Cell-free Preparations Of Rat Liver Research Articles

Enzyme induction and hepatic glycerolipid synthesis in rats treated with 3-methylcholanthrene.

Fasting male rats fed 3-methylcholanthrene in a daily dose of 40 mg . kg body weight-1 gave evidence of hepatic microsomal enzyme induction after 3 days through significantly increased hepatic aryl hydrocarbon hydroxylase activity, cytochrome P448 content, and characteristic changes in microsomal proteins analysed by sodium dodecyl sulfate--polyacrylamide gel electrophoresis. Concomitantly, activities of aminopyrine N-demethylase and microsomal gamma-glutamyltransferase, which are increased in phenobarbital-treated rats, significantly declined. In contrast to phenobarbital, which has been previously shown to increase hepatic triacylglycerol content and in vitro glycerolipid synthesis, 3-methylcholanthrene did not affect hepatic triacylglycerol content, but did inhibit glycerolipid synthesis by cell-free preparations of rat liver and significantly reduced serum triacylglycerol concentration. Thus, the two prototypical drugs inducing characteristically different changes in microsomal enzyme and hemoprotein response also seem to differ in their effect upon another important microsomal function, hepatic glycerolipid synthesis.

Inhibitors of sterol synthesis. Differential effects of 14 alpha-hydroxymethyl-5 alpha-cholest-7-ene-3 beta, 15 alpha-diol and 14 alpha-hydroxymethyl-5 alpha-cholest-6-ene-3 beta, 15 alpha-diol on sterol synthesis in cell-free homogenates of rat liver.

14 alpha-Hydroxymethyl-5 alpha-cholest-7-ene-3 beta, 15 alpha-diol and 14 alpha-hydroxymethyl-5 alpha-cholest-6-ene-3 beta, 15 alpha-diol have been shown to be potent inhibitors of the synthesis of digitonin-precipitable sterols in mouse L-cells and in primary cultures of fetal mouse liver cells and to cause a reduction in the levels of activity of 3-hydroxy-3-methylglutaryl-CoA reductase in the same cells (Schroepfer, G. J., Jr., Parish, E. J., Pascal, R. A., Jr., and Kandutsch, A. A. (1980) J. Lipid Res. 21, 571-584). In the present study, we have found that both sterols have a second, but distinct, site of action, distal to the formation of mevalonic acid. 14 alpha-Hydroxymethyl-5 alpha-cholest-7-ene-3 beta, 15 alpha-diol has been found to be a potent inhibitor of the synthesis of digitonin-precipitable sterols from labeled mevalonate in cell-free preparations of rat liver. This inhibition was accompanied by a striking accumulation of labeled lanosterol and 24,25-dihydrolanosterol. The latter sterols were fully characterized by the results of chromatographic and co-crystallization experiments. In contrast, 14 alpha-hydroxymethyl-5 alpha-cholest-6-ene-3 beta, 15 alpha-diol had only a slight effect on the synthesis of digitonin-precipitable sterols from labeled mevalonate in cell-free rat liver preparations. The delta 6-3 beta, 15 alpha, 32-triol had no apparent effect on the metabolism of lanosterol and 24,25-dihydrolanosterol but caused a substantial accumulation of labeled 5 alpha-cholest-8-en-3 beta-ol which was fully characterized by the results of chromatographic and co-crystallization experiments. These findings are compatible with a specific inhibition of the metabolism of lanosterol and 24,25-dihydrolanosterol by the delta 7-3 beta, 15 alpha, 32-triol and a specific inhibition of the delta 8 leads to delta 7 isomerase by the delta 6-3 beta, 15 alpha, 32-triol. [2,4]3H]14 alpha-Hydroxymethyl-5 alpha-cholest-7-ene-3 beta, 15 alpha-diol, prepared by chemical synthesis, was not convertible to cholesterol upon incubation, under aerobic conditions, with a cell-free homogenate preparation of rat liver. The labeled delta 7-3 beta, 15 alpha, 32-triol was, however, metabolized to several polar compounds.

