Molecular cloning and tissue expression of human mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase

Mitochondrial 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) synthase is a constituent of the HMG-CoA pathway responsible for ketone body synthesis and one of its main regulatory points. We report the isolation and characterization of a 2058-bp cDNA from human liver. This cDNA encodes a polypeptide of 508 residues and 56,635 Da. The homology with previously reported rat mitochondrial and human cytosolic HMG-CoA synthases is 88 and 66%, respectively. mRNA levels were high in liver and colon, low in testis, heart, skeletal muscle, and kidney, and faint in pancreas.     

Activation of purified 3-hydroxy-3-methylglutaryl-CoA reductase by phospholipids

3-Hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase), the enzyme that catalyzes the rate-limiting step in cholesterol biosynthesis, has been purified by two previously reported procedures. Enzyme purified by the method of Heller, R. and Shrewsbury, M. (1976) J. Biol. Chem. 251, 3815-3822) shows up to 3-fold enhancement of activity by various types of lipid dispersions while the enzyme purified by the procedure of Tormanen et al. ((1976) Biochem. Biophys. Res. Commun. 68, 754-762) shows no activation. These results suggest that interaction with microsomal membrane lipids may be important in determining the activity of this enzyme. Analysis of bound lipid showed that enzyme prepared by the procedure of Tormanen contained at last 50 times as much phospholipid on a weight basis as enzyme prepared by Heller and Shrewsbury. Analysis of both preparations by gel-electrophoresis indicates that enzyme activities of the two comigrate, but in neither case does activity coincide with the major protein species.     

Ubiquitin-mediated regulation of 3-hydroxy-3-methylglutaryl-CoA reductase

Regulation of the sterol-synthesizing mevalonate pathway occurs in part through feedback-regulated endoplasmic reticulum degradation of 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-R). In yeast, the Hmg2p isozyme of HMG-R is regulated in this manner. We have tested the involvement of ubiquitination in the regulated degradation of Hmg2p, by using both genetic and direct biochemical approaches. Hmg2p degradation required the UBC7 gene, and Hmg2p protein was directly ubiquitinated. Hmg2p ubiquitination was dependent on UBC7 and was specific for the degraded yeast Hmg2p isozyme. Furthermore, Hmg2p ubiquitination was regulated by the mevalonate pathway in a manner consistent with regulation of Hmg2p stability. Thus, regulated ubiquitination appeared to be the mechanism by which Hmg2p stability is controlled in yeast. Finally, our data indicated that the feedback signal controlling Hmg2p ubiquitination and degradation was derived from farnesyl diphosphate, and thus implied conservation of an HMG-R degradation signal between yeast and mammals.     

Synthesis and biological activity of new 3-hydroxy-3-methylglutaryl-CoA synthase inhibitors: 2-oxetanones with a meta-substituent on the benzene ring in the side chain

Isosteric side chain analogs of 3a were synthesized and tested for inhibitory activities towards 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase and upon cholesterol production in Hep G2 cells and in mouse liver. It became clear that the lipophilic substituent on the aromatic ring and the terminal hydrophilic group in the side chain were important in the enhancement of activity. 4-[2-(3-n-Hexyloxyphenyl)ethyl]-3-hydroxy-methyl-2-oxetanone (5a) showed equivalent inhibitory activity in vivo to that of 1233A.     

Short-term effects of 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor on cholesterol and bile acid synthesis in humans

Competitive inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase improve hypercholesterolemia. However, reports about the effects of these agents on bile acid synthesis, the metabolic pathway of cholesterol, are conflicting. We studied the short-term effect on one of these agents, pravastatin, on bile acid synthesis. Six male volunteers were given 40 mg of pravastatin. Plasma mevalonate level (which reflects cholesterol synthesis) and 7 alpha-hydroxy-4-cholesten-3-one level (which reflects bile acid synthesis) were measured every 2 h for 8 h. These plasma levels were compared to those of the same volunteers without pravastatin. Plasma mevalonate level after 2 h was lower than control (3.0 +/- 1.1 ng/mL vs. 6.7 +/- 2.5, mean +/- SD; P < 0.05). This decrease continued for 8 h (2.5 +/- 0.8 vs. 5.2 +/- 1.5; P < 0.05). On the other hand, plasma 7 alpha-hydroxy-4-cholesten-3-one level did not change until after 6 h; then at 8 h it was lower than control (15.7 +/- 11.8 ng/mL vs. 24.7 +/- 16.9; P < 0.05). According to three-way layout analysis of variance, mevalonate level was influenced by both pravastatin treatment (P < 0.01) and time-course (P < 0.01). On the other hand, the 7 alpha-hydroxy-4-cholesten-3-one level was affected by both individual difference (P < 0.01) and time course (P < 0.01), but pravastatin treatment did not influence this compound. This indicates that bile acid synthesis was influenced by pravastatin, although cholesterol synthesis was inhibited. The short-term inhibition of cholesterol synthesis did not affect bile acid synthesis.     

