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1.
Eugene E. Quist 《Archives of biochemistry and biophysics》1982,219(1):58-64
Incubation of rabbit erythrocyte ghosts at 25 °C with 1 mm [γ-32P]ATP and MgCl2 results in incorporation of 32P into diphosphoinositide and triphosphoinositide with initial rates of 15.6 and 1.8 nmol 32P/mg/h, respectively. Incorporation of 32P into diphosphoinositide plateaus after 20 min whereas incorporation into triphosphoinositide did not plateau until after 80 min. Diphosphoinositide and triphosphoinositide, prelabeled with 32P, did not undergo significant breakdown when incubated at 25 °C for 15 to 20 min. Turnover of 32P-labeled diphosphoinositide and triphosphoinositide was insignificant in the presence of MgCl2 and cold ATP. Diphosphoinositide is not phosphorylated to triphosphoinositide in the presence of Mg-ATP under conditions in which synthesis of these polyphosphoinositides can occur. In the presence of neomycin and Mg-ATP, labeled diphosphoinositide was rapidly phosphorylated to triphosphoinositide. Neomycin had no effect on labeled di- and triphosphoinositide content in the absence of ATP. Freeze-thawing the ghosts or the addition of Triton X-100 does not produce the same effect as neomycin. The results of this investigation suggest that diphosphoinositide and triphosphoinositide are normally synthesized from endogenous phosphatidylinositol in rabbit ghosts by separate enzymatic pathways. Neomycin an aminoglycoside which interacts with polyphosphoinositides may perturb the organization of substrates and kinase activities involved in polyphosphoinositide metabolism and alter these pathways. 相似文献
2.
1. Impermeable inside-out and right-side-out vesicles were prepared from membranes of human erythrocytes. During preparation of each kind of impermeable vesicle, permeable vesicles were also obtained. 2. Incubation of vesicles with [gamma-32P]ATP at 37 degrees C for periods of up to 1 hr did not change the topography or the permeability of the vesicles. 3. Vesicles incorporated labeled phosphate from [gamma-32P]ATP into both diphosphoinositide and triphosphoinositide, but impermeable inside-out vesicles incorporated significantly more nuclide than did right-side-out vesicles. 4. Permeable vesicles derived during the preparation of inside-out vesicles were as active as impermeable inside-out vesicles in the incorporation of labeled phosphate into the polyphosphoinositides. However, permeable vesicles derived during the preparation of right-side out vesicles were not as active. 5. Impermeable right-side-out vesicles, treated with 0.01 percent saponin, incorporated labeled phosphate into the polyphosphoinositides at a level comparable to that of impermeable inside-out vesicles. 6. These data show that the enzymes involved in metabolism of diphosphoinositide and triphosphoinositide are located on the cytoplasmic surface of the erythrocyte membrane. 相似文献
3.
The characteristics of ATP synthesis in cell envelope vesicles of Halobacterium halobium were further studied. The results confirmed the previous conclusion (Mukohata et al. (1986) J. Biochem. 99, 1-8) that the ATP synthase in this extremely halophilic archaebacterium can not be an ordinary type of F0F1-ATPase, which has been thought to be ubiquitous among all the aerobic organisms on our biosphere. The ATP synthesis was activated most in 1 M NaCl and/or KCl, and at 40 degrees C, and at 80 mM MgCl2 where F0F1-ATPase loses its activity completely. The synthesis was negligible at 10 degrees C, and at 5 mM MgCl2. The Km for ADP was about 0.3 mM in the presence of 20 mM Pi, 1 M NaCl, 80 mM MgCl2, and 10 mM PIPES at pH 6.8 and 20 degrees C. The ATP synthesis was not inhibited by NaN3 and quercetin (specific inhibitors for F0F1-ATPase) or vanadate (for E1E2-ATPase) or ouabain (for Na+,K+-ATPase) or P1,P5-di(adenosine-5')pentaphosphate (AP5A, for adenylate kinase). The ATP synthesis was not inhibited by modification (pretreatment) with NaN3 or 5'-p-fluorosulfonylbenzoyladenosine (FSBA). On the contrary, the ATP synthesis was rather non-specifically inhibited by N-ethylmaleimide (NEM), trinitrobenzenesulfonate (TNBS), phenylglyoxal, and pyridoxal phosphate. 7-Chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl) as well as N,N'-dicyclohexylcarbodiimide (DCCD) was found to be a specific inhibitor at least partly, because the NBD-Cl inhibition was partly prevented by ADP added to the modification mixture. 相似文献
4.
