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 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 . 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. several times with 0.01 M sodium phosphate buffer, Storage in deep freeze should be avoided, pH 6.8. This mixture was packed in a column with a size of 2 X 9 cm. Then 20 ml. of the because some loss of the activity was brought enzyme solution was applied on the column and about by freezing and thawing. immediatly eluted by linear gradient elution Reaction -The reaction product was system (The first reservoir contained 150 ml. of identified as D-ribulose 5-phosphate. The 0.01 M sodium phosphate buffer, pH 6.8, and the procedure of identification was described in second reservoir contained 150 tul. of 0.1 M sodium the previous saner (7). phosphate buffer, pH 6.8). Protein concentration Stoichiometry-The enzyme reaction was and enzyme activity were determined in each 5 ml. investigated stoichiometrically as follows. The fraction. complete reaction mixture contained 7.8 ƒÊmoles The activity on D-ribulose was determined by the standard assay condition. of D-ribulose, 13.5ƒÊmoles of ATP, 24 moles The activity on n-glucose was determined of MgClz, 30 ƒÊmoles of sodium phosphate under the same condition as on D-ribulose using buffer, pH 7.5, and 0.2 units of enzyme (no equimolar n-glucose as substrate, and the color ATPase activity) in a total volume of 3.0 ml. reaction was carried out by using S o m o g y i and In the control experiment, the enzyme solution Nelson's reagent (17). was added after the reaction was stopped. The activity on dihydroxyacetone was deter- Incubation was carried out in 37°C for 15 mined by Warburg's manometer under the condi minutes. After the incubation, (A) 1.0 ml. of tion described in "substrate specificity".

TABLE I Purification of Ribulokinase 342 T. KAMEYAMA and N. SHIMAZONO

the reaction mixture was immediately put into 2.0 ml. of 10% trichioroacetic acid, and at the same time, (B) another aliquot of 1.0ml. of the reaction mixture was transferred into 2.0 ml. of 5% ZnSO4.7H20 and then subjected to adding 2.0 ml. of 03N Ba (OH)2. The mixture obtained by procedure (A) was ex tracted several times with ether and then neutralized with 2 N NH4OH with phenol

phthalein an indicator. After dilution to about 100 ml., the solution was applied to Dowex-l-Cl- column according to a small FIG. 2. Effect of pH on ribulokinase. scale modification of C o h n and Carter's The assay mixture contained 2.6 pmoles of method (18). ADP and ATP were eluted D-ribulose, 4.5 ƒÊmoles of ATP, 8 ƒÊmoles of MgC12 , and their amounts were calculated on the 200ƒÊmoles of buffer (Tris - malate, Tris-HCI, basis of molar extinction coefficient at 260mƒÊ. glycine buffer) and 0.064 unit of ribulokinase in a The precipitate obtained by procedure (B) final volume of 1.0 ml. Incubation was carried was filtered off, and the decrease of D-ribulose out for 15 minutes at 37°C, after which the reac

in I.Oml. of the filtrate was measured by the tion was stopped by adding of 5% ZnSO4.7H20

orcinol reaction. The standard for this color and then 0.3 N Ba(OH)2, ahd 1.0 ml. of the super- reaction was determined by using D-arbinose natant was determined by orcinol reaction. The which was treated at the same conditions, control, in which ATP was omitted form the above reaction mixture, was incubated as above assuming that color development from keto and ATP was added after the addition of ZnSO4 pentose in orcinol reaction is 60% compared and Ba(OH)2. with aldopentose (19).

The results were as follows ; Decrease of ATP 0.97 ƒÊmoles Increase of ADP 1.1 ƒÊmoles Decrease of D-ribulose 0.85 femoles AMP formation was not detectable

Properties of Ribulokinase Effect of PH-The optimum pH for the ribulokinease activity was at about 8.5. The reaction velocity as a function of pH is shown in Fig. 2. Effect of Mg++-Mg++ is essential for the FIG. 3. Effect of Mg++ on ribulokinase. enzyme activity. The relationship between The reaction mixture contained 2.6 ƒÊmoles of the enzyme activity and Mg++ concentration D-ribulose, 5.0 moles of ATP, 20 ƒÊmoles of Tris- is shown in Fig. 3. When the ratio of Mg++ HCl buffer, pH 7.5, MgC12 as specified, and 0 .016 to ATP was 2: 1, the maximum activity was unit of ribulokinase in a final volume of 1.0 ml . Incubation was carried out for 15 rninntes at 37°C attained. Excess of Mg++ gave an inhibitory . effect. Following procedure was the same as in Fig . 2. Substrate Affinity Constant-The Michaelis

constants, calculated by the method of Substrate Specificity-Assay on various Lineup eaver and Burk (20), were found substrates was carried out by using Warburg's to be 7.15x 10-4 mole per liter for D-ribulose manometer. Condition was as follows . The and 2.86x l0-4 mole per liter for ATP (Fig . main compartment of the vessel contained 7 .8 4, A, B. ƒÊ moles of substrate, lO ƒÊmoles of MgCl2 30 Ribulokinase 343

FIG. 4. Effect of substrate concentration on ribulokinase activity.