Effects of diabetes on oxidative decarboxylation of branched-chain keto acids.

Oxidative decarboxylation is the first irreversible step in the degradation of leucine. The effect of streptozotocin diabetes on this reaction was studied in cell-free rat liver preparations, using [1-14C]alpha-ketoisocaproate as substrate. Diabetes increased the branched-chain ketoacid dehydrogenase (BCKD) activity (per g liver or per mg protein) of homogenates, but the ratios of homogenate BCKD activity to other mitochondrial markers remained unchanged. A cytosolic branched-chain ketoacid decarboxylase activity (15-22% of homogenate activity), which did not require NAD, CoA, or NADP, was also increased in diabetics. Insulin treatment of diabetics normalized enzyme activity in all fractions. The apparent Km of BCKD in homogenates was 43-45 microM; diabetes increased the apparent Vmax from 165 nmol x min-1 x g tissue-1 to 260 nmol x min-1 x g-1. In contrast, the Km for cytosolic alpha-ketoisocaproate decarboxylation was 270 microM in controls, and diabetes resulted in both a lower Km (210 microM) and a higher Vmax. Adrenalectomy did not affect activity in homogenates from controls, but partially reversed the diabetes-associated increase. Glucagon pretreatment of controls did not affect activity. In summary, distinct mitochondrial and cytosolic enzymes decarboxylate alpha-ketoisocaproate in liver. The increased hepatic capacity of diabetic rats to degrade the carbon skeleton of leucine is attributed mainly to a relative increase in mitochondrial mass.

Cycloheximide inhibits sterol biosynthesis in cell-free preparations of rat liver.

Cycloheximide, frequently used as an inhibitor of protein synthesis in vivo and in vitro, has been found to cause a significant reduction of the synthesis of digitonin-precipitable sterols from acetate, but not from mevalonate, at a concentration of 1 mM in the 10,000 x g supernatant fraction of rat liver homogenate preparations. The results of studies of the metabolism of labeled leucine under the same conditions indicated that the effect of cycloheximide on sterol synthesis from acetate was not related to an effect of cycloheximide on protein synthesis. Preincubation of the 10,000 x g supernatant fraction of rat liver homogenates with cycloheximide (1 mM) caused a significant reduction in the levels of acetate thiokinase and hydroxymethylglutaryl-CoA synthase activities but not of acetoacetyl-CoA thiolase activity. Preincubation of the 100,000 x g supernatant fraction of rat liver homogenates with cycloheximide (1 mM or 0.3 mM) also caused a significant reduction of the levels of hydroxymethylglutaryl-CoA synthase activity. When cycloheximide (1 mM) was preincubated with the 100,000 x g supernatant fraction, a reduction in the level of acetate thiokinase activity was observed. Preincubation of rat liver microsomes with cycloheximide (1 mM and 3 mM) had no effect on the level of hydroxymethylglutaryl-CoA reductase activity. These results suggest that biological effects observed upon exposure of cells or tissues to high concentrations of cycloheximide may not be exclusively due to effects of the cycloheximide on the synthesis of protein.

Metabolism of 2-amino-3-phosphono[3- 14C]propionic acid in cell-free preparations of rat liver

Incubation of 2-amino-3-phosphono[3- 14C]propionic acid with cell-free preparations of rat liver yielded labelled 3-phosphonopyruvic acid, 2-phosphonoacetaldehyde, 2-aminoethylphosphonic acid and acetaldehyde. No radioactivity was found in phospho enolpyruvate, pyruvic acid, alanine, and phosphonoacetic acid. When added to the cell-free preparations, 3-phosphonopyruvic acid trapped the radioactivity, resulting in decrease of incorporation of the radioactivity into 2-phosphonoacetaldehyde, 2-aminoethylphosphonic acid and acetaldehyde. Incorporation of the radioactivity into 2-aminoethylphosphonic acid and acetaldehyde was also decreased by 2-phosphonoacetaldehyde. Thus it appears that the main metabolic pathway of 2-amino-3-phosphonopropionic acid is deamination to produce 3-phosphonopyruvic acid which is, in turn, converted to 2-phosphonoacetaldehyde by decarboxylation, followed by both dephosphonylation and amination of the aldehyde to give acetaldehyde and 2-aminoethylphosphonic acid, respectively.

