Proc. Nati. Acad. Sci. USA Vol. 83, pp. 6267-6271, September 1986 Biochemistry 2'-Phosphoadenylylation of eukaryotic : A type of covalent modification (ADP-ribose/phosphoADP-ribosylation/NADP labeling/microsomes/mitochondria) HELMUTH HILZ, WERNER FANICK, AND KARIN KLAPPROTH Institut fUr Physiologische Chemie der Universit~it, 2000 Hamburg 20, Federal Republic of Germany Communicated by E. R. Stadtman, May 1, 1986

ABSTRACT An enzymatic system in rat liver microsomal NH2 * preparations has been detected that catalyzes the transfer ofthe 2'-phospho-AMP moiety from NADP to endogenous poly- peptides; the major acceptor is a polypeptide of about 40 kDa (p40). Modification of the acceptor by 2'-phospho-AMP resi- //0 dues was deduced from the simultaneous transfer of 2'- [33P]phosphate and [3H~adenine residues from double-labeled C - NH2 NADP, while no incorporation of radioactivity into p40 was seen with NADP species labeled in the NMN moiety. The true N I 0~~~~~~~~~~1** substrate of this phosphoadenylylation reaction was 2'- phospho-ADP-ribose rather than NADP, because labeled HO OH phospho-ADP-ribose was as efficient as or more efficient than I HO O NADP in forming modified p40. Also, NADP was rapidly converted to phospho-ADP-ribose during incubation with 0I * microsomes. Furthermore, isonicotinic acid hydrazide, an I civ- - -- inhibitor of NADP glycohydrolase, prevented phosphoadenyl- I ~~~~~Active P-AMP ylation from NADP, but not from phospho-ADP-ribose, and glycohydrolase-resistant NADPH could not substitute for Active P-ADP-Rib NADP. Transferase activity was found in liver and brain microsomes and, to a smaller extent, in the cytosol fractions. In FIG. 1. Active groups in NADP. The symbols 0 and * denote Ehrlich ascites tumor cells, most of the activity resided in the specific labeling of groups in NADP species used in this study. cytosol, from which it could be partially purified. The apparent Km for phospho-ADP-ribose was about 2 x 10-4 M, and the pH ribosyltransferases or adenylyltransferases. To date, howev- optimum was around 7. Divalent cations like Mg2+ and Mn2+ er, neither phosphoADP-ribosyl- nor phosphoadenylyltrans- inhibited the reaction. In all compartmental preparations, ferases have been described, except perhaps for a nonspecific activity was eliminated by heating or short treatment with ADP-ribosyltransferase from turkey erythrocytes that can alkali or acid. In submitochondrial particles from rat liver, a release nicotinamide from NAD and NADP with similar system with different characteristics led to the phosphoadenyl- efficiency in the presence (or absence) of guanidino com- ylation of several endogenous polypeptides. pounds (4). Here we report on phosphoadenylylation as a type of Post-translational modification is used by cells to expand and enzymatic covalent modification, which differs from the to modulate properties and functions ofproteins. Apart from ATP-dependent adenylylation of prokaryotic syn- hydrolytic processes, these modifications usually require thetase, described by Stadtman and co-workers (5) and by group transferring coenzymes, in which the modifying group Holzer and co-workers (6), and also from the autoadenylyla- is present in an activated form, such as "aktivierte Es- tion of bacterial DNA ligase as an intermediate step in DNA sigsaure" in acetyl-CoA or active sulfate in adenosine 3'- ligation (7). phosphate 5'-phosphosulfate (cf. refs. 1 and 2). This applies also to ADP-ribosylation and ADP-ribosylation polymerizing reactions, in which NAD as the cosubstrate represents an MATERIALS AND METHODS activated form of ADP-ribose (ADP-Rib) (3). Labeled Compounds. Synthesis of labeled NADP from Unlike NAD, the second coenzyme NADP has been found labeled NAD (8) will be described in detail elsewhere. so far to serve exclusively oxidoreduction reactions. Here, Briefly, 4 ,umol of [3H]NAD (282 x 106 cpm/,umol) was the additional phosphate group in the 2'-hydroxyl position of incubated in 2 ml containing 20 ymol of ATP, 40 jamol of the adenine-proximal ribose is used as a signal for MgSO4, 200 Amol ofTris HCl (pH 7.5), and NAD kinase (250 dehydrogenases serving primarily anabolic reactions. How- units) at 370C for 60 min. The mixture was chromatographed ever, NADP also has two energy-rich bonds that provide an on a Dowex 1/formate column (i x 4 cm), using a linear activated adenosine 2'-phosphate, 5'-diphosphate ribose (P- gradient with 250 ml each of H20 and 4 M HCOOH. The ADP-Rib) and an activated adenosine 2',5'-bisphQsphate [3H]NADP-containing fractions were pooled and evaporated (P-AMP) residue, respectively (cf. Fig. 1). Thus, the 2'- to dryness under reduced pressure. The residue was taken up phosphate group in NADP also could serve as a discriminat- in 1 ml of H20. The yield of chromatographically pure ing signal for transferases functionally different from ADP- [3H]NADP was 90%.

