the importance of nucleoside triphosphate inhibition of liver glycogen synthase phosphatase activity

3 4 0 Binchimlca et Biophyxica Aesa, 991 (1989) 340-346 Elsevier

B~A 23111

The importance of nucleoside triphosphate inhibition of liver glycogen synthase phosphatase activity

D a n i e l P. G i l b o e L2 a n d F r a n k Q. N u t t a l l L3

: V ~ t ~ n x Admintstrati~ ?,feStal Center, and DepoFsments of -" Biochemistry ~nd 3 Me~cin~ umr, ersi~ of Mirmesot~ Minneapolis, M N (~£S.A.)

(Received 8 September 1988) (Revised manuscript n:ceived 30 January 1989)

Key words: Liver 81ycogen-symhase phosphatase; Nucleoside triphosphate regulation; Glycogen syathase activation; Glycogen particle enzyme

The liver glycogen particle enntalns constitutive glycogen-symhese plm phalase activity which is inhibited by ATP-Mg in a concen/ration-depeadent numnes wlthhi the physlological renge (le~ = 0.1 raM), Thorefore, we d e ~ wbofla~ other nuclcosid~ tripbosphate-magnesium complexes also inhibit synthase pbosphatase activity. UTP-Mg, Ci 'P-Mg and GTP-Mg were all foond to be inhibitory. The maximmn iol~'bition was 85-90% which was greater than that for ATP-Mg. The le. s for UTP-Mg was comparable to that of ATP-Mg but it was greater for c r P - M g and for GTP-Mg. At in vivo physiological concentrations, both UTP and ATP are possible inbibitors of synthase phnsphatase activity. In the presence of a saturating concentration of ATP-Mg, added LrI'P, Mg increased the inhibition suggesting the presence of at least two distinct nuclcotide binding sites. Substitution of calcium for magnesium in an ATP complex had no effect on the 1o~ , but inemased the maximmn inhibition. The present studies also suggest that in the multlstep conversion of synthasa D to synthase !, ATP-Mg inliibition occurs early in the sequence. Addition of glycogen, a known inbibitor of synthase pbosphatese aetivity, to a reaction mixtme containing 3 mM ATP-Mg did not fro-thin" inhibit synthase pbosphataso ectivity when edded at concentrations up to 22 mg/ml . The latter data suggest that the presence of a nuclenside triphesphate may desensitize the pbosphatase to glycogen inhibition. ATP-Mg and, to a lesser extent, UTP-Mg and CTP-Mg al~ stimulated plmsphorylase phosphatese activity but GTP.Mg did not.

lntrodeetio,~

We have previously demonstrated that fiver synthase phosphatase (EC 3.1.3.16) activity in a glycogen particle preparation is inhibited by ATP-Mg [1]. The inhibition was concentration dependent end very sensitive (I0. 5 = 0.1 .,'aM). Maximum inhibition occurred at a physiological concentration o[ ATP ( - 4 -6 mM). However, at a maximal concentration of ATP-Mg, the reaction was inltibited only approx. 6070.

Modulation of the sensitivity to ATP-Mg inhibition also has been observed [1]. Glocagnn administration to intact rats increased the sensitivity while glucose administration had the opposite effect. The fraction of totai synthase phosphatase activity inhibitable hy ATP-Mg remained unchanged. Irrespective of the in vivo treatment, total phosphatase activity in the absence of ATP-

Correspondence: D.P. Gilbce, VA Medical Center (151). One Veterans Drive, Mineapofis, b/IN 55417, U.S.A.

Mg varied only slightly from preparation to prepara- lion.

In contrast to the inhibition of synthase phosphatase activity, ATP-Mg strongly stimulated phosphorylase phosphatase activity in all prcparation,~ [1].

in the present studies we have determined the effect of other nncleoside triphosphate-magnesium complexes known to be present in liver [2]. In addition, substitution of calcium for magnesium, both of which complex with ATP as diamagnetic ions [3] was tested for effects on symhase phnsphatase activity. The interaction of glycogen end ATP-Mg as inhlbitors of synthase phosphatase activity also was tested. Glycogen is known to inhibit sy~thase phesphatase activity in vitro [4-]. The influence of all these potential modifiers on glycogen particle phnsphnrylase phosphatase activity was determined simultaneously.