Chemical and enzymic studies on the characterization of intermediates during the removal of the 14alpha-methyl group in cholesterol biosynthesis. The use of 32-functionalized lanostane derivatives.

By using cell-free preparations of rat liver it was shown that the removal of the 14alpha-methyl group (C-32) of steroids containing either a delta7(8) or a delta8(9) double bond is attended exclusively by the formation of the corresponding 7,14- and 8,14-dienes respectively (structures of types III and VIII). Cumulative evidence from a variety of experimental approaches had led to the deduction that delta8(14)-steroids are not involved as intermediates on the major pathway of cholesterol biosynthesis. The metabolism of [32-3H]lanost-7-ene-3beta,32-diol (structure of type I) results in the formation of radioactive formic acid, no labelled formaldehyde being formed. By using appropriately labelled species of the compound (I) it was found that the release of formic acid and the formation of 4,4-dimethylcholesta-7,14-dien-3beta-ol (strurcture of type III) were closely linked processes, and that in the conversion of compound (I) into compound (III), 3-beta-hydroxylanost-7-en-32-al (II) is an obligatory intermediate. Both the conversion of lanost-7-ene-3beta,32-diol (I) into 3beta-hydroxylanost-7-en-32-al (II) and the further metabolism of the latter (II) to 4,4-dimethylcholesta-7,14-dien-3beta-ol (III) exhibited a requirement for NADPH and O2. This suggests that the oxidation of the 32-hydroxy group of compound (I) to the aldehyde group of compound (II) does not occur by the conventional alcohol dehydrogenase type of reaction, but may proceed by a novel mechanism involving the intermediacy of a gem-diol. A detailed overall pathway for the 14alpha-demethylation in cholesterol biosynthesis is considered, and proposals about the mechanism of individual steps in the pathway are made.

Hydrolysis of chyle cholesterol esters with cell-free preparations of rat liver

1. 1. The effect of pH on the hydrolysis of chylomicron and chylomicrom remnant cholesterol ester with rat liver homogenate was examined. The hydrolysis had three pH optima, at pH 4.5, at pH 6.0–6.5 and at pH 8.5. At the two upper pH optima extensive cholesterol ester hydrolysis occurred without simultaneous degradation of the triacylglycerol portion. 2. 2. Similarly, microsomes (at pH 6.5–8.0) and 100 000 × g supernatant (at pH 7.5–8.5) efficiently hydrolyzed the cholesterol ester but not the triacylglycerol of chylomicron remnants. 3. 3. With the same substrate no enrichment of neutral cholesterol esterase activity was seen in isolated plasma membranes. 4. 4. At pH 4.5 lysosomes efficiently hydrolyzed both the cholesterol ester and the triacylglycerol portion of chylomicron remnants. 5. 5. Three conclusions are drawn: (a) the study provides evidence against the existence of a plasma membrane-bound enzyme-hydrolyzing chylomicron cholesterol ester before or during its penetration into the cell; (b) enzymes of the cell sap and possibly of the endoplasmic reticulum can degrade cholesterol ester of chylomicron remnants without proceeding hydrolysis of the triacylglycerol core; and (c) lysosomal enzymes can degrade both the cholesterol ester and the triacylglycerol portion of chylomicron remnants if these are taken up as whole particles by endocytosis.

Selective inhibition of cholesterol synthesis by cell-free preparations of rat liver by using inhibitors of cytoplasmic acetoacetyl-coenzyme A thiolase.

