Diurnal changes in the fraction of 3-hydroxy-3-methylglutaryl-CoA reductase in the active form in rat liver microsomal fractions.

'Initial' and 'total' activities of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase) were measured in cold-clamped samples of liver from rats at 2h intervals throughout the 24h light/dark cycle. Initial activities were obtained in microsomes (microsomal fractions) isolated and assayed in the presence of 100mM-KF, whereas 'total' activities were measured in microsomes prepared from the same homogenates but washed free of KF and incubated with exogenous partially purified rat liver protein phosphatase. The initial/total-activity ratio for HMG-CoA reductase underwent a diurnal cycle, which had a nadir 4h into the light phase (when initial activity was 28% of total activity) and a peak 12h later, i.e. 4h into the dark phase (when initial activity was 80% of total activity). These low and high points of the cycle were separated by gradual steady changes in the ratio. The characteristics of this diurnal cycle were different from those of the cycle observed for total activity, which had a plateau of high activity between 2 and 10h into the dark cycle preceded and succeeded by a very rapid increase and decrease, respectively, in the total activity of HMG-CoA reductase. The combination of the two cycles resulted in the dampening of the resultant cycle for the initial or effective activity of HMG-CoA reductase, such that the changes in initial activity around the beginning and and end of the dark phase were more gradual than would otherwise have been the case if the initial/total-activity ratio for HMG-CoA reductase were constant throughout the diurnal cycle. The physiological implications of the observed diurnal variation in the fraction of hepatic HMG-CoA reductase in the active form are discussed.     

Properties of purified rat hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase and regulation of enzyme activity

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase from rat liver microsomes has been purified to apparent homogeneity with recoveries of approximately 50%. The enzyme obtained from rats fed a diet supplemented with cholestyramine had specific activities of approximately 21,500 nmol of NADPH oxidized/min/mg of protein. After amino acid analysis a specific activity of 31,000 nmol of NADPH oxidized/min/mg of amino acyl mass was obtained. The s20,w for HMG-CoA reductase was 6.14 S and the Stokes radius was .39 nm. The molecular weight of the enzyme was 104,000 and the enzyme subunit after sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 52,000. Antibodies prepared against the homogeneous enzyme specifically precipitated HMG-CoA reductase from crude and pure fractions of the enzyme. Incubation of rat hepatocytes for 3 h in the presence of lecithin dispersions, compactin, or rat serum resulted in significant increases in the specific activity of the microsomal bound reductase. Immunotitrations indicated that in all cases these increases were associated with an activated form of the reductase. However activation of the enzyme accounted for only a small percentage of the total increase in enzyme activity; the vast majority of the increase was apparently due to an increase in the number of enzyme molecules. In contrast, when hepatocytes were incubated with mevalonolactone the lower enzyme activity which resulted was primarily due to inactivation of the enzyme with little change in the number of enzyme molecules. Immunotitrations of microsomes obtained from rats killed at the nadir or peak of the diurnal rhythm of 3-hydroxy-3-methylglutaryl-CoA reductase indicated that the rhythm results both from enzyme activation and an increased number of reductase molecules.     

Diurnal variation of HMG-CoA reductase activity in rat liver peroxisomes

The specific activity of hepatic microsomal and peroxisomal 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) was determined at different times during a 24 hour cycle from cholestyramine treated rats. The microsomal HMG-CoA reductase activity displayed a peak at D-6 (6th hour of the dark cycle) as previously reported, whereas, the peroxisomal HMG-CoA reductase activity was the highest at L-2 (2nd hour of the light cycle). Immunoblots of the peroxisomal HMG-CoA reductase suggest that the increase in enzyme activity at L-2 is due to changes in enzyme mass. The different cyclic variations observed in microsomal and peroxisomal HMG-CoA reductase activity may suggest different mechanisms of regulation.     

Acyl-CoA: cholesterol acyltransferase and 3-hydroxy-3-methylglutaryl-CoA reductase in carp-liver microsomes: effect of cold acclimation on enzyme activities and on hepatic and plasma lipid composition.