Phosphomonoesterase hydrolysis of polyphosphoinositides in rat kidney: Properties and subcellular localization of the enzyme system.
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Tthe properties of diphosphoinositide and triphosphoinositide phosphatases from rat kidney homogenate were studied in an assay system in which non-specific phosphatase activity was eliminated. The enzymes were not completely metal-ion dependent and were activated by Mg2+. The detergent sodium deoxycholate, Triton X-100 and Cutscum inhibited the reaction; cetyltrimethylammonium bromide only activated when added with the subtrates and in the presence Mg2+. Both enzymes had a pH optimum of 7.5. Ca2+ and Li+ both activated triphosphoinositide phosphatase, but Ca2+ inhibited and L+ had little effect on diphosphoinositide phosphatase. Cyclic AMP had no effect on either enzyme. The enzymes were three times more active in kidney cortex than in the medulla. On subcellular fractionation of kidney-cortex homogenates by differential and density-gradient centrifugation, the distribution of the enzymes resembled that of thiamin pyrophosphatase (assayed in the absence of ATP), suggesting localization in the Golgi complex. However, the distribution differed from that of the liver Golgimarker galactosyltransferase. Activities of both diphosphoinositide and triphosphoinositide phosphatases and thiamin pyrophosphatase were low in purified brush-border fragments. Further experiments indicate that at least part of the phosphatase activity is soluble. 相似文献
5.
Acetylcholine increases the breakdown of triphosphoinositide of rabbit iris muscle prelabelled with [32P] phosphate.
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1. Paired iris smooth muscles from rabbits were incubated for 30 min at 37 degrees C in an iso-osmotic salt medium containg glucose, inositol, cytidine and [32P]phosphate. 2. One of the pair was then incubated at 37 degrees C for 10 min in unlabelled medium containing 10mM-2-deoxyglucose and the other was incubated in the presence of acetylcholine plus eserine (0.05mM each). 2-Deoxyglucose, which was included in the incubation medium to minimize the biosynthesis of triphosphoinositide from ATP and diphosphoinositide, decreased the amount of labelled ATP by 71% and inhibited further 32P incorporation from ATP into triphosphoinositide by almost 30%. 3. Acetylcholine (0.05mM) increased significantly the loss of 32P from triphosphoinositide (the 'triphosphoinositide effect') in 32P-labelled iris muscle. This effect was measured both chemically and radiochemically. It was also observed when 32Pi was replaced by myo-[3H]inositol in the incubation medium. 4. The triphosphoinositide effect was blocked by atropine but not by D-tubocurarine. Further, muscarinic but not nicotinic agonists were found to provoke this effect. 5. Acetylcholine decreased by 28% the 32P incorporation into triphosphoinositide, presumably by stimulating its breakdown. This decrement in triphosphoinositide was blocked by atropine, but not by D-tubocurarine. 6. The triphosphoinositide effect was accompanied by a significant increase in 32P labelling, but not tissue concentration, of phosphatidylinositol and phosphatidic acid. The possible relationship between the loss of 32P label from triphosphoinositide in response to acetylcholine and the concomitant increase in that of phosphatidylinositol and phosphatidic acid is discussed. 7. The presence of triphosphoinositide phosphomonoesterase, the enzyme that might be stimulated in the iris smooth muscle by the neurotransmitter, was demonstrated, and, under our methods of homogenization and assay, more than 80% of its activity was localized in the particulate fraction. 相似文献
6.
