Eur. J. Biochem. 68, 21 - 30 (1976)

The Purification and Properties of Rabbit Skeletal Muscle Glycogen Synthase Hugh G. NIMMO, Christopher G. PROUD, and Philip COHEN Department of Biochcmistry, University of Dundee (Rcceived February 2 9 / M a y 7, 1976)

Glycogen synthase u was purified over 500-fold by a procedure which involved solubilisation of the enzyme from a protein-glycogen complex by the action of endogenous phosphorylase and debranching enzyme, followed by DEAE-cellulose chromatography, and either gel filtration on Sepharose 4B or fractionation with polyethylene glycol. 15 mg of protein could be obtained from 1000 g of muscle in five days, corresponding to a yield of 20 The purity was over 90 % as judged by gel electrophoresis and ultracentrifugal analysis. The amino acid composition was determined and the absorption coefficient, A:% nm, measured refractometrically was 13.4. Glycogen synthase a sedimented as two major components, both of which were enzymatically active. The smaller species (13.3s) comprised 85 % and the larger species (19.0s) 15 % of the material. The molecular weight of the 13.343component was determined to be 377000 by high-speed sedimentation equilibrium centrifugation. The subunit molecular weight measured by gel electrophoresis in the presence of sodium dodecylsulphate was 88000 indicating that the 13.3-S species is a tetramer. The properties of the enzyme are compared to those obtained by other workers.

x.

Studies of the activities of enzymes involved in the synthesis of glycogen in skeletal muscle have suggested that glycogen synthase is the rate-limiting enzyme in the pathway, and considerable efforts have been made toward achieving an understanding of the mechanisms which might regulate the activity of the enzyme in vivo. The enzyme has been shown to exist in at least two forms, a phosphorylated b-form and a dephosphorylated a-form [l]. The b-form has a low activity but can be activated fully by the allosteric effector glucose6-P. This activation is antagonised by physiological concentrations of ATP, ADP and inorganic phosphate [2]. In the absence of ATP, the a-form is almost fully active without glucose-6-P. In the presence of ATP, the a-form is partially inhibited, but this inhibition is reversed by very low concentrations of glucose-6-P [2]. The a-form has therefore been considered to be the more active form in vivo under most physiological conditions [3].

Abhreviutions. Cyclic AMP, adenosine cyclic 3' : 5'-monophosphate; glucose-6-P, glucose 6-phosphate. Enzymes. Glycogen synthase (EC 2.4.1.1 1); protein kinase or ATP: protein phosphotransferase (EC 2.7.1.37); glycogen synthase phosphatase (EC 3.1.3.-); phosphorylase kinase or ATP: phosphorylase phosphotransferase (EC 2.7.1.38); phosphorylase (EC 2.4.1.1); debranching enzyme or amylo-I ,6-glucosidase (EC 3.2.1.33);B-galactosidase (EC 3.2.1.23).

The proportion of glycogen synthase in the a-form has been shown to be inversely related to the glycogen content of muscle [4]. The interconversion of glycogen synthases a and h is also under hormonal control. Adrenalin promotes a conversion of glycogen synthase a to h in skeletal muscle [4], while in the perfused diaphragm insulin (in the absence of adrenalin) increases the proportion of glycogen synthase a [5]. The effect of adrenalin is presumed to result from the activation of cyclic-AMP-dependent protein kinase which has been shown to catalyse the conversion of glycogen synthetase a to b in vitro [6,7]. The mechanism of action of insulin, on the other hand, has been much more difficult to understand. The effect is observed in the perfused diaphragm even in the absence of added glucose, indicating that it is not dependent on an increased transport of glucose into muscle which is also stimulated by insulin [4]. Neither has insulin been found to affect the tissue content of cyclic AMP [5]. The action of insulin does not therefore seem to be explainable in terms of a decrease in cyclic-AMP-dependent protein kinase activity caused by a fall in cyclic AMP. It has been claimed that insulin renders cyclic-AMP-dependent protein kinase activity, measured in crude cell extracts, less susceptible to activation by a given concentration of cyclic AMP [8]. However the molecular basis of this observation remains unclear.

Skeletal Muscle Glycogen Synthase

22

Recently the nature of the a-form to b-form transition and the hormonal regulation of the enzyme has started to come under a complete reappraisal, following preliminary reports that at least one further enzyme, distinct from cyclic-AMP-dependent protein kinase, is able to catalyse the phosphorylation of glycogen synthase in skeletal muscle and other tissues [9,10,11]. These discoveries have stressed the need to characterize the different forms of glycogen synthase produced by different glycogen synthase kinases, in order to investigate which kinase is important under different metabolic conditions. This paper reports on the first phase of this study, namely an improved procedure for isolating large quantities of homogeneous glycogen synthase a in a form which resembles the native configuration, and an analysis of its basic physico-chemical properties. The following paper describes the characterisation of a new activity termed glycogen synthase kinase-2 and the phosphorylation of glycogen synthase by glycogen synthase kinase-2 and cyclic-AMP-dependent protein kinase [ 121. A preliminary account of part of this work was presented at a meeting of the British Biochemical Society at Edinburgh in September 1975 [13], at the 4th International Interconvertable Enzyme Symposium at Arad, Israel in April 1975 [14] and at a CTBA Foundation Symposium at London in July 1975 [15]. EXPERIMENTAL PROCEDURE Ma terials

