Mechanism for the induction of 3-hydroxy-3-methylglutaryl coenzyme A reductase in HgCl2-treated sweet potaso root tissue

No 3-hydroxy-3-methylglutaryl coenzyme A reductase activitywas detected in microsomal fractions prepared from healthy andwounded sweet potato root tissues. However, there was considerableenzyme activity in the tissue discs when Hgcl₂ was applied afterincubation at 30?C for 18 hr. This increase in the enzyme activitywas followed by furano-terpene accumulation. Application ofcycloheximide to discs immediately after preparation completelyinhibited the increase in the enzyme activity when HgCl₂ wasapplied after incubation. In contrast, the increase was delayedfor about 4 hr, then the activity was enhanced, when CHI wasapplied after preliminary incubation. CHI completely inhibitedprotein synthesis when applied to the discs after the preliminaryincubation, as judged by the inhibition of the incorporationof ¹⁴C-leucine into protein and the inhibition of the increasein peroxidase activity which is synthesized de novo. These resultssuggest that the inactive precursor of HMG-CoA reductase issynthesized during the preliminary incubation in response onlyto wounding then it is converted into the active form aftertreatment with HgCl₂. (Received January 11, 1979; )     

Allosteric activation of rat liver cytosolic 3-hydroxy-3-methylglutaryl coenzyme A reductase kinase by nucleoside diphosphates

Extensively purified rat liver cytosolic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase kinase was used to examine the role of ADP in inactivation of HMG-CoA reductase (EC 1.1.1.34). Solubilized HMG-CoA reductase was a suitable substrate for HMG-CoA reductase kinase. At sufficiently high concentrations of solubilized HMG-CoA reductase, reductase kinase activity approached that measured using microsomal HMG-CoA reductase as substrate. Inactivation of solubilized HMG-CoA reductase by HMG-CoA reductase kinase required both MgATP and ADP. Other nucleoside diphosphates, including alpha, beta-methylene-ADP, could replace ADP. HMG-CoA reductase kinase catalyzed phosphorylation of bovine serum albumin fraction V by [gamma-32P]ATP. This process also required a nucleoside diphosphate (e.g. alpha, beta-methylene-ADP). Nucleoside diphosphates thus act on HMG-CoA reductase kinase, not on HMG-CoA reductase. For inactivation of HMG-CoA reductase, the ability of nucleoside triphosphates to replace ATP decreased in the order ATP greater than dATP greater than GTP greater than ITP, UTP. TTP and CTP did not replace ATP. Both for inactivation of HMG-CoA reductase and for phosphorylation of bovine serum albumin protein, the ability of nucleoside diphosphates to replace ADP decreased in the order ADP greater than CDP, dADP greater than UDP. GDP did not replace ADP. Nucleoside di- and triphosphates thus appear to bind to different sites on HMG-CoA reductase kinase. Nucleoside diphosphates act as allosteric activators of HMG-CoA reductase kinase. For inactivation of HMG-CoA reductase by HMG-CoA reductase kinase, Km for ATP was 140 microM and the activation constant, Ka, for ADP was 1.4 mM. The concentration of ADP required to modulate reductase kinase activity in vitro falls within the physiological range. Modulation of HMG-CoA reductase kinase activity, and hence of HMG-CoA reductase activity, by changes in intracellular ADP concentrations thus may represent a control mechanism of potential physiological significance.     

3-Hydroxy-3-methylglutaryl coenzyme A reductase from avian liver. Catalytic properties.

The catalytic properties of microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase from avian liver have been investigated. Solubilized and highly purified reductase preparations were not cold labile, and enzymic activity remained unchanged following preincubation at 37 degrees C. The pH optimum was 6.8--7.0 and maximal catalytic activity was achieved with 2 mM dithiothreitol and 0.75 M KCl. The heat stability of the enzyme was studied and the addition of 0.75 M KCl, 0.8 mg/ml bovine serum albumin and 5 mM NADPH reduced the inactivation of the purified reductase associated with heat treatment at 65 degrees C. At 37 degrees C, 0.8 mg/ml bovine serum albumin enhanced the purified reductase activity by 100 (+/- 20)%. An improved assay was developed for the avian hydroxymethylglutaryl-CoA reductase and the specific activity of the purified enzyme increased from 1550 to 3300 nmol . min-1 . mg-1. The Km values of solubilized and purified reductase for D-hydroxymethylglutaryl-CoA were 1.05 micrometer and 1.62 micrometer, and for NADPH, 1 mM and 263 micrometer, respectively. The activities of the reductase preparations were non-competitively inhibited by coenzyme A, acyl-CoA esters, and hydroxymethylglutarate. MgATP also reduced avian reductase activity. These modulators may play a role in the cellular regulation of the reductase activity.     

