Effects of glucose transport on the incorporation of 32P-inorganic phosphate into phospholipids in Hymenolepis diminuta (Cestoda)

1. The absorption of glucose and alpha-methyl-D-glucoside by Hymenolepis diminuta was concentration dependent. 2. alpha-Methyl-D-glucoside competitively inhibited glucose absorption and was not metabolised by the parasite. 3. alpha-Methyl-D-glucoside significantly lowered (P less than 0.05) the incorporation of 32Pi into all phospholipid classes. 4. Glucose inhibited (P less than 0.01) 32Pi incorporation into phosphatidylcholine only. 5. Phlorizin did not affect 32Pi labelling of phospholipids. 6. Serotonin and histamine stimulated (P less than 0.01) 32Pi labelling of all phospholipid classes. 7. Radioactivities in water soluble fractions were increased (P less than 0.01) in the presence of glucose, serotonin and histamine.     

Effects of 5-hydroxytryptamine (serotonin) on the incorporation of 32P-inorganic phosphate into phospholipids in Hymenolepis diminuta (Cestoda)

5-Hydroxytryptamine (5-HT) (1,2 and 5 mM) significantly stimulated the incorporation of radioactive inorganic phosphate (32Pi) into phosphatidylinositol, phosphatidylethanolamine, phosphatidic acid and also total phospholipid fraction of Hymenolepis diminuta after one hour of incubation. Such effect was both time and concentration dependent. In the presence of 5-HT early labelling of phosphatidylinositol was observed. Also, the percentage stimulation by 5-HT was the highest in this fraction under all experimental conditions. The inorganic, organic, total and phosphatidylcholine-bound phosphate of H. diminuta incubated with 5-HT were not significantly different from those of the control under all incubation conditions. Results reported herein suggest that messenger molecules that are derived from phosphoinositides may be involved in the stimulatory mechanism of 5-HT in H. diminuta.     

Brain cortex phosphatidylserine inhibits phosphatidylinositol turnover in rat anterior pituitary glands

The in vitro effect of bovine brain cortex phosphatidylserine on 32Pi incorporation into phosphatidylinositol, phosphatidylcholine, and phosphatidylethanolamine of rat anterior pituitary glands was studied. Phosphatidylserine (0.1 to 66.6 microM) decreased the incorporation of 32Pi into phosphatidylinositol, but not phosphatidylcholine or phosphatidylethanolamine, in a concentration-related manner. The inhibitory effect of phosphatidylinositol was similar to that of dopamine in the same experimental conditions. The combined effects of submaximal concentrations of dopamine and phosphatidylserine elicited an apparently additive inhibitory effect on phosphatidylinositol synthesis. The inhibitory effect of phosphatidylserine was completely reversed by haloperidol and sulpiride and only partially by pimozide, antidopaminergic agents which per se do not affect phosphatidylinositol synthesis. The stimulatory effect of TRH to increase 32Pi incorporation into phosphatidylinositol was decreased by phosphatidylserine. These observations suggest that the decrease in prolactin release in the presence of phosphatidylserine may be evoked through a dopaminergic mechanism.     

Modulation of 32Pi incorporation into phospholipids of polymorphonuclear leukocytes by ionophore A23187.

The effects of ionophore A23187 on the incorporation of 32Pi into phospholipids and on 45Ca2+ uptake and release by polymorphonuclear leukocytes were examined. A23187 increased 32Pi incorporation into phosphatidic acid, phosphatidylglycerol, phosphatidylserine, and the phosphoinositides. It also promoted a rapid burst uptake and release of 45Ca2+ by leukocytes. External Ca2+, but not Mg2+, was required for full stimulation of 32Pi incorporation into phosphatidic acid and the phosphoinositides. In the absence of external Ca2+, the increased radiophosphorus activity of phosphatidic acid, phosphatidylserine and the phosphoinositides was grossly reduced but not eliminated, and the decreased radiophosphorus activity of phosphatidylcholine became pronounced. In addition, the ionophore effect on 32Pi incorporation into leukocyte phospholipids was not abolished by ethyleneglycol bis(beta-amino-ethylether)-N,N'-tetraacetic acid. ATP radiophosphorus activity was also enhanced by the presence of A23187, but the enhancement was much less than that of the acidic phospholipids. Based on these findings, it is suggested that the increased 32Pi incorporation into the acidic phospholipids of leukocytes induced by A23187 was not solely derived from the higher radioactivity of ATP, increased Ca2+ fluxes and perturbation of cellular Ca2+ distribution of leukocytes exposed to A 23187 may trigger part of the altered 32Pi incorporation into phospholipids.     