Materials end Methods

Liver glycogen, ATP, UTP, GTP end CTP were obtained from Sigma Chemical Co. (St. Louis, MO).

0304-4165/89/$03.50 © 1989 Elsevier Science Publishers ELV. (Biomedical Division)

Uridine diphosphate glucose (UDPG) was obtained from Boehringer-Mannheim Biochemieals (Indianapo- lis, IN). The liver glycogen, prepared by a KOH extrac- tion method, was further purified with a mixed ion-ex- change resin colnnm (MallinkrodL MB-3) before use. All reagent chemicals were of the highest quality availa- ble.

Fed male rats obtained from Biolab (White Bear Lake, MN) weighing 150-200 g were used. Animal maintenance and their preparation for experimentation and the method for obtaining the fiver glycogen par- tides have been described previously [I].

Synthase phosphatase and phusphorylase phusphatase assays were conducted at the same time using a single reaction mixture. Activity was measured as an increase in synthuse I + R activity or a decrease in phosphorylase a activity, respectively. Standard assay methods have been described previously [5,6]. In some' experiments, a modh"ieation of the standard assay was used which is more spgeifie for synthase R. In the standard assay the UDPG concentration is 4.5 mM and the reaction is incubated at pH 8.8. In the modified assay, the UDPG concentration was increased to 13 mM and the reaction was carried out at pH 7.0 [7]. Otherwise the assay conditions were the same as for the standard assay. One unit of either synthase I or phusphorylase a activity is the amount of enzyme which incorporates 1 itmol of glucose into glycogen in 1 rain at 30°C under the assay conditions employed. Synthaso phosphatase activity is expressed as the number of units of synthase I + R generated per minute per gram wet weight of fiver. Reaction mixtures contained 0.2 mM glucose 6-phosphate m assure linear kinetics [1]. Phos- phorylase phosphatase activity is expressed as the decrease in units of pbospborylase u per minute per milliliter of glycogen particle suspension. Glycogen was assayed by a method developed in this laboratory which is a modification of two methods [8,9].

Results

Comparison of ATP-Mg, GTP-M~ CTP-Mg and UTP- Mg as effectom of synthase phasphatase and phasphory- lase phasphatase activities

As demonstrated previously, ATP-Mg inhibited synthase phosphatase activity with an 10. s of 0.1 mM and a maximum inhibition of 60% [1]. Phosphorylase phosphatase activity at 5 mM ATP-Mg was stimulated approx. 350% compared to control activity (Table I).

Synthase phosphatase also was inhibited by UTP-Mg with an In. s of 0.1 mM (Table l). The maximum inhibition was about 855 (Fig. 1). Maxir;,um inhibition of synthase phosphatase activity by GTP-Mg was similar to that of UTP-Mg. However, the 10.s for both was approximately an order of magnitude higher (Table I).

Substitution of calcium for magnesium as the diva- lent ion compleaed with ATP did not change the inhibi- tor constant, lo.s = 0.1 raM. The maximum inhibition of syntbase phosphatase activity increased, however, from approx. 60 to 80% (Table I).

Stimulation of phosphorylase phosphatase activity by UTP-Mg or CTP-Mg at 5 mM was substantially less than with ATP-Mg (Table I). OTP-Mg at this con- eentration did not stimulate phosphorylase phosphatase activity at all. The stimulation observed with these nuclenside triphosphates might be the result of their binding to the so-called nucle, otide binding site on phosphorylase a [10]. If so, the stimulation shcald be di- minished by AMP. When tested, the presence of 0.5 mM AMP rcdueed the stimulation [~y 3 mM ATP-Mg, UTP-Mg and CTP-Mg (data not shown). AMP binds to the nueicoside site with great afiinity and st~biFzes a conformer of phosphorylase a which is a poor phos- p'mtase substrate /11,12]. In contrast AMP sitmulates the synthase phosphatase reaction ~ut the mechanism i-.: not yet k~lnwn [13].

