(1/337) Point mutations in the guanine phosphoribosyltransferase from Giardia lamblia modulate pyrophosphate binding and enzyme catalysis.
Guanine phosphoribosyltransferase (GPRTase) from Giardia lamblia, an enzyme required for guanine salvage and necessary for the survival of this parasitic protozoan, has been kinetically characterized. Phosphoribosyltransfer proceeds through an ordered sequential mechanism common to many related purine phosphoribosyltransferases (PRTases) with alpha-D-5-phosphoribosyl-1-pyrophosphate (PRPP) binding to the enzyme first and guanosine monophosphate (GMP) dissociating last. The enzyme is a highly unique purine PRTase, recognizing only guanine as its purine substrate (K(m) = 16.4 microM) but not hypoxanthine (K(m) > 200 microM) nor xanthine (no reaction). It also catalyzes both the forward (kcat = 76.7 s-1) and reverse (kcat = 5.8.s-1) reactions at significantly higher rates than all the other purine PRTases described to date. However, the relative catalytic efficiencies favor the forward reaction, which can be attributed to an unusually high K(m) for pyrophosphate (PPi) (323.9 microM) in the reverse reaction, comparable only with the high K(m) for PPi (165.5 microM) in Tritrichomonas foetus HGXPRTase-catalyzed reverse reaction. As the latter case was due to the substitution of threonine for a highly conserved lysine residue in the PPi-binding loop [Munagala et al. (1998) Biochemistry 37, 4045-4051], we identified a corresponding threonine residue in G. lamblia GPRTase at position 70 by sequence alignment, and then generated a T70K mutant of the enzyme. The mutant displays a 6.7-fold lower K(m) for PPi with a twofold increase in the K(m) for PRPP. Further attempts to improve PPi binding led to the construction of a T70K/A72G double mutant, which displays an even lower K(m) of 7.9 microM for PPi. However, mutations of the nearby Gly71 to Glu, Arg, or Ala completely inactivate the GPRTase, suggesting the requirement of flexibility in the putative PPi-binding loop for enzyme catalysis, which is apparently maintained by the glycine residue. We have thus tentatively identified the PPi-binding loop in G. lamblia GPRTase, and attributed the relatively higher catalytic efficiency in the forward reaction to the unusual loop structure for poor PPi binding in the reverse reaction. (+info)
(2/337) Rickettsia prowazekii transports UMP and GMP, but not CMP, as building blocks for RNA synthesis.
Rickettsia prowazekii, the etiological agent of epidemic typhus, is an obligate intracellular bacterium and is apparently unable to synthesize ribonucleotides de novo. Here, we show that as an alternative, isolated, purified R. prowazekii organisms transported exogenous uridyl- and guanylribonucleotides and incorporated these labeled precursors into their RNA in a rifampin-sensitive manner. Transport systems for nucleotides, which we have shown previously and show here are present in rickettsiae, have never been reported in free-living bacteria, and the usual nucleobase and nucleoside transport systems are absent in rickettsiae. There was a clear preference for the monophosphate form of ribonucleotides as the transported substrate. In contrast, rickettsiae did not transport cytidylribonucleotides. The source of rickettsial CTP appears to be the transport of UMP followed by its phosphorylation and the amination of intrarickettsial UTP to CTP by CTP synthetase. A complete schema of nucleotide metabolism in rickettsiae is presented that is based on a combination of biochemical, physiological, and genetic information. (+info)
(3/337) Synthesis of adenine and guanine nucleotides at the 'inosinic branch point' in lymphocytes of leukemia patients.
The synthesis of purine nucleotides has been studied in human peripheral blood lymphocytes from healthy subjects and patients affected by B-cell chronic lymphocytic leukemia (B-CLL). The rate of the synthesis was measured by following the incorporation of 14C-formate into the nucleotides of lymphocyte suspensions. The whole sequence AMP-->ADP-->ATP was found reduced in B-CLL lymphocytes; in the case of guanylates only the last step of the sequence GMP-->GDP-->GTP was significantly lower in the same cells. From the analysis of these results, combined with previous data, we conclude that purine metabolism undergoes an imbalancement during CLL, which is partially compensated, and point out the importance of studying concomitantly purine metabolism and nucleic acid synthesis in leukemia cells. (+info)
(4/337) Virus-specific capping of tobacco mosaic virus RNA: methylation of GTP prior to formation of covalent complex p126-m7GMP.
