Specific induction of tumor neovasculature death by modified murine PPE-1 promoter armed with HSV-TK.

BACKGROUND: One strategy to increase tissue specificity of gene therapy is to use promoters or enhancers. OBJECTIVES: (1) To enhance the selectivity of a murine preproendothelin-1 (PPE-1) promoter in tumor angiogenesis by using a positive endothelial transcription-binding element. (2) To test the specificity and efficiency of the modified PPE-1 promoter [PPE-1(3X)] in vitro and in vivo by using reporter genes, and the therapeutic gene herpes simplex virus-thymidine kinase (HSV-TK) in a mouse model of Lewis lung carcinoma (LLC). RESULTS: The modified PPE-1 promoter specifically induced expression in the tumor angiogenic vascular bed with a 35-fold higher expression compared to the normal vasculare bed of the lung. Thus, when the HSV-TK gene controlled by the modified PPE-1 promoter was used systemically, it induced tumor-specific necrosis, apoptosis and mononuclear infiltrates, leading to massive destruction of the neovasculature of the pulmonary metastasis, which suppressed metastasis development. CONCLUSIONS: These results show that an adenoviral vector armed with HSV-TK controlled by the endothelial-selective murine PPE-1(3X) promoter is efficient and safe to target tumor neovasculature.

Interindividual variability in sensitivity to warfarin--Nature or nurture?

BACKGROUND: Interindividual variability in responses to warfarin is attributed to dietary vitamin K, drug interactions, age, or genetic polymorphism in the cytochrome P4502C9 enzyme (CYP2C9) (allelic variants 2C9*2 and 2C9*3 ) linked with impaired metabolism of the potent enantiomere S-warfarin. PATIENTS AND METHODS: We quantified the relative effects of age and of simultaneously determined CYP2C9 genotype, plasma warfarin and vitamin K concentrations, and concurrent medications on warfarin maintenance doses in 156 patients at optimized stable anticoagulation. RESULTS: Allele frequencies for CYP2C9*1, CYP2C9*2, and CYP2C9*3 were 0.84, 0.10, and 0.06. Warfarin doses were 6.5 +/- 3.2, 5.2 +/- 2.4, and 3.3 +/- 2.0 mg/d in the 3 genotype groups (P < .0001). Warfarin doses decreased with age as follows: 7.7 +/- 3.7 versus 4.9 +/- 2.9 mg/d at < 50 years and >66 years (P < .001), mainly as a result of decreased plasma warfarin clearance (2.8 +/- 1.4 mL/min versus 1.9 +/- 0.8 mL/min; P < .001). Vitamin K (1.6 +/- 1.1 ng/mL) did not differ among the age or genotype groups. Patients >or=66 years old with the CYP2C9*3 allele required only 2.2 +/- 1.2 mg/d compared with 7.9 +/- 3.7 mg/d in those

The ubiquitin-proteasome pathway mediates the regulated degradation of mammalian 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), the key regulatory enzyme in the mevalonate (MVA) pathway, is rapidly degraded in mammalian cells supplemented with sterols or MVA. This accelerated turnover was blocked by N-acetyl-leucyl-leucyl-norleucinal (ALLN), MG-132, and lactacystin, and to a lesser extent by N-acetyl-leucyl-leucyl-methional (ALLM), indicating the involvement of the 26 S proteasome. Proteasome inhibition led to enhanced accumulation of high molecular weight polyubiquitin conjugates of HMGR and of HMGal, a chimera between the membrane domain of HMGR and beta-galactosidase. Importantly, increased amounts of polyubiquitinated HMGR and HMGal were observed upon treating cells with sterols or MVA. Cycloheximide inhibited the sterol-stimulated degradation of HMGR concomitantly with a marked reduction in polyubiquitination of the enzyme. Inhibition of squalene synthase with zaragozic acid blocked the MVA- but not sterol-stimulated ubiquitination and degradation of HMGR. Thus, similar to yeast, the ubiquitin-proteasome pathway is involved in the metabolically regulated turnover of mammalian HMGR. Yet, the data indicate divergence between yeast and mammals and suggest distinct roles for sterol and nonsterol metabolic signals in the regulated ubiquitination and degradation of mammalian HMGR.

Impaired regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase degradation in lovastatin-resistant cells.

