Primary role of NADH formed by glucose dehydrogenase in ATP provision at the early stage of spore germination in Bacillus megaterium QM B1551

Metabolic events involved in energy metabolism were studied in order to evaluate the ATP-forming ability of Bacillus megaterium QM B1551 spores at the very early stage of germination. When heat-activated spores were germinated on glucose as a sole substrate, its oxidation into gluconate (catalyzed by glucose dehydrogenase, EC 1.1.1.47), the accompanying NADH formation, oxygen uptake, and RNA synthesis were initiated immediately after germination, even when anaerobic breakdown of 3-phosphoglycerate (an ATP source for spores) and the subsequent glucose metabolism via the phosphorylating pathway were impaired by potassium fluoride (KF). In contrast, fructose metabolism and the accompanying metabolic events did not begin until a few minutes after triggering of germination, and those events were entirely abolished by KF, indicating that fructose metabolism is initiated exclusively via its phosphorylation by the ATP derived from endogenous 3-phosphoglycerate. Thus those results provided further evidence for our previous proposal (Otani et al (1987) Microbiol. Immunol. 31: 967-974; Sano et al (1988) Biochem. Biophys. Res. Commun. 151: 48-52) that the first molecules of ATP in germinating spores can be efficiently generated via aerobic oxidation of NADH, which is formed by glucose dehydrogenase. Fluorescence monitoring of NADH in germinating spores also supported this conclusion.     

Stimulation of phosphate uptake in human platelets by thrombin and collagen. Changes in specific 32P labeling of metabolic ATP and polyphosphoinositides

The uptake of [32P]phosphate by human, gel-filtered blood platelets and its incorporation into cytoplasmic ATP and polyphosphoinositides was studied. In unstimulated platelets, uptake was Na+o-dependent and saturable at approximately 20 nmol/min/10(11) cells with a half-maximal rate at 0.5 mM extracellular phosphate. Upon stimulation with thrombin or collagen, net influx of [32P]Pi was accelerated 5- to 10-fold. With thrombin, [32P]Pi efflux was also increased. After the first 2 min, efflux exceeded influx, resulting in the net release of [32P]Pi from the platelets. Since the stimulus-induced burst in [32P]Pi uptake paralleled the secretory responses, it might be an integral part of stimulus-response coupling in platelets. The stimulus-induced burst in net [32P]Pi uptake led to an enhanced labeling of metabolic ATP, which was already detectable at 5 s after stimulation with thrombin. Concomitantly, the incorporation of [32P]Pi into phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate was accelerated. The thrombin-induced increase in specific 32P radioactivity of cytoplasmic ATP fully accounted for the simultaneous increase in specific 32P radioactivity of these phosphoinositides. In studying the extent of 32P labeling of phosphorylated compounds in response to a cellular stimulus, it is therefore essential to measure the effect of the stimulus on the specific radioactivity of cytoplasmic ATP.     

Effects of fructose on the energy metabolism and acid-base status of the perfused starved-rat liver. A 31phosphorus nuclear magnetic resonance study.

Fructose metabolism has been studied with 31P n.m.r. in perfused livers from rats starved for 48h. The time course of changes in liver ATP, Pi and sugar phosphate (fructose l-phosphate) concentrations, and intracellular pH were followed in each perfusion after infusion of fructose to give an initial concentration of either 5mM or 10mM. Rapid falls in the concentrations of ATP and Pi and intracellular pH occurred after infusion of fructose, reaching a minimum after 4-5 min, which was lower in the 10mM group than in the 5mM group. These changes were accompanied by a rapid rise in fructose 1-phosphate, reaching a plateau also after 4-5 min. At both concentrations of fructose, after the early falls, some recovery of ATP, Pi and intracellular pH occurred; this was complete for Pi and intracellular pH in the 5mM-fructose experiments (within 12-30 min). Complete restoration of ATP to the pre-fructose value was not achieved in either the 5mM of 10mM groups. Measurements of the uptake of lactate by the liver indicated that the fall in intracellular pH was caused primarily by production of protons accompanying the formation of lactate from fructose with possibly a transient contribution generated during the rise in fructose 1-phosphate.     