Hepatic microsomal activities of acyl-CoA:cholesterol acyltransferase (ACAT) and 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, rate-limiting enzymes in cholesterol esterification and cholesterol synthesis, and the concentration sand compartmentalization of esterified and unesterified cholesterol, were studied in carp acclimated to 10 and 30 degrees C. Irrespective of acclimation temperature, carp-liver ACAT is characterized by an apparent Km-value for oleoyl-CoA of 11-15 microM and displays an optimum activity at pH 7.4. The enzyme activity is reduced approx. 2-fold upon preincubation of microsomes with alkaline phosphatase. Arrhenius plots of ACAT-activity are curvilinear, with curvatures considerably affected by the acclimation temperature of the fish. Carp HMG-CoA reductase has been characterized previously by Teichert and Wodtke ((1987) Biochim. Biophys. Acta 920, 161-170). When measured at 30 degrees C, ACAT activities from 30 degrees C- and 10 degrees C-acclimated carp are identical (approx. 6 pmol/min per mg protein), whilst 'expressed' HMG-CoA reductase activity (18.1 +/- 12.2 pmol/min per mg protein for 30 degrees C-acclimated carp vs. 159.8 +/- 106.6 pmol/min per mg protein for 10 degrees C-acclimated carp) is enhanced 9-fold in the cold environment. This disparity indicates that cold-acclimation results in a massive increase in the capacity for hepatic cholesterol synthesis relative to hepatic cholesterol esterification. At the same time, hepatic compositional analysis reveals identical contents of unesterified cholesterol in either groups of carp but significantly decreased (3-fold) amounts in cholesterol ester (and also in triacylglycerol, 4-fold) in cold-acclimated carp. Moreover, microsomal fractions display lower cholesterol to phospholipid ratios in the cold. In contrast, concentrations of either cholesterol fractions (and of triacylglycerols) in plasma--the mobile compartment for lipoprotein transport--do not differ in cold- and warm-acclimated carp. Based on current concepts of cholesterol metabolism, it is concluded that the cold-enhanced expression of hepatic HMG-CoA reductase activity is a homeostatic response directed against and compensating for a cold-induced but not yet characterized deficiency in hepatic cholesterol availability.     

3-Hydroxy-3-methylglutaryl-coenzyme A reductase in normal and dystrophic hamsters.

The activity and diurnal variation of 3-hydroxy-3-methyglutaryl-CoA reductase (EC 1.1.1.34; HMG-CoA reductase), the rate-limiting enzyme in the cholesterol-biosynthetic pathway, of normal and dystrophic hamsters was determined. Liver enzyme activity showed a diurnal pattern in the normal male, but not in the dystrophic male. Enzyme values in normal males at the midpoint of the 12 h dark period were 10 times those in dystrophic males. No evidence for diurnal variation in the HMG-CoA reductase of the brain was observed, and similar activities were found for normal and dystrophic animals. The apparent Km for HMG-CoA reductase from the liver of normal or dystrophic hamsters was approx. 9 microM, and the Vmax. was 5.9 and 21.7 pmol/min per mg of protein for dystrophic and normal hamsters respectively.     

Treatment of CHO-K1 cells with 25-hydroxycholesterol produces a more rapid loss of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity than can be accounted for by enzyme turnover

A key enzyme in the regulation of mammalian cellular cholesterol biosynthesis is 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase). It is well established that treatment with the compound 25-hydroxycholesterol lowers HMG-CoA reductase activity in cultured Chinese hamster ovary (CHO-K1) cells. After brief incubation (0-4 h) with 25-hydroxycholesterol (0.5 microgram/ml), cellular HMG-CoA reductase activity is decreased to 40% of its original level. This also occurs in the presence of exogenous mevinolin, a competitive inhibitor of HMG-CoA reductase which has previously been shown to inhibit its degradation. The inhibition of HMG-CoA reductase activity by 25-hydroxycholesterol is complete after 2 h. Radio-immune precipitation analysis of the native enzyme under these conditions shows a degradation half-life which is considerably longer than that of the observed inhibition. Studies with sodium fluoride, phosphatase 2A, bacterial alkaline phosphatase and calf alkaline phosphatase indicate that the observed loss of activity is not due to phosphorylation. These data are not consistent with described mechanisms of HMG-CoA reductase activity regulation by phosphorylation or degradation but are consistent with a novel mechanism that regulates the catalytic efficiency of this enzyme.     

