Particle-free Supernatant Fraction Research Articles

Enzymic changes in rabbit and rat mammary gland during the lactation cycle.

1. Activities of glucose 6-phosphate dehydrogenase (EC 1.1.1.49), 6-phosphogluconate dehydrogenase (EC 1.1.1.44), isocitrate dehydrogenase (EC 1.1.1.42), malate dehydrogenase (EC 1.1.1.37), malate dehydrogenase (decarboxylating) (EC 1.1.1.40), and pyruvate carboxylase (EC 6.4.1.1) were determined in subcellular fractions of mammary gland from rabbits during pregnancy, at different stages of lactation and during weaning. The results were compared with those obtained in similar experiments with rat mammary gland. 2. Three bases of expression of the activity of enzymes in the particle-free supernatant fraction of mammary gland were compared. During lactation, activity expressed per mg. of particle-free supernatant protein (uncorrected for milk protein) correlated well with that expressed per mug. of DNA phosphorus. The disadvantages of expressing activities per g. wet wt. are discussed. 3. The major differences between the two tissues were: (a) neither malate dehydrogenase (decarboxylating) nor a soluble form of pyruvate carboxylase could be detected in rabbit mammary gland at any stage of the lactation cycle; (b) isocitrate dehydrogenase increased in activity during lactation in rabbit mammary gland, but not in that of the rat. 4. Pyruvate carboxylase in the mitochondrial fraction of rabbit mammary gland, and in both the mitochondrial and the soluble fractions of rat mammary gland, did not change in activity during lactation. 5. For each tissue, the NADP-dependent dehydrogenases studied had a high activity at all stages of the lactation cycle compared with the rate of fatty acid synthesis at mid-lactation. The significance of these results is discussed with respect to the supply of NADPH via NADH.

Pyruvate carboxylase in lactating rat and rabbit mammary gland.

1. Pyruvate carboxylase [pyruvate-carbon dioxide ligase (ADP), EC 6.4.1.1] was found in cell-free preparations of lactating rat and rabbit mammary glands, and optimum assay conditions for this enzyme were determined. 2. Subcellular-fractionation studies with marker enzymes showed pyruvate carboxylase to be distributed between the mitochondrial and soluble fractions of lactating rat mammary gland. Evidence is presented that the soluble enzyme is not an artifact due to mitochondrial damage. 3. In contrast, pyruvate carboxylase in lactating rabbit mammary gland is confined to the mitochondrial fraction. 4. The final product of pyruvate carboxylase action in the mitochondrial and particle-free supernatant fractions of lactating rat mammary gland was shown to be citrate. 5. The effects of freeze-drying, ultrasonic treatment and freezing-and-thawing on the specific activity of mitochondrial pyruvate carboxylase were investigated.

Biochemical effects of the hypoglycaemic compound pent-4-enoic acid and related non-hypoglycaemic fatty acids. Carbohydrate metabolism.

1. The effects of the hypoglycaemic compound, pent-4-enoic acid, and of four structurally related non-hypoglycaemic compounds (pent-2-enoic acid, pentanoic acid, cyclopropanecarboxylic acid and cyclobutanecarboxylic acid), on glycolysis, glucose oxidation and gluconeogenesis in some rat tissues were determined. 2. None of the compounds at low concentrations inhibited glycolysis by particle-free supernatant fractions from rat liver, skeletal muscle and intestinal mucosa, though there was inhibition by cyclopropanecarboxylic acid and cyclobutanecarboxylic acid at 3mm concentration. 3. Pent-4-enoic inhibited the oxidation of [1-(14)C]palmitate by rat liver slices, but did not increase the oxidation of [U-(14)C]glucose. 4. Pent-4-enoic acid (0.01mm) strongly inhibited gluconeogenesis by rat kidney slices from pyruvate or succinate, but none of the other compounds inhibited significantly at low concentrations. 5. There was also some inhibition of gluconeogenesis in kidney slices from rats injected with pent-4-enoic acid. 6. The mechanism of the hypoglycaemic effect of pent-4-enoic acid is discussed; it is suggested that there is an inhibition of fatty acid and ketone-body oxidation and of gluconeogenesis so that glucose reserves become exhausted, leading to hypoglycaemia. 7. The mechanism of the hypoglycaemic action of pent-4-enoic acid appears to be similar to that of hypoglycin.