Cytoplasmic acetoacetyl-CoA thiolase (acetyl-CoA C-acetyltransferase, EC 2.3.1.9) was partially purified from rat liver. The enzyme was irreversibly inactivated by 4-bromocrotonyl-CoA, but-3-ynoyl-CoA, pent-3-ynoyl-CoA and dec-3-ynoyl-CoA. In the case of the alk-3-ynoyl-CoA esters the potency as alkylating agents of acetoacetyl-CoA thiolase decreased with increased chain length of the alk-3-ynoyl moiety. Advantage was taken of the specific action of alk-3-ynoyl-CoA esters on acetoacetyl-CoA thiolase to show that in a postmitochondrial fraction from rat liver they are effective inhibitors of cholesterol synthesis from sodium [2-14C]acetate under conditions when mevalonate conversion into cholesterol and fatty acid synthesis are unafffected. Short-chain alk-3-ynoic acids have little effect on sterol synthesis, although dec-3-ynoic acid is an effective inhibitor owing to its conversion into the CoA ester by the microsomal fatty acyl-CoA synthetase.

Studies on the formation of C7-oxygenated cholesterol and β-sitosterol metabolites in cell-free preparations of rat liver

The microsomal fraction and the 18,000 g supernatant fluid obtained from livers from normal rats, cholestyraminetreated rats, or from rats with a bile fistula have been used to compare the 7alpha-hydroxylation of [4-(14)C]cholesterol and beta-[4-(14)C]sitosterol (24alpha-ethyl-cholesterol). It was not possible to increase the specific formation of 7alpha-hydroxy-beta-sitosterol above 0.05% with any of the preparations. This conversion was less than 1% of that found for cholesterol. The inhibitory effect of added 7-oxo- and 7beta-hydroxy-beta-sitosterol on the 7alpha-hydroxylation of cholesterol was found to be much less than that of the corresponding cholesterol compounds. 7alpha-Hydroxy-beta-sitosterol was without effect. It is concluded that the activity of the cholesterol 7alpha-hydroxylase is dependent upon the structure of the steroid side chain.

Inhibition of Sterol Biosynthesis by 2-Isopropyl-4-dimethylamino-5-methylphenyl-1-piperidine Carboxylate Methyl Chloride in Tobacco and Rat Liver Preparations

2-Isopropyl-4-dimethylamino-5-methylphenyl-1-piperidine carboxylate methyl chloride, 90%, applied to rootless tobacco (Nicotiana tabacum cv. Samson) seedlings inhibits the incorporation of (14)C-mevalonate into sterols. Concomitantly, the retardant causes the accumulation of squalene-2,3-epoxide, an intermediate in sterol biosynthesis. The results with tobacco are identical to those produced by the retardant in cell-free rat liver preparations.

Differential inhibition of rat liver cholesterol biosynthesis by plant growth retardants.

To investigate the action of plant growth retardants more precisely, cell-free rat liver preparations have been used as models of plant systems1. At least some of the retardants inhibited the enzymes of the isoprenoid biosynthesizing pathway by which radioactive acetate and mevalonate are incorporated into cholesterol. Inhibition of the isoprenoid biosynthetic route2 that leads to the formation of gibberellins3 thus seemed to be one of the main effects of applying the retardants to plants4. I have expanded the earlier results and here summarize the effects of five plant growth retardants.

Sites of Action of Plant Growth Retardants on Cholesterol Biosynthesis by Cell-Free Rat Liver .Preparations

Experiments with cell-free preparations of rat liver indicate that of the plant growth retardants Phosfon B, Phosfon D, B995, COlI, and Amo 1618 all but B995 inhibit at least one step between acetate and mevalonate in cholesterol biosynthesis. The results also demonstrate that all of the retardants, including B995, inhibit at least one step between mevalonate and cholesterol, and that several of them act at different points along this part of the biosynthetic sequence.