The publication costs of this article were defrayed in part by page charge Abbreviations: EAT, Ehrlich ascites tumor; P-ADP-Rib, adenosine payment. This article must therefore be hereby marked "advertisement" 2'-phosphate, 5'-diphosphate ribose; P-AMP, adenosine 2',5'-bis- in accordance with 18 U.S.C. §1734 solely to indicate this fact. phosphate; ADP-Rib, ADP-ribose. 6267 Downloaded by guest on September 28, 2021 6268 Biochemistry: Hilz et al. Proc. Natl. Acad. Sci. USA 83 (1986) Doubly labeled [2'-phosphate-33P, adenine-3H]NADP was buffer, pH 7.6 (18), incubated in the presence of 1 mM synthesized from [y_ 3P]ATP and [adenine-3H]NAD by in- dithiothreitol for 30 min at 37°C, and applied to 10% slab gels cubation with NAD kinase, using a modification ofthe above (sample buffer, pH 7.6). The gels were stained with Coomas- procedure (unpublished data). NAD and NADP labeled in sie blue, destained, and either dried for autoradiography other positions (cf. Fig. 1) were synthesized along the same (with the application of enhancer) or sliced (2-mm slices). To lines. [ribose(NMN)-14C]NAD was kindly provided by K. release the labeled components, slices were heated in 1 ml of Ueda (Kyoto) and by M. Jacobson (Fort Worth). 5% (vol/vol) trichloroacetic acid or 0.1 M NaOH (100'C, 60 P-[3H]ADP-Rib and P-ADP-Rib were prepared from la- min), and the radioactivity was measured. beled NADP by brief exposure to alkali (100 mM NaOH, 37TC, 10 min; M. Jacobson, personal communication), fol- lowed by chromatographic purification on Dowex 1/formate, RESULTS or by HPLC. P-AMP was obtained from Sigma or purified by Apparent PhosphoADP-Ribosyltransferase Activity in ion-exchange chromatography after treatment ofNADP with Microsomes from Rat Liver. When microsomal preparations phosphodiesterase. from rat liver were incubated with labeled NADP, a time- and Cellular Fractions and Extracts. Rat liver mitochondria, concentration-dependent incorporation of adenine equiva- mitoblasts, and submitochondrial particles were obtained as lents into the trichloroacetic acid-insoluble fraction was described (9). Extracts of submitochondrial particles were observed (Fig. 2). This activity was not caused by a phos- prepared by incubating submitochondrial particles (30 min, phatase-catalyzed breakdown of NADP to NAD and a 0C, 125 mg of ) with 7 ml of buffer containing 6% subsequent ADP-ribosylation, because NAD was much less (vol/vol) Triton X-100, 10 mM Tris HCl, 50 mM KCl, 10 mM active than NADP in this system. Due presumably to the potassium phosphate, 1 mM EDTA, 1 mM dithiothreitol (pH trapping of labeled substrates and/or breakdown products, 7.4). The suspension was centrifuged in the cold in a Ti-75 the incorporation of adenine equivalents into the acid- Rotor at 123,000 X g for 35 min, and aliquots of the extract insoluble fraction did not exactly reflect formation of cova- were frozen until use. lently modified polypeptides as analyzed by NaDodSO4 gel Microsomes of rat liver were obtained from the post- electrophoresis. When NADP was the substrate, and the mitochondrial (12,000 x g) supernatant by centrifugation at trichloroacetic acid-insoluble reaction products were sepa- 100,000 x g for 60 min. The pellet was resuspended in 20 ml rated by NaDodSO4 gel electrophoresis and subjected to of 10 mM Hepes (pH 7.2), and aliquots were kept frozen autoradiography, one major radioactive polypeptide with an (-300C) until use. Triton X-114-extracted microsomes were apparent molecular weight of 40,000 was seen. NAD was obtained by sonication (five times for 5 sec at maximal nearly ineffective in this test. The NADP-dependent labeling output, 0C) in 0.5% purified (10) Triton X-114/10 mM ofp40 was much more pronounced in Triton X-114-extracted Hepes, pH 7, and centrifugation (100,000 x g, 60 min). The microsomes, suggesting that there were Triton-extractable pellet was resuspended in 10 mM Hepes (pH 7.2). Rat liver inhibitors (Fig. 2, Inset). nuclei were isolated as described (11). Plasma membranes Characterization of the Transferase Reaction as 2'-Phos- from mouse liver were kindly provided by E. Steinhagen- phoadenylylation of p40. The precursor used in the initial Thiessen (Hamburg, F.R.G.). experiments was a [3H]NADP synthesized from [adenine- Electrophoresis samples were precipitated with cold 10% 3H]NAD. To show incorporation of the 2'-phosphate group (vol/vol) trichloroacetic acid, washed twice with the acid and into p40, doubly labeled NADP was prepared that contained twice with ether. The dry residue was dissolved in sample a [3H]adenine moiety and a 2'-[33P]phosphate group. When

