The complete nucleotide sequence for a chicken heat shock protein (hsp108) was determined from cDNA clones isolated from hen oviduct and bursal lymphoma recombinant DNA libraries. This protein has certain biochemical similarities to the progesterone receptor, but it is clearly distinct from it. The initial cDNA clone, isolated from a chicken oviduct cDNA library, was detected by antibody screening and hybrid-selected translation [Zarucki-Schulz, T., Kulomaa, M. S., Headon, D. R., Weigel, N. L., Baez, M., Edwards, D. P., McGuire, W. L., Schrader, W. T., & O'Malley, B. W. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 6358-6362]. The earlier clones were used to screen for additional cDNAs, and cDNAs that define the entire mRNA sequence of hsp108 have been obtained. The nucleotide sequence codes for peptides present in hsp108 as determined by protein microsequencing. The 5' end of the mRNA was determined by primer extension studies. The mRNA contains a noncoding region of 101 nucleotides upstream from the predicted initiation codon. The 3' untranslated region contains 244 nucleotides beyond the termination codon, and it contains a predicted polyadenylation signal 26 nucleotides from the end of the complete cDNA. The coding region of 2385 nucleotides corresponds to a polypeptide chain of 795 amino acids, giving a molecular weight of 91,555 for the hsp108 protein. In another paper, evidence is presented that hsp108 shows a high degree of amino acid sequence homology with two heat shock proteins, hsp90 (yeast) and hsp83 (Drosophila), and is indeed inducible by heat shock [Sargan, D. R., Tsai, M.-J., & O'Malley, B. W. (1986) Biochemistry (following paper in this issue)].
The natural gene for a steroid inducible 108K heat shock protein has been isolated from a lambda genomic library prepared from hen oviduct tissue. Genomic DNA blots indicate that it exists as a single copy gene in the chick oviduct haploid genome. The 9.9 kilobase gene codes for a messenger RNA of 2733bp (21) and is split into 18 exons as established by sequence comparison of cDNA and genomic clones. The 3' end of the gene contains a repetitive element which shares homology with the CR1 family of repeats. The first exon contains both the untranslated leader and coding regions of the gene. The promoter region is rich in G + C residues (70%) and the dinucleotide CG. This 5' flanking segment contains bases similar both in sequence and location to the Goldberg-Hogness TATA homology and consensus sequence CCAAT. A consensus sequence located upstream of steroid hormone responsive chicken genes is found at -267 and on a reverse orientation at -593. The structure of this gene is of interest since the presence of introns in heat shock genes is rare in any species examined to date. Furthermore, this gene lacks the previously described heat shock promoter consensus sequence (C-GAA-TTC-G) present in other species.
A procedure for the purification of 3-hydroxy-3-methylglutaryl-coenzyme A reductase [mevalonate: NADP+ oxidoreductase (CoA-acylating), EC 1.1.1.341 solubilized from rat liver microsomes is reported. This enzyme has a specific activity of 9,000-10,000 nmol of mevalonate formed per min/mg of protein. This represents a 4100 fold purification over the activity in microsomes, and a specific activity that is approximately 20-fold greater than the highest previously reported value. The enzyme is judged to be homogeneous on the basis of sodium dodecyl sulfate/polyacrylamide disc gel electrophoresis, polyacrylamide disc gel electrophoresis, and immunoanalysis. Data are also presented that indicate the separation of enzymatically active and inactive species of 3-hydroxy-3-methylglutaryl-coenzyme A reductase on affinity chromatography on a coenzyme A column.3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase [mevalonate:NADP+ oxidoreductase (CoA-acylating); EC 1.1.1.34] catalyzes the reduction of HMG-CoA to mevalonic acid, the rate-limiting step of cholesterol biosynthesis in liver (1-3). Therefore, a number of researchers have focused their attention on the regulation of this enzyme. However, to study the modulation of HMG-CoA reductase under various physiological states a method is required to quantitate the amount of enzyme present. This can be achieved by either direct isolation of the enzyme by a reproducible purification procedure or by immunoprecipitation using monospecific antiserum to the enzyme.A number of procedures have been reported for the solubilization and partial purification of HMG-CoA reductase from the microsomal membrane (4-8). In addition, workers in three laboratories have reported the preparation of enzyme that yields only one band on immunodiffusion or sodium dodecyl sulfate (NaDodSO4) disc gel electrophoresis (9-11). However, the enzyme activities of these preparations were very low (10-516 nmol of mevalonate formed per min/mg of protein).In a previous study (12) we succeeded in purifying yeast HMG-CoA reductase to homogeneity. This enzyme had a specific activity of approximately 10,000 nmol of mevalonate formed per min/mg of protein. In this paper we report the purification of HMG-CoA reductase from rat liver by a combination of standard protein fractionation steps and coenzyme A affinity chromatography. This preparation also has a specific activity of 9,000-10,000 nmol of mevalonate formed per min/mg of protein. This value is approximately 20-fold greater than the best value previously reported.As a part of this study we also show that enzymatically active and inactive species of HMG-CoA reductase are separated by affinity chromatography. This separation suggests the possibility that cholesterol synthesis may be regulated in vvo by the interconversion of these species.MATERIALS AND METHODS Materials. Chemicals were obtained from the following sources: 3-hydroxy-3-methyl[3-'4C]glutaric acid, New England Nuclear; coenzyme A, thioester-linked agarose-hexane-coenzyme A, and dithiothrei...
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