Acute effects of starvation and treatment of rats with anti-insulin serum, glucagon and catecholamines on the state of phosphorylation of hepatic 3-hydroxy-3-methylglutaryl-CoA reductase in vivo.

The fraction of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase in the dephosphorylated (active) form in rat liver in vivo was measured after various experimental treatments of animals. Intraperitoneal injection of glucose (to raise serum insulin concentrations) into rats 4 h into the light phase (L-4) resulted in a transient (30 min) increase in the expressed (E)/total (T) activity ratio of HMG-CoA reductase without any change in total activity (obtained after complete dephosphorylation of the enzyme). Conversely, intravenous injection of guinea-pig anti-insulin serum into rats 4 h into the dark phase (D-4) significantly depressed the E/T ratio within 20 min. Intravenous injection of glucagon into normal rats at this time point did not affect the degree of phosphorylation of the enzyme, in spite of a 10-fold increase in hepatic cyclic AMP concentration induced by the hormone treatment. A 3-fold increase in the concentration of the cyclic nucleotide induced by adrenaline infusion was similarly ineffective in inducing any change in expressed or total activities of hepatic HMG-CoA reductase. However, when insulin secretion was inhibited, either by the induction of streptozotocin-diabetes or by simultaneous infusion of somatostatin, glucagon treatment was able to depress the expressed activity of HMG-CoA reductase (i.e. it increased the phosphorylation of the enzyme). Therefore insulin appears to have a dominant role in the regulation of the phosphorylation state of hepatic HMG-CoA reductase. In apparent corroboration of this suggestion, short-term 4 h food deprivation of animals before D-4 resulted in a marked decrease in the E/T activity ratio of reductase, which was not affected further by an additional 8 h starvation. By contrast, the total activity of the enzyme was not significantly affected by 4 h starvation, but was markedly diminished after 12 or 24 h starvation. Longer-term starvation also produced a chronic increase in the degree of phosphorylation of the enzyme. These results are discussed in relation to the role of reversible phosphorylation in the control of hepatic HMG-CoA reductase activity in vivo.     

Involvement of 3-hydroxy-3-methylglutaryl coenzyme a reductase in the regulation of sesquiterpenoid phytoalexin synthesis in potato

The importance of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) in the regulation of sesquiterpenoid phytoalexin accumulation in potato (Solanum tuberosum L. cv Kennebec) was examined. Wounding of potato tubers produced a large temporary increase in HMG-CoA reductase activity of the microsomal and organelle fractions. Treatment of wounded tuber tissue with the sesquiterpenoid phytoalexin elicitor arachidonic acid further increased and prolonged the HMG-CoA reductase activity in the microsomal but not the organelle fraction. Incubation of elicitor-treated tuber tissue in white light reduced organelle and microsomal HMG-CoA reductase activity to 50% and 10%, respectively, of the activity of tissues held in darkness. Constant light also reduced overall phytoalexin accumulation 58% by greatly reducing levels of lubimin. Rishitin accumulation was not significantly altered by light. Application of nanomolar amounts of mevinolin, a highly specific inhibitor of HMG-CoA reductase, to elicitor-treated tuber tissue produced a large decline in lubimin accumulation and did not markedly alter rishitin accumulation. These results indicate that HMG-CoA reductase has a role in the complex regulation of sesquiterpenoid phytoalexin accumulation in potato.     