The mechanism of adenosine triphosphate depletion in the liver after a load of fructose. A kinetic study of liver adenylate deaminase.

1. The hepatic concentration of several nucleotides and metabolites was measured during the first few minutes after an intravenous load of fructose to mice. The first changes, observed at 30s, were a decrease in the concentration of Pi and a simultaneous accumulation of fructose 1-phosphate. The decrease in the concentrations of ATP and GTP proceeded more slowly. An increase in the concentration of IMP was detected only after 1 min and could therefore not be considered to be the cause of the accumulation of fructose 1-phosphate. 2. To explain the temporary burst of adenine nucleotide breakdown that occurs after a load of fructose, the kinetics of AMP deaminase (EC 3.5.4.6) from rat liver were reinvestigated at physiological (0.2 mM) concentration of substrate. For this purpose, a new radiochemical-assay procedure was developed. At 0.2mM-AMP a low activity could be measured, which was more than 90% inhibited by 5mM-Pi. ATP (3MM) increased the enzyme activity over 200-fold. Pi alone did not influence the ATP-activated enzyme, but 0.5mM-GTP caused a 60% inhibition. The combined effect of both inhibitors at their physiological concentrations reached 95%. 3. It is proposed that the rapid degradation of adenine nucleotides that occurs after a load of fructose is caused by a decrease in the concentration of both inhibitors, Pi and GTP, soon counteracted by the decrease in the concentration of ATP. 4. Some of the kinetic parameters of liver AMP deaminase were computed in terms of the concerted transition theory of Monod, Wyman & Changeux (1965) (J. Mol. Biol. 12, 88-118).     

Changes in 32P-phosphorus compounds during translocation in Laminaria digitata (L.) lamouroux

³²P was applied to a Laminaria digitata thallus and the pattern of ³²P phosphorylated compounds was studied, as a function of time, in the different tissues involved in translocation, i.e. source, pathway and sinks. The results showed that, 3 hours after absorption by the uptake region (lamina), the bulk of the radioactivity was incorporated into organic compounds (70 to 80% of total ³²P taken up), hexose monophosphates being the heaviest labelled. Further change in that region was marked by an accumulation of ³²P in the inorganic pool (65 to 70% after 13 days). Conversely, the ³²P pattern in the medulla of the stipe, which initially showed a similar pattern to the uptake region, did not vary during translocation. The pattern of ³²P distribution into sinks (growing stipe peripheral tissue or hapteron) leads to accumulation of the radioactive element in inorganic and acid-insoluble fractions. These results are discussed in terms of comparative distribution of ³²P in the different parts of the thallus and suggest that phosphate moves as Pi in that alga.Abbreviations TCA trichloroacetic acid - Po organic phosphate - Po sol acid-soluble organic phosphate fraction - Po insol acidinsoluble organic phosphate fraction - Pi morganic phosphate fraction - P lip lipidic phosphate - Np protein nitrogen - ATP adenosine triphosphate - ADP adenosine diphosphate - PEP phosphoenolpyruvic acid - PGA phosphoglyceric acid - G-1-P glucose-1-phosphate - G-6-P glucose-6-phosphate - UDPG uridine diphosphoglucose     

Acetylcholine increases the breakdown of triphosphoinositide of rabbit iris muscle prelabelled with [32P] phosphate.

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.     

Phosphatidylinositol metabolism in rat hepatocytes stimulated by vasopressin.

