共查询到20条相似文献,搜索用时 0 毫秒
1.
A protein that binds to the sterol regulatory region of the hamster promoter for 3-hydroxy-3-methylglutaryl-coenzyme A reductase has been identified. All of the DNA bases crucial to the binding of this protein were previously shown to be essential for sterol regulation of the intact promoter in cultured cells. This low abundance protein, called Red 25, has been purified from nuclear extracts of hamster liver by a series of standard chromatographic techniques coupled with a DNA affinity step. Its size has been estimated as approximately 42 kDa by gel electrophoresis, size exclusion chromatography, and protein-DNA cross-linking studies. Furthermore, it binds to its target site with a Kd = 6 x 10(-11) M. Red 25 does not bind to the sterol regulatory regions of the LDL receptor or 3-hydroxy-3-methylglutaryl-coenzyme A synthase. This is consistent with recent studies that show there is a unique site for sterol regulation in the reductase promoter. The identification and purification of this protein represents a significant step in the study of feedback regulation by cholesterol. 相似文献
2.
3.
Renu A. Heller Pamela Hoy Patricia P. Jones 《Biochemical and biophysical research communications》1982,106(2):412-421
This report describes the development of a series of monoclonal antibodies to rat liver 3-Hydroxy-3-methylglutaryl-CoA reductase (HMGR). Sera from hybridoma tumor-bearing mice were used to remove and characterize HMGR activity from a mixture of rat liver proteins. Two IgG2 monoclonal antibodies removed separately greater than 80% HMGR activity while non-immune mouse or negative hybridoma-derived sera were ineffective. Radiolabeled immunoprecipitates of enzyme preparations resolved in one- and two-dimensional SDS-PAGE showed two predominant subunits at Mr 52,000 and 54,000. Our results indicate that in these preparations of rat liver proteins HMGR exists as a heteropolymer with at least two distinct subunits of different molecular weights. 相似文献
4.
Subcellular localization of Arabidopsis 3-hydroxy-3-methylglutaryl-coenzyme A reductase 总被引:2,自引:0,他引:2
下载免费PDF全文
![点击此处可从《Plant physiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Leivar P González VM Castel S Trelease RN López-Iglesias C Arró M Boronat A Campos N Ferrer A Fernàndez-Busquets X 《Plant physiology》2005,137(1):57-69
Plants produce diverse isoprenoids, which are synthesized in plastids, mitochondria, endoplasmic reticulum (ER), and the nonorganellar cytoplasm. 3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) catalyzes the synthesis of mevalonate, a rate-limiting step in the cytoplasmic pathway. Several branches of the pathway lead to the synthesis of structurally and functionally varied, yet essential, isoprenoids. Several HMGR isoforms have been identified in all plants examined. Studies based on gene expression and on fractionation of enzyme activity suggested that subcellular compartmentalization of HMGR is an important intracellular channeling mechanism for the production of the specific classes of isoprenoids. Plant HMGR has been shown previously to insert in vitro into the membrane of microsomal vesicles, but the final in vivo subcellular localization(s) remains controversial. To address the latter in Arabidopsis (Arabidopsis thaliana) cells, we conducted a multipronged microscopy and cell fractionation approach that included imaging of chimeric HMGR green fluorescent protein localizations in transiently transformed cell leaves, immunofluorescence confocal microscopy in wild-type and stably transformed seedlings, immunogold electron microscopy examinations of endogenous HMGR in seedling cotyledons, and sucrose density gradient analyses of HMGR-containing organelles. Taken together, the results reveal that endogenous Arabidopsis HMGR is localized at steady state within ER as expected, but surprisingly also predominantly within spherical, vesicular structures that range from 0.2- to 0.6-microm diameter, located in the cytoplasm and within the central vacuole in differentiated cotyledon cells. The N-terminal region, including the transmembrane domain of HMGR, was found to be necessary and sufficient for directing HMGR to ER and the spherical structures. It is believed, although not directly demonstrated, that these vesicle-like structures are derived from segments of HMGR-ER. Nevertheless, they represent a previously undescribed subcellular compartment likely capable of synthesizing mevalonate, which provides new evidence for multiorganelle compartmentalization of the isoprenoid biosynthetic pathways in plants. 相似文献
5.
