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PubMed1976

Further studies on the possible compartmentation of the precursor pool of cholesterol for the biosynthesis of cholic acid in the rat.

In vivo and in vitro experiments with rats were carried out to investie precursor for the biosynthesis of cholic acid. When rats with a bile-fistula were given a mixture of [2-14C]mevalonate and [1,2-3H]cholesterol intravenously, the 14C:3H ratio in cholic acid in both whole homogenate and cytosol prepared from their lives was higher than that in free cholesterol in any subcellular fraction of the livers. When [2-14C] mevalonate was administered intravenously to bile-fistula rats, the specific radioactivity of free cholesterol in the hepatic microsomal fraction exceeded that in any other fraction, and the specific radioactivity of biliary cholic acid was remarkably high, exceeding that of microsomal free cholesterol. In similar experiments with [4-14C] cholesterol, the specific radioactivity of free cholesterol in the hepatic microsomal fraction exceeded that in any other subcellular fraction and the specific radioactivity of biliary cholic acid was lower than that of free cholesterol in any hepatic subcellular fraction. Tissue suspensions of rat livers in Krebs-Ringer bicarbonate (pH 7.4)-5.5 mM glucose were incubated with [2-14C]mevalonate in O2-CO2 (95:5, v/v) at 37 degrees. The specific radioactivity of free cholesterol in the microsomal fraction prepared from the incubated tissue exceeded the specific radioactivities of free cholesterol in the other subcellular fractions. The estimated specific radioactivity of taurocholate formed during the incubation was far higher than that of microsomal free cholesterol. These data indicate that hepatic microsomal free cholesterol which was newly synthesized in situ was preferentially incorporated into cholic acid.

Animals↗

PubMed1979

Hormone-stimulated phosphorylation of liver phosphofructokinase in vivo.

The effect of glucagon on the phosphorylation and the enzymic activity of phosphofructokinase in rat liver in vivo was investigated. Glucagon stimulated the phosphorylation of liver phosphofructokinase approximately 3- to 5-fold and increased cAMP levels 5-fold and blood glucose levels 2-fold over the values obtained for control animals. The specific radioactivity of ATP isolated from liver was the same in both control and hormone-treated animals. During the purification of the 32P-labeled enzyme from both animals, no difference was observed in the total or specific enzyme activities of the enzymes from the various fractions. Thus, phosphofructokinase appears to be phosphorylated in vivo by a cyclic AMP-dependent protein kinase. Although phosphorylation does not affect the maximum catalytic activity of the enzyme, it does render the enzyme significantly more sensitive to ATP inhibition. Thus, at a given concentration of ATP, the phosphorylated phosphofructokinase exhibits considerably lower activity than the unphosphorylated enzyme. The possible relationship between our observations and glucagon-mediated control of glycolysis is discussed.

Adenosine Triphosphate↗

PubMed1984

Synthesis of, and animal experiments with, N-isopropyl-p-123I-iodo-amphetamine (IMP) and 18F-3-deoxy-3-fluoro-D-glucose (3-FDG) as tracers in brain and heart diagnostic studies.

For the investigation of brain functions 18F-3-deoxy-3-fluoro-D-glucose (3-FDG) and N-isopropyl-p-123I-iodo-amphetamine (IMP) were synthesized and the course of radioactivity measured in several organs of mice. The results can be summarized as follows: IMP is rapidly extracted from the blood and reaches a value of less than 1% g within the first 15 min; 123I-radioactivity in the lungs shows a maximum of 76%/g as soon as half a minute after injection and decreases with a concomitant increase in the liver and brain; The maximum 123I-uptake in the brain of 11%/g is reached after 30 min and levels off at a constant value of 10%/g; 30 min after injection the brain/blood ratio for IMP is about 14; The time course of 3-FDG in the brain has a maximum of 4.8%/g as soon as 5 min after injection and decreases to a constant value of 3%/g within 1 hr; and Accumulation of 18F- radioactivity in the heart reaches a maximum of 14%/g after 1 hr and is eliminated with a half-life of 300 min. Comparative clinical studies with 3-FDG and 3-0-11C-methyl-D-glucose (CMG) have shown that 3-FDG can be considered as a CMG-analogue and thus can be used for the in-vivo determination of local glucose perfusion and transport rates.

Amphetamines↗

PubMed2001

Metabolism and distribution of [2,3-(14)C]acrolein in lactating goats.