3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Activity in Ochromonas malhamensis: A System to Study the Relationship between Enzyme Activity and Rate of Steroid Biosynthesis

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the key regulatory enzyme of the isoprenoid pathway, was found to be predominantly microsomal in Ochromonas malhamensis, a chrysophytic alga. Detection of HMG-CoA reductase requires the presence of 1% bovine serum albumin during cell homogenization, and the activity is stimulated by the presence of Triton X-100. The enzyme has a pH optimum of 8.0 and an absolute requirement for NADPH. When grown in 10 micromolar mevinolin, a competitive inhibitor of HMG-CoA reductase, O. malhamensis shows a 10- to 15-fold increase in HMG-CoA reductase activity (after washing) with little or no effect on cell growth rate. Cultures can be maintained in 10 micromolar mevinolin for months. O. malhamensis produces a large amount (1% dry weight) of poriferasterol, a product of the isoprenoid pathway. The addition of 10 micromolar mevinolin initially blocked poriferasterol biosynthesis by >90%; within 2 days the rate of synthesis returned to normal levels. Immediately after mevinolin was washed from the 2-day culture, there was a transient 2.5-fold increase in the rate of poriferasterol biosynthesis. The rate of poriferasterol biosynthesis and the level of HMG-CoA reductase activity both fell to control levels within hours.     

Properties of microsomal acyl coenzyme A reductase in mouse preputial glands

Synthesis of long-chain fatty alcohols in preputial glands of mice is catalyzed by an NADPH-dependent acyl coenzyme A (CoA) reductase located in microsomal membranes; sensitivity to trypsin digestion indicates that the reductase is on the cytoplasmic side of the membrane. Results with pyrazole and phenobarbital demonstrate the reaction is not catalyzed by a nonspecific alcohol dehydrogenase or an aldehyde reductase. Acyl-CoA reductase activity is sensitive to sulfhydryl and serine reagent modification, is stimulated by bovine serum albumin, and produces an aldehyde intermediate. The activity is extremely detergent sensitive and cannot be restored even after removal of the detergents. Phospholipase C or asolectin treatment does not release the acyl-CoA reductase from microsomal membranes, but causes a significant decrease in the activity recovered in the membrane pellet. Glycerol does not solubilize the reductase activity, nor does 3.0 m NaCl; however, the combination of glycerol and 3.0 m NaCl did release about 50% of the acyl-CoA reductase from the microsomal pellet. Substrate concentration curves obtained in the presence or absence of bovine serum albumin show significant differences in enzyme activities. The reductase is sensitive to the concentration of palmitoyl-CoA and is progressively inhibited at levels beyond the critical micellar concentration of the substrate. The apparent K_m for acyl-CoA reductase is 14 μm; however, the maximum velocity varies with the concentration of albumin used. Expression of enzyme activity in delipidated microsomes requires specific phospholipids, which suggests that in vivo regulation of acyl-CoA reductase activity could be achieved through modifications in membrane lipid composition.     

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.     

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.     

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)     

Modification of phospholipids fatty acid composition in reuber H35 hepatoma cells: effect on HMG-CoA reductase activity

There is controversy about the effect of saturated and polyunsaturated fats on 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, the main regulatory enzyme of cholesterogenic pathway. Results from dietary studies are difficult to interpret because diets normally contain a mixture of fatty acids. Therefore, we have used Reuber H35 hepatoma cells whose phospholipids were enriched in different individual fatty acids and have studied their effects on the cellular reductase activity. Lauric, myristic, eicosapentaenoic (EPA), and docosahexaenoic (DHA) acids were supplemented to the culture medium coupled to bovine serum albumin. The four fatty acids were incorporated into phospholipids from cells grown in media containing whole serum or lipoprotein-poor serum (LPPS). Reductase activity of cells cultivated in a medium with LPPS was three to four times higher than those cultivated in medium with whole serum. Saturated fatty acids increased reductase activity of cells grown in medium with whole serum, whereas n-3 polyunsaturated fatty acids (PUFA) decreased it. However, both saturated and polyunsaturated fatty acids increased reductase activity when serum lipoproteins were removed. In conclusion, this is one of the first reports demonstrating that saturated and n-3 PUFA only show differential effects on HMG-CoA reductase activity in the presence of lipoproteins.     