The relationship of polyphosphoinositide metabolism to erythrocyte ATP levels was examined. Turnover of polyphosphoinositides was not closely dependent on ATP as it is reported to be in yeast. Neomycin increased 32P incorporation into diphosphoinositide and to a lesser extent into triphosphoinositide without affecting intracellular ATP. Treatment of intact cells with ionophore A23187 resulted in a decrease of at least 80% in polyphosphoinositide levels which followed a decrease in cellular ATP and an increase in phosphatidate. The results indicate that polyphosphoinositide turnover does not regulate energy charge in the erythrocyte. However some of the events which follow treatment of erythrocytes with metabolic inhibitors or calcium and ionophore may be related to the accompanying decrease in polyphosphoinositide levels. 相似文献
7.
J T Buckley 《Canadian journal of biochemistry》1976,54(9):772-777
Homogenates of the free-living amoeba Acanthamoeba castellanii incorporate phosphate from [gamma-32P]ATP into a lipid which co-chromatographs with diphosphoinositide on one- and two dimensional chromatography. Incorporation into lipids similar in mobility to triphosphoinositide is not detected. The product co-chromatographs with diphosphoinositide whether exogenous phosphatidylinositol or total amoeba lipid is the substrate. The inositide kinase is almost entirely located in the supernatant fraction after centrifugation at 100 000 g. Incorporation of phosphate from [gamma-32P]ATP is linear for at least 15 min in the presence of 0.5 mM phosphatidylinositol. The enzyme requires Mg2+ of Mn2+ as well as ATP and it is not affected by low concentrations of Ca2+. The apparent Km for phosphatidylinositol in 2 mM. Both ADP and cAMP inhibit the reaction. 相似文献
8.
The relative distribution of phosphatidylinositol (PI) and phosphatidylinositol-4-phosphate (PIP) kinase activities in enriched cardiac sarcolemma (SL), sarcoplasmic reticulum (SR), and mitochondrial fractions was investigated. PI and PIP kinase activities were assayed by measuring 32P incorporation into PIP and phosphatidylinositol 4,5-bisphosphate (PIP2) from endogenous and exogenous PI in the presence of [gamma-32P]ATP. PI and PIP kinase activities were present in SL, SR, and mitochondrial fractions prepared from atria and ventricles although the highest activities were found in SL. A similar membrane distribution was found for PI kinase activity measured in the presence of detergent and exogenous PI. PI and PIP kinase activities were detectable in the cytosol providing exogenous PI and PIP and Triton X-100 were present. Further studies focused on characterizing the properties and regulation of PI and PIP kinase activities in ventricular SL. Alamethacin, a membrane permeabilizing antibiotic, increased 32P incorporation into PIP and PIP2 4-fold. PI and PIP kinase activities were Mg2+ dependent and plateaued within 15-20 min at 25 degrees C. Exogenous PIP and PIP2 (0.1 mM) had no effect on PIP and PIP2 labeling in SL in the absence of Triton X-100 but inhibited PI kinase activity in the presence of exogenous PI and Triton X-100. Apparent Km's of ATP for PI and PIP kinase were 133 and 57 microM, respectively. Neomycin increased PIP kinase activity 2- to 3-fold with minor effects on PI kinase activity. Calmidazolium and trifluoperazine activated PI kinase activity 5- to 20-fold and completely inhibited PIP kinase activity. Quercetin inhibited PIP kinase 66% without affecting PI kinase activity. NaF and guanosine 5'-O-(3-thiotriphosphate) had no effect on PI and PIP kinase activities, indicating that these enzymes were not modulated by G proteins. The probability that PIP and PIP2 synthesis in cardiac sarcolemma is regulated by product inhibition and phospholipase C was discussed. 相似文献
9.