DEAE-cellulose (Whatman DE 52) and Whatman GF/A glass fibre paper discs (2.1 cm diameter) were obtained from H. Reeve Angel & Co. Ltd (London, U. K.). Oyster glycogen, AMP, glycerol-2-phosphate (sodium salt), Amberlite monobed resin MB-3, bovine serum albumin (fraction V), polyethylene glycol 6000, and reagents for acrylamide gel electrophoresis were purchased from BDH'Chemicals Ltd (Poole, Dorset, U. K.). Rabbit liver glycogen (grade II), glucose-6-P (Na' salt), glucose-1-P (Na' salt) and /l-galactosidase (from E. coli) were obtained from Boehringer Corporation (London, U. K.). Uridine diphosphoglucose, 5,5'-dithiobis(2-nitrobenzoicacid) and Coomassie brilliant blue were from Sigma Chemical Co. (London, U . K.). [14C]Uridine diphosphoglucose (uniformly labelled in the glucose moiety) was obtained from the Radiochemical Centre (Amersham, Bucks., U . K.). Sepharose 4B was from Pharmacia (GB) Ltd (London, U. K.). Phosphorylase h was prepared from rabbit muscle by the method of Fischer and Krebs [16] and recrystallised three times. Phosphorylase a was prepared from phosphorylase h and also recrystallised three times [17]. Phosphorylase kinase was purified as described previously [IS].

Buffer Solutions The following solutions were used repeatedly in this work: Buffer A comprised 50 mM glycerol 2-phosphate, 2 mM EDTA and 30 mM mercaptoethanol adjusted to pH 7.0 with HCl. Buffer B comprised 5 mM Tris-HC1, 1 mM EDTA and 30 mM mercaptoethanol pH 7.5. Buffer C comprised buffer B plus 10 (v/v) glycerol. Purification of Glycogen Synthase (Method I ) The preparation was modified from that of Soderling et al. [7].A female New Zealand White rabbit was given a lethal dose of nembutal, exsanguinated, and the muscle from the hind limbs and back was rapidly removed and chilled in ice. All subsequent steps were performed at 0-4 "C. The muscle (800-900 g) was minced and then homogenised in 2.5 volumes of 4 mM EDTA pH 7.0 for 25 s at low speed in a Waring blender. The homogenate was centrifuged at 6000 x g for 45 min and the supernatant was decanted through glass wool (step 1). The solution was adjusted to pH 6.1 by the addition of 1 N acetic acid and the precipitate was collected by centrifugation at 6000 x g for 45 min. It was resuspended in 45 ml of 100mM glycerol 2-phosphate, 4 mM EDTA, 30 mM mercaptoethanol pH 8.2 using a hand homogeniser to disperse the pellet finely, and diluted to 120 nil with buffer A (step 2). This solution was centrifuged at 78 000 x g for 100 min and the supernatant was removed carefully and used in the preparation of phosphorylase kinase [I 81. The protein-glycogen pellets were resuspended in buffer A plus 5 mM MgCL to a volume of 120 ml using a hand homogeniser, incubated at 4 "C overnight, and then for one hour at 30 "C. The purpose of the addition of MgC12 was to promote dephosphorylation of the enzyme. The solution was then recentrifuged at 78000 x g for 100 min. The supernatant was discarded and the pellets were resuspended in 50 ml of 0.5 M potassium phosphate and 5 mM AMP, pH 7.5 (step 3). The suspension was then dialysed overnight against 20 volumes of buffer C. The solution was removed from the dialysis sac and 50 ml of 1.0 M potassium phosphate, 5 mM AMP pH 7.5 was added. The solution was then dialysed overnight against 20 volumes of buffer C with two changes of dialysis buffer. The purpose of this prolonged incubation with phosphate and AMP was to achieve complete degradation of glycogen using the phosphorylase and debranching enzyme activities present in the protein-glycogen complex [19]. If the glycogen is not removed glycogen synthase will not bind to DEAE-cellulose. The dialysate was centrifuged at 78000xg for 60 min. The supernatant (step 4) which contained at least 80% of the glycogen synthase activity was applied to a