Thyrotropin-releasing hormone and phorbol esters stimulate sphingomyelin synthesis in GH3 pituitary cells. Evidence for involvement of protein kinase C

Previous studies demonstrated that phorbol esters and thyrotropin-releasing hormone (TRH) stimulated phosphatidylcholine synthesis via protein kinase C in GH3 pituitary cells (Kolesnick, R. N. (1987) J. Biol. Chem. 262, 14525-14530). Since phosphatidylcholine may serve as the precursor for sphingomyelin synthesis, studies were performed to assess the effect of protein kinase C on sphingomyelin synthesis. The potent phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA), stimulated time- and concentration-dependent incorporation of 32Pi into the head group of sphingomyelin in cells short term labeled with 32Pi and resuspended in medium without radiolabel. TPA (10(-7) M) increased incorporation at a rate 1.4-fold of control after 2 h; EC50 congruent to 2 x 10(-9) M TPA. This correlated closely to TPA-induced phosphatidylcholine synthesis; EC50 congruent to 9 x 10(-10) M TPA. TRH (10(-7) M), which activates protein kinase C via a receptor-mediated mechanism, similarly stimulated 32Pi incorporation into sphingomyelin at a rate 1.5-fold of control; EC50 congruent to 5 x 10(-10) M TRH. This correlated closely with TRH-induced phosphatidylcholine and phosphatidylinositol synthesis; EC50 congruent to 2 x 10(-10) and 1.5 x 10(-10) M TRH, respectively. In cells short term labeled with [3H]palmitate, TRH induced a time- and concentration-dependent reduction in the level of [3H]ceramide and a quantitative increase in the level of [3H]sphingomyelin. Compositional analysis of the incorporated [3H]palmitate revealed that TRH increased radiolabel into both the sphingoid base and the fatty acid moieties of sphingomyelin. Similarly, TRH increased incorporation of [3H] serine into sphingomyelin to 145 +/- 8% of control after 3 h. TPA also stimulated these events. Like the effect of TRH on phosphatidylcholine synthesis, TRH-induced sphingomyelin synthesis was abolished in cells "down-modulated" for protein kinase C. In contrast, TRH-induced phosphatidylinositol synthesis still occurred in these cells. These studies suggest that protein kinase C stimulates coordinate synthesis of phosphatidylcholine and sphingomyelin. This is the first report of stimulation of sphingomyelin synthesis via a cell surface receptor.     

PHosphate metabolism and foetal growth in the rat. I. Net transfer of 31P and 32P phosphate from the maternal plasma to the normal placenta

Net transfer of 31P and 32P inorganic phosphate form the maternal plasma to the rat chorio-allantoic placenta has been studied after intraperitoneal injection of [32P] ortho-phosphate in primigravid females at the 12th day or late stages of gestation. The concentration and label uptake per unit weight of placenta of the inorganic phosphate (Pi), organic-bound acid-soluble phosphate (POAS) and organic-bound acid-insoluble phosphate (POAIS) fractions are negatively correlated with increasing placental weight, whereas their specific activities are independent of placental weight. The amount and label uptake per whole placenta of the Pi, POAS and POAIS fractions are positively correlated with increasing placental weight. The placental concentrations of inorganic phosphate and calcium are positively related without, however, any marked accumulation of calcium. The growing placenta is thus shown to reduce progressively, on a unit weight basis, both the inorganic phosphate uptake from the maternal plasma and its further incorporation into organic-bound fractions. There is no evidence of a control by the foetal weight, acting per se, on these placental activities.     