(A) : The main compartment of the vessel contained 16 ƒÊmoles of MgC12, 28 ƒÊmoles of NaHCO3, D- ribulose as specified and enzyme solution in a total volume of 2.6 ml. A side arm contained

9 ƒÊmoles of ATP (the pH of the solution was previously adjusted to 7.5), and 8 )ƒÊmoles of NaHCO3 in a total volume of 0.4 ml. Gas phase contained 5% CO2 and 95% N2 after equilibration at 37°C,

the content of the side arm was tipped. Readings were taken every 5 minutes starting at 3 minutes after tipping. (B) : In the above incubation mixture, ATP of the side arm and MgC12 of the main compartment were exchanged. Following procedure was the same as in (A). The reaction velocity V was calculated as ƒÊ1. of CO2 evolved per minute while the time course

remained linear.

ƒÊ moles of NaHCO3, and enzyme solution in a shown in Fig. 1. total volume of 2.6 ml. A side arm contained Activation and Inhibition-The enzyme re- 10 ƒÊmoles of ATP (the pH of the solution was action was completely inhibited with 10-5M

previously adjusted to 7.5) and 20ƒÊmoles of PCMB, and 10-2M iodoacetic acid showed NaHCO3 in total volume of 0.4 ml. Gas phase 64% inhibition. 10-2M cysteine caused 60% contained 5% CO2 and 95% N2. After activation. equilibration at 37°C, the content of the side arm was tipped. Readings were taken every DISCUSSION 5 minutes starting at 3 minutes after tipping. The properties of the relating to Among the substrates tested, only D-ribulose the pentose phospate pathway have been and dihydroxyacetone showed appreciable studied by many investigators. In this activity. However, as shown in Fig. 1, the metabolic system, glucose is at first phos

position of the peak of the activity for D- phorylated to glucose 6-phosphate, and then ribulose and that for dihydroxyacetone were the latter is dehydrogenated to 6-phospho- found in different fractions, although a part gluconolactone by glucose-6-phosphate dehydro of both activities overlapped. D-Glucose, D- genease [ECI. 1. 1.49]. 6-Phosphogluconolactone is then hydrolyzed to 6-phosphogluconic acid galactose, D-mannose, D-fructcse, D-arabinose, D-ribose, D-xylose, D-xylulose, D-gluconic acid, by its specific lactonase [an enzyme in EC 3. 1. 1 ribitol, xylitol, D-glucose i-phosphate, D-glucose group, different from EC 3.1.1.17] (21). On the 6-phosphate, D-fructose 1-phosphate amd D- other hand, there may be another pathway in fructose 6-phosphate were found to be inactive wich glucose is not phosphorylated directly . as substrate. The activity against D-glucose Glucose is oxidized at first to gluconolactone by in the liver supernatant could be separated glucose oxidase [EC 1. 1.3.4]. Then resulting from the activity against D-ribulose by the gluconolactone is hydrolyzed to gluconic acid hydroxylapatite colum chromatography as by its specific lactonase [another enzyme in EC 344 T. KAMEYAMA and N. SHIMAZONO

3. 1. 1 group, different from EC 3.1.1.17] (21). significance of ribulokinase and of the new Gluconic acid thus formed is phosphorylatnd shunt is not completely clarified by the present by gluconokinase [EC 2.7.1.12], and enters work, and the further investigation must be into the pentose phosphate pathway. However, awaited. Kagawa et al. (4) showed that there is another pathway for gluconic acid, namely, gluconic acid is metabolized to D-ribulose by 1. The presence of ribulokinase in animal oxidative decarboxylation by the action of liver was demonstrated. The enzyme was NAD-L-gulonate dehydrogenase (decarboxylat- purified about 100-fold from guinea pig liver. ine) [EC 1.1.1.451. 2. Mg++ and ATP were essential for the Futterman and Roe (1) reported that enzyme reaction. Maxinal activity was given at D-ribulose is present in the normal human the 2: 1 concentration ratio of Mg++ to ATP. urine. H i c k m a n and A s h w e l l (22) reported 3. D-ribulose 5-phosphate was produced the presence of xylulokinase [EC 2. 7. 1. 17] and by this reaction as reported previously. The A g r a n o f f and B r a d y (23) reported the stoichiometrical relationship was proved be presence of ribokinase [EC 2.7. 1. 15] in animal tween the production of ADP and the decrease tissues. Although these pentose phosphorylat of ATP and D-ribulose. ing enzyme were found in animal liver, they 4. This enzme was specific for ribulose could not phosphorylate D-ribulose. Therefore among the substrates tested. these reports suggested that D-ribulose might 5. The optimal pH of this enzme was be the metabolic end product in animal body. 8.5. The Michaelis constants were calculated But in the present paper, the presence of to be 7.15 X 10-4 and 2.86 x 10-4 mole per liter ribulokinase was demonstrated in animal for D-ribulose and ATP, respectively. The tissue, e.g., guinea pig liver. Thus the presence reaction was inhibited by the SH-blocking of another shunt of pentose phosphate pathway reagents. may illustrated as follows. The author wishes to express his gratitude to Dr. Y. Mano for his continuous interest and kind help throughout the investigation.

REFERENCES

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