Pyruvate carboxylase in lactating rat and rabbit mammary gland.

1. Pyruvate carboxylase [pyruvate–carbon dioxide ligase (ADP), EC 6.4.1.1] was found in cell-free preparations of lactating rat and rabbit mammary glands, and optimum assay conditions for this enzyme were determined. 2. Subcellular-fractionation studies with marker enzymes showed pyruvate carboxylase to be distributed between the mitochondrial and soluble fractions of lactating rat mammary gland. Evidence is presented that the soluble enzyme is not an artifact due to mitochondrial damage. 3. In contrast, pyruvate carboxylase in lactating rabbit mammary gland is confined to the mitochondrial fraction. 4. The final product of pyruvate carboxylase action in the mitochondrial and particle-free supernatant fractions of lactating rat mammary gland was shown to be citrate. 5. The effects of freeze-drying, ultrasonic treatment and freezing-and-thawing on the specific activity of mitochondrial pyruvate carboxylase were investigated.

Fatty acid biosynthesis. IV Properties of acetyl-CoA carboxylase in lactating-rabbit mammary gland

1. 1. The distribution of acetyl-CoA carboxylase between the microsomal and particle-free supernatant fractions of lactating-rabbit mammary gland has been investigated. Conditions are described which radically alter the distribution of the enzyme between the two fractions. The presence of acetyl-CoA carboxylase in the microsomal fraction is not an artefact of preparation. 2. 2. Repeated washing of the microsomal fraction with 0.3 M sucrose caused a preferential release of acetyl-CoA carboxylase of high specific activity. Addition of KCl to the sucrose did not increase the release. Attempts to release the particulate enzyme by sonication or detergents were unsuccessful. 3. 3. Sucrose density gradient studies showed a single acetyl-CoA carboxylase (which could be completely sedimented) in the microsomal plus particle-free supernatant fraction. This enzyme had a density lower than that of the majority of microsomal protein, but higher than that of the majority of soluble protein present. The enzyme in the microsomal fraction behaved as did the enzyme in the microsomal plus particle-free supernatant fraction. The enzyme recovered in the particle-free supernatant fraction ( i.e., after separation from microsomal material) had changed its molecular characteristics into those of a soluble enzyme. 4. 4. Acetyl-CoA carboxylase in the particle-free supernatant fraction of lactatingrabbit mammary gland was unusual in binding onto microsomal material from a number of tissues including lactating-rabbit mammary gland. In contrast, the microsomal fraction from lactating-rabbit mammary gland could bind much more soluble enzyme from this tissue than from the other tissues investigated.

The reactions of the pentose phosphate cycle in the particle-free supernatant fraction from the mucosa of the small intestine

1.1. The reactions of the pentose phosphate cycle were demonstrated manometrically in particle-free supernatant fractions from the mucosa of the small intestine, by coupling to oxygen with phenazine methosulphate.2.2. High activities were found in preparations from five mammalian species and a bird.3.3. Many preparations absorbed the theoretical amount of oxygen necessary to oxidize ribose 5-phosphate to carbon dioxide and water.

Fatty acid biosynthesis: III. Intracellular site of enzymes in lactating-rabbit mammary gland

1. 1. Subcellular fractions of lactating-rabbit mammary glands have been characterised by the use of “marker” enzymes and these fractions were then examined for the presence of enzymes involved in fatty acid synthesis. 2. 2. Acetate: CoA ligase (EC 6.2.1.1) was found to be confined to the particle-free supernatant fraction, whereas acetyl-CoA carboxylase (EC 6.4.1.2) was distributed between the microsomal and the particle-free supernatant fractions. Fatty acid synthetase was almost entirely situated in the particle-free supernatant fraction. Much lower levels of acetyl-CoA carboxylase and no fatty acid synthetase were observed in the mitochondrial fraction. The implications of these findings are discussed. 3. 3. Acetyl-CoA carboxylase is the rate-limiting step in fatty acid synthesis in this tissue under the conditions used.