Thyrotropin-releasing hormone and phorbol esters induce phosphatidylcholine synthesis in GH3 pituitary cells. Evidence for stimulation via protein kinase C

Phorbol esters have been shown to stimulate phosphatidylcholine synthesis via the CDP-choline pathway. The present study compares the effects of phorbol esters and thyrotropin-releasing hormone (TRH) on phosphatidylcholine metabolism in GH3 pituitary cells. In a previous study (Kolesnick, R.N., and Paley, A.E. (1987) J. Biol. Chem. 262, 9204-9210), the potent phorbol ester, 12-O-tetradecanoylphorbol 13-acetate (TPA) induced time- and concentration-dependent incorporation of 32Pi and [3H]choline into phosphatidylcholine in short-term labeling experiments. In this study, TPA is shown to activate choline-phosphate cytidylyltransferase (EC 2.7.7.15), the regulatory enzyme of the CDP-choline pathway, by stimulating redistribution of the inactive cytosolic form of the enzyme to the membrane. Redistribution was quantitative. TPA reduced cytosolic activity from 3.5 +/- 0.4 to 1.5 +/- 0.3 nmol . min-1 x 10(7) cells-1 and enhanced particulate activity from 2.5 +/- 0.4 to 4.9 +/- 0.6 nmol . min-1 x 10(7) cells-1. TRH also stimulated time- and concentration-dependent 32Pi and [3H]choline incorporation into phosphatidylcholine. An increase was detectable after 5 min; and after 30 min, the levels were 164 +/- 9 and 150 +/- 11% of control, respectively; EC50 congruent to 2 X 10(-10) M TRH. These events correlated directly with TRH-induced 32Pi incorporation into phosphatidylcholine. TRH also stimulated redistribution of cytidylyl-transferase specific activity. TRH reduced cytosolic activity 45% and enhanced particulate activity 51%. Neither TRH nor TPA stimulated phosphatidylcholine degradation. In cells down-modulated for protein kinase C (Ca2+/phospholipid-dependent protein kinase), the effects of TPA and TRH on 32Pi incorporation into phosphatidylcholine were abolished. However, TRH-induced incorporation into phosphatidylinositol still occurred. These studies provide evidence that hormones may regulate phosphatidylcholine metabolism via the protein kinase C pathway.     

Inhibitory effect of glucose on the maturation of rat liver mitochondria at birth. Phospholipid and oxidative metabolism

(1) The rate of ATP synthesis coupled with succinate oxidation in rat liver mitochondria is low at birth and increases rapidly during the first postnatal hours (Nakazawa, T., Asami, K., Suzuki, H. and Yakawa, O. (1973) J. Biochem. 73, 397-406). A glucose injection given to newborn rats immediately after birth seemed to delay this maturation process. (2) Glucose administration specifically diminished the rate of 32Pi incorporation into phosphatidylcholine both in microsomes and in mitochondria while other phospholipids remained unaffected. (3) In newborn rat liver, 32Pi incorporation into phospholipids can be explained by de novo synthesis of phospholipids in microsomes followed by transfer to mitochondria with two exceptions phosphatidylserine and sphingomyelin. Indeed, after a 20-min incorporation of 32Pi into phospholipids, the specific radioactivity of phosphatidylserine and sphingomyelin was higher in mitochondria than in microsomes. (4) As far as phospholipid synthesis is concerned, no precursor-product relationship could be observed between light and heavy mitochondria.     

Studies on the preferential incorporation of [3H]glycerol over [32P]phosphate into major phospholipids of human platelets

It is well known that platelets readily incorporate radioactive glycerol, but not radioactive phosphate into phosphatidylcholine (PC) and phosphatidylethanolamine (PE) in vitro, thus not in accordance with de novo synthesis according to the Kennedy pathway. In attempts to understand the reason for the discrepancy, gel-filtered platelets were incubated simultaneously with [32P]Pi and [3H]glycerol, and the specific and relative radioactivities of products and intermediates were determined. Both precursors were incorporated into phosphatidylinositol (PI) with a 32P/3H ratio similar to that in glycerol 3-phosphate (in accordance with the Kennedy pathway). However, PC and PE obtained a much lower ratio. The specific 32P radioactivity in phosphorylcholine was similar to that of the gamma-phosphoryl of ATP and 650-times higher than that of PC. The specific 32P radioactivity of phosphorylethanolamine was 20-times less than that of phosphorylcholine. Both mass and 32P labelling of CDP-choline were below the detection limits. It is concluded that the incorporation of [32P]Pi into PC via phosphorylcholine is insignificant while the preferential incorporation of [3H]glycerol could be explained by exchange of diacyl[3H]glycerol in the reversible choline phosphotransferase (CDP-choline: 1,2-diacylglycerol cholinephosphotransferase) reaction. The same mechanism would explain the preferential incorporation of 3H over 32P into PE, although dilution of 32P at the phosphorylethanolamine stage would account for part of the feeble 32P incorporation. Although other mechanisms are also possible, our results clearly show that the appearance of [3H]glycerol in PC and PE is not a reliable method of monitoring de novo synthesis of these phospholipids.     

