A regenerating system for studies of phosphoryl transfer from ATP

ANALYTICAL

BIOCHEIbfISTRY

22,

211-218 (1968)

A Regeneiating Phosphoryl G. WETTERMARK, Institute

System Transfer

for Studies from

E. BORGLUND,

of

ATP’ AND

S. E. BROLIN

of Physical Chemistry and Histological Department, University of Uppsala, Uppsala, Sweden Received May 2, 1967

In studies of phosphorylation by ATP the determinations are as a rule based on degradation of the phosphorylated product. The alternative possibility of measuring ADP formation has also been applied for various studies of phosphoryl transfer as in reactions catalyzed by adenosine phosphokinase (l), glucokinase (2) and. yeast (3, 4), brain (5) and muscle (6) hexokinases. Estimation of ADP formation is the main alternative for studies of the inhibition by the phosphorylated product. It should then be possible to maintain the level of ATP by the conversion of ADP, which would be favorable also in other kinetic studies. A further advantage of the ADP route would be that experiments extended to comparisons between phosphorylation of different compounds concern the assay of the same products. Among the sequential steps applicable for conversion and determination of ADP, the pyruvate kinase/lactate dehydrogenase reactions have been closely evaluated (7, 8). Measurements by these reactions lead to a more or less rapid reconstitution of ATP, which is well recognized (6, 9)) but the kinetics of complete systems have not been presented in detail. For this purpose, model studies of glucose phosphorylation by yeast hexokinase are chosen. THE

ANALYTICAL

AND

REGENERATING

SYSTEM

Principles. As seen in Figure 1, the phesphorylation, I, is measured by the auxiliary and indicating reactions, II and III, respectively. The measurements are based upon the conversion of NADH either by the disappearing native fluorescence or by the decreasing absorption at 346 rnp. It is necessary to provide for a rapid conversion in reactions II and III so that the over-all rate of the system depends on reactian I. Deter‘This study was supported No. 12X-525-03).

by the Swedish Medical 211

Research Council

(Project

212

WETTERMARK,

BORGLUND,

AND

BROLIN

minations of the native fluorescence of NADH makes it possible to reach about a RIO-fold increase in sensitivity as compared with spectrophotometry without special arrangements. As a consequence of this the added amount of hexokinase can be kept very low. The cycle operating in the system removes ADP and reconstitutes ATP. The properties desired for kinetic applications are thus reached in two ways: by limited ATP consumption and by a fast conversion of ADP.

Glucose

-6-P

elospkenolpyfuwtc FIQ. 1. S&heme of regenerating and analytical system, composed of three ensymic -reactioxq: -(I) hexokinaae, (II) pyruvate kinase, and (III) lactate dehydrogenase.

Reagents. ATP, ADP, PEP (phosphoenol pyruvate, tricyclohexylammonium) , NADH, and NADP were obtained from Boehringer und Soehne asswell as suspensions of the enzymes HK (hexokinase, EC 2.7.1.1), PK (pyruvate kinase, EC 2.7.1.40), LDH (lactate dehydrogenase, EC 1.1.1.27) and GGPDH (glucose-6-phosphate dehydrogenase, EC 1.1.1.49). Glucose was purchased from S. T. Baker Chemical Company, pyruvate jfrom Hopkins and Williams Ltd. Tris (Sigma 121) and G6P (glucose 6-phosphate, potassium salt) were obtained from Sigma Chemical Company.. Redistilled water and analytical-grade quality of salts were used throughout the experiments. Compositions. In the. fluorophotometric studies, the reaction mixture contained 0.1 M Tris, pH 7.6, 10 m2M KU, 3.4 n.&! MgC$, 220 miU glucose, 0.4 mM PEP, 45 ,uM NADH, 0.025 pLg HK, 40 pg PK and 60 pg LDH. The amounts of enzymes refer to 1 ml. PK and LDH were dialyzed against 0.05M Tris, pH 7.6, overnight at +4O. The suspension of HK was diIuted by lo/O glucose solution. Concentrations of KCl, MgC&, and the enzymes other than those given above were also used and are specified. Traces of pyruvates were consumed before the over-all reactions were started by continuous addition of a small amount of 58 mM ATP dissolved in 0.1 M Tris, pH 7.6. For. the spectrophotometric checks, the assay mixture contained 0.1 M Tris, pH 7.6, 3.4 mM MgCl,, 220 mM glucose, 0.8 mM ATP, 165 ,&f

