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1.
Tartrate-resistant acid phosphatase (TR-AcPh) from the ameba Amoeba proteus is represented by 3 bands (electromorphs) revealed after disk-electrophoresis in PAAG, using 2-naphthylphosphate as substrate. The presence of 50 mmol/l MgCl2 or CaCl2 in the incubation mixture increases activities of all electromorphs of TR-AcPh, while of ZnCl2, of two of them. The activity of the TR-AcPh electromorphs also rose after the 30-min incubation of the gels in MgCl2, CaCl2 or ZnCl2 (10 and 100 mM) before gel staining. However, 1 M ZnCl2, unlike 1 M CaCl2 or 1 M MgCl2, partly inactivated two out of three TR-AcPh electromorphs. The TR-AcPh electromorphs were inhibited by 1,10-phenanthroline (1,10-Ph), EDTA, and EGTA (all at a concentration of 5 mM) faster than by H2O2 (10 mM). The inactivation of the TR-AcPh electromorphs by the chelating agents did not depend (EGTA) or nearly did not depend (EDTA, 1,10-Ph) on their concentration (0.05, 0.5, and 5 mM). Out of 5 tested ions (Mg2+, Ca2+, Fe2+, Fe3+, and Zn2+), only Zn ions reactivated the TR-AcPh electromorphs inactivated by 1,10-Ph, EDTA or EGTA. The TR-AcPh electromorphs were reactivated worse after inactivation by EGTA than by EDTA or 1,10-Ph. It is suggested that the active site of TR-AcPh contains the zinc ion essential for catalytic activity of this enzyme, i.e., TR-AcPh of A. proteus is a metallophosphatase performing the phosphomonoesterase activity in acidic medium.  相似文献   

2.
Andreas Weith 《Chromosoma》1985,91(3-4):287-296
The fine structure of constitutive heterochromatin and euchromatin was compared in electron microscope whole-mount preparations of Tenebrio molitor (Insecta, Coleoptera) spermatocyte nuclei. Tenebrio molitor pachytene chromosomes display extended segments of centromeric heterochromatin and thus are especially suitable for this purpose. When nuclei were incubated in solutions containing different concentrations of NaCl or of MgCl2, two levels of chromatin fine structures were observed in the euchromatic segments: nucleosome fibers (0.1 mM–20 mM NaCl) and supranucleosomal fibers with 28 nm in diameter (40 mM–100 mM NaCl, 0.2 mM–1.0 mM MgCl2). The fine structure in the heterochromatic segments was the same as that in the euchromatic segments in all NaCl concentrations and in MgCl2 concentrations up to 0.4 mM. In higher MgCl2 concentrations the heterochromatin remained more compact than the euchromatin and consisted of 37-nm-thick fibers in 0.6 mM MgCl2 and of 65-nm-thick fibers in 1.0 mM MgCl2. After the 37-nm and the 65-nm fibers had been dispersed in Mg2+-free solutions they could be recondensed by incubation in 0.6 mM and 1.0 mM MgCl2, respectively. It is concluded that a Mg2+-sensitive component of the heterochromatin is responsible for the folding of the nucleosome chain to heterochromatin-specific supranucleosomal structures.  相似文献   

3.
The (Na+ + K+)-dependent ATPase exhibits substrate sites with both high affinity (K m near 1 µM) and low affinity (K m near 0.1 mM) for ATP. To permit the study of nucleotide binding to the high-affinity substrate sites of a canine kidney enzyme preparation in the presence as well as absence of MgCl2, the nonhydrolyzable - imido analog of ATP, AMP-PNP, was used in experiments performed at 0–4°C by a centrifugation technique. By this method theK D for AMP-PNP was 4.2 µM in the absence of MgCl2. Adding 50 µM MgCl2, however, decreased theK D to 2.2 µM; by contrast, higher concentrations of MgCl2 increased theK D until, with 2 mM MgCl2, theK D was 6 µM. The half-maximal effect of MgCl2 on increasing theK D occurred at approximately 1 mM. This biphasic effect of MgCl2 is interpreted as Mg2+ in low concentrations favoring AMP-PNP binding through formation at the high-affinity substrate sites of a ternary enzyme-AMP-PNP-Mg complex; inhibition of nucleotide binding at higher MgCl2 concentrations would represent Mg2+ acting through the low-affinity substrate sites. NaCl in the absence of MgCl2 increased AMP-PNP binding, with a half-maximal effect near 0.3 mM; in the presence of MgCl2, however, NaCl increased theK D for AMP-PNP. KCl decreased AMP-PNP binding in the presence or absence of MgCl2, but the simultaneous presence of a molar excess of NaCl abolished (or masked) the effect of KCl. ADP and ATP acted as competitors to the binding of AMP-PNP, although a substrate for the K+-dependent phosphatase reaction also catalyzed by this enzyme,p-nitrophenyl phosphate, did not. This lack of competition is consistent with formulations in which the phosphatase reaction is catalyzed at the low-affinity substrate sites.  相似文献   

