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1.
The bivalent-cation dependence of phosphatidylinositol synthesis in a cell-free system from lymphocytes. 总被引:1,自引:0,他引:1
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The bivalent-cation requirements of two enzymes involved in phosphatidylinositol synthesis were defined for pig lymphocyte membranes using a citric acid buffer. CTP:phosphatidic acid cytidylyltransferase (EC 2.7.7.41) is activated by free Mn2+ concentrations above 20nM and by free Mg2+ concentrations above 10 microM. When activated by Mg2+, the enzyme is weakly inhibited by Ca2+ (Ki greater than 250 microM), but Ca2+ has no effect when Mn2+ is used to stimulate CDP-diacylglycerol synthesis. The synthesis of phosphatidylinositol from phosphatidic acid is also stimulated by Mn2+ and Mg2+ concentrations similar to those above and is inhibited by free Ca2+ concentrations above 500nM, probably by its action on CDP-diacylglycerol:inositol 3-phosphatidyltransferase (EC 2.7.8.11). Taken together, these studies suggest that under physiological conditions phosphatidylinositol synthesis is activated by Mg2+ and it is possible that it is further regulated by the free concentrations of Ca2+ and/or Mn2+. 相似文献
2.
Biosynthesis of phosphatidylinositol in Crithidia fasciculata 总被引:1,自引:0,他引:1
Microsomal preparations from the protozoan (Crithidia fasciculata were shown to incorporate myo-[2-3H]inositol into phosphatidylinositol by both the CDPdiacylglycerol:myo-inositol phosphatidyltransferase reaction and by a myo-inositol exchange reaction. Non-ionic detergent and Mg2+ were necessary for the measurement of transferase activity. Untreated preparations could not be saturated with Mg2+, even at very high concentrations (50-75 mM). However, low concentrations of EGTA (75 micro M) both stimulated the activity 3-fold and reduced the Mg2+ required for saturation to 15-20 mM. EGTA also increased the apparent Km for CDPdiacylglycerol while increasing the sensitivity to substrate inhibition above 1 mM. The transferase activity was inhibited by relatively low concentrations of Ca2+ (50 micro M). This and the EGTA effect suggest a possible role for Ca2+ in the modulation of phosphatidylinositol synthesis. The myo-inositol exchange activity required Mn2+, was insensitive to Ca2+ inhibition and was only slightly stimulated by detergents and EGTA. This activity was preferentially inactivated by heating at 50 degrees C in the presence of Triton X-100. In a detergent solubilized preparation the exchange activity but not the transferase exhibited a non-specific requirement for phospholipid. The differences in properties of the two activities suggest the presence of a separate exchange enzyme. 相似文献
3.
Phosphatidylinositol-inositol exchange in a rabbit lung 总被引:3,自引:0,他引:3
A microsomal fraction prepared from rabbit lung tissue was found to catalyze CDPdiacylglycerol-independent incorporation of [3H]inositol into phosphatidylinositol. This incorporation resulted from CMP-dependent phosphatidylinositol-inositol exchange and did not constitute a net synthesis of phosphatidylinositol. The phosphatidylinositol-inositol exchange activity was distinct from the phospholipid-base exchange enzymes and was specific for inositol. Optimal in vitro phosphatidylinositol-inositol exchange activity was observed at pH 8.5--8.8 and either Mn2+ or Mg2+ was essential for activity. Mercaptoethanol stimulated phosphatidylinositol-inositol exchange and Hg2+ inhibited this activity. In the absence of CMP, no phosphatidylinositol-inositol exchange was observed. CDP (and to a smaller extent CTP) also supported phosphatidylinositol-inositol exchange and this appeared to occur via the generation of CMP during incubations. The apparent Km values of the phosphatidylinositol-inositol exchange enzyme for CMP and inositol were 0.4 mM and 11 microM, respectively. When CDPdiacylglycerol was present at a concentration optimal for CDPdiacylglycerol : inositol transferase activity, CMP-dependent phosphatidylinositol-inositol exchange activity was still observed. However, in the presence of Hg2+ CDPdiacylglycerol inhibited phosphatidylinositol-inositol exchange activity. Several properties of the phosphatidylinositol-inositol exchange enzyme resemble those of CDPdiacylglycerol : inositol transferase, but the two enzymes appear distinct on the basis of different degrees of inhibition by either Ca2+, Hg/+ or heat, and on the basis of different changes in activity during lung development. 相似文献
4.