L-90 cells were selected to grow in the presence of serum lipoproteins and 90 microM lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR). L-90 cells massively accumulate HMGR, a result of >10-fold amplification of the gene and 40-fold rise in mRNA, and also overexpress other enzymes of the mevalonate pathway. Western blot and promoter-luciferase analyses indicate that transcriptional regulation of sterol-responsive genes by 25-hydroxycholesterol or mevalonate is normal. Yet, none of these genes is regulated by lipoproteins, a result of severe impairment in the low density lipoprotein receptor pathway. Moreover, L-90 cells do not accelerate the degradation of HMGR or transfected HMGal chimera in response to 25-hydroxycholesterol or mevalonate. This aberrant phenotype persists when cells are grown without lovastatin for up to 37 days. The inability to regulate HMGR degradation is not due to its overproduction since in LP-90 cells, which were selected for lovastatin resistance in lipoprotein-deficient serum, HMGR is overexpressed, yet its turnover is regulated normally. Also, the rapid degradation of transfected alpha subunit of T cell receptor is markedly retarded in L-90 cells. These results show that in addition to gene amplification and overexpression of cholesterogenic enzymes, statin resistance can follow loss of regulated HMGR degradation.

Improved PCR amplification/Hhal restriction for unambiguous determination of apolipoprotein E alleles.

We present a modification to the polymerase chain reaction amplification/Hhal restriction isotyping method for human apolipoprotein (apo) E. This method includes a mutagenic forward primer and 5'-end labeling of both primers. These modifications of the original method described by Hixon and Vernier (J Lipid Res 1990;31:545-8) allow sensitive and unambiguous determination of apoE genotypes.

Direct plasma radioimmunoassay for rat amylin-(1-37): concentrations with acquired and genetic obesity.

Amylin (islet-associated polypeptide) is a 37-amino acid peptide that is cosecreted with insulin from the pancreatic beta-cell. Accurate measurement of its plasma levels is important for delineating the physiological range over which amylin acts. We describe a reproducible, highly specific, and sensitive radioimmunoassay for direct measurement of plasma amylin-(1-37). We measured changes in portal and systemic plasma amylin and insulin in three groups of anesthetized rats: lean young adult and old adult Wistar rats with acquired obesity, and Wistar fatty [WDF/TaFa (fa/fa)] rats, a model of genetic obesity and insulin resistance derived from the Wistar strain. Changes in response to fasting, feeding, and intravenous stimulation with glucose plus arginine were assessed. We find that the amylin-to-insulin ratio is constant in fasted or fed young and old rats because of proportionate increases in both entities with aging. In genetically obese Wistar rats, amylin and insulin levels are three- to tenfold higher than in lean young or obese old normal controls. Islet stimulation by feeding or intravenous glucose plus arginine resulted in a decreased amylin-to-insulin molar ratio in all groups. When normalized for the degree of islet stimulation, amylin-to-insulin ratios were significantly elevated in genetically obese vs. normal rats, both in the portal and systemic circulation. These results demonstrate that aging-related weight gain in normal rats is associated with moderate and proportional increases in amylin and insulin, whereas genetic obesity is characterized by elevated amylin and an increased amylin-to-insulin ratio. Implications for the pathogenesis of insulin resistance and obesity are discussed.

Lack of correlation between 3-hydroxy-3-methylglutaryl coenzyme A reductase activity and lovastatin resistance in nerve growth factor treated PC-12 cells.

1. The relationships among the mevalonic acid (MVA) forming enzyme, 3-hydroxy-3-methylglutaryl coenzyme A (CoA) reductase, cell growth and differentiation, and the cytotoxic effects of the reductase inhibitor lovastatin were studied in PC-12 cells, exposed to growth factors. 2. When added individually, nerve growth factor (NGF), basic fibroblast growth factor, and epidermal growth factor induce an increase in HMG-CoA reductase activity in cells grown in serum-containing medium. In the presence of serum, the effect of NGF on HMG-CoA reductase is persistent. 3. Short-term serum starvation and long-term NGF treatment, in combination, have an additive effect, resulting in a high reductase activity. 4. Unlike serum and MVA, which downregulate levels of HMG-CoA reductase by accelerating its degradation, NGF upregulates reductase by slowing the rate of its degradation. This mechanism, however, appears to operate only in the presence of serum, as after prolonged growth with NGF in serum-free medium, cells have a low reductase activity. 5. PC-12 cells grown in the absence of NGF are highly sensitive to lovastatin (25 microM) and more than 70% of the cells die after 48 hr. NGF confers lovastatin resistance on cells grown in the presence or in the absence of serum (only 30-40% cell death after 48 hr with lovastatin). 6. NGF-induced resistance on lovastatin develops with time and is apparent only in the well-differentiated PC-12 cells whether or not the cells express a high reductase activity.(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo inhibition of cyclin B degradation and induction of cell-cycle arrest in mammalian cells by the neutral cysteine protease inhibitor N-acetylleucylleucylnorleucinal.

