The sensitivity of acetyl-coenzyme A carboxylase to citrate stimulation in a homogenate of rat liver containing subcellular particles

1. Although citrate is known to activate purified preparations of acetyl-CoA carboxylase, it had no stimulatory effect on the incorporation of [¹⁴C]acetate into long-chain fatty acids in a whole homogenate of rat liver (S_0.7) under conditions in which the activity of acetyl-CoA carboxylase was rate-limiting for fatty acid synthesis. 2. The rate of incorporation of acetyl carbon into fatty acids was estimated in S_0.7 preparations incubated with [¹⁴C]acetate, by measuring the specific radioactivity of the acetyl carbon of acetyl-CoA and the incorporation of ¹⁴C into fatty acids. These estimates were compared with estimates of acetyl-CoA carboxylase activity in the S_0.7 preparation obtained by direct assay in conditions in which the enzyme was in the fully activated state. 3. In the absence of citrate, incorporation of acetyl carbon into fatty acids was about 75% of the value expected if the acetyl-CoA carboxylase in the S_0.7 preparation were in the fully activated state. 4. Incorporation of acetyl carbon into fatty acids in the S_0.7 preparation was stimulated by citrate, but the effect was many times less than the stimulation of [¹⁴C]acetate incorporation by citrate in particle-free preparations. 5. When the mitochondria and microsomes were removed from the S_0.7 preparation, [¹⁴C]acetate incorporation into fatty acids fell to a negligible value and the preparation became highly sensitive to stimulation by citrate. 6. It is suggested that in the presence of mitochondria and microsomes, and in the intact liver cell, the degree of activation of acetyl-CoA carboxylase is such that citrate activation may not be of physiological significance.     

Studies on the assay, activity and sedimentation behaviour of acetyl-CoA carboxylase from isolated hepatocytes incubated with insulin or glucagon.

The activity of acetyl-CoA carboxylase, measured in various ways, was studied in 15000g extracts of rat liver hepatocytes and compared with the rate of fatty acid synthesis in intact hepatocytes incubated with insulin or glucagon. Hepatocyte extracts were prepared by disruption of cells with a Dounce homogenizer or by solubilization with 1.5% (v/v) Triton X-100. Sucrose-density-gradient centrifugation demonstrated that the sedimentation coefficient of acetyl-CoA carboxylase from cell extracts was 30-35S, regardless of the conditions of incubation or disruption of hepatocytes. Solubilization of cells with 1.5% Triton X-100 yielded twice as much enzyme activity (measured by [14C]bicarbonate fixation) in the sucrose-gradient fractions as did cell disruption by the Dounce homogenizer. Analysis by high-performance liquid chromatography of acetyl-CoA carboxylase reaction mixtures showed that [14C]malonyl-CoA accounted for 10-60% of the total acid-stable radioactivity, depending on the method for disrupting hepatocytes and on the preincubation of the 15000g extract, with or without citrate, before assay. Under conditions in which incubation of cells with insulin or glucagon caused an activation or inhibition, respectively, of acetyl-CoA carboxylase, only 25% of the acid-stable radioactivity was [14C]malonyl-CoA and enzyme activity was only 13% (control), 16% (insulin), and 57% (glucagon) of the rate of fatty acid synthesis. Under conditions when up to 60% of the acid-stable radioactivity was [14C]malonyl-CoA and acetyl-CoA carboxylase activity was comparable with the rate of fatty acid synthesis, there was no effect of insulin or glucagon on enzyme activity.     

Induction of lipogenesis during differentiation in a "preadipocyte" cell line.

3T3-L1 fibroblasts differentiate in culture into cells having adipocyte character. This transition is accompanied by a 40- to 50-fold rise in the incorporation of [14C]acetate into triglyceride. The increase in lipogenic rate is exactly parallel to a coordinate rise in the activities of the key enzymes of the fatty acid biosynthetic pathway (ATP-citrate lyase, acetyl-CoA carboxylase, and fatty acid synthetase). Immunological studies indicate that the elevated acetyl-CoA carboxylase activity is the product of an increased cellular enzyme level.     