Characterization of rat kidney proximal tubule brush border membrane-associated phosphatidylinositol phosphodiesterase 总被引:1,自引:0,他引:1
D W Schwertz J I Kreisberg M A Venkatachalam 《Archives of biochemistry and biophysics》1983,224(2):555-567
Isolated rat kidney proximal tubule brush border membrane vesicles exhibit an increase in diacylglycerol levels (20- to 30-fold) and a concomitant decrease in phosphatidylinositol when incubated with [3H]arachidonate-labeled lipids, Ca2+, and deoxycholate. Levels of free arachidonate, triglyceride, and noninositol phospholipids are not altered. These results suggest phosphatidylinositol phosphodiesterase activity is associated with rat proximal tubule brush border membrane. Presence of both deoxycholate and certain divalent cations was necessary to demonstrate enzyme activity. Optimum pH ranged from 7.0 to 8.5. Ca2+, Mg2+, and Mn2+ stimulated diglyceride production while Ba2+, Zn2+, Hg2+, and K+ were ineffective. HgCl2 inhibited Ca2+-stimulated phosphatidylinositol phosphodiesterase. Mg2+ and deoxycholate-dependent enzyme activity was shown to be phosphatidylinositol specific. Sodium lauryl sulfate, tetradecyltrimethylammonium bromide, and Triton X-100 did not activate phosphatidylinositol phosphodiesterase in the presence of Ca2+. In combination with deoxycholate, diglyceride formation was not affected by sodium lauryl sulfate, partially inhibited by Triton X-100, and completely abolished by tetradecyltrimethylammonium bromide. Diglyceride kinase activity was not found associated with brush border membrane phosphatidylinositol phosphodiesterase. ATP (1-5 mM) inhibited Ca2+- or Mg2+-stimulated, deoxycholate-dependent phosphatidylinositol hydrolysis by chelating the required divalent cation. 相似文献
5.
J E Bleasdale P Wallis P C MacDonald J M Johnston 《Biochimica et biophysica acta》1979,575(1):135-147
CDPdiacylglycerol:inositol transferase activity in rabbit lung tissue has been characterized and the optimum conditions for assaying this enzyme in vitro were determined. Rabbit lung tissue CDPdiacylglycerol:inositol transferase activity was found primarily in the microsomal fraction. The pH optimum of the enzyme activity was between 8.8 and 9.4, and the reaction was dependent on either Mn2+ or Mg2+. Detergents and Ca2+ inhibited the activity of the enzyme. The apparent Km values of the enzyme for CDPdioleoylglycerol and myoinositol were 0.18 mM and 0.10 mM, respectively. The reversibility of the reaction catalyzed by CDPdiacylglycerol:inositol transferase in microsomes prepared from rabbit lung tissue was demonstrated by the synthesis of [3H]CMPdiacylglycerol when [3H]CMP and phosphatidylinositol were present in the incubation mixture. The reverse reaction was characterized and its importance in the regulation of the acidic phospholipid composition of surfactant during lung development is discussed. The pH optimum for the reverse reaction was 6.2, and the reverse reaction was also dependent on Mn2+ or Mg2+. The apparent Km value of CDPdiacylglycerol:inositol transferase for CMP was found to be 2.8 mM. 相似文献
6.
