A chemoenzymatic route to mannosamine derivatives bearing different N-acyl groups

The chemoenzymatic route to 2-deoxy-2-propionamido-D-mannose (1b), 2-butyramido-2-deoxy-D-mannose (2b) and 2-deoxy-2-phenylacetamido-D-mannose (3b) involved N-acylation of 2-amino-2-deoxy-D-glucose followed by alkaline C-2 epimerization and selective microbial removal of the epimers with gluco-configuration. The latter step employed whole cells of Rhodococcus equi A4 able to degrade 2-deoxy-2-propionamido-D-glucose (1a), 2-butyramido-2-deoxy-D-glucose (2a) and 2-deoxy-2-phenylacetamido-D-glucose (3a) but inactive towards the corresponding manno-isomers. The metabolism of the gluco-isomers probably involved phosphorylation and subsequent deacylation. 2-Acetamido-2-deoxy-6-O-phospho-D-glucose amidohydrolase [EC 3.5.1.25] but not 2-acetamido-2-deoxy-D-glucose amidohydrolase was detected in the cell extract, the former enzyme being partially purified (15.8-fold with an overall yield of 18.1% and a specific activity of 0.95 units mg-1 protein). According to SDS-PAGE electrophoresis, gel filtration and mass spectrometry, the enzyme was a monomer with an apparent molecular mass of approximately 42 kDa. The optimum temperature and pH of the enzyme were 60 degrees C and 8.0-9.0, respectively. 2-Acetamido-2-deoxy-6-O-phospho-D-glucose and 2-acetamido-2-deoxy-6-O-sulfo-D-glucose but not 2-acetamido-2-deoxy-1-O-phospho-D-glucose or 2-acetamido-2-deoxy-D-glucose were substrates of the enzyme. Its activity was slightly inhibited by the addition of 1 mM Al3+, Ca2+, Co2+, Cu2+, Mn2+ or Zn2+ and activated by 1 mM Mg2+. The concentrated enzyme is highly stable at 4 degrees C in the presence of 0.1 M ammonium sulfate.     

Cation and sequence effects on stability of intermolecular pyrimidine-purine-purine triplex.

A differential effect is found of various bivalent cations (Ba2+, Ca2+, Mg2+, Cd2+, Co2+, Mn2+, Ni2+, Zn2+ and Hg2+) on stability of intermolecular Py-Pu-Pu triplex with different sequence of base triads. Ca2+, Mg2+, Cd2+, Co2+, Mn2+, Ni2+ and Zn2+ do stabilize the d(C)n d(G)n d(G)n triplex whereas Ba2+ and Hg2+ do not. Ba2+, Ca2+, Mg2+ and Hg2+ destabilize the d(TC)n d(GA)n d(AG)n triplex whereas Cd2+, Co2+, Mn2+, Ni2+ and Zn2+ stabilize it. The complexes we observe are rather stable because they do not dissociate during time of gel electrophoresis in the co-migration experiments. Chemical probing experiments with dimethyl sulfate as a probe indicate that an arbitrary homopurine-homopyrimidine sequence forms triplex with corresponding purine oligonucleotide in the presence of Mn2+ or Zn2+, but not Mg2+. In the complex the purine oligonucleotide has antiparallel orientation with respect to the purine strand of the duplex. Specifically, we have shown the formation of the Py-Pu-Pu triplex in a fragment of human papilloma virus HPV-16 in the presence of Mn2+.     

The effect of divalent cations on bovine spermatozoal adenylate cyclase activity.