Acetyl-coenzyme A carboxylase in maize leaves

Purified chloroplasts from mesophyll and bundle sheath cells of maize leaves have been shown to be the location of acetyl-CoA carboxylase. In disrupted chloroplasts the enzyme was recovered in the stromal fraction, along with protein-bound biotin; acetyl-CoA carboxylase activity did not require a membrane component. Mg²⁺ and ATP are required for activity and sulfhydryl protecting agents enhance stability of the enzyme. Acetyl-CoA carboxylase activity was independent of leaf development in cell-free extracts of maize. Comparison of acetyl-CoA carboxylase activity with [¹⁴C]acetate incorporation into lipids, in isolated chloroplasts from developing leaves of maize, indicate that acetyl-CoA carboxylase is not limiting fatty acid synthesis.     

Affinity labelling of rat liver acetyl-CoA carboxylase by a 2',3'-dialdehyde derivative of ATP

The interaction of rat liver acetyl-CoA carboxylase with a 2',3'-dialdehyde derivative of ATP (oATP) has been studied. The degree of the enzyme inactivation has been found to depend on the oATP concentration and the incubation time. ATP was proved to be the only substrate which protected the inactivation. Acetyl-CoA did not effect inactivation, while HCO3- accelerated the process. Ki values for oATP in the absence and presence of HCO3- were 0.35 +/- 0.04 and 0.5 +/- 0.06 mM, and those of the modification constant (kmod) were 0.11 and 0.26 min-1 respectively. oATP completely inhibited the [14C]ADP in equilibrium ATP exchange and did not effect the [14C]acetyl-CoA in equilibrium malonyl-CoA exchange. Incorporation of approximately 1 equivalent of [3H]oATP per acetyl-CoA carboxylase subunit has been shown. No recovery of the modified enzyme activity has been observed in Tris or beta-mercaptoethanol containing buffers, and treatment with NaB3H4 has not led to 3H incorporation. The modification elimination of the ATP triphosphate chain. The results indicated the affinity modification of acetyl-CoA carboxylase by oATP. It was shown that the reagent apparently interacted selectively with the epsilon-amino group of lysine in the ATP-binding site to form a morpholine-like structure.     

Acute control of fatty acid synthesis by cyclic AMP in the chick liver cell: possible site of inhibition of citrate formation.

Glucagon and N,(6)O(2)-dibutyryl cyclic adenosine 3',5'-cyclic monophosphate (Bt(2)cAMP) inhibit fatty acid synthesis from acetate by more than 90% and prevent citrate formation in chick hepatocytes metabolizing glucose. With substrates that enter glycolysis at or below triose-phosphates, e.g., fructose, lactate, or pyruvate, Bt(2)cAMP has no effect on the citrate level and its inhibitory effect on fatty acid synthesis is substantially reversed. Because acetyl-CoA carboxylase requires a tricarboxylic acid activator for activity, it is proposed that regulation of fatty acid synthesis by Bt(2)cAMP is due, in part, to changes in the citrate level. Reduced citrate formation appears to result from a cAMP-induced inhibition of glycolysis. Bt(2)cAMP inhibits (14)CO(2) production from [1-(14)C]-, [6-(14)C]-, and [U-(14)C]glucose and has little effect on (14)CO(2) formation from [1-(14)C]- or [2-(14)C]pyruvate or from [1-(14)C]fructose. [(14)C]Lactate formation from glucose is depressed 50% by Bt(2)cAMP. In the presence of an inhibitor of mitochondrial pyruvate transport lactate accumulation is enhanced, but continues to be lowered 50% by Bt(2)cAMP. The activity of phosphofructokinase is greatly decreased in Bt(2)cAMP-treated cells while the activities of pyruvate kinase and acetyl-CoA carboxylase are unaffected. It appears that decreased glycolytic flux and decreased citrate formation result from depressed phosphofructokinase activity. Fatty acid synthesis from [(14)C]acetate is partially inhibited by Bt(2)cAMP in the presence of fructose, lactate, and pyruvate despite a high citrate level. Incorporation of [(14)C]fructose, [(14)C]pyruvate, or [(14)C]lactate into fatty acids is similarly depressed by Bt(2)cAMP. Synthesis of cholesterol from [(14)C]acetate or [2-(14)C]pyruvate is unaffected by Bt(2)cAMP. These results implicate a second site of inhibition of fatty acid synthesis by Bt(2)cAMP that involves the utilization, but not the production, of cytoplasmic acetyl-CoA.-Clarke, S. D., P. A. Watkins, and M. D. Lane. Acute control of fatty acid synthesis by cyclic AMP in the chick liver cell: possible site of inhibition of citrate formation.     