S Taniguchi S Morikawa H Hayashi K Fujii H Mori M Fujiwara M Fujiwara 《Journal of biochemistry》1986,100(2):485-491
The effects of Ca2+ on ethanolaminephosphotransferase [EC 2.7.8.1] and cholinephosphotransferase [EC 2.7.8.2] activities in rabbit platelet membranes were studied using endogenous diglyceride and CDP-[3H]ethanolamine or CDP-[14C]choline as substrates. Both transferases required Mn2+, Co2+, or Mg2+ as a metal cofactor and the optimal concentrations of the metals for both activities were about 5, 10, and 5 mM, respectively. When 5 mM Mg2+ was used as a cofactor, both transferase activities were inhibited by a low concentration of Ca2+ (half maximal inhibition at approx. 15 microM). In the presence of 5 mM Mn2+, however, approx. 5 mM Ca2+ was required to produce half maximal inhibition. The Ca2+-induced inhibition was reversible and the rate of the inhibition was not affected either by the concentrations of the CDP-compound or by exogenously added diacylglycerol. The relationship between Ca2+ and both Mg2+ and Mn2+ on the transferase activities was competitive. 45Ca2+ binding (and/or uptake) to the platelet membranes was inhibited by Mn2+, Mg2+, and Co2+, in a concentration-dependent manner. However, the inhibitory effects of the three metal ions on the total Ca2+ binding (and/or uptake) did not correlate with the activation of both transferase activities by the three metal ions in the presence of Ca2+. These results suggest that both transferase activities are regulated by low concentrations of Ca2+ in the presence of optimal concentrations of Mg2+, and that the inhibition is mediated directly by Ca2+, which interacts with a specific metal cofactor binding site(s) of the transferases. 相似文献
7.
Inositol Phospholipid Hydrolysis in Rat Cerebral Cortical Slices: II. Calcium Requirement 总被引:30,自引:17,他引:13
The calcium requirement for agonist-dependent breakdown of phosphatidylinositol and polyphosphoinositides has been examined in rat cerebral cortex. The omission of added Ca2+ from the incubation medium abolished [3H]inositol phosphate accumulation from prelabelled phospholipid induced by histamine, reduced that due to noradrenaline and 5-hydroxytryptamine, but did not affect carbachol-stimulated breakdown. EC50 values for agonists were unaltered in the absence of Ca2+. Removal of Ca2+ by preincubation with EGTA (0.5 mM) abolished all responses, but complete restoration was achieved by replacement of Ca2+. The EC50 for Ca2+ for histamine-stimulated [3H]inositol phosphate accumulation was 80 microM. Noradrenaline-stimulated breakdown was antagonised by manganese (IC50 1.7 mM), but not by the calcium channel blockers nitrendipine or nimodipine (30 microM). The calcium ionophore A23187 stimulated phosphatidylinositol/polyphosphoinositide hydrolysis with an EC50 of 2 microM, and this response was blocked by EGTA. Omission of Ca2+ or preincubation with EGTA or Mn2+ (EC50 = 230 microM) greatly enhanced the incorporation of [3H]inositol into phospholipids. The IC50 for Ca2+ in inhibiting incorporation was 25 microM. The results show that different receptors mediating phosphatidylinositol/polyphosphoinositide breakdown in rat cortex have quantitatively different Ca2+ requirements, and it is suggested that rigid opinions regarding phosphatidylinositol/polyphosphoinositide breakdown as either cause or effect of calcium mobilisation in rat cortex are inappropriate. 相似文献
8.
At low concentrations of Mg2+, incorporation of 32P from [gamma-32P]ATP into phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) in plasma membranes isolated from human polymorphonuclear leucocytes was enhanced 2-4-fold by the polyamines spermidine and spermine. Polyamines had no effects on inositol phospholipid phosphorylation at high concentrations of Mg2+. At 1 mM-Mg2+, [32P]PIP2 synthesis was maximally enhanced by 2 mM-spermine and 5 mM-spermidine, whereas putrescine only slightly enhanced synthesis. Spermine decreased the EC50 (concn. for half-maximal activity) for Mg2+ in [32P]PIP2 synthesis from 5 mM to 0.5 mM. Spermine did not modulate the Km for ATP for [32P]PIP or [32P]PIP2 synthesis. Spermine also decreased the EC50 for PI in [32P]PIP synthesis. In contrast, spermine elevated the apparent Vmax, without affecting the EC50 for PIP, for [32P]PIP2 synthesis. Spermine and spermidine also inhibited the hydrolysis of [32P]PIP2 by phosphomonoesterase activity. Therefore polyamines appear to activate inositol phospholipid kinases by eliminating the requirements for super-physiological concentrations of Mg2+. Polyamine-mediated inhibition of polyphosphoinositide hydrolysis would serve to potentiate further their abilities to promote the accumulation of polyphosphoinositides in biological systems. 相似文献
9.