P Louis-Flamberg C E Peishoff D L Bryan J Leber J D Elliott B W Metcalf R J Mayer 《Biochemistry》1990,29(17):4115-4120
The mechanism of slow binding inhibition of 3-hydroxy-3-methylglutaryl- coenzyme A reductase by lovastatin, fluindostatin, and related compounds was studied. Several of these compounds, including lovastatin, were found to be slow binding, while other less potent inhibitors were not. From a comparison of kinetic parameters obtained by steady-state measurements and progress curve analysis, it was concluded that the slow binding inhibitors bind by a mechanism which is more accurately described by biphasic binding than by single-step binding. The overall association rates of the slow binding inhibitors range from 1 x 10(6) to 4 x 10(-7) M-1 s-1, and the dissociation rates are in the range of 10(-3) s-1. The structures of slow binding and reversible inhibitors were compared by using molecular modeling methods. From these comparisons, it was proposed that the slow binding and very potent inhibition of, for instance, lovastatin, is not simply a result of binding of a transition state or reaction intermediate analogue. The various lipophilic groups of the inhibitors that do not seem to be related to structural features of the substrate may also play a crucial role in determining the mechanism of binding of HMGR inhibitors. 相似文献
6.
L Gondet T Weber P Maillot-Vernier P Benveniste T J Bach 《Biochemical and biophysical research communications》1992,186(2):888-893
In a tobacco mutant callus, containing up to tenfold more sterols than the wild-type genotype, HMG-CoA reductase activity is increased by a factor of approximately three, as is the case in mutant seedlings and plants. The rate of HMG-CoA synthesis from acetyl-CoA by the coupled enzyme system acetoacetyl-CoA thiolase/HMG-CoA synthase, as well as its conversion to acetyl-CoA plus acetoacetate by action of HMG-CoA lyase are not affected. These results confirm the key-regulating role of HMG-CoA reductase in sterol biosynthesis, which seems not to be confined only to the animal kingdom, but can also be extended to plants. 相似文献
7.
Both eukaryotes and archaebacteria use 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase to synthesize mevalonate, which eukaryotes employ in the production of sterols and archaebacteria need for the isoprenoid side chains of their unique and characteristic lipids. The drug mevinolin inhibits HMG-CoA reductase in eukaryotes and in the halophilic archaebacteria, and we have used a spontaneous mutation to mevinolin resistance in the construction of a selectable shuttle vector for Haloferax volcanii. Sequence analysis shows that this resistance determinant encodes an HMG-CoA reductase very like its eukaryotic homologs, but sharing with the one sequenced eubacterial HMG-CoA reductase (that of Pseudomonas mevalonii) few residues other than those common to all HMG-CoA reductases. Characterization of several spontaneous mevinolin-resistant mutants reveals that they are of two sorts: amplifications of the HMG-CoA reductase gene with varying amounts of flanking sequence, and point mutants upstream of the HMG-CoA reductase coding region. We compared sequence and expression of a mutant gene of the latter class to those of the wild-type gene. The point mutation found affects the TATA box-like "distal promoter element," results (like gene amplification) in resistance through the synthesis of excess gene product, and provides the first true genetic definition of an archaebacterial promoter. 相似文献
8.
9.
10.
11.
Evidence that barley 3-hydroxy-3-methylglutaryl-coenzyme a reductase kinase is a member of the sucrose nonfermenting-1-related protein kinase family. 总被引:3,自引:1,他引:2
下载免费PDF全文
![点击此处可从《Plant physiology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
J H Barker S P Slocombe K L Ball D G Hardie P R Shewry N G Halford 《Plant physiology》1996,112(3):1141-1149
A protein kinase was partially purified from barley (Hordeum vulgare L. cv Sundance) endosperm by ammonium sulfate fractionation, followed by ion-exchange, Reactive Blue, Mono-Q, and phosphocellulose chromatography. It was shown to phosphorylate Arabidopsis 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase and a synthetic peptide that was shown previously to act as a substrate for HMG-CoA reductase kinase purified from cauliflower, confirming it to be barley HMG-CoA reductase kinase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the partially purified preparation showed the presence of a polypeptide with an approximate relative molecular weight (M(r)) of 60,000, which is the size predicted for the barley sucrose nonfermenting-1 (SNF1)-related protein kinases BKIN2 and BKIN12. Antisera were raised to a rye (Secale cereale L.) SNF1-related protein kinase (RKIN1) expressed in Escherichia coli as a fusion with maltose-binding protein and to a synthetic peptide with a sequence that is conserved in, and specific to, plant members of the SNF1-related protein kinase family. The maltose-binding protein-RKIN1 fusion protein antiserum recognized a doublet of polypeptides with an approximate M(r), of 60,000 in crude endosperm extracts and a single polypeptide in root extracts, which co-migrated with the smaller polypeptide in the endosperm doublet. Both antisera recognized a polypeptide with an approximate M(r) of 60,000 in the partially purified protein kinase preparation, suggesting strongly that barley HMG-CoA reductase kinase is a member of the SNF1-related protein kinase family. 相似文献
12.