REGENERATING

SYSTEM

FOR

PHOSPHORYL

TRANSFFX

213

NADP, and 0.05 and 0.20 pg/ml of HK and GGPDH, respectively. The reactions were started by addition of ATP. In some experiments, accumulated amounts of G6P were estimated by a final addition of GGPDH. ARRANGEMENTS

AND PROCEDURES

Fluorophotometry. The reactions were performed in quartz cells with optical surfaces at right angles, a length of 50 mm, and a width of 10 mm. In each experiment the cuvet was loaded with 5 ml of the reaction mixture. ATP was added by an Agla syringe operated by a synchronous motor. The cuvet was fitted into a special adapter piece (Fig. 2) which replaced the cell carrier of a photoelectric fluorometer (Farrand Optical Company). The adapter was turned from brass and contained in addition

Fro. 2. Partial cut-away sketch of adapter piece. Light openings leading to and from the cell are exposed, whereas the apertures of the standard are not seen. Only connections to the channels drilled in the wall are shown, and the plastic support of the motor and inlet from the syringe are not outlined. The bottom plate is made to fit the turning mechanics of the fluorometer. C, cuvet containing reaction mixture. I, i&t from syringe. M, motor connected to stirrer. S, standard. T, tubings for supply of thermostated water.

214

WETTERMARK,

BORGLUND,

AND

BROLIN

,to the cuvet a fluorescing glass standard (Beckman Instruments, Ltd.). ,Their positions were easily interchangeable by a turn of the adapter. The ‘temperature in the cuvet was maintained at 38O by water from a thermostat bath, pumped through a closed system of channels in the adapter. The fluorescence was measured at right angles to the exciting light beam at the front part of the cuvet. The exciting light from the mercury vapor ‘lamp passed through filter No. 5840 and the measured emission through ,Nos. 4308 and 3387 (Corning Glass Works). The syringe inlet was located in the center of the cel1, and behind it was situated a glass stirrer driven by’ a very small electric motor. The tubing from the syringe and the motor were supported by a frame of dark plastic inserted in the opening above the cuvet. The consumption of NADH was continuously followed by the decrease of its native fluorescence. The photocell of the fluorometer was connected to a load resistance of 400 kn and the signal fed to a potentiometer recorder. The readings were checked by brief interpositions of the standard. Correction for quenching was based on calibration curves with known concentrations of NADH. In order to obtain an accurate value of low ATP concentrations, the reactions were started by a discrete addition of ATP and the rate assessed. This determination was carried out at an ATP concentration of 87 yikt. The remaining part of the curve was recorded by the gradient technique. The syringe delivered 5 $/min during a period of 10-20 min. Spectrophotometry. Differences in rate were estimated in cells with a path length of 10 mm at 10 times scale expansion by simultaneous measurements at 340 rnp with a double-beam recording spectrophotometer (Beckman DB) . Calculations. The fluorescence of NADH was continuously recorded as a function of the amount of ATP added and, after correction for quenching, curves were obtained with the concentration of NADH (c) as a function of the concentration of ATP (s). These curves were fitted to polynomials of the twelfth degree. Polynomials of higher order were also tried but did not yield any further advantage. From calculations of the first derivative of the polynomial, a Lineweaver-Burk diagram was drawn: [dc/dt]-1 vs. s- I. The singular value of the rate obtained from the initial addition of ATP was also included in the diagram. A regression line was calculated and its slope, k, and intercept, I, evaluated. The reported results refer to mean values (of k and 1) from all determinations on solutions having a specified composition. They were corrected for the variation in enzyme activity which occurred from day to day. All calculations were carried out with the help of a digital computer (Facit EDB and CD 3600).

