Enzymes of glycogen metabolism in white blood cells II. Activation and inactivation of glycogen phosphorylase of rat chloroma

BIOCHIMICA ET BIOPHYSICA ACTA BBA

65376

ENZYMES OF GLYCOGEN METABOLISM IN WHITE BLOOD CELLS II. ACTIVATION AND INACTIVATION OF GLYCOGEN PHOSPHORYLASE OF RAT CHLOROMA

ADEL A. YUNIS' AND GRACE K. ARIMURA Department of Medicine. University of Miami, Miami, Fla. (U.S.A.J (Received September 1St ,1965)

SUMMARY

1. The properties of glycogen phosphorylase (c-r.a-glucan :orthophosphate glucosyl transferase, EC 2+1.1) ofrat chloroma, a tumor composed entirely ofimmature granulocytes, and the interconversion of the active and inactive forms of the enzyme have been studied in crude chloroma extract. 2. Striking similarities in the properties of chloroma phosphorylase to liver phosphorylase have been noted. Thus, the following properties are shared by both enzymes: Two forms of enzyme can be identified, an active and an inactive form; the active form exhibits 65-80% of its activity in the absence of adenosine 5 '-phosphate, the inactive enzyme is not significantly active even in the presence of the nucleotide; the activity of the enzyme is not enhanced by cysteine; the inactive enzyme shows considerable activity when assayed in the presence of high salt concentration; the enzyme is activated by glucagon. 3. A phosphorylase kinase (ATP :phosphorylase phosphotransferase, EC 2.7.1.38) capable of converting rabbit-muscle phosphorylase b to a and a phosphatase (phosphorylase phosphohydrolase, EC 3.1.3.17) catalyzing the reverse reaction both active at neutral pH have been demonstrated in chloroma tissue.

INTRODUCTION

Phosphorylase isolated from skeletal muscle and from liver exists in two interconvertible forms designated a and b (a-1,4-glucan:orthophosphate glucosyl transferase a and b, EC 2+1.1) for muscle! and active and inactive for liver", Distinct differences have been found in the properties of the enzyme from these two tissues. Thus, muscle phosphorylase b requires AMP for activity- while inactive liver phosphorylase shows only IS % of its activity even in the presence of this nucleotide". Conversion of muscle phosphorylase b to a is accompanied by phosphorylation of the • Leukemia Society, Inc. Scholar.

Biochim, Biophys, Acta, 118 (1966) 325-334

A.

A. YUNIS,

G. K.

ARIMURA

enzyme with dimerization and doubling of the molecular weight"; the corresponding conversion of the liver enzyme is accompanied by phosphorylation but without dimerization-. Furthermore, in contrast to muscle phosphorylase the liver enzyme is activated by glucagon and its activity is not enhanced by cysteine-.s. Several studies dealing with glycogen phosphorylase (a-l,4-glucan:orthophosphate glucosyl transferase, EC 2-4-1.1) of leukocytesv" suggest that this enzyme is more closely related to liver phosphorylase; leukocyte phosphorylase is activated by glucagon" and its activity is not significantly enhanced by cysteine". However, detailed comparative studies on this enzyme have not been done. The present work was undertaken to study some of the properties of glycogen phosphorylase from rat chloromas-», a tumor having many similarities to human granulocytic leukemia and is composed entirely of immature granulocytes. Interconversion of the two forms of the enzyme and some oftheir properties have been studied in crude extracts. Striking similarities are noted in the properties of phosphorylase from rat chloroma and liver phosphorylase. Rat chloroma also possesses a very closely related phosphorylase activation-inactivation system. MATERIAL AND METHODS Yeast AMP crystalline sodium salt, ATP clisodium salt, a-D-glucose r-phosphate dipotassium salt, and glycogen (shell-fish) were obtained from Sigma Chemical Company, St. Louis, Missouri. Glucagon was a gift from Eli Lilly and Company, Indianapolis, Indiana. Epinephrine, I :1000 aqueous solution, and Pseudomonas polysaccharide (piromen, 10 Itg/ml) were obtained commercially. Three-times crystallized rabbit muscle phosphorylase b was prepared as described by FISCHER AND KREBS 12• Muscle phosphorylase a was prepared from the b form according to the method of FISCHER et al.13 . Two chloroma-bearing albino rats were initially obtained through the courtesy of Dr. M. Gruenstein of The Fels Institute of Philadelphia. Repeated transfer of the tumor was performed by the subcutaneous injection of whole cell suspension into 3- to 7-day-old Holtzman albino rats as described by MOLONEY et al. ll . Chloromas weighing up to 70 g were produced in 8-12 weeks. In some instances cells were transferred by intraperitoneal injection with subsequent production of intraperitoneal tumor masses with or without ascites. Preparation of chloroma extract Tumor-bearing rats were sacrificed by exsanguination and the chloroma dissected free of surrounding areolar tissue and rapidly cooled in ice. It was then sliced and homogenized in three volumes of ice-cold 100 mM NaF, I mM EDTA at 45 000 rev.jrnin for go sec in a Virtis 45 homogenizer. The homogenate was centrifuged at 15 000 X g for 30 min in a Sorvall centrifuge yielding a very slightly turbid supernatant. Phosphorylase assay Phosphorylase activity was measured by a modification of the method of SUTHERLAND 4 . The reaction mixture contained enzyme, 1% glycogen, 32 mM glucose r-phosphate, I mM AMP, 5 mM sodium glycerolphosphate (pH 6.3), 10 mM NaF, Biocbim, Biopliys. Acta, TIS (I966)