The effect of divalent cations on bovine sperm adenylate cyclase activity was studied. Mn2+, Co2+, Cd2+, Zn2+, Mg2+ and Ca2+ were found to satisfy the divalent cation requirement for catalysis of the bovine sperm adenylate cyclase. These divalent cations in excess of the amount necessary for the formation of the metal-ATP substrate complex were found to stimulate the enzyme activity to various degrees. The magnitude of stimulation at saturating concentrations of the divalent cations was strikingly greater with M2+ than with either Ca2+, Mg2+, Zn2+, Cd2+ or Co2+. The apparent Km was lowest for Zm2+ (0.1 - 0.2 mM) than for any of the other divalent cations tested (1.2 - 2.3 mM). The enzyme stimulation by Mn2+ was decreased by the simultaneous addition of Co2+, Cd2+, Ni2+ and particularly Zn2+ and Cu2+. The antagonism between Mn2+ and Cu2+ or Zn2+ appeared to have both competitive and non-competitive features. The inhibitory effect of Cu2+ on Mn2+-stimulated adenylate cyclase activity was prevented by 2,3-dimercaptopropanol, but not by dithiothreitol, L-ergothioneine, EDTA, EGTA or D-penicillamine. Ca2+ at concentrations of 1-5 mM was found to act synergistically with Mg2+, Zn2+, Co2+ and Mn2+ in stimulating sperm adenylate cyclase activity. The Ca2+ augmentation of the stimulatory effect of Zn2+, Co2+, Mg2+ and Mn2+ appeared to be specific.     

重金属对油菜种子萌发和胚根生长的影响

分析了Hg2 、Cd2 、Ni2 、Co2 、Zn2 5种重金属离子对油菜种子萌发和胚根伸长的影响,以及金属离子K 、Mg2 和Ca2 与重金属的交互作用。结果表明:(1)重金属对油菜种子萌发的抑制作用依次为Hg2 >Cd2 和Co2 >Ni2 >Zn2 ,而对胚根生长的毒害作用依次为Hg2 >Cd2 >Co2 >Ni2 >Zn2 。(2)萌发率为40%以上时,K 和Ca2 可以提高Ni2 、Zn2 和Co2 胁迫下油菜种子的萌发率,却进一步降低了Hg2 、Cd2 胁迫下种子的萌发;Mg2 可以提高Ni2 、Zn2 、Cd2 和Co2 胁迫下种子的萌发率,但对Hg2 毒害却没有缓解。(3)胚根伸长率达到60%以上时,K 和Mg2 增强了Ni2 、Hg2 、Cd2 和Co2 对胚根生长的抑制,而Ca2 则缓解了Zn2 、Ni2 和Co2 对胚根生长的抑制作用。研究结果对于重金属复合污染土壤中植物种子的萌发和定植具有理论和实践意义。     

Inhibition of photohemolysis induced by m-chloroperbenzoic acid by metal complexes with SOD-mimetic activity

Red blood cells (RBCs) are probably the most common target through the damaging action of reactive oxygen species on the cells. The photohemolysis activity of m-chloroperbenzoic acid (CPBA) was concentration- and exposure time-dependent. Twenty minutes photo exposure time and 200 μm of CPBA concentration were optimum to study the effect of generated superoxide (O_{2-) and hydroxyl (&bull•OH) radicals on RBCs. RBCs lysis photosensitized by CPBA was investigated in the presence of [(VL2O)(VL2H2O)]Cl6, [MnL2O]2Cl42H2O, [FeL2Cl2]Cl H2O, [CoL2Cl2]4H2O or [ZnL2Cl2]H2O respectively, where L is 2-methylaminopyridine, with SOD-mimetic activities with the aim of ascertaining their protective activity towards the photo induced cell damage. The decrease of photolytic activity caused by these complexes was concentration-dependent and the maximum percentage of protective activity was 75, 70, 68, 57 or 24% for [(VL2O)(VL2H2O)]Cl6, [MnL2O]2Cl4 2H2O, [FeL2Cl2]Cl H2O, [CoL2Cl2]4H2O or [ZnL2Cl2]H2O complex respectively, against the cell irradiated without addition of metal complexes. The comparison between the decrease of photolytic activity caused by these complexes and their SOD-mimetic activity of these metal complexes showed an appreciable correlation.}     

磷脂酶A2在诱导红豆杉细胞产生活性氧中的作用

对磷脂酶A2(PLA2)在真菌诱导中国红豆杉细胞产生活性氧中的作用进行研究,结果表明：PLA2非特异抑制剂可降低真菌诱导子诱导产生的H2O22通过钙离子螯合和PLA2特异抑制剂实验,表明参与H2O2产生的PLA2为胞质CaO^2 依赖型2对PLA2诱导H202产生的机理进行分析,发现亚油酸可缓解PLA2抑制剂对诱导的活性氧的抑制作用,而且亚油酸单独处理可导致H2O2的发生,其它的脂肪酸也具有类似诱导H2O2发生的作用.不同离子型的脂肪酸对H2O2产生的影响不同,阴离子型脂肪酸较非离子型脂肪酸更能促进活性氧的发生.这些结果表明,PLA2可能通过产生脂肪酸或其衍生物激活H2O2的产生酶系.     

