Purification and Characterization of 3-Hydroxy-3-Methylglutaryl CoA Reductase from Potato Tubers

Abstract

3-Hydroxy-3-methylglutaryl coenzyme A reductase (NADPH) was solubilized by trypsin digestion from sliced potato tuber microsomes, and purified to apparent homogeneity in the absence of detergent with a recovery of 1.8%. The enzyme had a specific activity of 7,910 nmol of mevalonate formed per min per mg of protein. On molecular-sieving high-performance liquid chromatography, the activity was coincident with the single protein peak corresponding to a molecular weight of approximately 110 kDa. On SDS-polyacrylamide gel electrophoresis, the purified enzyme showed only one protein staining band corresponding to a molecular weight of approximately 55 kDa. The apparent Km value for S-HMG-CoA was 6.4 microM and that for NADPH was 25 microM.

Bacterial Expression of the Catalytic Domain of 3–Hydroxy‐3–Methylglutaryl‐CoA Reductase (Isoform HMGR1) from Arabidopsis thaliana, and Its Inactivation by Phosphorylation at Ser577 by Brassica oleracea 3‐Hydroxy‐3‐Methylglutaryl‐CoA Reductase Kinase

“…7 pM and that for (S)-HMGCoA was 8.3 2 1 .5 pM. Table 2 shows the apparent K,,, values of the HMGRlcd for both substrates compared with those obtained in previous studies performed either with purified HMGR from radi5h [35], potato [45] and Hevea brasiliensis [44] or partially purified HMGR from maize [S].…”

Differential induction and suppression of potato 3-hydroxy-3-methylglutaryl coenzyme A reductase genes in response to Phytophthora infestans and to its elicitor arachidonic acid.

“…(1985) suggest a transcriptional component to the induction of potato HMGR activity. The instability of HMGR enzymes (Kondo and Oba, 1986) precluded purification of the different isoforms and gene isolation by oligonucleotides derived from amino acid sequence information. Therefore, we determined whether a highly conserved C-termina1 region of an Arabidopsis HMGR cDNA (Learned and Fink, 1989) would hybridize with potato nuclear DNA.…”

Induction Mechanism of 3-Hydroxy-3-methylglutaryl-CoA Reductase in Potato Tuber and Sweet Potato Root Tissues

“…HMGR was solubilized from the microsomal fraction by using trypsin and puriˆed by a‹nity chromatography (2?,5?-ADP-Sepharose and HMG-CoA-HexaneAgarose) as described previously. 25) Preparation of polyclonal antibody against HMGR. Three male BALB W c mice received subcutaneous injection of 5-10 mg protein of puriˆed HMGR in 50 ml of 10 mM potassium phosphate buŠer (pH 7.5) emulsiˆed in an equal volume of complete Freund's adjuvant four times, i.e., on d 0,11, 21, and 45.…”

“…This molecular mass is close to the size of the puriˆed HMGR from the microsomal fraction of cut potato tuber tissue as previously described. 25) This monoclonal antibody also recognized a single band with a molecular mass of 64.5 kDa corresponding to SDS-solubilized fraction from microsomal suspen- The puriˆed enzyme (ng) was incubated in wells previously coated with monoclonal antibody (mAb-5A5) and then the labeled probe, 125 I-labeled monoclonal antibody (mAb-1H5) was added. The radioimmunoassay in a well was assayed as described in the text.…”

“…The enzyme is induced in potato tissue in response to cutting. 25) In this paper, we describe a method for preparing the speciˆc monoclonal and polyclonal antibodies against HMGR puriˆed from cut tissue of potato tuber, and the changes in levels of the immunoreactive proteins of HMGR (HMGR protein) in potato tubers and sweet potato roots after cutting and pathogen infection. We propose a hypothesis on the mechanism of inducing of HMGR after cutting and fungal infection in potato tubers and sweet potato roots on the basis of the immunochemical results.…”