- Email: [email protected]
On the nature of cellular ADP-ribosyltransferase from rat liver specific for elongation factor 2
Vol. 139, No. 3, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
September 30, 1986
Pages 1210-1214
ON THE NATURE OF CELLULAR ADP-RIBOSYLTRANSFERASE FROM RAT LIVER SPECIFIC FOR ELONGATION FACTOR 2 O. Sayhan ÷,
M. Ozdemirli t,
R. Nurten ÷
and E. Bermek +~
+Biyofizik Bilim Dalz, Istanbul Tip FakOItesi ~apa , Istanbul ~Biyoloji BSI~mH, TUBITAK Temel Bilimler Ara@tzrma EnstitHsH, Gebze, Kocaeli, Turkey Received August I, 1986 SUMMARY: A cellular ADP-ribosyltransferase, specific for elongation factor 2 (EF-2), is found in extracts from rat liver. Co-migrating with EF-2 throughout purification, this activity is, moreover, located in the protein bands corresponding to EF-2 after native or sodium dodecyl sulfate polyacrylamide gel electrophoresis. The observed activity is thus implicated to be an inherent property of EF-2. Preincubation of EF-2 with GuoPPCH2Pox inhibits endogenous, but not diphtheria toxin catalyzed ADP-ribosylation. © 1986 Academic Press, Inc.
Various bacterial toxins exert their effect in the eukaryotic cell through (mono-)ADP-ribosylation of the representants of a special class of regulatory proteins. Having the property of interaction with guanine nucleotides in common, these proteins are generally called G proteins (for recent reviews see references 1,2). Elongation factor2(EF-2) has been the first of G proteins, shown to undergo ADP-ribosylation which is catalyzed by the fragment A of diphtheria toxin (3) and toxin A of P.aeruginosa
(4). Bacterial toxin-catalyzed
ADP-ribosylation reactions have been once regarded as simulations of some physiological events in the eukaryotic cell and these toxins as products of genes accidentally introduced into bacterial chromosome (5,6). The presence of a cytosolic ADP-ribosyltransferase catalyzing the (mono-)ADP-ribosylation of several endogenous proteins has provided the first evidence supporting this view (7). Moreover, a cellular ADP-ribosyltransferase specific for EF-2 and its diphthamide residue (8) has been, recently, found in polyoma virus transformed baby hamster kidney cells (9), beef liver (i0) and rabbit reticulocytes (ll). Despite its regular presence in EF-2 containing protein fractions, this activity has been regarded as pertaining to a protein distinct from EF-2 (i0,ii). The findings of the present report suggest, however, that the observed endogenous ADP-ribosyltransferase is an inherent property of EF-2.
Abbreviations: EF-2, eukaryotic elongation factor 2; ADP-ribose, adenosine diphosphate ribose; GuoPPCH2Pox, guanosine 5'-(~,y-methylene) triphosphate periodate-oxidized; NaDodSO 4, sodium dodecyl sulphate. 0006-291 X/86 $1.50
Copyright © 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.