3~5-·334

327

ACTIVATION-INACTIVATION OF CHLOIWMA PHOSPHORYLASE

and 0.5 mM EDTA in a total volume of 0-4 ml. Substrate was prepared by mixing equal parts of a 4% solution of glycogen and a solution containing 128 mM glucose r-phosphate and 4 mM AMP (pH 6.3). The extract was diluted in a buffer containing 10 mM sodium glycerolphosphate, 20 mM NaF, I mM EDTA (pH 6.3). The reaction was started by the addition of 0.2 ml of dilute extract to 0.2 ml of substrate at 37°. The reaction was terminated in 30 min by the addition of 2. I ml of 5 %trichloroacetic acid and the precipitate eliminated by centrifugation. Pi was determined on the clear supernatant by the method of FISKE AND SUBBARoW14 . A blank in which the enzyme was added after the trichloroacetic acid accompanied each assay. The activity was expressed as flmoles Pi liberated per 30 min per ml of extract or per mg of protein. Protein concentration was determined by the method of LOWRY et al.15 using crystalline bovine albumin as a standard. EXPERIMENTAL AND RESULTS

Phosphorylase activity in chloroma extracts The phosphorylase activities determined on extracts from 12 chloromas are shown in Table 1. The activity ranged between 3.64 and 5.30 ,umoles Pi per 30 min per mg of protein with AMP and between 2.84 and 4.30 ,umoles Pi per 30 min per mg TABLE I PHOSPHORYLASE CONTENT 01·' RAT CHLOROMA

Phosphorylase activity is given for protein.

With AMP Range

12

tumors and expressed as Ilffioles

PI

per 30 min per mg

Without AMP Mean

Range

Mean 3·35

without the nucleotide with an average activity ratio without AMP(with AMP of 0.8; the ratio for active liver phosphorylase is 0.75 (ref. 4). The phosphorylase activity in fresh chloroma extracts was not enhanced by IS mM cysteine. This finding indicated that the enzyme in the extract was primarily in the active form. If one assumes that like inactive liver phosphorylase, inactive chloroma phosphorylase shows no significant activity even with AMP, then the inactive enzyme can be assayed only after activation or conversion to the active form. Conversion of inactive to active liver phosphorylase has been shown to occur readily in crude liver extracts and requires Mg2+ and ATplG. In order to test for inactive chloroma phosphorylase extracts were preincubated with 0.01 M Mg2+- 0 . 0 1 M ATP for 30 min. This procedure increased the activity by 5-35 % further indicating that only a small portion of the glycogen phosphorylase in the extract existed in the inactive state. Conversion to the inactive form A water extract of chloroma was prepared as described and aliquots of 2 ml were distributed in separate test tubes. To one aliquot was added 0.5 ml of o.S M NaF Biochim, Biophys. Acta, IlS (1966) 325-334