Modulation in vitro of 3-hydroxy-3-methylglutaryl coenzyme A reductase in brain microsomes: Evidence for the phosphorylation and dephosphorylation associated with inactivation and activation of the enzyme

The activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase in brain microsomes was modified in vitro. The inactivation of the enzyme required Mg²⁺ and ATP or ADP, and an inactivator present both in S₁₀₅ and microsomes. Inactivation was dependent on inactivator concentration and time of preincubation. The inactive reductase in brain microsomes could be completely reactivated by a factor present in brain S₁₀₅. Reactivation of the enzyme also depended on incubation time and the activator concentration. Activator activity was inhibited by NaF, a phosphatase inhibitor. Both the inactivator and the activator appear to be proteins. Our data thus suggest that the inactivation and the reactivation of the reductase in brain microsomes occurs via protein-mediated interconversion to phosphorylated and dephosphorylated forms of the enzyme with differing catalytic activity. The HMG-CoA reductase activity increases almost two-fold during isolation of the brain microsomes. This increase in activity is blocked when brain tissue is homogenized in the medium containing NaF. In rat brain about 50% of the reductase exists in an inactive form in both young and adult rats. The low reductase activity in brain of adult animals does not appear to be related to an increase in the proportion of an inactive phosphorylated form of the enzyme. This suggests that developmental change in the reductase activity is not associated with the change in the proportion of phosphorylated and dephosphorylated forms of the enzyme.     

Subcellular localization of 3-hydroxy-3-methylglutaryl coenzyme A reductase and other membrane-bound enzymes in sweet potato roots

The subcellular localization of 3-hydroxy-3-methylglutaryl coenzymeA reductase and other membrane-bound enzymes in fresh, cut anddiseased sweet potato root tissues was resolved by differentialcentrifugation and sucrose density gradient centrifugation.In fresh, cut and diseased tissues, cytochrome c oxidase wasalmost localized in mitochondria, and NADH cytochrome c reductasewas in mitochondria in fresh and cut tissues, but in both mitochondriaand microsomes in diseased tissue. NADPH cytochrome c reductaseand antimycin A insensitive NADH cytochrome c reductase weremainly associated with microsomes. Catalase was dominantly foundin the mitochondrial fraction. 3-Hydroxy-3-methylglutaryl coenzymeA reductase was localized only in mitochondria and not in microsomaland supernatant fractions in both fresh and cut tissues. Indiseased tissue (infected with Ceratocystis fimbriata), in additionto being present in mitochondria, the enzyme was also localizedin microsomes. These results indicate that microsomal 3-hydroxy-3-methylglutarylcoenzyme A reductase whose activity rapidly increased in responseto the infection, predominandy participates in the formationof terpenes such as ipomeamarone. ¹ This paper constitutes Part 122 in the Series "The PhytopadiologicalChemistry of Sweet Potato with Black Rot and Injury." (Received March 1, 1976; )     

Use of protein in extraction and stabilization of nitrate reductase

The in vitro instability of nitrate reductase (EC 1.6.6.1) activity from leaves of several species of higher plants was investigated. Decay of activity was exponential with time, suggesting that an enzyme-catalyzed reaction was involved. The rate of decay of nitrate reductase activity increased as leaf age increased in all species studied. Activity was relatively stable in certain genotypes of Zea mays L., but extremely unstable in others. In all genotypes of Avena sativa L. and Nicotiana tabacum L. studied, nitrate reductase was unstable. Addition of 3% (w/v) bovine serum albumin or casein to extraction media prevented or retarded the decay of nitrate reductase activity for several hours. In addition, the presence of bovine serum albumin or casein in the enzyme homogenate markedly increased nitrate reductase activity (up to 15-fold), especially in older leaf tissue.     

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.     

The in vivo regulation of rat liver 3-hydroxy-3-methylglutaryl coenzyme A reductase. Phosphorylation of the enzyme as an early regulatory response following cholesterol feeding

Although substantial evidence supports the conclusion that 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase is the major regulatory enzyme in cholesterol biosynthesis, the molecular events involved in the in vivo regulation of this enzyme have remained obscure. In order to study this problem, rats received a single meal consisting of either rat chow or rat chow containing 2% cholesterol. The rats were killed 60 or 120 min after the beginning of feeding, and liver microsomes were prepared by ultracentrifugation. Two phases of inhibition of microsomal HMG-CoA reductase were observed. The first phase of inhibition, observed 60 min after the beginning of cholesterol feeding, was completely reversed by preincubation of the microsomes with purified phosphoprotein phosphatase. The second phase of inhibition, observed 120 min after the beginning of cholesterol feeding, was not reversed by phosphoprotein phosphatase. These results are consistent with the conclusion that phosphorylation of HMG-CoA reductase is the first step in a series of in vivo regulatory events which produce inactivation and ultimately degradation of the enzyme.     

