HMG-COENZYME A REDUCTASE KINASE: PURIFICATION AND CHARACTERIZATION OF A UNIQUE PROTEIN KINASE WITH AN ABSOLUTE REQUIREMENT FOR ADP
Abstract
The activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR; E.C. 1.1.1.34), the catalyst for the first committed step in polyisoprenoid biosynthesis, is modulated by covalent modification by ATP. Phosphorylation lowers HMGR activity. HMGR kinase (RK) catalyzes this phosphorylation. We previously described an assay for RK activity using heated microsomal HMGR (mHMGR) as substrate (J. Lipid Res. 23: 745-761, 1982). RK was purified 1,250 fold from rat liver cytosol in 8% overall yield to an average final specific activity of 3,000 nU/mg. Purification utilized (NH(,4))(,2)SO(,4) precipitation and DEAE-Sephacel, Blue Sepharose, Sephacryl S-300, and hydroxylapatite chromatography. Purified RK contained one major protein (nondenaturing PAGE). Activity comigrated with over 90% of the protein on Sephacryl S-300. SDS-PAGE suggested multiple subunits of M(,r) 56,500, 53,500 and 12,500 (1:1:2 ratio). The M(,r) of RK was 145,000 (+OR-) 3,300 (three independent techniques). At sufficently high solubilized HMGR (sHMGR) concentrations, inactivation of sHMGR occurred at the same rates as with mHMGR. Half maximal velocity was observed with 885 pU sHMGR, six times the amount of mHMGR required for maximal velocity of the kinase reaction. Inactivation of sHMGR followed Michaelis-Menten kinetics. {('32)P}Orthophosphate incorporated into purified sHMGR by purified RK was time-dependent and accompanied inactivation. Phosphorylation of phosphorylase kinase, histone 2A, casein and bovine serum albumin fraction V occurred, but to less than 2% that of sHMGR. RK may therefore be unique to the regulation of HMGR. Both ADP and ATP are required for inactivation of sHMGR and for phosphorylation of alternate substrates by RK. (alpha),(beta)-Methylene-ADP substituted for ADP for both processes. ADP thus does not act to transfer phosphate. The RK nucleotide binding sites for ADP and ATP are different. GDP cannot replace ADP, GTP replaces ATP. CTP cannot replace ATP; CDP replaces ADP. ADP thus acts as an allosteric activator of RK. K(,m) of RK for ATP was 144 (mu)M. K(,m) for ADP was 1.4 mM (twice intracellular ADP concentrations). Allosteric regulation of RK by ADP thus represents a potential physiological regulatory mechanism. A requirement for ADP has not previously been reported for a protein kinase. Thus, RK represents a novel protein kinase.
Degree
Ph.D.
Subject Area
Biochemistry
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