H. G . Nimmo, C. G . Proud, and P. Cohen

column of DEAE-cellulose (8 x 3 cm) equilibrated with buffer C (50 ml) and then with 175 mM Tris-HCI, 1 mM EDTA, 30 mM mercaptoethanol, 10% (v/v) glycerol pH 7.5 until the absorbance at 280 nm of the effluent fell below 0.01. Glycogen synthase activity was then eluted from the column with 300 mM TrisHCI, 1 mM EDTA, 30 mM mercaptoethanol 10% (v/v) glycerol pH 7.5. Fractions with an absorbance at 280 nm of greater than 0.25 were pooled (step 5) and concentrated to about 1.5 ml by vacuum dialysis. The material was dialysed for 2 h against buffer A containing 10 "/: (v/v) glycerol and then passed through a column of Sepharose 4B (200 x 1.6 cm) which was equilibrated in buffer A containing 10 % (v/v) glycerol. The fractions of highest specific activity were pooled and concentrated by vacuum dialysis (step 6). The enzyme was either used directly or dialysed against a solution formed by mixing 2 volumes of buffer A with 1 volume of glycerol and then stored at - 15 'C. Method 2

The 300 mM Tris-HCI eluate from DEAE-cellulose (method 1) was treated with polyethylene glycol. One volume of 50% w/v polyethylene glycol was added to nine volumes of the eluate, and the mixture was allowed to stand at room temperature for one hour. The precipitate containing phosphorylase kinase and other impurities was then removed by centrifugation at 15000 x g for 10 min. Further polyethylene glycol was added to the supernatant to increase its concentration to 10%. After standing at 0 "C overnight, or until the precipitate had flocculated, the suspension was again centrifuged at 15000 x g. The supernatant was discarded and the precipitate redissolved in a solution composed of two parts of buffer A and one part of glycerol. The solution was centrifuged at 15000xg for 2 min to remove any turbidity and stored at -1 5 "C. Most of the studies described in this paper were carried out using enzyme prepared by method 1. Studies of the effect of phosphorylation on the activity of glycogen synthase were carried out with enzyme prepared by method 2, and are described in the following paper [12].

23

which has been used by some workers [21], was omitted from all solutions. The activity ratio of the enzyme is defined as its activity in the absence of glucose-6-P divided by its activity in the presence of 6.7 mM glucose-6-P. In studies of the reactivation of glycogen-depleted glycogen synthase by added glycogen, the enzyme was assayed in two ways. Firstly, its activity was measured as described above except that glycogen was omitted from the enzyme diluent. Secondly, the synthase was preincubated with 10 mg/ml rabbit liver glycogen at 30 "C for 5 min and then at 4 "C for a further 12-24 h, and then it was analysed in the standard assay with glycogen in the diluent. Phosphorylase kinase [ 181 and phosphorylase h [22] were assayed by standard procedures. Acrylamidr Gel Electrophoresis This was carried out on 5 gels in the presence of sodium dodecylsulphate [23] as described previously [18]. The samples were heated at 100 "C for 5 min in the presence of 1 % sodium dodecylsulphate and 100 mM mercaptoethanol to achieve complete reduction and denaturation. The marker proteins used in the estimation of the subunit molecular weight of glycogen synthase were the a, fi and y subunits of phosphorylase kinase (molecular weights 145000, 128000 and 45 000 respectively [ 1S]), glycogen phosphorylase (100000 [17]) and bovine serum albumin (68000 [24]). Ultracrntrifugat ion This was carried out in a Spinco analytical ultracentrifuge (model E) as described previously [18]. Sedimentation velocity experiments were carried out at 4 f 1 mg/ml in buffer A and high-speed sedimentation equilibrium runs were carried out at an initial concentration of 0.4 & 0.1 mg/inl in buffer A. The partial specific volume of glycogen synthase calculated from its amino acid composition [25] was 0.732 at 20 "C. The temperature correction factor d7,qdT = 0.0005 m l x g - l x K - ' was applied. The relative viscosity of buffer A was 1.037 and its density was 1.005 g/ml at 20 "C [17].

Enzyme Assays

Absorption Coejj'k ient

Glycogen synthase was assayed using the method of Thomas et al. [20]. The enzyme was diluted in 50 mM Tris-HCI pH 7.8 containing 5 mM EDTA, 30 mM mercaptoethanol and 3 mg/ml rabbit liver glycogen: it was prewarmed in this diluent at 30 "C for 5 min before being assayed at 30 "C. Activity was measured either in the absence of glucose-6-P or in the presence of 6.7 mM glucose-6-P. Sodium sulphate,

The absorbance and protein concentration of glycogen synthase were measured in buffer A using the refractometric method of Babul and Stellwagen [26] as described previously [18]. The absorbance coefficient of glycogen synthase, A;& ",, was found to be 33.4 f 0.1 (from two difTerent preparations). This value is also consistent with protein determination carried out by the procedure of Lowry

24

et al. [27] using bovine serum albumin (A;% nm = 6.0) as the protein standard. The ratio of the absorbance of the enzyme at 280 nm to its absorbance at 260 nm was 1.83.

