Abstract
Assay of HMG-CoA reductase kinase activity requires HMG-CoA reductase (reductase, E.C. 1.1.1.34) free of associated reductase kinase. Microsomal reductase insensitive to inactivation by Mg-nucleotides alone may be prepared by heating microsomes at 50 degrees C for 15 min. The reductase in these microsomes may subsequently be inactivated by Mg-nucleotides only after addition of reductase kinase. Inactivation is a linear function of time and of cytosol protein concentration and may be reversed by treatment with a phosphoprotein phosphatase. The extent of inactivation observed under standard conditions provides an assay for reductase kinase activity. Factors present in cytosol that hinder measurement of either reductase or reductase kinase activity must be removed or inhibited. Reductase phosphatase is inhibited by 50 mM NaF. Reductase kinase kinase activity is not expressed under the assay conditions used. Mg-Nucleotide-independent inhibitors of reductase activity are removed by chromatography on DEAE-Sephacel or Blue Sepharose. Mevalonate kinase and reductase kinase are separable by chromatography on DEAE-Sephacel or Sephadex G-200. We describe a rapid chromatographic procedure for separating reductase kinase of crude fractions from mevalonate kinase and from Mg-nucleotide-independent inhibitors of reductase activity. The 1.0 M KCl eluate from DEAE-Sephacel contains all of the cytosol reductase kinase activity. This method is applicable to measurement of reductase kinase activity in cytosol or more purified fractions.
Highlights
Assay of HMG-CoA reductase kinase activity requires HMG-CoA reductase free of associated reductase kinase
As noted by George et al [19], the reductase of microsomes heated at 50°C for 15 min is no longer inactivated by Mgnucleotides alone
Reductase in heated microsomes is, inactivated by addition of Mg-nucleotides plus cytosol (Table 1). This observation forms the basis for assay of reductase kinase activity
Summary
Assay of HMG-CoA reductase kinase activity requires HMG-CoA reductase (reductase, E.C. 1. l .1.34) free of associated reductase kinase. Preparation of kinase-free reductase has previously involved repetitive washing of microsomes [7, 10, 12, 13], incubation of microsomes at 37°C [12,13,14], or solubilization of microsomal proteins with subsequent resolution of reductase kinase from reductase [2, 4,5, 13, 15]. Repetitive washing or incubation of microsomes at 37°C extracts or inactivates variable quantities of reductase kinase [13], while solubilizing reductase is time-consuming
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
