[69a] Hexokinase

[69a]

HEXOKINASE

371

[69a] Hexokinase I. Brain

B y MIRA D. JOSHI and V. JAGANNATHAN

Hexose + ATP --* hexose 6-phosphate -{- ADP The preparation of a particulate form of brain hexokinase has been described. 1 A method for obtaining this enzyme in soluble form 2 and its purification are described here.

Assay

Method

Principle. Glucose 6-phosphate formed by the hexokinase reaction is measured by adding glucose 6-phosphate dehydrogenase and NADP and following N A D P H formation2 This method minimizes inhibition due to glucose 6-phosphate by oxidizing it to 6-phosphogluconie acid. Reagents

Glucose, 0.15 M MgC12" 6 H20, 0.2 M Tris-HC1 buffer, 0.2 M, pH 7.6 NADP, 0.0013 M Disodium ethylenediaminetetraacetate (EDTA), 0.0001 M ATP, sodium salt, 0.3 M, pH 7.6 Glucose 6-phosphate dehydrogenase, 2 units/ml (for activity determination, see Vol. I [42]). It should have negligible glucose 6phosphatase, NADPase, N A D P H oxidase, hexokinase, glucoseNADP reductase, and 6-phosphogluconic dehydrogenase. ~ Procedure. Mix 0.3 ml each of glucose, MgC12, Tris, EDTA, NADP, and glucose 6-P dehydrogenase and 1.0 ml of water in a cuvette (1-cm light path) : add 0.1 ml of hexokinase and then 0.1 ml of ATP and note the rate of change in optical density at 340 m~. The change in optical density should be between 0.005 and 0.020 per minute and is measured from the second to the tenth minute after adding ATP. Correct for glu-

1See Vol. I [33]. ~V. Jagannathan, Indian J. Chem. 1, 192 (1963). 3M. W. Slein, G. T. Cori, and C. F. Cori, J. Biol. Chem. 186, 763 (1950). 4Glucose 6-phosphate dehydrogenase, Type V, Sigma Chemical Co., St. Louis, Missouri, has been routinely used.

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KINASES AND TRANSPHOSPHORYLASE8

[59a]

cose-NADP reductase and hexokinase activities of the glucose 6-P dehydrogenase by measuring NADP reduction in controls without ATP and without brain hexokinase, respectively. (aO.D. of the blanks should not be more than 0.001 per minute.) Crude hexokinase is diluted with 0.l M phosphate, pH 7.6, and the purified enzyme with buffer containing glucose (0.5M), serum albumin (0.01%), and thioethanol (0.001 M). Definition o] Unit and Specific Activity. One unit is defined as that amount of enzyme which catalyzes the formation of 1 micromole of glucose 6-P (which corresponds to the reduction of 1 micromole of NADP) per minute at 30 °. Specific activity is defined as the units of enzyme per milligram of protein. Protein is determined either by Warburg's ultraviolet absorption method or Lowry's colorimetric procedure (see Vol. I I I [73]). Dialysis is required to remove thioethanol (TE), phosphate (more than 0.1M), glucose, and ammonium sulfate which interfere in Lowry's method. The Warburg method is used for routine determinations. A blank with the same concentration of TE and buffer as in the enzyme solution is used for correction for the UV absorption of TE. Purification Procedure All operations are carried out at 0-4 ° unless stated otherwise. All solutions are made with glass-distilled water, and phosphate buffers are prepared from potassium salts. Step 1. Preparation o] Particulate Brain Hexokinase. The particulate enzyme is prepared according to the method of Crane and Sols2 Wash fresh ox brains with water and scrape the cortex free from the white matter. Homogenize 400g of cortex with 800 ml of 0.1M phosphate, pH 6.8, for 3 minutes in a Waring blendor in two lots (maximum temperature 10-12°). Mix with 400 ml of the same buffer and centrifuge the homogenate at 800 g for 20 minutes. Mix the residue with 1200 ml of the same buffer and reeentrifuge as before. Centrifuge the combined supernatants at 3500 g for 30 minutes. Suspend the sediment in 150 ml of 0.05M phosphate, pH 6.2, and centrifuge at 6000g for 25 minutes. Disperse the residue in 0.05 M phosphate, pH 7.2, to give a final volume of 90 ml. Step 2. Elastase Treatment. Mix 90 ml of the particulate enzyme with 18 ml of 0.09 M sodium pyrophosphate-HC1 buffer, pH 8.4, and 2.6 mg of twice crystallized pancreatic elastase and keep at 0 ° for 24 hours with occasional mixing. Freeze and thaw the enzyme 5 or 6 times by keeping at --20 ° for 24 hours and then thawing slowly at 5 ° . Centrifuge the enzyme, which becomes a thick jelly at this stage, at 14,000g for 50