De novo fatty acid synthesis in developing rat lung

The rate of de novo fatty acid synthesis in developing rat lung was measured by the rate of incorporation of 3H from 3H2O into fatty acids in lung slices and by the activity of acetyl-CoA carboxylase in fetal, neonatal and adult lung. Both tritium incorporation and acetyl-CoA carboxylase activity increased sharply during late gestation, peaked on the last fetal day, and declined by 50% 1 day after birth. In the adult, values were only one-half the peak fetal rates. In vitro regulation of acetyl-CoA carboxylase activity in fetal lung was similar to that described in adult non-pulmonary tissues: activation by citrate and inhibition by palmitoyl-CoA. Similarly, incubation conditions that favored enzyme phosphorylation inhibited acetyl-CoA carboxylase activity in lung while dephosphorylating conditions stimulated activity. Incorporation of [U-14 C]glucose into lung lipids during development was influenced heavily by incorporation into fatty acids, which generally paralleled the rate of tritium incorporation into fatty acids. The relative utilization of acetyl units from exogenous glucose for overall fatty acid synthesis was greater in adult lung than in fetal or neonatal lung, suggesting that other substrates may be important for fatty acid synthesis in developing lung. In fetal lung explants, de novo fatty acid synthesis was inhibited by exogenous palmitate. Taken together, these data suggest that de novo synthesis may be an important source of saturated fatty acids in fetal lung but of lesser importance in the neonatal period. Furthermore, the regulation of acetyl-CoA carboxylase activity and fatty acid synthesis in lung may be similar to non-pulmonary tissues.     

Insulin activation of lipogenesis in isolated mammary acini from lactating rats fed on a high-fat diet. Evidence that acetyl-CoA carboxylase is a site of action.

Feeding lactating rats on high-fat cheese crackers in addition to laboratory chow increased the dietary intake of fat from 2 to 20% of the total weight of food eaten and decreased mammary-gland lipogenesis in vivo by approx. 50%. This lipogenic inhibition was also observed in isolated mammary acini, where it was accompanied by decreased glucose uptake. These inhibitions were completely reversed by incubation with insulin. Insulin had no effect on the rate of glucose transport into acini, nor on pyruvate dehydrogenase activity as estimated by the accumulation of pyruvate and lactate, suggesting that these are not the sites of lipogenic inhibition. Insulin stimulated the incorporation of [1-14C]acetate into lipid in acini from high-fat-fed rats. In the presence of alpha-cyanohydroxycinnamate, a potent inhibitor of mitochondrial pyruvate transport, and with glucose as the sole substrate, neither [1-14C]glucose incorporation into lipid nor glucose uptake were stimulated by insulin. Insulin did stimulate the incorporation of [1-14C]acetate into lipid in the presence of alpha-cyanohydroxycinnamate, and this was accompanied by an increase in glucose uptake by the acini. This indicated that increased glucose uptake was secondary to the stimulation of lipogenesis by insulin, which therefore must occur via activation of a step in the pathway distal to mitochondrial pyruvate transport. Insulin stimulated acetyl-CoA carboxylase activity measured in crude extracts of acini from high-fat-fed rats, restoring it to values close to those of chow-fed controls. The effects of insulin on acetyl-CoA carboxylase activity and lipogenesis were not antagonized by adrenaline or dibutyryl cyclic AMP.     