The activity of 3-hydroxy-3-methylglutaryl-CoA reductase and acyl-CoA: cholesterol acyltransferase in hepatic microsomes from male, female and pregnant rats. The effect of cholestyramine treatment and the relationship of enzyme activity to microsomal lipid composition

The relationship of microsomal cholesterol and phospholipid fatty acid composition to the activities of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase and acyl-CoA: cholesterol acyltransferase was investigated in male, female virgin and pregnant rats when hepatic cholesterogenesis was stimulated by cholestyramine. Cholestyramine increased HMG-CoA reductase activity in both sexes but had no effect on microsomal free cholesterol level or acyl-CoA: cholesterol acyltransferase activity. The data suggest that during cholestyramine treatment high rates of bile acid synthesis are supported by preferential channelling of cholesterol into this pathway, whilst the substrate pool and activity of acyl-CoA:cholesterol acyltransferase are maintained unaltered. The lack of a consistent relationship among enzyme activities and microsomal lipid composition infers that HMG-CoA reductase and acyl-CoA:cholesterol acyltransferase are regulated in vivo by independent mechanisms which are unlikely to involve modulation by the physical properties of the microsomal lipid.     

Effects of diabetes on the expressed and total activities of 3-hydroxy-3-methylglutaryl-CoA reductase in rat liver in vivo. Reversal by insulin treatment.

The expressed and total activities of HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) reductase (EC 1.1.1.34) were measured in microsomal fractions prepared from cold-clamped liver samples [Easom & Zammit (1984) Biochem. J. 220, 733-738] from control or insulin-treated diabetic animals. Streptozotocin-induced diabetes resulted in a marked decrease in total activity of HMG-CoA reductase and in the fraction of the enzyme in the active form, but appreciable effects were only observed in the liver of animals in which the blood glucose was above 20 mM. Intravenous infusion of insulin into diabetic rats resulted in a rapid (less than 20 min) and total dephosphorylation of the enzyme in vivo without any change in total activity. Longer-term (4 h) treatment with insulin (injected intraperitoneally) produced a rapid increase in expressed/total HMG-CoA reductase activity ratio to about 90%, followed, after a lag of 2-3 h, by a 5-6-fold increase in total activity. These observations are discussed with respect to the possible role of insulin in generating and maintaining the respective diurnal rhythms in total and in expressed/total HMG-CoA reductase activity ratio observed for normal animals in vivo [Easom & Zammit (1984) Biochem. J. 220, 739-745].     

Characterization of active and inactive forms of rat hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase

Rat hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase was purified to homogeneity using agarose-HMG-CoA affinity chromatography. Additional protein was isolated from the affinity column with 0.5 M KCl that demonstrated no HMG-CoA reductase activity, yet comigrated with purified HMG-CoA reductase on sodium dodecyl sulfate-polyacrylamide gels. This protein was determined to be an inactive form of HMG-CoA reductase by tryptic peptide mapping, reaction with anti-HMG-CoA reductase antibody, and coelution with purified HMG-CoA reductase from a molecular-sieving high-performance liquid chromatography column. This inactive protein was present in at least fourfold greater concentration than active HMG-CoA reductase, and could not be activated by rat liver cytosolic phosphoprotein phosphatases. Immunotitration studies with microsomal and solubilized HMG-CoA reductase isolated in the presence and absence of proteinase inhibitors suggested that the inactive protein was not generated from active enzyme during isolation of microsomes or freeze-thaw solubilization of HMG CoA reductase.     

Role of reversible phosphorylation in mediating changes in the activity of hepatic 3-hydroxy-3-methylglutaryl-CoA reductase during pregnancy and lactation in the rat.

1. The expressed and total (completely dephosphorylated) activities of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase were measured in microsomal fractions isolated from cold-clamped liver samples from female rats in various stages of the reproductive cycle. 2. There was little change in total HMG-CoA reductase activity during pregnancy and early lactation, but after 2 days post partum there was a marked increase in total activity. 3. The expressed/total activity ratio of HMG-CoA reductase showed a profound decrease during the last 2 days of pregnancy. The fraction of the enzyme in the active form increased progressively during the first 2 days of lactation. 4. The combined effect of these changes was that the expressed activity of HMG-CoA reductase changed in parallel with the known changes in the hepatic rate of cholesterogenesis during pregnancy and lactation in vivo.     