Density Gradient Centrfugation

Glycerol density gradient centrifugation was performed using a linear gradient of 5-15% (w/v) glycerol in buffer A. 0.5 ml of glycogen synthase (5 mg/ml in buffer A) was loaded on the gradient (final volume 22.5 ml). Centrifugation was carried out at 4 ° C and 25000 rev./min for 17 h in a 3 x 2 5 ml swingout rotor using an MSE Superspeed 65 centrifuge. At the completion of each run the bottom of each tube was pierced and fractions of 11 drops were collected. Estimates of S Z O , ~ were obtained using phosphorylase b (szO,w = 8.8 S [17]) and P-galactosidase ( S ~ O = , ~ 15.95 S [28]) as marker proteins.

Chemical Analysis of Glycogen Syrithase

Glycogen synthase was dialysed against 10-fold diluted buffer A and freeze-dried. Samples were hydrolysed in vucuo in 6 N HCl containing 10 mM phenol for 24 h, 48 h and 72 h at 110 "C. The samples were analysed with a Beckman "Multichrom" amino acid analyser [18]. Cysteine was determined after oxidation with performic acid [29] and also by titration of sodium dodecylsulphate-denatured glycogen synthase with 5,5'-dithiobis(2-nitrobenzoicacid) [30] after freeing the enzyme from mercaptoethanol by gel filtration on Sephadex G25 [17]. Tryptophan was determined spectrophotometrically [31] and also by procedure K of the colorimetric method described by Spies and Chambers [32]. Glucosamine and galactosamine were measured after 16 h hydrolysis by amino acid analysis using a modified programme [33]. Carbohydrate was estimated by the phenol-sulphuric acid method [34] using glucose as a standard. For the determination of alkali-labile phosphate, protein samples (3 - 15 nmol) were precipitated by the addition of trichloracetic acid to a final concentration of 5 % (w/v). The samples were kept at 0 "C for 10 min and then centrifuged at 15 000 x g for 2 min. The supernatants were discarded and the pellets redissolved in 0.5 ml portions of 0.1 N NaOH at 0 "C. The proteins were reprecipitated by the addition of 0.5 ml portions of 10 % trichloroacetic acid, kept at 0 ° C for 10 min and then centrifuged at 15000 x g for 2 min. The pellets were washed twice with 1 ml portions of 5 % trichloroacetic acid and then incubated for 18-20 h at 37 "C with 0.3 ml portions of 1.0 N NaOH, in order to hydrolyse alkali-labile bonds. After the hydrolysis, 0.1 ml of 100% trichloro-

Skelatal Muscle Glycogen Synthase

acetic acid was added to each sample, and the samples were kept at 0 "C for 10 min, and then centrifuged at 15000xg for 2 min. 0.3 ml of each supernatant was removed for the assay of inorganic phosphate, the pellets were washed once with 0.2-ml portions of 25 % trichloroacetic acid, and the supernatants were removed. The two supernatants were then assayed separately for Pi by the method of Ames r351 - - using KHzP04- in 25 irichloroacetic acid as a standardThe entire procedure was carried out using a single plastic microcentrifuge tube for each sample. Each assay was carried out in duplicate. 3 x recrystallised phosphorylase b and phosphorylase a were used as control proteins in each determination. Phosphorylase b always contained < 0.03 mol of alkali-labile phosphate per 100000 protein and phosphorylase a was found to contain 1.O f 0.1 mol of phosphate per 100000 g as measured by the procedure described above or by 32P-incorporation.

RESULTS Enzyme Purification and Reactivation

A summary of the purification of glycogen synthase by method 1 from two rabbits is shown in Table 1. Usually 15- 20 mg of glycogen synthase with a specific activity in the range 10- 20 U/mg could be obtained in 7- 8 days from 1500 g muscle. Recently the shorter procedure (method 2) has been developed. This allows the purification to be completed within 5 days. The specific activity is usually near 10 U/mg. The activity ratio of the enzyme was near 0.3 in the extract (step 1). It had risen t,o 0.5 by the time the washed proteinglycogen pellets (step 3) had been prepared and to 0.8 after the incubation with 5 mM Mg2+. The majority of the glycogen synthase activity was recovered in the protein-glycogen complex (step 3) indicating that the enzyme binds extremely tightly to glycogen, even in comparison with several other enzymes of glycogen metabolism [ 191. Degradation of the glycogen at this stage, using the endogenous phosphorylase b and debranching enzyme, resulted in a decrease in the glycogen synthase activity to 25 - 30 of the previous value. The enzyme could be reactivated by incubating it with rabbit liver glycogen and the extent of reactivation that could be obtained increased with each subsequent step of purification after step 3 (see Table 1). However, only 55- 65 % of the original activity could be restored indicating that the final specific activity of the enzyme preparation (18 units/mg) may be only about half the true specific activity of the enzyme in its native state. Making allowances for this, glycogen synthase is only about 0.1 by weight of the soluble muscle protein, over 50-fold lower than glycogen phosphorylase. Maximal