4.
Summary The effect of adenosine triphosphate (ATP), magnesium chloride (MgCl2) and phospholipids on the calcium-binding activity and crystal formation within the decalcified shell-repair membrane of the snail, Helix pomatia, was studied in vitro. The application of ATP produced a characteristic dual effect on calcification: (1) It strongly inhibited the formation of inorganic calcium carbonate (CaCO3) crystals. (2) It stimulated the development of organic crystalline bodies and induced deposition of amorphous calcium carbonate. The demineralized shell-repair membranes became white and rigid after incubation for 7 days in the medium containing 1.0mM ATP. The inhibitory effect of Mg2+ on CaCO3 crystal formation was diminished by reduction of the concentration of MgCl2 in the incubation solution. Thus, after incubation for only 24h, 1.0mM MgCl2 promoted the formation of birefringent CaCO3 crystals within the repair membranes. The principal effect of phospholipids on the demineralized shell-repair membrane was stimulatory, but after application of phospholipids to the medium, the formation of crystals proceeded slowly. The very large, composite crystals that were formed within the repair membranes showed strong birefringence. In all cases the development of the crystals and the organic crystalline bodies occurred in close vicinity to the amoebocytes. The role of ATP, MgCl2 and phospholipids in the recalcification of shell-repair membrane is discussed.The author wishes to thank Mrs. E. Hellmén for valuable technical assistance  相似文献   

5.
The activities of DNA polymerization and DNA ligation in extract of Chinese hamster ovary cells were both stimulated by MgCl2. DNA polymerization was stimulated by MgCl2 above 0.25 mM, whereas, MgCl2 above 2 mM was required to stimulate DNA ligation. The activity of DNA polymerization maintained a plateau at MgCl2 1–12 mM, whereas DNA ligation reached a maximal activity at MgCl2 6 mM and decreased thereafter. NiCl2 0.1-0.2 mM also had a stimulatory effect on DNA polymerization, but was much less potent than MgCl2. However, nickel ion (Ni2+) had no detectable stimulating effect on the activity of DNA ligation. In the presence of MgCl2, the activities of DNA polymerization and DNA ligation decreased with increasing concentration of NiCl2. Ni2+ inhibition of DNA polymerization was reduced by increasing the concentration of MgCl2, but increasing the concentration of MgCl2 did not reduce Ni2+ inhibition of DNA ligation. Preincubating cell extract with MgCl2 decreased the Ni2+ inhibition of DNA polymerization but not DNA ligation. These results suggest that Ni2+ may compete with magnesium ion (Mg2+) to reduce DNA polymerization, but this mechanism seems not applicable to Ni2+ inhibition of DNA ligation.  相似文献   

