The Journal of Biochemistry, Vol. 57 , No. 3, 1965 Studies on Ribulokinase from Liver By TSUNEO KAMEYAMA and NORIO SHIMAZONO (From the Department of Biochemistry, Faculty of Medicine, University of Tokyo, Tokyo) (Received for publication, August 11, 1964) It has been reported by F utter m a n shakings and after cooling, the red precipitate and and Roe (1) that D-ribulose was found in remaining benzaldehyde were removed by repeated the normal human urine. Although little extraction with chloroform. A small amount of has been known about the formation of D- chloroform was removed by airation and stored frozen ribulose in animal tissue, one can suppose the at pH about 3.5~6.0 (to prevent its becoming alkaline). formation of D-ribulose from ribitol as The concentration of D-ribulose was calculated based on the amount of hydrazone hydrolyzed assuming the Hollmann and Touster. (2) and Mc- hydrolysis was complete. C o r k i n d a l e and E d s o n. (3) reported. On D-Xylulose was prepared according to the method the other hand, we (4) have reported the described by T o u s t e r (10). Separation of D-xylulose formation of D-ribulose from D-gluconic acid from D-xylose was carried out by a cellulose column in guinea pig liver preparation. On the chromatography using water saturated n - butanol. metabolism of D-ribulose, Burma and Each fraction was subjected to a paper chromatography H o r e c k e r. (5) reported the presence of with the solvent described by Touster (10), and ribulokinase [EC 2.7.1.16] in Lactobacillus the parts which showed a single spot different from that of standard D-xylose, were collected and evapo plantarum grown on L-arabinose and F r o m m rated under reduced pressure. The resulted syrup (6) has reported the presence of specific D ribulokinase [EC 2.7. 1.47] in Aerobactor was kept dry in a desiccator. D-Glucose 1-phosphate was prepared according to aerogenes ; all the enzyme sources reported so the method described by M c C r e a d y and Hassid far were bacteria. (11). a-Glucose 6-phosphate was a gift from Dr. K. However, the fate of D-ribulose in the Iwasaki. D-Gluconic acid was prepared from D-gluco- animal tissue has not been clarified yet. Our nolactone by quantitative hydrolysis with dilute NaOH recent studiet on the D-ribulose metabolism solution, and it was confirmed that any residual in guinea pig liver (7) showed that D-ribulose lactone was not present in the solution by testing with is phosphorylated to form D-ribulose 5- the hydroxamate formation described by E i s e n b e r g phosphate. The present paper deals with the and Field (12). purification and characterization of the enzyme Hydroxylapatite was prepared according to the which promotes this reaction. method of Tiselius et al. (13). Other chemicals used in the experiments were MATERIALS AND METHODS commercial products. Materials-n-Ribulose was prepared by decom Determination of the Enzyme Activity posing D-ribulose o-nitrophenylhydrazone with benzal dehyde. The hydrazone was synthesized according to The enzyme activity was determined by the the method of Glatthaar and Reichstein (8) following two methods. and its melting point was 164°C, which coincided with 1. Colorimetric Assay-The disappearance of free the report of H o r e c k e r et at. (9). The decomposi D-ribulose was measured by the orcinol reaction after tion with benzaldehyde was carried out as follows; the precipitation with 5% ZnS04-7H,O and 0.3N the reaction mixture containing crystalline D-ribulose Ba(OH)2 (it is not necessary to prepare the solutions o-nitrophenylhydrazone, benzaldehyde, water, and one in accurate concentration, but they should be equiva drop of dilute acetic acid was heated on a boiling lent to each other as titrated with phenolphthalein) . water bath during about 2 hours with occasional The reagent concentration of the orcinol reaction 339 340 T. KAMEYAMA and N. SHIMAZONO followed the recomendation of M i 11 e r et al. (14, 15). centrifugation at 105,000 Xg for 60 minutes. The standard assay mixture contained 2.6 ƒÊmoles of Step 2. Heat Tteatment-The pH of the D-ribulose, 5 ƒÊmoles of ATP, 10 ƒÊmoles of MgC12, supernatant was adjusted to 7.0 with 2 N 20 ƒÊmoles of Tris-HCI buffer, pH 7.5, and 0.1--0.2 ml. NH4OH and the solution was placed in a of enzyme solution in a total volume of 1.0 ml. The 500 ml. Erlenmeyer flask. The temperature mixture incubated at 37°C for 15 minutes. After the of the solution was rapidly raised to 50°C incubation, the reaction was stopped by addition of and kept at this temperature for 5 minutes 2.0 ml. of 