1210
VOI. 139, No. 3, 1 986
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
MATERIALS AND METHODS: All reagent grade chemicals were obtained from Sigma, Chem. Comp. St.Louis. Diphtheria toxin was a gift from Behring Werke, Marburg. GuoPPCH2P was periodate-oxidized according to (12). EF-2 was purified as described (12). Shortly, EF-2 was isolated from the rat liver postribosomal supernatant (S-100) by adsorption to hydroxyapatite and elution with 150 mM potassium phosphate, pH 7.0. It was further purified by chromatography on DEAE-cellulose (DE-52 Whatman). EF-2 activity in the 15-65 mM KCI eluate at pH 7.4 was purified by isoelectric focusing in an LKB 8100-1 column in the presence of Ampholine carrier ampholytes, pH range 5-8. The EF-2 fraction obtained from the pI 6.6 region was subsequently separated from ampholytes by filtration through a Sephadex G-100 column. EF-2 obtained by this procedure was more than 85 percent pure as analyzed by NaDodSO4/polyacrylamide gel electrophoresis (13). ADP-ribosylation was performed in 0.05 ml reaction mixtures containing 50 mM Tris-HCl, pH 7.4, 7 mM 2-mercaptoethanol and 7 pM (adenosine-U-14C)NAD ÷ , specific activity 260 mCi/mmol, Radiochemical Center, Amersham, in the presence or absence of diphtheria toxin and the extent of ADP-ribosylation of EF-2 determined as described (14). If not otherwise indicated, incubation time was 15 min for diphtheria toxin-catalyzed reaction (i.e. ADP-ribosyl acceptor activity assay of EF-2) and 2 h for that catalyzed by endogenous ADP-ribosyltransferase. Electrophoresis in native polyacrylamide gels was performed according to (15). EF-2 in NaDodSO 4 polyacrylamide gels was renatured as described (16). RESULTS: As shown in Fig.l, ADP-ribosyltransferase activity found in extracts from rat liver co-migrated with EF-2 activity throughout purification. The
8000~ cpm
A280 2.0
A
4000
8000
1.0
B15~3045
60
PH 8 6
4000
4 2
1'0
20 3() 40 Froction number Fi$.l. Co-migration of EF-2-specific ADP-ribosyltransferase activity with EF-2 during different purification steps. (A), Elution of EF-2-ADP-ribosyl acceptor and-transferase activities from DE-52 column (2x15 cm) between 15-65 mM KCI in standard (dialysis) buffer (12). Elution buffers containing 15 mM and 65 mM KCI, respectively, were added as indicated with arrows. EF-2 fraction used in this experiment was obtained by hydroxyapatite fractionation and contained total EF-2-specific endogenous ADP-ribosyltransferase activity present in the S-100 fraction. (B), Electrofocusing of EF-2-ADP-ribosyl acceptor and-transferase activities at pH 6.6. EF-2-ADP-ribosyl acceptor andtransferase activities obtained from the electrofocusing step eluted also from Sephadex G-100 column together (not shown). Principally, the same profiles of activity were obtained in all experiments performed in parallel where the indicated fractions were assayed for ADP-ribosyltransferase activity in the presence of EF-2, added as ADP-ribose acceptor. Details of experimental procedure was as described under Methods. cO---O), A280; (I~---O), ADPribosylation in the presence and (~--~), in the absence of diphtheria toxin; (I--l~, pH.
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Vol. 139, No. 3, 1986
BIOCHEMICAL AND BIOPH~'SlCAL RESEARCH COMMUNICATIONS
specific activity of ADP-ribosyltransferase with that of EF-2:
increased accordingly
in comparison to that of the starting fraction
in parallel (S-100),
it showed a nearly 3000 fold increase after G-100 step (not shown). The specific activity of the ADP-ribosyltransferase this final step (4.7 pmol ADP-ribose
in the EF-2 fraction after
incorporated EF-2 protein
(ug) -I
time (hr) -I) was comparable to that in polyoma virus transformed baby hamster kidney cells
(9). Unlike activities
ADP-ribosyltransferase
in other systems
over months at -70°C. The ADP-ribosyltransferase located in the protein bands corresponding polyacrylamide
(9-11), the rat liver
activity was fairly stable and could be preserved activity was, moreover,
to EF-2 in both native and NaDodSO 4
gels (Fig.2)-a finding strongly suggesting that this activity
is an inherent property of EF-2.
cpm A
800
400
5
10
15
cpm 800
400
5 Froction
10
1S
number
Fig.2, Localization of ADP-ribosyl acceptor and-transferase activities in EF-2 bands in polyacrylamide gels. (A) 70 ~g EF-2 protein was electrophoresed in polyacrylamide gels at 4°C according to (15). Control sample was stained and destained. Samples electrophoresed in parallel were cut into 1 mm slices and homogenized in 300 ~i dialysis buffer in Eppendorf tubes. ADP-ribosyl acceptor (2 ug diphtheria toxin present) (O--~) and transferase (no diphtheria toxin) (0---0) activities in 50 U1 eluates were assayed as described under Methods. (B), Electrophoresis of EF-2 protein in NaDodSO 4 polyacrylamide gel electrophoresis. Samples containing I0 ~g EF-2 protein in 0.5 percent NaDodSO 4 and 140 mM 2-mereaptoethanol were electrophoresed according to (13). Control samples was stained and destained. Unstained gel samples were cut into 1 mm slices which were treated for renaturation as described (16). I00 U1 eluates were assayed for ADP-ribosylationo (O---O), ADP-ribosylation with 2 ug diphtheria toxin present; (~---Q),ADP-ribosylation without diphtheria toxin; ( x ~ × ) ADP-ribosylation without diphtheria toxin but in the presence of EF-2. a, EF-2; b, bovine serum albumin; c, trypsinogen; d, cytochrome c.