Development of a noncompetitive, solid phase, bridged biotin-avidin enzyme immunoassay for measurement of human leukocyte microsomal HMG-CoA reductase protein concentration

Methods were developed for determination of human mononuclear leukocyte HMG-CoA reductase protein concentration by a noncompetitive, solid phase, bridged biotin-avidin enzyme immunoassay procedure. Leukocyte microsomal HMG-CoA reductase, first immobilized onto a nitrocellulose filter, is sequentially reacted with 1) monospecific, polyclonal rabbit anti-rat liver HMG-CoA reductase antiserum, which crossreacts with the human liver and leukocyte enzymes; 2) biotinylated donkey anti-rabbit immunoglobulin; 3) a streptavidin-horseradish peroxidase conjugate; and 4) 4-chloro-1-naphthol and H2O2 to visualize the quantity of horseradish peroxidase bound to the immunocomplex. Color development was proportional to the quantity of either purified liver or leukocyte microsomal HMG-CoA reductase applied to the nitrocellulose. Color development was not observed, however, when HMG-CoA reductase was omitted from the nitrocellulose, when one of the reactant species was omitted from the incubation reactions, or when anti-rat liver HMG-CoA reductase antiserum was pre-absorbed with either rat liver or human leukocyte HMG-CoA reductase. Immunoreactivity of microsomal HMG-CoA reductase was independent of the phosphorylation state of the enzyme, but was inversely related to the concentration of thiol-reducing agents present in the microsomal preparation up to 4 mM. Further increases in thiol-reductant failed to produce changes in immunoreactivity. Freshly isolated mononuclear leukocyte microsomal HMG-CoA reductase protein concentration in leukocytes from 31 healthy, normocholesterolemic subjects was a linear function of HMG-CoA reductase activity (R = 0.65; P less than 0.001). The catalytic efficiency of the freshly isolated mononuclear leukocyte enzyme was 313 +/- 34 pmol of mevalonate formed per min of incubation at 37 degrees C per mg immunoreactive protein. This methodology, in conjunction with that recently developed to measure human leukocyte HMG-CoA reductase activity (1984. J. Lipid Res. 25: 967-978), should prove useful in discriminating between HMG-CoA reductase regulatory mechanisms involving changes in enzyme protein concentration and those resulting from changes in enzyme catalytic efficiency.     

In vivo regulation of human leukocyte 3-hydroxy-3-methylglutaryl coenzyme A reductase: increased enzyme protein concentration and catalytic efficiency in human leukemia and lymphoma.

The activity of microsomal HMG-CoA reductase in freshly isolated leukocytes from patients with a variety of hematologic malignancies was significantly increased (up to 20-fold) when compared to enzyme activity in leukocytes from normal subjects (average 10.3 +/- 0.8 pmol/min per mg). Increased enzyme activity was not due to nonspecific leukocyte stimulation or to the presence of a malignancy, since normal enzyme activity was observed in subjects with either viral illnesses or solid tumors. Increased HMG-CoA reductase activity accompanying hematologic malignancy could also not be attributed to alterations in enzyme-substrate kinetic parameters (Km), or to alterations in the phosphorylation state or thiol-disulfide status of the enzyme, nor was it correlated with differences in serum lipid or lipoprotein concentrations. The increase (3.6-fold) in HMG-CoA reductase activity in leukocytes from patients with preleukemia was due entirely to a rise in enzyme catalytic efficiency (specific activity), whereas the increase (4.3-fold) observed in leukocytes from patients with overt leukemia or non-Hodgkin's lymphoma was due to a concomitant increase in both enzyme catalytic efficiency (2.5-fold) and enzyme protein concentration (1.6-fold). Similar increases in HMG-CoA reductase activity and catalytic efficiency were also noted for both transformed, nonmalignant, and malignant cultured leukocytes, suggesting that increased enzyme catalytic efficiency is not a nonspecific consequence of physiological changes occurring in response to the malignancy but may be an integral aspect of the malignant phenotype. HMG-CoA reductase protein concentrations, however, were not elevated in either transformed, nonmalignant, or malignant cultured leukocytes, suggesting that increases in enzyme protein levels may be secondary to other physiological changes that occur during the development of overt leukemia. Taken together, these observations suggest that an increase in the activity of HMG-CoA reductase, the rate-controlling enzyme in cholesterol synthesis, is a common occurrence in human hematologic malignancies and that a biphasic elevation of enzyme activity may exist in malignant leukocytes, such that changes in catalytic activity may occur early in tumorigenesis and may be followed by secondary changes in enzyme levels.     