[69a]

HEXOKINASE

373

minutes to obtain soluble hexokinase. The enzyme is not sedimentable at 100,000 g in 2 hours. In all subsequent steps the enzyme is stored at --20 ° when not in use. Step 3. Protamine Sul]ate and Ammonium Sul]ate Treatment. Mix the soluble enzyme with 0.1 volume of 1 M phosphate, pH 7.5, and then with 0.2 volume of 5% protamine sulfate (final pH about 7.2-7.3). Centrifuge at 14,000 g for 30 minutes. Add ammonium sulfate to the supernatant (22.5 g for 100 ml) and centrifuge as before. Precipitate the enzyme by further addition of 33.4 g of ammonium sulfate for every 100 ml of supernatant. Collect the precipitate by centrifugation, dissolve it in 0.04 M phosphate-0.001 M TE, pH 7.5, and dialyze with gentle stirring against three changes of the same buffer for 3-4 hours. Step 4. Calcium Phosphate Gel Treatment. Centrifuge the dialyzed enzyme if it is turbid, and add calcium phosphate gel and phosphate buffer (final concentrations, 2 units of enzyme and 30 mg of gel per milliliter of 0.05 M phosphate, pH 7.5). Stir occasionally for 30 minutes, centrifuge, and wash the gel 3 or 4 times with 100-ml lots of 0.05 M phosphate, pH 7.5. Elute the gel successively with 90-100 ml and 30-40 ml of 0.2 M phosphate, pH 7.5. Add 3.6 g of glucose and 60.2 g of ammonium sulfate to every 100 ml of the combined eluates, and after 40 minutes centrifuge at 14,000 g for 60 minutes. Dissolve the precipitate in 5-10 ml of 0.005 M phosphate-0.001 M TE, pH 7.5, dialyze against three changes of the same buffer, and clarify by centrifugation if necessary. Step 5. DEAE-CeUulose Chromatography. Wash DEAE-cellulose successively with 1 N NaOH, water, 1 N HC1, water, 1 N NaOH and finally with water until free from alkali. Equilibrate with 0.5 M phosphate, pH 7.5, wash 3 or 4 times with water and finally equilibrate with 0.005 M phosphate-0.001 M TE-0.2 M glucose, pH 7.5, and suspend in the same buffer. Prepare a 5 X 9-cm column containing about 25 g of cellulose with a layer of acid-washed glass wool at the bottom, and pass 400 ml of the same buffer through the column under gentle pressure. Subsequent operations are carried out without applying pressure. Add about 5-10 ml of enzyme (about 25 mg of total protein) to the column, rinse the sides twice with 5 ml and then wash with 400 ml of 0.01 M phosphate-0.001 M TE-0.2 M glucose, pH 7.5. Elute with 0.07 M phosphate-0.001 M T E 0.2 M glucose, pH 7.5, and collect 5-ml fractions. Assay each fraction for activity and protein (by the Warburg method), and pool fractions having a specific activity of more than 25. Precipitate the enzyme by adding 60.2 g of ammonium sulfate per 100 ml and after 1 hour centrifuge at 14,000 g for 1 hour. Dissolve the precipitate in 0.1 M phosphate0.01 M T E - 0 . 5 M glucose, pH 7.5, and centrifuge to remove any in-

374

KINASES AND TRANSPHOSPHORYLASES

[69a]

soluble material. The enzyme can be dialyzed against buffer of the same composition to remove ammonium sulfate. I t is stored at --20. ° A summary of the purification procedure is given in the table. PURIFICATION PROCEDURE FOR BRAIN HEXOKINASE