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Vol. 139, No. 3, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
cpm
I000
600
200 30
60
go
120
150 t(min)
Fig.3. The effect of the binding of GuoPPCH2Pox to EF-2 on the ADP-ribosyl acceptor and-transferase activities. EF-2 was incubated with or without i00 ~M GuoPPCH2Pox for 15 min at 37°C and dialyzed for 2 hours at 4°C against dialysis buffer. ADP-ribosylation was assayed in 120 ~i reaction mixtures containing 8.4 ~g EF-2 protein in the presence or absence of 2 ~g diphtheria toxin as described under Methods. After indicated periods I0 ~i aliquots were plated on GF/C (Whatman) filters and the extent of ADP-ribosylation determined as described. (e~---O), EF-2 treated with G~oPPCH2Pox ; (~ ~), EF-2 treated with GuoPPCH2Pox plus equal amount of control EF-2; ([]--[]), control EF-2; (m--~), control EF-2 (twice amount); (~--f~), EF-2 treated with GuoPPCH2Pox plus diphtheria toxin; (x--x), control EF-2 plus diphtheria toxin; ((~----O),no EF-2.
The incubation of EF-2 with GuoPPCH2Pox (which has been shown to bind specifically to EF-2 (12) and inhibit its activity in polymerization and ribosome-dependent GTPase reactions (17))
depressed the ADP-ribosylation
to 30 percent of its control value (Fig.3). The addition of EF-2-GuoPPCH2Pox conjugate had also a rate-limiting effect on ADP-ribosylation of control EF-2. However, EF-2 incubated with GuoPPCH2Pox retained its ADP-ribosyl acceptor activity in the reaction catalyzed by diphtheria toxin. DISCUSSION: This study attests, consistent with the recent findings (9-Ii), to the presence of an endogenous (EF-2 specific) ADP-ribosyltransferase activity in highly purified EF-2 fractions from rat liver. The strict co-migration of ADP-ribosyltransferase with EF-2 throughout purification and during native and NaDodSO 4 polyacrylamide gel electrophoresis suggests that this activity is an intrinsic property of EF-2. The partial inhibition of ADP-ribosyltransferase activity observed after treatment with GuoPPCH2Pox implicates that the binding of oxidized guanine nucleotides to EF-2 affects the ADP-ribosyltransferase site and/or the interaction of ADP-ribosyltransferase and acceptor sites of EF-2 molecules through some conformational changes. Previously guanine nucleotides have been shown to depress the rate of diphtheria toxin-promoted ADP-ribosylation (18,19). ADP-ribosylated proteins constitute a sub-group of the G proteins which fulfill important regulatory tasks in cellular processes (2). That EF-2, a typical
1213
Vol. 139, No. 3, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
representative of G protein~ possesses an intrinsic ADP-ribosyltransferase activity, suggests the existence of a cellular control mechanism based on the autoregulation of the activity of these proteins in the cell. Since, EF-2 with intrinsic ADP-ribosyltransferase
activity should mean a constitutive
depression of protein synthesis, the requirement arises that this activity is under stringent control in the cell (9). Indeed, an ADP-ribosyl-EF-2 glycohydrolase activity present in the cell may play a role in the extent of ADP-ribosyl-EF-2
formed and in the rate of protein synthesis. Alternatively,
(or additionally) some effectors present in the cell sap may modulate the ADP-ribosyltransferase
activity.