Phosphate metabolism and foetal growth in the rat. III. Net transfer of 31P and 32P inorganic phosphate from the maternal plasma to the placenta after foetectomy

In primigravid rat females at the 12th day or later stages of gestation one foetus is surgically removed and the amniotic sac allowed to retract freely around the remaining placenta in situ. One hour to 10 days after foetectomy [32P]ortho-phosphate is injected intraperitoneally. The post-foetectomy placenta and matched normal placenta and foetus in the contralateral horn are taken after labelling periods of one or three hours and analysed for calcium, inorganic phosphate, organic-bound acid-soluble and organic-bound acid-insoluble phosphates in their [31P] and [32P] forms. The acutely foetectomized placenta--up to 24 hours after surgery--may serve as an appropriate model of the physiologically functioning syncytiotrophoblast: it is indeed noteworthy that organic-bound phosphate placental concentrations and labelling are little affected in the early post-foetectomy stages. On the other hand the placental inorganic phosphate concentration and labelling are profoundly affected by foetectomy. A surplus radioactive incorporation in the Pi fraction is observed which is strongly related to the weight of the control foetus and may represent up to twelve times the label incorporation in the control placenta; it decreases as the time interval since foetectomy lengthens. A cumulative increase in Pi concentration is observed which is strongly related to calcium concentration and may reach twenty-five times the maternal plasma Pi concentration. The presence of two independent pools of Pi is suggested: one geared to the metabolic needs of the placenta itself, the other representing the Pi normally transported to the foetus under metabolic control by the latter. The possibility of an electroneutral PO4(2-)/Ca2+ co-transport is evoked.     

Effect of Auxin,Kinetin and Monosaccharide on Callus Cell wall Composition of Abutilon avicennae Gaertn

The effects of exogenous kinetin, IAA, glucose (the component of cellulose) and galactose (one of tile inhibiting compounds of cellulose synthesis) on callus cell wall composition of China jute (Abutilon avicennae Gaertn) were investigated. When the China jute callus materials were treated with 2 mg/l IAA and H3-glucose, there was a 437% increase of the Ha-glucose, as compared with control. When galactose was added simultaneously with H3-glucose, there was a 138% increase of the H3-glucose as compared with control. Galactose inhibited H3-glucose incorporate content in cellulose, and also in pectin and hemicellulose. In the experiment with 10 mg/l kinetin treatment of the callus materials, when galactose was added, the incorporation H3-glucose was inhibited. It is interesting that the kinetin enhanced H3-galactose incorporation into all cell wall components. When it was added simultaneously with glucose, glucose inhibited H3-galactose into the cell Wall components. Results showed that the hormones effect cell wall component, not only concerned with hormones, but also concerned with exogenous monosaccharides. When exogenous monosacchrides were added to the culture medium (as galactose, glucose) in combination or respectively, they are quite different in effecting hormone induced China jute callus cell wall. When the callus was treated by IAA, galactose inhibited H3-glucose incorporation into cell wall components. When it was treated by kinetin, glucose inhibited H3-galactose incorporation into cell wall components.     

Phosphatidylserine biosynthesis in cultured Chinese hamster ovary cells. I. Inhibition of de novo phosphatidylserine biosynthesis by exogenous phosphatidylserine and its efficient incorporation