The effects of dibutyryl cyclic adenosine 3':5'-monophosphate and ATP on isotope incorporation into phospholipids and the release of beta-glucuronidase into the extracellular medium were studied in polymorphonuclear leukocytes from guinea pig peritoneal exudates. Exogenous dibutyryl cyclic adenosine 3':5'-monophosphate (0.1--1.0 mM) reduced beta-glucoronidase release induced by cytochalasin B in the absence of inert particles. It selectively inhibited 32Pi incorporation into phosphatidic acid and the phosphoinositides and the incorporation of myo-[2-3H]inositol into the phosphoinositides. Added ATP (0.1--1.0 MM), but not other nucleotides, was found to potentiate beta-glucuronidase release provoked by cytochasin B, but it impaired the labeling of the phosphoinositides by myo-[2-3H]inositol. The mechanism of the inhibition the isotope incarparation into these acidic phospholipids by the two mucleotides has not been defined. Dibutyryl cyclic adenosine 3':5'-monophosphate at 2--4 mM concentration was not found to appreciably alter the incorporation of [gamma-32P]ATP into phosphatidic acid, phosphatidylinositol, diphosphoinositide, and triphosphoinositide. 相似文献
10.
Relation between phosphorylation and adenosine triphosphate-dependent Ca2+ binding of swine and bovine erythrocyte membranes 总被引:2,自引:0,他引:2
The correlation between the ATP-dependent Ca2+ binding and the phosphorylation of the membranes from swine and bovine erythrocytes was studied. The Ca2+ binding was measured by using 45CaCl2, and the phosphorylation by [gamma-32P]ATP was studied with the technique of SDS polyacrylamide gel electrophoresis. 200 mM NaCl and KCl markedly repressed the Ca2+ binding of swine erythrocyte membranes. The radioactivity of 32P-labelled membranes was revealed mainly in 250,000 dalton protein and a lipid fraction. NaCl and KCl also repressed the phosphorylation of the lipid which was identified as triphosphoinositide by paper chromatography. The membranes prepared from trypsin-digested erythrocytes completely retained the Ca2+-binding activity, and lost 30% of (Ca2+ + Mg2+)-ATPase activity. The Ca2+-binding and ATPase activity of isolated membranes decreased to 55% and to 0%, respectively, by tryptic digestion. Neither the Ca2+ binding nor the phosphorylation of polyphosphoinositides were detected in bovine erythrocyte membranes. These results suggest that the formation of triphosphoinositide rather than the (C2+ + Mg2+)-ATPase of membranes is linked to the ATP-dependent Ca2+ binding of erythrocyte membranes. 相似文献
11.
High heat of enzymatic hydrolysis of triphosphoinositide phosphate bonds and of high-energy phosphates is observed using microcalorimetry. Heats of hydrolysis of triphosphoinositide, ADP and ATP sharply increase with increasing pH values from 6.6 to 7.4. Heat of hydrolysis of diphosphoinositide correlates with that of low-energy phosphates, pK4 and pK5 values for triphosphoinositide are found to be 7.4 and 9.3 respectively by means of potentiometric titration deltaGo' values for diphosphoinositide and triphosphoinositide are -3.5 and -7.1 kcal/mole respectively, taking into consideration the correction for heat neutralization-ionization during hydrolysis. Rapid triphosphoinositide hydrolysis takes place in 1% aqueous pyridine solution at 100 degrees C. In contrast to diphosphoinositide and monophosphoinositide, infrared spectra of triphosphoinositide have an additional absorption band at 930 cm(-1). 31P NMR method has revealed the presence of one diester and two monoester groups in the molecule of triphosphoinositide. The differences described between triphosphoinositide and other compounds with phosphomonoester groups are suggested to be due to electrostatic nonbounded interaction of vicinal diequatorial phosphate groups. 相似文献
12.
The role of pH, KCl, ATP, water activity, and temperature in ATP synthesis from ADP and Pi was investigated in sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle. In totally aqueous medium, the synthesis of ATP was inhibited by ATP, KCl, and pH values above 6.5. When the water activity of the medium was decreased by the addition of 30% (v/v) dimethyl sulfoxide, the synthesis of ATP was no longer inhibited by ATP; it was activated by KCl and the optimum pH changed from 6.5 to 7.5. In totally aqueous medium, the concentration of MgCl2 needed for half-maximal synthesis of ATP was found to vary with the temperature of the assay medium; at 35 degrees C it was 1 mM and increased to a value higher than 10 mM when the temperature was decreased to 15 degrees C. In the presence of 30% dimethyl sulfoxide, maximal synthesis of ATP was attained in presence of 0.05 mM MgCl2 at both 15 and 35 degrees C. The hypothesis is raised that in the living cell water structure may play a role in regulating the synthesis of ATP observed during the reversal of the Ca2+ pump of the sarcoplasmic reticulum. 相似文献
13.
