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Effects of nucleotides on activity of a purified ADP-ribosyltransferase from turkey erythrocytes
OF BIOCHEMISTRY AND BIOPHYSICS Vol. 216, No. 1, June, pp. 74-80,1982
ARCHIVES
Effects of Nucleotides
on Activity of a Purified ADP-Ribosyltransferase from Turkey Erythrocytes
PAUL Laboratoq
A. WATKINS’
AND JOEL
MOSS
of Cellular hfetabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland
2&‘&i
Received September 1, 1981, and in revised form January 15, 1982
A transferase purified from turkey erythrocytes catalyzed the NAD-dependent ADPribosylation of proteins in the supernatant, particulate, and detergent-solubilized fractions of bovine thymus as well as several purified proteins. Nucleoside triphosphates increased the rate of ADP-ribosylation of multiple soluble proteins from thymus and several purified proteins by about twofold. With lysozyme as substrate and 10 mM nucleotide, the order of effectiveness was ATP > ITP = GTP > CTP = UTP. Half-maximal stimulation of ADP-ribose incorporation into lysozyme was observed with 2.5 mM ATP. App(NH)p and inorganic tri- and tetrapolyphosphate were less effective than ATP; ADP, AMP, CAMP, and inorganic pyrophosphate were ineffective. Enhancement of transferase-catalyzed ADP-ribosylation by ATP was observed only at low (20-200 pM) NAD concentrations; with lysozyme as substrate, however, the effect of ATP was not due to prevention of NAD hydrolysis during the assay, nor was it due to an effect on ionic strength. The transferase catalyzed the ADP-ribosylation of several purified proteins and, depending on the protein substrate, ATP either increased, decreased, or did not alter the rate of ADP-ribosylation. It appears that ADP-ribosylation of cellular proteins by endogenous ADP-ribosyltransferases may be subject to regulation by nucleoside triphosphates.
Regulation of enzyme activity by posttranslational covalent modification of proteins has been observed in several systems. One such modification is ADP-ribosylation. Several bacterial toxins, e.g., choleragen (l-4), diphtheria toxin (5), and Escherichia coli heat-labile enterotoxin (l), catalyze the NAD-dependent ADP-ribosylation of cellular proteins. We recently observed that the ADP-ribosylation of both cellular and purified proteins by choleragen can be modified by GTP (6). We have now found, as reported here, that ATP enhances the ADP-ribosylation of many cellular and purified proteins catalyzed by a highly purified ADP-ribosyltransferase from turkey erythrocytes 1 Author dressed.
to whom correspondence
ooo3-9861/82/070074-07%02.00/O Copyright All rights
Q 1982 by Academic PM, Inc. of reproduction in any form reserved.
which uses the guanidino nine and other guanidinyl ribose acceptors (7). MATERIALS
moiety of argigroups as ADP-
AND METHODS
The ADP-ribosyltransferase from turkey erythrocytes was purified as described previously (7, 8), with modification (9). Lysozyme, ovalbumin, histones, poly-L-arginine, DNase I, ,T-lactoglobulin, tripolyphosphate, tetrapolyphosphate, AMP, CAMP, ADP, and nucleoside triphosphates were purchased from Sigma Chemical Company (ATP was Sigma A233.3, prepared by phosphorylation of adenosine); NAD from P-L. Biochemicals; snake venom phosphodiesterase from Worthington; Gpp(NH)p* from Boehringer; human (Y-,j%, and y-globulins from Miles Laboratories; crystalline bovine plasma albumin
should be ad’ Abbreviation 74
used: p, phosphoric residue.
NUCLEOTIDE
EFFECTS
from Armour Pharmaceutical Company; soybean trypsin inhibitor from Calbiochem; [@PINAD and App(NH)p from ICN, [cur&&-“C]NAD from Amersham Corporation; [2,8-*HlNAD from New England Nuclear; polyethyleneimine-cellulose thin-layer chromatography plates from Brinkmann Instruments, AG l-X2 (100-200 mesh, chloride form) and reagents for gel electrophoresis from Bio-Rad. NAD hydrolysis was assessed as [“Clnicotinamide release from [co&&-“C]NAD (10). Protein was determined by the method of Lowry et al (ll), using bovine plasma albumin as standard. Fractions from bovine thymus or brain homogenates were prepared as previously described (6). The thymus 20,OOOgsupernatant was brought to 80% saturation with solid (NH4)zS04; after 30 min, precipitated proteins were collected by centrifugation and dialyzed against 20 mM Tris(Cl-), pH 8.0. After dialysis, insoluble material was removed by centrifugation. ADP-ribosylation assay. Assays contained 50 mM potassium phosphate (pH 7.0), 200 PM NAD, 0.2 &i [(~-8pplNAD or [2,8-*HlNAD, 100 to 250 ng of protein substrate, and 0.3 ng of transferase or an equivalent amount of transferase buffer (50 mM potassium phosphate, pH 7.0, containing 0.1% propylene glycol and 2 mM NaCl) in a total volume of 0.1 ml. After incubation for 60 min at 30°C, 2 ml of 5% trichloroacetic acid was added. Precipitated protein was collected on a Millipore filter (0.45 pm) and washed four times with 2 ml of 5% trichloroacetic acid before radioassay. Data are reported as nanomoles of ADP-ribose incorporated per hour per milligram of protein substrate. ADP-ribose incorporation was constant for at least 90 min under these assay conditions.
Snake venom phosphodiesterase digestion After precipitation and washing with trichloroacetic acid, [S2P]ADP-ribosyl lysozyme was dissolved and incubated for 4 h at 3O’C in 0.3 ml of 160 mM Tris (Cl-) (pH 8.0) containing 10 mM MgClz, 40 mM a-glycerophosphate, 1.0 mM 5’-AMP, and 6 to 7 units of snake venom phosphodiesterase. After the addition of trichloroacetic acid, the soluble fraction was chromatographed on PEI-cellulose thin-layer sheets with a solvent that separates 5’-AMP and 2’-(B-phosphoribosyl)-5’-AMP (12). More than 90% of the lysozyme radioactivity was released by this treatment, and all of it cochromatographed with 5’-AMP (data not shown): no radioactivity was recovered with 2’-(5”phophoribosyl)-5’-AMP, indicating that the product of the transferase reaction was ADP-ribosyl-, rather than poly(ADP-ribosyl)-, lysozyme. Polyacrglumide slab gel electrophoresis. ADP-ribosylated proteins were precipitated with 10% trichloroacetic acid, solubilized in 1% sodium dodecyl sulfate, and subjected to electrophoresis on 12% slab gels according to the method of Laemmli (13). Phosphorylase b, serum albumin, ovalbumin, carbonic anhydrase, trypsin inhibitor, and a-lactalbumin were used as molecular-weight markers. Slabs were dried and exposed to Kodak X-Omat R film for 24 to 48 h.