In capping cellular mRNAs, a covalent GMP-enzyme intermediate leads to formation of G(5')ppp(5')N at the 5' end of the RNA, which is modified by methylation catalyzed by guanine-7-methyltransferase. Here we show that isolated membranes from tobacco mosaic virus (TMV)-infected plant or insect cells expressing TMV replicase protein p126, synthesized m7GTP using S-adenosylmethionine (AdoMet) as the methyl donor, and catalyzed the formation of a covalent guanylate-p126 complex in the presence of AdoMet. The methyl group from AdoMet was incorporated into p126, suggesting that the complex consisted of m7GMP-p126. Thus, TMV and alphaviruses, despite their evolutionary distance, share the same virus-specific capping mechanism. (+info)
(5/337) Reversal of HO-1 related cytoprotection with increased expression is due to reactive iron.
It is often postulated that the cytoprotective nature of heme oxygenase (HO-1) explains the inducible nature of this enzyme. However, the mechanisms by which protection occurs are not verified by systematic evaluation of the physiological effects of HO. To explain how induction of HO-1 results in protection against oxygen toxicity, hamster fibroblasts (HA-1) were stably transfected with a tetracycline response plasmid containing the full-length rat HO-1 cDNA construct to allow for regulation of gene expression by varying concentrations of doxycycline (Dox). Transfected cells were exposed to hyperoxia (95% O(2)/5% CO2) for 24 h and several markers of oxidative injury were measured. With varying concentrations of Dox, HO activity was regulated between 3- and 17-fold. Despite cytoprotection with low (less than fivefold) HO activity, high levels of HO-1 expression (greater than 15-fold) were associated with significant oxygen cytotoxicity. Levels of non-heme reactive iron correlated with cellular injury in hyperoxia whereas lower levels of heme were associated with cytoprotection. Cellular levels of cyclic GMP and bilirubin were not significantly altered by modification of HO activity, precluding a substantial role for activation of guanylate cyclase by carbon monoxide or for accumulation of bile pigments in the physiological consequences of HO-1 overexpression. Inhibition of HO activity or chelation of cellular iron prior to hyperoxic exposure decreased reactive iron levels in the samples and significantly reduced oxygen toxicity. We conclude that there is a beneficial threshold of HO-1 overexpression related to the accumulation of reactive iron released in the degradation of heme. Therefore, despite the ready induction of HO-1 in oxidant stress, accumulation of reactive iron formed makes it unlikely that exaggerated expression of HO-1 is a cytoprotective response. (+info)
(6/337) Chemosensory signal transduction in paramecium.
Paramecia are ciliated single-cell eukaryotic organisms that can respond to chemical cues in their environment. Glutamate is among those cues, which attract cells. We describe briefly here the following attributes of glutamate chemoresponse: 1) Cells are attracted to L-glutamate relative to KCl at high concentrations of glutamate. 2) There are at least two specific, relatively low affinity glutamate binding sites on the cell surface. Glutamate can be displaced from only one of the binding sites by inosine monophosphate (IMP), and quisqualate displaces glutamate from the second site, which is likely to be the glutamate receptor involved in attraction to glutamate. 3) IMP is a repellent and does not act synergistically with glutamate, whereas guanosine monophosphate (GMP) does. 4) Similarly, glutathione is an attractant, but glutamate and glutathione appear to use different transduction pathways. 5) Glutamate hyperpolarizes the cell. The ionic mechanism is not yet verified, but is likely to involve a K conductance. 6) Glutamate induces a rapid and robust increase in cAMP in the cell. Protein kinase A (PKA) is possibly involved in the transduction pathway because kinase inhibitors such as H7 and H8 inhibit glutamate response, but do not affect responses to other attractants, such as acetate and ammonium. Activation of PKA by the rapid rise in cAMP may sustain the hyperpolarization phosphorylation and activation of the plasma membrane calcium pump. 7) Candidate glutamate binding proteins are being identified among the cell surface proteins with the use of affinity chromatography. (+info)
(7/337) A sequence motif involved in the donor substrate binding by alpha1,6-fucosyltransferase: the role of the conserved arginine residues.