A. A. YUNIS, G. K. ARIMURA

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Fig. r a. Inactivation of chloroma phosphorylase at 0° and reactivation with Mg2+-ATP. Aliquots of 2 ml of a water extract of chloroma were placed in two separate tubes. To one tube was added 0.5 ml of 0.5 M NaF and to the other 0.5 ml of water. Both samples were stored in ice and their phosphorylase activity was followed over a period of I week. Reactivation was accomplished by preincubating stored extracts with 0.01 M Mg 2 L O. O I M ATP at 37° for 30 min followed by pnosphorylase assay in the presence of I mM AMP. Fig. lb. Inactivation of chloroma phosphorylase at 37° and reactivation with MgH_ATP. Experiment carried out exactly as in Fig. ra to test the effect of NaF and EDTA on the rate of enzyme inactivation.

and to the other 0.5 ml of water. The extracts were stored in ice and their activity assayed with AMP at the indicated intervals (Fig. raj. By the seventh day of storage the activity in the water extract had dropped to approx. 30% of its original level. This inactivation could be largely prevented in the presence of o.r M NaF. Virtually complete restoration of activity could be accomplished by incubating the stored extract with o.or M MgClg-o.or M ATP at pH 7.0. Activation could be readily blocked by 0.01 M EDTA. A similar experiment wherein the extracts were incubated at 37° (Fig. rb) gave similar results with loss of over 80% of the activity in the water extract in 60 min. The presence of EDTA alone did not prevent inactivation. It is of interest that incubation of the water extract at 37° results in Some degree of irreversible inactivation as shown by failure to restore complete activity with Mg'i!.LATP. The effect of various additions made before storage upon the course of enzyme activity is shown in Fig. 2. Cysteine, EDTA, or AMP had no significant effect. The activity was significantly preserved by NaF or a combination of NaF and EDTA. The addition of Mg2+-ATP with NaF resulted in approx. 30% increase in activity after r day of storage followed by a slow decline to a level slightly higher than the original. Substituting the Mg2+ with r mM EDTA prevented activation ofthe enzyme. The above experiments demonstrate: (1) The presence in chloroma extracts of phosphorylase activating and inactivating enzymes both active at neutral pH. Biochim. Hiophys. Acta, rrS (r966) 325-334

AC1'IVATlON-INACTIVATION OF CHLOROMA PHOSPHORYLASE

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(2) The activating enzyme requires Mg2+ and is completely inhibited by EDTA. (3) The inactivating enzyme is inhibited by F-. (4) The activity of inactive chloroma phosphorylase is not brought out significantly with AMP.

I nierconoereion of muscle phosphorylase a and b with chloroma extract The phosphorylase activating and inactivating enzymes of rat chloroma were further testecl using crystalline phosphorylase b and a as substrates. For the activating enzyme the reaction mixture contained: 0.2 ml of AMP-free rabbit-muscle phosphorylase b (8000 Cori units}!", 0.2 ml of 0.125 M glycerolphosphate-o.rzg M Tris at the indicated pH and o.I ml of extract; the reaction was started by the addition of 0.1 ml of 0.1 M MgCl~-O.I M ATP at 30°. A blank containing all the components ofthe reaction mixture except extract accompanied each experiment. Aliquots of 0.1 ml were taken at intervals and diluted 1 :20 in cold 0.04 M glycerolphosphate-o.oj 1\1 cysteine (pH 6.8), and phosphorylase activity assayed without AMP by the method of Biochim, Biopliys . Acta, lIS (1966) 325-334

A. A. YUNIS, G. K ARIMURA

33°

ILLINGWORTH AND CORIl? The conversion of rabbit-muscle phosphorylase b to a followed a linear course as shown in Fig. 3a. Conversion occurred readily at pH 7.34 but was more rapid at alkaline pH. The conversion of rabbit-muscle phosphorylase a to b using chloroma extract as the converting enzyme is shown in Fig. 3b. The reaction mixture contained 0.2 ml of muscle phosphorylase a (8000 Cori units), 0.3 ml of 0.01 M Tris buffer (pH 7.5), and