Requirement of Ca2+ for aflatoxin production: inhibitory effect of Ca2+ channel blockers on aflatoxin production by Aspergillus parasiticus NRRL 2999

Aflatoxin production by Aspergillus parasiticus NRRL 2999 was inhibited when Ca2+ channel blockers, i.e., verapamil and diltiazem (> 1 mmol 1(-1)), were included in the culture medium. Inhibition was not accompanied by growth inhibition, nor was the [14C]-glucose uptake by the organism altered. However, both the compounds inhibited [14C]-acetate incorporation into aflatoxin B1 in a dose-dependent manner and decreased sporulation of the organism. Even though a nutritional role for Ca2+ has not been demonstrated unequivocally in fungi, the present study suggests the importance of Ca2+ in the production of these secondary metabolites.     

Biochemical analysis of oxidative stress in the production of aflatoxin and its precursor intermediates

The relevance of oxidative stress in the production of aflatoxin and its precursors was examined in different mutants of Aspergillus parasiticus, which produce aflatoxin or its precursor intermediates, and compared with results obtained from a non-toxigenic strain. In comparison to the non-toxigenic strain (SRRC 255), an aflatoxin producing strain (NRRL 2999) or mutants that accumulate aflatoxin precursors such as norsolorinic acid (by SRRC 162) or versicolorin (by NRRL 6196) or O-methyl sterigmatocystin (by SRRC 2043) had greater oxygen requirements and higher contents of reactive oxygen species. These changes were in the graded order of NRRL 2999 > SRRC 2043 > NRRL 6196 > SRRC 162 > SRRC 255, indicating incremental accumulation of reactive oxygen species, being least in the non-toxigenic strain and increasing progressively during the ternary steps of aflatoxin formation. Oxidative stress in these strains was evident by increased activities of xanthine oxidase and free radical scavenging enzymes (superoxide dismutase and glutathione peroxidase) as compared to the non-toxigenic strain (SRRC 255). Culturing the toxigenic strain in presence of 0.1–10 μM H₂O₂ in the medium resulted in enhanced aflatoxin production, which could be related to dose-dependent increase in [¹⁴C]-acetate incorporation into aflatoxin B₁ and increased acetyl CoA carboxylase activity. The combined results suggest that formation of secondary metabolites such as aflatoxin and its precursors by A. parasiticus may occur as a compensatory response to reactive oxygen species accumulation.     

The effect of hypolipidemic drugs on plant lipid metabolism

The effect of hypolipidemic drugs, WY14643 and DH990, on plant lipid metabolism has been studied. The total incorporation of [14C]acetate into lipids was inhibited by addition of both drugs to aged potato (Solanum tuberosum) tuber discs, spinach (Spinacia oleracea) leaves, and spinach chloroplasts, while the incorporation in Chlorella vulgaris cells was affected only by DH990. Moreover, DH990 inhibited the incorporation of 14C-labeled fatty acids into phosphatidylcholine and phosphatidylethanolamine of potato discs, and decreased the incorporation into phosphatidylglycerol of Chlorella cells. DH990 inhibited the formation of polyunsaturated fatty acids in potato discs, Chlorella cells, and spinach leaves, whereas WY14643 had no effect on the formation of these fatty acids. Stearoyl-ACP desaturase from safflower (Carthamus tinctorius) seeds was very sensitive to both drugs, especially DH990, which completely blocked the activity at 2 mM levels. When safflower lysophospholipid acyltransferases were solubilized by detergent treatment, only DH990 inhibited the incorporation of [14C]oleoyl-CoA into lysophosphatidylcholine or lysophosphatidylethanolamine. Both drugs inhibited fatty acid synthesis from [14C]malonyl-CoA in the microsomal fraction from safflower seeds, but only DH990 inhibited FAS activity in the soluble fraction; both drugs inhibited severely the formation of stearic acid. Both acetyl-CoA carboxylase and acetyl-CoA synthetase were sensitive to both drugs.     