The effect of phosphatidylserine exogenously added to the medium on de novo biosynthesis of phosphatidylserine was investigated in cultured Chinese hamster ovary cells. When cells were cultured for several generations in medium supplemented with phosphatidylserine and 32Pi, the incorporation of 32Pi into cellular phosphatidylserine was remarkably inhibited, the degree of inhibition being dependent upon the concentration of added phosphatidylserine. 32Pi uptake into cellular phosphatidylethanolamine was also partly reduced by the addition of exogenous phosphatidylserine, consistent with the idea that phosphatidylethanolamine is biosynthesized via decarboxylation of phosphatidylserine. However, incorporation of 32Pi into phosphatidylcholine, sphingomyelin, and phosphatidylinositol was not significantly affected. In contrast, the addition of either phosphatidylcholine, sphingomyelin, phosphatidylethanolamine, or phosphatidylinositol to the medium did not inhibit endogenous biosynthesis of the corresponding phospholipid. Radiochemical and chemical analyses of the cellular phospholipid composition revealed that phosphatidylserine in cells grown with 80 microM phosphatidylserine was almost entirely derived from the added phospholipid. Phosphatidylserine uptake was also directly determined by using [3H]serine-labeled phospholipid. Pulse and pulse-chase experiments with L-[U-14C] serine showed that when cells were cultured with 80 microM phosphatidylserine, the rate of synthesis of phosphatidylserine was reduced 3-5-fold whereas the turnover of newly synthesized phosphatidylserine was normal. Enzyme assaying of extracts prepared from cells grown with and without phosphatidylserine indicated that the inhibition of de novo phosphatidylserine biosynthesis by the added phosphatidylserine appeared not to be caused by a reduction in the level of the enzyme involved in the base-exchange reaction between phospholipids and serine. These results demonstrate that exogenous phosphatidylserine can be efficiently incorporated into Chinese hamster ovary cells and utilized for membrane biogenesis, endogenous phosphatidylserine biosynthesis thereby being suppressed.     

Galactose utilization by the rat tapeworm, Hymenolepis diminuta.

1. Hymenolepis diminuta incorporated label from 14C-galactose into glycogen, but the sugar would not support net glycogen synthesis. Glucose stimulated the incorporation of label from 14C-galactose into glycogen, while glycerol did not. 2. During incubations in galactose, large internal pools of galactose and galactose 1-P accumulated, while the concentration of glucose 6-phosphate remained unchanged. 3. In vitro culture experiments indicated that galactose would not support worm growth. Therefore, while galactose can be metabolized to a limited extent, it cannot substitute for glucose as a nutrient source.     

Studies on the energy metabolism of opossum (Didelphis Virginiana) erythrocytes. I. Utilization of carbohydrates and purine nucleosides

Opossum erythrocytes filtered through cellulose columns were used to estimate their permeability to D-glucose and optimum inorganic phosphate requirement for D-glucose utilization at pH 7.4 and 8.1. D-Glucose readily penetrated opossum red cells; there was no measurable difference whether plasma or electrolyte solution served as the suspending medium. Optimum extracellular inorganic phosphate concentration for glucose utilization as indicated by red cell lactate production was pH-dependent, with a sharp optimum of 30 mmol/liter at pH 8.1. Whereas glucose, fructose, mannose, dihydroxyacetone, adenosine, and inosine were readily utilized at pH 7.4 and Pi 30 mmol/liter as shown by net lactate and ATP production by the red cells, galactose and ribose as substrates were not metabolized. In electrolyte, Pi 30 mmol/liter, and pH 7.4 glucose utilization by opossum red cells averaged 3.5 mumol, at pH 8.1, 9.5 mumol/ml cells/hr were utilized. Red cells suspended in leukocyte-free plasma utilized D-glucose at a rate of 3.0 mumol/ml/hr at pH 7.5. Seven percent of D-glucose flowed through the pentose phosphate pathway; this rate increased 11-fold by methylene blue stimulation. The amount of D-glucose recycled through the pentose phosphate pathway increased 300-fold in the presence of the redox dye.     

Influence of potassium and calcium ions on the phosphatidylinositol and phosphatidylcholine metabolism in rat fibroblasts after growth stimulation by calf serum.

In secondary cultures of embryonic rat fibroblasts which were arrested in G1 (G0) by serum depletion and subsequently triggered into the cell cycle by readdition of growth factors isolated from fetal calf serum the influence of the potassium and calcium concentrations in the medium on phosphatidylinositol and phosphatidylcholine metabolism was investigated. The incorporation of inorganic [32P]phosphate into phosphatidylinositol is dependent on the potassium content of the culture medium. The specific activity of 32P in phosphatidylinositol is increased at K+ concentrations between 0.1 and 1 mM. Also calcium (between 0.01 and 2 mM) slightly stimulates phosphatidylinositol metabolism. Also the incorporation of myo-[3H]inositol is increased at potassium concentrations between 0.2 and 1 mM, whereas calcium is slightly inhibitory. The labelling of phosphatidylcholine with either [32P]phosphate or [3H]choline is not dependent on the potassium and calcium concentrations of the culture medium. Moreover, the phospholipid metabolism of permanently growing epithelioid and fibroblastoid cells lines, which were investigated, is considerably less dependent on the K+ and Ca2+ ions.     