A B 800- 7.5- NADP E 0 0. / 6)600-

0 92

400- .)_ 45

._ I ,0-~ NAD U 2.5 / M _OM o ~~~~~31- 040 gOo '03 C 1)

I I 1 20 40 60 20 40 Incubation time, min Microsomes, ,ul FIG. 2. Incorporation of adenine equivalents from labeled pyridine into the microsomal acid-insoluble fraction. (A) Rat liver microsomes (240 ,Ag of protein) were incubated in 100 mM Tris/acetate, pH 6.0, containing 100 ,uM [3H]NAD or [3H]NADP (4 x 10i cpm) for the times indicated. (B) Different amounts of microsomes (24 ,ug/,u) were incubated with [3H]NADP in 100 mM Hepes (pH 7.0) at 37°C for 45 min. Inset: NaDodSO4 gel electropherogram of incubations with [3H]NAD (lanes A and C) or [3H]NADP (lanes B and D). Coomassie Blue stain (lanes A and B) and autoradiogram (lanes C and D) are shown. Rat liver microsomes extracted with Triton X-114 (500 ,g ofprotein) were incubated in 100 Al containing 100 mM Tris/acetate buffer (pH 7.0) and 100 ,uM [3H]NAD or [3H]NADP (3 x 106 cpm each) at 37C for 120 min. Downloaded by guest on September 28, 2021 Biochemistry: Hilz et al. Proc. Nati. Acad. Sci. USA 83 (1986) 6269 this compound was used as a substrate for the microsomal reaction, 3H and 33P were incorporated into p40 at the same ratio present in the labeled NADP (Fig. 3A). Thus the modified acceptor contained at least the 2'-phosphate group, the associated ribose, and the adenine ring of NADP. ~100-o V ' However, when NADP labeled in the nicotinamide-proximal ~~~~~O~.4 ribose was the substrate, virtually no labeling ofthe acceptor o was seen (Fig. 3C). To show that the [14C]NADP containing 25 50 75 the label in the nicotinamide-proximal ribose did not contain a ~~~~~~~~0 inhibiting material, it was also used as a substrate for an CZ ADP-ribosyltransferase from turkey erythrocytes (4). This ItP-ADP-Rib 50 was able to transfer phosphoADP-ribosyl groups Q from the [NMN-14C]NADP to H1 at a rate somewhat higher than that observed with labeled NAD (unpublished P-ADP experiments), thus demonstrating intact substrate properties < 25 of the [14C]NADP. Our data then show that the reaction - catalyzed by the microsomal enzyme is a phosphoadenylyla- tion rather than a phosphoADP-ribosylation. NADP To gain further support for this interpretation, we have also synthesized NADP labeled with 32P at the 5'-phosphate group 0 25 50 75 ofthe AMP moiety or at the 5'-phosphate group of the NMN Time, min moiety. The former substrate again yielded a labeled deriv- ative migrating to the position ofp40 (Fig. 3C), while p40 was FIG. 4. Kinetics of NADP degradation in microsomes extracted not labeled when NADP labeled in the NMN-linked 5"- with Triton X-114. Double-labeled NADP (65 nmol) ([2'-phosphate- phosphate group was used (Fig. 3B), thus confirming the data 32P, adenine-3H]NADP: 298 cpm of33P per pmol ofNADP, 1.7 X 10' cpm; 250 cpm of3H per pmol ofNADP, 1.5 x 107 cpm) was incubated obtained with NADP labeled at the NMN-linked ribose. in 650 ul containing 100 mM Hepes, pH 7.0, and the microsomal P-ADP-Rib Is the True Substrate of the Phosphoadenylyla- pellet remaining after extraction with 0.5% Triton X-114 (1.6 mg of tion Reaction. The reaction with [3H]NADP was analyzed for protein). Aliquots were removed at the indicated times and pro- inhibition by various phosphoAMP analogs. No significant cessed. The trichloroacetic acid-insoluble residue was dissolved in interference ofp40 phosphoadenylylation was seen by any of sample buffer and analyzed for p40 labeling by gel electrophoresis. these derivatives when added at concentrations equimolar to Aliquots ofthe trichloroacetic acid-soluble fraction were analyzed by HPLC using ,Bondapak C18 columns and 20 mM phosphate to 20 the NADP, except for P-ADP-Rib. When free P-[3H]ADP- mM phosphate/50%o (vol/vol) methanol gradients. Inset: Time Rib was used as a precursor instead of NADP, incorporation course of p40 labeling. ofadenine equivalents into p40 was equal or even higher than with NADP (cf. Fig. 5). Incorporation from labeled P-AMP was insignificant. This suggested that P-ADP-Rib rather than min, NADP was degraded to negligible concentrations (Fig. NADP was the true substrate. The following experiments 4); yet, incorporation of P-AMP residues into p40 continued strongly support this interpretation: (i) [3H]NADP was incu- linearly for at least 75 min (Fig. 4, Inset). The only substrate bated with Triton X-114-extracted microsomes. Within 10 present in high amounts throughout the entire incubation 600- 600- A Cc

