Neurospora crassa glycogen phosphorylase: Characterization and kinetics via a new radiochemical assay for phosphorolysis

ARCHIVES

OF

BIOCHEMISTRY

Neurospora Kinetics DAVID

Department

AND

crassa Via

BIOPHYSICS

Glycogen

a New

SHEPHERD,2

136, 334-349 (1969)

Radiochemical

SHEILA

of Biochemistry

Phosphorylase:

for

ROSENTHAL, GERALD IRWIN H. SEGEL4

and Biophysics,

Received

Assay

Characterization

University

June 16, 1969; accepted

Phosphorolysis’ T. LUNDBLADP

of California, September

and

Davis,

California

AND

95618

19, 1969

Purified glycogen phosphorylase (a-1,4-glucan:orthophosphate glucosyl transferase, E.C.2.4.1.1.) from Neurospora crassa shows a specific activity of 51 units/mg protein when measured in the direction of phosphorolysis by a new radiochemical assay. Thii compares to 148 units/mg protein when assayed in the direction of glycogen synthesis. The optimum pH of the enzyme is 6.5-8.0. Fluoride and pCMB are inhibitory. Glycogen, dextrin, and amylopectin are equally good substrates in the direction of phosphorolysis. In the synthesis direction glycogen was a superior primer. The enzyme follows normal Michaelis-Menten kinetics with respect to phosphate and glycogen. The K, values for phosphate and glycogen are 2.6-3.1 X 1O--2M and 4.0 mg/ml respectively. Each K, value was independent of the concentration of the second substrate. AMP at 1.5 X lO+ M increased the V,,, 30% but had no effect on the K, values. Glucose-6-phosphate (Kd = 2.65.0 X 1(r3 M) and UDPG (Ka = 4.4-5.5 X l&3 M) are competitive inhibitors with respect to phosphate, as are ADPG and TDPG. UDPG and glucose-6-phosphate are both noncompetitive inhibitors with respect to glycogen. The inhibition of phosphorylase by UDPG and glucosedphosphate may play an important regulatory role in vivo. Arsenate was competitive with phosphate and noncompetitive with glycogen (K; = 4-9 X 10-3 M). The enzyme was stabilized by glycogen and phosphate (but not by glucose-l-phosphate) against denaturation at 30”. Rabbit muscle phosphorylase 01 phosphatase failed to convert the enzyme into an AMP-dependent form.

In a previous paper (1) we described the isolation and purification of a glycogen phosphorylase from the filamentous fungus Neurospora crassa. The enzyme activity in that study was assayed in the (nonphysiological) direction of glycogen synthesis. In the present study we describe a new radiochemical assay for the enzyme in the direction

of

pbospborolysis

and

the

the reaction in the physiological

kinetics

of

direction.

1 This research was supported by Unit.ed States Public Health Service Research Grant AI-07560. z Recipient of a Wellcome Trust Travel Grant. 3 Recipient of Shell Summer Research Fellowship. 4 Author to whom reprint requests should be sent.

MATERIALS The growth

AND

METHODS

of the organism,

the enzyme puriwere performed by methods described earlier (1). The specific act.ivity of the 1344-fold purified enzyme was 51 units/mg protein in the phosphorolysis direction. Phosphorylase assay. Phosphorylase activity was routinely determined by following the glycogen-dependent rate of 32P incorporation from orthophosphate-32P into glucose-1-phosphate-32P. The reaction mixtures contained: 2 mg glycogen, 2 rmoles phosphate-32P (pH 7.0, specific act.ivity 4-9 X lo5 cpm//lmole), 0.10 rmoles AMP, and 20 bl enzyme in a final volume of 0.1 ml. For many of the kinetics experiments, the incubation volume was increased to 0.3 ml and the amounts of assay components increased proportionately. The mixture was incubated at 30” for 20 min in a conical