RESULTS
Turkey erythrocyte transferase catalyzed the ADP-ribosylation of proteins in the supernatant, particulate, and Lubrol PX-solubilized particulate fractions from bovine thymus (Table I). In the presence
TABLE EFFECTOF
75
ON ADP-RIBOSYLATION
I
ATP ONADP-RIBOSYLATIONOFBOVINETHYMUSANDBRAINFRACTIONS BYTURKEYERYTHROCYTETRANSFERASE
ADP-ribosylation
(nmol/h/mg
protein)
Thymus Homogenate fraction
Transferase
-ATP
Brain +ATP
-ATP 0.20 + 0.01
20,006g supernatant
-
0.31 * 0.02
Particulate
+ +
0.95 + 0.03 0.41 f 0.01
0.33 t 0.02 1.76 t 0.01 0.50 i 0.01
1.10 f 0.07
1.40 + 0.04
+ +
0.57 f 0.03
0.52 +_ 0.02 2.68 + 0.09
Lubrol-solubilized particulate (NH&S04-precipitate from supernatant
1.74 f 0.07 f 0.01 0.99 * 0.14 0.62
0.59 f 0.01 2.16 f 0.03
1.59 i 0.01 0.05 * 0.01
0.07 * 0.01 ‘- 0.01 F 0.01 -
0.28 0.45
+ATP 0.10 1.93 0.04 0.07 0.20 0.64
+f f * -+ t
0.01 0.05 0.02 0.01 0.01 0.02
-
Note. ADP-ribosylation of thymus or brain fractions was assayed as described under Materials and Methods. Turkey erythrocyte transferase (0.3 ng) and 10 mM ATP were present where indicated. Means f standard error of values from triplicate assays are reported as nmol of ADP-ribose incorporated/h/mg protein substrate.
76
WATKINS
AND MOSS
of transferase, ATP enhanced ADP-ribosylation to different degrees in all fractions. In the absence of the erythrocyte enzyme, ATP had no effect. The transferase also catalyzed the ADP-ribosylation of supernatant and detergent-solubilized fractions prepared from bovine brain, and this was enhanced by ATP; there was very little ADP-ribosylation of the crude brain particulate fraction (Table I). With an ammonium sulfate-precipitated fraction of thymus proteins as substrate, ATP increased transferase-dependent ADP-ribosylation by >lOO% (Table I). This type of preparation, which could be stored without alteration in activity, was used as substrate in a number of experiments. The ammonium sulfate-precipitated thymus proteins, incubated with [=P]NAD in the absence of transferase, exhibited one major radioactive band (J& -42,000) after electrophoresis on slab gels containing sodium dodecyl sulfate (Fig. 1). Labeling of this band was decreased by ATP or CTP; however, increased labeling of proteins not entering the gel was observed under these conditions. In samples that had been incubated with the transferase, multiple radioactive protein bands were seen and nucleoside triphosphates appeared to increase the labeling of all (Fig.
94,00067.00043,wo3o,ow-
20,10014,400
DYE>
FIG. 1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of thymus proteins ADP-ribosylated by turkey erythrocyte transferase. Ammonium sulfate-precipitated proteins from thymus were incubated with [a-3”PlNAD and other additions as indicated. Electrophoresis and radioautography were carried out as described under Materials and Methods. Lanes l-3, no transferase: 1, no nucleotide; 2, 10 mM ATP, 3,10 mM CTP. Lanes 4-80.3 ng transferase: 4, no nucleotide; 5, 10 mM ATP; 6, 10 mM CTP, '7, 10 mM GTP, 8, 10 mM UTP.
1). After incubation of lysozyme with the transferase and [=P]NAD (with or without nucleoside triphosphate), only one radioactive protein was detected, M, - 14,000 (data not shown). Transferase-catalyzed ADP-ribosylation of the thymus protein fraction was
TABLE
II
EFFEC~OFNUCLEOSIDETRIPHOSPHATESONNICOTINAMIDERELEASEANDADP-RIBOSYLATIONOFTHYMUS PROTEINSANDLYSOZYMEINTHEPRESENCEOFTURKEYERYTHROCYTETRANSFERASE ADP-ribosylation Additions (10 mM) None ATP CTP GTP ITP UTP
Thymus 0.21 0.41 0.33 0.45 0.44 0.39
f + * * + +
0.01 0.03 0.01 0.02 0.03 0.04
(nmol/h)
Nicotinamide
release (nmol/h)
Lysozyme
Thymus
Lysozyme
0.26 0.60 0.44 0.52 0.53 0.44
15.1 10.7 13.7 10.0 10.4 14.2
0.50 0.93 0.71 0.66 0.84 0.71
k + f f f +
0.01 0.01 0.01 0.02 0.01 0.02
f + + f + f
0.3 0.4 0.3 0.2 0.2 0.4
+ k f f + *
0.08 0.02 0.02 0.01 0.02 0.03
Note. ADP-ribosylation of ammonium sulfate-precipitated proteins from thymus or lysozyme was assayed as described under Materials and Methods with 0.3 ng of transferase, 250 r(8 of protein substrate, and 10 mM nucleoside triphosphate where indicated. Data are presented as in Table I. Nicotinamide release was assayed under identical conditions except that [carbonyl-‘“CjNAD (20 nmol per assay) replaced [2,8-%JNAD. After terminating the reaction with 0.2 ml of ice-cold 20 mM Tris(Cl-), pH 8.0, duplicate O.l-ml samples were applied to AG l-X2 columns and [co&c&-“Cjnicotinamide was collected for radioassay. Means * standard error of values from duplicate incubations are recorded.