Effects of polyamines and analogs on staphylococcal nuclease.

The promoting activity of polyamine analogs (IV approximately XV) on staphylococcal nuclease with DNA as the substrate was compared with that of natural polyamines (I APPROXIMATELY III): I. NH2(CH2)3NH(CH2)4NH(CH2)3NH2(spermine); II. NH2(CH2)3NH(CH2)3NH(CH2)3NH2(thermine); III. NH2(CH2)4NH2 (putrescine); IV. CN(CH2)2NH(CH2)4NH(CH2)2CN; V. HOOC(CH2)2NH(CH2)4NH(CH2)2COOH; VI. C2H5OOC(CH2)2NH(CH2)4NH(CH2)2COOC2H5; VII. HO(CH2)3NH(CH2)4HH(CH2)3OH; VIII. CH3COHH(CH2)3NH(CH2)4NH(CH2)3NHCOCH3; IX. C2H5NH(CH2)3NH(CH2)4NH(CH2)3NHC2H5; X. NH2(CH2)3S(CH2)4S(CH2)3NH2; XI. NH2(CH2)3NH(CH2)2O(CH2)2NH(CH2)3NH2; XII. NH2(CH2)3NCH3(CH2)4HCH3(CH2)3NH2; XIII. CN(CH2)2NCH3(CH2)4NCH3(CH2)2CN; XIV. (CH3)2N(CH2)3NCH3(CH2)4NCH3(CH2)3N(CH3)2; XV. NH2(CH2)2O(CH2)2NH2 Replacement of the terminal groups by CN, COOH, COOEt, NHAc, NHEt, or N(CH3)2 remarkably decreased the activity. The compound VII with terminal hydroxyl groups had a lower promoting activity at low concentrations, but revealed higher activity at higher concentrations and, in contrast to spermine, no inhibition at all even at very high concentrations. Replacement of both internal amino groups by sulfur or NCH3 decreased the activity. The introduction of an ether bond into the internal methylene groups (compound XI) highly decreased the activity. Based upon these findings the possible relationship between structure and activity is discussed.     

铜、铅、镉、锌、汞和银离子复合污染对水螅的急性毒性效应

以水螅(Hydrasp)为例,通过单因子静态急性毒性试验方法和等毒性溶液法,分别研究Hg2 、Cu2 、Cd2 、Ag 、Zn2 和Pb2 对其单一和复合毒性效应。单一实验结果表明,它们对水螅毒性大小顺序为Hg2 >Cu2 >Cd2 >Ag >Zn2 >Pb2 。复合毒性实验表明,Zn2 与Cu2 、Hg2 、Pb2 、Ag ;Pb2 与Cu2 ;Hg2 与Ag ;Pb2 与Ag 这些组合对水螅联合急性毒性总体上表现出拮抗作用,Cd2 与Cu2 、Hg2 、Pb2 、Ag 组合总体上则是协同作用,Zn2 与Cd2 、Pb2与Hg2 、Cu2 与Hg2 ,Ag 在不同的浓度水平组合下明显表现出不同的毒性效应。     

Transition metal complexes of diclofenac with potentially interesting anti-inflammatory activity

An overview is given of the results of metal ion-diclofenac interactions. Several complexes have been synthesized at the University of Ioannina. Binuclear complexes, [Cu(L)2(H2O)]2 x 2H2O and [CuL2(S)]2 where S is H2O, EtOH, DMSO, (CH3)2CO and DMF, and mononuclear complexes, [MnL2(H2O)], [FeL2(H2O)2], [CoL2(H2O)2] x 0.5H2O, [CoL2(H2O)], [NiL2(H2O)2] x 2H2O, [NiL2] and [PdL2] x 2H2O, have been characterized by spectroscopy, X-ray crystallography and electrochemical studies. The catalytic activity of these complexes was correlated to the reduction potential. Some of the complexes of diclofenac exhibit very promising anti-inflammatory activity and act as antioxidant compounds, a property that is absent from diclofenac.     