Studies on (Na+ + K+)-activated ATPase. XXXVIII. A 100 000 molecular weight protein as the low-energy phosphorylated intermediate of the enzyme.

Phosphorylation of NaI-treated bovine brain cortex microsomes by inorganic phosphate in the presence of Mg2+ and ouabain has been studied at 0 degrees C (pH 7.4) and 20 degrees C (pH 7.0). Nearly maximal (90%) and half-maximal phosphorylation are achieved at 20 degrees C within 2 min with 50--155 and 5.6--17 muM 32Pi, respectively, and at 0 degrees C within 75 s with 300--600 and 33--66 muM 32Pi, respectively. Maximal phosphorylation yields 146 pmol 32P - mg-1 protein. Without ouabain (20 degrees C, pH 7.0) less than 25% of the incorporation observed in the presence of ouabain is reached. Preincubation of the native microsomes with Mg2+ and K+, in order to decompose possibly present high-energy phosphoryl-bonds prior to ouabain treatment, does not affect the maximal phosphate incorporation. This indicates that the inorganic phosphate incorporation is not due to an exchange with high-energy phosphoryl-bonds, which might have been preserved in the microsomal preparations. Phosphorylation of the native microsomes by ATP in the presence of Mg2+ and Na+ reaches 90 and 50% maximal levels within 15--30 s at 0 degrees C and pH 7.4 at concentrations of [gamma-32P]ATP of 5--32 and 0.5--3.5 muM, respectively. The maximal phosphorylation level is 149 pmol 32P-mg-1 protein, equal to that of ouabain-treated microsomes phosphorylated by inorganic phosphate. Both inorganic phosphate and ATP phosphorylate on site per active enzyme subunit of 135 000 molecular weight. From the equilibrium constants for the phosphorylation of ouabain-treated microsomes by inorganic phosphate at 0 degrees C and 20 degrees C standard free-energy changes of --5.4 and --6.8 kcal/mol, respectively, are calculated. These values yield a standard enthalpy change of 14 kcal/mol and an entropy change of 70 cal/mol - degree K. This characterizes the reaction as a process driven by an entropy change. The intermediate formed by phosphorylation with Pi has maximal stability at acidic pH, as is the case for the intermediate formed with ATP. Solubilization in sodium dodecyl sulfate stabilizes the phosphoryl-bond in the pH range of 4--7. The non-solubilized preparation has optimal stability at pH 2--4, the level of which is equal to that of detergent-solubilized intermediate. Sodium dodecyl sulfate gel electrophoresis of the microsomes at pH 3, following incorporation of 32Pi yields 11 protein bands, only one of which (mol. wt 100 000--106 000) carries the radioactive label. This protein has the same molecular weight as the protein, which is phosphorylated by ATP in the presence of Mg2+ and Na+.     

Agonist-stimulated inositol phosphate metabolism in avian erythrocytes.