Fatty acid biosynthesis: II. Fate of fatty acids synthesised by subcellular fractions of lactating-rabbit mammary gland

1. 1. Subcellular fractions of lactating-rabbit mammary gland have been incubated under optimum conditions for fatty acid synthesis and the nature and subcellular site of the products examined. 2. 2. When “microsomal + particle-free supernatant” fractions were incubated with labelled acetate, a large proportion of the radioactivity was associated with newly synthesised glycerides in the microsomal fraction. Radioactive unesterified fatty acids were found to be located in the particle-free supernatant. 3. 3. Particle-free supernatant fractions alone synthesised exclusively unesterified fatty acids. 4. 4. Though both the mitochondrial and microsomal fractions bind synthesised fatty acids, the microsomal fraction stimulated both fatty acid and glyceride synthesis to a greater extent than did the mitochondrial fraction. 5. 5. It has been shown that whereas certain free fatty acids inhibited fatty acid synthesis, the corresponding glycerides had little inhibitive effect. 6. 6. The results support the view that the microsomal stimulation of fatty acid synthesis is due, at least in part, to a relief of the feedback inhibition excercised by the products of fatty acid synthesis.

Studies on the incorporation of CO 2 by cells and cell-free extracts of Nitrosomonas europaea

Whole cells of Nitrosomonas, when oxidizing hydroxylamine, incorporated about three times more 14CO 2 than when equivalent amounts of ammonia were utilized, although nitrite production was similar in both cases. When cells supplied with ammonia were exposed to 14CO 2 for 1 min, the following incorporation (per cent) was observed: serine 40, phosphopyruvate 25, sugar phosphates 9, succinic acid 8 and phosphoglyceric acid 3. After a 2-min exposure, however, the values were: serine 24, phosphoglyceric acid 25, sugar phosphates 22 and phosphoglycollic acid 8. Cell-free extracts incorporated CO 2 when ribulose diphosphate or ribose 5-phosphate and ATP were added and most of the tracer was recovered in phosphoglyceric acid. The extracts also fixed CO 2 in the presence of phosphoenolpyruvate. The D-ribose-5-phosphate ketol-isomerase (EC 5.3.1.6) enzyme purified 40-fod from particle-free extracts has an optimum pH between 7 and 7.4 and a K m value of 1.6 · 10 −6 M. The following enzymes of the photosynthetic carbon-reduction cycle, were detected in the particle-free supernatant fraction: D-ribose-5-phosphate ketol-isomerase, ATP: D-ribulose-5-phosphate 1-phosphotransferase (EC 2.7.1.19), carboxydismutase, ATP: D-3-phosphoglycerate 1-phosphotransferase (EC 2.7.2.3), D-glyceraldehyde-3-phosphate:NAD oxidoreductase (phosphorylating) (EC 1.2.1.12), D-glyceraldehyde-3-phosphate ketol-isomerase (EC 5.3.1.1), fructose-1,6-diphosphate aldehyde-lyase (EC 4.1.2.7), D-ribulose-5-phosphate 3-epimerase (EC 5.1.3.1), D-sedoheptulose-7-phosphate: D-glyceraldehyde-3-phosphate glycolaldehydetransferase (EC 2.2.1.1) and orthophosphate: oxaloacetate carboxy-lyase (phosphorylating) (EC 4.1.1.31). Enzymes involved in the oxidation of hydroxylamine, the generation of ATP and NADH 2 are, however, associated with particles.