Alpha1,6-fucosyltransferase catalyzes the transfer of fucose to the innermost GlcNAc residue of an N-linked oligosaccharide. In order to identify the amino acid residue(s) which are associated with the enzyme activity and to investigate their function, we prepared a series of mutant human alpha1,6-fucosyltransferases in which the conserved residues in the region homologous to alpha1,2-fucosyltransferase had been replaced. These proteins were then characterized by kinetic analyses. The wild-type and mutant alpha1,6-fucosyltransferases were expressed using a baculovirus-insect cell system. The activity assay showed that replacement of Arg-365 by Ala or Lys led to a complete loss of activity while substitution of Ala or Lys for the neighboring Arg-366 decreased the activity to about 3% that of the wild type. Kinetic analyses revealed that the replacements of Arg-366 lead to an increase in the apparent K (m) value for both GDP-fucose and the acceptor oligosaccharide but did not markedly affect the apparent V (max). When these mutants were inhibited by GDP in a competitive manner with respect to the donor substrate, the K (i) values were found to be 50-100 times higher than the value in the wild type. On the other hand, in the inhibition by GMP, the K (i) values for the mutants were very similar to that of the wild type. These findings suggest that Arg-366 contributes to the binding of GDP-fucose via an interaction with the beta-phosphoryl group of the GDP moiety of the donor, and that Arg-365 may also play an essential role in substrate binding. The results suggest that the motif common to alpha1,2- and alpha1,6-fucosyltransferases is critical for binding of the donor substrate, GDP-fucose. (+info)
(8/337) Preferential inhibition by a novel Na(+)/Ca(2+) channel blocker NS-7 of severe to mild hypoxic injury in rat cerebrocortical slices: A possible involvement of a highly voltage-dependent blockade of Ca(2+) channel.
The hypoxic injury was induced in rat cerebrocortical slices by the exposure to hypoxia for 45 min in the absence or presence of 3 mM glucose, followed by reoxygenation for 5 h. The injury was more pronounced in the absence of glucose (severe hypoxic injury) than in the presence of glucose (mild hypoxic injury). A novel Na(+)/Ca(2+) channel blocker, NS-7 [4-(4-fluorophenyl)-2-methyl-6-(5-piperidinopentyloxy) pyrimidine hydrochloride], at 3 to 30 microM inhibited preferentially the severe hypoxic injury, whereas MK-801, omega-conotoxin GVIA (omega-CTX), and N(G)-nitro-L-arginine methylester suppressed preferentially the mild hypoxic injury. The extracellular cyclic GMP formation, a marker of nitric oxide synthesis, was enhanced during hypoxia, although the extent was greater in the absence of glucose. As observed in the hypoxic injury, NS-7 preferentially inhibited the cyclic GMP formation induced by severe hypoxic insults, whereas MK-801 or omega-CTX reduced it under mild hypoxic condition. When 30 to 50 mM KCl was applied to normoxic slices, a concentration-dependent increase in the extracellular cyclic GMP formation was observed. NS-7 blocked the cyclic GMP formation induced by 50 mM KCl but not by 30 to 40 mM KCl, whereas omega-CTX suppressed only the 30 mM KCl-evoked response. In primary neuronal culture, NS-7 reversed KCl-induced increase in intracellular Ca(2+) in which the inhibition was marked when the KCl concentration was increased. These findings suggest that NS-7, unlike other neuroprotective compounds used in this study, is more effective in severe hypoxic injury. The highly voltage-dependent Ca(2+) channel blockade may contribute to the mode of neuroprotective action of NS-7. (+info)