The activity and kinetic properties of mevalonate kinase in superovulated rat ovary

Methods are described for the assay and partial purification of mevalonate kinase from superovulated rat ovary. The total activity of mevalonate kinase in superovulated rat ovary was 1.6+/-0.14units/g wet wt.; it was unchanged by the administration of luteinizing hormone in vivo. The K(m) of a partially purified preparation of mevalonate kinase for dl-Mevalonate was 3.6+/-0.5mum; its K(m) for MgATP(2-) was 120+/-7.7mum. The enzyme was inhibited by geranyl pyrophosphate and farnesyl pyrophosphate, but not by isopentenyl pyrophosphate or 3,3'-dimethylallyl pyrophosphate. dl-mevalonate 5-phosphate inhibited at high concentrations. With both geranyl pyrophosphate and farnesyl pyrophosphate the inhibition was competitive with respect to MgATP(2-). The K(i) for inhibition by geranyl pyrophosphate was 1.3+/-0.2mum; the K(i) for inhibition by farnesyl pyrophosphate was 1.0+/-0.3mum. These findings are discussed with reference to the control by luteinizing hormone of steroidogenesis from acetate.     

Free and esterified cholesterol concentration and cholesteryl ester composition in the ovaries of maturing and superovulated immature rats

The cholesterol and cholesteryl ester concentration and cholesteryl ester composition were determined in the ovaries of immature rats, sexually mature rats and superovulated immature rats. The immature rat ovary accumulated cholesteryl esters, and long-chain polyunsaturated fatty acids were preferentially incorporated into these esters. The cholesteryl esters decreased in concentration and changed in composition with the onset of the first estrous cycle. Superovulation of immature rats, by injection of 50 I.U. pregnant mare serum gonadotropin, caused a decrease in the cholesteryl ester concentration of the ovary within 24 h and specific depletion of some esters, particularly those of 20 : 1 and 22 : 6 acids. Human choriogonadotropin, administered 54 h later, induced synchronous luteinization of the ovaries and was followed by increases in the concentration of free and esterified cholesterol and preferential accumulation of the esters of 20 : 4, 22 : 4, 4, 22 : 5 and 22 : 6 acids. Acute stimulation of luteinized ovaries by a second injection of the rats with 25 I.U. of human choriogonadotropin resulted in preferential hydrolysis of the esters of 18 : 1, 20 : 4, 22 : 4 and 22 : 5 acids.     

The effect of calcium on cholesterol side-chain-cleavage enzyme in superovulated rat ovaries

Cholesterol side-chain-cleavage enzyme activity in mitochondria isolated from heavily luteinized rat ovaries was determined using isocitrate as an electron donor and [4-14C]cholesterol to start the reaction. The activity of the enzyme was reduced when more than 10 microM calcium was added to the mitochondrial preparations. When 100 microM EGTA or 2 mM ATP was added to the reaction an increase in enzyme activity was observed and ATP was able to partially overcome the calcium-induced inhibition.     

Differential uptake and metabolism of free and esterified cholesterol from high-density lipoproteins in the ovary

Rat luteal cells utilize high-density lipoproteins (HDL) as a source of cholesterol for steroid synthesis. Both the free and esterified cholesterol of HDL are utilized by these cells. In this report, we have examined the relative uptake of free and esterified cholesterol of HDL by cultured rat luteal cells. Incubation of the cells with HDL labeled with [3H]cholesterol or [3H]cholesteryl linoleate resulted in 4-6-fold greater uptake of the free cholesterol compared to esterified cholesterol. The increased uptake of free cholesterol correlated with its utilization for progestin synthesis: utilization of HDL-derived free cholesterol was 3-6-fold higher than would be expected from its concentration in HDL. The differential uptake and utilization of free and esterified cholesterol was further examined using egg phosphatidylcholine liposomes containing cholesterol or cholesteryl linoleate as a probe. Liposomes containing free cholesterol were able to deliver cholesterol to luteal cells and support steroid synthesis in the absence of apolipoproteins, and the addition of apolipoprotein A-I (apo A-I) moderately increased the uptake and steroidogenesis. Similar experiments using cholesteryl linoleate/egg phosphatidylcholine liposomes showed that inclusion of apo A-I resulted in a pronounced increase in the uptake of cholesteryl linoleate and progestin synthesis. These experiments suggest that free cholesterol from HDL may be taken up by receptor-dependent and receptor-independent processes, whereas esterified cholesterol uptake requires a receptor-dependent process mediated by apolipoproteins.     

Degradation of non-esterified and esterified xanthophylls by free radicals

beta-Carotene and other xanthophylls present in pepper fruit as both free and esterified forms were oxidized using a free radical initiator (2,2'-azo-bis-isobutyronitrile). Capsorubin was degraded most slowly, followed by zeaxanthin, capsanthin, and beta-carotene. The presence of keto groups at the ends of the polyene chain could be a structural factor contributing to this difference in reactivity. It was also shown that whereas capsanthin and its esters and capsorubin and its esters were degraded at the same rate, zeaxanthin esters responded differently to the oxidation process, and were degraded more quickly than free zeaxanthin. The presence of unsaturated fatty acids (mainly linoleic) that esterify zeaxanthin help to accelerate the degradation of this xanthophyll and decreasing its antioxidant action. The antioxidant capacity of capsorubin and capsanthin (both in free and esterified form) exclusive to the genus capsicum should be taken into account.     