6.
Na+-ATPase activity of a dog kidney (Na+ + K+)-ATPase enzyme preparation was inhibited by a high concentration of NaCl (100 mM) in the presence of 30 μM ATP and 50 μM MgCl2, but stimulated by 100 mM NaCl in the presence of 30 μM ATP and 3 mM MgCl2. The K0.5 for the effect of MgCl2 was near 0.5 mM. Treatment of the enzyme with the organic mercurial thimerosal had little effect on Na+-ATPase activity with 10 mM NaCl but lessened inhibition by 100 mM NaCl in the presence of 50 μM MgCl2. Similar thimerosal treatment reduced (Na+ + K+)-ATPase activity by half but did not appreciably affect the K0.5 for activation by either Na+ or K+, although it reduced inhibition by high Na+ concentrations. These data are interpreted in terms of two classes of extracellularly-available low-affinity sites for Na+: Na+-discharge sites at which Na+-binding can drive E2-P back to E1-P, thereby inhibiting Na+-ATPase activity, and sites activating E2-P hydrolysis and thereby stimulating Na+-ATPase activity, corresponding to the K+-acceptance sites. Since these two classes of sites cannot be identical, the data favor co-existing Na+-discharge and K+-acceptance sites. Mg2+ may stimulate Na+-ATPase activity by favoring E2-P over E1-P, through occupying intracellular sites distinct from the phosphorylation site or Na+-acceptance sites, perhaps at a coexisting low-affinity substrate site. Among other effects, thimerosal treatment appears to stimulate the Na+-ATPase reaction and lessen Na+-inhibition of the (Na+ + K+)-ATPase reaction by increasing the efficacy of Na+ in activating E2-P hydrolysis.  相似文献   

7.
Arrhenius plots of rabbit skeletal muscle sarcolemmal Na+,K+-ATPase contain no temperature breaks. The apparent activation energy (22.8 kcal/mole in the presence of 1 mM MgCl2 or 15.9 kcal/mole in the presence of 3 mM MgCl2) does not depend on the Na+/K+ ratio in the incubation medium, but decreases in the presence of anserine (instead of Tris buffer).  相似文献   

8.
Trehalose-6-phosphate synthase, catalyzing the reaction between UDP-glucose and glucose 6-phosphate and forming trehalose 6-phosphate, was isolated and partially purified (30-fold) from the phototrophic, haloalkaliphilic bacteriumEctothiorhodospira halochloris. The activity is stabilized by 20mM MgCl2, 50mM NaCe and 2M glycine betaine. The molecular weight was 63000.The enriched enzyme had a MgCl2 optimum at 3–6mM, a pH optimum at 7.5 (in Tris-HCl buffer) and a temperature optimum at 50°C. The Km-values were 1.5×10–3M for UDP-glucose and 2×10–3M for glucose 6-phosphate. The enzyme showed a salinity dependence with optimal concentrations between 100 and 300mM salt. Higher concentrations of salt resulted in a decrease in activity. In the presence of inhibitory salt concentrations the compatible solute glycine betaine had a protective effect with a maximum between 0.5 and 2.0M.  相似文献   

9.
In the presence of 1.0 mM ATP and MgCl2, the specific viscosity of suspensions of human erythrocyte ghosts decreases 35% in 20 minutes at 22°C. The changes in viscosity are a sensitive index of Mg-ATP dependent shape changes in these membranes. Low concentrations of Ca2+ (1 to 5 μM) inhibit Mg-ATP dependent viscosity changes. If ghosts were preincubated with 1 mM Mg-ATP and 20 μM A23187 to produce a maximal decrease in viscosity, addition of 10 μM Ca2+ to the preincubated ghosts increased the viscosity to levels observed in ghosts preincubated without ATP. Ca2+ (1 to 5 μM) also inhibited Mg2+ dependent phosphorylation 30% and stimulated dephosphorylation 25% in ghost membranes. These effects of Ca2+ on viscosity and phosphorylation may be due to a membrane bound Ca2+ phosphatase activity which dephosphorylates membranes phosphorylated by a Mg2+ dependent kinase activity.  相似文献   