TABLE i Comparison of effects of A TP-Mg and other nucleoside triphosphate.nmgneaium complexes on liver s),nthase phoxphatase activity

Complex In rive Estimated Approx imate Phospholylase phosphatasc Liver treatment 10. ~ (raM) maximum activity (mM) Cone.

inhibition (~) p mol/g ATP-Mg g]ocagon 0.I 60 350~g of control at 5 2.94 " LYIP-M 8 glucagon 0.I 85 176~ of control at 5 0.23 b GTP-Ms glucagon 0.9 80 no stimulation up to 5 0.32 a CTP-M g 81ucagon 1.4 90 19g~ of control at 5 0.083 ~ ATP-Ca 81ucagon 0.l 80 229¢~ of control at 5

a Grabe~, W., Molkfin$, H. and Bersmeyer, H.U. (I~6) Esu~ym. BioL Clln, 7,115. The value for GTP represents the combination of GTP and ITP which are not conveniently discerned.

b Burch+ H.B., Max, P., Chyu, K. and Lowly, O.H. (1969) Biochem. Biophys. Res. CoramutL 34,169. © Domschke, w., Keppler, D., Bischoff~ E. =nd Decker, K. (1971) Hoppe-Seyler's Z. Physiol. Chem. 352, 275.

342

2 o o I 0 0 • o

/ / . t °

4 0 s e ~ :~ 501- o , ~ "-1140 ~. , /

~") I " ~ o. 0.~'5 0.51 2o FI -l mo

"° : :,l t.) o . IOO

o 5 IO 15

UTP-Mg,(mM) Fig. 1. Effect of UTP-Mg on glycogen symhase phosphatase ~nd phospho~lase phosphatase activities. Reaction mixtures contained 0.4 ml of resuspended glycogen particles prepared from glucagon=treated r,~ts as described in Methods, Each mixture contained, in addition, 0.2 mM glucose 6 phosphate and varying amounts of an eqalmolar mixture oi UTP and Mg~2, as indite, feeL The final volume was 0.5 ml. A mixture without UTP-Mg served as a control. Mixtut~ were incubated at 25°C and aliquots were withdrawn at appropriate intervals, dflutad with a stopping reagnet and assayed for synthase (It) or phoephorylase (o). The synthase acfivatio~ rate is fastest in the interval from 2.5 to 10 mill. Phosphofylase phosphatase activity was estimated as the rate of decrease in phosphorylase a in the interval from 0-5 rain. In each case, the phosphalase rates of four separate experiments were meaned and compared to controls prep~.xad without added UTP-Mg. Results ate expressed as a percentage of the control. Data used to ~timate the lo~ for UTP-ME inhibition of synthase phosphatase activity are shown in the inset and were oblalned in the same manner as described above. The data represent results froth three separate experiments. In these experiments, the initial $ synthase I was 12~

~ad the mean total synthas¢ was 0.8 ultils/g ,,vet wL

It is apparen t tha t broth U T P an d A T P at concentra- t ions expected wi th in the li-¢ez cell (Table I) arc signifi- can t inhibi tors o f synthase phospha tase activity. I n ad-

TABLE II

The additive effects of ATP-Mg and UTP-Mg on synthoxe phosphatase ccrivity

Additions to Synthase phosphatase • of reaction mixture reaction rate b maximum

4 units/min per g wet wt, rate

1 Control-none ' 0.044 100 2 +0.25 mM UTP-Mg 0.020 45 3 +3 mM ATP-Mg 0.018 40 4 +0.25 mM UTP-Mg

3 |ttM ATP-M[~ 0*01O 22

= Reaction mixture,~ were prepared from glycogen particles as described in the Legend for Fig, 1 with each mixture containing 0.2 mM glucuse 6*phosphate and addition as indicated. The synthase pho~phatase rate was estinmtad in intervals from 2.5 to 10 mln. The incubation was conducted at 25°C.

b ReSults represent the mean of four sl~arate determinations.

d i t ion , the m a x i m u m inh ib i t ion of sya these phospba tese b y U T P - M g exceeded tha t for A T P - M g . Therefore , the effect o f these inhib i tors in combina t ion , each a t a low phys io |ogica l concentra t ion , was compa red to the indi- v idual effects. I nh ib i t i on wi th A T P - M g was m a x i m a l at 3 m M [ l ] a n d w as greater t han that wi th 0.25 m M U T P - M g as expected (Tab le II). However , when bo th were ad d ed in co mbina t i on the inh ib i t ion was greater and , thus, the effects were addit ive. In a separate experi- m e n t inh ib i t ion o f synthase phaspha ta se activity b y 0.5 m M U T P - M g was readi ly reversed by 20 m M glucose (da ta n o t shown) . Add i t i ona l inh ib i t ion also was observed w h e n 0.25 m M G T P - M g was added together wi th 3 m M A T P - M g (da ta no t shown). Inh ib i t ion by the co mb i n a t i o n of 0.25 m M C T P - M g and 3 r aM A T P - M g was n o t d i f ferent f r om tha t of A T P - M g alone.

