ISOLATION OF HEXOKINASE FROM BAKER'S YEAST

作者：Louis Berger, Milton W. Slein, Sidney P. Colowick, Carl F. Cori · 发表于：The Journal of General Physiology · 年份：1946 · DOI：10.1085/jgp.29.6.379 · 被引用次数：285 · 研究领域：Enzyme Structure and Function、Metabolism, Diabetes, and Cancer、Glycogen Storage Diseases and Myoclonus

1. A method is described for the isolation of hexokinase from baker's yeast. The method is based mainly on fractionation with alcohol and results See PDF for Structure in a 30-fold increase in specific activity. The final product could be crystallized from ammonium sulfate without change in specific activity. 2. The enzyme catalyzes a transfer of phosphate from adenosinetriphosphate to glucose, fructose, or mannose, the relative rates with these three sugars being 1:1.4:0.3. 3. With glucose as substrate, the turnover number for the crystalline enzyme is 13,000 moles of substrate per 10(5) gm. of protein per minute at 30 degrees and pH 7.5. The temperature coefficient (Q(10 degrees )) between 0 and 30 degrees is 1.9. 4. Magnesium ions are necessary for the activity, the dissociation constant for the Mg(++) -protein complex being 2.6 x 10(-3). Fluoride in concentrations as high as 0.125 M has no inhibitory effect on the enzyme when the Mg(++) and orthophosphate concentrations are 6.5 x 10(-3)M and 1 x 10(-3)M, respectively. 5. The crystalline enzyme shows a loss in activity when highly diluted. This loss in activity can be prevented by diluting in the presence of small amounts of other proteins. Of the various protective proteins tested, insulin was the most effective, providing complete protection in a concentration of 6 micrograms per cc.; with serum albumin, a concentration of 60 micrograms per cc. was necessary. Thiol compounds (cysteine, glutathione) exerted no protective ...