Metabolism of [3H]2-hydroxyestradiol by cultured porcine granulosa cells: evidence for the presence of a catechol-O-methyltransferase pathway and a direct stimulatory effect of 2-methoxyestradiol on progesterone production

Porcine granulosa cells synthesize and respond to catecholestrogens, but the stimulatory potency of catecholestrogens on progesterone production is much less than that of estradiol (E2). Therefore, to determine if metabolism of catecholestrogens by granulosa cells could account for the reduced potency of 2-hydroxyestradiol (2-OH-E2) observed in vitro, porcine granulosa cells were cultured with [3H]2-OH-E2 and medium collected at 0, 0.5, 1, 2, 4, 6, or 12 h in the presence or absence of 1 microgram/ml 2-OH-E2, 0.5 mM L-ascorbate or 10 microM U-0521 (a specific catechol-O-methyltransferase inhibitor). Metabolism of [3H]2-OH-E2 was very rapid with only 16% of the original [3H]2-OH-E2 remaining after 4 h exposure to cells. The main metabolite comigrated with 2-methoxyestradiol (2-MeO-E2) on thin-layer chromatography. Although appreciable degradation of [3H]2-OH-E2 occurred with time in the absence of cells, formation of the O-methyl derivative was minimal. Rather, formation of polar metabolites occurred in the absence of cells. Ascorbate dramatically reduced this noncellular degradation. Ascorbate added to cell cultures had no effect on the rate of formation of O-methyl products but slowed the formation of polar compounds as well as the overall rate of degradation of [3H]2-OH-E2 by nearly 2-fold. U-0521 completely blocked the formation of O-methyl products, slowed the overall rate of degradation of [3H]2-OH-E2 by half and resulted in an increase in polar metabolites. The effects of U-0521 and ascorbate on 2-OH-E2-stimulated progesterone production in vitro was also examined. Ascorbate (0.5 mM) enhanced the effect of 2-OH-E2 (but not E2) on progesterone production by 2-fold (p less than 0.05). The addition of 10 microM U-0521 in the presence of 0.5 mM ascorbate had no effect on 1 microgram/ml 2-OH-E2-stimulated progesterone production, but it increased (p less than 0.05) the response to 4 micrograms/ml 2-OH-E2. The effects of 2-MeO-E2, 2-OH-E2, and E2 on progesterone production by cultured granulosa cells were then compared. The ED50 of E2 was 6- to 8-fold lower than that of 2-OH-E2 and 2-MeO-E2, whereas the ED50 of 2-OH-E2 was 15% lower than that of 2-MeO-E2. In the presence of ascorbate (0.5 mM), the maximal effect of E2 and 2-OH-E2 was approximately equal, whereas 2-OH-E2 was nearly 2-fold more efficacious than 2-MeO-E2.(ABSTRACT TRUNCATED AT 400 WORDS)     

Implications on zinc binding to S100A2

Human S100A2 is an EF-hand calcium-binding S100 protein that is localized mainly in the nucleus and functions as tumor suppressor. In addition to Ca2+ S100A2 binds Zn2+ with a high affinity. Studies have been carried out to investigate whether Zn2+ acts as a regulatory ion for S100A2, as in the case of Ca2+. Using the method of competition with the Zn2+ chelator 4-(2-pyridylazo)-resorcinol, an apparent Kd of 25 nM has been determined for Zn2+ binding to S100A2. The affinity lies close to the range of intracellular free Zn2+ concentrations, suggesting that S100A2 is able to bind Zn2+ in the nucleus. Two Zn2+-binding sites have been identified using site directed mutagenesis and several spectroscopic techniques with Cd2+ and Co2+ as probes. In site 1 Zn2+ is bound by Cys21 and most likely by His 17. The binding of Zn2+ in site 2 induces the formation of a tetramer, whereby the Zn(2+) is coordinated by Cys2 from each subunit. Remarkably, only binding of Zn2+ to site 2 substantially weakens the affinity of S100A2 for Ca2+. Analysis of the individual Ca2+-binding constants revealed that the Ca2+ affinity of one EF-hand is decreased about 3-fold, whereas the other EF-hand exhibits a 300-fold decrease in affinity. These findings imply that S100A2 is regulated by both Zn2+ and Ca2+, and suggest that Zn2+ might deactivate S100A2 by inhibiting response to intracellular Ca2+ signals.     