fi cation, and other procedures

334

Neurospora

GLYCOGEN

centrifuge tube. The reaction was stopped by adding 1 ml of 0.1 M cadmium acetate solution. The suspension was mixed for 10 sec. We found that other compounds usually used for phosphate precipitation (e.g., barium or ammonia-magnesium) were not so effective as cadmium in precipitating the phosphate-32P. The cadmium phosphate precipitate was removed by centrifuging at top speed in a clinical centrifuge for 5 min. The supernatant fluid was decanted to another centrifuge tube. To the supernatant fluid 1 ml of 0.01 M KxHPOd solution (pH 9.1) was added. The suspension w&s mixed for 10 sec. The precipitate was again. removed by centrifuging as above. A 0.5-ml aliquot of the supernatant fluid was taken and mixed with 5 ml of Beckman scintillation fluid (6 g 2,5-diphenyloxazole, 100 g naphthalene, and dioxane to 1 liter) for liquid scintillation counting. One unit, of enzyme activity is defined as the am.ount. which catalyzed the formation of 1 Nmole of glucose-i-phosphate-32P per minute. The react#ion was always started with enzyme. Tubes with enzyme added just before the addition of the cadmium acetate served as blanks. Recoveries of added glucose-l-phosphate-l% were 8590% over a lOO-fold concentration range (0.044 pmoles added to a standard reaction mixture containing unlabeled phosphate and enzyme added immediately before the cadmium acetate). Blanks generally counted 400-2000 cpm per 0.5-ml aliquot of supernatant fluid (from a O.l-ml assay sample), indicating that more than 99% of the unreacted phosphateJ2P was removed. In comparison, 20 ~1 of crude extract generally catalyzed the formation of 5,000-25,000 cpm glucose-lphosphateJ2P per 20 min per 0.5-ml aliquot of supernatant material. The addition of phosphoglucomutase or phosphohexoisomerase to the crude or purified enzyme preparations had no effect on the result.s, indicating that if glucose-6phosphateJ2P or fructose-6-phosphate-32P were formed they would not be removed by the precipitation procedure. This was further verified by adding phosphoglucomutase and phosphohexoisomerase to a solution of glucose-1-phosphateJ4C and running the mixture through the assay procedure. The recovery of radioactivity was 85’%,. RESULTS

AND

CONCLUSIONS

Heat stability of the enzyme. When the purified enzyme was incubated at 30” for 2 hr, more than 90% of the activity was lost. However, when the enzyme was incubated with assay concentrations of glycogen (20 mg/ml) or phosphate (0.02 M) then only about 15 % of the activity was lost. As pre-

PHOSPHORYLASE

I

0

20

PROTEIN

335

40

60

30

CONCENTRATlON(~G/ASSAY)

FIG. 1. Effect of protein concentration on the incorporation of phosphate-asp into glucose-lphosphate. Each incubation mixture (total volume 0.3 ml) contained 6 rcmoles phosphate-“P (sp act, 9.0 X lo6 cpm per pmole), pH 7.0, 0.3 rmole AMP, 6 mg glycogen, and varying amounts of crude cell-free extract (diluted with 0.05 M 8-glycerophosphate buffer, pH 7). The mixtures were incubated at 30” for 20 min. The intercept on the Y-axis represents the blank (no enzyme) value.

viously reported (l), glucose-l-phosphate at assay concentrations failed to protect the enzyme. E$ect of protein concentration and incubation time on phosphate-32P incorporation. The linearity of the assay is shown in Figs. 1 and 2 (with different cell-free, crude extract preparations). The higher blank value in Fig. 1 results from the fact that the sample size was increased to 0.3 ml while the precipitant (cadmium acetate) volume was maintained at 1.0 ml. E$ect of different polyglucose primers on the incorporation of phosphate-32P. The primers used were amylopectin, dextrin, and glycogen (shellfish, liver, and E. coli) at near-satu-

SHEPHERD

ET AI,. TABLE

I

VELOCITY OF Neurospora GLYCOGEN PHOSPHORYLASE IN BOTH REACTION DIRECTIONS WITH DIFFERENT POLYGLUCOSE PRIMERS Velocity &rnoles/min/mg protein)

Substratel”

Glycogen synthesis*

Shellfish glycogen E. coli glycogen Liver glycogen Bacteriological dextrin Amylopectin

15 t

8.26 8.12 8.23 0.68 0.54

Phosphorolysis’

2.75 2.81 2.68 2.77 2.71

a All polyglucose substrates were present at a final concentration of 20.0 mg/ml. b Glucose-1-phosphate-‘4C concentration used was 0.08 m. c PhosphateJ2P concentration used was 0.06 M.