Inactivation of mitochondrial 2-oxoglutarate dehydrogenase complex as a result of phospholipid degradation induced by freeze-thawing

The inactivation of 2-oxoglutarate dehydrogenase complex by freeze-thawing was examined along with alterations of membrane phospholipids, in order to elucidate the mechanism of freezing injury in mitochondria.The dehydrogenase complex activity in slowly frozen and thawed mitochondria decreased to 70% as compared to intact mitochondria and further decreased during incubation. This inactivation during incubation was temperature dependent, i.e., at temperatures up to 25°C there was a slight decrease, while at higher temperatures there was a marked decrease in the dehydrogenase complex activity. Simultaneously, there was a significant accumulation of free fatty acids, generated from mitochondrial phospholipids, which inhibited 2-oxoglutarate dehydrogenase and subsequently enzyme complex activity. Oxoglutarate dehydrogenase activity in mitochondria was markedly inhibited by exogenous phospholipase A, and this inhibition was partially prevented with bovine serum albumin. Furthermore, when intrinsic phospholipase A was either inhibited or stimulated, there was a respective decrease or increase in the enzyme complex inactivation.The activity of the purified enzyme complex decreased slightly after slow freezing, but remained constant even when incubated at temperatures up to 32°C. However, the activity of this enzyme complex was markedly reduced when incubated either in the presence of venom phospholipase A or with exogenous fatty acid.The relationship between inactivation of the 2-oxoglutarate dehydrogenase complex, phospholipase A activation and production of free fatty acids in frozen and thawed mitochondria is discussed.     

Stimulation of hepatic cholesterol biosynthesis by fatty acids. Effects of oleate on cytoplasmic acetoacetyl-CoA thiolase, acetoacetyl-CoA synthetase and hydroxymethylglutaryl-CoA synthase.

The effects of oleic acid on the activities of cytosolic HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) synthase, AcAc-CoA (acetoacetyl-CoA) thiolase and AcAc-CoA synthetase, as well as microsomal HMG-CoA reductase, all enzymes in the pathway of cholesterol biosynthesis, were studied in the isolated perfused rat liver. Oleic acid bound to bovine serum albumin, or albumin alone, was infused for 4 h at a rate sufficient to sustain an average concentration of 0.61 +/- 0.05 mM fatty acid during the perfusion. Hepatic cytosol and microsomal fractions were isolated at the termination of the perfusion. Oleic acid simultaneously increased the activities of the cytosolic cholesterol-biosynthetic enzymes 1.4-2.7-fold in livers from normal fed rats and from animals fasted for 24 h. These effects were accompanied by increased net secretion by the liver of cholesterol and triacylglycerol in the very-low-density lipoprotein (VLDL). We confirmed the observations reported previously from this laboratory of the stimulation by oleic acid of microsomal HMG-CoA reductase. In cytosols from perfused livers, the increase in AcAc-CoA thiolase activity was characterized by an increase in Vmax. without any change in the apparent Km of the enzyme for AcAc-CoA. In contrast, oleic acid decreased the Km of HMG-CoA synthase for Ac-CoA, without alteration of the Vmax. of the enzyme. The Vmax. of AcAc-CoA synthetase was increased by oleic acid, and there was a trend towards a small increase in the Km of the enzyme for acetoacetate. These data allow us to conclude that the enzymes that supply the HMG-CoA required for hepatic cholesterogenesis are stimulated, as is HMG-CoA reductase, by a physiological substrate, fatty acid, that increases rates of hepatic cholesterol synthesis and cholesterol secretion. Furthermore, we suggest that these effects of fatty acid on hepatic cholesterol metabolism result from stimulation of secretion of triacylglycerol in the VLDL by fatty acids, and the absolute requirement of cholesterol as an important structural surface component of the VLDL necessary for transport of triacylglycerol from the liver.     

Regulation of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase. In vitro inhibition by a protein present in bile.

The activity of rat hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34), the rate-limiting enzyme of cholesterol biosynthesis, is inhibited in vitro by factors present in both rat and bovine bile. The inhibitory factor from bovine bile has been purified to near homogeneity and is a high molecular weight lipoprotein with a density (p = 1.024) and lipid composition similar to serum β-lipoprotein. Analysis of the interaction of the enzyme and inhibitor demonstrate that the observed inactivation/inhibition is a function of lipoprotein concentration, microsomal protein concentration and duration of interaction. The observed inhibition is apparently irreversible and while neither substrate alone protects the enzyme, both substrates decrease the rate of inactivation several fold.     