The vanadium nitrogenase of Azotobacter chroococcum. Reduction of acetylene and ethylene to ethane.

1. The vanadium (V-) nitrogenase of Azobacter chroococcum transfers up to 7.4% of the electrons used in acetylene (C2H2) reduction for the formation of ethane (C2H6). The apparent Km for C2H2 (6 kPa) is the same for either ethylene (C2H4) or ethane (C2H6) formation and much higher than the reported Km values for C2H2 reduction to C2H4 by molybdenum (Mo-) nitrogenases. Reduction of C2H2 in 2H2O yields predominantly [cis-2H2]ethylene. 2. The ratio of electron flux yielding C2H6 to that yielding C2H4 (the C2H6/C2H4 ratio) is increased by raising the ratio of Fe protein to VFe protein and by increasing the assay temperature up to at least 40 degrees C. pH values above 7.5 decrease the C2H6/C2H4 ratio. 3. C2H4 and C2H6 formation from C2H2 by V-nitrogenase are not inhibited by H2. CO inhibits both processes much less strongly than it inhibits C2H4 formation from C2H2 with Mo-nitrogenase. 4. Although V-nitrogenase also catalyses the slow CO-sensitive reduction of C2H4 to C2H6, free C2H4 is not an intermediate in C2H6 formation from C2H2. 5. Propyne (CH3C identical to CH) is not reduced by the V-nitrogenase. 6. Some implications of these results for the mechanism of C2H6 formation by the V-nitrogenase are discussed.     

Evaluation of platelet calcium ion mobilization by the use of various divalent ions.

Divalent ion mobilization in human platelets was evaluated with Fura-2 fluorescence changes induced by Ca2+, Sr2+, Ba2+ and Mn2+. Extracellular Ca2+, Sr2+ and Ba2+ all entered thrombin-stimulated platelets. These divalent ions were also able to refill the intracellular Ca2+ storage sites which had been depleted of Ca2+ by ionomycin treatment, and were released from the storage sites upon thrombin stimulation. However, only the refilling of the storage sites with Ca2+ and Sr2+, but not with Ba2+, were capable of suppressing the opening state of Ca2+ channels assessed with Mn2+ influx. Efflux of intracellularly accumulated divalent ions was observed with Ca2+ and Sr2+ but not with Ba2+. These findings indicate that there are subtle differences in the Ca(2+)-binding domains of the various systems involved in Ca2+ mobilization in platelets, some of which discriminate Ba2+ while accepting Sr2+.     

MEK kinase 2 binds and activates protein kinase C-related kinase 2. Bifurcation of kinase regulatory pathways at the level of an MAPK kinase kinase

MEK kinase 2 (MEKK2) is a 70-kDa protein serine/threonine kinase that has been shown to function as a mitogen-activated protein kinase (MAPK) kinase kinase. MEKK2 has its kinase domain in the COOH-terminal moiety of the protein. The NH(2)-terminal moiety of MEKK2 has no signature motif that would suggest a defined regulatory function. Yeast two-hybrid screening was performed to identify proteins that bind MEKK2. Protein kinase C-related kinase 2 (PRK2) was found to bind MEKK2; PRK2 has been previously shown to bind RhoA and the Src homology 3 domain of Nck. PRK2 did not bind MEKK3, which is closely related to MEKK2. The MEKK2 binding site maps to amino acids 637-660 in PRK2, which is distinct from the binding sites for RhoA and Nck. This sequence is divergent in the closely related kinase PRK1, which did not bind MEKK2. In cells, MEKK2 and PRK2 are co-immunoprecipitated and PRK2 is activated by MEKK2. Similarly, purified recombinant MEKK2 activated PRK2 in vitro. MEKK2 activation of PRK2 is independent of MEKK2 regulation of the c-Jun NH(2)-terminal kinase pathway. MEKK2 activation of PRK2 results in a bifurcation of signaling for the dual control of MAPK pathways and PRK2 regulated responses.     