REGENERATING

SYSTEM

EVALUATION

FOR

PHOSPHORYL

OF THE

TkANSFER

215

REACTIONS

The over-all reaction consists of three enzymic steps as’ previously specified in Figure 1. Our prime interest is the kinetics of reaction I. Reactions II and III are merely auxiliary and indicating, but must meet certain requirements. In order to ascertain the adequacy of ,reaction III, small amounts of pyruvate were added to the system containing the standard amount of LDH, but 20 yg PK/ml and 80 mM KCl. ATP was not added. Within the concentration range studied (45-10 & NADH), the reduction of NADH was proportional to the added amounts of pyruvate. After each addition, the system relaxed with a half-life of about 4 sec. Reaction II coupled to III was tested by repeated additions of small amounts of ADP to the system containing 80 mM KC1 and lacking HK. No ATP was added. The response showed the same pattern of NADH consumption as in the preceding experiment. For different concentrations of PK the following half-lives of relaxing were obtained: 2, 10, and 20 pg/ ml gave 14,5, and 4 set, respectively. In order to check the capacity of the coupled reactions, the rate of phosphorylation was increased by keeping the concentration of ATP as high as 0.55 mM and by adding HK in steps. The initial concentration of HK was 0.005 pg/ml. Linearity was obtained over a %O-fold concentration range, corresponding to an activity of 96 pmoles/min/mg for the enzyme preparation. Repeated experiments of this kind indicated that the linearity of the response was not influenced by the concentration of KCl. It was also possible to substitute (NH,),S04 for KCl. The salt concentration appeared to be more critical for the rate. Phosphoenolpyruvate and KC1 (or some substitute) are required for the auxiliary reaction but may inhibit HK. It was therefore considered essential to evaluate their effects. In these experiments, the rate of the HK reaction was followed by the absorption of NADPH in a coupled GGPDH reaction. The results are shown in Figure 3. For a proper functioning of the analytical system (Fig. l), phosphoenolpyruvate may obviously be allowed in appropriate concentrations while (NH,) $0, should be essentially eliminated and KC1 minimized. The suspensionsof PK and LDH were therefore dialyzed but the minute amounts of hexokinase were not. A concentration of 10 mM KC1 appeared to be a satisfactory compromise as the activity of PK is about one-half of the maximal rate and that of HK is not seriously affected. As accounted for, 20 fig PK/ml was found to be adequate at optimal concentrations of KCl. To compensate for the loss of PK activity at 10 mM KCl, the

-216

WETTERMARK,

Per cent

BORGLUND,

AND

BROLIN

per cent

%

FIQ. 3. Effects on HK reaction by added KC1 (01, (NH&SO, (01, and PEP (0) ‘evaluated spectrophotometricallyby formation of NAPDH in a coupled G6PDH reaction. The valuesrepresentrates given in per cent of the rates in reaction mixtures without additives. amount of PK was doubled when the system finally was composed for the application. With the final composition of the system, the activity of HK was found to be in the range of 120-160 ,umoles/min/mg. The high concentration of glucose (220 mM) ensured that substrate utilization couId not affect the kinetics. When applying the analytical system, an appropriate response of the coupled reactions was frequently ascertained by addition of ADP as soon as the curve had been recorded. This is of particular importance for studies of inhibitors because they may delay the response despite the excess of PK and LDH as compared to HK. APPLICATION OF THE SYSTEM

The applicability of the system depends on the accuracy with which various rates of the HK steps are det.ermined. A satisfactory discrimination of different rates was indicated by the experiments with increasing amounts of HK. A systematic variation in the deflection of the system generally appeared from day to day and could not be eliminated, aIthough the dilution of HK was performed uniformly and carefully. Because of this, corrections were made as specified in the section presenting the calculations. K,,, for ATP was estimated to be 0.25 -e 0.02 m&f (mean & S.E.M.), which is in the same range as that obtained by measurements of G6P formation (4). Many publications present data from which Km can easily be derived, although not explicitly given. It should be emphasized thatthese values would cover an extensive range. The daily .determined K, values yielded a standard deviation of 0.06 mJf from the .accumulated mean value. As an example of the applicability of the system, the inhibitory effect of G6P is shown in Figure 4, in which each line represents the average