3-Hydroxy-3-methylglutaryl coenzyme A reductase activity in beef adrenal cortex.

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) activity (mevalonate:NADP+ oxidoreductase )CoA-acylating) EC 1.1.1.34) was demonstrated in beef adrenal cortex. Most of the HMG-CoA reductase activity is in the microsomal fraction while a small percentage of the activity is associated with the mitochondria, Mitochondria purified on a linear sucrose gradient are enriched in HMG-CoA reductase and cytochrome c oxidase activities. The reductase present in microsomal preparations from the whole adrenal cortex has an apparent Km of 5.6 X 10(-5) M for (R,S)-HMG-CoA. Reductase activities found in the microsomal fractions from the zona glomerulosa, the zona fasciculata, and the zona reticularis were 1.32, 7.37, and 9.74 nmol mevalonate formed per milligram protein in 30 min respectively.     

3-hydroxy-3-methylglutaryl-CoA reductase from neonatal chick

The optimal conditions for identification of mevalonic acid as the product of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase are described, as well as the effect of different buffer constituents on the enzyme activity. Under the chosen assay conditions, reductase activity from neonatal chick liver increased with the incubation time up to 60 min and was proportional to the amounts of protein added in a range of 0.1-0.5 mg. The specific activity was maximal in brain and liver and lower in intestine of 6-day-old chicks. Thermostability of hepatic reductase was studied. When microsomal preparations were maintained at 4 degrees C, reductase activity remained unchanged for 6 hr and decreased afterwards. Addition of 50 mM KF to the homogenization medium had no effect on the reductase activity. Similarly, preincubation of microsomal preparations with 105,000 g supernatants in the presence or absence of KF did not significantly increase the reductase activity. These results suggest that HMG-CoA reductase was isolated from neonatal chick in the fully activated form.     

Regulation of HMG-CoA reductase: identification of the site phosphorylated by the AMP-activated protein kinase in vitro and in intact rat liver.

The intact, 100 kd microsomal enzyme and the 53 kd catalytic fragment of rat HMG-CoA reductase are both phosphorylated and inactivated by the AMP-activated protein kinase. Using the catalytic fragment, we have purified and sequenced peptides containing the single site of phosphorylation. Comparison with the amino acid sequence predicted from the cDNAs encoding other mammalian HMG-CoA reductases identifies this site as a serine residue close to the C-terminus (Ser872 in the human enzyme). Phosphopeptide mapping of native, 100 kd microsomal HMG-CoA reductase confirms that this C-terminal serine is the only major site phosphorylated in the intact enzyme by the AMP-activated protein kinase. The catalytic fragment of HMG-CoA reductase was also isolated from rat liver in the presence of protein phosphatase inhibitors under conditions where the enzyme is largely in the inactive form. HPLC, mass spectrometry and sequencing of the peptide containing Ser872 demonstrated that this site is highly phosphorylated in intact liver under these conditions. We have also identified by amino acid sequencing the N-terminus of the catalytic fragment, which corresponds to residue 423 of the human enzyme.     

Phosphorylation and modulation of the enzymic activity of native and protease-cleaved purified hepatic 3-hydroxy-3-methylglutaryl-coenzyme A reductase by a calcium/calmodulin-dependent protein kinase

