The purification in high yield and characterization of rat hepatic glucokinase

Abstract

A new improved procedure for the purification of rat hepatic glucokinase (ATP-d-glucose 6-phosphotransferase, EC 2.7.1.2) is given. A key step is affinity chromatography on Sepharose-N-(6-aminohexanoyl)-2-amino-2-deoxy-d-glucopyranose. A homogeneous enzyme, specific activity 150 units/mg of protein, is obtained in about 40% yield. The molecular weight of the pure enzyme was determined by several procedures. In particular, sedimentation-equilibrium studies under a variety of conditions indicate a molecular weight of 48000 and no evidence for dimerization; reports in the literature of other values are discussed in the light of this evidence on the pure enzyme. The amino acid composition suggests that hepatic glucokinase is closely related to rat brain hexokinase and also the wheat "light" hexokinases.

Purification and properties of rat skeletal muscle hexokinase

“…of 96 000. This value is the same as that obtained for the hexokinase type I isolated from rat brain [2], rat kidney (M. J. Holroyde and I. P. Trayer, unpublished observations) and pig heart [ 131 but is twice that obtained for the rat liver type IV (glucokinase) isoenzyme [6]. No evidence of high mol.…”

“…The slightly different assay conditions almost certainly account for the minor differences noted between our figures and those reported by others for impure preparations of the enzyme [ 1, 151. In table 2 the amino acid composition of rat muscle hexokinase, type II, is compared with published values for the rat brain type I isoenzyme [2] and rat hepatic glucokinase [6]. The similarities are quite striking and are emphasized when the 'difference indices of compositional relatedness' of Metzger et al [ 161 are calculated.…”

“…a ligand concentration on the column is chosen then the enzyme will begin to leak from the column prior to introducing the glucose. The need for adjustment of ligand concentration on the Sepharose support to achieve maximum purification of hepatic glucokinase has already been discussed [5,6] and the same principles also apply to this related enzyme.…”

“…The active fractions emerging from the affinity column were concentrated on a small DEAE-cellulose column [6] prior to gel filtration on Sephadex G-200. This column (90 X 3 cm) was operated in 20 mM triethanolamine-HCl buffer, pH 7.2, containing 100 mM KCl, 100 mM glucose, 1 mM EDTA, 0.5 mM dithiothreitol and 5% (v/v) glycerol.…”

Hepatic Glucokinase Turnover in Intact and Adrenalectomized Rats in vivo,

“…Glucokinase was purified as described previously by Holroyde et al [9] with some modifications. After extraction and centrifugation (step 1) and batchwise chromatography on DEAE-cellulose DE 52 (step 2) (see [9]), the pooled, concentrated ( 1 2 -15 nil) fractions were applied directly to a Sephacryl S-200 column (90 x 5 cm), previously equilibrated with buffer B (20 m M triethanolamine/HCl pH 7.0, containing 50 mM KCI, 4 mM EDTA, 7.5 mM MgC12. 1 mM dithiothreitol, and 5 0 4 (v/v) glycerol).…”