Decarboxylation of malonyl-CoA and biosynthesis of mevalonic acid in rat liver]

Biosynthesis of mevalonic acid (MVA), total formation of 14CO2 from [1,3-14C]malonyl-CoA and the activity of malonyl-CoA decarboxylase in subcellular fractions of rat liver were studied. The dependence of the rate of MVA biosynthesis on malonyl-CoA concentration was found to be linear both in 140,000 g supernatant and solubilized microsomal fractions. It was shown that in a composite system (140,000 g supernatant fraction added to washed microsomes, 10 : 1) the optimal concentration ratio for the substrates of MVA biosynthesis (malonyl-CoA and acetyl-CoA) is 1 to 2. In the absence of acetyl-CoA decarboxylation of [1,3-14C]malonyl-CoA was prevalent. In all subcellular fractions studied decarboxylation of [1,3-14C]malonyl-CoA prevailed over its incorporation into MVA, total non-saponified lipid fraction and fatty acids. The degree of malonyl-CoA, decarboxylation was not correlated with the rate of its incorporation into MVA, i. e. the increase in the 14CO2 formation was not accompanied by stimulation of [1,3-14C]malonyl-CoA incorporation either into MVA or into total non-saponified lipid fractions. The incorporation of [1-14C]acetyl-CoA into MVA under the same conditions was considerably lower than that of [1,3-14C]malonyl-CoA. In all subcellular fractions under study the activity of malonyl-CoA decarboxylase was found. The experimental data suggest that a remarkable part of malonyl-CoA is incorporated into MVA without preliminary decarboxylation. A possible role of malonyl-CoA decarboxylase as an enzyme which protects the cell against accumulation of malonyl-CoA and its immediate metabolites -- malonate and methylmalonyl-CoA is disucssed.     

Addendum to a new mathematical approach to metabolism of [14C]glucose: the pyruvate carboxylase reaction

—Previously published equations for analysis of [¹⁴C]glucose metabolism assumed that products of glycolysis enter the citric acid cycle only through acetyl-CoA (Larrabee , 1978). These equations are now extended to include entrance into the citric acid cycle through the pyruvate carboxy-lase reaction as well as via acetyl-CoA and are applied to previously reported data from dorsal root ganglia of 15-day-old chicken embryos. The rate of output of labelled CO₂ in the presence of [2-¹⁴C] glucose could not be accounted for if the flux rate into the citric acid cycle through the pyruvate carboxylase reaction was assumed to be more than about 10–15% of that through acetyl-CoA. It is concluded (1) that the pyruvate carboxylase reaction is a relatively minor source of material for the citric acid cycle in these ganglia and (2) that the previous conclusions about [¹⁴C]glucose metabolism, which ignored the pyruvate carboxylase reaction, need not be modified in the light of this reanalysis.     

De novo fatty acid synthesis by freshly isolated alveolar type II epithelial cells

Fatty acid synthesis was studied in freshly isolated type II pneumocytes from rabbits by 3H2O and (U-14C)-labeled glucose, lactate and pyruvate incorporation and the activity of acetyl-CoA carboxylase. The rate of lactate incorporation into fatty acids was 3-fold greater than glucose incorporation; lactate incorporation into the glycerol portion of lipids was very low but glucose incorporation into this fraction was approximately equal to incorporation into fatty acids. The highest rate of de novo fatty acid synthesis (3H2O incorporation) required both glucose and lactate. Under these circumstances lactate provided 81.5% of the acetyl units while glucose provided 5.6%. Incubations with glucose plus pyruvate had a significantly lower rate of fatty acid synthesis than glucose plus lactate. The availability of exogenous palmitate decreased de novo fatty acid synthesis by 80% in the isolated cells. In a cell-free supernatant, acetyl-CoA carboxylase activity was almost completely inhibited by palmitoyl-CoA; citrate blunted this inhibition. These data indicate that the type II pneumocyte is capable of a high rate of de novo fatty acid synthesis and that lactate is a preferred source of acetyl units. The type II pneumocyte can rapidly decrease the rate of fatty acid synthesis, probably by allosteric inhibition of acetyl-CoA carboxylase, if exogenous fatty acids are available.     

