Continuous Enzymatic Assay for Phosphorylase Kinase in a Monocascade Enzyme System

Share Embed


Descrição do Produto

ANALYTICAL BIOCHEMISTRY ARTICLE NO.

244, 45–49 (1997)

AB969862

Continuous Enzymatic Assay for Phosphorylase Kinase in a Monocascade Enzyme System1 Natalya B. Livanova,2 Iraida E. Andreeva, Valentina F. Makeeva, and Boris I. Kurganov Bach Institute of Biochemistry, Russian Academy of Sciences, Leninskii pr. 33, Moscow 117071, Russia

Received April 18, 1996

A turbidimetric method for continuous monitoring of the enzymatic reaction catalyzed by rabbit skeletal muscle phosphorylase kinase has been developed. The reaction mixture contained the substrates of glycogen phosphorylase a, i.e., glycogen and glucose 1-phosphate (or Pi), in addition to the usual components of the kinase reaction. The kinetics of the cascade enzyme system were followed by the change in glycogen concentration over time, as measured by the absorbance of the reaction medium at 360 nm. The reliability of this turbidimetric method for measuring phosphorylase kinase activity was proven by comparison with a commonly used radiochemical assay. We present here a newly developed method for calculating the initial rate of phosphorylase kinase reaction in our conjugated system. We demonstrate that our procedure is applicable for investigating the hysteretic properties of phosphorylase kinase. q 1997 Academic Press, Inc.

Phosphorylase kinase (EC 2.7.1.38) is a key enzyme in the regulation of glycogenolysis in skeletal muscle, heart, and liver. This enzyme catalyzes the phosphorylation of phosphorylase b, converting it into phosphorylase a, a form which is active in the absence of AMP. Skeletal muscle phosphorylase kinase is an oligomeric enzyme with the subunit formula (abgd)4 and a total molecular mass of 1,300,000. Current assay procedures for phosphorylase kinase are based on the estimation of accumulated phosphorylase a during a certain period of the enzymatic reaction. Phosphorylase a formation is monitored by measuring either inorganic phosphate release (1) or 32P incorporation into the enzyme (2, 3). 1 This work was supported by the Russian Foundation for Fundamental Research (Grants 96-04-49243, 96-04-50819, and 96-0400016C). 2 To whom correspondence should be addressed. Fax: (7-095) 95427-32. E-mail: [email protected].

Jennissen and Heilmeyer (4, 5) developed an automated phosphorylase kinase assay in the presence of glycogen, based on inorganic phosphate release from glucose 1-phosphate. Malencik et al. (6) originated a method based on continuous measurement of the increase in the intensity of 1-anilinonaphthalene-8-sulfonic acid (ANS)3 fluorescence during the transformation of phosphorylase b into phosphorylase a. In the present study, we have developed a new and improved method suitable for continuous monitoring of the phosphorylase kinase reaction in a cascade enzyme system, which contains, in addition to phosphorylase kinase, phosphorylase b, ATP–MgCl2 , glycogen, and a low-molecular-weight substrate of phosphorylase (glucose 1-phosphate or Pi). Our method is based on the turbidimetric determination of phosphorylase activity (7). The kinetics of the cascade enzyme system are monitored by the increase (or decrease) in absorbance of the glycogen solution with time at 360 nm. EXPERIMENTAL PROCEDURES

Materials. Hepes, Tris, ATP, and glucose 1-phosphate were purchased from Sigma Chemical Co. (USA), [g-32P]ATP was from Obninsk (Russia), and glycogen was from Biolar (Latvia). The average molecular mass of glycogen from pig liver was equal to 5.5 1 106 Da. Other reagents (of high purity) were from Russian suppliers. Protein preparation. Phosphorylase kinase was obtained from rabbit skeletal muscle according to Cohen (1) using ion-exchange chromatography on DEAE-Toyopearl at the final step of purification (8). Rabbit skeletal muscle phosphorylase b was prepared by the method of Fischer and Krebs (9) using dithiothreitol instead of cysteine and was recrystallized at least three times. The enzyme was freed of AMP by treatment with Norit A. The preparation of phosphorylase b used here 3

Abbreviation used: ANS, 1-anilinonaphthalene-8-sulfonic acid. 45

0003-2697/97 $25.00 Copyright q 1997 by Academic Press, Inc. All rights of reproduction in any form reserved.