The cytotoxic neutral cysteine protease inhibitor N-acetylleucylleucylnorleucinal (ALLN) inhibits cell-cycle progression in CHO cells, affecting the G1/S and metaphase-anaphase transition points, as well as S phase. Mitotic arrest induced by ALLN is associated with the inhibition of cyclin B degradation. At mitosis-inhibiting concentrations of ALLN, cells undergo nuclear-envelope breakdown, spindle formation, chromosome condensation, and congression to the metaphase plate. However, normal anaphase events do not occur, and cells arrest in a metaphase configuration for a prolonged period. Steady-state levels of cyclin B increase to greater than normal mitotic levels, and cyclin B is not degraded for an extended period. Histone H1 kinase activity remains elevated during mitotic arrest. Duration of mitotic arrest depends on ALLN concentration; high concentrations (> 50 micrograms/ml) produce a prolonged mitotic arrest, whereas at lower concentrations, cells are transiently delayed through mitosis (up to 4-12 hr), after which they undergo aberrant cell division resulting in randomly sized daughter cells containing variable amounts of DNA. Cyclin B degradation fails to occur, and histone H1 kinase remains activated for the duration of mitotic arrest at all ALLN concentrations.

Distinct sterol and nonsterol signals for the regulated degradation of 3-hydroxy-3-methylglutaryl-CoA reductase.

The in vivo turnover rate of the endoplasmic reticulum protein 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in the mevalonate (MVA) pathway, is accelerated when excess MVA or sterols are added to the growth medium of cells. As we have shown recently (Roitelman, J., Bar-Nun, S., Inoue, S., and Simoni, R. D. (1991) J. Biol. Chem. 266, 16085-16091), perturbation of cellular Ca2+ homeostasis abrogates the MVA-accelerated degradation of HMG-CoA reductase and HMGal. Here we show that, in contrast, the sterol-accelerated degradation of HMG-CoA reductase is unaffected by Ca2+ perturbation achieved either by Ca2+ ionophore or by inhibitors of the endoplasmic reticulum Ca(2+)-ATPase. The differential effects of Ca2+ perturbation can be attributed neither to global alteration in protein synthesis nor to inhibition of MVA conversion to sterols. Yet, such manipulations markedly reduce the incorporation of MVA into cellular macromolecules, including prenylated proteins. Furthermore, we directly demonstrate that MVA gives rise to at least two distinct signals, one that is essential to support the effect of sterols and another that operates independently of sterols. Our results indicate that the cellular signals operating in the MVA-accelerated turnover of HMG-CoA reductase are distinct from those involved in the sterol-regulated degradation. A working model for the degradation pathway is proposed.

Immunological evidence for eight spans in the membrane domain of 3-hydroxy-3-methylglutaryl coenzyme A reductase: implications for enzyme degradation in the endoplasmic reticulum.

We have raised two monospecific antibodies against synthetic peptides derived from the membrane domain of the ER glycoprotein 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate limiting enzyme in the cholesterol biosynthetic pathway. This domain, which was proposed to span the ER membrane seven times (Liscum, L., J. Finer-Moore, R. M. Stroud, K. L. Luskey, M. S. Brown, and J. L. Goldstein. 1985. J. Biol. Chem. 260:522-538), plays a critical role in the regulated degradation of the enzyme in the ER in response to sterols. The antibodies stain the ER of cells and immunoprecipitate HMG-CoA reductase and HMGal, a chimeric protein composed of the membrane domain of the reductase fused to Escherichia coli beta-galactosidase, the degradation of which is also accelerated by sterols. We show that the sequence Arg224 through Leu242 of HMG-CoA reductase (peptide G) faces the cytoplasm both in cultured cells and in rat liver, whereas the sequence Thr284 through Glu302 (peptide H) faces the lumen of the ER. This indicates that a sequence between peptide G and peptide H spans the membrane of the ER. Moreover, by epitope tagging with peptide H, we show that the loop segment connecting membrane spans 3 and 4 is sequestered in the lumen of the ER. These results demonstrate that the membrane domain of HMG-CoA reductase spans the ER eight times and are inconsistent with the seven membrane spans topological model. The approximate boundaries of the proposed additional transmembrane segment are between Lys248 and Asp276. Replacement of this 7th span in HMGal with the first transmembrane helix of bacteriorhodopsin abolishes the sterol-enhanced degradation of the protein, indicating its role in the regulated turnover of HMG-CoA reductase within the endoplasmic reticulum.