In isolated rat hepatocytes, vasopressin evoked a large increase in the incorporation of [32P]Pi into phosphatidylinositol, accompanied by smaller increases in the incorporation of [1-14C]oleate and [U-14C]glycerol. Incorporation of these precursors into the other major phospholipids was unchanged during vasopressin treatment. Vasopressin also promoted phosphatidylinositol breakdown in hepatocytes. Half-maximum effects on phosphatidylinositol breakdown and on phosphatidylinositol labelling occurred at about 5 nM-vasopressin, a concentration at which approximately half of the hepatic vasopressin receptors are occupied but which is much greater than is needed to produce half-maximal activation of glycogen phosphorylase. Insulin did not change the incorporation of [32P]Pi into the phospholipids of hepatocytes and it had no effect on the response to vasopressin. Although the incorporation of [32P]Pi into hepatocyte lipids was decreased when cells were incubated in a Ca2+-free medium, vasopressin still provoked a substantial stimulation of phosphatidylinositol labelling under these conditions. Studies with the antagonist [1-(beta-mercapto-beta, beta-cyclopentamethylenepropionic acid),8-arginine]vasopressin indicated that the hepatic vasopressin receptors that control phosphatidylinositol metabolism are similar to those that mediate the vasopressor response in vivo. When prelabelled hepatocytes were stimulated for 5 min and then subjected to subcellular fractionation. The decrease in [3H]phosphatidylinositol content in each cell fraction with approximately in proportion to its original phosphatidylinositol content. This may be a consequence of phosphatidylinositol breakdown at a single site, followed by rapid phosphatidylinositol exchange between membranes leading to re-establishment of an equilibrium distribution.     

Phosphatidic acid and phosphatidylinositol labelling in adipose tissue. The role of endogenously formed adenosine.

Incorporation of [32P]Pi into phosphatidic acid and phosphatidylinositol of hamster epididymal adipocytes was partially inhibited by 3-isobutyl-1-methylxanthine. This effect of 3-isobutyl-1-methylxanthine was antagonized by isopropyl-N6-phenyladenosine but not by 2',5'-dideoxyadenosine, prostaglandin E1 or clonidine. N6-Phenylisopropyladenosine did not affect incorporation of [32P]Pi into phosphatidic acid or phosphatidylinositol when 3-isobutyl-1-methylxanthine was not present. In contrast with 3-isobutyl-1-methylxanthine inhibition of [32P]Pi incorporation into phospholipids, which was blocked only by N6-phenylisopropyladenosine, accelerated lipolysis was blocked by prostaglandin E1, clonidine and 2',5'-dideoxyadenosine as well as by N6-phenylisopropyladenosine. Phospholipid labelling was also decreased in the presence of adenosine deaminase, but not in the presence of isoprenaline (isoproterenol). The stimulatory effect of N6-phenylisopropyladenosine on [32P]Pi incorporation into phospholipids in cells exposed to 3-isobutyl-1-methylxanthine was evident as soon as 3 min after addition of the adenosine analogue and maximum 10 min after its addition. As observed by others, [32P]Pi incorporation into phospholipids was increased by the alpha 1-selective agonist methoxamine. The stimulatory effect of methoxamine occurred with a time course similar to that of N6-phenylisopropyladenosine and was present at nearly equal magnitude in the absence or presence of 3-isobutyl-1-methylxanthine. The inhibitory effects of 3-isobutyl-1-methylxanthine and adenosine deaminase on phospholipid labelling are attributed to blockade of the action, or to the enzymic removal, of adenosine formed in and released from the fat-cells during their incubation. Supporting this view is the selective reversal of the actions of 3-isobutyl-1-methylxanthine and of adenosine deaminase by N6-phenylisopropyladenosine. These findings suggest an important role for endogenous adenosine in regulation of phospholipid turnover in adipocytes.     