The metabolism and distribution of [2,3-(14)C]acrolein were studied in a lactating goat orally administered 0.82 mg/kg of body weight/day for 5 days. Milk, urine, feces, and expired air were collected. The goat was killed 12 h after the last dose, and edible tissues were collected. The nature of the radioactive residues was determined in milk and tissues. All of the identified metabolites were the result of the incorporation of acrolein into the normal, natural products of intermediary metabolism. There was evidence that the three-carbon unit of acrolein was incorporated intact into glucose, and subsequently lactose, and into glycerol. In the case of other natural products, the incorporation of radioactivity appeared to result from the metabolism of acrolein to smaller molecules followed by incorporation of these metabolites into the normal biosynthetic pathways.

Acrolein↗

PubMed1994

Acetoacetate metabolism in AS-30D hepatoma cells.

Metabolic characteristics of experimental hepatoma cells include elevated rates of glycolysis and lipid synthesis. However, pyruvate derived from glucose is not redily oxidized, and the source of acetyl CoA for lipid synthesis in As-39D cells has not been characterized. In this study ketone bodies were examined as a possible source of acetyl CoA in AS-30D hepatoma cells. The major findings were: 1. Acetoacetate was utilized by AS-30D cells, with 14C-lipid and 14CO2 as major products of [3-14C] acetoacetate. 2. Lipid synthesis from acetoacetate was dependent on the presence of glucose in the medium. 3. Acetoacetate supported rapid respiration by AS-30D mitochondria in the presence of 0.1 mM malate. 4. Succinyl CoA acetoacetyl CoA transferase activity in AS-30D mitochondria was approximately 40 fold greater than that found in rat liver mitochondria. 5. Addition of acetoacetate, but not beta-hydroxybutyrate decreased conversion of [1-14C] acetate to 14CO2, presumably by diluting the specific radioactivity of the acetyl CoA derived from the acetate tracer. 6. In the presence of glucose, approximately one fourth of acetoacetate utilized was converted to lipid. This result is consistent with elevated lipogenesis postulated by the truncated TCA cycle hypothesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetoacetates↗

PubMed2002

Anomeric specificity of the stimulatory effect of D-glucose on D-fructose phosphorylation by human liver glucokinase.

D-Glucose was recently reported to stimulate d-fructose phosphorylation by human B-cell glucokinase. The present study aims at investigating the anomeric specificity of such a positive cooperativity. The alpha-anomer of D-glucose was found to increase much more markedly than beta-D-glucose the phosphorylation of D-fructose by human liver glucokinase. Such an anomeric preference diminished at high concentrations of the D-glucose anomers, i.e. when the effect of the aldohexose upon d-fructose phosphorylation became progressively less marked. A comparison between the effects of the two anomers of D-glucose and those of equilibrated D-glucose upon D-fructose phosphorylation by human liver glucokinase indicated that the results obtained with the equilibrated aldohexose were not significantly different from those expected from the combined effects of each anomers of D-glucose. In isolated rat islets incubated for 60 min at 4 degrees C, alpha-D-glucose (5.6 mm), but not beta-D-glucose (also 5.6 mm), augmented significantly the conversion of D-[U-(14)C]fructose (5.0 mm) to acidic radioactive metabolites. Likewise, in islets prelabeled with (45)Ca and perifused at 37 degrees C, D-fructose (20.0 mm) augmented (45)Ca efflux and provoked a biphasic stimulation of insulin release from islets exposed to alpha-D-glucose (5.6 mm), while inhibiting (45)Ca efflux and causing only a sluggish and modest increase in insulin output from islets exposed to beta-D-glucose (also 5.6 mm). The enhancing action of D-glucose upon D-fructose phosphorylation by glucokinase thus displays an obvious anomeric preference for alpha-D-glucose, and such an anomeric specificity remains operative in intact pancreatic islets.

Animals↗

PubMed1991

Hyperglycemia-induced cholestasis in the isolated perfused rat liver.