The influence of ATP.Mg on 1he jbrmation of intermediate forms of glycogen synth~z~e

T h e dephosphory la t ion of synthase D to fo rm synthase I potent ia l ly involves a n u m b e r of in termediates

which at present are designated collectively as synthase R [7]. The inabili ty to observe nearly complete i~thibi- floP. of synthasa con,~ersion in the presence of ATP-Mg as was seen with UTP-Mg, may reflect the complexity of the process and the inability to measure the change in concentrations of intermediate forms accurately. Therefore, several factors which might influence these measurements were examined in a series of experiments.

In this laboratory, total synthase has been measured a t p H 8.8 since this is the p H maximum for synthase D activity. Synthase I has a broad p H opt imum and, thus, also could be measured efficiently at p H 8.8. Reccofly we have determined that the p H opt imum for the R form is in the range of p H 7 - 7 . 4 and at p H 8.8 the activity is greatly reduced. Thus, the s tandard assay considerably underestimates the activity of synthase R. The estimated Km for U D P G also is lower a t p H 7.0 than a t p H 8.8 [7,14].

fn order to determine the effect that a more effective measurement of synthasa R has on the observed kinetics of ATP- M g initibition, the conversion of synthase D ~o the active species was monitored using the modified essay (Fig. 2A). The rate of the control reaction without added ATP-Mg was greater using the modified assay.

343

This most likely is due to the faster rate of synthase R formation relative to synthase 1. This would he expected i f synthase R formation preceded the formation of synthase I. In the presence of 3 m M ATP-Mg, lit t le difference in ~ e p r o ~ s of the reaction was detected by the two assays. Since the overall reaction is inhibited, the best explanation for these results is that A T P - M g inhibits an early step in the multistep conversion of synthase D to I, resulting in a reduced rate of R formation. Using this assay the reaction was maximally inhibited by about 85~ compared to 60~o usually ob- • ~ e d [1]. This occurred without a change in the lo.s for ATP-Mg (data not shown).

The effects of UTP-Mg on the formation of intermediate forms of glycogen synfhase

Since ATP-Mg appeared to inhibit early in the de- phusphorylation sequence which converts synthase D to I we considered i t important to determine the effects of U T P - M g with the same assay. Experiments were con- dneted as described for ATP-Mg. With the modified assay, the synthase phosphatase reaction was inhibited by 85~ (Fig. 2B) compared to 8090 with the s tandard assay with UTP-Mg. However, after 10 rain, the two

A 0,7

0.6

£ =

o.t

5 tO 15

'rneubotioe Time Imin)

B

l0 20 30

l"ncubotion Time (rain)

Fig, 2. The kinetics of the synthas¢ pho~phetase reaction in Ihe absence and presence of ATP-Mg or UTP-Mg using two separate assays for the measurement of gbcogen synthase. (A) Two separate reaction mLxtures were prepared from each liver tissue preparation. One contained resuspandcd glycogen particles and 0.2 mM glucose 6-phosphate (o, 0). The other had the same composition but 3 mM ATP-Mg was also added (•, a). Samples were incubated at 25°C with aliquots withdrawn at appropriate intervals. Each sample was assayed both with the standard assay and with the modified assay. The standard assay mixture contained 4.5 toM UDPG and was buffered at pH 8.8 to, ~,). The modified assay contained 13 toM UDPG and was buffered at pH 7.0 (0, &). Total synthase was dete,'mined using the standard assay in the presence of 7 mM glucose 6.phosphat~, ~'mh data point repress;As fl:.e mean of four separate determinations with separat¢~ tissue preparations. The mean total synthese was 0.94 units/g wet wt. and the mean initial % syntha.ce 1 was 2t~ measured by the standard ~say. The percent active syntha~ determined by the modified assay was not notably differenL (BI The e~parlment was conducted essentially as described in (A), except that 3 mM UTP-Mg was substituted for ATP-Mg, Curve designations are, fihewise, the same as those in (A), except for the substitution of UTP-MB for ATP-Mg, Each data point represents the mean froto four separate tissue preparations. The total syathase taean concentration was 0.88 units/g wet wt. The mean initial 5g synthase I was 17~ me~ured by the standard assay and the Sg active synthase was not notably different using the

modified assay.

