ANALYTICAL BIOCHEMISTRY 173,235-240 (1988)

A Stopped-Flow Assay for Glycogen Phosphorylase Appropriate to Measure Catalytic Activity at High Enzyme Concentrations

M. COMPANY, J. ZULUAGA, P. MART~NEZ,ANDJ.S.JIM~NEZ'

Department of Chemistry, Faculty of Sciences, Universidad Autbnoma de Madrid, Madrid, Spain

Received December 22, 1987

Glycogen phosphorylase (EC 2.4.1.1) may be assayed in the glycogen degradation direction by a continuous spectrophotometric method. The formation ofglucose l-phosphate from glyco- gen and phosphate produces a controlled change of pH which can be measured by the changes in absorbance of phenol red added to the system. The procedure may be conveniently applied to a stopped-flow spectrophotometer to measure the rate of the reaction. Therefore the activity of the enzyme may be determined at low conventional concentrations and, by the same tech- nique, at high enzyme concentrations approaching those supposed to exist in vivo. 0 1988 ACB

demic Ress, Inc.

KEY WORDS: stopped-flow; glycogen; phosphorylase; assay; catalytic activity.

Glycogen phosphorylase (EC 2.4.1.1) cata- lyzes the glycogen degradation according to the reaction ( 1,2)

~dycogen)n+~ + Pi =

(glycogen), + glucose 1 -phosphate.

Free phosphate and glucose l-phosphate possess different values for the second acid dissociation constant (3) and therefore, dur- ing the course of the reaction, there is a change in the proton concentration of the system. This change has been previously used to develop a titrimetric method (4) to assay the enzyme and also a spectrophotometric procedure using the change in absorbance of phenol red added to the medium (5). This last procedure was described for the glycogen syn- thesis direction, and the specific activity of the enzyme obtained by the spectrophoto- metric method agreed well (5) with the values obtained by the usual procedure of Hedrick and Fischer (6), based on the Fiske and Sub- barow (7) determination of free phosphate. At neutrality, the equilibrium of the phos- phorylase-catalyzed reaction is largely dis-

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played in the glycogen synthesis direction. We show here that the spectrophotometric method is also valid to assay the enzyme in the glycogen degradation direction which is the one catalyzed by the enzyme in vivo. The method has also been applied to measure the rate of the reaction in a stopped-flow spectro- photometer. Therefore the same procedure can be used to assay the enzyme at variable concentrations, ranging from micrograms to milligrams per milliliter. Taking into account that the enzyme concentration supposed to exist in the cell is within the range of a few milligrams per milliliter (8), it may be valu- able to have an assay method which enables one to measure the rate of glycogen degrada- tion when the enzyme concentration is much higher than that used in conventional assays and close to the in vivo concentration. Thus for example glycogen phosphorylase has been shown to exist in two different aggregation states (2) and thereby, the increase of the en- zyme concentration would favor the propor- tion of the high-molecular-weight form of the enzyme. It would be then most interesting to test whether the known behavior of phos- phorylase b in connection with allosteric

235 0003-2697/88 $3.00 Copyright 0 1988 by Academic Press, Inc. All rights of reproduction in any form reserved.

236 COMPANY ET AL.

properties, interaction with glycogen, specific activity, etc., as acquired from experiments carried out at low concentrations, is attribut- able to the enzyme at those high in vivo con- centrations at which a different aggregation state probably exists.

MATERIALS AND METHODS

Materials. Glycogen phosphorylase b was prepared from rabbit skeletal muscle accord- ing to the method described by Fischer et al. (9,lO) with the modifications introduced by Krebs et al. (11). The method of Hedrick and Fischer (12) was used to routinely verify the activity of the enzyme. Traces of AMP-ami- nohydrolase were removed by incubation with alumina C, after the third crystallization of the enzyme (13). AMP and Alumina C, were purchased from Sigma; phenol red and phosphate were from Merck. Glycogen from Sigma was purified by successive precipita- tions with ethanol. All other chemicals were of the highest available purity. Distilled, de- ionized water was used throughout.

Methods. A Beckman spectrophotometer equipped with a water-jacketted cell holder at 30°C was used to monitor the change in the absorbance of phenol red. When the enzyme concentration was too high to follow the change in absorbance by conventional proce- dures, the reaction was carried out in a DURRUM-D 110 stopped-flow apparatus equipped with Tektronik R5 103N oscillo- scope.

The relation between the changes in absor- bance registered and the consumption of pro- tons was established in a similar way to that described in a preceding paper (5). At any time during the course of the reaction, the concentration of phosphate as a function of the proton concentration is given by

PI = PI0

x(K,I(K,+ [ll

(K,I(K, + [H+IN - K/W, + W+lN where [P] stands for the concentration of free phosphate remaining, and the zero subscript

indicates the initial conditions when time t = 0. Kp and Kg stand for the second acid dis- sociation constants of free phosphate and glu- cose 1 -phosphate, respectively.