Acid-soluble phosphorus compounds in mammalian semen

1. A method is described for the extraction, purification and separation of acid-soluble phosphorus compounds from mammalian semen. [8-(14)C]ATP and [8-(14)C]AMP were used as internal recovery standards to measure the breakdown and loss of these nucleotides in the procedure. 2. Bull, ram, boar and stallion semen was separated into seminal plasma and spermatozoa and the two fractions were examined separately. The overall composition of the mixture of the phosphorus compounds extracted from the two fractions was similar for the four species. 3. Glycerylphosphorylcholine and glycerylphosphorylinositol were the two phosphorus compounds identified in extracts of seminal plasma. ATP, ADP, AMP, GTP, GDP, NAD, fructose 1,6-diphosphate and glucose 6-phosphate were identified in extracts prepared from spermatozoa.     

Correlation of Carbohydrate Catabolism and Synthesis of Macromolecules During Enzyme Synthesis in Pseudomonas fluorescens.

Kirkland, Jerry J. (Oklahoma State University, Stillwater), and Norman N. Durham. Correlation of carbohydrate catabolism and synthesis of macromolecules during enzyme synthesis in Pseudomonas fluorescens. J. Bacteriol. 90: 23-28. 1965.-Glucose, ribose, and fructose shorten the lag period required for synthesis of protocatechuate oxygenase. Radioactivity from uracil-2-C(14) is incorporated into the hot trichloroacetic acid-soluble fraction after a lag period of approximately 20 min after addition of protocatechuic acid. Addition of glucose or ribose simultaneously with the inducer shortens the lag period to approximately 5 min and increases the rate of uracil incorporation. The inducer must be present to initiate incorporation of radioactivity, and the exogenous carbon source accelerates incorporation but is not sufficient to initiate synthesis by itself. The addition of protocatechuic acid increases the rate and total incorporation of radioactivity from uniformly labeled glucose or ribose-1-C(14) into the hot trichloroacetic acid-soluble fraction. Ribose decreases the incorporation of radioactivity from uniformly labeled glucose into the hot trichloroacetic acid-soluble fraction, and glucose shows a similar effect on incorporation of radioactivity from ribose-1-C(14), indicating the two sugars are serving in the same capacity to enhance enzyme synthesis. Radioactivity from glucose-1-C(14) is not incorporated into the hot trichloroacetic acid-soluble fraction. The results suggest that glucose and ribose shorten the lag period for inducible enzyme formation by serving as a "specific" carbon source for synthesis of macromolecules such as ribonucleic acid.     

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.     

Bovine spermatozoa incorporate 32Pi into ADP by an unknown pathway.

Intact ejaculated bovine sperm incorporate 32Pi into ADP to a specific activity two to three times higher than into ATP. This contrasts with other cell types where ATP specific activity is higher than that of ADP. Predominant labeling of ADP may be partially due to compartmentation of ATP, but removal of cytosolic ATP does not change the relative labeling of ADP and ATP. Dilution of extracellular 32Pi following labeling resulted in loss of 70% of label from ADP but only 50% loss from gamma-ATP at 26 min. ADP was labeled in the absence of detectable ATP in the presence of rotenone plus antimycin. Fractionation of ejaculated sperm yielded midpieces that are depleted of adenylate kinase and have coupled respiration. ATP was labeled with 32Pi, but ADP was not in midpieces. Evidence for mitochondrial substrate level phosphorylation-supported incorporation of 32Pi into nucleotides was observed for intact sperm incubated with pyruvate and inhibitors of oxidative phosphorylation, but this activity did not occur in midpieces and does not appear to explain disproportionate labeling of ADP. We conclude that labeling of ADP in intact and permeabilized cells occurs by two pathways; one involves adenylate kinase, and the other is an unknown pathway which may be independent of ATP.     

Failure of adrenaline to induce hyperglycaemia after fructose injection in young mice.