Factors affecting the chain length of fatty acids synthesised by lactating-rabbit mammary glands

1. 1. Gas-liquid radiochromatography has been used to determine the chain lengths of fatty acids synthesised by extracts of lactating-rabbit mammary glands. 2. 2. The particle-free supernatant fraction synthesised mainly short-chain fatty acids. Addition of “microsomal” protein (and to a lesser extent mitochondrial protein) increased the overall rate of synthesis, particularly that of long-chain fatty acids. 3. 3. The effects of time of incubation, ATP, CoA, bivalent cations, reduced pyridine nucleotides, HCO 3- and avidin on the chain lengths of the fatty acids synthesised has been investigated. Results indicate that butyrate can be synthesised by two separate mechanisms. 4. 4. Synthesis was stimulated by a number of dicarboxylic acids, malonate and succinate being the most effective. Citrate caused a greater stimulation, which was competitive with that of malonate. Stimulation by citrate and malonate was greatest for synthesis of longer chain-length fatty acids (C 14:0 and C 16:0) using the microsomal plus particle-free supernatant fraction, but greatest for C 8:0, C 10:0 and C 10:0 using the particle-free supernatant fraction. 5. 5. Rates of incorporation of acetate, citrate and malonate into individual fatty acids have been compared. 6. 6. The concentration of malonyl-CoA in incubation systems has been found to directly influence the chain lengths of fatty acids synthesised.

Metabolism of phospholipids IX. Phosphatidate phosphohydrolase in rat liver

1. 1. Rat-liver homogenates were fractionated into mitochondrial, lysosomal, microsomal, and particle-free supernatant fractions which were characterised by specific marker enzymes, and the intracellular distribution of phosphatidate phosphohydrolase ( l-α-phosphatidate phosphohydrolase, EC 3.1.3.4) was studied. 2. 2. The lysosomal fraction had the highest specific activity with respect to phosphatidate phosphohydrolase activity but the mitochondrial and microsomal fractions contained the relatively highest proportions of this enzyme. It was concluded that phosphatidate phosphohydrolase is a true constituent of all three particulate sub-cellular fractions, with some activity occurring in the supernatant. 3. 3. Repeated extraction of the three particulate subcellular fractions by freezing and thawing in 0.3 M sucrose solubilised 60, 25 and 16% of the mitochondrial, lysosomal, and microsomal phosphatidate phosphohydrolase activity, respectively. 4. 4. Investigation of Michaelis constants, pH optima, and the effect of detergents and cations revealed significant differences between the mitochondrial enzyme which had been extracted by freezing and thawing and that which remained insoluble. 5. 5. Examination of an extract obtained from a mitochondrial preparation by repeated freezing and thawing showed that of a number of important enzymes in glyceride and phospholipid biosynthesis only phosphatidate phosphohydrolase was extracted.

Oxidation of reduced nicotinamide-adenine dinucleotide phosphate by soluble rat muscle α-glycerophosphate dehydrogenase. A comparison with purified lactate dehydrogenase and malate dehydrogenase

1. 1. NADPH was observed to disappear from a Tris-histidine medium (pH 7·5) in the presence of glucose 6-phosphate, ATP, magnesium ion and a particle-free supernatant fraction obtained from a rat-muscle homogenate. This phenomenon can be explained by direct oxidation of the NADPH by the soluble α-glycerohosphate dehydrogenase ( l-glycerol-3-phosphate:NAD + oxidoreductase, EC 1.1.18). From measurements of α-glycerophosphate dehydrogenase activity in the muscle preparation in the presence of NADPH, we can conclude that the capacity of this dehydrogenase to oxidize NADPH exceeds by thr the capacities of the dehydrogenases that act on glucose 6-phosphate and 6-phosphogluconate to generate NADPH. 2. 2. Kinetic characteristics of a α-glycerophosphate dehydrogenase of the particle-free fraction obtained from rat muscle and commercially purified (a) rabbit-muscle α-glycerophosphate dehydrogenase, (b) rabbit-muscle lactate dehydrogenase ( l-lactate:NAD + oxidoreductase, EC 1.1.1.27), and (c) porcine-heart malate dehydrogenase ( l-malate:NAD + oxidoreductase, EC 1.1.1.37) were investigated and found to be quite similar: for each enzyme, pH-optima were higher for the reaction with NADH than for that with NADPH; the substrate inhibited the enzymes when NADH acted as the coenzyme (especially at low pH's), but not when NADPH did; and the apparent Km values for NADH were considerably lower than those for NADPH. 3. 3. Inhibition of rabbit-muscle α-glycerophosphate dehydrogenase by NAD + was observed with NADPH as the coenzyme, but not with NADH, NADP + produced no inhibition with either coenzyme.