On the other hand, the changes in the pH of the system were followed by registering the absorbance of phenol red at 558 nm accord- ing to

A pH = pK+ log-

A,,-A’ PI

where pK, A, and Ab stand for the pK of phe- nol red, the absorbance, and the absorbance of the basic form of the indicator.

Combining Eq. [l] with Eq. [2] would yield an expression relating directly phosphate consumption with the decrease of phenol red absorbance. Despite the apparent complexity of Eqs. [I] and [2], a computer simulation with a BASIC program showed that, under the experimental conditions used in the as- says, there exists a linear relationship between the phosphate consumption and the diminu- tion of absorbance, with an error lower than 0.5%. The BASIC program was designed to generate small pH changes which produced variations in absorbance and phosphate con- centrations according to expressions [I] and [2]. The program then looked for the linear equation relating both magnitudes, yielding the proportional factor between them. An in- spection of Equation [l] shows that the con- stant factor should be directly proportional to the phosphate concentration used, within the theoretical error of the linear adjustment and the experimental error involved in the fact that, on changing the phosphate concentra- tion, the ionic strength must be kept constant by changing the concentration of an inert salt. Thus, for example, in a series of experi- ments in which the millimolar phosphate concentrations used were 55, 50, 30, 15, and 10, the constant factors displayed by the com- puter program were, respectively, 0.120, 0.109, 0.066, 0.034, and 0.024 M phosphate per absorbance unit. Therefore, the introduc- tion in the program of the pK values for phos- phate, glucose l-phosphate, and phenol red,

STOPPED-FLOW ASSAY FOR GLYCOGEN PHOSPHORYLASE 237

as well as the experimental conditions of the assay, i.e., initial phosphate concentration, pH, and phenol red concentration, permits us to readily obtain the proportional ratio be- tween dP/dt and dA/dt, the latter being the magnitude recorded directly.

The ionic strength was adjusted in all the assays by KCl. The calculation of the ionic strength and the corresponding pK values for phosphate and glucose 1 -phosphate were made using

1.544ql ~K=pK(~=0)-1+1~6~’ [31

. P

where the pK was calculated by a method of successive approach by a BASIC program and using the pK at zero ionic strength as the starting value (4). For phosphate and glucose l-phosphate the values of pK at P = 0 were 7.19 and 6.5 1, respectively (3). The concen- tration of phenol red was determined by us- ing an absorption coefficient of 41 rnM-’ cm-’ at 560 nm (14). The pK of the indicator used in all the experiments was 7.785 (5).

RESULTS AND DISCUSSION

Figure 1 displays some examples of the progress of phosphate consumption obtained as described under Materials and Methods. The phosphate concentration, represented on the ordinate axis of this figure, has been obtained by multiplying the variation in ab- sorbance by the constant factor mentioned in the previous section. Therefore the initial rates are directly obtained from the initial slopes of the progression curves. The average value of 12 determinations of the specific ac- tivity of the enzyme by this procedure was 30 & 1.1 pmol of phosphate converted to glucose 1 -phosphate per minute and milligram of en- zyme. This value is in good agreement with the reported values for the specific activity of the enzyme when measured in the glycogen degradation direction (2,4).

The change in pH during the course of the assay was never higher than 0.05 pH unit, as estimated from the change in absorbance of

I I I I L I I I 0 2 4 6

timelmin

FIG. 1. Time course of phosphate consumption. The assays were initiated by adding 100 ~1 of enzyme to a substrate solution at 30°C. The reference sample pos- sessed the same composition except for the enzyme. The final composition of the assay in the sample cell was 1% glycogen, 50 mM phosphate, 1 mM AMP, 2 IrIM mercap- toethanol, 1 mg/ml albumin, 100 mM KCl. and the fol- lowing enzyme concentrations: (a) 5 &ml; (b) 15 pg/ml; (c) 20 &ml. The initial pH was 6.89 and the concentra- tion of phenol red was 86 PM. The calculated conversion factor for this case (see Materials and Methods) was0.123 M phosphate per absorbance unit.

phenol red. According to the pH profile for the specific activity of phosphorylase ( 15- 17) that change of pH should not provoke changes in the activity of the enzyme higher than the experimental error, when measuring around neutrality. In fact the average value mentioned above was obtained from assays run at different initial values of pH around 6.8. It is evident that undesirable changes in the pH of the system might be produced if the reaction were allowed to proceed in a suffi- ciently high extension. In such a case it would be possible, however, to use an additional buffer in order to increase the buffer capacity and then, while the conversion of phosphate to glucose 1 -phosphate may be allowed to be as extensive as desired, the corresponding pH changes would be conveniently constrained to the least possible value necessary to pro- voke changes in the absorbance of the indica- tor. All the assays described here, however, have been carried out using the substrate, i.e., free phosphate, as the buffer of the medium, to make the theoretical calculation of the ini- tial rates simpler.