In situ measurement of HMG-CoA reductase activity in digitonin-permeabilized hepatocytes

An assay procedure for HMG-CoA reductase is described which allows rapid measurement of the activity of this enzyme in isolated rat hepatocytes. In a one step procedure digitonin permeabilizes the plasma membrane and at the same time HMG-CoA reductase activity is measured. Digitonin at a concentration of 64 micrograms per mg of cell protein was found to be optimal for exposing microsomal HMG-CoA reductase to the assay components. The enzyme assay is linear with time up til 5 min and with protein concentrations in the range of 0.06-0.6 mg of cell protein per assay. It is shown that cellular enzyme activity is affected by preincubation of intact hepatocytes with a variety of short-term modulators of hepatic cholesterogenesis.     

In Vitro Studies of Nitrate Reductase Activity in Cotton Cotyledons: Effects of Dowex 1-Cl and BSA

Germinating cotton (Gossypium hirsutum L. cv. Deltapine 16) cotyledons developed two peaks of in vitro nitrate reductase activity; the first was stable in vitro and appeared 24 hours after imbibition; and the second, which was extremely labile in vitro, began to develop after the seedlings had emerged and developed chlorophyll. Nitrite reductase activity peaked only after the seedlings had emerged. Dowex 1-Cl (10%, w/v) and bovine serum albumin (3%, w/v) significantly improved the activity of extracted enzyme; greater improvement occurred as expansion of the cotyledons progressed. The major effect of bovine serum albumin on nitrate reductase activity in cotyledon extracts appeared to be that of making the extracted enzyme more active rather than increasing the amount of nitrate reductase extracted or improving the stability of the extracted enzyme.     

Inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in hepatoma tissue culture cells by pure cholesterol and several cholesterol derivatives. Evidence supporting two distinct mechanisms.20l.

Pure cholesterol associated in complexes with lipoproteins (whole serum and human low density lipoproteins) or esterified with succinic acid (cholesteryl succinate) and bound to albumin effectively suppresses 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity in hepatoma tissue culture (HTC) cells grown in lipoprotein-poor serum medium during short 4-hour) incubation periods. Simultaneous measurments of the kinetics of uptake of radioactive unesterified cholesterol of whole serum and cholesteryl succinate, their conversion to lipid products, and the decay in enzyme activity, suggest that the cholesterol-induced suppression is mediated by the sterol itself rather than by inhibitory lipid products derived from its metabolism. Several cholesterol derivatives such as cholestenone, 7-ketocholesterol, and 7alpha-and 25-hydroxycholesterol also suppress reductase activiy in HTC cells and are significantly more inhibitory than the pure cholesterol preparations. The decrease in enzyme activity produced by cholesterol and its derivatives is concentration-dependent and specific. [1-14C]Oleate incorporation experiments indicate that cholesterol ester formation in HTC cells is not increased at inhibitory concentrations of the steroids. These data suggest that sterol ester formation is not an obligatory process in the feedback control of HMG-CoA reductase activity. The half-life of the reductase (3 to 4 hours) is not significantly changed by cycloheximide, plus or minus whole serum, and cholesteryl succinate. In contrast, the half-life is strongly reduced when HTC cells are incubated with cycloheximide plus maximal concentrations of 25-hydroxycholesterol, 7-ketocholesterol, or cholestenone, resulting in t1/2 values of 24, 36, and 60 min, respectively. Increasing concentrations of whole serum and cholesteryl succinate have no significant effect on the apparent rate constant of inactivation of the enzyme, whereas its apparent rate of synthesis is decreased 3- and 10-fold, respectively. These results are reversed with oxygenated steroid inhibitors. The rate of synthesis of reductase is essentially unchanged as the concentrations of 25-hydroxycholesterol, 7-ketocholesterol, and cholestenone are increased in the culture medium, whereas the apparent rate constant for degradation is increased 9-, 7-, and 3-fold, respectively. HMG-CoA reductase activity in HTC cells thus appears to be modulated by two different mechanisms in which steroid structure is important. Whole serum and cholesteryl succinate specifically decrease the rate of enzyme synthesis, while 25-hydroxycholesterol, 7-ketocholesterol, and cholestenone increase the rate of inactivation of the reductase.