KATP channels were reconstituted in COSm6 cells by coexpression of the sulfonylurea receptor SUR1 and the inward rectifier potassium channel Kir6.2. The role of the two nucleotide binding folds of SUR1 in regulation of KATP channel activity by nucleotides and diazoxide was investigated. Mutations in the linker region and the Walker B motif (Walker, J.E., M.J. Saraste, M.J. Runswick, and N.J. Gay. 1982. EMBO [Eur. Mol. Biol. Organ.] J. 1:945–951) of the second nucleotide binding fold, including G1479D, G1479R, G1485D, G1485R, Q1486H, and D1506A, all abolished stimulation by MgADP and diazoxide, with the exception of G1479R, which showed a small stimulatory response to diazoxide. Analogous mutations in the first nucleotide binding fold, including G827D, G827R, and Q834H, were still stimulated by diazoxide and MgADP, but with altered kinetics compared with the wild-type channel. None of the mutations altered the sensitivity of the channel to inhibition by ATP4−. We propose a model in which SUR1 sensitizes the KATP channel to ATP inhibition, and nucleotide hydrolysis at the nucleotide binding folds blocks this effect. MgADP and diazoxide are proposed to stabilize this desensitized state of the channel, and mutations at the nucleotide binding folds alter the response of channels to MgADP and diazoxide by altering nucleotide hydrolysis rates or the coupling of hydrolysis to channel activation. 相似文献
Members of the transketolase group of thiamine-diphosphate-dependent enzymes from 17 different organisms including mammals,
yeast, bacteria, and plants have been used for phylogenetic reconstruction. Alignment of the amino acid and DNA sequences
for 21 transketolase enzymes and one putative transketolase reveals a number of highly conserved regions and invariant residues
that are of predicted importance for enzyme activity, based on the crystal structure of yeast transketolase. One particular
sequence of 36 residues has some similarities to the nucleotide-binding motif and we designate it as the transketolase motif.
We report further evidence that the recP protein from Streptococcus pneumoniae might be a transketolase and we list a number of invariant residues which might be involved in substrate binding. Phylogenies
derived from the nucleotide and the amino acid sequences by various methods show a conventional clustering for mammalian,
plant, and gram-negative bacterial transketolases. The branching order of the gram-positive bacteria could not be inferred
reliably. The formaldehyde transketolase (sometimes known as dihydroxyacetone synthase) of the yeast Hansenula polymorpha appears to be orthologous to the mammalian enzymes but paralogous to the other yeast transketolases. The occurrence of more
than one transketolase gene in some organisms is consistent with several gene duplications. The high degree of similarity
in functionally important residues and the fact that the same kinetic mechanism is applicable to all characterized transketolase
enzymes is consistent with the proposition that they are all derived from one common ancestral gene. Transketolase appears
to be an ancient enzyme that has evolved slowly and might serve as a model for a molecular clock, at least within the mammalian
clade.
Received: 13 September 1995 / Accepted: 14 November 1996 相似文献
Thiamine phosphate esters (thiamine monophosphate-TMP; thiamine diphosphate-TDP and thiamine triphosphate-TTP) were measured as their thiochrome derivatives by High Performance Liquid Chromatography in the brains of pyrithiamine-treated rats at various stages during the development of thiamine deficiency encephalopathy. Severe encephalopathy was accompanied by significant reductions of all three thiamine phosphate esters in brain. Neurological symptoms of thiamine deficiency appeared when brain levels of TMP and TDP fell below 15% of normal values. Activities of the TDP-dependent enzyme -ketoglutarate dehydrogenase were more severely reduced in thalamus compared to cerebral cortex, a less vulnerable brain structure. On the other hand, reductions of TTP, the non-cofactor form of thiamine, occurred to a greater extent in cerebral cortex than thalamus. Early reductions of TDP-dependent enzymes and the ensuing metabolic pertubations such as lactic acidosis impaired brain energy metabolism, and NMDA-receptor mediated excitotoxicity offer rational explanations for the selective vulnerability of brain structures such as thalamus to the deleterious effects of thiamine deficiency. 相似文献
The rat liver microsomal enzyme CTP: phosphatidate cytidylyltransferase (EC 2.7.7.41) which catalyzes the formation of CDP-diacylglycerol has been found to be markedly stimulated by GTP. The requirement for GTP is absolute, the novel GTP analogues such as guanosine 5′-[β,γ-methylene]-triphosphate, guanosine 5′-[α,β-methylene]-triphosphate, guanosine 5′-[β,γ-imido]-triphosphate and guanosine 3′-diphosphate 5′-diphosphate are without significant effect. Maximal stimulation occurs at 1 mM GTP. ATP at a concentration of 5 mM totally inhibits the formation of CDP-diacylglycerol even in the presence of optimal GTP concentration. Analogues of ATP such as adenosine 5′-[α,β-methylene]-triphosphate, adenosine 5′-[β,γ-methylene]-triphosphate and adenosine 5′-[β,γ-imido]-triphosphate are without effect on the reaction. The addition of fluoride (8 mM) likewise abolishes the stimulatory effect of GTP. 相似文献