Sequence determinants for regulated degradation of yeast 3-hydroxy-3-methylglutaryl-CoA reductase, an integral endoplasmic reticulum membrane protein

The degradation rate of 3-hydroxy-3-methylglutaryl CoA reductase (HMG-R), a key enzyme of the mevalonate pathway, is regulated through a feedback mechanism by the mevalonate pathway. To discover the intrinsic determinants involved in the regulated degradation of the yeast HMG-R isozyme Hmg2p, we replaced small regions of the Hmg2p transmembrane domain with the corresponding regions from the other, stable yeast HMG-R isozyme Hmg1p. When the first 26 amino acids of Hmg2p were replaced with the same region from Hmg1p, Hmg2p was stabilized. The stability of this mutant was not due to mislocalization, but rather to an inability to be recognized for degradation. When amino acid residues 27-54 of Hmg2p were replaced with those from Hmg1p, the mutant was still degraded, but its degradation rate was poorly regulated. The degradation of this mutant was still dependent on the first 26 amino acid residues and on the function of the HRD genes. These mutants showed altered ubiquitination levels that were well correlated with their degradative phenotypes. Neither determinant was sufficient to impart regulated degradation to Hmg1p. These studies provide evidence that there are sequence determinants in Hmg2p necessary for degradation and optimal regulation, and that independent processes may be involved in Hmg2p degradation and its regulation.     

Inactivation of pyridoxal phosphate dependent enzymes by mono- and polyhaloalanines

beta,beta-Dichloro- and beta,beta,beta-trifluoroalanine irreversibly inactivate a number of pyridoxal phosphate dependent enzymes which catalyze beta- or gamma-elimination reactions. The inactivation is time dependent and the rate of inactivation is first order in enzyme concentration. This suggests that inactivation is due to covalent modification of the enzyme by a species generated at the active site from the polyhaloalanine (i.e., suicide inactivation). Monohaloalanines are substrates and do not inactivate. For gamma-cystathionase, covalent and stoichiometric attachment of [1-14C]beta,beta,beta-trifluoroalanine was shown. It is proposed that the mechanism of inactivation involves Schiff base formation between inactivator and enzyme-bound pyridoxal and subsequent elimination of HC1 from dichloroalanine or HF from trifluoroalanine. This results in the formation of a beta-halo-alpha,beta unsaturated imine, an activated Michael acceptor. Michael addition of a nucleophile at the active site leads to covalent labeling of the enzyme and inactivation. Alanine racemase is also inactivated by the two polyhaloalanines. Glutamate-pyruvate and gultamate-oxaloacetate transaminase are inactivated by monohaloalanines but not by polyhaloalanines.     

Fluvastatin, an inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase, scavenges free radicals and inhibits lipid peroxidation in rat liver microsomes

A total 17,121 patients with pancreatic cancer have been collected by the Pancreatic Cancer Registration Committee of the Japan Pancreas Society. Significant differences in the postoperative prognosis were observed between patients with tumor limited to the pancreas and with tumor extending to surrounding tissues or adjacent organs. The lymph node metastases and distant metastases were definitive factors on the prognosis after resection. It might was possible for the subdivision of regional lymph nodes as Japanese classification to provide the Stage classification. Further development of the TNM system is necessary for assessing the outcome of most advanced cancers.     

Comparison of cytochrome P-450-dependent metabolism and drug interactions of the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors lovastatin and pravastatin in the liver