Phospholipid base exchange activity using choline as substrate was detected in plasma membranes (PM) and other subcellular fractions of rat liver, with microsomes (MS) showing the highest specific activity. In contrast, phospholipase D activity was only detected in PM. In PM, choline exchanged for phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS), whereas ethanolamine exchanged for PE and PS, and serine exchanged for PS. Ca2+ (10 microM or higher) stimulated choline incorporation into PC in MS and PM, whereas Mg2+ (10 microM or higher) stimulated it only in PM. Ethanolamine and serine incorporation into PM phospholipids was also stimulated by Ca2+, and inositol incorporation by Mn2+. Phospholipase D activity was substantial in the presence of EGTA and was slightly stimulated by Ca2+ concentrations less than 500 microM. It was undetectable without Mg2+. Low concentrations of oleate (1 mM or less) stimulated phospholipase D activity. These concentrations inhibited choline base exchange activity, whereas higher concentrations (3-8 mM) were stimulatory. Comparison of the subcellular distribution and Ca2+, Mg2+, and oleate effects on choline base exchange and phospholipase D activities supports the view that they are catalyzed by different enzymes. The incorporation of choline, but not ethanolamine or serine, into the phospholipids of PM, but not MS, was stimulated by micromolar concentrations of guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) and other slowly hydrolyzable analogues of GTP. GDP, GMP, and other nucleoside triphosphates and their analogues were ineffective. GTP gamma S stimulation of base exchange activity was dependent upon Mg2+ and was inhibited by high concentrations of guanosine 5'-O-2-(thio)diphosphate. In the presence of low concentrations of GTP gamma S, ATP and its slowly hydrolyzable analogues stimulated base exchange activity. Dose-response curves for these nucleotides revealed a potency order consistent with mediation by purinergic receptors of the P2Y type. Base exchange activity stimulated by ATP plus GTP gamma S or GTP gamma S alone was not altered by treatment with pertussis or cholera toxins. These results suggest that the choline base exchange activity of liver PM is regulated by a pertussis toxin-insensitive G-protein linked to P2Y purinergic receptors. 相似文献
10.
A membrane-bound phospholipase C with an apparent specificity for lysophosphatidylinositol in porcine platelets 总被引:2,自引:0,他引:2
A membrane preparation from porcine platelets catalyzed the hydrolysis of [2-3H]glycerol-labeled lysophosphatidylinositol to form monoacylglycerol and inositol phosphates. The hydrolysis was optimal at pH 9. The addition of Ca2+ did not enhance the hydrolysis, but the enzyme was inhibited completely by EGTA. The EGTA-inactivated enzyme was partially reactivated by Ca2+; Mn2+, Mg2+, and Zn2+ were much less effective or ineffective for the reactivation. The phospholipase C was apparently specific for lysophosphatidylinositol; phosphatidylinositol, phosphatidylcholine, phosphatidylethanolamine, lysophosphatidylcholine, lysophosphatidylethanolamine, phosphatidic acid, and lysophosphatidic acid were not hydrolyzed at significant rates under the conditions used. Phospholipase C with these properties has not been reported previously. 相似文献
11.
Isolation of a phosphomonoesterase from human platelets that specifically hydrolyzes the 5-phosphate of inositol 1,4,5-trisphosphate 总被引:30,自引:0,他引:30
Platelets, and a variety of other cells, rapidly hydrolyze the phosphoinositides in response to stimulation by agonists. One of the products of hydrolysis of phosphatidylinositol 4,5-diphosphate is inositol 1,4,5-trisphosphate, which recently has been suggested to mediate intracellular Ca2+ mobilization. We have found that human platelets contain an enzyme that degrades inositol 1,4,5-trisphosphate. We have isolated this soluble enzyme and find that it hydrolyzes the 5-phosphate of inositol 1,4,5-trisphosphate (Km = 30 microM, Vmax = 5.3 microM/min/mg of protein). The products of the reaction are inositol 1,4-diphosphate and phosphate. The apparent molecular weight of the enzyme is 38,000 as determined both by gel filtration and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence and absence of 2-mercaptoethanol. This enzyme is specific for inositol 1,4,5-trisphosphate. Other water soluble inositol phosphates as well as phosphorylated sugars are not hydrolyzed, while the only inositol containing phospholipid hydrolyzed is phosphatidylinositol 4,5-diphosphate at a rate less than 1% that for inositol 4,5-trisphosphate. The inositol 1,4,5-trisphosphate 5-phosphomonoesterase requires Mg2+ for activity and is inhibited by Ca2+, Ki = 70 microM. Li+, up to 40 mM, has no effect on enzyme activity. The duration and magnitude of any inositol 1,4,5-trisphosphate response in stimulated platelets may be determined by the activity of this enzyme. 相似文献
12.