3 4 4

^ 061

~o.o

0.2

O.t

5 I0 15 Incubollon Time (~r,)

ig. 3. The effect of glucose on synthase phosphalase inhibition by ~TP-Mg and UTP-Mg. Two reaction mixtures containing glycogen

~articles obtained as described in Materials and Methods were prepared containing 0.2 mM glucose 6-phosphate and 3 mM ATP-Mg and 025 mM UTP-Mg. A control mixture contained glucose 6-phos- ahate but no other additions (@). Mixtures wel~ warmed to 25°C to ~Jtiate phosphatase activity. At 6 rain. glucose was added (F ina l ~nc.. 20 raM) to one mixture containing ATP and UTP (o) and an ual volume of 50 mM imidazule (pH 7.0) was added to the other i×ture containing the nucleotides (~.). Af iquots were removed at the

ltervals indicated to assay for synthase activity by the standard assay. Each data point repres~ts the mean results from four separate issue preparations. The mean total syndiase was about 0.92 uaits/g e t wl. and was stable during the incubation. The initial estimated

synthase I was about 20%.

plots diverge wi th the a m o u n t of actiw: synthase in the reaction mixture con ta in ing U T P - M g measured b y the modif ied assay increas ing progressively, sugges t ing that yn thase R was accumula t ing . Since U T P - M g is a po ten t n h i b i t o r o f the overall react ion, this resul t indicates ~hat U T P - M g inhibi ts the R to I convers ion mo re s trongly than the synthase D to R conversion.

~he influence of glucose on UTP-Mg inhibition We have previously demons t ra ted tha t g lucose over-

comes A T P - M g inhib i t ion [15]. Present s tudies sug- gested that the U T P - M g and A T P - M g b i n d i n $ sites ~nay be different. Therefore, we were interested in de- t e rmin ing whether glucose wou ld overcome the inhibi -

FABLE Ill

ze effect of UDPG on ATP-inhibited phosphatase reaction

~esuhs represent m e n of three separate determinations.

Sample Addition % control activity

0.3 mM GIc-6-P 100 0.3 mM GIc-6-P, 3 mM ATP, 10 mM Mg 2+ 40 0.3 mM GIc-6-P, 3 mM Mg 2 ÷. t0 mM UDPG 107 0.3 mM GIc-6-P, 3 mM Mg 2÷, 20 mM UDPG 1(30 0.3 mM GIc-6-P, 3 mM Mg 2+,

3 mM ATP, l0 mM UDPG 56 0.3 mM GIe-6-P, 3 mM Mg 2+,

3 mM ATP, 20 mM UDPG 45 0.3 mM GIc-6-P, 3 mM Mg 2+ 105

t ion w h en b o t h A T P - M g and U T P - M g were present at app rox ima te physiological concentrat ions. Add i t i on of glucose at a 20 m M concent ra t ion complete ly overcame the inh ib i t ion (Fig . 3).

The UDPG binding site on syntbase as a possible site for A TP-Mg binding

I t has been repor ted tha t A T P was compet i t ive wi th U D P G for the subs t ra te b ind ing site on g lycogen synthase [16]. T h e impor tance of this si te to A T P - M g inhib i t ion of synthase phospha tase activity was investi- ga ted by de t e rmin ing whether U D P G a t concent ra t ions up to 20 m M affected A T P - M g inhibi t ion. In bo th the presence an d absence of g lucose f -phospha t e (GIc-6-P) there was n o effect o n A T p o M g inhib i t ion (Tab!e II1).