The mannose/glucose-binding Dolichos lablab lectin (designated DLL) has been purified from seeds of Dolichos lablab (hyacinth bean) to electrophoretic homogeneity by affinity chromatography on an ovalbumin-Sepharose 4B column. The purified lectin gave a single symmetric protein peak with an apparent molecular mass of 67 kDa on gel filtration chromatography, and five bands ranging from 10 kDa to 22 kDa upon SDS-PAGE. N-Terminal sequence analysis of these bands revealed subunit heterogeneity due to posttranslational proteolytic truncation at different sites mostly at the carboxyl terminus. The carbohydrate binding properties of the purified lectin were investigated by three different approaches: hemagglutination inhibition assay, quantitative precipitation inhibition assay, and ELISA. On the basis of these studies, it is concluded that the Dolichos lablab lectin has neither an extended carbohydrate combining site, nor a hydrophobic binding site adjacent to it. The carbohydrate combining site of DLL appears to most effectively accommodate a nonreducing terminal alpha-d-mannosyl unit, and to be complementary to the C-3, C-4, and C-6 equatorial hydroxyl groups of alpha-d-mannopyranosyl and alpha-d-glucopyranosyl residues. DLL strongly precipitates murine IgM but not IgG, and the recent finding that this lectin interacts specifically with NIH 3T3 fibroblasts transfected with the Flt3 tyrosine kinase receptor and preserves human cord blood stem cells and progenitors in a quiescent state for prolonged periods in culture, make this lectin a valuable tool in biomedical research.     

Isolation and characterization of a cDNA encoding Arabidopsis thaliana 3-hydroxy-3-methylglutaryl-coenzyme A synthase

An 1.7-kb Arabidopsis thaliana (At) cDNA was isolated by complementation of a bap1 mutation affecting the transport of branched-chain amino acids (aa) in the yeast Saccharomyces cerevisiae. The determination of the nucleotide (nt) sequence revealed an open reading frame of 1383 nt which may encode a protein of 461 aa with a predicted molecular mass of 51,038 Da. The deduced aa sequence exhibited strong similarities with mammalian 3-hydroxy-3-methylglutaryl-coenzyme A synthase (HMGS) sequences. Although former biochemical studies have suggested that acetoacetyl-coenzyme A thiolase (AACT) and HMGS activities were carried by a single protein in plants, complementation studies and measurements of enzymatic activities clearly showed that the At HMGS is devoid of AACT activity.     

Inactivation of rabbit muscle glycogen synthase by glycogen synthase kinase-3. Dominant role of the phosphorylation of Ser-640 (site-3a)

Rabbit skeletal muscle glycogen synthase, a rate-limiting enzyme for glycogen biosynthesis, is regulated by multisite phosphorylation. The protein kinase glycogen synthase kinase 3 (GSK-3) phosphorylates 4 Ser residues (Ser-640, Ser-644, Ser-648, and Ser-652; also known as sites 3a, 3b, 3c, and 4, respectively) at the COOH terminus of the subunit. Phosphorylation of these sites by GSK-3 is sequential, from COOH- to NH2-terminal, and is wholly dependent on prior phosphorylation by casein kinase II at Ser-656 (site 5). Expression in Escherichia coli was used to generate mutant forms of glycogen synthase, S640A, S644A, and S648A, in which site 3a, site 3b, or site 3c was changed to Ala, respectively. The purified enzymes had -/+ glucose-6-P activity ratios in the range of 0.8-0.9. Phosphorylation by casein kinase II and GSK-3 gave results consistent with the model of obligate sequential action of GSK-3. Phosphorylation at site 5, sites 4 + 5, or sites 3c + 4 + 5 had no measurable effect on activity. When sites 3b + 3c + 4 + 5 were phosphorylated, modest inactivation resulted. Additional phosphorylation at site 3a, however, was potently inactivating, reducing the -/+ glucose-6-P activity ratio to 0.1 and increasing the glucose-6-P concentration needed for half-maximal activation by an order of magnitude. Introduction of each additional phosphate, in the order site 4, 3c, 3b, and 3a, caused an incremental reduction in the mobility of the subunit when analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The results of this study demonstrate that GSK-3 phosphorylation of site 3a (Ser-640), and to a lesser extent, site 3b, correlates with inactivation of glycogen synthase by GSK-3. Evidence is also presented for an allosteric mechanism of inactivation whereby modification of one subunit influences the activity state of adjacent subunits.     

The active site of hamster 3-hydroxy-3-methylglutaryl-CoA reductase resides at the subunit interface and incorporates catalytically essential acidic residues from separate polypeptides

We employed site-directed mutagenesis based on sequence comparisons and characterization of purified mutant enzymes to identify Glu558 and Asp766 of Syrian hamster 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.34) as essential for catalysis. Mutant enzymes E558D, E558Q, and D766N had wild-type Km values for (S)-HMG-CoA and NADPH, but exhibited less than 0.5% of the wild-type catalytic activity. The inactive mutant polypeptides E558Q and D766N nevertheless can associate to generate an active enzyme. In vitro, 6% of the wild-type activity was observed when mutant polypeptides E558D and D766N were mixed in the absence of chaotropic agents. When mutant polypeptides E558Q and D766N were co-expressed in Escherichia coli, the resulting purified enzyme had 25% of wild-type activity. Hamster HMG-CoA reductase thus is a two-site, dimeric enzyme whose subunits associate to form an active site in which each monomer contributes at least one residue (e.g. Glu558 from one monomer and Asp766 from the other). The wild-type enzyme behaves as a dimer during size exclusion chromatography and has one HMG-CoA binding site per monomer. Syrian hamster HMG-CoA reductase thus appears to be a homodimer with two active sites which are located at the subunit interface.     