In a previous report we showed that cholestasis in diabetic rats is due in part to hyperglycemia. To gain information about the mechanism responsible for this phenomenon, bile flow was studied in isolated perfused rat livers. The perfusion media were modified erythrocyte-free Krebs-Henseleit solutions. Under these experimental conditions, no cholestasis was observed in isolated rat livers obtained from rats treated with streptozotocin (6 mg/100 gm body wt) 6 days before the experiments. We then proceeded to use normal animals. The composition of the perfusion media was modified to maintain the osmolality even after increasing D-glucose concentrations from 0 to 35 mmol/L. Bile flow was not affected with doses up to 15 mmol/L D-glucose. Beyond a threshold value for plasma D-glucose concentrations between 15 and 20 mmol/L, cholestasis was observed. Using D-glucose analogs such as L-glucose and 3-O-methyl-D-glucose, bile flow was also reduced (by 0.54 and 0.53 microliters/min/gm liver, respectively). Isosmotic sucrose-containing perfusion media were also observed to impair bile flow (by 0.66 microliters/min/gm liver). However, i-erythritol and D-mannitol failed to inhibit bile formation. The study of bile/plasma concentration ratios determined using tracer amounts of radioactive sugars indicated that this value was much lower for cholestatic sucrose (0.11) and L-glucose (0.31) than for noncholestatic i-erythritol (0.99) and D-mannitol (0.98). Cholestasis was partly reversed after induction by 35 mmol/L D-glucose if perfusion media were replaced by sugar-free ones, but also by media containing 25 mmol/L D-glucose. Insulin given during the perfusion with sugar-free media was observed to have no effect on bile flow.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1974

Biosynthesis of fosfomycin by Streptomyces fradiae.

The antibiotic fosfomycin was produced as a secondary metabolite in a glucose-asparagine medium containing citrate, l-methionine, and l-glutamate. The citrate requirement for antibiotic synthesis was related to its requirement for growth. In contrast, l-methionine and l-glutamate caused a marked stimulation of fosfomycin production and had no effect on growth. l-Methionine had to be added early to effect maximal antibiotic synthesis later in the fermentation. The l-glutamate requirement was not specific, since several tricarboxylic acid cycle intermediates could replace this amino acid. l-Asparagine was the most effective nitrogen source for growth and production of fosfomycin. Glycine, an alternate nitrogen source, supported fosfomycin synthesis only when added in excess of that needed for growth. Cobalt and inorganic phosphate were required also for antibiotic production at concentrations exceeding those supporting maximal growth. Radioactive incorporation studies showed that the methyl carbon of methionine was the precursor of the methyl of fosfomycin. Carbon 1 of fosfomycin was derived from glucose carbons 1 and 6, whereas glucose-2-(14)C labeled fosfomycin carbon 2. Radioactivity from acetate-2-(14)C was distributed equally between fosfomycin carbons 1 and 2. No incorporation of acetate-1-(14)C, asparagine-U-(14)C, citrate-1,5-(14)C, or glutamate-U-(14)C occurred. The labeling pattern of fosfomycin carbons 1 and 2 was similar to that found in 2-aminoethylphosphonate from Tetrahymena.

Acetates↗

PubMed1968

Studies on the enzymic N-acylation of amino sugars in the sheep colonic mucosa.

1. d-[2-(14)C]Glucose, [2-(14)C]acetate, hydroxy[3-(14)C]pyruvate, [3-(14)C]pyruvate and [U-(14)C]glycine were incorporated by surviving scrapings of sheep colonic mucosal tissue into glycoprotein. 2. d-[2-(14)C]Glucose, [2-(14)C]acetate, incorporated hydroxy-[3-(14)C]pyruvate and [3-(14)C]pyruvate resulted in labelling of each of the monosaccharide residues of the glycoprotein, namely N-glycollylneuraminic acid, N-acetylneuraminic acid, galactose, fucose, glucosamine and galactosamine. [U-(14)C]Glycine was incorporated as glycyl and seryl residues of the glycoprotein. 3. Despite N-glycollylneuraminic acid being quantitatively the predominant sialic acid (N-glycollylneuraminic acid and N-acetylneuraminic acid were 8.5 and 5.2% by weight of the glycoprotein respectively) the corresponding ratio of the radio-active labelling from d-[2-(14)C]glucose in N-glycollylneuraminic acid to that in N-acetylneuraminic acid was 1.00:7.27 (expressed as percentages of the total radioactivity in the glycoprotein). Neutral sugar, hexosamine and N-acetylneuraminic acid residues of the mucoprotein were each labelled to a similar extent. 4. Similarly, the ratio of the radioactivity in N-glycollylneuraminic acid to that in N-acetylneuraminic acid in the mucoprotein from tissue incubations with [2-(14)C]-acetate was 1.0:4.0. 5. Both [2-(14)C]acetate and [2-(14)C]glucose with whole tissue led to labelling of the N-glycollyl substituent and of the main nonose skeleton of the N-glycollylneuraminic acid. In whole-tissue incubations, [3-(14)C]pyruvate was also a precursor of radioactive N-glycollylneuraminic acid. 6. Hydroxy[3-(14)C]-pyruvate and [U-(14)C]glycine caused labelling of the carbohydrate and peptide residues of the glycoprotein, but did not give rise to labelling in the N-glycollylneuraminic acid residues. 7. With a wide variety of possible N-glycollyl precursors (fructose 6-phosphate, hydroxypyruvate, glycollate and chemically synthesized glycollyl-CoA) biosynthesis of N-glycollylglucosamine was not observed in cell-free preparations.