IO

20

INCUBATION

30

40

TIMEtMIN.)

FIG. 2. Effect of time on the incorporation of phosphateJ2P into glucose-l-phosphate. The concentrations of assay components were the same as those described in Materials and Methods and Fig. 1. Each incubation mixture (total volume 2.0 ml) contained 800 pg of protein (crude cellfree extract,). At the times indicated a O.l-ml aliquot was removed and added to 1.0 ml of cadmium acetate as described in Materials and Methods. The intercept on the Y-axis represents the blank (no enzyme) value, which was identical to the zero-time value.

rating assay concentrations. All the primers showed about the same activity. These results are quite different from those obtained when the enzyme was assayed in the direc-

tion of synthesis where glycogen was clearly the better substrate. The results of the assays in both directions are summarized in Table I. The enzyme used for this experiment was about 50-fold purified. In another experiment at 1 mg/ml primer, soluble corn amylose was also shown to be an effective substrate in the phosphorolysis reaction. pH Profile of glycogen phosphorylase aetivity. The pH optimum for glycogen phosphorolysis is 6.5-8.0. The decrease in activity above and below the pH optimum was

subsequently shown to result from an irreversible inactivation of the enzyme. E$ect of nucleotides on the enzyme activity. The enzyme was activated by 5’-AMP. The maximum activation (ca. 30%) was obtained by 1.5 X lOA M AMP. Above this concentration the AMP became slightly inhibitory. This amount of stimulation is similar to that obtained with muscle phosphorylase a. On the other hand, this small stimulation is different from the effect observed when the Neurospora enzyme was assayed in the direction of glycogen synthesis. In the synthesis direction a 300 % stimulation was obtained with 5.4 X low4 M AMP. These results are shown in Fig. 3. Further experiments established that AMP affected the V,,, of the reaction and not the K,. Other experiments carried out at various glycogen and phosphate concentrations gave the same result. Several other nucIeotides were tested as potential activators or inhibitors of the enzyme. Those tested were: ADP, ATP, UMP, UDP, UTP, TMP, TDP, TTP, CDP, CTP, GMP, GDP, IMP, 3’AMP, 2’-AMP, and 3’5’-cyclic AMP. All the nucleotides were added at 1.5 X 1O-4 M final concentration. None of these produced any stimulation

or inhibition

of the enzyme.

The effect of salts on enzyme activity. Figure 4 shows the effect of fluoride on enzyme activity. There is a striking difference in the

Neurospora

0

I

2

3 AMP

4

5

GLYCOGEN

6

7

8

(MX104)

FIG. 3. Effect of different AMP concentrations on enzyme activity when assayed in the direction of synthesis and phosphorolysis. Standard assay conditions were used except that the AMP concentration was varied.

effect of fluoride depending on the direction of the assay. Using the new assay, relatively low concentrations of fluoride caused a marked inhibition of the enzyme, whereas in the direction of synthesis there is some stimulation before fluoride becomes inhibitory. The inhibition seems to be an effect on the enzyme rather than an artifact of the assay procedure. It was shown that at the concentrations used, fluoride did not form a precipitate with cadmium nor did it reduce the recovery of glucose-i-phosphate-32P. Other work in this laboratory with a bacterial enzyme showed no inhibition at all with fluoride. The other salts used were sulfate, bromide, chloride, and iodide at 0.1 M final concentration. Bromide and chloride had no effect on the enzyme activity, whereas sulfate inhibited the enzyme by 50% and iodide inhibited 70 %. The e$crct of sulfhydryl inhibitors. pCMB6 inhibited phosphorolysis 25% at 6 X lop4 M 5 Abbreviations used: UDPG, uridinediphosphoglucose; ADPG, adenosinediphosphoglucose; TDPG, thymidinediphosphoglucose; P (on vertical axes of figures), protein; pCMB, parachloromercuribenzoate.