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Fig. 3a. Conversion of rabbit-muscle phosphorylase b to a with converting enzyme from chloroma extract. Reaction mixture contained: 0, I ml of dilute extract, 0.2 ml of 3 times crystallized AMPfree rabbit-muscle phosphorylase b (8000 Cori units); 0.2 ml of 0.125 1\1 Tris-o.125 M sodiumglycerolphosphate at the indicated pH; the reaction was started by the addition of o. I ml of 0.1 M MgCl.-LO M ATP at 30°. Aliquots were taken at intervals for phosphorylase assay without AMP. Fig. 3b. Inactivation of rabbit-muscle phosphorylase a to b by chloroma extract. The reaction mixture contained 0.2 ml muscle phosphorylase a (8000 Cori units) 0.3 ml of o.or M Tris (pH 7.5), and 0,1 ml of dilute extract. Aliquots were taken for phosphorylase assay with and without AMP. Control tube contained all reagents except extract. 0.1 ml of extract. The control reaction mixture contained all the components except extract. Inactivation of phosphorylase a to b could readily be demonstrated by a drop in the ratio of activity without AMP/with AMP.

Activation by glucagon. and epinephrine Rats with intraperitoneal chloromas were sacrificed and the ascites fluid rich in chloroma cells was aspirated into a heparinized syringe and distributed in z-ml aliquots into 25-ml incubation flasks. After 30-min incubation at 37° glucagon or other test material was added in a volume of 0.1 ml and incubation continued an additional 30 min. The control flask received 0.1 ml saline. At the end of incubation z ml of cold 0.15 M NaF-o.OOI M EDTA was added to each flask and the cell suspensions transferred into pre-cooled centrifuge tubes. The cells were collected by centrifugation at 1800 X g for IS min and each cell pellet homogenized in 5 ml of 0.1 M Naf'-o.oor M Biochim, Biophys. Acta, lI8 (1966) 325-334

ACTIVATlON-1NAC1'lVATlON OF CHLOROMA PHOSPHORYLASE

331

EDTA in a Virtis 45 homogenizer as previously described. Phosphorylase activity in the homogenates was then assayed with and without AMP. The results are shown in Fig. 4- Glucagon in a concentration of 200 f-lg/ml gave approx. 33 % activation. Epinephrine, 20 f-lg/ml, gave a similar effect. It is of interest that a similar degree of activation was also brought on by piromin, a Pseudomonas polysaccharide.

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Effect oj salt It has been demonstrated that inactive liver phosphorylase will show significant activity if assayed in the presence of high salt concentrations's. A water extract of chloroma was prepared and stored at 0°. Phosphorylase activity was assayed at intervals in the presence of AMP before and after Mg2+-ATP activation. Assay was also performed in the presence of 0.5 M Na 2S04 . The results are illustrated in Fig. 5. Initially when the enzyme consisted largely of the active form, Na2S0 4 had a mild inhibitory effect. As the enzyme became inactivated a marked stimulatory effect of salt became evident. On the tenth day of storage when only about 25 % of the activity had remained, a 3-fold increase in the activity was noted in the presence of 0.5 M Na 2S04' That the loss of activity during storage was due to conversion of the enzyme to the inactive form is shown by the complete restoration of activity by incubating the stored extract with Mg2+-ATP. It should be noted, however, that the stimulation of activity by salt is probably not brought on by the same mechanism as that of Mg2+-ATP activation, e.g. conversion of inactive to active phosphorylase, since Biochim. Biophys, Acta, lIS (1966) 325-334

33 2

A. A. YUNIS, G. K. ARIMURA

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lowering the salt concentration by dialysis or dilution reduces the activity to its original level. The effect of increasing salt concentrations on the activity of a ro-days old extract is shown in Fig. 6. A concentration of 0.5 M of Na2S04 gave optimum stimulation. (NH4)2S04 in equimolar concentration was much less effective. Among other salts tested sodium acetate was also slightly stimulatory but NaCl, MgS0 4, and NH 4Cl were inhibitory (Fig. 7).