Acetates↗

PubMedDate not supplied

Biosynthesis of acid mucopolysaccharides by the surviving new born rat skin. I. - Kinetics of the biosynthesis at the polymer level.

1) The amounts of individual mucopolysaccharides in the new born rat skin have been estimated and their specific rates of labelling assessed in vitro. Total and percentage amounts of these polymers agree satisfactorily with previously published data. 2) Relative rates of labelling from [U14C]-glucose have been estimated by combining column chromatography separation and electrophoresis on cellulose acetate strips. Specific radioactivities have been measured either with respect to the total uronic acid content of the fractions or with respect to their quantitative staining with Alcian Blue. The two methods agreed satisfactorily. 3) Average biosynthetic rates almost identical for hyaluronic acid and the total sulfated mucopolysaccharides. However, within the latter fraction, heparin + heparan sulfate incorporate [U14C]-glucose about 4 to 5 times more rapidly than the chondroitin sulfates. This result could not be expected from previous data obtained in vivo and is discussed with reference to a possible heterogeneity of the cell material whence the various mucopolysaccharides originate. 4) In the presence of puromycin, labelling of the sulfated mucopolysaccharides stops almost immediately, indicating a stringent requirement for protein primers. Biosynthesis of hyaluronic acid is affected only after preincubation of tissue with puromycin (one hour) and subsequent incubation of two hours with [U14C]-glucose.

Animals↗

PubMed1977

An effect of extracellular redox state on the glucagon-stimulated glucose release by rat hepatocytes and perfused liver.

The effect of changes in the extracellular redox-state on glucagon-stimulated glucose release by intact isolated rat hepatocytes and the perfused liver was examined. For hepatocytes from the fed rat an increase in pyruvate, ammonium ion or oxygen concentration or a decrease in the lactate/pyruvate or sorbitol/fructose ratios decreased the ability of 1 microM-glucagon to stimulate glucose release without significantly altering the control rate. These changes coincided with a decrease in the lactate/pyruvate ratio of the cell suspension. A decrease in the lactate/pyruvate ratio also decreased the ability of 1 microM-glucagon to stimulate glycogen breakdown measured by loss of contained radioactivity. For the isolated perfused rat liver (fed rat) maximal effects of glucagon as a stimulant of glucose release occurred when lactate instead of pyruvate was present in the perfusion medium. It is concluded that the efficacy of glucagon as a stimulant of glucose release by isolated hepatocytes and the perfused liver depends upon the cytoplasmic redox-state represented by the intracellular lactate/pyruvate ratio.

Ammonium Chloride↗

PubMed1980

Effects of 2-deoxy-D-glucose on the synthesis of RNA and protein in Saccharomyces carlsbergensis G-517.

When Saccharomyces carlsbergensis G-517 was grown in 10 mM galactose as the carbon source, the addition of 2-deoxy-D-glucose restricted the uptake of galactose, [3H]uridine and [3H]leucine, and restricted invertase synthesis (beta-D-fructofuranoside fructohydrolase; EC 3.2.1.26) for a period of 60-90 min. During this time, the radioactive antimetabolite was taken up by the cells; afterwards, invertase synthesis was enhanced, and the utilizaton rate of galactose, [3H]uridine and [3H]leucine increased until it reached that of the control culture. When glucose was used as a carbon source, sugar utilization and uptake of radioactive precursors were unaffected by addition of the deoxysugar.

Deoxy Sugars↗

PubMed1992

Cerebral responses to acute maternal alcohol intoxication in immature fetal sheep.