Peptide transport by the multidrug resistance pump.

The membrane P-glycoprotein (P170) is an ATP-hydrolyzing transmembrane pump, and elevated levels of P170, due to higher expression with or without amplification of the multidrug resistance gene (mdr1), result in resistance to a variety of chemotherapeutic agents in mammalian cells. The function of the P170 pump has been proposed as a protection against toxic substances present in animal diets. Here we describe a Chinese hamster ovary cell line that was selected for resistance to a synthetic tripeptide, N-acetyl-leucyl-leucyl-norleucinal (ALLN). This ALLN-resistant variant shows the classical multidrug resistance (MDR) phenotype, including overexpression and amplification of the mdr1 gene. Additionally, a mouse embryo cell line overexpressing the transfected mdr1 gene is likewise resistant to ALLN. Our results demonstrate that P170 is capable of transporting peptides and raise the possibility that the mdr1 gene product or other MDR-like genes, present in the genome of mammalian cells, may be involved in secretion of peptides or cellular proteins as is the case with the structurally similar hylB and ste6 gene products of Escherichia coli and yeast, respectively.

Involvement of calcium in the mevalonate-accelerated degradation of 3-hydroxy-3-methylglutaryl-CoA reductase.

3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase), the rate-limiting enzyme in the biosynthesis of cholesterol and isoprenoids, is subject to rapid degradation which is regulated by mevalonate (MVA)-derived metabolic products. HMG-CoA reductase is an integral membrane protein of the endoplasmic reticulum, the largest nonmitochondrial pool of cellular Ca2+. To assess the possible role of Ca2+ in the regulated degradation of HMG-CoA reductase, we perturbed cellular Ca2+ concentration and followed the fate of HMG-CoA reductase and of HMGal, a fusion protein consisting of the membrane domain of HMG-CoA reductase and the soluble bacterial enzyme beta-galactosidase. The degradation of HMGal mirrors that of HMG-CoA reductase, demonstrating that the membrane domain of HMG-CoA reductase is sufficient to confer regulated degradation (Skalnik, D.G., Narita, H., Kent, C., and Simoni, R.D. (1988) J. Biol. Chem. 263, 6836-6841; Chun, K.T., Bar-Nun, S., and Simoni, R.D. (1990) J. Biol. Chem. 265, 22004-22010). In this study we show that the MVA-dependent accelerated rates of degradation of HMG-CoA reductase and HMGal in cells maintained in Ca(2+)-free medium are 2-3-fold slower than the rate of degradation in cells grown in high (1.8-2 mM) Ca2+ concentration. This effect is reversed upon addition of Ca2+ to the medium. Furthermore, when cells maintained in high Ca2+ are treated with 1 microM ionomycin, the MVA-dependent accelerated degradation of HMG-CoA reductase and HMGal is also reduced about 2-3-fold. This inhibition is not due to a Ca(2+)-dependent uptake or incorporation of MVA into sterols, since these processes are not affected in the absence of external Ca2+. In addition, cobalt, a known antagonist of Ca(2+)-dependent cellular functions, totally abolishes (IC50 = 520 microM in the presence of 1.8 mM extracellular Ca2+) the MVA-accelerated degradation of HMGal. These results suggest that Ca2+ plays a major role in the regulated degradation of HMG-CoA reductase.

Inhibition of degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in vivo by cysteine protease inhibitors.