G V Kiselev A N Minenko P Oeme 《Biulleten' eksperimental'no? biologii i meditsiny》1986,102(11):566-567
2- and 4-month-old male spontaneously hypertensive rats (SHR) were injected fusaric acid at a dose of 50 mg/kg body weight. Fusaric acid increased diphosphoinositide (DPI) and triphosphoinositide (TPI) levels in erythrocyte membranes of 4-month-old SHR by 41% and 20%, respectively. 32P incorporation into TPI decreased by 24% in 2- and by 20% in 4-month-old SHR. Phosphatidylinositol metabolism remained unchanged. The results also suggest that fusaric acid normalized DPI and TPI metabolism in erythrocyte membranes of SHR. 相似文献
14.
M R Hokin 《Journal of neurochemistry》1970,17(3):357-364
(1) Dopamine–In slices from guinea pig corpus striatum, dopamine significantly inhibited incorporation of 32P into phosphatidylethanolamine-plus-phosphatidylserine at a concentration of 0001 mM, and into phosphatidylinositol and phosphatidylcholine at 001 mM. In eight areas of the guinea pig brain in which the effects of 01 mM-dopamine were studied, the only significant increase in incorporation of 32P into phosphatides was into phosphatidic acid in the hypothalamus; there was significant inhibition of incorporation of 32P into phosphatidylcholine in cerebellar cortex and thalamus, and into phosphatidylethanolamine-plus-phosphatidylserine in the olfactory bulbs. (2) Gamma-aminobutyric acid—In slices of guinea pig cerebral cortex, GABA (1 mM) significantly inhibited incorporation of 32P into only phosphatidic acid, diphosphoinositide and phosphatidylinositol and did not significantly affect the level or the specific activity of the nucleotide ~P. GABA (10 mM), significantly inhibited incorporation of 32P into diphosphoinositide, phosphatidylinositol and phosphatidylcholine, and significantly lowered the specific activity of the nucleotide ~P. (3) 5-Hydroxytryptamine—In slices of guinea pig cerebral cortex, 5HT, (1 mM) significantly increased incorporation of 32P into phosphatidic acid; in a concentration of 10 mM, 5HT increased incorporation of 32P into phosphatidic acid four-fold and into both diphosphoinositide and phosphatidylinositol two-fold; other phosphatides were not significantly affected and the specific activity of the nucleotide ~P was not significantly different. In eight brain areas studied, 5HT (10 mM) significantly increased incorporation of 32P into phosphatidic acid in all areas; into phosphatidylinositol in six areas (excepting cerebellar cortex and hypothalamus); and into diphosphoinositide in the olfactory bulbs, cerebral cortex, hypothalamus and corpus striatum. Incorporation of 32P into triphosphoinositide was not significantly affected in any area. Incorporation of 32P into phospha-tidylethanolamine-plus-phosphatidylserine was significantly greater than the control in the olfactory bulbs and incorporation of 32P into phosphatidylcholine was significantly less than the control in the cerebellar cortex, olfactory bulbs and hypothalamus. (4) The possibility is discussed that increased incorporation of 32P into phosphatidic acid and/or phosphatidylinositol in response to neurotransmitters might be associated with excitatory, but not inhibitory, neurotransmission; and that inhibition of incorporation of 32P into various phosphatides may be associated with inhibitory neurotransmission or neuromodulation. 相似文献
15.