A Ca2+/calmodulin-dependent kinase has been purified which catalyzed the phosphorylation and concomitant inactivation of both the microsomal native (100,000 Da) and protease-cleaved purified 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase) (53,000 Da) fragments. This low molecular weight brain cytosolic Ca2+/calmodulin-dependent kinase phosphorylates histone H1, synapsin I, and purified HMG-CoA reductase as major substrates. The kinase, purified by sequential chromatography on DEAE-cellulose, calmodulin affinity resin, and high performance liquid chromatography (TSKG 3000 SW) is an electrophoretically homogeneous protein of approximately 110,000 Da. The molecular weight of the holoenzyme, substrate specificity, subunit protein composition, subunit autophosphorylation, subunit isoelectric points, and subunit phosphopeptide analysis suggest that this kinase of Mr 110,000 may be different from other previously reported Ca2+/calmodulin-dependent kinases. Maximal phosphorylation by the low molecular form of Ca2+/calmodulin-dependent kinase of purified HMG-CoA reductase revealed a stoichiometry of approximately 0.5 mol of phosphate/mol of 53,000-Da enzyme. Dephosphorylation of phosphorylated and inactivated native and purified HMG-CoA reductase revealed a time-dependent loss of 32P-bound radioactivity and reactivation of enzyme activity. Based on the results reported here, we propose that HMG-CoA reductase activity may be modulated by yet another kinase system involving covalent phosphorylation. The elucidation of a Ca2+/calmodulin-dependent HMG-CoA reductase kinase-mediated modulation of HMG-CoA reductase activity involving reversible phosphorylation may provide new insights into the molecular mechanisms involved in the regulation of cholesterol biosynthesis.     

Mechanism of Photoregulated Carotenogenesis in Rhodotorula minuta V. Photoinduction of 3-Hydroxy-3-Methyl Glutaryl Coenzyme A Reductase

The effect of light on the activity of 3-hydroxy-3-methylglutarylCoenzyme A (HMG-CoA) reductase in Rhodotorula minuta was studiedin cell-free extracts prepared from cells grown under variouslight conditions. HMG-CoA reductase activity in cells grown under continuous illuminationwas higher than that in cells grown in the dark, and dependedon the light intensity used during incubation. The relationshipbetween activity [A (nmol/mg-N/min)] and light intensity [I(erg/cm²/sec)] was expressed by the equation A=0.72 log I$0.80. Illumination at –1.5?C followed by dark incubation at26?C resulted in a rapid increase in HMG-CoA reductase activityimmediately after the beginning of incubation. This photoinducedHMG-CoA reductase activity was regulated by the light dose andfollowed the Roscoe-Bunsen reciprocity law. When cycloheximide was added immediately after the beginningof incubation in the dark, the increase in HMG-CoA reductaseactivity was completely inhibited. The inhibitory effect ofcycloheximide, however, gradually decreased with the delay ofthe addition. On the basis of these results we have postulated that the photoregulationof carotenogenesis in Rh. minuta results from the photoregulationof HMG-CoA reductase synthesis. (Received November 7, 1981; Accepted March 19, 1982)     

Phosphorylation of native 97-kDa 3-hydroxy-3-methylglutaryl-coenzyme A reductase from rat liver. Impact on activity and degradation of the enzyme

Immunoprecipitation of native rat liver microsomal 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, phosphorylated by [gamma-32P]ATP in the presence of reductase kinase, revealed a major 97-kDa 32P band which disappeared upon competition with pure unlabeled 53-kDa HMG-CoA reductase. A linear correlation between the expressed/total HMG-CoA reductase activity ratio (E/T) and the fraction of 32P released from the 97-kDa enzyme established the validity of the E/T ratio as an index of HMG-CoA reductase phosphorylation state in isolated microsomes. Incubation of rat hepatocytes with mevalonolactone resulted in a rapid increase in phosphorylation of microsomal reductase (decrease in E/T) followed by an enhanced rate of decay of total reductase activity which was proportional to the loss of 97-kDa enzyme mass determined by immunoblots. Inhibitors of lysosome function dampened both basal and mevalonate-induced reductase degradation in hepatocytes. In an in vitro system using the calcium-dependent protease calpain-2, up to 5-fold greater yields of soluble 52-56-kDa fragments of reductase (immunoblot and total activity) were obtained when the substrate 97-kDa reductase was phosphorylated before proteolysis. Immunoblots of unlabeled phosphorylated reductase compared with gels of immunoprecipitated 32P-labeled reductase resolved a 52-56-kDa doublet which contained 32P solely in the upper band. These data suggest that a major phosphorylation site of HMG-CoA reductase lies within the "linker" segment joining the membrane spanning and cytoplasmic domains of the native 97-kDa protein.