Characteristics of rat liver microsomal 3-hydroxy-3-methylglutaryl-coenzyme A reductase. 总被引:4,自引:0,他引:4
下载免费PDF全文
![点击此处可从《The Biochemical journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
A procedure for the preparation of rat liver microsomal fractions essentially devoid of contaminating lysosomes is described. When this preparation was examined by immunoblotting with a rabbit antiserum to rat 3-hydroxy-3-methylglutaryl-CoA reductase, a single band corresponding to an Mr of 100000 was observed. No evidence was found for glycosylation of rat liver-3-hydroxy-3-methylglutaryl-CoA reductase. Native rat liver microsomal 3-hydroxy-3-methylglutaryl-CoA reductase differs from the purified proteolytically modified species in that it displays allosteric kinetics towards NADPH. 相似文献
13.
We have studied the regulated degradation of the enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase within the endoplasmic reticulum in cells permeabilized with digitonin. Using Chinese hamster ovary cells transfected with a plasmid encoding HMGal, a chimeric protein containing the membrane domain of HMG-CoA reductase coupled to beta-galactosidase, we have demonstrated mevalonate and sterol-stimulated loss of beta-galactosidase activity. In pulse-chase experiments we have demonstrated mevalonate-stimulated degradation of both HMGal and HMG-CoA reductase. The rate of mevalonate-stimulated degradation observed in permeabilized cells tends to be slightly slower than that observed in intact cells treated with mevalonate and is dependent upon incubation of cells with mevalonate prior to permeabilization. The degradation process measured in this report extends a previous report of HMG-CoA reductase degradation in digitonin-permeabilized cells (Leonard, D. A., and Chen, H. W. (1987) J. Biol. Chem. 262, 7914-7919) by mimicking key physiological features of the in vivo process, including: stimulation by regulatory molecules, specifically mevalonate and sterols; inhibition by cycloheximide; and inhibition by an inhibitor of neutral cysteine proteases. 相似文献
14.
Heise N Opperdoes FR 《Zeitschrift für Naturforschung. C, Journal of biosciences》2000,55(5-6):473-477
Contrary to Leishmania spp. and Trypanosoma cruzi, Trypanosoma brucei bloodstream forms do not synthesise their own sterols but take these compounds in the form of cholesterol directly from the mammalian host. However, procyclic insect stages synthesise ergosterol rather than cholesterol. Here the sub-cellular localisation of the first committed enzyme of this pathway of isoprenoid synthesis 3-hydroxy-3-methylglutaryl-coenzyme A reductase in T. brucei procyclics (0.9 nmol x min(-1) x mg(-1) protein) was carried out using both cell-fractionation by isopycnic centrifugation and digitonin-titration experiments. The majority of the NADP+-linked 3-hydroxy-3-methylglutaryl-coenzyme A reductase is a soluble enzyme present in the mitochondrial matrix with some additional membrane-associated activity in glycosomes and possibly in the endoplasmic reticulum. It is suggested that the active metabolism of threonine and/or leucine as preferred 2-carbon source for the incorporation of acetyl units into lipids and/or sterols in the mitochondrion of T. brucei procyclics is the explanation for a high 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity in these protozoan organelles. 相似文献
15.