Triton X-100 is known to affect phospholipid metabolism and the generation of various signal molecules from cellular phospholipids. In the present work the effect of Triton X-100 on phospholipid metabolism of human decidua and of the primordial placenta (chorion frondosum) was studied. Triton X-100 (0.05%, v/v) added to tissue mince 30 min before the end of a 60 min incubation stimulated 2-4-fold (decidua) and 4-6-fold (placenta) the incorporation of [32P]phosphate ([32P]Pi) into phosphatidic acid, while markedly decreasing the labeling of phosphatidylcholine. Triton X-100 had no effect on the labeling of phosphatidylinositol in the decidua, and only a slight increase was observed in the placenta. When labeled glucose was used to assess phospholipid synthesis, the addition of Triton had no effect on phosphatidic acid, while decreasing the synthesis of phosphatidylcholine. Incorporation of [32P]Pi into phosphatidic acid was not accelerated by a submicellar concentration (0.01%) of Triton, whereas the synthesis of phosphatidylcholine was decreased irrespective of detergent concentration. Anionic or cationic detergents could not mimic the action of Triton on phosphatidic acid synthesis. Although Triton inhibited the synthesis of ATP in a dose-dependent manner, this could not account for the above results. Instead, it is suggested that diacylglycerol kinase and phosphocholine:CTP cytidylyltransferase are possible targets of the action of Triton X-100. 相似文献
16.
Human erythrocyte membranes (ghosts) prepared from fresh blood changed in shape from spherical to crenated, when suspended in 10(-7)-10(-6) M Ca2+-EGTA buffers. Although the ghosts from long-stored ACD blood (10 weeks) were less sensitive to 10(-7)-10(-6) M Ca2+, the ghosts obtained from this blood after it had been preincubated with adenine and inosine for 3 h at 37 degrees C were highly sensitive to Ca2+. When these highly sensitive ghosts were incubated in 10 mM Tris-Cl buffer (pH 7.4) or 1 mM MgCl2 (pH 7.4) at 0 degrees C, they gradually lost Ca2+ sensitivity within 60 min, but they recovered Ca2+ sensitivity again after re-incubation with 2 mM Mg-ATP for 20 min at 37 degrees C followed by washing with 1 mM MgCl2 (pH 7.4). The shape of these highly Ca2+-sensitive ghosts immediately changed from crenate to disc on addition of 1 mM Mg-ATP even at 6 degrees C in the presence of 10(-7)-10(-6) M Ca2+. A similar shape change was also observed when ghosts treated with 0.5% Triton X-100 (Triton shells) were used. Triton shells from fresh blood ghosts or from long-stored blood ghosts which had been preincubated with 2 mM Mg-ATP for 20 min at 37 degrees C shrank immediately in the presence of 10(-6) M Ca2+ and then swelled on addition of 1 mM Mg-ATP. The specificity to ATP and the dependency on ATP concentration are in agreement with those of the ghost shape change at step 2 (Jinbu, Y. et al., Biochem biophys res commun 112 (1983) 384-390) [18]. These results suggest that cytoskeletal protein phosphorylation enhances sensitivity to Ca2+ and induces erythrocyte shape change in the presence of physiological concentrations of ATP and Ca2+. 相似文献
17.
J D Robinson 《Biochimica et biophysica acta》1981,642(2):405-417
MnCl2 was partially effective as a substitute for MgCl2 in activating the K+- dependent phosphatase reaction catalyzed by a purified (Na+ + K+)-ATPase enzyme preparation from canine kidney medulla, the maximal velocity attainable being one-fourth that with MgCl2. Estimates of the concentration of free Mn2+ available when the reaction was half-maximally stimulated lie in the range of the single high-affinity divalent cation site previously identified (Grisham, C.M. and Mildvan, A.S. (1974) J. Biol. Chem. 249, 3187--3197). MnCl2 competed with MgCl2 as activator of the phosphatase reaction, again consistent with action through a single site. However, with MnCl2 appreciable ouabain-inhibitable phosphatase activity occurred in the absence of added KCl, and the apparent affinities for K+ as activator of the reaction and for Na+ as inhibitor were both decreased. For the (Na+ + K+)-ATPase reaction substituting MnCl2 for MgCl2 was also partially effective, but no stimulation in the absence of added KCl, in either the absence or presence of NaCl, was detectable. Moreover, the apparent affinity for K+ was increased by the substitution, although that for Na+ was decreased as in the phosphatase reaction. Substituting MnCl2 also altered the sensitivity to inhibitors. For both reactions the inhibition by ouabain and by vanadate was increased, as was binding of [48V] -vanadate to the enzyme; furthermore, binding in the presence of MnCl2 was, unlike that with MgCl2, insensitive to KCl and NaCl. Inhibition of the phosphatase reaction by ATP was decreased with 1 mM but not 10 mM KCl. Finally, inhibition of the (Na+ + K+)-ATPase reaction by Triton X-100 was increased, but that by dimethylsulfoxide decreased after such substitution. These findings are considered in terms of Mn2+ at the divalent cation site being a better selector than Mg2+ of the E2 conformational states of the enzyme, states also selected by K+ and by dimethylsulfoxide and reactive with ouabain and vanadate; the E1 conformational states, by contrast, are those selected by Na+ and ATP, and also by Triton X-100. 相似文献
18.
