role in Fis activation of the P2 promoter, it functions as a repressor of transcription initiating from the P1 promoter by either sigma70 or sigma38.
J Mol Biol 1997 Jul 18
PMID:Activation of RpoS-dependent proP P2 transcription by the Fis protein in vitro. 923 2

The histamine H2-receptor antagonists have been identified as inhibitors of human liver aldehyde dehydrogenase (EC 1.2.1.3) isozymes, E1, E2, and E3. Inhibition was strongest with the E3 isozyme, whose substrates include gamma-aminobutyraldehyde, the aldehyde metabolites of polyamines, and betaine aldehyde. Burimamide, metiamide, cimetidine guanidine, cimetidine, and tiotidine were competitive with aldehyde substrates and noncompetitive with the coenzyme, binding to both the free E3 isozyme and the enzyme-coenzyme binary complex. Cimetidine and tiotidine were the best inhibitors, with Ki values of 1.1 +/- 0.2 microM and 1.0 +/- 0.0 microM, respectively; both are the first ever described potent and selective inhibitors of the E3 isozyme. Examination of the H2-receptor antagonist structures for insight into the moieties accounting for E3 isozyme inhibition pointed to the side-chain polar groups as strongly influencing inhibition, with the cyanoguanidine side chain of cimetidine and tiotidine having the strongest influence. The Ki value of the E3 isozyme for cimetidine was the same as the in vitro dissociation constant for the H2-receptor.
Mol Pharmacol 1997 Aug
PMID:Cimetidine and other H2-receptor antagonists as inhibitors of human E3 aldehyde dehydrogenase. 927 49

The tissue distribution of the E3 isozyme of human aldehyde dehydrogenase has been investigated by three methods: enzyme activity assay employing betaine aldehyde as substrate, Western blotting employing E3 isozyme-specific antibodies, and Northern blotting using a human liver E3 cDNA as probe. All three methods showed that E3 isozyme was universally distributed among all tissues tested. The highest levels of the E3 isozyme activity were found in liver, adrenal gland, and kidney. These same tissues also showed highest levels of the E3 protein via the Western blot. This distribution is consistent with the possible physiological role of E3 isozyme in the synthesis of the osmolyte, betaine, and the neurotransmitter, GABA. Northern blot analysis, however, differed from that of enzyme assay and the Western blot in that it showed highest mRNA levels in skeletal and heart muscles, which had low enzyme activities and E3 protein levels.
Comp Biochem Physiol B Biochem Mol Biol 1997 Sep
PMID:Tissue distribution of human aldehyde dehydrogenase E3 (ALDH9): comparison of enzyme activity with E3 protein and mRNA distribution. 941 93


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