Factors effecting the chain-length of fatty acids synthesized by lactating-rabbit mammary glands

Abstract 1. 1. Gas-liquid radiochromatography has been used to determine the chain lengths of fatty acids synthesised by extracts of lactating-rabbit mammary glands. 2. 2. The particle-free supernatant fraction synthesised mainly short-chain fatty acids. Addition of “microsomal” protein (and to a lesser extent mitochondrial protein) increased the overall rate of synthesis, particularly that of long-chain fatty acids. 3. 3. The effects of time of incubation, ATP, CoA, bivalent cations, reduced pyridine nucleotides, HCO3- and avidin on the chain lengths of the fatty acids synthesised has been investigated. Results indicate that butyrate can be synthesised by two separate mechanisms. 4. 4. Synthesis was stimulated by a number of dicarboxylic acids, malonate and succinate being the most effective. Citrate caused a greater stimulation, which was competitive with that of malonate. Stimulation by citrate and malonate was greatest for synthesis of longer chain-length fatty acids (C14:0 and C16:0) using the microsomal plus particle-free supernatant fraction, but greatest for C8:0, C10:0 and C10:0 using the particle-free supernatant fraction. 5. 5. Rates of incorporation of acetate, citrate and malonate into individual fatty acids have been compared. 6. 6. The concentration of malonyl-CoA in incubation systems has been found to directly influence the chain lengths of fatty acids synthesised.

Effect of Dietary Carbohydrate on Glucokinase and Mannokinase Activities of Various Rat Tissues

Glucokinase activities were determined in 9 rat tissues — liver, brain, muscle, kidney, small intestine, heart, spleen, adipose tissue and mammary gland. Significant amounts of glucokinase activity were particle-bound in brain (40 to 55%), in heart (50 to 60%) and in mammary gland (40%). In the other tissues, almost all of the activity appeared in the particle-free supernatant fraction. Except for a minor response by small intestine, liver was the only tissue in which glucokinase activity responded significantly to nutritional treatment of the rat. Evidence for the existence of 2 distinct enzymes capable of phosphorylating glucose was obtained from a study of the Km (glucose) values for the enzymes in the particle-free supernatant fraction of rat liver. The hepatic enzyme with a high Km value for glucose was shown to adapt to dietary treatment, whereas the hepatic glucokinase with a low Km for glucose was non-adaptable. Mannokinase activities were determined in brain, liver and mammary gland. The rates at which brain, liver and mammary gland phosphorylated mannose were 33, 27 and 26%, respectively, of those observed for glucose.

Dietary control of liver glucokinase activity in the normal rat

Adult male rats were fasted 48 hours and then fed a diet containing either: (a) 60% glucose, (b) 60% fructose, or (c) no utilizable carbohydrate. The rats were killed at various times after the onset of the feeding, and glucokinase activity was measured in the particle-free supernatant fractions of their livers. An existing method for preparation of the particle-free, supernatant fraction of the liver and assay of its glucokinase activity was modified to yield activities 2 to 3 times higher than those previously reported. Glucokinase activity increased rapidly in response to the feeding of both the high-glucose and high-fructose diets. The response to glucose was consistently greater than that to fructose. No change from fasting levels was observed in the groups fed diets containing no utilizable carbohydrate. In rats fed the high glucose diet, liver glucokinase levels increased as early as 4 hours after the start of feeding. In the fructose-fed rats, the first change was noted at 12 hours. Glucokinase levels continued to increase over a 24-hour period in the glucose-fed rats but not in the fructose-fed rats. There was no apparent correlation between the increase in glucokinase activity and the level of protein in the supernatant fraction. Decreases in glucokinase activity observed in fasted rats seemed to follow similar changes in the protein content of the supernatant fraction.