3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase is a key regulatory enzyme of cholesterol biosynthesis and is located in the endoplasmic reticulum (ER). A fusion protein, HMGal, consisting of the membrane domain of HMG-CoA reductase fused to Escherichia coli beta-galactosidase and expressed in Chinese hamster ovary (CHO) cells from the SV40 promoter, was previously constructed and was found to respond to regulatory signals for degradation in a similar fashion to the intact HMG-CoA reductase. Degradation of both HMG-CoA reductase and HMGal in CHO cells was enhanced by addition of mevalonate or low density lipoprotein (LDL). In this report we show that 2 cysteine protease inhibitors, N-acetyl-leucyl-leucyl-norleucinal (ALLN) and N-acetyl-leucyl-leucyl-methioninal (ALLM), completely inhibit the mevalonate- or LDL-accelerated degradation of HMG-CoA reductase and HMGal and also block the basal degradation of these enzymes. It has been shown that in vitro these protease inhibitors inhibit the activities of Ca(2+)-dependent neutral proteases as well as lysosomal proteases, including cathepsin L, cathepsin b, and cathepsin D. However, the mevalonate-accelerated degradation of HMG-CoA reductase and HMGal is not affected by lysosomotropic agents, suggesting that the site of action of these inhibitor peptides in preventing the degradation is not the cathepsins. In brefeldin A-treated cells, where protein export from the ER is blocked, ALLN is still effective in inhibiting the degradation of HMG-CoA reductase and HMGal. These results indicate the involvement of non-lysosomal Ca(2+)-dependent proteases in the basal and the accelerated degradation of HMG-CoA reductase and HMGal. Enzymatic assays in vitro and immunoblot analyses have revealed calpain- and calpastatin-like proteins in CHO cells. The activities and the amount of these proteins do not change under conditions of enhanced degradation, indicating that the levels of these proteins are not subject to mevalonate regulation.

Studies on the catalytic site of rat liver HMG-CoA reductase: interaction with CoA-thioesters and inactivation by iodoacetamide.

The localization of reactive cysteines and characterization of the HMG-CoA binding domain of rat liver HMG-CoA reductase were studied using iodoacetamide (IAAD) and short-chain acyl-CoA thioesters. Freeze-thaw-solubilized HMG-CoA reductase is irreversibly inactivated by IAAD with a second order rate constant of 0.78 M-1 sec-1 at 37 degrees C and pH 7.2. This IAAD inactivation is slowed down by pretreatment of the enzyme with disulfides, indicating that inactivation of HMG-CoA reductase occurs mainly through alkylation of specific cysteine residues in the protein. The substrate HMG-CoA, but not NADP(H), effectively protects the reductase from IAAD inactivation. When both HMG-CoA and NADP(H) are present, the reductase is inactivated by IAAD at a rate much faster than the inactivation in the presence of HMG-CoA alone. Of the two moieties of the HMG-CoA thioester, the CoA moiety confers protection from IAAD inactivation whereas HMG is totally ineffective. A series of CoA-thioesters of mono- and dicarboxylic acids of various size were tested for their effect on the activity of HMG-CoA reductase. The CoA analog, desulfo-CoA (des-CoA), and all CoA-thioesters of monocarboxylic acids of up to 6 carbons in length exhibit mixed-type inhibition of reductase activity. The competitive inhibition constants (Ki) for these compounds vary between 1 and 2 mM, whereas the noncompetitive component (K'i) is relatively constant (540 +/- 20 microM). As the acyl chain length increases beyond 6 carbons, the thioesters of monocarboxylic acids become more potent and acquire the characteristics of pure noncompetitive inhibitors. In contrast, the monothioesters of dicarboxylic acids are pure competitive inhibitors with Ki values which are similar to the Ki values of the corresponding thioesters of monocarboxylates. HMG does not affect reductase activity in concentrations of up to 2 mM, yet it greatly enhances the inhibition of the enzyme by des-CoA. Specifically, HMG affects only the Ki value of des-CoA by decreasing it from 1030 microM to 280 microM. The results indicate that reactive cysteine(s) are localized in the catalytic site of HMG-CoA reductase. Within the active site, these cysteines are closely associated with and probably participate in the binding of the CoA moiety of the substrate HMG-CoA. The results are also consistent with the existence of a noncatalytic hydrophobic site in HMG-CoA reductase.

Allosteric and non-allosteric forms of rat liver 3-hydroxy-3-methylglutaryl coenzyme A reductase: differential inhibition of activity by adenosine 2'-monophospho-5'-diphosphoribose.