REGENERATING

SYSTEM

FOR

PHOSPHORYL

t min.yM

lOmM

-I

TRANSFER

217

6mM OmM

0.6-

I o 2 FIQ. 4. Reciprocal of reaction

1 4 velocity

* I I I 6 8 IO m/T’ versus reciprocal of ATP concentration

with and without G6P. Except for MgCL, which wa4 kept at 2 m&f, the reaction mixture was as described in the section on composition. It may be pointed out that the mode of calculation does not lead to the conventional presenting of lines fitted to reciprocal plots. of several determinations. G6P was added as the potassium salt and the amount of KC1 adjusted so that a 10 miW concentration of potassium was obtained. The maximum amount of G6P accumulated from the hexokinase reaction corresponded to about 1% of the lowest inhibitor concentration. The inhibitor constant, Ki, was evaluated using the relation rC= K,,,V1(l

+ [i]/Ki)

where [i] is the concentration of inhibitor. For each concentration, V-l was set equal to LK, was obtained from the line representing the uninhibited reaction, where -K,,,-l was identified by the intercept on the s-l axis. From, altogether, seven analyses with 6 and 10 mM G6P, a common Ki value of 24 + 4 mM (mean r+ S.E.M.) was obtained. In agreement with data from other kinetic studies of yeast hexokinase (4) and of brain hexokinase (5)) the inhibition of HK seemed to be competitive with respect to ATP. On the other hand, G6P is claimed to inhibit muscle hexokinase in a noncompetitive way (6). Attempts to evaluate our observations as compared to the reports of others are postponed, since research in progress, comprising various inhibitors and the relations between ATP and Mg, may facilitate a closer interpretation. The data computation carried out as described should minimize any errors due to subjective handling of the material. Systematic errors may be introduced but, if so, to a large extent cancel each other in studies of the effect of inhibitors.

218

WETTERMARK,

BORGLUND,

AND

BROLIN

Our experiences from the application of the system have further supported the view that the ratio between the rates of phosphorylation and coupled reactions must be kept low to provide for reliable response. It might be difficult to meet this requirement in studies of certain inhibitors by ordinary spectrophotometry due to the lower sensitivity. In our spectrophotometric experiments, scale expansion was used in order to facilitate measurements at small amounts of HK. The main limitation for the applicability of the system seems to be disturbing effects of adenosine triphosphatase, which it is essential to recognize when crude preparations of enzymes are involved. SUMMARY

A new cuvet adapter designed for the Farrand A photoelectric fluorometer is found to be suitable for kinetic studies of enzymes. Added reactants are rapidly mixed with the substrate. The temperature is kept constant, and brief interposition of the standard is easily accomplished. The discharge of the fluorometer is automatically recorded. The curves after correcting for quenching are subjected to data computation and reproduced by a polynomial of the twelfth degree. The program includes derivation and calculations of Lineweaver-Burk diagrams. The experimental model is tested by studies on glucose phosphorylation by coupling to the pyruvate kinase/lactate dehydrogenase reactions. The consumption of NADH in the latter is followed by the decrease of the fluorescence. The ADP-ATP cycle operating in the system facilitates measurements at low ATP concentration since this can be maintained by appropriate amounts of enzymes. Further advantages of the system are the improved possibilities of measuring product inhibition from the phosphorylated compound and comparisons related to the same measuring product, when different compounds are phosphorylated. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.

KORNBERQ,A., AND PRICER,W. E., JR., J. Biol. Chem. 193,481 (1951). SALAS, J., Sms, M., VIRUELA, E., AND SOLS, A., J. Biol. Chem. 240, 1014 (1965). lihss, L. F., BOYER, P. D., AND REYNARD, A. M., J. Biol. Chem. 236, 2284 (1961). FROMM, H. J., AND ZEWE, V., J. Biol. Chem. 237,3027 (1962). FROMM, H. J., AND ZEWE, V., J. Biol. Chem. 237,166l (1962). TOEWS, C. J., Biochem. J. 100,739 (1966). MCQUATE, J. T., AND UTTER, M. F., J. Biol. Chem. 234,215l (1959). Bomb, P. D., in “The Enzymes” (Boyer, P. D., Lardy, H., and Myrbiick, K., eds.), 2nd ed., Vol. 6, p. 95. Academic Press, New York-London, 1962. WELLNER, V. P., ZOUKIS, M., AND MEISTER, A., Biochemistry 5, 3509 (1966).