NUCLEOTIDE
fraction exhibited NAD glycohydrolase activity (nicotinamide released minus ADP-ribosylation), and this was significantly decreased by ATP, GTP, and ITP (Table II). However, when lysozyme was the substrate for the transferase, glycohydrolase activity was less than ADP-ribosylation and was relatively unaffected by nucleoside triphosphates (Table II). Since NAD hydrolysis (nicotinamide release) during assay was insignificant with lysozyme (~5%) as compared to thymus proteins (>50%), lysozyme was used as a model substrate in subsequent experiments. ADP-ribosylation of lysozyme by turkey erythrocyte transferase was enhanced by ATP; GTP and ITP were somewhat less effective (Table II). With both thymus proteins and lysozyme, CTP and UTP were less effective than the purine nucleotides (Table II). Half-maximal stimulation of ADP-ribosylation was observed with 8 mM ATP when the thymus protein fraction served as substrate and with 2.5 mM ATP when lysozyme was the substrate (Fig. 2). ATP increased ADP-ribosylation of both thymus proteins and lysozyme in the presence of 20 or 200 PM NAD (Table III). With 2 mM NAD, ATP had no effect and with 10 InM NAD, ATP decreased labeling of lysozyme (Table III). The nucleotide effect on transferasecatalyzed ADP-ribosylation was specific for the triphosphate; ADP, AMP, CAMP,
[AT+‘] ~MI FIG. 2. Effect of ATP concentration on ADP-ribosylation of lysozyme and thymus proteins by turkey erythrocyte transferase. ADP-ribosylation of lysozyme (0) or ammonium sulfate-precipitated proteins from thymus (0) was assayed as described under Materials and Methods with 0.3 ng of turkey erythrocyte transferase and ATP as indicated. Digestion of lysozyme, ADP-ribosylated with or without 10 mM ATP, with snake venom phosphodiesterase released only one labeled product with a mobility on thin-layer chromatograms identical to that of 5’AMP.
increased to about the same extent by 10 mM GTP, ITP, or ATP in the experiments shown in Table II. With several other preparations of thymus proteins, ATP was clearly most effective. The thymus protein TABLE EFFECT
OF NAD
III
CONCENTRATION ON TRANSFERASE-CATALYZED OF THYMUS PROTEINS AND LYSOZYME
ADP-ribosylation NAD concentration (mM) 0.02 0.2 2.0 10.0
77
EFFECTS ON ADP-RIBOSYLATION
ADP-RIBOSYLATION
(nmol/h/mg
protein)
Thymus proteins -ATP 0.07 0.72 3.52 11.5
+ + k +
Lysozyme +ATP
0.01 0.04 0.02 0.01
0.28 1.24 3.51 10.2
+ + f +
-ATP 0.03 0.06 0.02 0.01
0.48 0.81 2.70 8.53
* + f +
+ATP 0.19 0.03 0.01 0.01
0.62 1.44 2.56 4.37
-t * + +
0.18 0.07 0.01 0.01
Note. ADP-ribosylation of ammonium sulfate-precipitated proteins from thymus and lysozyme was assayed as described under Materials and Methods with 0.3 ng of transferase; 10 mM ATP was present where indicated. Data are presented as in Table I.
78
WATKINS
and inorganic pyrophosphate were not effective (Table IV). App(NH)p, a nonhydrolyzable analog of ATP, was half as effective as ATP, but the corresponding guanosine derivative, Gpp(NH)p, was ineffective (Table V). Inorganic tri- and tetrapolyphosphate were also half as effective as ATP (Table V). Increasing concentrations of NaCl up to 100 MM did not enhance ADP-ribosylation; however, the magnitude of the ATP effect was decreased in the presence of 100 mM NaCl (Table VI). A number of purified proteins were ADP-ribosylated to different degrees by the transferase (Table VII). ATP enhanced ADP-ribosylation of P-globulin, DNase I, and trypsin inhibitor (as well as lysozyme), decreased ADP-ribosylation of several others (e.g., bovine plasma albumin, polyarginine), and had no effect on others (e.g., histone fza, a-globulin). DISCUSSION
ADP-ribosylation of multiple cellular proteins and purified proteins, catalyzed by a highly purified ADP-ribosyltransferase from turkey erythrocytes, was enhanced by nucleoside triphosphates at low (20-200 PM) NAD levels. The intracellular TABLE
IV
EFFECT OF ADENINE NUCLEOTIDES ON ADP-RIBOSYLATION OF THYMUS PROTEINS AND LYSOZYME BY TURKEY ERYTHROCYTETRANSFERASE ADP-ribosylation (nmol/h/mg protein) Additions (10 mM) None ATP ADP AMP CAMP NaPz07
Thymus 0.90 1.60 0.85 0.98 0.94 1.02
* + f + f *
0.02 0.04 0.09 0.02 0.01 0.03
Lysozyme 0.82 1.73 1.06 0.77 0.76 0.92
* + + + f k
0.04 0.03 0.04 0.08 0.01 0.09
Note. ADP-ribosylation of iysozyme and ammonium sulfate-precipitated proteins from thymus was assayed as described under Materials and Methods with 0.3 ng of transferase and other additions as indicated. Data are presented as in Table I.
AND MOSS TABLE
V
EFFECT OF NONHYDROLYZABLE ANALOGS OF NUCLEOSIDE TRIPHOSPHATES AND POLYPHOSPHATES ON ADP-RIBOSYLATION OF LYSOZYME BY TURKEY ERYTHROCYTETRANSFERASE
Additions
(10 mM)
None ATP APP(NH)P GTP GPP(NH)P Tripolyphosphate Tetrapolyphosphate
ADP-ribosyiation (nmol/h/mg protein) 1.10 2.38 1.62 1.81 0.79 1.75 1.74
+ + f + f + +
0.03 0.12 0.13 0.06 0.01 0.08 0.04
Note. ADP-ribosylation of lysozyme was assayed as described under Materials and Methods with 0.3 ng of transferase and additions as indicated. Data are presented as in Table I.
NAD concentration in various tissues has been reported to be between 60 and 800 PM (14-17). Sols and Marco (15) and Kaplan (18) have postulated, however, that most of the intracellular NAD exists bound to proteins, particularly dehydrogenases. In support of this hypothesis, Solti and Friedrich (17) studied the reactivity of NAD toward extracellular NAD glycohydrolase and found that about two-thirds of human erythrocyte NAD was protein bound. Bernofsky and Pankow (16), however, using techniques of ultrafiltration and gel permeation chromatography, reported that only 20% of rabbit muscle NAD was protein bound. Thus, there is a wide variation both in the NAD level in a specific tissue and in the estimate of free NAD concentration. It appears, therefore, that the free NAD concentration is not significantly higher than either the NAD concentration at which ATP enhances transferase-catalyzed ADP-ribosylation of proteins or the Km of transferase for NAD of 30 PM (7). The thymus protein fraction used as substrate for transferase in several experiments exhibited significant NAD glycohydrolase activity which was partially inhibited by nucleoside triphosphates. Enhanced ADP-ribosylation in the presence
NUCLEOTIDE
TABLE
EFFECTS
VI
EFFECT OF IONIC STRENGTH ON TURKEY ERYTHROCYTE TRANSFERASE-CATALYZED ADP-RIBOSYLATION OF LYSOZYME NaCl concentration (m@ 0 1
10 100
ADP-ribosylation (nmol/h/mg protein) -ATP 2.01 1.91 1.86 1.88
f f f +
+ATP 0.03 0.03 0.17 0.13
4.13 3.81 2.75 2.93
+ k f f
0.16 0.12 0.14 0.04
Note. ADP-ribosylation was assayed as described under Materials and Methods with 0.3 ng of transferase; 10 mM ATP was present where indicated. Data are presented as in Table I.