The rate of phosphate consumption was found to be directly proportional to the en-

238 COMPANY ET AL.

zyme concentration (Fig. 2) within the same range of concentration which is usual in the assay of Hedrick and Fischer (12). Also the activation of the enzyme by AMP was ob- tained by this procedure (Fig. 3) yielding a sigmoidal behavior with a Hill index of 1.5, which is in agreement with the value cur- rently accepted ( 18,19). Likewise, the K, for phosphate and V,, were 2.8 and 34, respec- tively, also in good agreement with the re- ported values for these parameters (2,4).

The procedure described here to assay phosphorylase b has been found to be appro- priate to follow the activity of the enzyme in a stopped-flow spectrophotometer. The pro- tonation reactions are very fast when com- pared with the rate of the enzyme catalyzed reaction-for example, when using 1.6 mg/ ml of enzyme, the time scale used to follow the progress of the reaction was between 0 and 10 s-and therefore the protonation rates of the indicator and substrates can be ignored. On the other hand, the concentra- tion of both phosphate and glucose l-phos- phate bound to the enzyme can be considered negligible as compared to the free concentra- tion of both substrates and, consequently, the

0 __I_ . l

.

“,k , , 0 5 10 15

enzyme concentration (@ml -’ )

FIG. 2. Glucose l-phosphate formation at different en- zyme concentrations. The initial rates were determined at 30°C as described under Materials and Methods. The composition of the assay was 50 mM phosphate, 1% gly- cogen, 1 mM AMP. 1.5 mM mercaptoethanol. and 1 mg/ ml albumin. In all the assays the initial pH was around 6.8 and the phenol red concentration was between 73 and 86 FM. KC1 was 100 mM.

AMP concentrationlmbl

FIG. 3. Phosphorylase activity as a function of AMP concentrations. The assay composition was 50 mM phos- phate, 1% glycogen, 100 mM KCl, 1 m&ml albumin, 2 mM mercaptoethanol, 85 PM phenol red, 12.5 &ml phosphorylase, and the indicated AMP concentrations.

changes of the pH must reflex the formation of glucose l-phosphate from phosphate. Most of the measurements of the phosphory- lase-catalyzed reaction have been tradition- ally made in the glycogen synthesis direction, due to the fact that at pH values close to neu- trality the equilibrium is largely displayed in this direction. This difficulty is overcome with the procedure described here and, at the same time, it allows one to carry out the assay at high enzyme concentrations using phos- phate as the initial substrate instead of glu- cose l-phosphate, making the assay much less expensive. The glycogen degradation is also precisely the direction in which the en- zyme acts in vivo.

Figure 4 depicts the specific activity of phosphorylase b as a function of enzyme con- centration. As can be seen, the activity ob- tained when the enzyme concentration is close to 100 pg/ml is practically the same that that obtained by the conventional procedure described before. When the enzyme concen- tration is higher, however, a progressive dimi- nution in the activity can be seen. A plausible source of error when using this kind of assay procedure, especially important when the en- zyme concentration is high, proceeds from the possible buffer capacity of the enzyme it- self. An “undetected” increase in the buffer capacity of the system would give rise to an

STOPPED-FLOW ASSAY FOR GLYCOGEN PHOSPHORYLASE 239

0 0.5 1 1.6

enzyme concentrationlmg ml -’

FIG. 4. Stopped-flow initial rates of glycogen degrada- tion as a function of enzyme concentration. The reaction was initiated by the stopped-flow system by mixing two enzyme solutions. One of them contained 68 pM phenol red, 100 mM KCI, 100 mM phosphate, 1 mM AMP, 1 mg/ml albumin, 2 mM mercaptoethanol, and enzyme at the concentrations indicated. The second syringe con- tamed the same composition except for 2% of glycogen instead of 100 mM phosphate. The temperature was 30°C and the initial pH was 6.9. The solid circles (O), at low enzyme concentrations, correspond to the specific activ- ity obtained by the conventional procedure. The empty circles (0) are those corresponding to the specific activity as obtained by the stopped-flow technique.

apparent diminution of the reaction rate. The buffer capacity of a 6 mg/ml solution of phos- phorylase in 50 IIIM phosphate, however, was found to be the same that the same solution of phosphate without the enzyme. Even in the presence of 10 mM phosphate, a solution of 3 mg/ml of enzyme did not show any in- crease in buffer capacity. Therefore we rule out the possibility that the diminution in the specific activity of the enzyme results as a consequence of the increasing buffer capacity of the system, due to the enzyme itself. Also the influence of the increasing enzyme con- centration on the pK of the indicator was found to be negligible in the range of enzyme concentration shown in Fig. 4.