Enterococcus faecalis acetoacetyl-coenzyme A thiolase/3-hydroxy-3-methylglutaryl-coenzyme A reductase, a dual-function protein of isopentenyl diphosphate biosynthesis
下载免费PDF全文
![点击此处可从《Journal of bacteriology》网站下载免费的PDF全文](/ch/ext_images/free.gif)
Hedl M Sutherlin A Wilding EI Mazzulla M McDevitt D Lane P Burgner JW Lehnbeuter KR Stauffacher CV Gwynn MN Rodwell VW 《Journal of bacteriology》2002,184(8):2116-2122
Many bacteria employ the nonmevalonate pathway for synthesis of isopentenyl diphosphate, the monomer unit for isoprenoid biosynthesis. However, gram-positive cocci exclusively use the mevalonate pathway, which is essential for their growth (E. I. Wilding et al., J. Bacteriol. 182:4319-4327, 2000). Enzymes of the mevalonate pathway are thus potential targets for drug intervention. Uniquely, the enterococci possess a single open reading frame, mvaE, that appears to encode two enzymes of the mevalonate pathway, acetoacetyl-coenzyme A thiolase and 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. Western blotting revealed that the mvaE gene product is a single polypeptide in Enterococcus faecalis, Enterococcus faecium, and Enterococcus hirae. The mvaE gene was cloned from E. faecalis and was expressed with an N-terminal His tag in Escherichia coli. The gene product was then purified by nickel affinity chromatography. As predicted, the 86.5-kDa mvaE gene product catalyzed both the acetoacetyl-CoA thiolase and HMG-CoA reductase reactions. Temperature optima, DeltaH(a) and K(m) values, and pH optima were determined for both activities. Kinetic studies of acetoacetyl-CoA thiolase implicated a ping-pong mechanism. CoA acted as an inhibitor competitive with acetyl-CoA. A millimolar K(i) for a statin drug confirmed that E. faecalis HMG-CoA reductase is a class II enzyme. The oxidoreductant was NADP(H). A role for an active-site histidine during the first redox step of the HMG-CoA, reductase reaction was suggested by the ability of diethylpyrocarbonate to block formation of mevalonate from HMG-CoA, but not from mevaldehyde. Sequence comparisons with other HMG-CoA reductases suggest that the essential active-site histidine is His756. The mvaE gene product represents the first example of an HMG-CoA reductase fused to another enzyme. 相似文献
16.
We identify His381 of Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase as the basic residue functional in catalysis. The catalytic domain of 20 HMG-CoA reductases contains a single conserved histidine (His381 of the P. mevalonii enzyme). Diethyl pyrocarbonate inactivated the P. mevalonii enzyme, and hydroxylamine partially restored activity. We changed His381 to alanine, lysine, asparagine, and glutamine. The mutant proteins were overexpressed, purified to homogeneity, and characterized. His381 mutant enzymes were not inactivated by diethyl pyrocarbonate. All four mutant enzymes exhibited wild-type crystal morphology and chromatographed on substrate affinity supports like wild-type enzyme. The mutant enzymes had low catalytic activity (Vmax 0.06-0.5% that of wild-type enzyme), but Km values approximated those for wild-type enzyme. For wild-type enzyme and mutant enzymes H381A, H381N, and H381Q, Km values at pH 8.1 were 0.45, 0.27, 3.7, and 0.71 mM [(R,S)-mevalonate]; 0.05, 0.03, 0.20, and 0.11 mM [coenzyme A]; 0.22, 0.14, 0.81, and 0.62 mM [NAD+]. Km values at pH 11 for wild-type enzyme and mutant enzyme H381K were 0.32 and 0.75 mM [(R,S)-mevalonate]; 0.24 and 0.50 mM [coenzyme A]; 0.15 and 1.23 mM [NAD+]. Both pK values for the enzyme-substrate complex increased relative to wild-type enzyme (by 1-2.5 pH units for pK1 and by 0.5-1.3 pH units for pK2). For mutant enzyme H381K, the pK1 of 10.2 is consistent with lysine acting as a general base at high pH. His381 of P. mevalonii HMG-CoA reductase, and consequently the histidine of the consensus Leu-Val-Lys-Ser-His-Met-Xaa-Xaa-Asn-Arg-Ser motif of the catalytic domain of eukaryotic HMG-CoA reductases, thus is the general base functional in catalysis. 相似文献
17.