AID

AB 9862

/

6m24$$$161

11-25-96 07:45:14

aba

46

LIVANOVA ET AL.

did not contain any phosphorylase a. The concentrations of phosphorylase kinase and phosphorylase b were determined spectrophotometrically using extinction coefficients of 12.4 and 13.2, respectively, for 1% solutions (1, 11). Phosphorylase kinase assay. Phosphorylase kinase activity in the cascade enzyme system was measured by continuous monitoring of the change in absorbance of the glycogen solution at 360 nm. The enzymatic reaction was initiated by the addition of ATP (1.5 mM) to the reaction mixture containing phosphorylase kinase, phosphorylase b, 0.1 mM CaCl2 , 10 mM MgCl2 , either 6 mM glucose 1-phosphate or 6 mM Pi , and, for the reverse and forward phosphorylase reactions, 0.25 and 0.5 mg/ml glycogen, respectively. The reaction mixture was incubated for 2 min at 307C before initiation of the reaction. Activity measurements were carried out at 307C in 0.02 M Hepes at pH 6.8. Absorbance changes were determined on a Hitachi 557 spectrophotometer (Japan) equipped with a thermostated cell holder and 1-cm optical path-length cuvettes. RESULTS

Substantiation of the method. Consider a monocascade enzyme system where the enzyme E1 (phosphorylase kinase) catalyzes the covalent modification of the enzyme E2 (glycogen phosphorylase). This modification results in transformation of the initial inactive form E2 (E2(i); phosphorylase b) into the active form (E(a) 2 ; phosphorylase a). If the monocascade enzyme system contains the substrate (S; glycogen / glucose 1-phosphate or glycogen / inorganic phosphate) of the enzyme which is a subject of modification, then it is possible to follow the E(a) 2 accumulation by measuring the formation of the product (P) of the enzymatic reaction catalyzed by the form E(a) 2 . In the presence of excess substrate S, relative to the enzyme form E(a) 2 , the rate of chemical transformation of S into P is proportional (a) to E(a) 2 : d[P]/dt Å a2[E2 ], where t is the time of reaction. The proportionality coefficient a2 represents the specific activity of the form E(a) 2 under the conditions of the enzyme assay. Denote as v0 the initial rate of the enzymatic reaction catalyzed by the enzyme E1 , the first enzyme in the cascade system: v0 Å lim(d[E(a) 2 ]/dt). At tr0

relatively short time periods of the reaction catalyzed by the enzyme E1 , accumulation of E(a) 2 with time is proportional to t: [E(a) 2 ] Å v0t. The following expression is valid for the initial rate of the product (P) formation: lim (d[P]/dt) Å a2v0t

[1]

tr0

By integrating this equation, we obtain an expression for the accumulation of P with time at relatively small values of t: [P] Å a2v0t2/2.

AID

AB 9862

/

6m24$$$162

11-25-96 07:45:14

Thus, for calculation of the initial rate (v0) of the reaction catalyzed by E1 , the enzyme which begins the cascade, the kinetic data should be presented in the coordinates {[P]; t2}. The slope of the resulting straight line is equal to a2v0/2. The a2 value necessary for v0 calculation must be obtained from an independent experiment. If the experimental method used for the assay of enzyme form E(a) 2 allows the continuous measurement of the accumulation of product P, it is possible to use Eq. [1] to calculate v0 . For this purpose, we differentiate the [P] versus t curve with respect to time and create the plot with the coordinates {d[P]/dt; t}. The initial slope of the dependence of d[P]/dt on t corresponds to the value a2v0 . Transformation of the [P] versus t curve into the dependence of d[P]/dt on t allows construction of the kinetic curve of accumulation of the product of enzyme E1 because [E(a) 2 ] is proportional to d[P]/dt. This approach allows study of deviations from steady-state accumulation of the form E(a) 2 with time, which could result, for instance, from hysteretic properties of the enzyme initiating the cascade. Estimation of the specific activity of phosphorylase a. To evaluate the specific activity (a2) of the product of phosphorylase kinase reaction (phosphorylase a), it is necessary to measure the velocity of the phosphorylase reaction after phosphorylase kinase has acted. To achieve quick completion of the kinase reaction, the phosphorylase kinase concentration should be significantly higher than the concentration of phosphorylase b. Figure 1 shows the experimental data which allow us to calculate the value of a2 . The reaction mixture contained all the components of the kinase reaction (except for ATP), as well as glucose 1-phosphate, the low-molecular-weight substrate of the phosphorylase reaction, and was incubated for 2 min at 307C. The concentration of phosphorylase kinase was held fixed at 6.4 mg/ml, and the concentration of phosphorylase b varied from 0.33 to 1.65 mg/ml. After initiation of the kinase reaction by the addition of ATP, this mixture was incubated for 5 min at 307C, during which full conversion of phosphorylase b into phosphorylase a took place. The initial slope of the dependence of DA360 on time, which characterizes the initial rate of the phosphorylase reaction, stops changing with an increase in time of preincubation of the reaction mixture before initiation of the phosphorylase reaction by addition of glycogen. Thus, we can assume that the phosphorylase a concentration after the completion of the phosphorylase kinase reaction corresponds exactly to the initial concentration of the substrate of the kinase reaction 0 phosphorylase b. The values of the initial rate of the phosphorylase