Purification and characterization of phosphatidylinositol kinase from porcine liver microsomes 总被引:5,自引:0,他引:5
Phosphatidylinositol kinase was solubilized and purified from porcine liver microsomes to apparent homogeneity. The purification procedure includes: solubilization of microsomes by 2% Triton X-100, ammonium sulfate precipitation (20-35% saturation), Reactive blue agarose chromatography, DEAE-Sephacel chromatography and two consecutive hydroxyapatite chromatographies. A total of 4900-fold purification with 8% recovery of enzyme activity was achieved. The molecular weight of the enzyme as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 55000. The enzyme is stimulated in a decreasing order by Mg2+, Fe2+, Mn2+, Fe3+ and Co2+. Ca2+ inhibited Mg2+-stimulated activity with an I50 of 0.4 mM. Apparent Km values for phosphatidylinositol and ATP are 120 and 60 microM, respectively. The enzyme is inhibited by adenosine (I50 = 70 microM), ADP (I50 = 120 microM) and quercetin (I50 = 100 microM). The enzyme is also sensitive to sulfhydryl inhibitors. Using the purified enzyme as an immunogen, we have successfully prepared antibodies for phosphatidylinositol kinase in rabbits. The antibodies appear to recognize an antigen of Mr 55000 on SDS-polyacrylamide gel electrophoresis from various porcine tissues in Western blot analysis. 相似文献
13.
I A Sokolova N N Khodarev S S Aleksandrova I I Votrin 《Biokhimii?a (Moscow, Russia)》1988,53(7):1163-1173
A scheme for the isolation of Ca,Mg-dependent endonuclease from human spleen lymphocyte nuclei has been developed. The isolation procedure resulted in protein preparations (Mr = 57 kD) possessing an enzymatic activity and stable upon storage for over a period of one year. The enzyme is an endonuclease which predominantly cleaves double-stranded DNA by a mixed single- and double-hit mechanism with the formation of 5'-phosphate and 3'-OH terminal groups. Its maximal activation is induced by Ca2+ plus Mg2+. The enzyme is also active in the presence of Mn2+, Ca2+, Mg2+ and Zn2+ and is inhibited by Co2+. NaCl and KCl (0.15-0.2 M) and p-chloromercuribenzoate (1 mM) also inhibit the enzyme. ATP has no activating effect. 相似文献
14.
I Morishima 《Journal of biochemistry》1978,84(6):1495-1500
Adenylate cyclase was assayed in a sonicated preparation of silkworm pupal fat body. The adenylate cyclase was found mostly in the particulate fraction. The activity depended upon either Mg2+ or Mn2+, and the degree of stimulation by Mn2+ was 2 times greater than that by Mg2+ compared at the saturating concentrations. In the presence of Mg2+, the enzyme was inhibited by both EGTA and high concentrations of Ca2+, showing biphasical response to Ca2+. The enzyme was stimulated several-fold by NaF. The enzyme exhibited typical Michaelis-Menten kinetics and Km values were 0.13 mM for MgATP and 0.086 mM for MnATP. 相似文献
15.