Inactivation of glyceraldehyde 3-phosphate dehydrogenase by sugars, prednisolone-21-hemisuccinate, cyanate and other small molecules

Diabetes, diarrhoea, renal failure and glucocorticoid therapy have all been identified as independent risk factors for cataract. Increased post-translational modification of proteins, leading to inactivation of enzymes and induction of conformational changes within proteins could result in lens opacification and cataract. Aspirin has been associated with many beneficial effects, including protection against cataract, in-vivo. alpha-Crystallin has been shown to act as a molecular chaperone in-vitro. This lenticular protein prevented the thermal aggregation of other lens proteins in-vitro and has sequence and functional homology with the small heat shock proteins. Glyceraldehyde 3-phosphate dehydrogenase (GAP-DH) is constitutively expressed in tissues and is susceptible to chemical modification in-vivo. In-vitro incubations of GAP-DH with sugars, cyanate and prednisolone-21-hemisuccinate, all led to significant loss of enzyme activity with time in two buffer systems. Rapid inactivation occurred when GAP-DH was incubated with fructose 6-phosphate or prednisolone-21-hemisuccinate. Slower inactivation was observed when GAP-DH was incubated with fructose, glucose 6-phosphate or potassium cyanate. Glucose did not inactivate GAP-DH under the conditions of our experiments. Aspirin and ibuprofen were shown to inactivate GAP-DH very rapidly in-vitro. Bovine lenticular alpha-crystallin conferred no protection against GAP-DH inactivation. This is the first occasion that alpha-crystallin has been demonstrated to be unable to protect against inactivation in our chemical enzyme inactivation system. This may have implications for the susceptibility of lenticular GAP-DH to post-translational inactivation.     

3-Hydroxy-3-methylglutaryl-CoA reductase gene of Gibberella fujikuroi: isolation and characterization

PURPOSE: Cardiovascular disease is a major cause of morbidity and death in non-insulin-dependent diabetes mellitus (NIDDM). While hyperglycemia is clearly related to diabetic microvascular complications, it contribution to large-vessel atherosclerosis is controversial. PATIENTS AND METHODS: We performed an analysis of the association between glycemic control and prevalent cardiovascular disease in 1,539 participants in the NIDDM Patient Outcomes Research Team study who were under usual care in a health maintenance organization. Prevalent cardiovascular disease and its risk factors were identified by self-administered questionnaire. Cardiovascular disease was defined by the presence of coronary heart disease, peripheral vascular disease, and/or cerebrovascular disease. Glycohemoglobin and lipid levels were obtained from a computerized laboratory database. RESULTS: The mean age of participants was 63 years (range 31 to 91); 51% were women. The mean duration of NIDDM was 9 years (range < 1 to 50), 35% took insulin, and 48% took sulfonylureas. Mean glycohemoglobin was 10.6%. Sixty percent had hypertension, 16% currently smoked cigarettes, and the mean total high-density lipoprotein (HDL) cholesterol ratio was 5.7. Fifty-one percent had cardiovascular disease. Cardiovascular disease prevalence remained constant across increasing quartiles of glycohemoglobin for both men and women. In contrast, prevalent cardiovascular disease was associated with established cardiovascular disease risk factors including age (67 versus 59 years, P < 0.0001), hypertension (66% versus 54%, P < 0.0001), current cigarette smoking (17% versus 13%, P < 0.005), and total/HDL cholesterol ratio (5.9 versus 5.6, P < 0.005). Cardiovascular disease was also associated with duration of NIDDM (11 versus 8 years, P < 0.0001). In multiple logistic regression analysis controlling for established cardiovascular disease risk factors and diabetes duration and therapy, glycohemoglobin remained unassociated with cardiovascular disease. CONCLUSIONS: Glycemic control is not associated with prevalent cardiovascular disease in this large population of individuals with NIDDM. Conventional cardiovascular disease risk factors are independently associated with cardiovascular disease and be a more promising focus for clinical intervention to reduce atherosclerotic complications in NIDDM.     