of nucleoside triphosphate could, therefore, be interpreted as resulting from preservation of NAD during assay. To avoid difficulty with the interpretation of results obtained with the thymus fraction, we, therefore, employed lysozyme as a purified model substrate in subsequent experiments. With lysozyme, >95% of the NAD in the assay was present at the end of assays with or without nucleoside triphosphate. We recently observed that the choleragen-catalyzed ADP-ribosylation of several purified proteins and multiple cellular proteins was enhanced by GTP; ATP was almost totally ineffective when the thymus protein fraction served as substrate (6). With both choleragen and the turkey erythrocyte transferase, the effects of nucleotides on ADP-ribosylation were dependent on the protein substrate. Lysozyme was ADP-ribosylated by both enzymes, but enhancement by nucleoside triphosphate was observed only with the erythrocyte transferase; the choleragencatalyzed reaction was unaffected by either GTP or ATP ((6) and unpublished observations). Conversely, ADP-ribosylation of histone fi, by the transferase was unaffected by ATP and inhibited by GTP, whereas the choleragen-catalyzed reaction was enhanced by either ATP or GTP ((6) and unpublished observations). Thus, the responses of choleragen and the turkey
79
ON ADP-RIBOSYLATION
erythrocyte transferase to nucleoside triphosphates differ, as do their substrate specificites (8). The effects of nucleotides on the transferase-catalyzed reaction could result from interactions with the substrate protein that induce a conformational change which might either expose reactive guanidino groups and thereby increase ADP-ribosylation or make an otherwise accessible guanidino group inaccessible and lead to decreased ADP-ribosylation. This would mean that for different proteins different nucleotides and probably other molecules that can induce alterations in tertiary structure would be more or less effective in increasing or decreasing ADP-ribosylation by specific transferases. Alternatively, the phosphate groups of nucleoside triphosphates or inorganic tri- and tetrapolyphosphate may interact with guanidino moieties, thereby rendering a particular site either more or less accessible for TABLE
VII
EFFECT OF ATP ON ADP-RIBOSYLATION OF PURIFIED PROTEINS CATALYZED BY TURKEY ERYTHROCYTE TRANSFERASE ADP-ribosylation (nmol/h/mg protein) Substrate protein (250 pg)
Bovine plasma albumin #l-Lactoglobulin Histone (crude) Histone f,” Histone f%” Histone far,” Histone fs’ Ovalbumin Human o-globulin Human P-globulin Human y-globulin DNase
I
Trypsin inhibitor Lysozyme Poly-L-arginine
-ATP 0.24 0.20 0.41 0.40 0.44 0.54
+ + + k + t
+ATP 0.03 0.03 0.02 0.01 0.02 0.02
0.59 + 0.01 0.26 0.28 0.76 0.24 0.38 0.64 0.56 2.07
If: + +z + t 2 k
0.03 0.03 0.02 0.02 0.02 0.02 0.03
+ 0.10
0.13 0.11 0.29 0.26 0.40 0.33 0.26 0.19 0.22 1.01 0.19 0.67 1.27 1.23 0.26
f i + k * + + + * + + + + + +
0.01 0.01 0.01 0.01 0.01 0.01 0.09 0.03 0.04 0.05 0.03 0.03 0.04 0.07 0.01
Note. ADP-ribosylation was assayed as described in “Methods” with 0.3 ng of transferase; 10 mM ATP was present where indicated. Data are reported as in Table I. a 100 pg.
80
WATKINS
ADP-ribosylation, depending on the microenvironment of the site. Transferase-catalyzed ADP-ribosylation of lysozyme was half-maximally stimulated by 2.5 mM ATP. With the thymus fraction as substrate, higher ATP levels were necessary to achieve half-maximal enhancement, perhaps because of the presence of ATPases and other ATP-utilizing enzymes in the thymus protein fraction. Cellular ATP concentrations are reported to be in the millimolar range (14), and there are likely localized areas of higher concentrations. Thus, ATP (and perhaps other small molecules that interact with proteins) may play a role in the regulation of intracellular ADP-ribosylation. ACKNOWLEDGMENTS The authors thank Dr. Martha Vaughan for many useful discussions and critical reading of the manuscript and Mrs. D. Marie Sherwood for expert secretarial assistance.
AND
MOSS
4. JOHNSON, G. L., KASLOW, H. R., AND BOURNE, H. R. (1978) J. Bid Chem 253.7120-7123. 5. PAPPENHEIMER, A. M., JR. (1977) Annu. Rev.
Biochem 46.69-94. 6. WATKINS, P. A., Moss, J., AND VAUGHAN, M. (1980) J. BioL Chem 255,3959-3963. 7. Moss, J., STANLEY, S. J., AND OPPENHEIMER, N. J. (1979) J. BioL Chem 254,8891-8894. 8. Moss, J., AND VAUGHAN, M. (1978) Proc Nut.
Acad Sci USA 75,3621-3624. 9. Moss,
J., STANLEY,
S., AND WATKINS,
P. A. (1980)
J. BioL Chem 255.5838-5840. 10. Moss, J., MANGANIELLO, V. C., AND VAUGHAN, M. (1976) Proc Nat. Acad Sci. USA 73,4424-4427. 11. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. BioL Chem. 193, 265-275. 12. LEHMANN, A. R., KIRK-BELL, S., SHAU, S., AND WHISH, W. J. D. (1974) Exp. Cell Re.s. 83, 6372. 13. LAEMMLI, U. K. (1970) Nature &m&m) 227,680685. 14. WILLIAMSON, D. H., ANDBROSNAN, J. T. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.), 2nd ed., pp. 22662302, Academic Press, New York. 15. SOLS, A., AND MARCO, R. (1970) Curr. Top. CelL
ReguL 2,227-273. REFERENCES 1. Moss, J., AND VAUGHAN, M. (1979) Annu. Bi4Jck 48,581~600. 2. CASSEL, D., AND PFEUFFER, T. (1978) Pm.
Ad 3. GILL,
Rev.
D. M.,
AND MEREN,
R. (1978)
C., AND PANKOW,
M.
(1973)
Arch.
Biophys. 166,143-153. AND
FRIEDRICH,
P. (1979)
Eur. J.
Biochem 95.551-559. Nut.
Sci. USA 75.2669-2673.
Acad Sk USA 75,3050-3054.
16. BERNOFSKY, B&hem 17. SOLTI, M.,
Proc Nat.