S B Hwang 《Biochimica et biophysica acta》1991,1064(2):351-359
Specific, saturable and reversible binding of tritium-labeled inositol 1,4,5-trisphosphate [( 3H]Ins(1,4,5)P3) to human platelet membranes is demonstrated. The Ins(1,4,5)P3-binding sites are abundant and display high selectivity for Ins(1,4,5)P3. Other inositol phosphates exhibit much lower affinity for this site. The specific [3H]Ins(1,4,5)P3 binding was found to be modulated by pH, monovalent and divalent cations, and GTP. A sharp increase in binding occurs at slightly alkaline pH. The monovalent cations, Na+, K+ and Li+ almost double the binding at 30 mM. Mg2+ inhibits the specific [3H]Ins(1,4,5)P3 binding. At low concentrations of Ca2+, the binding is inhibited, but at concentrations higher than 5 mM the binding is potentiated and increases by almost 5-fold at 100 mM. Similar pattern of the effects is also observed for Mn2+ and Sr2+. The specific [3H]Ins(1,4,5)P3 binding is specifically inhibited by GTP. Other nucleotides also inhibit the binding but at higher concentrations. From saturation binding studies, Ca2+ potentiation seems to be due to the conversion of the receptor from the low-affinity state to the high-affinity one. In the absence of Ca2+, the Scatchard plot is nonlinear and concave, and statistically can be fitted best with two equilibrium dissociation constants (Kd values), 0.19 +/- 0.11 and 13.2 +/- 18.1 nM, respectively, for high- and low-affinity binding sites. However, in the presence of 100 mM CaCl2, the Scatchard plot reveals only the high-affinity binding sites with a Kd value of 0.32 +/- 0.15 nM. The specific Ins(1,4,5)P3 receptor in human platelets could therefore exist in multiple conformational states to regulate the intracellular Ca2+ concentration. 相似文献
16.
The pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40) from Streptococcus lactis C10 had an obligatory requirement for both a monovalent cation and divalent cation. NH+4 and K+ activated the enzyme in a sigmoidal manner (nH =1.55) at similar concentrations, whereas Na+ and Li+ could only weakly activate the enzyme. Of eight divalent cations studied, only three (Co2+, Mg2+ and Mn2+) activated the enzyme. The remaining five divalent cations (Cu2+, Zn2+, Ca2+, Ni2+ and Ba2+) inhibited the Mg2+ activated enzyme to varying degrees. (Cu2+ completely inhibited activity at 0.1 mM while Ba2+, the least potent inhibitor, caused 50% inhibition at 3.2 mM). In the presence of 1 mM fructose 1,6-diphosphate (Fru-1,6-P2) the enzyme showed a different kinetic response to each of the three activating divalent cations. For Co2+, Mn2+ and Mg2+ the Hill interaction coefficients (nH) were 1.6, 1.7 and 2.3 respectively and the respective divalent cation concentrations required for 50% maximum activity were 0.9, 0.46 and 0.9 mM. Only with Mn2+ as the divalent cation was there significatn activity in the absence of Fru-1,6-P2. When Mn2+ replaced Mg2+, the Fru-1,6-P2 activation changed from sigmoidal (nH = 2.0) to hyperbolic (nH = 1.0) kinetics and the Fru-1,6-P2 concentration required for 50% maximum activity decreased from 0.35 to 0.015 mM. The cooperativity of phosphoenolpyruvate binding increased (nH 1.2 to 1.8) and the value of the phosphoenolpyruvate concentration giving half maximal velocity decreased (0.18 to 0.015 mM phosphoenolyruvate) when Mg2+ was replaced by Mn2+ in the presence of 1 mM Fru-1,6-P2. The kinetic response to ADP was not altered significantly when Mn2+ was substituted for Mg2+. The effects of pH on the binding of phosphoenolpyruvate and Fru-1,6-P2 were different depending on whether Mg2+ or Mn2+ was the divalent cation. 相似文献
17.
G P Diadiusha V A Tuga? N N Zemlianaia A N Zakharchenko 《Biokhimii?a (Moscow, Russia)》1988,53(5):832-837
It was found that the initial rate of passive KC1-stimulated Ca2+ influx into sarcoplasmic reticulum (SR) vesicles follows the saturation kinetics at Ca2+ concentrations of 8-10 mM. The inhibitory effect of Ca2+ channel blockers (La3+, Mn2+, Co2+, Cd2+, Mg2+) on passive Ca2+ influx into SR vesicles is competitive with respect to Ca2+. These blockers also inhibit the initial fast phase of Ca2+ efflux from Ca2+-loaded SR vesicles. Verapamil (0.1-0.5 mM) added to the incubation mixture has no effect on passive Ca2+ fluxes across the SR vesicle membrane or on Ca2+ binding and ATP-dependent Ca2+ accumulation. However, preincubation of SR vesicles with verapamil (18 hours, 4 degrees C) or its introduction into the medium for SR vesicle isolation leads to the inhibition of passive Ca2+ fluxes. 相似文献
18.