REFERENCES i) Ueda, K., and Hayaishi, O. (1985) Ann.Rev. Biochem. 54, 73-100. 2) Hughes, S.M. (1983) FEBS Letters 164, 1-8. 3) Honjo, T., Nishizuka, Y., Hayaishi, O., and Kato, I. (1968), J.Biol. Chem. 243, 3553-3555. 4) Iglewski, B.H. and Kabat, D. (1975) Proc.Natl.Acad.Sci. USA, 72, 2284-2288. 5) Pappenheimer, Jr.A.M., and Gill, D.M. (1973) Science 182, 353-358. 6) Pappenheimer, Jr.A.M., (1977) Ann.Rev. Biochem. 46, 69-94. 7) Moss, J., and Vaughan, M. (1978) Proc.Natl.Acad. Sci. USA 75, 3621-3624. 8) Van Ness, B.G., Howard, J.B. and Bodley, J.L. (1980) J.Biol. Chem. 255, 10710-10716. 9) Lee, H., and Iglewski, W.J., (1984) Proc.Natl.Acad.Sci. USA 81, 2703-2707. i0) Iglewski, W.J., Lee, H., and MHller, P. (1984) FEBS Letters 173, 113-118. ii) Sitikov, A.S., Davydova, E.K., and Ovchinnikov, L.P. (1984) FEBS Letters 176, 261-263. 12) Nurten, R., and Bermek, E. (1980) Eur.J.Biochem. 103, 551-555. 13) Laemmli, U., (1970) Nature 227 , 680-685. 14) Bermek, E. (1976) J.Biol.Chem. 251, 6544-6549. 15) Davis, B.J. (1964) Ann NY Acad. Sci. 121, 404-427. 16) Hager, D.A., and Burgess, R.R. (1980) Anal.Biochem. 109, 76-86. 17) Bilgin, N., Nurten, R. and Bermek, E. (in preparation). 18) Raeburn, S., Goor, R.S., Schneider, J.A. and Maxwell, E.S. (1968), Proc.Natl.Acad. Sci. USA 61, 1428-1434. 19) Goor, R.S., and Maxwell, E.S. (1969) Cold Spring Harbor Symp. Quant.Biol. 3__44,609-610.
1214
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
September 30, 1986
Pages 1210-1214
ON THE NATURE OF CELLULAR ADP-RIBOSYLTRANSFERASE FROM RAT LIVER SPECIFIC FOR ELONGATION FACTOR 2 O. Sayhan ÷,
M. Ozdemirli t,
R. Nurten ÷
and E. Bermek +~
+Biyofizik Bilim Dalz, Istanbul Tip FakOItesi ~apa , Istanbul ~Biyoloji BSI~mH, TUBITAK Temel Bilimler Ara@tzrma EnstitHsH, Gebze, Kocaeli, Turkey Received August I, 1986 SUMMARY: A cellular ADP-ribosyltransferase, specific for elongation factor 2 (EF-2), is found in extracts from rat liver. Co-migrating with EF-2 throughout purification, this activity is, moreover, located in the protein bands corresponding to EF-2 after native or sodium dodecyl sulfate polyacrylamide gel electrophoresis. The observed activity is thus implicated to be an inherent property of EF-2. Preincubation of EF-2 with GuoPPCH2Pox inhibits endogenous, but not diphtheria toxin catalyzed ADP-ribosylation. © 1986 Academic Press, Inc.
Various bacterial toxins exert their effect in the eukaryotic cell through (mono-)ADP-ribosylation of the representants of a special class of regulatory proteins. Having the property of interaction with guanine nucleotides in common, these proteins are generally called G proteins (for recent reviews see references 1,2). Elongation factor2(EF-2) has been the first of G proteins, shown to undergo ADP-ribosylation which is catalyzed by the fragment A of diphtheria toxin (3) and toxin A of P.aeruginosa
(4). Bacterial toxin-catalyzed
ADP-ribosylation reactions have been once regarded as simulations of some physiological events in the eukaryotic cell and these toxins as products of genes accidentally introduced into bacterial chromosome (5,6). The presence of a cytosolic ADP-ribosyltransferase catalyzing the (mono-)ADP-ribosylation of several endogenous proteins has provided the first evidence supporting this view (7). Moreover, a cellular ADP-ribosyltransferase specific for EF-2 and its diphthamide residue (8) has been, recently, found in polyoma virus transformed baby hamster kidney cells (9), beef liver (i0) and rabbit reticulocytes (ll). Despite its regular presence in EF-2 containing protein fractions, this activity has been regarded as pertaining to a protein distinct from EF-2 (i0,ii). The findings of the present report suggest, however, that the observed endogenous ADP-ribosyltransferase is an inherent property of EF-2.