5% ZnSO4.7H20 and 2.O ml. of 0.3N with continuous stirring. After this treatment, (OH)2. D-Ribulose in 1.0 ml. of the clear supernatant it was cooled sufficiently in an ice, bath, and was measured by the orcinol reaction and compared with the control. The control in which ATP was denatured protein was centrifuged off. omitted from the above reaction mixture, was incu Step 3. Fractionation with Ammonium bated as above and ATP was added after the addition Sulfate (I)-To the above svpernatant solution, of ZnSO4 and Ba(OH)2. solid ammonium sulfate in the amount of The unit of the enzyme activity was defined as 15.2g. per 100 ml. was added to give 0.2 the amount of enzyme which causes the disappearance saturation under continuous stirring during of I ƒÊmole of free D-ribulose per minute under the about 30 minutes, and the precipitate was above standard condition. Specific activity was removed by centrifugation. The supernatant expressed as unit tier me, protein. fluid was brought to 0.35 saturation by adding 2. Manometric Assay-The reaction mixture was 10.5g. of solid ammonium sulfate per 100 ml. incubated in sodium bicarbonate buffer, and CO, After centrifugation the 0.2 to 0.35 saturation evolved was measured, which indicated the amount of phosphate transferred from ATP to substrate. fraction was dissolved in a small volume of 0.01 M sodium phosphate buffer, pH 6.7, and Detailed conditions will be given in each place. This method was mainly used in the experiments for the the solution was dialyzed overnight against a substrate specificity because phosphate ester of sugar large volume of the same buffer. and some other substrates could not be assayed by Step 4. Negative Adsorption with Carboxy the colorimetric method. methyl Cellulose-A small amount of precipitate Protein Concentration-Protein concentration of the formed during dialysis was centrifuged off enzyme solution was calculated by estimating the and the supernatant fluid was diluted with optical density of the solution at 280 and 260 my, 0.01 M sodium phosphate buffer, pH 6.7, to according to the method of W a r burg and Chris about 80 ml. To this solution one half volume tian (16). of carboxymethyl cellulose which was buffer ized in advance with 0.01 mM sodium RESULTS phosphate buffer, pH 6.7 was added. The Purificationof the Enzyme mixture was placed in an ice bath and stood All operations were carried out at ap- for about 40 minutes with occasional stirring. proximately 2°C unless otherwise indicated. The cellulose was then removed by centrifuga The pH of the buffer solutions were adjusted tion and washed with 0.01 M sodium phosphate at room temperature and subsequently cooled buffer, pH 6.7. to 2°C. A summary of purification procedure Step 5. Precipitation at pH 6.0-The pH is shown in Table I. of combined supernatant was brought to about Step 1. Preparationof IsotonicKCl Supernatant 5 by adding a small amount of N acetic acid -Guinea pigs weighing from 280--320 g. were and then gradually brought to 6 by adding sacrificed by decapitation under ether anaes a small amount of 2 N NH4OH. Resulting thesia. About 70g. of fresh liver (livers from precipitate was immediately centrifuged off. 5 guinea pigs) were cut with scissors and The pH of the supernatant fluid was brought homogenized by a Potter and Elvehjem glass to 7.0 by adding 2 N NH4OH. homogenizer with about 300 ml. of isotonic Step 6. Fractionation with AmmoniumSulfate KCl solution (0.154 M). After the total volume (II)-The supernatant fluid was brought to was adjusted to 425 ml. by addition of isotonic 0.35 saturation by adding 26.3 g. of solid KCl solution, the supernatant was gained by ammonium sulfate per 100 ml. of the solution Ribulokinase 341 for about 30 minutes , and the precipitate formed was dissolved in a small volume of 0.01 M sodium phosphate buffer pH 6.8. The solution was dialyzed against a large volume of the same buffer. Step 7. Hydroxylapatite Column Chromato- graphy-The dialyzed solution was diluted to 20 ml. with 0.01 M sodium phosphate buffer, pH 6.8, and was subjected to column chromato- graphy. Conditions of the column chromato graphy is showen in Fig. 1. The fractions of high specific activity were pooled and some properties of the enzyme FIG. 1. Chromatographic purification of were examined on this preparation. ribulokinase on hydroxylapatite column. This preparation was fairly stable and Hydroxylapatite was mixed with hyflosupercel could be stored in a refrigerator for several in the ratio of 1 :5 in dry weight, and washed weeks without any noticeable loss of activity.