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Biochim. Biophys. Acta, lI8 (Ig66) 32;;-334

ACTIVATI0N-!NACTIVATlON OF CHLOROMA PHOSPHORYLAsE

333

Effect of heavy metals

The enzyme was strongly inhibited by heavy metals. Approx. 80% inhibition resulted from the addition of 50 flM Hg2+, 0.5 mM Ag+ or Cu2+, to the reaction mixture. DISCUSSION

SUTHERLAND and co-workers characterized two forms of phosphorylase from Iiver originally designated inactive and active liver phosphorylase-. The inactive form is converted to the active form by a specific kinase in a reaction requiring Mg2+ and ATP involving the phosphorylation of serine residues in the protein-". Active liver phosphorylase is inactivated by phosphorylase phosphatase (phosphorylase phosphohydrolase, EC 3.I.3.I7) which releases Pi from the protein" and hence the name dephosphophosphorylase for the inactive liver enzyme. Both phosphorylase kinase (ATP:phosphorylase phosphotransferase, EC 2.7.1.38) and phosphorylase phosphatase are active at neutral pH. Active liver phosphorylase exhibits 60-85 % of its activity in the absence of AMP while inactive liver enzyme shows very little activity even in the presence of the nucleotide. The results of the present study show that glycogen phosphorylase of chloroma exists largely in the active form. This is in contrast to extracts of rabbit and human skeletal muscle and of rabbit-heart muscle in all of which the enzyme is primarily in the inactive form 19 - 21 . It is also of interest that the phosphorylase kinase of chloroma is active at neutral pH, a property shared by phosphorylase kinase ofliver but not of muscle; when assayed in resting muscle phosphorylase kinase is essentially inactive at pH 7.0 but shows its maximal activity at pH 8.6 (ref. 2:2). 'WILLIAMS AND FIELD 6 have shown that glycogen phosphorylase of human leukocytes was activated by glucagon. A similar activation though less striking, could be demonstrated with chloroma phosphorylase. The activation by piromin, a Pseudomonas polysaccharide, is of interest since this material has been shown to cause marked increase in lactic acid production by leukocytes 23 ,24 . Of particular interest is the ready reversible conversion of rabbit-muscle phosphorylase b to a by chloroma extract. While both phosphorylase kinase and phosphatase are highly specific in that they do not act on proteins other than glycogen phosphorylase, neither enzyme appears to possess a high degree of organ or species specificity. Thus, beef spleen contains an enzyme which converts rabbit-muscle phosphorylase a to b (ref. 25). Dog-liver phosphorylase phosphatase catalyzes the release of Pi from dog-heart phosphorylase a (ref. 26). Phosphorylase kinase from human skeletal muscle will convert rabbit-muscle phosphorylase b to a (ref. 27) and rabbit-muscle phosphorylase kinase will convert human-muscle phosphorylase b to a (ref. 20). However, one exception to this pattern has been observed; lobster-muscle phosphorylase kinase does not act on rabbit-muscle phosphorylase b (ref. 28). COWGILL 28 reported that lobster phosphorylase b was activated by high salt concentration. RILEY AND HAYNES 29 described a similar salt effect on dephosphophosphorylase of adrenal cortex; these authors showed that Na ZS0 4 activation was 93 % as effective as kinase activation. Activation of dephosphophosphorylase ofliver by Na ZS0 4 has also been described". The mechanism of activation by salt has not been elucidated. In all instances the activation has been reversible with removal of Biochim, Biophys. Acta, !IS (1966) 325-334

334

A. A. YUNIS, G. K. ARIM UR A

the high sa lt concentration suggesting that the mechanism of activation is probably different from t hat of kinase-Mgvc-A'Tf' activ ation. The present st udies demonstrate that inactive chloroma phosphorylase shares this property with inactive lobst er, adrenal cortex, and liver phosphorylase. It is interesting to note that t he active form of the enzyme is inhibited by high salt concentration and therefore in order to obtain significant activation by salt the major part of the enzyme must be in the inactive form . It would thus be of great inter est to exam in e the effect of high salt concen tration on the behavior of purified inactive phosphorylase and phosphorylase kinase.