Step No. 1 2 3 4 5

Fraction Crude extract Particulate preparation Elastase treatment Frotamine and ammonium sulfate treatment Calcium phosphate gel treatment DEAE-cellulose

Volume (ml)

Total units

Specific activity (units/mg protein)

2100 90 76 11

2100 610 296 272

0.05-0.08 0.3 0.8 1.1

12 2

146 108

6 31

Properties The enzyme obtained after gel elution can be kept at - - 2 0 ° (or at 0 ° in 0.5 saturated ammonium sulfate) for a few days, but more purified preparations rapidly lost activity even at - - 2 0 °. Further work was possible only when it was found t h a t high concentrations of glucose or sucrose (0.5-1.0 M) have a marked stabilizing effect on the enzyme. The purified enzyme (25 units/ml) is stable in 0.5 M glucose, 0.1 M phosphate-0.01 M TE, p H 7.5 for at least 2 weeks at --20 °. The properties of the soluble enzyme are in general very similar to those of the particulate preparation with regard to its activity with different substrates, K~ values for ATP, Mg ÷÷, and hexose and inhibition by glucose 6-P and A D P (see Vol. I [33]). Fructose is phosphorylated at 1.4 times the rate with glucose, and the rate with I T P is about 12% that with ATP. G T P and U T P are inactive as phosphate donors. Iodoacetate (8 X 10-a M) and p-chloromercuribenzoate (2.5 X 10 -7 M) cause complete inhibition of the enzyme. Cysteine, insulin, and avidin show no significant effect on enzyme activity. Notes on Purification Procedure 1. Reproducible results have been obtained with commercial samples of crystalline elastase and with enzyme prepared according to the procedure of Lewis e t a l 2 ~U. J. Lewis, D. E. Williams, and N. G. Brink, J. Biol. Chem. 222, 705 (1956).

[69a]

HEXOKINASE

375

2. Freezing and thawing are necessary in addition to elastase treatment for obtaining the soluble enzyme. About 40-60% of the activity is obtained in soluble form. The insoluble residue can be rehomogenized with phosphate and pyrophosphate buffer and treated with more elastase to obtain an additional 20-30% of the enzyme in soluble form, but it has not been used for further purification. 3. Enzyme with a specific activity of 5 or more is highly susceptible to inhibition or inactivation by contact with several commonly used materials (glassware washed with chromic acid, some varieties of rubber or plastic tubing, stopcock grease, sintered glass, and detergents). Columns are set up with only ground glass joints and stopcocks without grease. All glassware is successively washed with sodium carbonate solution, water, dilute nitric acid, and finally with glass-distilled water. Dialysis tubing is boiled with water for 10 minutes and rinsed with water. 4. Calcium phosphate gel is prepared according to Swingle and Tiselius2 A preliminary experiment with a small amount of enzyme should be carried out with calcium phosphate gel since the amount of gel required for adsorption and of buffer for elution varies slightly with different batches of gel. Protamine sulfate (ex-herring) (Koch-Light Laboratories Ltd., Colnbrook, Bucks, England) and salmine sulfate (British Drug Houses Ltd., Poole, England) have been routinely used. Other samples of protamine sulfate should be tested for their suitability. 5. DEAE-cellulose prepared according to Peterson and Sober 7 (100200 mesh, 0.50 meq/g) and Cellex-D (0.62 meq/g) (Bio-Rad Laboratories, Richmond, California) have been used. A column of the size described in the text is used for a load of about 25 mg of total protein and several columns are run simultaneously for processing larger batches. DEAE-cellulose is not re-used. 6. Ammonium sulfate precipitations are carried out by adding small amounts of solid at a time without too rapid stirring. Care is necessary to avoid loss of enzyme during transfers, since the precipitate tends to float to the surface in concentrated ammonium sulfate. 7. The first four steps of the above procedure have been repeated about 30 times with reproducible results and ten DEAE-cellulose fractionations gave enzyme of specific activity varying from 25 to 51. Ultracentrifugally the preparation is about 85% pure.

6S. W. Swingle and A. Tiselius, Biochem. J. 48, 171 (1951). TE. A. Peterson and H. A. Sober, J. Am. Chem. Soc. 78, 751 (1956).