18. KAPLAN, N. 0. (1966) in Current chemical Energetics (Kaplan, nedy, E. P., eds.), pp. 447-458, New York.
Aspects of BioN. O., and KenAcademic Press,
ARCHIVES
Effects of Nucleotides
on Activity of a Purified ADP-Ribosyltransferase from Turkey Erythrocytes
PAUL Laboratoq
A. WATKINS’
AND JOEL
MOSS
of Cellular hfetabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland
2&‘&i
Received September 1, 1981, and in revised form January 15, 1982
A transferase purified from turkey erythrocytes catalyzed the NAD-dependent ADPribosylation of proteins in the supernatant, particulate, and detergent-solubilized fractions of bovine thymus as well as several purified proteins. Nucleoside triphosphates increased the rate of ADP-ribosylation of multiple soluble proteins from thymus and several purified proteins by about twofold. With lysozyme as substrate and 10 mM nucleotide, the order of effectiveness was ATP > ITP = GTP > CTP = UTP. Half-maximal stimulation of ADP-ribose incorporation into lysozyme was observed with 2.5 mM ATP. App(NH)p and inorganic tri- and tetrapolyphosphate were less effective than ATP; ADP, AMP, CAMP, and inorganic pyrophosphate were ineffective. Enhancement of transferase-catalyzed ADP-ribosylation by ATP was observed only at low (20-200 pM) NAD concentrations; with lysozyme as substrate, however, the effect of ATP was not due to prevention of NAD hydrolysis during the assay, nor was it due to an effect on ionic strength. The transferase catalyzed the ADP-ribosylation of several purified proteins and, depending on the protein substrate, ATP either increased, decreased, or did not alter the rate of ADP-ribosylation. It appears that ADP-ribosylation of cellular proteins by endogenous ADP-ribosyltransferases may be subject to regulation by nucleoside triphosphates.
Regulation of enzyme activity by posttranslational covalent modification of proteins has been observed in several systems. One such modification is ADP-ribosylation. Several bacterial toxins, e.g., choleragen (l-4), diphtheria toxin (5), and Escherichia coli heat-labile enterotoxin (l), catalyze the NAD-dependent ADP-ribosylation of cellular proteins. We recently observed that the ADP-ribosylation of both cellular and purified proteins by choleragen can be modified by GTP (6). We have now found, as reported here, that ATP enhances the ADP-ribosylation of many cellular and purified proteins catalyzed by a highly purified ADP-ribosyltransferase from turkey erythrocytes 1 Author dressed.
to whom correspondence
ooo3-9861/82/070074-07%02.00/O Copyright All rights
Q 1982 by Academic PM, Inc. of reproduction in any form reserved.
which uses the guanidino nine and other guanidinyl ribose acceptors (7). MATERIALS
moiety of argigroups as ADP-
AND METHODS
The ADP-ribosyltransferase from turkey erythrocytes was purified as described previously (7, 8), with modification (9). Lysozyme, ovalbumin, histones, poly-L-arginine, DNase I, ,T-lactoglobulin, tripolyphosphate, tetrapolyphosphate, AMP, CAMP, ADP, and nucleoside triphosphates were purchased from Sigma Chemical Company (ATP was Sigma A233.3, prepared by phosphorylation of adenosine); NAD from P-L. Biochemicals; snake venom phosphodiesterase from Worthington; Gpp(NH)p* from Boehringer; human (Y-,j%, and y-globulins from Miles Laboratories; crystalline bovine plasma albumin
should be ad’ Abbreviation 74
used: p, phosphoric residue.
NUCLEOTIDE
EFFECTS
from Armour Pharmaceutical Company; soybean trypsin inhibitor from Calbiochem; [@PINAD and App(NH)p from ICN, [cur&&-“C]NAD from Amersham Corporation; [2,8-*HlNAD from New England Nuclear; polyethyleneimine-cellulose thin-layer chromatography plates from Brinkmann Instruments, AG l-X2 (100-200 mesh, chloride form) and reagents for gel electrophoresis from Bio-Rad. NAD hydrolysis was assessed as [“Clnicotinamide release from [co&&-“C]NAD (10). Protein was determined by the method of Lowry et al (ll), using bovine plasma albumin as standard. Fractions from bovine thymus or brain homogenates were prepared as previously described (6). The thymus 20,OOOgsupernatant was brought to 80% saturation with solid (NH4)zS04; after 30 min, precipitated proteins were collected by centrifugation and dialyzed against 20 mM Tris(Cl-), pH 8.0. After dialysis, insoluble material was removed by centrifugation. ADP-ribosylation assay. Assays contained 50 mM potassium phosphate (pH 7.0), 200 PM NAD, 0.2 &i [(~-8pplNAD or [2,8-*HlNAD, 100 to 250 ng of protein substrate, and 0.3 ng of transferase or an equivalent amount of transferase buffer (50 mM potassium phosphate, pH 7.0, containing 0.1% propylene glycol and 2 mM NaCl) in a total volume of 0.1 ml. After incubation for 60 min at 30°C, 2 ml of 5% trichloroacetic acid was added. Precipitated protein was collected on a Millipore filter (0.45 pm) and washed four times with 2 ml of 5% trichloroacetic acid before radioassay. Data are reported as nanomoles of ADP-ribose incorporated per hour per milligram of protein substrate. ADP-ribose incorporation was constant for at least 90 min under these assay conditions.
Snake venom phosphodiesterase digestion After precipitation and washing with trichloroacetic acid, [S2P]ADP-ribosyl lysozyme was dissolved and incubated for 4 h at 3O’C in 0.3 ml of 160 mM Tris (Cl-) (pH 8.0) containing 10 mM MgClz, 40 mM a-glycerophosphate, 1.0 mM 5’-AMP, and 6 to 7 units of snake venom phosphodiesterase. After the addition of trichloroacetic acid, the soluble fraction was chromatographed on PEI-cellulose thin-layer sheets with a solvent that separates 5’-AMP and 2’-(B-phosphoribosyl)-5’-AMP (12). More than 90% of the lysozyme radioactivity was released by this treatment, and all of it cochromatographed with 5’-AMP (data not shown): no radioactivity was recovered with 2’-(5”phophoribosyl)-5’-AMP, indicating that the product of the transferase reaction was ADP-ribosyl-, rather than poly(ADP-ribosyl)-, lysozyme. Polyacrglumide slab gel electrophoresis. ADP-ribosylated proteins were precipitated with 10% trichloroacetic acid, solubilized in 1% sodium dodecyl sulfate, and subjected to electrophoresis on 12% slab gels according to the method of Laemmli (13). Phosphorylase b, serum albumin, ovalbumin, carbonic anhydrase, trypsin inhibitor, and a-lactalbumin were used as molecular-weight markers. Slabs were dried and exposed to Kodak X-Omat R film for 24 to 48 h.