Adenosine 2'-monophospho-5'-diphosphoribose (P-ADP-Rib) is a structural analog of NADPH which was reported to competitively inhibit (Kiapp = 21.7 microM) solubilized rat liver 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (Tanazawa, K., and A. Endo. 1979. Eur. J. Biochem. 98: 195-201). However, microsomal HMG-CoA reductase, which at low thiol concentrations exhibits allosteric properties, is only poorly inhibited by P-ADP-Rib (Kiapp = 550 microM at 4.5 mM GSH). Gradual shift of the microsomal reductase towards a non-allosteric form by increasing glutathione (GSH) concentrations resulted in a higher inhibition by P-ADP-Rib. Under these conditions, Ki values for P-ADP-Rib were 165 microM and 53 microM at 9 mM and 27 mM GSH, respectively. The largest change in the degree of inhibition by P-ADP-Rib was observed within the 10 mM range of GSH. By contrast, freeze-thaw solubilized HMG-CoA reductase, which does not display allosteric properties, is readily inhibited by P-ADP-Rib, even when assayed at a low concentration of GSH (Kiapp = 50 microM at 4.5 mM GSH). Assaying the solubilized reductase in the presence of increased thiol concentration results in a minor decrease in the apparent Ki for P-ADP-Rib (22 microM at 27 mM GSH). Microsomal HMG-CoA reductase is allosterically activated by various nucleotides. When activated by NADH, the enzyme is effectively inhibited by P-ADP-Rib even at a 4.5-mM GSH concentration (Kiapp = 175 microM in the presence of 300 microM NADH).(ABSTRACT TRUNCATED AT 250 WORDS)

Altered kinetic properties of rat liver 3-hydroxy-3-methylglutaryl coenzyme A reductase following dietary manipulations.

The microsomal enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase catalyzes the rate-limiting step in the cholesterogenic pathway and was proposed to be composed in situ of 2 noncovalently linked subunits (Edwards, P.A., Kempner, E.S., Lan, S.-F., and Erickson, S.K. (1985) J. Biol. Chem. 260, 10278-10282). In the present report, the activities and kinetic properties of HMG-CoA reductase in microsomes isolated from livers of rats fed on diets supplemented with either ground Amberlite XAD-2 ("X"), cholestyramine/mevinolin ("CM"), or unsupplemented, normal rat chow ("N"), were compared. The specific activities of HMG-CoA reductase in X and CM microsomes were, respectively, 5- and 83-fold higher than that of N microsomes. In NADPH-dependent kinetics of HMG-CoA reductase activated with 4.5 mM GSH, the concentration of NADPH required for half-maximal velocity (S0.5) was 209 +/- 23, 76 +/- 23, and 40 +/- 4 microM for the N, X, and CM microsomes, respectively. While reductase from X microsomes displays cooperative kinetics toward NADPH (Hill coefficient (nH) = 1.97 +/- 0.07), the enzyme from CM microsomes does not (nH = 1.04 +/- 0.07). Similarly to HMG-CoA reductase from CM microsomes, the freeze-thaw solubilized enzyme ("SOL") displays no cooperativity toward NADPH and its Km for this substrate is 34 microM. At 4.5 mM GSH, HMG-CoA reductase from X, CM, and SOL preparations has a similar Km value for [DL]-HMG-CoA, ranging between 13-16 microM, while reductase from N microsomes had a higher Km value (42 microM) for this substrate. No cooperativity towards HMG-CoA was observed in any of the tested enzyme preparations. Immunoblotting analyses of the different preparations demonstrated that the observed altered kinetics of HMG-CoA reductase in the microsomes is not due to preferential proteolytic cleavage of the native 97-100 kDa subunit of the enzyme to the noncooperative 50-55 kDa species. Moreover, it was found that the ratio enzymatic activity/immunoreactivity of the reductase increased in the order N less than X less than CM approximately equal to SOL, indicating that the activity per reductase molecule increases with the induction of the enzyme. These results are compatible with a model suggesting that dietary induction of hepatic HMG-CoA reductase may change the state of functional aggregation of its subunits.

Structural requirements for allosteric activators of rat liver microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase.