Enzymic Studies of Bile Acid Metabolism: I. 6β-HYDROXYLATION OF CHENODEOXYCHOLIC AND TAUROCHENODEOXYCHOLIC ACIDS BY MICROSOMAL PREPARATIONS OF RAT LIVER

Abstract 6β-Hydroxylation of chenodeoxycholic acid-24-14C and taurochenodeoxycholic acid-24-14C was demonstrated in vitro by cell-free preparations of rat liver. The enzymic activity, requiring O2 and NADPH, was located in the microsomes prepared in 0.1 m phosphate buffer and could be stimulated by addition of the 105,000 x g supernatant fluid. The microsomal fraction prepared in 0.01 m phosphate buffer had full activity and was not further stimulated by the 105,000 x g supernatant fluid. A clear extract made by suspending this microsomal pellet in 1.0 m phosphate and subsequently centrifuging it at 105,000 x g contained the complete 6β-hydroxylase system. The activity of the 1.0 m phosphate extract was inhibited by sulfhydryl reagents, Cu++, cytochrome c, and CO. The inhibition by CO could be reversed by light. The presence of cytochrome b5 and the CO-binding pigment, P-450, in the extract was detected by differential spectrophotometry.

Incorporation of 14C-leucine into serum transferrin by slices and cell-free preparations of rat liver.

Der Einbau von14C-Leucin in Serumtransferrin in vitro in Leberschnitten und in isolierte Lebermikrosomen der Ratte wurde gezeigt und mit demjenigen in Serumalbumin verglichen. Die gereinigten Proteine wurden immunoelektrophoretisch ausgefallt und ihre Radioaktivitat bestimmt. Ebenso wurde die Reindarstellung von Transferrin aus Rattenserum beschrieben.

COMPARATIVE EFFECTS OF VARIOUS ANALOGUES OF THYROXINE ON AMINO ACID INCORPORATION INTO PROTEIN.

The effects of a number of analogues with varying degrees of thyroxinelike biological activity in vivo have been compared with those of L-thyroxine on amino acid incorporation into protein in cell-free rat liver preparations. D-Thyroxine, administered in vivo in doses at which L-thyroxine clearly stimulates amino acid incorporation, is without effect. When added directly to the reaction mixtures containing the cell-free preparations, D-thyroxine is as effective as Lthyroxine in stimulating amino acid incorporation over a range of concentrations. LTriiodothyronine and tetraiodothyroacetic acid stimulate amino acid incorporation after both in vivo and in vitro administration. In vivo they are both at least as active as Lthyroxine. In vitro, however, triiodothyronine has only a fractional part of the activity of L-thyroxine, but tetraiodothyroacetic acid is considerably more active than L-thyroxine, and its optimal concentration is one fourth that of L-thyroxine. The in vitro effect of tetraiodothyroacetic acid exhibits the same 5 min lag period preceding the stimulation of amino acid incorporation observed with Lthyroxine. The results of experiments with miscellaneous analogues indicate the following order of effectiveness in stimulating amino acid incorporation in vitro as regards the degree of iodination: tetraiodo >triiodo>diiodo. The completely deiodinated analogue, thyronine, is without any significant effect. No great differences in activities of analogues with acetic acid and propionic acid side chains were noted; the propionic acid side chain may, perhaps, confer slightly greater activity. (Endocrinology75: 304, 1964)

Fatty acid synthesis in cell-free preparations of rat liver containing phosphorylating mitochondria.

Fatty acid synthesis in cell-free preparations of rat liver containing phosphorylating mitochondria.

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.

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.

Biotin in the synthesis of fatty acid and cholesterol by mammalian liver.

Wakil, Titchener and Gibson1 have shown that the formation of malonate from acetate and carbon dioxide is an early step in the synthesis of fatty acids in cell-free preparations of pigeon liver. They have also shown that biotin is necessary for this reaction, since it is inhibited by avidin, a specific inhibitor of biotin. By observing the effect of avidin, we have been able to show that, in cell-free preparations of rat liver, biotin is also required for the synthesis from acetate of fatty acids, but is not required for synthesis of cholesterol. It is thus possible to inhibit one biosynthetic pathway without influencing the other.