Delta12-prostaglandin D2 is a potent and selective CRTH2 receptor agonist and causes activation of human eosinophils and Th2 lymphocytes

Prostaglandin D2 (PGD2) is a lipid mediator produced by mast cells, macrophages and Th2 lymphocytes and has been detected in high concentrations in the airways of asthmatic patients. There are two receptors for PGD2, namely the D prostanoid (DP) receptor and the chemoattractant receptor-homologous molecule expressed on Th2 cells (CRTH2). The proinflammatory effects of PGD2 leading to recruitment of eosinophils and Th2 lymphocytes into inflamed tissues is thought to be predominantly due to action on CRTH2. Several PGD2 metabolites have been described as potent and selective agonists for CRTH2. In this study we have characterized the activity of delta12-PGD2, a product of PGD2 isomerization by albumin. Delta12-PGD2 induced calcium mobilization in CHO cells expressing human CRTH2 receptor, with efficacy and potency similar to those of PGD2. These effects were blocked by the TP/CRTH2 antagonist ramatroban. delta12-PGD2 bound to CRTH2 receptor with a pKi of 7.63, and a 55-fold selectivity for CRTH2 compared to DP. In Th2 lymphocytes, delta12-PGD2 induced calcium mobilization with high potency and an efficacy similar to that of PGD2. delta12-PGD2 also caused activation of eosinophils as measured by shape change. Taken together, these results show that delta12-PGD2 is a potent and selective agonist for CRTH2 receptor and can cause activation of eosinophils and Th2 lymphocytes. These data also confirm the selective effect of other PGD2 metabolites on CRTH2 and illustrate how the metabolism of PGD2 may influence the pattern of leukocyte infiltration at sites of allergic inflammation.     

Dechlorination of chlorophenols found in pulp bleach plant E-1 effluents by advanced oxidation processes

Studies were conducted on the response of 2,4,6-trichlorophenol (1), 2,3,4,5-tetrachloro-phenol (2) and 4,5-dichloroguaiacol (3) toward advanced oxidation processes, such as UV-, O2/UV-, H2O2/UV-, O3/UV- and O3-H2O2/UV-photolyses with irradiation of 254 nm photons. The compounds 1-3 are among the chlorophenols found in the Kraft-pulp bleach plant E-1 effluents. The studies were extended to treatment of these compounds with ozonation and O3-H2O2 oxidation systems in alkaline aqueous solution. Except for the O2/UV-photolysis of 1 and H2O2/UV-photolysis of 2, the dechlorination of 1-3 by O2/UV- and H2O2/UV-potolyses were less effective than the corresponding N2UV-potolysis of 1-3. Guaiacol-type chlorophenols were more readily able to undergo dechlorination than non-guaiacol type chlorophenols by N2/UV-, O2/UV- and H2O2/UV-potolyses. In addition, the efficiency for the dechlorination of 1-3 by N2/UV-, O2/UV- and H2O2/UV-potolyses appeared to be dependent upon the inductive and resonance effects of substituents as well as number and position of chlorine substituent in the aromatic ring of the compounds. The dechlorination of 2 by treatment with O3 alone is slightly more effective than the corresponding the O3/UV-photlysis, whereas the dechlorination of 2 by treatment with the combination of O3 and H2O2 was slightly less effective than the corresponding O3-H2O2/UV-photolysis. In contrast, the dechlorination of 3 on treatment with O3 alone was slightly less effective than the corresponding the O3/UV-photolysis, whereas the dechlorination of 3 on treatment with the combination of O3 and H2O2 was slightly more effective than the corresponding the O3-H2O2/UV-photolysis. In the dechlorination of 2 and 3, chemical species derived from ozone and hydrogen peroxide in alkaline solution were dominant reactions in the O3/UV- and O3-H2O2/UV-photolysis systems as in the O3 and O3-H2O2 oxidation systems. Possible dechlorination mechanisms involved were discussed on the basis of kinetic data.     