RESULTS
Turkey erythrocyte transferase catalyzed the ADP-ribosylation of proteins in the supernatant, particulate, and Lubrol PX-solubilized particulate fractions from bovine thymus (Table I). In the presence
TABLE EFFECTOF
75
ON ADP-RIBOSYLATION
I
ATP ONADP-RIBOSYLATIONOFBOVINETHYMUSANDBRAINFRACTIONS BYTURKEYERYTHROCYTETRANSFERASE
ADP-ribosylation
(nmol/h/mg
protein)
Thymus Homogenate fraction
Transferase
-ATP
Brain +ATP
-ATP 0.20 + 0.01
20,006g supernatant
-
0.31 * 0.02
Particulate
+ +
0.95 + 0.03 0.41 f 0.01
0.33 t 0.02 1.76 t 0.01 0.50 i 0.01
1.10 f 0.07
1.40 + 0.04
+ +
0.57 f 0.03
0.52 +_ 0.02 2.68 + 0.09
Lubrol-solubilized particulate (NH&S04-precipitate from supernatant
1.74 f 0.07 f 0.01 0.99 * 0.14 0.62
0.59 f 0.01 2.16 f 0.03
1.59 i 0.01 0.05 * 0.01
0.07 * 0.01 ‘- 0.01 F 0.01 -
0.28 0.45
+ATP 0.10 1.93 0.04 0.07 0.20 0.64
+f f * -+ t
0.01 0.05 0.02 0.01 0.01 0.02
-
Note. ADP-ribosylation of thymus or brain fractions was assayed as described under Materials and Methods. Turkey erythrocyte transferase (0.3 ng) and 10 mM ATP were present where indicated. Means f standard error of values from triplicate assays are reported as nmol of ADP-ribose incorporated/h/mg protein substrate.
76
WATKINS
AND MOSS
of transferase, ATP enhanced ADP-ribosylation to different degrees in all fractions. In the absence of the erythrocyte enzyme, ATP had no effect. The transferase also catalyzed the ADP-ribosylation of supernatant and detergent-solubilized fractions prepared from bovine brain, and this was enhanced by ATP; there was very little ADP-ribosylation of the crude brain particulate fraction (Table I). With an ammonium sulfate-precipitated fraction of thymus proteins as substrate, ATP increased transferase-dependent ADP-ribosylation by >lOO% (Table I). This type of preparation, which could be stored without alteration in activity, was used as substrate in a number of experiments. The ammonium sulfate-precipitated thymus proteins, incubated with [=P]NAD in the absence of transferase, exhibited one major radioactive band (J& -42,000) after electrophoresis on slab gels containing sodium dodecyl sulfate (Fig. 1). Labeling of this band was decreased by ATP or CTP; however, increased labeling of proteins not entering the gel was observed under these conditions. In samples that had been incubated with the transferase, multiple radioactive protein bands were seen and nucleoside triphosphates appeared to increase the labeling of all (Fig.
94,00067.00043,wo3o,ow-
20,10014,400
DYE>
FIG. 1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of thymus proteins ADP-ribosylated by turkey erythrocyte transferase. Ammonium sulfate-precipitated proteins from thymus were incubated with [a-3”PlNAD and other additions as indicated. Electrophoresis and radioautography were carried out as described under Materials and Methods. Lanes l-3, no transferase: 1, no nucleotide; 2, 10 mM ATP, 3,10 mM CTP. Lanes 4-80.3 ng transferase: 4, no nucleotide; 5, 10 mM ATP; 6, 10 mM CTP, '7, 10 mM GTP, 8, 10 mM UTP.
1). After incubation of lysozyme with the transferase and [=P]NAD (with or without nucleoside triphosphate), only one radioactive protein was detected, M, - 14,000 (data not shown). Transferase-catalyzed ADP-ribosylation of the thymus protein fraction was
TABLE
II
EFFEC~OFNUCLEOSIDETRIPHOSPHATESONNICOTINAMIDERELEASEANDADP-RIBOSYLATIONOFTHYMUS PROTEINSANDLYSOZYMEINTHEPRESENCEOFTURKEYERYTHROCYTETRANSFERASE ADP-ribosylation Additions (10 mM) None ATP CTP GTP ITP UTP
Thymus 0.21 0.41 0.33 0.45 0.44 0.39
f + * * + +
0.01 0.03 0.01 0.02 0.03 0.04
(nmol/h)
Nicotinamide
release (nmol/h)
Lysozyme
Thymus
Lysozyme
0.26 0.60 0.44 0.52 0.53 0.44
15.1 10.7 13.7 10.0 10.4 14.2
0.50 0.93 0.71 0.66 0.84 0.71
k + f f f +
0.01 0.01 0.01 0.02 0.01 0.02
f + + f + f
0.3 0.4 0.3 0.2 0.2 0.4
+ k f f + *
0.08 0.02 0.02 0.01 0.02 0.03
Note. ADP-ribosylation of ammonium sulfate-precipitated proteins from thymus or lysozyme was assayed as described under Materials and Methods with 0.3 ng of transferase, 250 r(8 of protein substrate, and 10 mM nucleoside triphosphate where indicated. Data are presented as in Table I. Nicotinamide release was assayed under identical conditions except that [carbonyl-‘“CjNAD (20 nmol per assay) replaced [2,8-%JNAD. After terminating the reaction with 0.2 ml of ice-cold 20 mM Tris(Cl-), pH 8.0, duplicate O.l-ml samples were applied to AG l-X2 columns and [co&c&-“Cjnicotinamide was collected for radioassay. Means * standard error of values from duplicate incubations are recorded.