'x

H. G. Nimmo, C. G. Proud, and P. Cohen

25 A

I

20

10

B

I

30 Fraction number

LO

50

Fig. 1. Gel fi'ltration ofglycogen synthase on Sepharose 48 (200 x 1.6 mi).The material from step 5 (Table 1) (1.5 ml) was applied to the column and fractions of 6 mi were collected. (0-0) Absorbance at 280 n m ; (0-0) glycogen synthase activity; (V -V) phosphorylasc activity. The two arrows denote the positions at which phosphorylase kinase (A) and b-galactosidase (B) elute from the column

Table 1. Purification q/'rahhif muscle glyc~ogensynthase by method I At steps 1 - 4 protein was measured by the procedure of Lowry et al. [27]. At steps 5,6, the absorption coefficient Azsoof 13.4 found for purified glycogen synthase was used. 1600 g muscle was used in this preparation. n. d., not determined Step

Protein

Activity"

mg

U

79 400

2580

n.d.

2. pH 6.1 precipitation

5 640

2000

n.d.

3. 75000 x g pellet

3 596

1710

1.5

4. 78 000 x g supernatant after glycogenolysis

2 570

1000

2.3

0.39

1. Extract

5. DEAE-cellulose pH 7.5 6. Sepharose 4 B eluate

Reactivation' ratio

Specific activity

Purification

U/mg

-fold

0.032

Yield

Activity ratio glucose-6-P

(X

1

100

0.35

11

78

n.d.

0.48

15

66

0.61

12

39

n.d.

0.31

10s

760

2.1

7.2

225

30

0.74

25

470

4.1

18.6

582

18

0.75

Measured after reactivation with glycogen from step 3 onwards. with glycogen relative to that measured without this prior reactivation.

' The activity measured after preincubation

reactivation of glycogen synthase was obtained only at glycogen concentrations of 5 mg/ml or greater. N o detectable reactivation was achieved with glucose, fructose, lactose, maltose or maltotriose, each at a concentration of 10 mg/ml. Reactivation with glycogen did not affect the activity ratio of the glycogen synthase preparation. Glycogen synthase could be stored in buffer A plus 5 mg/ml glycogen at 4 "C for up to one week with no detectable change in activity or activity ratio. It could also be stored in 33 %glycerol at - 15 "C. Under these conditions there was no significant loss of activity in one month if 5 mg/ml glycogen was included, and only about a 20 loss in activity in the absence of glycogen. Glycogen synthase stored in the cold in the presence of glycogen forms a flocculant precipitate. This redissolves completely when warmed at 30 "C for 15 min.

Criteria qf Purity

At the final step in the purification by method 1, the Sepharose 4 B column, absorbance at 280 nm and glycogen synthase activity closely paralleled each other and the last traces of phosphorylase b were removed at this step (Fig. 1). Contamination of glycogen synthase with phosphorylase kinase was still about 0.5 by weight, as judged by enzyme activity measurements. When the active fractions were concentrated and analysed by sedimentation velocity experiments in the ultracentrifuge, two major peaks with sedimentation coefficients of 13.3 S and 19.0 S were observed (Fig. 2). The proportion of the 13.3-S species was five or six times greater than that of the 19.0-S species. In addition a minor component (6.0 S) amounting to about 5 % of the total material was usually, but not

Skeletal Muscle Glycogen Synthase

26 r

100

-

- 80 -

5

5

z

60-

-?

1,L O c

?

u

20-

0

b 10 20 Fraction number

30

Fig. 3. Sedirnetztution uf' pur$ed glycogen synthase in u glycerol density grudirnt. (O- -0) Glycogen synthase activity; 0-0, phosphorylase activity (8.8 S). The arrow denotes the peak tube of B-galactosidase activity (16.0 S). The direction of sedimentation is from right to left

Fig. 2. Srciimrwtutioii oJ puriJkd ~ I J U J @ ? I .s}wilmsi~in huffiv A . The photograph was taken 20 min after reaching a speed of 56000 rev./ min at 18 "C. The enzyme concentration was 3.5 mg/ml. Migration is from left to right

always, present. Whether this is an impurity or a dissociated form of glycogen synthase is not known. The nature of these multiple peaks was investigated further using glycerol density gradient centrifugation. The results (Fig. 3) showed that there were two active species of glycogen synthase with sedimentation coefficients of 13.5 S and 18.8 S. The sedimentation coefficients and relative proportions of these two components indicated that they corresponded to the two major components observed by analytical ultracentrifugation (Fig. 2). The relative proportions of the 13.3 S and 19.0 S forms of glycogen synthase did not alter significantly between 5 "C and 20 "C, nor on storage at 4 "C for one week, nor in the presence of 5 mM glucose-6-P. However prolonged storage did result in the appearance of a small amount of aggregated material which sedimented more rapidly than the 19.0-S component (not illustrated). These species also appeared to be enzymically active as judged by glycerol density gradient centrifugation. The glycogen synthase preparation showed one major band on acrylamide gel electrophoresis in the presence of sodium dodecylsulphate (Fig. 4) and the mobility of this band corresponded to a subunit molecular weight of 88000 (not illustrated). This value is very similar to that obtained in two other labo-