Malonate as a precursor in the biosynthesis of aflatoxins.

Incorporation of [I-14C]acetate and [2-14C]malonate into aflatoxins by resting mycelia of Aspergillus parasiticus resuspended in different buffers was studied. A decrease in pH from 5-8 to 2-8, as well as addition of EDTA, markedly stimulated the incorporation of malonate but the effect on acetate incorporation was less pronounced. Mycelia took up comparatively more acetate than malonate, but more malonate (4-3%) entering mycelia was incorporated into aflatoxins than was acetate (1-6%). Furthermore, the addition of unlabelled acetate reduced the incorporation of label from [I-14C]acetate by 75% but from [2-14C]malonate by only 25%. These results suggest that malonate is an intermediate in aflatoxin synthesis and that is can be incorporated without prior conversion to acetate.     

Enzymatically inactive forms of acetyl-CoA carboxylase in rat liver mitochondria.

Biotinyl proteins were labelled by incubation of SDS-denatured preparations of subcellular fractions of rat liver with [14C]methylavidin before polyacrylamide-gel electrophoresis. Fluorographic analysis showed that mitochondria contained two forms of acetyl-CoA carboxylase [acetyl-CoA:carbon dioxide ligase (ADP-forming) EC 6.4.1.2], both of which were precipitated by antibody to the enzyme. When both forms were considered, almost three-quarters of the total liver acetyl-CoA carboxylase was found in the mitochondrial fraction of liver from fed rats while only 3.5% was associated with the microsomal fraction. The remainder was present in cytosol, either as the intact active enzyme or as a degradation product. The actual specific activity of the cytosolic enzyme was approx. 2 units/mg of acetyl-CoA carboxylase protein while that of the mitochondrial enzyme was about 20-fold lower, indicating that mitochondrial acetyl-CoA carboxylase was relatively inactive. Fractionation of mitochondria with digitonin showed that acetyl-CoA carboxylase was associated with the outer mitochondrial membrane. The available evidence suggests that mitochondrial acetyl-CoA carboxylase represents a reservoir of enzyme which can be released and activated under lipogenic conditions.     

Formation of mevalonic acid, sterols and bile acids from [1-14C]acetyl-CoA and [2-14C]malonyl-CoA in the liver of rabbits with experimental hypercholesterolemia

The effect of cholesterol diet on the rate of mevalonic acid biosynthesis from 1-14C acetyl-CoA, 2-14C malonyl-CoA and the incorporation of these substrates into sterols and bile acids in rabbit liver were studied. Simultaneously, the activities of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) and acetyl-CoA carboxylase and the biosynthesis of fatty acids from acetyl-CoA and malonyl-CoA were measured. Hypercholesterolemia was found to be concomitant with the inhibition of acetyl-CoA carboxylase activity only in cell-free (700 g) and mitochondrial fractions and slightly decreased the incorporation of acetyl-CoA and malonyl-CoA into fatty acids in the postmitochondrial fraction. The HMG-CoA reductase activity in all subcellular fractions except for the postmicrosomal one was inhibited under these conditions. A significant decrease of acetyl-CoA incorporation and an increase in malonyl-CoA incorporation into mevalonic acid in all liver fractions except for microsomal one were observed in rabbits with hypercholesterolemia. These data provide evidence for the existence of two pathways of mevalonic acid synthesis from the above-said substrates that are differently sensitive to cholesterol. Cholesterol feeding resulted in a decreased synthesis of the total unsaponified fraction including cholesterol from acetyl-CoA, malonyl-CoA and mevalonic acid. The rate of incorporation of these substrates into lanosterol was unchanged. All the indicated substrates (acetyl-CoA, malonyl-CoA, mevalonic acid) are precursors of bile acid synthesis in rabbit liver. Cholesterol feeding and the subsequent development of hypercholesterolemia resulted in bile acid synthesis stimulation, preferentially in the formation of the cholic + deoxycholic acids from these precursors.     