Metabolism of endogenous sterol ester by the superovulated rat ovary in vitro

Sterol, glyceride and phospholipid were found to account for more than 90% (w/w) of the lipid extracted from whole superovulated rat ovaries. These lipids, together with non-esterified fatty acids, were assayed in slices of the tissue after incubation for various times. Whereas the concentrations of triglyceride, diglyceride and phospholipid did not change significantly during incubation, that of sterol ester markedly decreased and those of free sterol, monoglyceride and non-esterified fatty acid increased. Evidence is presented that in this tissue (in contrast with other mammalian tissues) the main endogenous substrate for respiration is fatty acid derived from sterol ester.     

Correlation of rat liver chromatin-bound free and esterified cholesterol with the circadian rhythm of cholesterol biosynthesis in the rat.

Cholesterol has been shown to be present in rat liver chromatin isolated by methods designed to avoid contamination by membrane fragments. Evidence that the cholesterol was actually a component of chromatin includes (a) the constancy of the amount (1.30 +/- 0.14 mug per mg DNA), (b) the striking difference in the ratio of free (i.e. unesterified) to esterified cholesterol between that in chromatin and that in membrane, and (c) the rapid and marked changes which occurred in this ratio during the circadian cycle in chromatin but not in membranes. Although the total amount of chromatin-bound cholesterol did not change throughout the circadian cycle, the concentration of free cholesterol increased sharply a short time before the peak of cholesterol synthetic activity was reached at about midnight; it reached a basal level about 6 h later at approximately the same time the rate of synthesis returned to its basal level. When labelled cholesterol was administered by stomach tube, it was detectable within 2 h in whole nuclei and in chromatin, indicating that chromatin-bound cholesterol is rapidly exchangeable with that in liver cytoplasm and in blood plasma. Removal of basic proteins from chromatin did not result in the loss of any cholesterol, but removal of most of the acidic as well as the basic proteins resulted in loss of most of the chromatin-bound cholesterol. These results indicate that cholesterol is bound either to the acidic proteins or to both the acidic proteins and DNA. The data are compatible with the hypothesis that cholesterol biosynthesis controlled at the nuclear level and suggest that the relative amounts of free and esterified cholesterol associated with chromatin may play a role.     

The activity of an esterified cholesterol transferring factor in human and rat serum.

In incubations containing mixtures of lipoproteins isolated either from humans or from rats, the addition of human lipoproteins-free serum (the dialysed 1.25 g/ml infranatant solution) was more than five times more effective than that of the rat in promoting the transfer of esterified [3H]cholesterol from high density lipoproteins to very low density lipoproteins, regardless of the species origin of the lipoproteins, implying the existence of an esterified cholesterol transferring factor of much greater activity in human than in rat serum.     

Interactions between ATP and cholesterol side-chain cleavage in mitochondria isolated from superovulated rat ovaries

ATP stimulated the rate of [4-14C]cholesterol side-chain cleavage in mitochondria isolated from superovulated rat ovaries. The effect of ATP was apparently similar to the stimulatory effect of choriogonadotropin on mitochondrial [4-14C]cholesterol utilization. Enhancement of the rate of steroidogenesis by ATP and choriogonadotropin were not additive. ATP seemed to promote both cholesterol uptake into the inner mitochondrial membrane and the supply of electrons for [4-14C]cholesterol utilization from both endogenous substrate and succinate.     

In vivo study of free and esterified cholesterol turnover in various tissues of the rat.

Rats were infused for 3.5 to 10 hrs with either red cells or plasma previously labelled in vivo by [3H]-cholesterol. Cholesterol specific radioactivities were measured in plasma, HDL, LDL and VLDL, and various tissues. Red cell infusions led to a higher labelling of free than of esterified cholesterol in the plasma of infused rats. The opposite situation was observed following plasma infusion. Comparison of free and esterified cholesterol specific radioactivities in each tissue showed that esterified cholesterol was transferred from plasma to all the tissues, except the adrenals. Study of the ratios of cholesterol specific radioactivities from one experimental group to the other in each tissue, made it possible to demonstrate clearly the occurence of hydrolysis within all the studied tissues except 5 of them where its existence remains uncertain (lung, heart, kidney, tendon, muscle) and of esterification in 3 tissues (adrenal, liver lung). In addition, ratios of cholesterol radioactivities (free/ester) were found to be identical in plasma and in 4 tissues, where neither hydrolysis nor esterification were detected (heart, muscle, kidney, tendon). This finding is an argument in favor of a simultaneous transport of free and esterified cholesterol from plasma into these 4 tissues and suggests that the entire lipoprotein particles can penetrate these tissues, with no specificity of one special class. In adrenal, unlike all other tissues: 1) the turnover of esterified cholesterol was achieved mostly by hydrolysis and esterification in situ; 2) a preferential lipoprotein class (LDL) was responsible for the transport of free cholesterol from the plasma.     