ACKNOWLEDGEMENTS

This work was supported by R esear ch Grant AMo900I-01 from th e National Institut e of Health U.S. Public H ealth Servic e and Research Gran t P-39 1 from the American Cancer Society. R E F E REN CES I C. F. CaRL, G. T . CaRL AND A. A. GR EEN, J. B ioi. Chem .• 151 (1943) 39 · 2 W . D . 'W OSILAIT AND E . "V. SUTHERLAND, j. B ioi . Ch em .• 218 (1956) 4 69. 3 P .]. KELLER AND G . T. CaRL . Biochim. Bioplz)'s . A eta, 12 (19 53) 35 · 4 E . W. S UTHERLAND AND W . D. WOSILAIT, j. B ioi. cue«; 218 (19 5 6) 459· 5 "V. C. HiiLS~1AN N, T. L. DEI AND S . V. CREVELD, Lancet. 9 (J96I) 5 8 1. 6 H . E. WILLIAMS AND J. B. FIELD, ] . cu« Ln uest., 40 (1961) 184 1. 7 A . A . YUNIS AND G . K . A RIMURA. Cancer Res.. 24 (19 64) 489. 8 H . SHAY, M. GRUENSTEIN. H. E . M ARKS AND L. GLAZER. Cancer R es .• I I (195 1) 29· 9 H. SHAY, 1'1, GRUE NSTEIN, C. H ARRIS AND L. GL AZER, Blood, 7 (195 2) 622 . 10 H . SHAY, M. GR UENSTEIN AND C. HARRIS, Acta Haema toi ., 14 (1955) 337 · I I W. C. MOLONEY, A . D . DORR. G . Down AND A. BOS CHETTI, Blood, 19 (19 6 2 ) 45 · 12 E . H . FISCH ER AND E . G . KREBS, j. Bioi. Chem ., 23 1 (1958) 65 · 13 E . H . FISCHER , E . G . KRE BS AND A . B . KENT. B iochem . P I·ep n., 6 (1 958) 68 . 14 C. H . F ISIm AND Y . SUBBAHo w , j. B ioI. 66 (19 25) 3 75 · 15 O. H . LOWRY, N . J. ROSEBROUGH, A. L. FARR AND R . J. R ANDALL. j. B ioI . Chem., 193 (195 1) 26 5. 16 T . "V. RALL, E . W . SUTHERLAND AND W . D . WOSILAIT, ]. B ioi. ctu-«, 218 (195 6) 4 8 3 . 17 B . ILLINGWORTH AND G. T . Conr , Biochem , P rep-n. , 3 (195 3) l. 18 E . H . FISCHER , M . M. ApPLEMAN AND E . G . KREBS , Ciba Found. Symp . Control , Glycogen Metab., 1964, Chur chill, L o ndon. 1964. p . 94 . 19 E . G. KREBS AND E . H . F ISCHER. ] . BioI. Chem ., 216 (195 5) II3· 235 (1960) 3 16 3 . 20 A. A. YU NIS. E. H . FISCHER AND E . G . K REBS. j. B ioI. 21 A. A . YUNIS, E . I-I. FISCHER AND E . G . KREBS, ]. Bioi , Chem ., 237 (1962) 2809· 22 E . G . KREBS, D . ] . GRANES AND E. H . FISCHER , j. Bioi. Chen:.• 2 34 (1959) 286 7. 23 S. P . M ARTIN. G. R. McKINNEY AND R. GREE N. Ann. N. Y . A cad. Sci., 59 (19 55) 99 6 . 24 A . A . YUNIS AND W. J, HAHRINGTON. ]. L ab. cu« Med .. 55 (19 60) 5 84 . 25 G . T . Con i AND A . A. GREEN .]. B ioi. Chem ., 151 (1943) 3 1 . 26 T . W. R ALL. W. D . WOSILAIl' AND E. W. SUTHERLAND, Biochim . B iopbys. Acta. 20 (195 6) 69· 27 R. SCHMID, P . W . R OBBINS AND R. R. TRA U1', P roc. N atl. A cad. S ci. U .S ., 45 (19 59) 1 236. 28 R. W . COWGILL, ]. Bioi. Ch em ., 234 (19 59) 3146. 29 G . A. HILEY AND R. C. H AYNES JR" ]. B ioi . Chem .• 23S (1963) 15 63 .

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B iochim, B iophys , A cta , rIS (1 966) 3 2 5-334