Inhibition of fatty acid synthesis by RMI 14,514 (5-tetradecyloxy-2-furoic acid)

RMI 14,514 strongly inhibited the incorporation of label from [1-¹⁴C]acetyl-CoA into fatty acids by rat liver homogenates. No inhibition was observed when [2-¹⁴C]malonyl-CoA was used as the labeled fatty acid precursor. These results suggest that the drug inhibits $\text{de novo}$ fatty acid biosynthesis at the step mediated by acetyl-CoA carboxylase. The data presented in this communication support earlier reports that RMI 14,514 probablyexerts its hypolipidemic effects by inhibition of fatty acid biosynthesis.     

Alterations in the lipid content of pituitary gland and serum prolactin and growth hormone in cadmium treated rats

The present study was undertaken to assess whether chronic exposition to cadmium (Cd, 0.133 mM per liter for 2 months) through drinking water may affect the lipid contents in the pituitary anterior lobe (PAL) of adult male Wistar rats. As compared to metal non-exposed controls, PALs exposed to cadmium showed an increase in total phospholipid contents, which was associated to an increase of the incorporation of [1–¹⁴C]-methyl choline into phosphatidylcholine and of [U–¹⁴C]-glucose into total phospholipids. The incorporation of [1–¹⁴C]-methyl choline into sphingomyelin was not changed. Incorporation of [1–¹⁴C]-acetate into total fatty acids also increased but incorporation of [1–¹⁴C]-acetate into cholesterol did not change. The activity of phospholipase D decreased both in PALs from Cd exposed rats and in PAL dispersed cells treated with Cd in the culture medium from Cd non-exposed rats. In PALS from Cd exposed rats, a decrease of serum prolactin and growth hormone concentrations was determined. The results shown that cadmium modifies the lipid contents of pituitary gland and directly or indirectly the levels of prolactin and growth hormone in serum.     

Acetyl-CoA-carboxylase: modification of ATP-binding site of the active center by 2',3'-dialdehyde derivative of ATP

The interaction of rat liver acetyl-CoA carboxylase with a 2',3'-dialdehyde derivative of ATP (oATP) has been studied. The degree of the enzyme inactivation has been found to depend on the oATP concentration and the incubation time. ATP was the only reaction substrate which provided protection from inactivation. Acetyl-CoA did not affect inactivation, while HCO3- accelerated the process. Ki values for oATP in the absence and the presence of HCO3- were 0.35 +/- 0.04 and 0.5 +/- 0.06 mM, and those of the modification constant (k) were 0.11 and 0.26 min-1, respectively. oATP completely inhibited the reaction of [14C]ADP in equilibrium ATP exchange, whereas produced actually no effect on [14C]acetyl-CoA equilibrium with malonyl-CoA exchange. Incorporation of about one equivalent of [3H]oATP per acetyl-CoA carboxylase subunit has been shown. No restoration of the modified enzyme activity has been observed in Tris or beta-mercaptoethanol containing buffers, and treatment with NaB[3H]4 has not led to 3H incorporation. The modification process involves elimination of the triphosphate chain of oATP. The results obtained indicate the affinity character of oATP-mediated modification of acetyl-CoA carboxylase. The reagent apparently interacts selectively with the epsilon-amino group of lysine in the ATP-binding site to form a morpholine-like structure.