Fatty acid synthesis by complex systems The possibility of regulation by microsomes

1. 1. Fatty acid synthesis from malonyl-CoA catalyzed by rat-liver microsomal protein does not seem to be due to contamination with enzymes from the particle-free supernatant or mitochondrial fractions. 2. 2. Optimum conditions for the conversion of malonyl-CoA to fatty acids by rat-liver microsomes with respect to substrate concentration, protein concentration, and incubation time were established. The enzyme systems concerned with fatty acid synthesis from malonyl-CoA in the particle-free supernatant fraction were 4–5 times more active than those in the microsomes. 3. 3. Preincubation of the microsomes caused a release of malonyl-CoA decarboxylase (EC 4.1.1.9) but not of fatty acid synthetase. This resulted in a greater conversion of the available malonyl-CoA to fatty acids. 4. 4. The products of synthesis in the supernatant system were free fatty acids bound to protein, whereas the products of the microsomal system were predominantly complex lipids (phospholipid). 5. 5. The predominant fatty acid synthesized from malonyl-CoA by the supernatant fraction was palmitate, and that synthesized by the microsomes was stearate. 6. 6. In the system composed of the supernatant fraction plus microsomes, the fatty acids synthesized were complex lipids (phospholipids), and the pattern of fatty acid synthesis resembled that found in the liver slice. 7. 7. The mechanism of the stimulatory effect of microsomes on fatty acid synthesis by the supernatant fraction is discussed and tentatively localized at the level of acetyl-CoA carboxylation. The stimulation may be due to a release of feedback inhibition.

Fatty acid synthesis by complex systems The possibility of regulation by microsomes

1. 1. Fatty acid synthesis from malonyl-CoA catalyzed by rat-liver microsomal protein does not seem to be due to contamination with enzymes from the particle-free supernatant or mitochondrial fractions. 2. 2. Optimum conditions for the conversion of malonyl-CoA to fatty acids by rat-liver microsomes with respect to substrate concentration, protein concentration, and incubation time were established. The enzyme systems concerned with fatty acid synthesis from malonyl-CoA in the particle-free supernatant fraction were 4–5 times more active than those in the microsomes. 3. 3. Preincubation of the microsomes caused a release of malonyl-CoA decarboxylase (EC 4.1.1.9) but not of fatty acid synthetase. This resulted in a greater conversion of the available malonyl-CoA to fatty acids. 4. 4. The products of synthesis in the supernatant system were free fatty acids bound to protein, whereas the products of the microsomal system were predominantly complex lipids (phospholipid). 5. 5. The predominant fatty acid synthesized from malonyl-CoA by the supernatant fraction was palmitate, and that synthesized by the microsomes was stearate. 6. 6. In the system composed of the supernatant fraction plus microsomes, the fatty acids synthesized were complex lipids (phospholipids), and the pattern of fatty acid synthesis resembled that found in the liver slice. 7. 7. The mechanism of the stimulatory effect of microsomes on fatty acid synthesis by the supernatant fraction is discussed and tentatively localized at the level of acetyl-CoA carboxylation. The stimulation may be due to a release of feedback inhibition.