NUCLEOTIDE
fraction exhibited NAD glycohydrolase activity (nicotinamide released minus ADP-ribosylation), and this was significantly decreased by ATP, GTP, and ITP (Table II). However, when lysozyme was the substrate for the transferase, glycohydrolase activity was less than ADP-ribosylation and was relatively unaffected by nucleoside triphosphates (Table II). Since NAD hydrolysis (nicotinamide release) during assay was insignificant with lysozyme (~5%) as compared to thymus proteins (>50%), lysozyme was used as a model substrate in subsequent experiments. ADP-ribosylation of lysozyme by turkey erythrocyte transferase was enhanced by ATP; GTP and ITP were somewhat less effective (Table II). With both thymus proteins and lysozyme, CTP and UTP were less effective than the purine nucleotides (Table II). Half-maximal stimulation of ADP-ribosylation was observed with 8 mM ATP when the thymus protein fraction served as substrate and with 2.5 mM ATP when lysozyme was the substrate (Fig. 2). ATP increased ADP-ribosylation of both thymus proteins and lysozyme in the presence of 20 or 200 PM NAD (Table III). With 2 mM NAD, ATP had no effect and with 10 InM NAD, ATP decreased labeling of lysozyme (Table III). The nucleotide effect on transferasecatalyzed ADP-ribosylation was specific for the triphosphate; ADP, AMP, CAMP,
[AT+‘] ~MI FIG. 2. Effect of ATP concentration on ADP-ribosylation of lysozyme and thymus proteins by turkey erythrocyte transferase. ADP-ribosylation of lysozyme (0) or ammonium sulfate-precipitated proteins from thymus (0) was assayed as described under Materials and Methods with 0.3 ng of turkey erythrocyte transferase and ATP as indicated. Digestion of lysozyme, ADP-ribosylated with or without 10 mM ATP, with snake venom phosphodiesterase released only one labeled product with a mobility on thin-layer chromatograms identical to that of 5’AMP.
increased to about the same extent by 10 mM GTP, ITP, or ATP in the experiments shown in Table II. With several other preparations of thymus proteins, ATP was clearly most effective. The thymus protein TABLE EFFECT
OF NAD
III
CONCENTRATION ON TRANSFERASE-CATALYZED OF THYMUS PROTEINS AND LYSOZYME
ADP-ribosylation NAD concentration (mM) 0.02 0.2 2.0 10.0
77
EFFECTS ON ADP-RIBOSYLATION
ADP-RIBOSYLATION
(nmol/h/mg
protein)
Thymus proteins -ATP 0.07 0.72 3.52 11.5
+ + k +
Lysozyme +ATP
0.01 0.04 0.02 0.01
0.28 1.24 3.51 10.2
+ + f +
-ATP 0.03 0.06 0.02 0.01
0.48 0.81 2.70 8.53
* + f +
+ATP 0.19 0.03 0.01 0.01
0.62 1.44 2.56 4.37
-t * + +
0.18 0.07 0.01 0.01
Note. ADP-ribosylation of ammonium sulfate-precipitated proteins from thymus and lysozyme was assayed as described under Materials and Methods with 0.3 ng of transferase; 10 mM ATP was present where indicated. Data are presented as in Table I.
78
WATKINS
and inorganic pyrophosphate were not effective (Table IV). App(NH)p, a nonhydrolyzable analog of ATP, was half as effective as ATP, but the corresponding guanosine derivative, Gpp(NH)p, was ineffective (Table V). Inorganic tri- and tetrapolyphosphate were also half as effective as ATP (Table V). Increasing concentrations of NaCl up to 100 MM did not enhance ADP-ribosylation; however, the magnitude of the ATP effect was decreased in the presence of 100 mM NaCl (Table VI). A number of purified proteins were ADP-ribosylated to different degrees by the transferase (Table VII). ATP enhanced ADP-ribosylation of P-globulin, DNase I, and trypsin inhibitor (as well as lysozyme), decreased ADP-ribosylation of several others (e.g., bovine plasma albumin, polyarginine), and had no effect on others (e.g., histone fza, a-globulin). DISCUSSION
ADP-ribosylation of multiple cellular proteins and purified proteins, catalyzed by a highly purified ADP-ribosyltransferase from turkey erythrocytes, was enhanced by nucleoside triphosphates at low (20-200 PM) NAD levels. The intracellular TABLE
IV
EFFECT OF ADENINE NUCLEOTIDES ON ADP-RIBOSYLATION OF THYMUS PROTEINS AND LYSOZYME BY TURKEY ERYTHROCYTETRANSFERASE ADP-ribosylation (nmol/h/mg protein) Additions (10 mM) None ATP ADP AMP CAMP NaPz07
Thymus 0.90 1.60 0.85 0.98 0.94 1.02
* + f + f *
0.02 0.04 0.09 0.02 0.01 0.03
Lysozyme 0.82 1.73 1.06 0.77 0.76 0.92
* + + + f k
0.04 0.03 0.04 0.08 0.01 0.09
Note. ADP-ribosylation of iysozyme and ammonium sulfate-precipitated proteins from thymus was assayed as described under Materials and Methods with 0.3 ng of transferase and other additions as indicated. Data are presented as in Table I.
AND MOSS TABLE
V
EFFECT OF NONHYDROLYZABLE ANALOGS OF NUCLEOSIDE TRIPHOSPHATES AND POLYPHOSPHATES ON ADP-RIBOSYLATION OF LYSOZYME BY TURKEY ERYTHROCYTETRANSFERASE
Additions
(10 mM)
None ATP APP(NH)P GTP GPP(NH)P Tripolyphosphate Tetrapolyphosphate
ADP-ribosyiation (nmol/h/mg protein) 1.10 2.38 1.62 1.81 0.79 1.75 1.74
+ + f + f + +
0.03 0.12 0.13 0.06 0.01 0.08 0.04
Note. ADP-ribosylation of lysozyme was assayed as described under Materials and Methods with 0.3 ng of transferase and additions as indicated. Data are presented as in Table I.
NAD concentration in various tissues has been reported to be between 60 and 800 PM (14-17). Sols and Marco (15) and Kaplan (18) have postulated, however, that most of the intracellular NAD exists bound to proteins, particularly dehydrogenases. In support of this hypothesis, Solti and Friedrich (17) studied the reactivity of NAD toward extracellular NAD glycohydrolase and found that about two-thirds of human erythrocyte NAD was protein bound. Bernofsky and Pankow (16), however, using techniques of ultrafiltration and gel permeation chromatography, reported that only 20% of rabbit muscle NAD was protein bound. Thus, there is a wide variation both in the NAD level in a specific tissue and in the estimate of free NAD concentration. It appears, therefore, that the free NAD concentration is not significantly higher than either the NAD concentration at which ATP enhances transferase-catalyzed ADP-ribosylation of proteins or the Km of transferase for NAD of 30 PM (7). The thymus protein fraction used as substrate for transferase in several experiments exhibited significant NAD glycohydrolase activity which was partially inhibited by nucleoside triphosphates. Enhanced ADP-ribosylation in the presence
NUCLEOTIDE
TABLE
EFFECTS
VI
EFFECT OF IONIC STRENGTH ON TURKEY ERYTHROCYTE TRANSFERASE-CATALYZED ADP-RIBOSYLATION OF LYSOZYME NaCl concentration (m@ 0 1
10 100
ADP-ribosylation (nmol/h/mg protein) -ATP 2.01 1.91 1.86 1.88
f f f +
+ATP 0.03 0.03 0.17 0.13
4.13 3.81 2.75 2.93
+ k f f
0.16 0.12 0.14 0.04
Note. ADP-ribosylation was assayed as described under Materials and Methods with 0.3 ng of transferase; 10 mM ATP was present where indicated. Data are presented as in Table I.