337

PHOSPHORYLASE

and 80% at 3 X 10u3 M. Iodoacetate, iodoacetamide, and N-ethylmaleimide showed less than 10% inhibition at either concentration. Kinetic constants of phosphate and glycoyen. The reciprocal plot of velocity vs. phosphate concentration at varying glycogen concentrations is shown in Fig. 5. The K, value for phosphate was 2.6-3.1 X 1O-2 ELIand was essentially independent of the glycogen concentration in the range studied. Arsenate was a competitive inhibitor with respect to phosphate and noncompetitive with respect to glycogen (Ki = 4-9 X 10d3 11).The reciprocal plot of velocity vs. glycogen concentration at varying phosphate concentrations is shown in Fig. 6. The K, for glycogen was 4.0 mg/ml and was independent of the phosphate concentration in the range studied. The enzyme used for these and most of the other kinetics studies was about 300-fold purified. Phosphate content of the mycelium. One gram (wet weight) of mycelium was extracted for 10 min in about 50 ml of boiling deionized water. The extract was filtered, diluted back to 50 ml, and the phosphate content determined by the method of Fiske and SubbaRow (2). The extract contained a total of 15 pmoles of phosphate. Wet

0

I

2

3

FLUOR!DE

4

5

6

(M X IO)

FIG. 4. Concentration-dependence of fluoride effect in the direction of glycogen synthesis and glycogen phosphorolysis. Standard assay conditions were used except that varying amounts of fluoride were added.

SHEPHERD

338

^a P f f

K,= 2.6-3.1 X 16’ i-l6

I

0

- 50

ET AL.

50

100

(PHOSPHATE j’

150 w-

FIG. 5. Reciprocal

plot of velocity against concentration of phosphate. Standard assay conditions were used except that the amount of phosphate was varied. The glycogen concentration was maintained at 3.2 mg/ml, 8.0 mg/ml, 16.0 mg/ml and 24.0 mg/ml in four separate experiments,

0.7 -

-a.3

-0.2

-&I

0

0.1

UYCOGENI-

FIG. 6. Reciprocal

a2

0.3

~~G/ML)-’

plot of velocity against glycogen concentration. Standard assay conditions were used except that the amount of glycogen was varied. The phosphate concentration was maintained at 0.006 M, 0.015 M, 0.03 M, and 0.045 M in four separate experiments,

Neurospora GLYCOGEN

7,

L4 _

a

-3

-2

339

K,=2.5X163M 8.0 MG,ML GLYCOGEN

I: I z I

-4

PHOSPHORYLASE

1.2 .

-I

0

I

2

3

4

5

6

(MXIO?

(GLUCOSE-~-PHOSPHATE)

7. Competitive inhibition of N. crassa glycogen phosphorylase by glucose-6-phosFIG. phate. The standard assay procedure was used except that varying amounts of glucose-gphosphate were added, the phosphate-32P concentration was varied, and the total incubation volume increased to 0.3 ml. The glycogen concentration was maintained at 8.0 mg/ml.