10.
Bovine chromaffin secretory granules were purified by isopycnic Metrizamide gradient centrifugation and their Ca2+ sequestration pathways were characterized. The rate of Ca2+ sequestration at 37°C was first order, with a maximal uptake of 26.9 ±0.46 (mean ± S.D., n = 3) nmol Ca2+/mg protein and a first order rate constant (k) of 0.046 ± 0.002 min–1. At 4°C the rate of uptake was substantially attenuated, with only 2.47 ± 0.2 (mean ± S.D, n = 3) nmol Ca2+/mg protein sequestered in 60 min. Ca2+ sequestration was 93% inhibited by 180 mM NaCl [I50% of 78.7 ± 9.3 mM NaCl (mean ± S.D., n = 11)] but only slightly inhibited by KCl or MgCl2. Ca 2+ sequestration was not stimulated by incubation with MgATP but was inhibited by 57% after incubation with 30 M monensin. Ca 2+ sequestration was dependent on extravesicular Ca 2+ with half-maximal sequestration at pCa2+ 6.81 ± 0.028 (mean ± S.D., n = 3). Sequestered Ca2+ could be exchanged with external 45Ca2+, the exchange rate was first order (k of 0.042 ± 0.004: mean ± S.D., n = 3) and saturated at 27.7 ± 1.1 nmol Ca2+/mg (mean ± S.D., n = 3). The Ca2+/Ca2+ exchange system was totally inhibited by NaCl or KCl but only slightly by MgCl2. About 75% of sequestered 45Ca2+ could be released by incubation with NaCl, but only 8% was released by incubation with KCI. Half-maximal release of sequestered 45Ca2+ required 69.3 ± 12.2 mM NaCl (mean ± S.D., n = 3). The Na+-induced release of sequestered 45Ca2+ was rapid, t0.5 of 2.80 ± 0.63 min (mean ± S.D., n = 3) and inhibited at 4°C. The concurrent incubation of chromaffin granules with 45Ca2+ and either annexin proteins V or VI resulted in attenuated uptake of 45Ca2+. These results suggest that Ca2+ uptake in adrenal chromaffin granules is regulated by Na+ and Ca2+ gradients and also possibly by annexins V and VI.Abbreviations EGTA ethylene glycol bis (-aminoethyl ether)-N,-N,N,N-tetraacetic acid - SDS Sodium dodecyl sulphate - PAGE Polyacrylamide gel electrophoresis - BSA bovine serum albumin - AI Annexin I - AIIt Annexin II tetramer - AIII Annexin III - AIV Annexin IV - AV Annexin V - AVI Annexin VI - k first order rate constant - AT total extent of Ca2+ uptake (nmol) - BufferA 300 mM sucrose, 10 mM potassium phosphate (pH 7.0), 5 mM EGTA - Buffer B 300 mM sucrose, 10 mM potassium phosphate (pH 7.0) and 1 mM EGTA - Buffer C 300 mM sucrose, 10 mM potassium phosphate (pH 7.0) - Buffer D 300 mM sucrose, 10 mM potassium phosphate (pH 7.0), 0.5 mM EGTA and 0.65 MM CaCl2 - Buffer E 300 mM sucrose, 10 mM potassium phosphate (pH 7.0), 0.25 mM EGTA and 0.325 mM CaCl2  相似文献   

11.
The effects of sodium, lithium, and magnesium on the in vitro binding properties of the D1 antagonist [3H]SCH23390 were examined with membrane preparations from rat neostriatum (CPU; caudate-putamen) and cerebral cortex (CTX). The saturation binding isotherms for both tissues performed in the presence of 120 mM of either Na+ or Li+ revealed an increase in the affinity, as compared to that observed when the incubation buffer was composed of Tris-Cl 50 mM with MgCl2 1 mM alone. For the CPU there were no changes in the maximum binding capacity (B max) in the different buffers used. In the case of the CTX, there was a loss of [3H]SCH23390 binding sites when either Na+ or Li+ 120 mM were added to the incubations, suggesting a lack of selectivity of this ligand in the absence of group IA cations. The agonist state of the [3H]SCH23390 binding site was studied in competition experiments with dopamine. The highest agonist affinity was obtained in 50 mM Tris-Cl buffer with 1 mM MgCl2 while the addition of 120 mM of either Na+ or Li+ caused a 3- to 5-fold decrease in the potency of dopamine to compete with specific [3H]SCH23390 binding in both CPU and CTX. The presence of magnesium was essential for the competition experiments; i.e.: a concentration of 1 mM MgCl2 was optimum to obtain dopamine antagonism of ligand binding, while increasing Mg2+ to 2 or 5 mM did not appear to further improve the inhibitions. The results support both agonist and antagonist affinity shifts for the dopamine D1 receptor labeled with [3H]SCH23390. Receptor affinity studies should take into account that pharmacological specificity may vary with the incubation buffer utilized, especially when comparing binding data from different laboratories performed under varying ionic conditions.  相似文献   