aba

PHOSPHORYLASE KINASE ASSAY

47

milligram of phosphorylase a or (3.30 { 0.08) 1 1006 unit of absorbancerliter/s per nanomol of phosphorylase a monomer (or 39 { 1 mmol Pi/min per milligram of phosphorylase a). For these calculations, we assumed that an increase in 1 unit in the absorbance at 360 nm corresponds to release of 19 mM Pi for the glycogen preparation with a molecular mass of 5.5 1 106 Da (7). The a2 value that we obtained characterizes the specific activity of a phosphorylase subunit in a completely phosphorylated dimer molecule. It is apparent, however, that at the initial stage of the kinase reaction, hybrid forms of phosphorylase containing phosphorylated and nonphosphorylated subunits were produced. In all calculations, we assumed that the specific activity of the phosphorylated subunit in the hybrid molecule coincides with that in the completely phosphorylated dimer. Available data (11) suggest that there are only minor differences in the specific activities of phosphorylated subunits of the ab and aa forms of phosphorylase at saturating concentrations of the low-molecular-weight substrate of phosphorylase.

FIG. 1. Determination of the specific activity of phosphorylase a. (a) Kinetic curves of the enzymatic reaction catalyzed by phosphorylase a (increase in absorbance of glycogen solution nA360 in time). A reaction mixture containing phosphorylase kinase (6.4 mg/ml), phosphorylase b (0.33, 0.66, 0.99, 1.32, and 1.65 mg/ml for curves 1–5, respectively), 1.5 mM ATP, 0.1 mM CaCl2 , 10 mM MgCl2 , and 6 mM glucose 1-phosphate was incubated at 307C for a time (5 min) sufficient for completion of the kinase reaction. The phosphorylase reaction was initiated by addition of glycogen (final concentration, 0.25 mg/ml). (b) Dependence of the initial rate of the phosphorylase reaction w0 (absorbance unit per minute) divided by phosphorylase a (Pha) concentration (mg/ml) on [Pha].

reaction (w0) at different concentrations of phosphorylase a were calculated from the data presented in Fig. 1a. Figure 1b shows that the ratio of w0 to phosphorylase a concentration decreases when the phosphorylase a concentration increases. The value of the ratio of w0 to phosphorylase a concentration, obtained by extrapolation to zero phosphorylase a concentration, corresponds most closely to conditions of the phosphorylase kinase reaction ([PhK] @ [Phb]), which favors complete transformation of phosphorylase b into phosphorylase a. Therefore, this extrapolation procedure gives the real value of the specific activity of phosphorylase a: a2 Å (2.03 { 0.05) 1 1003 unit of absorbancerliter/min per

AID

AB 9862

/

6m24$$$162

11-25-96 07:45:14

Estimation of the initial rate of the phosphorylase kinase reaction. Typical curves of the increase in absorbance of the glycogen solution (DA360) in the monocascade enzyme system containing phosphorylase kinase and phosphorylase b are presented in Fig. 2a with the coordinates {DA360 ; t2}. Glucose 1-phosphate was used as a low-molecular-weight substrate of phosphorylase a. The kinetic curves show the glycogen synthesis catalyzed by phosphorylase a—the product of the kinase reaction. Given the value of a2 , it is not difficult to estimate the initial rate (v0) of the phosphorylase kinase reaction from the initial slope of the kinetic curves in these coordinates (or in the coordinates {d(DA360)/dt; t}; data not shown). It can be seen from Fig. 2b that the dependence of v0 on phosphorylase kinase concentration is linear, as is usually observed when an enzymatic reaction is carried out in the presence of excess substrate. The slope of the dependence of v0 on phosphorylase kinase concentration allows us to determine the specific activity of phosphorylase kinase, which is 1.01 { 0.06 mol phosphorylase b/s per mol of protomer abgd. We also performed a series of experiments to study the kinetics of the monocascade enzyme system, using inorganic phosphate as the low-molecular-weight substrate of phosphorylase. The decrease in absorbance of the glycogen solution was registered by the turbidimetric method. For evaluation of the initial rate of the kinase reaction, the initial parts of the kinetic curves were presented in the coordinates {DA360 ; t2} (Fig. 3). The specific activity of phosphorylase a under these conditions was found to be (0.36 { 0.01) 1 1003 absorbance unitrliter/min per milligram of phosphory-

aba

48

LIVANOVA ET AL.