Previous studies in mature fetal sheep have shown that alcohol depresses cerebral blood flow (CBF), cerebral O2 consumption (CMRO2), and cerebral glucose consumption (CMRglu). This effect earlier in gestation might contribute to the pathogenesis of fetal alcohol syndrome. Physiologic studies of immature fetal sheep have demonstrated lower CBF, CMRO2, and CMRglu as well as a blunted vasodilatory response to hypoxia compared with mature fetal sheep. The purpose of this study was to determine whether immature fetal responses to alcohol are blunted compared with near-term fetal responses. We studied seven immature fetal sheep in utero at 92 +/- 1 d gestation (term = 147 d) 2 d after placement of vascular catheters. Pure ethanol (1 g/kg) was infused i.v. to the mother over 1 h. We measured CBF and myocardial blood flow by radioactive microspheres and calculated CMRO2 and CMRglu using arterial and sagittal sinus O2 and glucose concentrations. At a fetal ethanol concentration of 33 + 8 mmol/L (150 +/- 37 mg/dL), there were no significant changes in CBF, CMRO2, or CMRglu. There was mild hypoglycemia (glucose concentration = 1.05 +/- 0.2 versus 1.33 +/- 0.2 mM baseline) and lactic acidemia (lactate concentration = 1.29 +/- 0.3 versus 1.07 +/- 0.2 mM baseline). Cardiovascular variables were unchanged as was myocardial blood flow. The immature fetal sheep brain shows no significant cerebrovascular and metabolic response to acute alcohol intoxication compared with mature fetal sheep. Mild hypoglycemia and lactic acidemia did develop. The reason for the developmental differences in response to alcohol and their relationship to fetal alcohol syndrome remain to be elucidated.

Alcoholic Intoxication↗

PubMed1977

Binding of mescaline with subcellular fractions upon incubation of brain cortex slices with [14C] mescaline.

Incubation of brain cortex slices in the presence of glucose resulted in the permeation of about 65% of [14C] mescaline into slices. Of this, about one-third radioactivity was bound with nuclei, mitochondria, microsomes, and ribosomes. Dialysis of subcellular fractions did not markedly reduce the amounts of radioactivity bound to the fractions. The permeation into slices and the binding of mescaline to subcellular fractions were fairly time-dependent, but were inhibited by the presence of potassium cyanide, or by the absence of glucose and by heating to 80 degrees C for 1 min.

Animals↗

PubMed1992

Gastrointestinal morphology and absorption of monosaccharides in fowls conditioned to different types and levels of dietary fibre.

To test a possible influence of dietary fibre on intestinal sugar uptake, rates of absorption of 10 mM-D-[U-14C] glucose and 10 mM-D-[U-14C] xylose were measured in either jejunum or (distal) caecum, by in vivo lumen perfusion, in immature female fowls preconditioned to a standard diet containing (g/kg) either 0, 100, 200 or 400 added dried grass, 200 powdered cellulose, or 200 grass with a polysaccharidase enzyme supplement. When birds were killed after perfusion, dimensions of (unperfused) parts of their alimentary tracts were determined, and recoveries of 14C radioactivity in some body tissues were compared with measured activities absorbed. On average, absorption of glucose was 1.9 and 1.2 times faster than xylose in jejunum and caecum respectively, although these differences varied with dietary treatment and order of perfusion. Increasing grass in the diet caused significant changes in xylose absorption rate in both jejunum and caecum, but only when it was perfused before glucose. With any one sugar and intestinal segment, mean rates of absorption were correlated positively with corresponding mean rates of fluid loss from perfusate. Although their influence on sugar absorption was not well defined, the dietary fibre treatments had more pronounced effects on gross dimensions of parts of the alimentary tract and, hence, potentially on total rates of absorption. Compared with the basal diet, addition of 100, 200 or 400 g grass/kg or 200 g cellulose/kg caused significant increases in small intestine length while 200 g grass/kg with supplementary enzyme did not, and combined caecal length increased with the 400 g grass/kg and the supplementary enzyme treatments. Absorbed 14C activity was recovered in plasma after jejunal perfusions but not caecal ones, whereas it was recovered in liver and in the flushed perfused segment after both types of perfusion. Since there was overlap in absorption rates between jejunum and caecum, this result suggests that the liver may be able to distinguish and treat differently compounds absorbed in the two regions.

Animals↗

PubMed1997

Ceramide glucosylation in bean hypocotyl microsomes: evidence that steryl glucoside serves as glucose donor.