Ignacio E. Maldonado-Mendoza Ronald J. Burnett Melina Lòpez-Meyer Craig L. Nessler 《Plant Cell, Tissue and Organ Culture》1994,38(2-3):351-356
HMGR (3-hydroxy-3-methylglutaryl-coenzyme A reductase; E.C.1.1.1.34) supplies mevalonate for the synthesis of many plant primary and secondary metabolites, including the terpenoid component of indole alkaloids. Suspension cultures of Camptotheca acuminata and Catharanthus roseus, two species valued for their anticancer indole alkaloids, were treated with the elicitation signal transducer methyl jasmonate (MeJA). RNA gel blot analysis from MeJA treated cultures showed a transient suppression of HMGR mRNA, followed by an induction in HMGR message. Leaf disks from transgenic tobacco plants containing a chimeric hmgl::GUS construct were also treated with MeJA and showed a dose dependent suppression of wound-inducible GUS activity. The suppression of the wound response by MeJA was limited to the first 4 h post-wounding, after which time MeJA application had no effect. The results are discussed in relation to the differential regulation of HMGR isogenes in higher plants.Abbreviations GUS
-glucuronidase
-
hmg
gene of hmgr
- HMGR
3-hydroxy-3-methylglutaryl-coenzyme A reductase
- JA
jasmonic acid
- MeJA
methyl jasmonate
- MUG
methylumbelliferyl--d-glucuronide
- TDC
tryptophan decarboxylase
- SDS
sodium dodecyl sulfate
- SS
strictosidine synthase 相似文献
18.
Regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity in mouse peritoneal macrophages.
下载免费PDF全文
![点击此处可从《The Biochemical journal》网站下载免费的PDF全文](/ch/ext_images/free.gif)
B Angelin 《The Biochemical journal》1988,255(2):529-534
The lipoprotein-mediated regulation of 3-hydroxy-3-methylglutaryl-(HMG-) CoA reductase in cultured mouse peritoneal macrophages has been investigated. In contrast to what has been reported for other cells, HMG-CoA reductase activity is not suppressed by normal serum or by normal low density lipoproteins (LDL) from humans or dogs. Suppression of reductase activity occurred when cells were cultured in the presence of beta-migrating very low density lipoproteins (beta-VLDL) or LDL from hypercholesterolaemic dogs, or LDL modified by acetoacetylation. Human beta-VLDL from an atypical type III hyperlipoproteinaemic patient was also effective, as was apolipoprotein (apo) E-containing high density lipoproteins (HDL) from cholesterol-fed dogs (apo-E HDLc). The results indicate that cholesterol biosynthesis in mouse peritoneal macrophages is regulated by lipoprotein cholesterol entering via receptor-mediated endocytosis. Normal LDL were not effective because of the poor binding and uptake of these lipoproteins by the apo-B, E (LDL) receptor. Only beta-VLDL, apo-E HDLc, and hypercholesterolaemic LDL were avidly taken up by this receptor and were able to suppress HMG-CoA reductase. Acetoacetylated LDL were internalized via the acetyl-LDL (scavenger) receptor. Thus, mouse macrophages differ from human fibroblasts and smooth muscle cells in their physiological regulation of cholesterogenesis. 相似文献
19.
20.
A protein with an estimated subunit mass of 19 kDa was isolated and purified from perfused rat liver cytosol. This protein activates hydroxymethylglutaryl-coenzyme A (HMG-CoA) reductase (NADPH) (EC 1.1.1.34), the rate-limiting enzyme in the cholesterol biosynthetic pathway. The activation process by this HMG-CoA reductase activating protein (RAP) is time-dependent and requires NADPH. Maximal activity of HMG-CoA reductase induced by RAP is comparable to that obtained in the presence of thiols, such as GSH, and can exceed 100-fold the activity obtained when thiols are omitted. Purified RAP lacks ability to reduce 5,5'-dithiobis-(2-nitrobenzoic acid). RAP was purified to homogeneity utilizing DEAE- and phenyl-Sepharose CL-4B column chromatography. The purified RAP migrates as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and shows multiple interconvertible aggregational forms on native polyacrylamide gel electrophoresis. A monospecific antibody against RAP was prepared by immunization of hens and extracted from either their egg yolks or serum. The catalytic activity of RAP might be responsible for the physiological activation of HMG-CoA reductase and regulation of its activity. 相似文献