12.
(1) Calmodulin-depleted red cell membranes catalyse a Ca2+, Mg2+-dependent ATP-[3H]ADP exchange at 37° C. The Ca2+, Mg2+-dependent exchange, measured at 20 μM CaCl2, 1.5 mM MgCl2, 1.5 mM ADP and 1.5 mM ATP, is comparable to the (Ca2+ + Mg2+)-ATPase activity, between 0.3 and 0.8 mmol/litre original cells per h. (2) EDTA-washed membranes present a Ca2+-dependent ATP-ADP exchange whose rate is not more than 7% of that found in a Mg2+-containing medium, while their Ca2+-dependent ATPase is essentially zero. Addition of 1.5 mM MgCl2 to the medium restores both activities to the levels found with membranes not treated with EDTA. (3) Calmodulin (16 μg/ml) produces an eight-fold stimulation of the Ca2+-dependent ATP-ADP exchange, slightly less than it stimulates the Ca2+-dependent ATP hydrolysis. The effect of 1.5 mM MgCl2 on the exchange is greater in the presence than in the absence of calmodulin. (4) It is proposed that the reversal of the initial phosphorylation of the Ca2+ pump, occurring at a fast rate at 37° C, involves a conformational change in the phosphoenzyme. Thus, it would be an ADP-liganded phosphoenzyme of the form EP(ADP) that would experience the fast conformational transition at 37° C. The great difficulty in producing an overall reversal of the Ca2+ pump should then be due to one or more reaction steps later than and including Ca2+ release and dephosphorylation.  相似文献   

13.
1-adrenaline, ACTH and glucagon activate the adenylate cyclase of rat adipocytes by decreasing its S0.5(Mg2+) (concentration yielding 0.5 Vmax) from its basal value of 11.5 to 1.2, 0.3 and 1.8 mM and by increasing its Ki(ATP4?) from 0.03 to 0.25; 0.62 and 0.16 mM respectively. The kinetic properties of the enzyme are regulated by its state of saturation with ATP4? or Mg2+; its saturation with ATP4? and citrate3? suppressed its basal and hormone-dependent activities. The hormone-dependent decrease in Km and increase in Vmax of the enzyme occur when shifting from suboptimal low concentrations of hormone and Mg2+ to optimal conditions, i.e., high concentration of hormone and low concentration of Mg2+. The increase in the state of saturation of the enzyme with Mg2+ decreases the hormone-dependent effects on Vmax and results in identical values of Km (0.14 mM) for its basal and 1-adrenaline dependent activities. CaCl2 saturation curves at 5 mM ATP with either 5, 10 or 20 mM MgCl2 show that the substitution of 5 mM MgCl2 by 10 mM and 20 mM MgCl2 increased the Ki(Ca2+) of the enzyme from 0.19 to 0.49 and 0.94 mM but decreased its Ki(CaATP) from 0.42 to 0.19 and 0.14 mM respectively. Only when the concentration of MgCl2 exceeded that of ATP did 1-adrenaline and ACTH activate the enzyme by increasing its Ki(Ca2+), although only ACTH increased its Ki(CaATP). An increase in energy charge would decrease the intracellular concentrations of Mg2+ and Ca2+ because ATP4? has stronger binding constants for Mg2+ and Ca2+ than ADP3? and AMP2?. Hence, the reported properties of the enzyme suggests that changes in energy charge may allow for metabolic feedback control of the hormonal responsiveness of the Mg2+, Ca2+, ATP4? -sensitive adenylate cyclase.  相似文献   