In control animals a 2-fold increase in liver phosphorylase activity 10min after adrenaline treatment was associated with a 55% increase in plasma glucose (P less than 0.001); at 20 min plasma glucose was 247% of the control value (P less than 0.001). Liver phosphorylase activity was decreased by 74%, 20 min after fructose injection (P less than 0.001), and, although phosphorylase activity increased 5-fold within 5 min of adrenaline injection, no increases in plasma glucose concentration over that found in fructose-injected animals which did not receive adrenaline occurred at either 5, 10 or 20 min. The data confirm inactivation of liver phosphorylase after fructose injection and suggest inhibition of the adrenaline-activated enzyme by the decrease in Pi and elevation of fructose 1-phosphate concentrations produced by the injection of fructose. These findings may be causally related to the hypoglycaemia and the lack of response to glucagon seen in patients with hereditary fructose intolerance after fructose ingestion.     

Metabolism and the triggering of germination of Bacillus megaterium. Concentrations of amino acids, organic acids, adenine nucleotides and nicotinamide nucleotides during germination.

A considerable amount of evidence suggests that metabolism of germinants or metabolism stimulated by them is involved in triggering bacterial-spore germination. On the assumption that such a metabolic trigger might lead to relatively small biochemical changes in the first few minutes of germination, sensitive analytical techniques were used to detect any changes in spore components during the L-alanine-triggered germination of Bacillus megaterium KM spores. These experiments showed that no changes in spore free amino acids or ATP occurred until 2-3 min after L-alanine addition. Spores contained almost no oxo acids (pyruvate, alpha-oxoglutarate, oxaloacetate), malate or reduced NAD. These compounds were again not detectable until 2-3 min after addition of germinants. It is suggested, therefore, that metabolism associated with these intermediates is not involved in the triggering of germination of this organism.     

Action of insulin on the subcellular metabolism of polyphosphoinositides in isolated rat hepatocytes

The effect of insulin on 32Pi incorporation into phospholipids in various subcellular sites of isolated rat hepatocytes was investigated. After labeling the phospholipids of hepatocytes from rats previously starved for 24 h with 32Pi (10 mu Ci/10(6) cells) for 90 min, either saline or insulin (32 nM) was added. Following incubations of 1, 5, and 30 min, chilled cells were rapidly washed, homogenized in the presence of inhibitors of phospholipid degradation, and fractionated into the major subcellular organelles. Phospholipids were extracted from plasma membranes, microsomes, lysosomes, mitochondria, and nuclei with acidic chloroform:methanol. The aqueous deacylation products were separated by anion exchange high performance liquid chromatography, and the 32Pi incorporated into all the major diacylglycerophospholipids was determined. In parallel experiments, the specific radioactivity of 32Pi and [gamma-32P]ATP was determined. The results revealed that insulin had no effect on the turnover of the major phospholipids, including the polyphosphoinositides, of all subcellular compartments analyzed relative to the control. In addition, there were no significant differences in the amount and 32P labeling of cellular orthophosphate between saline- and insulin-treated cells. The specific radioactivity of [gamma-32P]ATP was increased by 20% after 30-min treatment with insulin, requiring appropriate correction of 32P-labeled phosphatidic acid, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate for estimation of mass changes at near steady-state labeling of cellular ATP.     

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.     

Fructose protects rat hepatocytes from anoxic injury. Effect on intracellular ATP, Ca2+i, Mg2+i, Na+i, and pHi.