The formation of glucosylceramide, the predominant sphingolipid in plant tissues, was examined in microsomes from wax bean hypocotyls. Membranes were incubated with UDP-[14C]glucose in an assay mixture. The lipid extracts obtained from the assays were separated by thin-layer chromatography, and the radioactivity incorporated into glucosylceramide, steryl glucoside, and acylated steryl glucoside was determined. Although the formation of glucosylceramide was detected and characterized, several lines of evidence contradicted the assumption that UDP-glucose is the immediate glucose donor for glucosylceramide formation in plants: PDMP (DL-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol), an inhibitor of ceramide glucosyltransferase in animal tissues, did not inhibit glucosylceramide formation in bean microsomes. Addition of UDP-glucose pyrophosphorylase during the assay to degrade UDP-[14C]glucose blocked the further production of labeled steryl glucoside, but did not prevent the continued formation of labeled glucosylceramide. Omitting UDP-[14C]glucose and including steryl [14C]glucoside in the assay resulted in the formation of labeled glucosylceramide. Collectively, these results suggest that glucosylceramide formation in plants does not utilize UDP-glucose as the immediate glucose donor, as has been demonstrated for the reaction in animal tissues, and that steryl glucoside serves as glucose donor for ceramide formation. This study, the first to examine glucosylceramide formation in plants, provides evidence for a novel enzymatic reaction in sphingolipid synthesis as well as a new, metabolic role for steryl glucoside in plant tissues.

Ceramides↗

PubMed1986

Ascorbate transport in cultured cat retinal pigment epithelial cells.

Transport of ascorbate by primary cultures of cat retinal pigment epithelial cells (RPE) was studied. Confluent primary cultures were incubated with 10-500 microM L-[carboxyl-14C] ascorbic acid in balanced salt solution (BSS) at 37 degrees C for 1 to 40 min. The uptake of radioactive ascorbate followed saturation kinetics with a Km of 42 microM and Vmax of 117 pmol min-1 microgram-1 DNA. Cells incubated with 10 microM radioactive ascorbate for 40 min showed a ratio of intracellular to extracellular radioactive ascorbate of greater than 40. The transport of ascorbate was sodium- and energy-dependent. Replacement of 150 mM NaCl in BSS with 150 mM LiCl reduced ascorbate uptake significantly. Ouabain, 2,4-dinitrophenol, alpha-D-glucose, 3-O-methyl-D-glucose, and the ascorbate analogues, D-isoascorbate and dehydroascorbate, each inhibited ascorbate uptake into RPE cells. The efflux of radioactivity into the incubation media was slow when cells were preloaded with either 50- or 500 microM radioactive ascorbate, but increased when cells preloaded with 50 microM ascorbate were incubated in the presence of excess non-radioactive ascorbate. These studies demonstrated that a sodium-dependent carrier system is involved in transport of ascorbate in primary cultures of cat RPE.

3-O-Methylglucose↗

PubMed1980

Endogenous phosphorylation of soluble enzymes in human red cells. Cyclic 3',5'-AMP-dependent phosphorylation of phosphofructokinase without detectable regulatory effect.

ATP-depleted human red cells have been incubated in a glucose-containing medium with [32P]orthophosphate in the presence and in the absence of cyclic 3',5'-AMP and dibutyril cyclic 3',5'-AMP. Spectrin, pyruvate kinase, phosphofructokinase, glucose-6-phosphate dehydrogenase and hemoglobin A1 have been purified and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Protein-bound radioactivity has been measured from the sodium dodecyl sulfate polyacrylamide gels and the trichloroacetic acid-precipitated proteins. In the cytosol, the most intense phosphorylation was found for pyruvate kinase whose, in the presence of cyclic AMP, specific radioactivity was comparable to that of the membrane protein and spectrin. In the absence of cyclic nucleotides it was five times less phosphorylated. Phosphofructokinase was only phosphorylated when the red cells were incubated with cyclic nucleotides; the extent of phosphorylation was four times less than for pyruvate kinase. Hemoglobin, glucose-6-phosphate dehydrogenase and a contaminant protein copurified with phosphofructokinase were not phosphorylated: the 'background' of the radioactivity found for these proteins was 100 times less than for pyruvate kinase and spectrin, and 20 times less than for phosphofructokinase (+cyclic AMP).

Cyclic AMP↗