K,=5.0Xl&4 0.03M PHOSPHATE

I

2

3

(GLUCOSE+PH~SPHATE)

4

5

6

(MXl07

FIG. 8. Noncompetitive inhibition of N. crassa glycogen phosphorylase by glucosedphosphate. The standard assay procedure was used except that varying amounts of glucose6-phosphate were added, the glycogen concentration was varied, and the total incubation volume increased to 0.3 ml. The phosphate concentration was maintained at 0.03 M.

mycelium contains about 80% water. Consequently, the intracellular inorganic phosphate level is about 0.02 M (assuming no compartmentation). This level is in the same range as the K, value for phosphorylase. Equilibrium constant. The enzyme-catalyzed reaction was allowed to come to equilibrium from both directions. Glycogen

phosphorolysis was measured as described in Materials and Methods. Glycogen synthesis was measured as described previously (1). In the direction of phosphorolysis, the equilibrium ratio of inorganic orthophosphate glucose-l-phosphate was calculated to be about 3 (25% conversion of the 32Porthophosphate to glucose-i-phosphate-32P).

340

SHEPHERD

When measured in the direction of glycogen synthesis, the equilibrium ratio was about 2.5 (70% incorporation of the glucose-14C from glucose-1-phosphate-14C into glycogen). When the experimentally determined V mS.xand K, values were substituted into the Haldane equation (3), the K,, was calculated as 6. E$ect of glucoseB-phosphate. Glucose-6phosphate is a competitive inhibitor with respect to phosphate. The Ki value was 2.53.2 X 10e3 M with glycogen concentrations ranging from 3.2 mg/ml to 16 mg/ml. Figure 7 shows the results obtained at 8.0 mg/ ml glycogen. Glucose-6-phosphate is a noncompetitive inhibitor with respect to glycogen. The Ki value was 4.0-5.0 X lOA M at 0.006 M to 0.03 31 phosphate. Figure 8 shows the results obtained at 0.03 M phosphate. Effect of nucleotide diphosphak glucose compounds. UDPG is a competitive inhibitor with respect to phosphate (Ki = 4.4 X 1O-3 M). ADPG and TDPG were similarly inhibitory. UDPG is a noncompetitive inhibitor (Ki 5.2-5.5 X low3 M). The Ki values were essentially constant (4.4-5.5 X 10e3 M) at all assay concentrations of glycogen (3.2-16 mg/ml) and phosphate (0.006-0.03 M). Treatment of the enzyme with phosphorylasea phosphatase. The 1300-fold purified enzyme was incubated with rabbit muscle phosphorylase- a phosphatase for varying lengths of time. In a parallel experiment muscle phosphorylase- a was also treated with the phosphatase. The muscle phosphorylase was converted to an AiVIP-dependent form whereas the Neurosproa phos-

ET AL.

phorylase remained active in the absence of AMP. DISCUSSION

Our in vitro studies suggest a logical regulatory scheme by which the glycogen cycle may be regulated in N. crassa: Under conditions of high intracellular ATP the level of glucose-6-phosphate, which is an activator of Neurospora glycogen synthetase (5), and UDPG, which is the substrate of the synthetase (5) will increase. Glycogen synthesis will be stimulated, while simultaneously the increased levels of glucose-6-phosphate and UDPG will inhibit glycogen phosphorolysis. Under conditions of low intracellular ATP the UDPG and glucose-6-phosphate levels will fall, thereby decreasing the rate of glycogen synthesis and simultaneously releasing the inhibition of phosphorylase. Also, an increase in the intracellular AMP level would stimulate phosphorylase slightly. ACKNOWLEDGMENTS We thank Miss Marilyn Holby and Miss Lynn Hackette for their technical assistance in various phases of this work. We also thank Dr. D. Walsh and Dr. E. H. Fischer for t,he gift of muscle phosphorylase a phosphatase. REFERENCES D., AND SEGEL, I. H., Arch. Bio1. SHEPHERD, them. Biophys. 131, 609 (1969). 2. FISKE, C. H., AND Su~saRow, Y., J. Biol. Chem. 66, 375 (1925). 3. HALDANE, J. B. C., “Enzymes.” Longmans, Green, New York (1930). 4. SIGAL, N., CATTANEO, J., AND SEGEL, I. H., Arch. Biochem. Biophys. 180, 440 (1964). 5. TRAUT, R. R., AND LIPM.~N, F., J. Biol. Chem. 238, 1213 (1963).