Abbreviations: EF-2, eukaryotic elongation factor 2; ADP-ribose, adenosine diphosphate ribose; GuoPPCH2Pox, guanosine 5'-(~,y-methylene) triphosphate periodate-oxidized; NaDodSO 4, sodium dodecyl sulphate. 0006-291 X/86 $1.50
Copyright © 1986 by Academic Press, Inc. All rights of reproduction in any form reserved.
1210
VOI. 139, No. 3, 1 986
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
MATERIALS AND METHODS: All reagent grade chemicals were obtained from Sigma, Chem. Comp. St.Louis. Diphtheria toxin was a gift from Behring Werke, Marburg. GuoPPCH2P was periodate-oxidized according to (12). EF-2 was purified as described (12). Shortly, EF-2 was isolated from the rat liver postribosomal supernatant (S-100) by adsorption to hydroxyapatite and elution with 150 mM potassium phosphate, pH 7.0. It was further purified by chromatography on DEAE-cellulose (DE-52 Whatman). EF-2 activity in the 15-65 mM KCI eluate at pH 7.4 was purified by isoelectric focusing in an LKB 8100-1 column in the presence of Ampholine carrier ampholytes, pH range 5-8. The EF-2 fraction obtained from the pI 6.6 region was subsequently separated from ampholytes by filtration through a Sephadex G-100 column. EF-2 obtained by this procedure was more than 85 percent pure as analyzed by NaDodSO4/polyacrylamide gel electrophoresis (13). ADP-ribosylation was performed in 0.05 ml reaction mixtures containing 50 mM Tris-HCl, pH 7.4, 7 mM 2-mercaptoethanol and 7 pM (adenosine-U-14C)NAD ÷ , specific activity 260 mCi/mmol, Radiochemical Center, Amersham, in the presence or absence of diphtheria toxin and the extent of ADP-ribosylation of EF-2 determined as described (14). If not otherwise indicated, incubation time was 15 min for diphtheria toxin-catalyzed reaction (i.e. ADP-ribosyl acceptor activity assay of EF-2) and 2 h for that catalyzed by endogenous ADP-ribosyltransferase. Electrophoresis in native polyacrylamide gels was performed according to (15). EF-2 in NaDodSO 4 polyacrylamide gels was renatured as described (16). RESULTS: As shown in Fig.l, ADP-ribosyltransferase activity found in extracts from rat liver co-migrated with EF-2 activity throughout purification. The
8000~ cpm
A280 2.0
A
4000
8000
1.0
B15~3045
60
PH 8 6
4000
4 2
1'0
20 3() 40 Froction number Fi$.l. Co-migration of EF-2-specific ADP-ribosyltransferase activity with EF-2 during different purification steps. (A), Elution of EF-2-ADP-ribosyl acceptor and-transferase activities from DE-52 column (2x15 cm) between 15-65 mM KCI in standard (dialysis) buffer (12). Elution buffers containing 15 mM and 65 mM KCI, respectively, were added as indicated with arrows. EF-2 fraction used in this experiment was obtained by hydroxyapatite fractionation and contained total EF-2-specific endogenous ADP-ribosyltransferase activity present in the S-100 fraction. (B), Electrofocusing of EF-2-ADP-ribosyl acceptor and-transferase activities at pH 6.6. EF-2-ADP-ribosyl acceptor andtransferase activities obtained from the electrofocusing step eluted also from Sephadex G-100 column together (not shown). Principally, the same profiles of activity were obtained in all experiments performed in parallel where the indicated fractions were assayed for ADP-ribosyltransferase activity in the presence of EF-2, added as ADP-ribose acceptor. Details of experimental procedure was as described under Methods. cO---O), A280; (I~---O), ADPribosylation in the presence and (~--~), in the absence of diphtheria toxin; (I--l~, pH.