The effect of Mg2+ on hepatic microsomal Ca2+ and Sr2+ transport

The ATP-dependent uptake of Ca2+ by rat liver microsomal fraction is dependent upon Mg2+. Studies of the Mg2+ requirement of the underlying microsomal Ca2+-ATPase have been hampered by the presence of a large basal Mg2+-ATPase activity. We have examined the effect of various Mg2+ concentrations on Mg2+-ATPase activity, Ca2+ uptake, Ca2+-ATPase activity and microsomal phosphoprotein formation. Both Mg2+-ATPase activity and Ca2+ uptake were markedly stimulated by increasing Mg2+ concentration. However, the Ca2+-ATPase activity, measured concomitantly with Ca2+ uptake, was apparently unaffected by changes in the Mg2+ concentration. In order to examine the apparent paradox of Mg2+ stimulation of Ca2+ uptake but not of Ca2+-ATPase activity, we examined the formation of the Ca2+-ATPase phosphoenzyme intermediate and formation of a Mg2+-dependent phosphoprotein, which we have proposed to be an attribute of the Mg2+-ATPase activity. We found that Ca2+ apparently inhibited formation of the Mg2+-dependent phosphoprotein both in the absence and presence of exogenous Mg2+. This suggests that Ca2+ may inhibit (at least partially) the Mg2+-ATPase activity. However, inclusion of the Ca2+ inhibition of Mg2+-ATPase activity in the calculation of Ca2+-ATPase activity reveals that this effect is insufficient to totally account for the stimulation of Ca2+ uptake by Mg2+. This suggests that Mg2+, in addition to stimulation of Ca2+-ATPase activity, may have a direct stimulatory effect on Ca2+ uptake in an as yet undefined fashion. In an effort to further examine the effect of Mg2+ on the microsomal Ca2+ transport system of rat liver, the interaction of this system with Sr2+ was examined. Sr2+ was sequestered into an A23187-releasable space in an ATP-dependent manner by rat liver microsomal fraction. The uptake of Sr2+ was similar to that of Ca2+ in terms of both rate and extent. A Sr2+-dependent ATPase activity was associated with the Sr2+ uptake. Sr2+ promoted formation of a phosphoprotein which was hydroxylamine-labile and base-labile. This phosphoprotein was indistinguishable from the Ca2+-dependent ATPase phosphoenzyme intermediate. Sr2+ uptake was markedly stimulated by exogenous Mg2+, but the Sr2+-dependent ATPase activity was unaffected by increasing Mg2+ concentrations. Sr2+ uptake and Sr2+-dependent ATPase activity were concomitantly inhibited by sodium vanadate. In contrast to Ca2+, Sr2+ had no effect on Mg2+-dependent phosphoprotein formation. Taken together, these data indicate that Mg2+ stimulated Ca2+ and Sr2+ transport by increasing the Ca2+ (Sr2+)/ATP ratio.(ABSTRACT TRUNCATED AT 400 WORDS)     

ATP binding site mutagenesis reveals different subunit stoichiometry of functional P2X2/3 and P2X2/6 receptors

The aim of the present experiments was to clarify the subunit stoichiometry of P2X2/3 and P2X2/6 receptors, where the same subunit (P2X2) forms a receptor with two different partners (P2X3 or P2X6). For this purpose, four non-functional Ala mutants of the P2X2, P2X3, and P2X6 subunits were generated by replacing single, homologous amino acids particularly important for agonist binding. Co-expression of these mutants in HEK293 cells to yield the P2X2 WT/P2X3 mutant or P2X2 mutant/P2X3 WT receptors resulted in a selective blockade of agonist responses in the former combination only. In contrast, of the P2X2 WT/P2X6 mutant and P2X2 mutant/P2X6 WT receptors, only the latter combination failed to respond to agonists. The effects of α,β-methylene-ATP and 2-methylthio-ATP were determined by measuring transmembrane currents by the patch clamp technique and intracellular Ca(2+) transients by the Ca(2+)-imaging method. Protein labeling, purification, and PAGE confirmed the assembly and surface trafficking of the investigated WT and WT/mutant combinations in Xenopus laevis oocytes. In conclusion, both electrophysiological and biochemical investigations uniformly indicate that one subunit of P2X2 and two subunits of P2X3 form P2X2/3 heteromeric receptors, whereas two subunits of P2X2 and one subunit of P2X6 constitute P2X2/6 receptors. Further, it was shown that already two binding sites of the three possible ones are sufficient to allow these receptors to react with their agonists.