Perhaps the most simple explanation for the diminution in the activity observed at high enzyme concentrations is the existence of an equilibrium between two forms of the enzyme with different molecular weights, similar to the situation described for the a form of phosphorylase (2,20), except that in the case of the b form, the aggregation equi- librium constant would be lower. In fact, a

tetrameric form of phosphorylase b has been reported to be induced by the presence of AMP and phosphate (2). The lower specific activity of the tetrameric form as compared with the dimeric one (2) would explain the diminution in specific activity when, upon increasing the enzyme concentration, the for- mation of the tetrameric species would be fa- vored according to the mass action law. Pres- ently we are carrying out light scattering mea- surements in order to find a correlation between specific activity and molecular weight of the enzyme.

Considering that the enzyme concentra- tion supposed to exist under in vivo condi- tions is within the milligram per milliliter range (8), the existence of an equilibrium be- tween dimers and tetramers may be of special significance since, at those concentrations, a considerable amount of both forms of the en- zyme may exist in vivo. The assay described here provides an appropriate method to study the general kinetic and allosteric properties of this enzyme under these concentrations and overcomes the usual difficulties of the cur- rently available conventional assays.

ACKNOWLEDGMENTS

We gratefully acknowledge the Comision Asesora of the Spanish government for the financial support to this laboratory. We also thank Dr. M. Cortijo for critical reading of the manuscript.

REFERENCES

1. Fischer, E. H., Packer A., and Saary, J. C. (1970) in Essays in Biochemistry (Campbell, P. N., and Dickens, F., Eds.), Vol. 6, pp. 23-68, Academic Press, London.

2. Graves, D. J., and Wang, J. H. (1972) in The En- zymes (Boyer, P. D., Ed.), 3rd ed., Vol. 7, pp. 435- 482, Academic Press, New York.

3. Sober, H. A., Ed. (1970) Handbook of Biochemistry 2nd ed.

4. Palter, K., and Lukton, A. (1973) Anal. Biochem. 53, 613-623.

5. Company, M., Zuluaga, J., and Jimenez, J. S. (1988) Int. J. Biol. Macromol. 10,2 1-24.

6. Hedrick, J. L., and Fischer, E. H. (1965) Biochemis- try4, 1337-1343.

240 COMPANY ET AL.

7. Fiske, C. H., and Subbarow, J. (1925) J. Biol. Chem. 14. 66,375400.

8. Cohen, P. (1976) Control of Enzyme Activity, pp. 32-50, Chapman & Hall, London. 15.

9. Fischer, E. H., Krebs, E. G., and Kent, A. D. (1958) Biochem. Prep. 6,68-73. 16.

10. Fischer, E. H., and Krebs, E. G. (1962) in Methods in Enzymology, (Colowick, S. P., and Kaplan, N. O., Eds.), Vol. 5, pp. 369-373, Academic

17.

Press, New York. 11. Krebs, E. G., Love, D. S., Bratvold, G. E., Trayser, 18.

K. A., Meyer, W. L., and Fischer, E. H. (1964) Biochemistry3, 1022-1036. 19.

12. Hedrick, J. L., and Fischer, E. H. (1965) Biochemis- try4, 1337-1343.

Scarpa, A. (1979) in Methods in Enzymology (Fleischer, S., and Packer, L., Eds.), Vol. 56, pp. 3 10-3 13, Academic Press, New York.

Helmreich, E., and Cori, C. F. (1964) Proc. Nat/. Acad. Sci. USA 52,647-654.

Cortijo, M., Steinberg, I. Z., and Shahiel, S. (1971) J. Biol. Chem. 246,933-938.

Uhing, R. J., Lentz, S. R., and Graves, D. J. (1981) Biochemistry 20,2537-2544.

Danforth, W. H., Helmreich, E., and Cori, C. F. ( 1962) Proc. Natl. Acad. Sci. USA 48,119 1 - 1199.

Mateo, P. L., Baron, C., Lopez-Mayorga, O., Jime- nez, J. S., and Cortijo, M. (1984) J. Biol. Chem. 259,9384-9389.

13. Baron, C., Mateo, P. L., Cortijo, M., and Jimenez, 20. Huang, C. Y., andGraves, D. J. (1970) Biochemistry J. S. (1982) Anal. Biochem. 124,84-87. 9,660-67 1.