Inactivation of glyceraldehyde-3-phosphate dehydrogenase by ferrylmyoglobin

This article describes the development and psychometric evaluation of self-report measurement instruments in (nursing) research. The aim of this study is to gain more insight into, and understanding of the use of such instruments. To be more specific, this paper deals with: (1) What is a self-report measurement instrument?; (2) How to develop such an instrument?; (3) What is its psychometric quality in terms of validity and reliability?; (4) How to analyze an instrument statistically?; (5) Which are the pros en cons of a self-report measurement instrument in general?; and (6) Where do we find examples of good measurement instruments? These six questions will be answered with the help of several practical research examples. The article concludes with a few suggestions for literature concerning existing measurement instruments and their psychometric qualities.     

Purification of amylases and other enzymes by a forced-affinity chromatography method

An affinity matrix of soluble starch gel was prepared by cross-linking catalyzed by epichlorohydrin. The elution pattern of Taka-amylase A (TAA) indicated that the amount of enzyme bound to the starch gel column increased with increases in the ammonium sulfate (AmS) concentration in the equilibrating buffer. TAA had an affinity for the gels with a starch structure, and desorbed from the column with the buffer containing no AmS. Bound TAA was also eluted with starch and cyclodextrin solution. The AmS stimulative effect was partially replaced by polyethylene glycol and surfactants. Besides TAA, various, other amylases bound satisfactorily to the starch gel. Moreover, affinity purifications of dextranase, cellulase, and pectinase were done by gels with dextran, cellulose, and pectin structures, respectively. By the aid of forced effects of AmS, various carbohydrases could be purified by the affinity gels of polysaccharide linked by epichlorohydrin.     

Multiple DNA elements for sterol regulatory element-binding protein and NF-Y are responsible for sterol-regulated transcription of the genes for human 3-hydroxy-3-methylglutaryl coenzyme A synthase and squalene synthase

Plasmin triggers a strong metabolic activation in human platelets, leading to shape change and granule exocytosis. However, its capacity to induce cell aggregation remains discussed and, when observed, this aggregation is preceded by a remarkable lag phase. We have thus investigated the effect of plasmin on the adhesive proteins which can be secreted by isolated platelets and mediate cell-to-cell interactions, but are also substrates for the enzyme. Immunoblot analysis of fibrinogen (Fg), thrombospondin-1 (TSP-1), fibronectin (Fn) and von Willebrand factor (vWf) was performed on extracts of platelets exposed under stirring to increasing concentrations of plasmin for up to 10 min at 37 degrees C. Under conditions leading to formation of large aggregates, Fg, Fn and TSP-1 are extensively degraded concomitantly with their secretion, and readily lost from the surface of aggregated cells. Part of the monomers in the platelet vWf are cleaved during secretion into two main fragments with Mr approximately 180,000 and approximately 145,000. However, multimer distribution analysis shows only a slight decrease in the very high molecular weight multimers, and most of the fragmented as well as intact vWf remains associated with the platelet surface when aggregation is maximal. That indeed vWf largely supports plasmin-induced aggregation is suggested by the observation that platelets from a patient with type 3 von Willebrand's disease, who totally lacks vWf, show little aggregation in response to the enzyme. Finally, plasmin-induced aggregation can be totally inhibited by antagonists of the alpha(IIb)beta3 integrin. The present study thus indicates a major role for secreted vWf in platelet aggregation induced by plasmin, through its likely interaction with the multifunctional receptor alpha(IIb)beta3.     

Inactivation and conformational changes of fatty acid synthase from chicken liver during unfolding by sodium dodecyl sulfate

Fatty acid synthase is an important enzyme participating in energy metabolism in vivo. The inactivation and conformational changes of the multifunctional fatty acid synthase from chicken liver in SDS solutions have been studied. The results show that the denaturation of this multifunctional enzyme by SDS occurred in three stages. At low concentrations of SDS (less than 0.15 mM) the enzyme was completely inactivated with regard to the overall reaction. For each component of the enzyme, the loss of activity occurred at higher concentrations of SDS. Significant conformational changes (as indicated by the changes of the intrinsic fluorescence emission and the ultraviolet difference spectra) occurred at higher concentrations of SDS. Increasing the SDS concentration caused only slight changes of the CD spectra, indicating that SDS had no significant effect on the secondary structure of the enzyme. The results suggest that the active sites of the multifunctional fatty acid synthase display more conformational flexibility than the enzyme molecule as a whole.