ratories [7,36]. Densitometric tracing indicated that this component comprised 85-90% of the protein staining material on the gel (not illustrated). Three minor protein staining bands, termed A, B and C , with subunit molecular weights of 80000,71000 and 44000 daltons respectively were also observed (Fig. 4). These minor components represent 10- 15 % of the proteinstaining material on the gel and several lines of evidence indicate that they are derived from the 88 000dalton component and are not simply impurities. Firstly, they are not visible after step 3 of the purification, although the 88 000-dalton component is clearly visible [19], but rather they appear to form during chromatography on DEAE-cellulose (step 5). Secondly, all three minor bands are present in both the 13.5 S and 18.8 S species of glycogen synthase which are resolved by glycerol density gradient centrifugation (Fig. 3). Thirdly, appreciable conversion of the 88000dalton component to components A and B occurred on prolonged storage at room temperature (not illustrated) : this was presumably catalysed by trace endogenous proteolytic activity. Since components A and B are usually present in only trace amounts and are sometimes absent, and some preparations lacking component C have been obtained, it seems clear that the component of molecular weight 88000 is indeed the synthase protein. This is strongly supported by studies of the phosphorylation of the enzyme which are described in the following paper [12]. Thus the studies described in this section indicate that at least 90 0 4of the isolated enzyme is active glycogen synthase, but that this protein tends to aggregate and is very susceptible to proteolytic digestion. In this respect it is very similar to phosphorylase kinase [18]. The formation of components A and B can be minimized

21

H. G. Nimmo, C. G. Proud, and P. Cohen Table 2. Amino m i d composition of glycogen s y t h u s e Amino acid

Residues per 88000 g enzyme"

Aspartic acid Threonine Serine Glutdmic acid Proline Glycine Alanine Valine' Methionine Isoleucine' Leucine Tyrosine Phenylalaninc Histidine Lysine Arginine Cysteine Tryptophan

68 48

Total

59 94

38 43 51 50

9 30 74 2x 40 23 44 44 12", 10'

16', 16K 711

* Average of determinations at three times of hydrolysis (24-

12 h).

Fig. 4. Polj~ucrl,br,lio'c.luniide gel ~lrctrol,hore.sis of glrcogen s,ynlhnse in the presence of sociium dodecylsulphute. (I) A mixture of glycogen synthase and the two marker proteins, phosphorylase and serum albumin; (11) glycogen synthase, showing the minor components A, B and C. The migration is from top to bottom, and the gels were stained with Coomassie brilliant blue

by careful sterilisation of all materials used in the preparation. Although the specific activity of glycogen synthase prepared by method 2 is slightly lower than that of the enzyme prepared by method 1, its purity is identical as judged by sodium dodecylsulphate gel electrophoresis.

Amino Acid Composition, Carbolzydrute Content and Phosphate Content The amino acid composition of purified glycogen synthase is shown in Table 2. The number of cysteine residues obtained by perforinic acid oxidation was in good agreement with the thiol group content of the enzyme obtained by titration with 5,5'-dithiobis(2nitrobenzoic acid) and this suggests that there are few, if any, disulphide bridges in the molecule. In comparison with some of the other enzymes of glycogen metabolism, phosphorylase [37], phosphorylase kinase [18] and debranching enzyme [19], the most striking features of the amino acid composition of glycogen synthase are its methionine content and its

Obtained by extrapolation to t = 0. Obtained from 72 h hydrolysis time. Measured after performic acid oxidation (three samples). Obtained by titration with 5,S'-dithiobis(2-nitrobenzoicacid). Determined spectrophotometrically. Determined colorimetrically.

tyrosine : phenylalanine ratio. The methionine content of glycogen synthase is less than half those of the other proteins, on a weight basis : this suggests that cleavage with CNBr may be a suitable method for obtaining information about the chemical structure of the enzyme. The tyrosine :phenylalanine ratio of glycogen synthase is 0.7 whereas this ratio is essentially 1.0 for these other enzyme of glycogen metabolism. The partial specific volume of glycogen synthase calculated from its amino acid composition is 0.732 ml/g at 20 "C. The composition reported here is in reasonable agreement with that described by Takeda et af. [38], except that these authors obtained a significantly higher methionine and lower glutamic acid content while their argininellysine ratio was much higher than in the present work. 1.3 mg of purified glycogen synthase (1 5 nmol) was analysed for the presence of amino sugars 1331. No galactosamine was detected and the amount of ninhydrin-positive material eluting in a position corresponding to glucosamine was about 0.3 mol per 88 OOOg of enzyme. Since this trace of material could well represent a peptide rather than the amino sugar and it was present at a stoicheiometry of considerably less than one inolecule per subunit it seems likely that glycogen synthase contains no covalently attached amino sugars. Purified glycogen synthase was ana-