Effect of unsaturated fatty acids and Ca2+ on phosphatidylinositol synthesis and breakdown 总被引:1,自引:0,他引:1
CDP-diglyceride : inositol transferase was inhibited by unsaturated fatty acids. The inhibitory activity decreased in the following order: arachidonic acid greater than linolenic acid greater than linoleic acid greater than oleic acid greater than or equal to palmitoleic acid. Saturated fatty acids such as myristic acid, palmitic acid, and stearic acid had no effect. Calcium ion also inhibited the activity of CDP-diglyceride : inositol transferase. In rat hepatocytes, arachidonic acid inhibited 32P incorporation into phosphatidylinositol and phosphatidic acid without any significant effect on 32P incorporation into phosphatidylcholine, phosphatidylethanolamine and phosphatidylserine. Ca2+ ionophore A23187 also inhibited 32P incorporation into phosphatidylinositol. However, 32P incorporation into phosphatidic acid was stimulated with Ca2+ ionophore A23187. Phosphatidylinositol-specific phospholipase C was activated by unsaturated fatty acids. Polyunsaturated fatty acids such as arachidonic acid and linolenic acid had a stronger effect than di- and monounsaturated fatty acids. Saturated fatty acids had no effect on the phospholipase C activity. The phospholipase C required Ca2+ for activity. Arachidonic acid and Ca2+ had synergistic effects. These results suggest the reciprocal regulation of phosphatidylinositol synthesis and breakdown by unsaturated fatty acids and Ca2+. 相似文献
19.
M Sumida M Hamada H Takenaka Y Hirata K Nishigauchi H Okuda 《Journal of biochemistry》1986,100(3):765-772
The effects of various divalent cations on the Ca2+ uptake by microsomes from bovine aortic smooth muscle were studied. High concentrations (1 mM) of Co2+, Zn2+, Mn2+, Fe2+, and Ni2+ inhibited neither the Ca2+ uptake by the microsomes nor the formation of the phosphorylated intermediate (E approximately P) of the Ca2+,Mg2+-ATPase of the microsomes. The cadmium ion, however, inhibited both the Ca2+ uptake and the E approximately P formation by the microsomes. Dixon plot analysis indicated Cd2+ inhibited (Ki = 135 microM) the Ca2+ dependent E approximately P formation in a non-competitive manner. The inhibitory effect of Cd2+ was lessened by cysteine or dithiothreitol. The strontium ion inhibited the Ca2+ uptake competitively, while the E approximately P formation increased on the addition of Sr2+ at low Ca2+ concentrations. At a low Ca2+ concentration (1 microM), Sr2+ was taken up by the aortic microsomes in the presence of 1 mM ATP. It is thus suggested that Sr2+ replaces Ca2+ at the Ca2+ binding site on the ATPase. 相似文献
20.
Upon stimulation with 10(-6) -10(-3) M ATP, A-431 human epidermoidal carcinoma cells incorporated radioactive calcium from their medium in a temperature-dependent manner. The rate of incorporation of 45Ca2+ was rapid for the initial 5 min, but decreased immediately thereafter. The preincubation of cells for 2 h in medium depleted of both Ca2+ and Mg2+ abolished the ATP-dependent 45Ca2+ incorporation, irrespective of whether or not the subsequent incubation medium contained Mg2+ ions. ATP-dependent 45Ca2+ incorporation could be restored by a second preincubation (1 h) in medium containing 1 mM Mg2+, but no Ca2+. The Mg2+ ions in the second preincubation medium could be replaced by Ca2+, Co2+, or Cu2+ for restoration of such activity. Elevation of inositol trisphosphate (InsP3) was observed in cells depleted of either Ca2+ or Mg2+, but not in cells depleted of both ions. A parallel effect was observed in changes in [Ca2+]i. Since the concentration of cytosolic calcium ions does not change by incubation of cells in medium depleted of and (or) restored with calcium ions, we conclude that either calcium or magnesium ions associated with some cellular component(s) are responsible for production of InsP3, which then supposedly mobilizes Ca2+ and provokes 45Ca2+ influx. 相似文献