The metabolism of esterified cholesterol in rabbit plasma low density lipoproteins

The metabolism of esterified cholesterol in plasma low density lipoproteins (LDL) has been studied in rabbits. LDL labelled with ³H in the esterified and free cholesterol moieties was isolated from the serum of donar rabbits which has been injected with [³H]mevalonic acid, and subsequently either incubated at 37°C in vitro with unlabelled rabbit serum or unlabelled rabbit lipoprotein fractions, or reinjected into other rabbits.In vitro there was found to be a transfer of 40–60% of the esterified [³H]-cholesterol out of LDL into both the very low density lipoprotein (VLDL) and high density lipoprotein (HDL) fractions which could not be explained in terms of net transfer of esterified cholesterol mass. In the incubations of labelled LDL with either of the other unlabelled lipoprotein fractions, transfers were apparent only if the dialysed 1.21 g/ml infranatant of rabbit serum was also present. The transfer of esterified [³H]cholesterol out of LDL was enhanced when lecithin:cholesterol acyltransferase was active.After reinjecting labelled LDL into other rabbits, it was found that more than half of the esterified [³H]cholesterol removed from the recipient LDL fraction during the first 30 min was not lost from the plasma compartment, but rather was recovered in HDL. There was only minimal in vivo transfer of LDL esterified [³H]cholesterol into VLDL.It has been concluded that in vitro the esterified cholesterol in LDL exchanges with that in both the VLDL and HDL, and that in vivo the esterified cholesterol pools in LDL and HDL may represent parts of a progressively equilibrating plasma pool.     

Glucose metabolism in the superovulated rat ovary in vitro. Effects of luteinizing hormone and the role of glucose metabolism in steroidogenesis

1. Superovulated rat ovary slices from rats treated with 20μg. of luteininzing hormone/100g. body wt. 2hr. before death and from control animals have been incubated in vitro. Output of Δ⁴-3-oxo steroids (0·2μmole/g. wet wt./hr. in control tissue) was linear for 4hr., and was increased by approx. 70% in slices from luteinizing hormone-treated rats. Rate of oxygen consumption (90·0±4·6μmoles/g. wet wt./hr.) was linear for 3hr. and unaltered by luteinizing hormone treatment or addition of glucose (1mg./ml.) to the medium. 2. In slices from control animals, steady-state rate of glucose uptake was 78·0±2·9μg. atoms of carbon/g. wet wt./hr.; steady-state rates of lactate output, pyruvate output and incorporation of [U-¹⁴C]-glucose carbon atoms into carbon dioxide and total lipid extract were 60·7±0·9, 2·4±0·1, 18·0±1·1 and 0·7±0·1μg. atom of carbon/g. wet wt./hr. and accounted for 104·5±1·9% of the glucose uptake. In slices from luteinizing hormone-treated rats, glucose uptake and outputs of lactate, pyruvate and [¹⁴C]carbon dioxide were increased by approx. 25%, and 108·4±3·2% of the glucose uptake could be accounted for. 3. The total lipid extract was separated by thin-layer chromatography and saponification. Of the ¹⁴C incorporated into this fraction during incubation with [U-¹⁴C]glucose 97% was found in the fractions containing glyceride glycerol and less than 3% in the fractions containing sterols, steroids or fatty acids. Appreciable quantities of ¹⁴C were incorporated into these lipid fractions from [1-¹⁴C]acetate. 4. From a consideration of the tissue glycogen content, the specific activities of [¹⁴C]lactate and glucose 6-phosphate (C-1) derived from [1-¹⁴C]-, [6-¹⁴C]- and [U-¹⁴C]-glucose, and the ratio of [¹⁴C]carbon dioxide yields from [1-¹⁴C]glucose and [6-¹⁴C]glucose, it was concluded that there was no appreciable glycogenolysis or flow through the pentose phosphate cycle. 5. In ovary slices from both control and luteinizing hormone-treated animals, glucose in vitro raised the incorporation rate of ¹⁴C from [1-¹⁴C]acetate into sterols and steroids. Luteinizing hormone in vivo stimulated the incorporation rate in vitro but only in the presence of glucose. 6. In slices incubated in medium containing [³H]water, [¹⁴C]sorbitol and glucose (1mg./ml.), the total water space (865±7·1μl./g.) and the extracellular water space (581±22μl./g.) were unchanged by luteinizing hormone treatment in vivo but the glucose space was raised from 540±23·6μl./g. to 639±31·3μl./g. 7. Luteinizing hormone treatment was found to lower the tissue concentration of the hexose monophosphates and to increase the total activity of hexokinase, glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase and possibly of phosphofructokinase. 8. The kinetic properties of a partially purified preparation of phosphofructokinase were found to be qualitatively similar to those from other mammalian tissues. 9. The results are discussed with reference to both the role of glucose metabolism in steroidogenesis and the mechanism by which luteinizing hormone increases the rate of glucose uptake.