28

Skeletal Muscle Glycogen Synthase

3.0

2.8 /

- 2.6 E

i’

I

- 2.4 C

/

I

C

The molecular weight of 377000 is consistent with the gel filtration behaviour of the enzyme, and its migration during glycerol density gradient centrifugation. Using the marker proteins phosphorylase kinase, molecular weight 1280000 [18], P-galactosidase, molecular weight 520000 [28] and phosphorylase b, molecular weight 200000 [17], the size of glycogen synthase was estimated as 420 000 by filtration on Sepharose 4 B (Fig. 1). The estimated molecular weight by density gradient centrifugation was 390000 using the same marker proteins (Fig.3).

/ /

22

2.c

DISCUSSION

50.5

51.0

51.5

r2(crn*)

Fig. 5. High-speed sdimeutaiion equilihtium of glycogen synthase in huf;f;.rA at 10 “C. The initial protein concentration was 0.35 mg/ml. The plot of log fringe displacement versus square of distance from the centre of rotation was obtained from interference patterns, photographed after 24 h at 9000 rev./min

lysed for carbohydrate by the phenol-sulphuric acid method [34] and the results indicated that the preparation contained less than 0.2 % carbohydrate by weight, corresponding to about one molecule of glucose per subunit. Alkali-labile phosphate covalently bound to glycogen synthase preparations which possessed an activity ratio in the range 0.7-0.8 was about 0.3 mol per 88000 g of enzyme. Molecular Weight and Subunit Structure

The molecular weight of purified glycogen synthase was estimated by the high-speed sedimentation equilibrium technique, using a preparation of the enzyme which was devoid of the 6-S component observed in some sedimentation velocity experiments (Fig. 2). The plot of log fringe displacement against the square of the distance from the centre of rotation was linear through much of the solution column, but it showed upward curvature near the bottom of the cell (Fig. 5). This indicates that, as would be expected from the sedimentation velocity experiment, the major component of glycogen synthase was contaminated with some material of higher molecular weight. The molecular weight of the enzyme estimated from the linear portion of the curve was 377000. Since the subunit molecular weight is 88 000, this result indicates that the major species of glycogen synthase is a tetramer formed from subunits of uniform size. The 19-S species probably represents an aggregate of the major species of the enzyme, and it is presumably an octamer.

This paper describes simple procedures for obtaining nearly homogeneous glycogen synthase a, which can be scaled up several fold without any increase in preparation time to yield about 50 mg of enzyme from 3000 g muscle (four rabbits) in one week. The final specific activity after reactivation with glycogen was 10 - 20 U/mg. Since the enzyme was assayed in the absence of sodium sulphate which approximately doubles the activity [39], this value is very similar to the specific activity of 30 - 35 U/mg recently reported by Takeda et al. [38]. The major difficulty in the purification is to free glycogen synthase a from glycogen without destroying its activity and without altering its regulatory behaviour. Smith et al. [40] reported a 350-fold purification of glycogen synthetase a in a 300/, overall yield. This procedure did not free the enzyme from glycogen, nor was the preparation homogeneous. Attempts to remove glycogen by digestion with purified salivary amylase, followed by gel filtration, led to considerable losses of activity [40,41]. More recently, the inclusion of 25% glycerol at this step was reported to stabilise the enzyme, but the yield they obtained (20 mg from 3-4 kg muscle) was only about half that obtained in the present work [38]. The novel feature of the present procedure is the use of glycogen phosphorylase and debranching enzyme, present endogeneously in the protein-glycogen complex, to degrade glycogen. Although this treatment caused a decline to about 30 of the original activity, significant reactivation could be achieved by incubation with rabbit liver glycogen. Previous methods of freeing the enzyme from glycogen have employed storage of protein-glycogen pellets at - 15 “C for several months [7] and/or the use of purified salivary amylase [38,40-421. As is discussed in detail in the following paper, these treatments appear to generate forms of glycogen synthase a with modified regulatory properties. In contrast, the present method of degrading glycogen by the action of endogenous enzymes yield synthase preparations that do not show these “desensitization” phenomena [ 121.