lase a (or 6.8 { 0.2 mmol Pi/min per milligram of phosphorylase a). Using this value of a2 , we calculate the specific activity of phosphorylase kinase as 0.110 { 0.006 mol phosphorylase a monomer/s per mol of abgd promoter. The value of the specific activity of phosphorylase kinase is an order of magnitude less than the corresponding value measured using glucose 1-phosphate as a low-molecular-weight substrate of the phos-

FIG. 3. The functioning of phosphorylase kinase in the monocascade enzyme system (in the direction of glycogen degradation). Dependencies of the absolute value of nA360 on t2. Reaction conditions: 33 mg/ml phosphorylase b; 0.53, 1.07, 2.14, and 3.20 mg/ml phosphorylase kinase (curves 1–4, respectively); 6 mM KH2PO4 ; 0.5 mg/ml glycogen. Other conditions are as described in the legend to Fig. 2.

FIG. 2. The functioning of phosphorylase kinase in the monocascade enzyme system (in the direction of glycogen synthesis). (a) Time course of the increase in absorbance of the glycogen solution at 360 nm (nA360) in the coordinates {nA360 ; t2}. (b) Dependence of the initial rate of the phosphorylase kinase (PhK) reaction v0 (nmol phosphorylase a monomer/s per liter) on [PhK]. For calculation of v0 the slope of the kinetic curves in the coordinates {nA360 ; t2} (1) and {d(nA360)/ dt; t} (2) was used. Reaction conditions: 33 mg/ml phosphorylase b; 0.014, 0.027, 0.045, and 0.063 mg/ml phosphorylase kinase (curves 1–4, respectively); 0.1 mM CaCl2 ; 10 mM MgCl2 ; 6 mM glucose 1phosphate; 0.25 mg/ml glycogen; 0.02 M Hepes buffer (pH 6.8), 307C. The reaction was initiated by the addition of ATP to a final concentration of 1.5 mM.

AID

AB 9862

/

6m24$$$162

11-25-96 07:45:14

phorylase reaction. This difference is due to the inhibition of the phosphorylase kinase reaction by Pi (12). Comparison with the direct method based on the detection of the 32P incorporation into phosphorylase b. To test the reliability of our proposed method for calculating the initial rate of the phosphorylase kinase reaction, we compared the values of the initial rate of the kinase reaction calculated by our turbidimetric method and by measuring the number of phosphate groups transferred from ATP to phosphorylase using [g-32P]ATP. The initial rate of 32P incorporation into phosphorylase, detected at concentrations of phosphorylase kinase and phosphorylase b of 0.4 and 128 mg/ml, respectively (Fig. 4a), was 0.97 { 0.04 nmol of 32P incorporated/s per liter. From the kinetic curve of phosphorylase a formation (Fig. 4b), the initial rate of the kinase reaction detected under the same conditions was calculated as 1.01 { 0.04 nmol phosphorylase b monomer/s per liter. The correspondence of these two values proves the reliability of the turbidimetric method for registration of the phosphorylase kinase reaction. Hysteretic properties of phosphorylase kinase. Phosphorylase kinase b shows a distinct lag period on the curves of the time-dependent accumulation of reaction product. Kim and Graves (13) proposed that hysteretic properties of phosphorylase kinase resulted from dissociation of the complete kinase oligomer into smaller, more active components. Carlson and Graves (14) later suggested that the lag period is due to enzyme auto-