Hydrogen transfer in fatty acid synthesis by rat liver and mammary-gland cell-free preparations studied with tritium-labelled pyridine nucleotides and glucose

Incorporation of tritium from position 4 of the pyridine ring of TPNH and DPNH into fatty acids was studied in systems composed of particle-free supernatant fractions plus microsomes prepared from rat livers and in particle-free supernatant fractions from lactating rat mammary glands. 1. 1. Tritium from both nucleotides was incorporated into fatty acids, but that from TPNH was preferentially incorporated. 2. 2. Tritium transfer from both α- and β-configurations on position 4 of TPNH was demonstrated, but that from the α- exceeded that from the β-configuration. 3. 3. DPN+inhibited tritium transfer from [β- 3H]TPNH but not from [α- 3H]TPNH. The transfer from [β- 3H]DPNH was inhibited by TPN+, and the transfer was neglible from [α- 3H]DPNH. 4. 4. Two methods are presented for calculating integral changes in tritium activities on TPN+ when the nucleotide is continuously oxidized and reduced. 5. 5. Under certain conditions, in the mammary-gland system the value for tritium transfer from TPNH to fatty acids 14 C conversion from [I- 14C]acetate to fatty acids approached the theoretical value of two. 6. 6. No tritium from pyridine nucleotides nor 14C from acetate was incorporated into fatty acids in the absence of citrate. 7. 7. Tritium from [I- 3H]glucose 6-phosphate was efficiently transferred through TPN + to fatty acids by lactating rat mammary-gland preparations.

Hydrogen transfer in fatty acid synthesis by rat liver and mammary-gland cell-free preparations studied with tritium-labelled pyridine nucleotides and glucose

Incorporation of tritium from position 4 of the pyridine ring of TPNH and DPNH into fatty acids was studied in systems composed of particle-free supernatant fractions plus microsomes prepared from rat livers and in particle-free supernatant fractions from lactating rat mammary glands. 1. 1. Tritium from both nucleotides was incorporated into fatty acids, but that from TPNH was preferentially incorporated. 2. 2. Tritium transfer from both α- and β-configurations on position 4 of TPNH was demonstrated, but that from the α- exceeded that from the β-configuration. 3. 3. DPN+inhibited tritium transfer from [β- 3H]TPNH but not from [α- 3H]TPNH. The transfer from [β- 3H]DPNH was inhibited by TPN+, and the transfer was neglible from [α- 3H]DPNH. 4. 4. Two methods are presented for calculating integral changes in tritium activities on TPN+ when the nucleotide is continuously oxidized and reduced. 5. 5. Under certain conditions, in the mammary-gland system the value for ▪ approached the theoretical value of two. 6. 6. No tritium from pyridine nucleotides nor 14C from acetate was incorporated into fatty acids in the absence of citrate. 7. 7. Tritium from [I- 3H]glucose 6-phosphate was efficiently transferred through TPN + to fatty acids by lactating rat mammary-gland preparations.

The role of microsomes in fatty acid synthesis from acetate by cell-free preparations of rat liver and mammary gland

1. 1. The conversion of acetate to fatty acids by particle-free supernatant and microsomal fractions of rat livers and of lactating and non-lactating rat-mammary glands was studied. 2. 2. Stimulation of fatty acid synthesis by addition of microsomes to the supernatant fraction was related to the amounts of microsomal protein and ATP added to the incubation mixtures. Excessive amount of either one depressed the level of synthesis, which could be reversed by addition of larger amounts of the other. 3. 3. Microsomal stimulation cannot be explained solely by the presence of microsomal ATPase since the highest level of synthesis was observed at a critical ratio of microsomal protein to ATP concentration. This suggests that microsomal stimulation is related to an ATP-dependent process. 4. 4. The newly synthesized fatty acids were firmly bound to the incubated protein. Upon re-isolation of microsomes at the end of incubation, it was found that 80% of the newly synthesized fatty acids was bound to these particles. However, microsomal stimulation cannot be explained solely on the basis of a binding phenomenon. 5. 5. The possibility that microsomal stimulation of fatty acid synthesis from acetate or acetyl-CoA is due to an effect on acetyl-CoA carboxylase present in the supernatant fraction is discussed.