1211
Vol. 139, No. 3, 1986
BIOCHEMICAL AND BIOPH~'SlCAL RESEARCH COMMUNICATIONS
specific activity of ADP-ribosyltransferase with that of EF-2:
increased accordingly
in comparison to that of the starting fraction
in parallel (S-100),
it showed a nearly 3000 fold increase after G-100 step (not shown). The specific activity of the ADP-ribosyltransferase this final step (4.7 pmol ADP-ribose
in the EF-2 fraction after
incorporated EF-2 protein
(ug) -I
time (hr) -I) was comparable to that in polyoma virus transformed baby hamster kidney cells
(9). Unlike activities
ADP-ribosyltransferase
in other systems
over months at -70°C. The ADP-ribosyltransferase located in the protein bands corresponding polyacrylamide
(9-11), the rat liver
activity was fairly stable and could be preserved activity was, moreover,
to EF-2 in both native and NaDodSO 4
gels (Fig.2)-a finding strongly suggesting that this activity
is an inherent property of EF-2.
cpm A
800
400
5
10
15
cpm 800
400
5 Froction
10
1S
number
Fig.2, Localization of ADP-ribosyl acceptor and-transferase activities in EF-2 bands in polyacrylamide gels. (A) 70 ~g EF-2 protein was electrophoresed in polyacrylamide gels at 4°C according to (15). Control sample was stained and destained. Samples electrophoresed in parallel were cut into 1 mm slices and homogenized in 300 ~i dialysis buffer in Eppendorf tubes. ADP-ribosyl acceptor (2 ug diphtheria toxin present) (O--~) and transferase (no diphtheria toxin) (0---0) activities in 50 U1 eluates were assayed as described under Methods. (B), Electrophoresis of EF-2 protein in NaDodSO 4 polyacrylamide gel electrophoresis. Samples containing I0 ~g EF-2 protein in 0.5 percent NaDodSO 4 and 140 mM 2-mereaptoethanol were electrophoresed according to (13). Control samples was stained and destained. Unstained gel samples were cut into 1 mm slices which were treated for renaturation as described (16). I00 U1 eluates were assayed for ADP-ribosylationo (O---O), ADP-ribosylation with 2 ug diphtheria toxin present; (~---Q),ADP-ribosylation without diphtheria toxin; ( x ~ × ) ADP-ribosylation without diphtheria toxin but in the presence of EF-2. a, EF-2; b, bovine serum albumin; c, trypsinogen; d, cytochrome c.
1212
Vol. 139, No. 3, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
cpm
I000
600
200 30
60
go
120
150 t(min)
Fig.3. The effect of the binding of GuoPPCH2Pox to EF-2 on the ADP-ribosyl acceptor and-transferase activities. EF-2 was incubated with or without i00 ~M GuoPPCH2Pox for 15 min at 37°C and dialyzed for 2 hours at 4°C against dialysis buffer. ADP-ribosylation was assayed in 120 ~i reaction mixtures containing 8.4 ~g EF-2 protein in the presence or absence of 2 ~g diphtheria toxin as described under Methods. After indicated periods I0 ~i aliquots were plated on GF/C (Whatman) filters and the extent of ADP-ribosylation determined as described. (e~---O), EF-2 treated with G~oPPCH2Pox ; (~ ~), EF-2 treated with GuoPPCH2Pox plus equal amount of control EF-2; ([]--[]), control EF-2; (m--~), control EF-2 (twice amount); (~--f~), EF-2 treated with GuoPPCH2Pox plus diphtheria toxin; (x--x), control EF-2 plus diphtheria toxin; ((~----O),no EF-2.