The effect of glucose 6-phosphate on synthase phosphatast actipily in the prezence of ATP-Mg measured by the modified a~ay

Glucose 6 -phospha te causes a change in the kinet ics of the synthase phospha tase activity measured in the presence o f A T P - M g b u t the effect is no t under s tood [13]. N o rma l l y , the react ion course is biphasic , ba t g lucose 6 -phospha te causes it to proceed finearly. U n - l ike glucose, g lucose 6-phosphate nei ther prevents nor reverses A T P - M g inhibi t ion. However , because glucose 6 -phuspha te b o t h s t imulates the synthase phuspha tase react ion a n d acts to mod i fy A T P - M g inhib i t ion o f the react ion, it was i mp or t an t to test the effect o f glucose 6 -phospha te us ing the modi f i ed synthase assay. In the presence of 0.6 m M glucose 6-phosphate , which is near ly

o~

~ ' ~ o.2 ,

¢~ at

4o 20 Zncubotion Time (mln)

Fig. 4. The kinetics of synthase phosphatase activity in die presence of glucose 6-phosphate determined by the n-,cdified synthase assay. Two identical reaction mixtures prepared as described in die legend fo~ Fig. 4 contained 0.6 mM glucose 6-phosphate and 3 mM ATP-Mg (z~, A). Identical control mixtures contained only glucose 6-phosphate (0, 0). Mixtures were incubated at 25°C. After 5 rain, glucose (20 raM, final concentration) wz~ added one mixture of each of the pairs (o. 4) and an equal volume of 50 mM imulazolc (pH 7.0) was added to each of the remaining mixtures (0, A). At various intervals~ afiquots were withdrawn for the determination of active syndiase using the modified synthase assay at pH 7.0. Results represent the mean of three separate determinations. In these experiments the mean initial percent active

synthase was 14% and total syndiase was 0.72 units/g wet wt.

t20

g ioo

~ eo

20

I I t I

tO t5 16 t9 22

Glycogen in Reot:tlon Mixture Img/ml ) Fig. 5. Glycogea inhibition of glycogen synthas¢ phosphatase acti~Ry Without and with ATP-Mg. Centre; reaction mixtures which Con- lathed 0.2 mM glucose 6 - p h ~ t e w ~ found to contain about 10 mg of glycogen per ml. in c, ther mixturc'h the glyco~n ¢onc~atraficm was incrcmcd by the addition of KOH-cxtracted rabbit liver glycogen. Two sets of reaction mixtuxcs were plcpated which were identical. except thai to one was added 3 mM ATP-Mg (@). Mixtures were incubated at 250C. Sytlthase phosphatese activity plotted was the greatest measured rate in the incubation interval from 2.5 to l0 rain. All mulls have been compared to the rate of the control mixture to which neither glycogen nor ATP-Mg were added. Results are re]rotted aS a perc~tage of that rate. Each data point represents the mean of three sepia'ate tissue pfeparatioas, o, activity in the abumce of added

ATP-Mg; O, activity in the presence of ATP-Mg.

saturating (Arts = 0.14 mM), the synthasc phosphatasc reaction was 83% inhibited by ATP-Mg (Fig. 4). Simi- larly, glucose readily overcame ATP-Mg inhibition. Thus, there was tittle difference in the results when the reaction was monitored using the standard or modified assay.

The effect of glycogen on ATP-Mg inhibition At low glycogen concentrations (< 3 mg/ml) syn-

thase phusphatase activity is stimulated [17[ but at high concentrations it is inhibitory [4]. Therefore, the effects of increasing amounts of glycogen and a constant amount of ATP-Mg on synthase phosphatasc activity was tested (Fig. 5). Reaction mixtures were prepared with and without 3 mM ATP-Mg and contained, in addition to a standard amount of a glycogen particle preparation, various additional amounts of liver glycogen. Without added glycogen, the typical reaction mixture contains 10 mg of glycogen per ml. Inhibition of the synthase phosphatasc reaction by ATP-Mg was unaffected by increasing glycogen concentrations up to 22 mg/ml. In the absence of ATP-Mg there was a progressive decrease in activity. At approx. 34 mg/ml glycogen in the presence of ATP-Mg the reaction was 85% inhibited.