Nuclei isolated from Yoshida sarcoma cells had activity for conversion of dGTP to dGMP dependent on DNA synthesis. The ratio of nucleotide generation/generation + incorporation was 0.4 ± 0.1, indicating that approx. 40% of the incorporated dGMP was excised. Two lines of evidence indicated the dependence of this activity on DNA synthesis. (1) The activity was observed only in the presence of ATP, which is essential for nuclear DNA synthesis. (2) Inhibitors of DNA synthesis, such as N-ethylmaleimide, aphidicolin, spermine and KCl, also inhibited ATP- or DNA synthesis-dependent dGMP generation. Although nuclei contain nucleoside triphosphatase (N-nucleotidase), this enzyme was not involved appreciably in DNA synthesis-dependent dGMP generation. The reason for this was explained by the following findings. (a) Inhibitors did not decrease dGMP production in the complete absence of DNA synthesis. (b) Inhibitors did not inactivate N-nucleotidase to the same degree as they inhibited DNA synthesis-dependent dGMP generation. (c) Addition of ATP reduced dGTP hydrolysis catalyzed by N-nucleotidase. (d) GDP had no appreciable effect on DNA synthesis-dependent dGMP generation, but had a diluting effect on dGMP production catalyzed by N-nucleotidase. These results show that the pathway of dGMP generation in isolated nuclei was switched on addition of ATP from a N-nucleotidase-catalyzed one to a DNA polymerase-exonuclease-catalyzed one. 相似文献
Showdomycin inhibited pig brain ( with pseudo first-order kinetics. The rate of inhibition by showdomycin was examined in the presence of 16 combinations of four ligands, i.e., Na+, K+, Mg2+ and ATP, and was found to depend on the ligands added. Combinations of ligands were divided into five groups in terms of the magnitude of the rate constant; in the order of decreasing rate constants these were: (1), (2) , , and none, (3) , and , (4) and , , , and ATP. The highest rate was obtained in the presence of Na+, Mg2+ and ATP. The apparent concentrations of Na+, Mg2+ and ATP for half-maximum stimulation of inhibition () were 3 mM, 0.13 mM and 4μM, respectively. The rate was unchanged upon further increase in Na+ concentration from 140 to 1000 mM. The rates of inhibition could be explained on the basis of the enzyme forms present, including E1, E2, ES, E1-P and E2-P, i.e., E2 has higher reactivity with showdomycin than E1, while E2-P has almost the same reactivity as E1-P. We conclude that the reaction of ( proceeds via at least four kinds of enzyme form (E1, E2, E1 · nucleotide and EP), which all have different conformations. 相似文献
1. 1. Tightly bound ATP and ADP, found on the isolated mitochondrial ATPase, exchange only slowly at pH 8, but the exchange is increased as the pH is reduced. At pH 5.5, more than 60% of the bound nucleotide exchanges within 2.5 min.
2. 2. Preincubation of the isolated ATPase with ADP leads to about 50% inhibition of ATP hydrolysis when the enzyme is subsequently assayed in the absence of free ADP. This effect, which is reversed by preincubation with ATP, is absent on the membrane-bound ATPase. This inhibition seems to involve the replacement of tightly bound ATP by ADP.
3. 3. Using these two findings, the binding specificity of the tight nucleotide binding sites was determined. iso-Guanosine, 2′-deoxyadenosine and formycin nucleotides displaced ATP from the tight binding sites, while all other nucleotides tested did not. The specificities of the tight sites of the isolated and membrane-bound ATPase were similar, and higher than that of the hydrolytic site.
4. 4. The nucleotide specificities of ‘coupled processes’ nucleoside triphosphate-driven reversal of electron transfer, nucleoside triphosphate-32Pi exchange and phosphorylation were higher than that of the hydrolytic site of the ATPase and similar to that of the tight nucleotide binding sites.
5. 5. The different nucleotide specificities of uncoupled ATP hydrolysis and coupled processes can be explained even if both processes involve a single common site on the ATPase molecule. This model requires that energy can be ‘coupled’ only when it is released/utilised in the nucleotide binding steps of the mechanism.
6. 6. Adenosine β,γ-imidotriphosphate (AMP-PNP) is not a simple reversible inhibitor of the ATPase, since incubation requires preincubation and is not reversed when the compound is diluted out, or by addition of ATP. This compound inhibits the isolated and membrane-bound ATPase equally well. Its guanosine analogue does not act in this way.
7. 7. In submitochondrial particles, ADP inhibited uncoupled hydrolysis of ATP much more effectively than coupled hydrolysis, the latter being measured both directly (from ATP hydrolysis in the absence of uncoupler) or indirectly, by monitoring ATP-driven reduction of NAD+ by succinate.
8. 8. The effects of ADP and AMP-PNP were interpreted as providing evidence for two of the intermediates in the proposed scheme for coupled triphosphate hydrolysis.