14.
In Acetobacter aceti growing on pyruvate as the only source of carbon and energy, oxaloacetate (OAA) is produced by a phosphoenolpyruvate (PEP) carboxylase (EC 4.1.1.31). The enzyme was purified 122-fold and a molecular weight of about 380,000 was estimated by gel filtration.The optimum pH was 7.5 and the K m values for PEP and NaHCO3 were 0.49 mM and about 3 mM, respectively. The enzyme needed a divalent cation; the K m for Mn2+, Co2+ and Mg2+ were 0.12, 0.26 and 0.77 mM, respectively. Maximal activity was only obtained with Mg2+. Mn2+ and Co2+ became inhibitory at high concentrations.The activity was inhibited by succinate and, to a lesser extent, by fumarate, citrate, -ketoglutarate, aspartate and glutamate.As compared with the corresponding enzyme from A. xylinum, the PEP carboxylase of A. aceti showed the following differences: a) It had an absolute requirement for acetyl CoA (K a 0.18 mM) or propionyl CoA (K a 0.2 mM). b) It was not affected by ADP. c) It was sensitive to thiol blocking agents.Abbreviations PEP phosphoenolpyruvate - OAA oxaloacetate - MW molecular weight - TEMG buffer 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 5 mM MgCl2, 1 mM glutathione - HEPES N-2-hydroxyethylpiperazine-N-ethanesulfonic acid  相似文献   

15.
A particulate (Na + K)-ATPase preparation from dog kidney bound [48V]-ortho-vanadate rapidly at 37°C through a divalent cation-dependent process. In the presence of 3 mM MgCl2 theK d was 96 nM; substituting MnCl2 decreased theK d to 12 nM but the maximal binding remained the same, 2.8 nmol per mg protein, consistent with 1 mol vanadate per functional enzyme complex. Adding KCl in the presence of MgCl2 increased binding, with aK 0.5 for KCl near 0.5 mM; the increased binding was associated with a drop inK d for vanadate to 11 nM but with no change in maximal binding. Adding NaCl in the presence of MgCl2 decreased binding markedly, with anI 50 for NaCl of 7 mM. However, in the presence of MnCl2 neither KCl nor NaCl affected vanadate binding appreciably. Both the nonhydrolyzable, ,-imido analog of ATP and nitrophenyl phosphate, a substrate for the K-phosphatase reaction that this enzyme also catalyzes, decreased vanadate binding at concentrations consistent with their acting at the low-affinity substrate site of the enzyme; the presence of KCl increased the concentration of each required to decrease vanadate binding. Oligomycin decreased vanadate binding in the presence of MgCl2, whereas dimethyl sulfoxide and ouabain increased it. With inside-out membrane vesicles from red blood cells vanadate inhibited both the K-phosphatase and (Na + K)-ATPase reactions; however, with the K-phosphatase reaction extravesicular K+ (corresponding to intracellular K+) both stimulated catalysis and augmented vanadate inhibition, whereas with the (Na + K)-ATPase reaction intravesicular K+ (corresponding to extracellular K+) both stimulated catalysis and augmented vanadate binding.  相似文献   

16.
Endogenous phosphorylation was studied with highly purified fractions of the plasma membrane and the endoplasmic reticulum of SV40-transformed mouse fibroblasts using [γ-32P]ATP and [γ-32P]GTP as precursors. With ATP maximum overall incorporation of 32P into both membrane fractions occured at pH 7.8 in the presence of 10 mM MgCl2 after incubation for 1 min. GTP could be utilized only by the plasma membrane fraction showing maximum incorporation of 32P at pH 7.8 and 10 mM MgCl2 after incubation for 3 min.The pattern of phosphoproteins of the plasma membrane is represented by more than 15 proteins whereas the endoplasmic reticulum essentially contained only one phosphorylated component of 35 000 molecular weight. The comparison of ATP- and GTP-specific phophorlation of the plasma membrane revealed GTP to be a less efficient precursor yielding a similar phosphoprotein pattern with one significant difference: the GTP-specific main component exhibited a molecular wieght of about 100 000 and the ATP-specific main component a molecular weight of 110 000.The relative distribution of individual phosphoproteins in the pattern of the plasma membrane was dependent on pH but not on MgCl2 concentration or time of incubation. Increasing concentrations of plasma membrane protein altered the patterns of phosphoproteins dramatically: At high protein concentrations the ATP-specific main component (Mr = 110 000) was no more phosphorylated whereas with GTP the main component Mr = 100 000 was essentially the sole phosphorylated protein.  相似文献   