Several compounds containing various structural moieties of NAD(P)(H), were examined as possible effectors of rat liver 3-hydroxy-3-methylglutaryl coenzyme A reductase activity. Microsomal reductase was activated with 4.5mM GSH, assayed with subsaturating NADPH concentration and increasing amounts of the tested compounds. Under these conditions, the essential and sufficient structure required to allosterically enhance the activity of the reductase is that of 5'-AMP. When the 2' position of the nucleotide is phosphorylated, this allosteric activation is diminished.

Allosteric activation of rat liver microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase by nicotinamide adenine dinucleotides.

NADH and NAD+ are neither substrates nor inhibitors of 3-hydroxy3-methylglutaryl coenzyme A (HMG-CoA) reductase in concentrations up to 1 mM. Addition of either NADH or NAD+ enhanced the activity of rat liver microsomal reductase, yet NADH failed to affect the activity of the freeze-thaw solubilized enzyme. The degree of enhancement of enzyme activity by NADH decreased as GSH concentration in the assay increased. Addition of 500 microM NADH to the assay converted the sigmoidal (Hill coefficient = 2.0) NADPH-dependent kinetic curve of the microsomal reductase into Michaelis-Menten kinetics (Hill coefficient = 1.1). Furthermore, the kinetic curves were shifted to the left, resulting in an up to 35% decrease in the concentration of NADH required to obtain half-maximal velocity (S0.5) in the presence of 500 microM NADH. Again, this effect of NADH was diminished as GSH concentrations increased. These results demonstrate that NAD(H) is an allosteric activator of HMG-CoA reductase. These results also indicate that HMG-CoA reductase has NAD(H) binding site(s) distinct from the catalytic NADPH site(s).

Regulation of rat liver 3-hydroxy-3-methylglutaryl coenzyme A reductase. Evidence for thiol-dependent allosteric modulation of enzyme activity.

Rat liver microsomes devoid of free thiols were prepared in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer in the presence of 30 microM leupeptin. The activation of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase by GSH and dithiothreitol (DTT) in these microsomes was studied and compared to the activation by these thiols of enzyme that was solubilized by freeze-thawing. An increase of Vmax was observed, for the two enzyme preparations, with increasing concentrations of the two activating thiols. Reactions of GSH-activated microsomal enzyme with increasing concentrations of NADPH show sigmoidal kinetics with a Hill coefficient of 2.01 +/- 0.07 at 2-4 mM GSH. Increase of the activating GSH concentrations resulted in a gradual change towards Michaelis-Menten kinetics, and a Hill coefficient of 1.08 +/- 0.03 for NADPH was calculated at 25 mM GSH. Activation of the microsomal enzyme by DTT yielded similar results except that a Hill coefficient of 1.1 was observed already at 2.5 mM DTT. Normal Michaelis-Menten kinetics were observed for HMG-CoA at all GSH concentrations. Solubilization of HMG-CoA reductase by the widely used freeze-thaw procedure abolished the cooperative pattern, and normal Michaelis-Menten kinetics with a Hill coefficient of 1.0 was observed regardless of GSH concentration. These results are compatible with a model in which HMG-CoA reductase activity is GSH-dependent, allosterically modulated under physiological hepatic conditions. In addition, the widely used assay conditions, using high DTT concentrations or employment of highly purified soluble enzyme precluded the observation of sigmoidal kinetics and the suggested model.

Photoactivated aflatoxins are mutagens to Salmonella typhimurium and bind covalently to DNA in vitro.

The mutagenesis of Salmonella typhimurium TA100 and covalent binding in vitro of photoactivated aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), aflatoxin G1 (AFG1) and aflatoxin G2 (AFG2) were investigated. Covalent binding levels of 1140, 225, 330 and 8 pmol aflatoxin per mumol nucleotide phosphate were obtained for AFB1, AFG1, AFB2 and AFG2, respectively, at 50 microM mycotoxin after 2 h of irradiation. Mutant frequencies to histidine prototrophy wre 97, 19, 49 and 0 x 10(-6) for AFB1, AFG1, AFB2 and AFG2 respectively, after 2 h irradiation at 100 microM mycotoxin in the surviving fraction of the mutagenized cultures. Toxicity to Salmonella was 0.59, 0.03, 0.31 and 0 lethal hits under the conditions specified for mutagenesis for AFB1, AFG1, AFB2 and AFG2, respectively.