1. A screen for agonists capable of stimulating the formation of inositol phosphates in erythrocytes from 5-day-old chickens revealed the presence of a population of phosphoinositidase C-linked purinergic receptors. 2. If chicken erythrocytes prelabelled with [3H]Ins were exposed to a maximal effective dose of adenosine 5'-[beta-thio]diphosphate for 30 s, the agonist-stimulated increment in total [3H]inositol phosphates was confined to [3H]Ins(1,4,5)P3, Ins(1,3,4,5)P4 and InsP2. After 40 min stimulation, the radiolabelling of nearly all of the [3H]inositol phosphates that have been detected in these extracts [Stephens, Hawkins & Downes (1989) Biochem. J. 262, 727-737] had risen. However, some of these increases [especially those in Ins(3,4,5,6)P4 and Ins(1,3,4,5,6)P5] were accountable for almost entirely by increases in specific radioactivity rather than in mass. 3. The effect of purinergic stimulation on the rate of incorporation of [32P]Pi in the medium into the gamma-phosphate group of ATP and InsP4 and InsP5 was also measured. After 40 min stimulation, the incorporation of 32P into Ins(1,3,4,6)P4, Ins(1,3,4,5)P4, Ins(3,4,5,6)P4 and Ins(1,3,4,5,6)P5 was significantly elevated, whereas the mass of the last two and the specific radioactivity of the gamma-phosphate of ATP were unchanged compared with control erythrocyte suspensions. 4. In control suspensions of avian erythrocytes, the specific radioactivity of the individual phosphate moieties of Ins(1,3,4,6)P4 increased through the series 1, 6, 4 and 3 [Stephens & Downes (1990) Biochem. J. 265, 435-452]. This pattern of 32P incorporation is not the anticipated outcome of 6-hydroxy phosphorylation of Ins(1,3,4)P3 [the assumed route of synthesis of Ins(1,3,4,6)P4]. Although adenosine [beta-thio]diphosphate significantly stimulated the accumulation of [3H]Ins(1,3,4)P3, and despite the fact that avian erythrocyte lysates were shown to possess a chromatographically distinct, soluble, ATP-dependent, Ins(1,3,4)P3 6-hydroxykinase activity, purinergic stimulation of intact cells did not significantly alter the pattern of incorporation of [32P]Pi into the individual phosphate moieties of Ins(1,3,4,6)P4. These results suggest that the route of synthesis of this inositol phosphate species is not changed during the presence of an agonist.     

Glycogen synthase of Hymenolepis diminuta. II. Nutritional state, interconversion of forms, and primer glycogen molecular weight as control factors.

Glycogen synthase I (UDP glucose: glycogen alpha-4-glycosyltransferase, EC2.4.1.11) of the tapeworm Hymenolepis diminuta is the form of the enzyme which is active in vivo, while the D-form represents an inactive "storage form." Utilizing the differential effect of inorganic phosphate (Pi) on the I and D-forms, the ratio of the 2 forms in vivo has been determined under conditions of starvation of the host and refeeding of the parasite with glucose. This procedure reveals that conversion of the inactive D-form to the active I-form takes place when glycogen-depleted worms are incubated in glucose. The activity of glycogen synthase I also is affected by the molecular weight of the primer glycogen. With certain molecular weight fractions, enzymatic activity is higher than with others. This specificity of the glycogen primer could explain the relatively low concentrations of those molecular weight fractions which confer the highest synthase activity.     

Triton X-100 promotes the accumulation of phosphatidic acid and inhibits the synthesis of phosphatidylcholine in human decidua and chorion frondosum tissues in vitro

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.     

Factors which affect the amount of inorganic phosphate, phosphorylcholine, and phosphorylethanolamine in xylem exudate of tomato plants

Phosphate in the xylem exudate of tomato (Lycopersicon esculentum) plants was 70 to 98% inorganic phosphate (Pi), 2 to 30% P-choline, and less than 1% P-ethanolamine. Upon adding ³²Pi to the nutrient, Pi in xylem exudate had the same specific activity within 4 hours. P-choline and P-ethanolamine reached the same specific activity only after 96 hours. The amount of Pi in xylem exudate was dependent on Pi concentration in the nutrient and decreased from 1700 to 170 micromolar when Pi in the nutrient decreased from 50 to 2 micromolar. The flux of 0.4 nmoles organic phosphate per minute per gram fresh weight root into the xylem exudate was not affected by the Pi concentration in the nutrient solution unless it was below 1 micromolar. During 7 days of Pi starvation, Pi in the xylem exudate decreased from 1400 to 130 micromolar while concentrations of the two phosphate esters remained unchanged.

The concentration of phosphate esters in the xylem exudate was increased by addition of choline or ethanolamine to the nutrient solution, but Pi remained unchanged. Upon adding [¹⁴C]choline to the nutrient, 10 times more [¹⁴C]P-choline than [¹⁴C]choline was in the xylem exudate and 85 to 90% of the ester phosphate was P-choline. When [¹⁴C]ethanolamine was added, [¹⁴C]P-ethanolamine and [¹⁴C]ethanolamine in the xylem sap were equal in amount. P-choline and P-ethanolamine accumulated in leaves of whole plants at the same time and the same proportion as observed for their flux into the xylem exudate. No relationship between the transport of P-choline and Pi in the xylem was established. Rather, the amount of choline in xylem exudate and its incorporation into phosphatidylcholine in the leaf suggest that the root is a site of synthesis of P-choline and P-ethanolamine for phospholipid synthesis in tomato leaves.