The effects of fructose on the intracellular ionic changes evoked by anoxia were studied in freshly isolated rat hepatocytes maintained in agarose gel threads and perfused with Krebs-Henseleit bicarbonate buffer (KHB). Cytosolic free calcium (Ca2+i) was measured with aequorin, intracellular sodium (Na+i) with sodium-binding benzofuran isophthalate, intracellular pH (pHi) with 2'-7'-bis(carboxyethyl)-5,6-carboxyfluorescein, lactic dehydrogenase (LDH) by the increase in NADH absorbance during lactate oxidation to pyruvate, and viability by trypan blue exclusion. ATP, Pi, phosphomonoesters, and the cell phosphorylation potential assessed by the reciprocal of the Pi/ATP ratio were measured by 31P NMR spectroscopy in real time. Intracellular free Mg2+ (Mg2+i) was calculated from the chemical shift of beta-ATP relative to alpha-ATP in the NMR spectra. Anoxia was induced by perfusing the cells with KHB saturated with 95% N2, 5% CO2. When the perfusate contained 5 mM glucose as substrate, anoxia caused a fall in ATP, a rise in Pi, and in the Pi/ATP ratio, a biphasic increase in Ca2+i that reached 1.45 +/- 0.42 microM and a 6-fold increase in LDH. When 15 mM fructose was used as substrate during the anoxic period, intracellular ATP decreased much faster than with glucose, Pi did not increase, and the concentration of phosphomonoesters increased 2.5-fold. During the first hour of anoxia, the Pi/ATP ratio was higher in the fructose than in the glucose group indicating that the hepatocyte phosphorylation potential and ATP decreased faster and to lower levels with fructose than with glucose. On the other hand, ATP and the phosphorylation potential of the fructose group increased during the second hour of anoxia, in contrast to their continuous decline in the glucose group. The major surge in Ca2+i was depressed 52% when glucose was replaced by fructose: Ca2+i reached only 0.7 +/- 0.2 microM instead of 1.45 +/- 0.42 microM (p less than 0.01). Anoxia also caused an increase in Na+i and an intracellular acidosis. The rise in Na+i was significantly greater with fructose than with glucose. Na+i rose from a control value of 15.9 +/- 2.4 to 32.2 +/- 0.4 mM with glucose and to 48.7 +/- 0.7 mM with fructose (p less than 0.001). The decrease in pHi from a control value of 7.43 +/- 0.03 was consistently greater and faster with fructose than with glucose: 6.59 +/- 0.03 and 7.04 +/- 0.01, respectively. At the same time, fructose completely suppressed LDH release and reduced the loss of viability produced by anoxia from 27.7 +/- 2.9 to 14 +/- 3.1% (p less than 0.05).     

Phospholipid metabolism of 3T3 mouse fibroblasts after serum stimulation and through the Gl and S cell cycle phases : incorporation and disappearance of P

Phospholipid metabolism of 3T3 mouse fibroblasts has been studied after serum stimulation of arrested cells.The study of [³²Pi]incorporation shows : a) in the case of PE and PC an early peak of incorporation in the Gl phase of the cell cycle, 6 hours after serum addition ; b) in the case of PI an intense initial increasing of the incorporation which continues up to a S phase peak.The study of the disappearance of [³²P] Phosphate from the different phospholipids points out : a) at the beginning of serum stimulation, an intense breakdown of PI, that continues through the Gl and S phases. Except at the onset, the breakdown of PI, is at any time exactly compensated for by synthesis : the two phenomena are closely linked ; b) a synthesis of PE, from PC probably, at the Gl phase, 4 hours after the serum addition and the beginning of the chase experiment.     

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.     

Effects of L-thyroxine on incorporation of (32)P into phospholipids of freshwater eels

The effects of L-thyroxine on phospholipid biosynthesis, via (32)P incorporation, were studied in gill, kidney, liver and muscle tissue of eels acclimatized at 11 degrees C. L-thyroxine treatment had no effect on tissue content of lipid, inorganic and organic acid-soluble phosphorus. Only an increase of the specific radioactivities of lipid, inorganic and organic acid-soluble phosphorus was observed in the muscle. Percentage distribution of (32)P among classes of phospholipid were significantly altered in liver and muscle, without change in phospholipid composition. A specific effect of L-thyroxine on (32)P incorporation into phosphatidic acid in muscle and liver has been shown. As expected by the higher specific radioactivity of muscle inorganic and organic acid-soluble phosphorus, the increased incorporation of (32)P into phosphatidic acid probably results from a higher specific radioactivity of muscle ATP phosphorus.