H. G. Nimmo, C. G. Proud, and P. Cohen

The sedimentation coefficient of 13.3 S for the major species of glycogen synthase a (Fig. 2) is lower than the value of 14.3 S originally reported by Soderling et al. [7]. However, this has recently been revised to 13.3 S [45]. The molecular weight of native glycogen synthase was difficult to measure accurately by sedimentation equilibrium because of significant amounts of an active 19-Sspecies (Fig. 2,3). However the molecular weight of 377000 was supported by the more empirical methods of gel filtration and density gradient centrifugation (Fig. 1,3). A molecular weight of 360- 370000 was recently estimated by Soderling, by density gradient analysis [45]. The results indicate that the major 13.3-S species of glycogen synthase a is formed from four subunits of molecular weight 88000. Larner and coworkers recently estimated the molecular weight of native glycogen synthase a by acrylamide gel electrophoresis at pH 8.4 [38]. This empirical method showed the presence of two active species of the enzyme with apparent molecular weights of 340000 and 155000. The component of molecular weight 340000 is presumably the tetrameric 13.3-S species, and the results suggest that this species may partially dissociate to active dimers under the conditions of electrophoresis that were employed. Glycogen synthase a has a tendency to undergo proteolytic modification during its isolation. In the present work, the formation of subfragments A and B (molecular weights 80000 and 71 000 respectively) was limited to less than 5 % of the major species (molecular weight 88000). This is much lower than previous preparations of glycogen synthase a in which the proportion of component A approached 50 % [38,42]. The reason for the greater fragmentation of glycogen synthase a in these preparations is unclear, although one possibility is that it is catalysed by trace proteinase activities present in the purified salivary amylase used to degrade glycogen; it is known that saliva contains such activities [43]. On the other hand glycogen synthase b appears much less susceptible to proteolysis during its isolation [38,42]. Incubation of purified glycogen synthase a with trypsin also results in the formation of subfragments which appear to be similar or identical to components A and B. There is now general agreement that the formation of component B by trypsin is associated with a decline in the activity ratio T glucose-6-P [38,42,45]. Takeda and Larner [44] recently reported that the conversion of glycogen synthase a into coniponents A and B involves cleavage near the Cterminus of the protein. This was based on the finding of a single amino terminal sequence (Pro-Leu ----) when either glycogen synthase a, or component B produced by tryptic attack was used [44]. However, as virtually all the enzymes of carbohydrate metabolism in muscle are N-acetylated [46], and the glycogen synthase a preparations they used contained signif-

29

icant amounts of component A, and also component C (see Fig.4), further experiments seem necessary to confirm this idea. The minor 19-S species of glycogen synthase observed in the present work (Fig.2,3) may be an associated form of the major 13.3-S species, in which case it is probably an octamer. However, since no interconversion of these two forms has been observed (see Results), it remains possible that it is a distinct isoenzyme. The presence of multiple forms of two other enzymes of glycogen metabolism in mixed skeletal muscle fibres, cyclic-AMP-dependent protein kinase [47] and phosphorylase kinase [48] is now well documented. Glycogen synthase binds to concanavalin-A-Sepharose columns and can be eluted with a-methyl glucoside [49] (H. G. Nimmo, unpublished observations). It also stains weakly for carbohydrate when sodium dodecylsulphate polyacrylamide gels are stained with the periodate-Schiffs reagent method of Glossmann and Neville [50], whereas two other enzymes of glycogen metabolism, phosphorylase and phosphorylase kinase, do not (H. G. Nimmo, unpublished observations). This raises the possibility that glycogen synthase might be a glycoprotein. However, significant amounts of amino sugars could not be detected by amino acid analysis and estimations for carbohydrate suggested the presence of no more than the equivalent of about one glucose residue per subunit (see Results). This has been confirmed by a gas chromatographic analysis carried out for us by Professor J. Clamp (University of Bristol) in which small amounts of free glucose was the only sugar that could be positively identified. The anomalous “glycoprotein” behaviour of glycogen synthase therefore remains to be clarified. The results presented in this paper provide the basis for more detailed studies of the structure and regulation of the enzyme. The following paper describes the phosphorylation of glycogen synthase a by glycogen synthase kinase-2 and cyclic-AMP-dependent protein kinase [12]. We are very grateful to Mrs Carol Taylor and Mrs Linda Coutie for expert technical assistance, and to Mr Barry Caudwell for making some of the glycogen synthase preparations. This work was supported by research grants from the Medical Research Council, the British Diabetic Association, The Wellcome Trust and the Science Research Council. Philip Cohen is currently the recipient of a Wellcome Trust Special Research Fellowship and Christopher Proud is a postgradudle student of the Science Research Council.

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H. G. Nimmo, C. G. Proud, and P. Cohen: Skeletal Muscle Glycogen Synthase

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H. G . Nimmo, C. G . Proud, and P. Cohen, Department of Biochemistry, Medical Sciences Institute, University of Dundee, Dundee, Great Britain, DD1 4HN

The purification and properties of rabbit skeletal muscle glycogen synthase.

Eur. J. Biochem. 68, 21 - 30 (1976) The Purification and Properties of Rabbit Skeletal Muscle Glycogen Synthase Hugh G. NIMMO, Christopher G. PROUD,...
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