345

Discussion

We have previously reported that constitutive glycogen-synthase phosphatase associated with the liver glycogen particle is inhibited by physiological concentrations of ATP-Mg (Io. s =0.1 raM) [1]. However, under the conditions of the assay employed, maximal inhibition was only about 60%. The ATP-caleium complex structurally comparable to ATP-Mg [3], also inhibits synthase phosphatase activity with the same approxi- mate 10s, 0.1 mM, but with greater maximum inhibition.

The present data also clearly indicate that UTP, CTP and GTP in the presence of excess magnesium all inhibit synthas~ phosphatase activity. At maximal con- ccntratious the inhibition was the same for all three ( - 85-90%). The 10. 5 for UTP was similar for that of ATP and both were considerably lower than those for the magnesium complexes of GTP and CTP. Thus, at the usual tissue concentrations (see Table I) ATP attd UTP have the greatest potential as nncleotide modula- tors of sypthase phosphatase activity in rive. Of consid- erable interest was the increased inhibition of synthas¢ phosphatase by the addition of UTP-Mg in the presence of a saturating concentration of ATP-Mg (see Table II), indicating that the effects were additive. This suggests the presence of at least one binding site which differen- tiates between the two complexes.

As noted previously, phospborylase phosphatase activity was strongly stimulated by ATP-Mg in contrast to the inhibition of synthase phosphatase activity [1]. In the present study, magnesium complexes of UTP and CTP were much less potent stimulators of phosphorylase phosphatas¢ activity and GTP-Mg had no effect. Thus, there was little correlation between the stimula- tory effects on phosphorylase phosphatase and the inhibitory effects on synthase phosphatase activity of these nucleotides. This is further evidence for a dissocia- tion of the regulation of the two phosphatase activities. The/~TP-Ca complex also was less potent than ATP-Mg in stimdating phosphorylase phosphatase activity (Ta- ble I). Stimulation of phosphnrylase phosphatase activity by ATP-Mg most likely occurs because ATP binds to the nuclcoside site on phosphorylase a [10]. As a result, the "R" conformer of phosphorylase a is stabilized which, at once, reduces the catalytic capability and makes the protein a better phosphorylase phosphatase substrate [12,18,19]. The nucleotide site binds AMP with greater affinity than ATP and AMP is able to reduce ATP stimulation ]10,20]. in the present studies, AMP also reduces UTP and CTP stimulation. Presuma- bly the same site that binds ATP and AMP also accom- modates UTP and CTP and thus, can account for their respective effects.

The nuclentide effects on the phosphorylase phosphatase reaction observed in vitro are not likely to be

346

physiologically important . The nucleotide site has much greater affinity for A M P tllan for A T P [10,19]. At the average tissue concentration of A T P (3 m m o l / g wet wt.) and A M P (0.2 m m o l / g wet wt.) [21], the ' R ' conformer stabilized by A M P predominates [22]. In contrast physiological concentrations of A M P have been shown not to prevent inhibit ion o f the synthase phosphatase reaction by ATP-Mg. I f A M P and A T P com- pete for the same site, the relative affinity strongly favors A T P binding [13,15]. Thus, synthase phosphatase activity should remain inhibited in vivo at physiological concentrations of both ligands.

The present data suggest that the conversion of synthase D to the intermediate synthase R is inhibited to a greater degree by A T P - M g than the conversion of synthase R to syuthase I. In contrast , U T P - M g may more strongly inhibit the conversion o f synthase R to 1. In any case the inhibit ion by both figands can be overcome by physiological concentrations of glucose. This may be mechanistically important in the regulation of synthase phosphatase activity in vivo.

We have confirmed thai increasing glycogen concentrat ions reduce synthase phosphatase activity [4]. However, in the presence of 3 m M ATP-Mg, the already part ial ly inhibited synthase phosphatase activity shows litt le or no response to increasing glycogen concentra- t ions up to a concentrat ion of 22 m g / m L Thus, the presence o f ATP-Mg appears to desensitize the synthase phusphatase to further glycogen addition. This also could be a potentially important mechanism for regulat- ing glycogen synthesis in vivo,

Acknowledgements

The authors wish to thank Ms. Georjean Madery for excellent technical assistance. This work was supported by grants from the Veterans Administration.

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the importance of nucleoside triphosphate inhibition of liver glycogen synthase phosphatase activity

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