of nucleoside triphosphate could, therefore, be interpreted as resulting from preservation of NAD during assay. To avoid difficulty with the interpretation of results obtained with the thymus fraction, we, therefore, employed lysozyme as a purified model substrate in subsequent experiments. With lysozyme, >95% of the NAD in the assay was present at the end of assays with or without nucleoside triphosphate. We recently observed that the choleragen-catalyzed ADP-ribosylation of several purified proteins and multiple cellular proteins was enhanced by GTP; ATP was almost totally ineffective when the thymus protein fraction served as substrate (6). With both choleragen and the turkey erythrocyte transferase, the effects of nucleotides on ADP-ribosylation were dependent on the protein substrate. Lysozyme was ADP-ribosylated by both enzymes, but enhancement by nucleoside triphosphate was observed only with the erythrocyte transferase; the choleragencatalyzed reaction was unaffected by either GTP or ATP ((6) and unpublished observations). Conversely, ADP-ribosylation of histone fi, by the transferase was unaffected by ATP and inhibited by GTP, whereas the choleragen-catalyzed reaction was enhanced by either ATP or GTP ((6) and unpublished observations). Thus, the responses of choleragen and the turkey
79
ON ADP-RIBOSYLATION
erythrocyte transferase to nucleoside triphosphates differ, as do their substrate specificites (8). The effects of nucleotides on the transferase-catalyzed reaction could result from interactions with the substrate protein that induce a conformational change which might either expose reactive guanidino groups and thereby increase ADP-ribosylation or make an otherwise accessible guanidino group inaccessible and lead to decreased ADP-ribosylation. This would mean that for different proteins different nucleotides and probably other molecules that can induce alterations in tertiary structure would be more or less effective in increasing or decreasing ADP-ribosylation by specific transferases. Alternatively, the phosphate groups of nucleoside triphosphates or inorganic tri- and tetrapolyphosphate may interact with guanidino moieties, thereby rendering a particular site either more or less accessible for TABLE
VII
EFFECT OF ATP ON ADP-RIBOSYLATION OF PURIFIED PROTEINS CATALYZED BY TURKEY ERYTHROCYTE TRANSFERASE ADP-ribosylation (nmol/h/mg protein) Substrate protein (250 pg)
Bovine plasma albumin #l-Lactoglobulin Histone (crude) Histone f,” Histone f%” Histone far,” Histone fs’ Ovalbumin Human o-globulin Human P-globulin Human y-globulin DNase
I
Trypsin inhibitor Lysozyme Poly-L-arginine
-ATP 0.24 0.20 0.41 0.40 0.44 0.54
+ + + k + t
+ATP 0.03 0.03 0.02 0.01 0.02 0.02
0.59 + 0.01 0.26 0.28 0.76 0.24 0.38 0.64 0.56 2.07
If: + +z + t 2 k
0.03 0.03 0.02 0.02 0.02 0.02 0.03
+ 0.10
0.13 0.11 0.29 0.26 0.40 0.33 0.26 0.19 0.22 1.01 0.19 0.67 1.27 1.23 0.26
f i + k * + + + * + + + + + +
0.01 0.01 0.01 0.01 0.01 0.01 0.09 0.03 0.04 0.05 0.03 0.03 0.04 0.07 0.01
Note. ADP-ribosylation was assayed as described in “Methods” with 0.3 ng of transferase; 10 mM ATP was present where indicated. Data are reported as in Table I. a 100 pg.
80
WATKINS
ADP-ribosylation, depending on the microenvironment of the site. Transferase-catalyzed ADP-ribosylation of lysozyme was half-maximally stimulated by 2.5 mM ATP. With the thymus fraction as substrate, higher ATP levels were necessary to achieve half-maximal enhancement, perhaps because of the presence of ATPases and other ATP-utilizing enzymes in the thymus protein fraction. Cellular ATP concentrations are reported to be in the millimolar range (14), and there are likely localized areas of higher concentrations. Thus, ATP (and perhaps other small molecules that interact with proteins) may play a role in the regulation of intracellular ADP-ribosylation. ACKNOWLEDGMENTS The authors thank Dr. Martha Vaughan for many useful discussions and critical reading of the manuscript and Mrs. D. Marie Sherwood for expert secretarial assistance.
AND
MOSS
4. JOHNSON, G. L., KASLOW, H. R., AND BOURNE, H. R. (1978) J. Bid Chem 253.7120-7123. 5. PAPPENHEIMER, A. M., JR. (1977) Annu. Rev.
Biochem 46.69-94. 6. WATKINS, P. A., Moss, J., AND VAUGHAN, M. (1980) J. BioL Chem 255,3959-3963. 7. Moss, J., STANLEY, S. J., AND OPPENHEIMER, N. J. (1979) J. BioL Chem 254,8891-8894. 8. Moss, J., AND VAUGHAN, M. (1978) Proc Nut.
Acad Sci USA 75,3621-3624. 9. Moss,
J., STANLEY,
S., AND WATKINS,
P. A. (1980)
J. BioL Chem 255.5838-5840. 10. Moss, J., MANGANIELLO, V. C., AND VAUGHAN, M. (1976) Proc Nat. Acad Sci. USA 73,4424-4427. 11. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. BioL Chem. 193, 265-275. 12. LEHMANN, A. R., KIRK-BELL, S., SHAU, S., AND WHISH, W. J. D. (1974) Exp. Cell Re.s. 83, 6372. 13. LAEMMLI, U. K. (1970) Nature &m&m) 227,680685. 14. WILLIAMSON, D. H., ANDBROSNAN, J. T. (1974) in Methods of Enzymatic Analysis (Bergmeyer, H. U., ed.), 2nd ed., pp. 22662302, Academic Press, New York. 15. SOLS, A., AND MARCO, R. (1970) Curr. Top. CelL
ReguL 2,227-273. REFERENCES 1. Moss, J., AND VAUGHAN, M. (1979) Annu. Bi4Jck 48,581~600. 2. CASSEL, D., AND PFEUFFER, T. (1978) Pm.
Ad 3. GILL,
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16. BERNOFSKY, B&hem 17. SOLTI, M.,
Proc Nat.
18. KAPLAN, N. 0. (1966) in Current chemical Energetics (Kaplan, nedy, E. P., eds.), pp. 447-458, New York.
Aspects of BioN. O., and KenAcademic Press,