The effect of isobutylmethylxanthine on the photoresponse and phosphorylation of phosphatidylinositol in octopus retina 总被引:2,自引:0,他引:2
The effect of isobutylmethylxanthine on the photoresponse and the phosphorylation of phosphatidylinositol was studied. The recovery of photoresponse after a bright flash was greatly altered by the addition of isobutylmethylxanthine. Phosphatidylinositol phosphorylation, assessed by the incorporation of label from [gamma-32P]ATP, was influenced by isobutylmethylxanthine. Incorporation of 32P was decreased into diphosphoinositide and triphosphoinositide, and increased into phosphatidic acid. These changes were interpreted by introducing a parameter, phi, calcium charge of the plasma membrane. 相似文献
19.
Jen-sie Tou Christine Maier 《Biochimica et Biophysica Acta (BBA)/General Subjects》1976,451(2):353-362
The effects of dibutyryl cyclic adenosine 3′ : 5′-monophosphate and ATP on isotope incorporation into phospholipids and the release of β-glucuronidase into the extracellular medium were studied in polymorphonuclear leukocytes from guinea pig peritoneal exudates. Exogenous dibutyryl cyclic adenosine 3′ : 5′-monophosphate (0.1–1.0 mM) reduced β-glucoronidase release induced by cytochalasin B in the absence of inert particles. It selectively inhibited 32Pi incorporation into phosphatidic acid and the phosphoinositides and the incorporation of myo-[2-3H]inositol into the phosphoinositides. Added ATP (0.1–1.0 mM), but not other nucleotides, was found to potentiate β-glucuronidase release provoked by cytochasin B, but it impaired the labeling of the phosphoinositides by myo-[2-3H]inositol. The mechanism of the inhibition of the isotope incorporation into these acidic phospholipids by the two nucleotides has not been defined. Dibutyryl cyclic adenosine 3′ : 5′-monophosphate at 2–4 mM concentration was not found to appreciably alter the incorporation of [γ-32P]ATP into phosphatidic acid, phosphatidylinositol, diphosphoinositide, and triphosphoinositide. 相似文献
20.
Incubation of rabbit erythrocytes with 32Pi resulted in labeling of membrane diphosphoinositide, triphosphoinositide, and phosphatidic acid. Hypotonic lysis at 37 degress C resulted in an extremely rapid breakdown of the labeled polyphosphoinositides. This breakdown could be retarded by lysis in the presence of EDTA and by lowering the temperature to 0 degrees thus allowing preparation of membranes with minimum breakdown of the labeled lipids. Rapid breakdown of di- and triphosphoinositide in isolated membranes could be initiated by Ca++ or to a lesser extent by Mg++ and prevented by detergents and by heating to 75 degrees C. Assay of radiolabeled lipid was carried out by a method which bypassed prior lipid extraction and which enabled sequential sampling of reactions at 10-second intervals. This method was more convenient than standard procedures and gave yields of di- and triphosphoinositide equivalent to that obtained by the method of Folch. 相似文献