aba

PHOSPHORYLASE KINASE ASSAY

49

phosphorylase (glucose 1-phosphate or Pi). It is known that kinetic properties of the enzymes adsorbed by glycogen particles are quite different from those of the isolated enzymes (15). With regard to the method of continuous monitoring of phosphorylase kinase activity based on measurement of the increment in the intensity of ANS during conversion of phosphorylase b into phosphorylase a (6), it should be noted that ANS is not an ‘‘inert’’ substance for phosphorylase. It is known that ANS can influence the affinity of the enzyme for its specific ligands (16, 17). This circumstance could be responsible, in particular, for the experimentally observed discrepancy between the curves of 32P incorporation into phosphorylase and the curves of increase in ANS fluorescence (6). ACKNOWLEDGMENT We are grateful to Professor J. Collins for help in editing the manuscript. FIG. 4. Comparison of the turbidimetric method of phosphorylase kinase assay with the method based on the measurement of 32P incorporation into phosphorylase. (a) Dependence of the concentration of incorporated 32P on time. (b) Dependence of phosphorylase a (Pha) formation on time. Reaction conditions: phosphorylase kinase, 0.4 mg/ml; phosphorylase b, 128 mg/ml; glucose 1-phosphate, 6 mM; [g-32P]ATP, 0.1 mM (100 counts/min per picomol of ATP). Other conditions are as described in the legend to Fig. 2.

phosphorylation. Our results show that, in accordance with data obtained by Graves and co-workers (13, 14), initiation of the phosphorylase kinase reaction by addition of kinase or CaCl2 / MgCl2 results in the appearance of a distinctive lag period on the kinetic curves of accumulation of phosphorylase a. It is obvious that the continuous turbidimetric assay is very useful for detailed studies of hysteretic properties of phosphorylase kinase. DISCUSSION

The phosphorylase kinase assay described in this paper is based on continuous recording of a kinetic curve. This method has the following advantages over methods that detect the reaction product in samples withdrawn from the reaction mixture at different time intervals: (i) the method is more reliable for calculation of the initial rate of the enzymatic reaction; (ii) the method is especially convenient for studying hysteretic properties of phosphorylase kinase; and (iii) the phosphorylase kinase assay is carried out under conditions similar to physiological ones, namely, in the presence of glycogen and a low-molecular-weight substrate of

AID

AB 9862

/

6m24$$$162

11-25-96 07:45:14

REFERENCES 1. Cohen, P. (1973) Eur. J. Biochem. 34, 1–14. 2. Reinmann, E. M., Walsh, D. A., and Krebs, E. G. (1971) J. Biol. Chem. 246, 1986–1995. 3. Kee, S. M., and Graves, D. J. (1987) J. Biol. Chem. 262, 9448– 9453. 4. Jennissen, H. P., and Heilmeyer, L. M. G., Jr. (1974) Anal. Biochem. 57, 118–126. 5. Haschke, R. H., and Heilmeyer, L. M. G., Jr. (1972) Anal. Biochem. 47, 451–456. 6. Malencik, D. A., Zhao, Z., and Anderson, S. R. (1991) Biochem. Biophys. Res. Commun. 174, 344–350. 7. Sugrobova, N. P., Lissovskaya, N. P., and Kurganov, B. I. (1983) J. Biochem. Biophys. Methods 8, 299–310. 8. Morozov, V. E., Eronina, T. B., Andreeva, I. E., Silonova, G. V., Solovyeva, N. V., Shchors, E. I., Livanova, N. B., and Poglazov, B. F. (1989) Biokhimiya 54, 448–455. 9. Fischer, E. H., and Krebs, E. G. (1962) in Methods in Enzymology (Colovik, S. P., and Kaplan, N. O., Eds.), Vol. 5, pp. 369– 372, Academic Press, New York/London. 10. Kastenschmidt, L. L., Kastenschmidt, J., and Helmreich, E. (1968) Biochemistry 7, 3590–3607. 11. Vereb, G., Fodor, A., and Bot, G. (1987) Biochim. Biophys. Acta 915, 19–27. 12. Carlson, G. M., Bechtel, P. J., and Graves, D. J. (1979) in Advances in Enzymology (Meister, A., Ed.), Vol. 50, pp. 41–115, Wiley, New York. 13. Kim, G., and Graves, D. J. (1973) Biochemistry 12, 2090–2095. 14. Carlson, G. M., and Graves, D. J. (1976) J. Biol. Chem. 251, 7480–7486. 15. Meyer, F., Heilmeyer, L. M. G., Haschke, R. H., and Fischer, E. H. (1970) J. Biol. Chem. 245, 6642–6648. 16. Seery, V. L., and Anderson, S. R. (1972) Biochemistry 11, 707– 712. 17. Stryer, L. (1965) J. Mol. Biol. 13, 482–495.

aba

Lihat lebih banyak...

Comentários

Copyright © 2017 DADOSPDF Inc.