17.
Na+, K+-ATPase activities of the membranes obtained from intact red cells that are exposed to ouabain, digoxin, and digitoxin are inhibited. The extent of inhibition of each enzyme sample can be found by the following two assays: 1) Activity is measured by the addition of enzyme to a buffered solution containing 2 mM ATP, 3 mM Mg2+, 1 mM EDTA, 100 mM Na+, and 25 mM K+. Since little regeneration of the inhibited enzyme occurs under these conditions, the measured activity is that of the partially inhibited enzyme. 2) Enzyme is preincubated for ten minutes in the same solution from which Mg2+ and K+ are omitted, and then assayed by the addition of Mg+ and K+. Since the inhibited enzyme is completely regenerated during the preincubation period, the activity measured here serves as a control for that determined in the first assay.  相似文献   

18.
An ATPase activity stimulated by divalent ions (Mg2+, Ca2+, Mn2+, Zn2+) has been observed in intact hamster fibroblasts cultured in vitro (BHK line). Such activity has been determined by the incubation (30 min at 37°C) of washed cell suspensions (about 1 mg of proteins) in a medium containing 100 mM NaCl, 20 mM KCl, 15 mM Tris—HCl (pH 7.4), 10 mM NaHCO3, 5 mM glucose and equimolar concentrations of ATP and divalent cation. Mg2+-ATPase activity is insensitive to ouabain and lacks specificity towards nucleoside triphosphate substrates. AMP and ADP are not hydrolyzed under these conditions. Apparent Km of 0.76 mM and Vmax of 1.46 μmol Pi · mg proteins?1 · h?1 have been calculated for Mg-ATP complex. This ATPase is an ectoenzyme, therefore its activity could be used as a suitable index of the action of chemicals like chromium compounds known for their cytotoxic effects on membrane functions.Salts of trivalent (CrCl3) and hexavalent (K2Cr2O7) chromium at concentrations ranging from 1 mM to 5 mM inhibit Mg2+-ATPase. The inhibition by K2Cr2O7 is observed after pretreatment of the cells with this compound followed by its absence from the assay medium “per se” for Mg2+-ATPase, and it is referred to the alterations of membrane bound enzyme structures by the oxidizing hexavalent chromium. The inhibition by CrCl3 is mainly evident when this compound is present in the incubation medium, and is referred to the interaction of trivalent chromium with Mg2+-ATP as it is partially reversed by increasing Mg2+-ATP concentration.  相似文献   

19.
Fatty acid synthesis from Na (1-14C) acetate in leucoplasts isolated from developing seeds of Brassica compestris was found to be maximum when leucoplasts were supplied with 0.8 mM acetate, 20 mM NaHCO3, 8 mM ATP, 8 mM MgCl2, 4 mM MnCl2, 0.6 mM CoA, 1 mM NADH, 1 mM NADPH and 0.2 M sorbitol and incubated at 30°C for 2 h. The rate of fatty acid synthesis was highest at pH 8.5 In presence of 0.4 M Bistris-propane buffer and linear for upto 4 h at 30°C with 80–110 μg plastid protein. Sorbitol was an essential requirement as it prevented the rupturing of leucoplasts by osmosis. ATP and divalent cations were almost absolute requirements, whereas nucleotides, CoA and bicarbonate improved the rate of fatty acid synthesis by two to ten folds. Mg2+ and NADH were the preferred cation and nucleotide, respectively. High concentration of dithiothreltol inhibited the incorporation of (14C) acetate Into fatty acids. The system developed as above could be used for in vitro studies.  相似文献   

20.
Jajoo  Anjana  Bharti  Sudhakar 《Photosynthetica》2000,37(4):529-535
Cations such as Mg2+ regulate spillover of absorbed excitation energy mainly in favour of photosystem (PS) 2. Effect of low concentration (<10 mM) of the monovalent cation Na+ on chlorophyll (Chl) a fluorescence was completely overridden by divalent cation Mg2+ (5 mM). Based on Chl a fluorescence yield and 77 K emission measurements, we revealed the role and effectiveness of anions (Cl-, SO4 2-, PO4 3-) in lowering the Mg2+-induced PS2 fluorescence. The higher the valency of the anion, the lesser was the expression of Mg2+ effect. Anions may thus overcome Mg2+ effects up to certain extent in a valency dependent manner, thereby diverting more energy to PS1 even in the presence of MgCl2. They may do so by reversing Mg2+-induced changes.  相似文献   

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