The incubation of EF-2 with GuoPPCH2Pox (which has been shown to bind specifically to EF-2 (12) and inhibit its activity in polymerization and ribosome-dependent GTPase reactions (17))
depressed the ADP-ribosylation
to 30 percent of its control value (Fig.3). The addition of EF-2-GuoPPCH2Pox conjugate had also a rate-limiting effect on ADP-ribosylation of control EF-2. However, EF-2 incubated with GuoPPCH2Pox retained its ADP-ribosyl acceptor activity in the reaction catalyzed by diphtheria toxin. DISCUSSION: This study attests, consistent with the recent findings (9-Ii), to the presence of an endogenous (EF-2 specific) ADP-ribosyltransferase activity in highly purified EF-2 fractions from rat liver. The strict co-migration of ADP-ribosyltransferase with EF-2 throughout purification and during native and NaDodSO 4 polyacrylamide gel electrophoresis suggests that this activity is an intrinsic property of EF-2. The partial inhibition of ADP-ribosyltransferase activity observed after treatment with GuoPPCH2Pox implicates that the binding of oxidized guanine nucleotides to EF-2 affects the ADP-ribosyltransferase site and/or the interaction of ADP-ribosyltransferase and acceptor sites of EF-2 molecules through some conformational changes. Previously guanine nucleotides have been shown to depress the rate of diphtheria toxin-promoted ADP-ribosylation (18,19). ADP-ribosylated proteins constitute a sub-group of the G proteins which fulfill important regulatory tasks in cellular processes (2). That EF-2, a typical
1213
Vol. 139, No. 3, 1986
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
representative of G protein~ possesses an intrinsic ADP-ribosyltransferase activity, suggests the existence of a cellular control mechanism based on the autoregulation of the activity of these proteins in the cell. Since, EF-2 with intrinsic ADP-ribosyltransferase
activity should mean a constitutive
depression of protein synthesis, the requirement arises that this activity is under stringent control in the cell (9). Indeed, an ADP-ribosyl-EF-2 glycohydrolase activity present in the cell may play a role in the extent of ADP-ribosyl-EF-2
formed and in the rate of protein synthesis. Alternatively,
(or additionally) some effectors present in the cell sap may modulate the ADP-ribosyltransferase
activity.
REFERENCES i) Ueda, K., and Hayaishi, O. (1985) Ann.Rev. Biochem. 54, 73-100. 2) Hughes, S.M. (1983) FEBS Letters 164, 1-8. 3) Honjo, T., Nishizuka, Y., Hayaishi, O., and Kato, I. (1968), J.Biol. Chem. 243, 3553-3555. 4) Iglewski, B.H. and Kabat, D. (1975) Proc.Natl.Acad.Sci. USA, 72, 2284-2288. 5) Pappenheimer, Jr.A.M., and Gill, D.M. (1973) Science 182, 353-358. 6) Pappenheimer, Jr.A.M., (1977) Ann.Rev. Biochem. 46, 69-94. 7) Moss, J., and Vaughan, M. (1978) Proc.Natl.Acad. Sci. USA 75, 3621-3624. 8) Van Ness, B.G., Howard, J.B. and Bodley, J.L. (1980) J.Biol. Chem. 255, 10710-10716. 9) Lee, H., and Iglewski, W.J., (1984) Proc.Natl.Acad.Sci. USA 81, 2703-2707. i0) Iglewski, W.J., Lee, H., and MHller, P. (1984) FEBS Letters 173, 113-118. ii) Sitikov, A.S., Davydova, E.K., and Ovchinnikov, L.P. (1984) FEBS Letters 176, 261-263. 12) Nurten, R., and Bermek, E. (1980) Eur.J.Biochem. 103, 551-555. 13) Laemmli, U., (1970) Nature 227 , 680-685. 14) Bermek, E. (1976) J.Biol.Chem. 251, 6544-6549. 15) Davis, B.J. (1964) Ann NY Acad. Sci. 121, 404-427. 16) Hager, D.A., and Burgess, R.R. (1980) Anal.Biochem. 109, 76-86. 17) Bilgin, N., Nurten, R. and Bermek, E. (in preparation). 18) Raeburn, S., Goor, R.S., Schneider, J.A. and Maxwell, E.S. (1968), Proc.Natl.Acad. Sci. USA 61, 1428-1434. 19) Goor, R.S., and Maxwell, E.S. (1969) Cold Spring Harbor Symp. Quant.Biol. 3__44,609-610.
1214