400- 500-

E 200- -6c) -o 400- -

0. C) 300 -

0

200- ._cu

100-

! _1 I I 10 20 30 40 50 Slice number FIG. 3. Incorporation of radioactivity from differently labeled NADP species into microsomal polypeptides. (A) [3H]Adenine (o) and 2'-[33P]phosphate (e) groups from doubly labeled NADP. (B) [3H]Adenine (o) vs. 5"-[33P]phosphate NMN (o) groups in NADP. (C) [a-32P]AMP (0) vs. [ribose-'4C]NMN (o) groups in NADP. The differently labeled NADP species were incubated at 100 AM concentrations under standard conditions, with slight variations in time and pH. Downloaded by guest on September 28, 2021 6270 Biochemistry: Hilz et al. Proc. Nati. Acad. Sci. USA 83 (1986) period was P-ADP-Rib, which had been formed at the Table 2. Inhibition of phosphoadenylylation in EAT cell cytosol expense of NADP. (ii) Preventing NADP glycohydrolase by P-ADP-Rib analogs activity by isonicotinic acid hydrazide blocked the transfer P-AMP incorporation, reaction from NADP, but not from P-ADP-Rib (Table 1). Also, [3H]NADPH, which is quite resistant to NADP Additions pmol % of control glycohydrolase (12), could not serve as a substrate. (iii) The None 9.29 100 significantly higher heat sensitivity of the reaction when ADP-Rib at 1 mM 9.22 100 [3H]NADP was the precursor (Table 1) also suggested in- ADP-Rib at 10 mM 3.33 36 volvement of an additional, more labile enzyme. ATP at 1 mM 7.23 78 Transfer of P-AMP residues from P-ADP-Rib appeared to P-AMPat 10 mM 1.35 14 be enzymatic in nature as indicated by the sensitivity towards CoA at 1 mM 2.55 27 heating and towards short exposure to NaOH or trichloro- HPO2- at 10 mM 3.34 36 acetic acid (Table 1). H2P2Oq- at 10 mM 1.58 17 Phosphoadenylyltrans- Preliminary Characterization of the EAT cytosol (500 .ug ofprotein) was incubated in Tris/acetate, pH ferase System. PhosphoAMP transferase activity was not 6.8, with 100 A&M P-[3H]ADP-Rib (282 cpm/pmol) for 2 hr and only present in rat liver microsomes, but also in Ehrlich analyzed for p40-45 modification. ascites tumor (EAT) cells, where most activity was found in the cytosol (see below). In both systems, transferase activity was specific for P-ADP-Rib. Although ADP-Rib was able to to phosphodiesterase I, more detailed studies are required to interfere with the transfer reaction, concentrations 100-fold elucidate the true nature of the linkage involved. higher than the substrate were required to inhibit the reaction Subcellular Distribution of Phosphoadenylyltransferase. to 36% (Table 2). ATP had also little effect at 1 mM When rat liver was fractionated into subcellular compart- concentrations. The importance ofa second phosphate group ments, no significant endogenous P-AMP transfer reaction is further by the was found in nuclei or plasma membranes, but high specific in the AMP moiety ofthe substrate indicated activity was found in the microsomal fraction, especially significant inhibitory potential ofcoenzyme A (3' phosphate) when inhibitory factors were removed by treatment with and ofP-AMP. It should be pointed out, that P-AMP, which Triton X-114, and small amounts of transferase activity were lacks an energy-rich bond, could not serve as a substrate for also observed in the cytosol. Microsomal preparations from the transferase reaction (not shown). Phosphate and pyro- mouse brain and from EAT cells also exhibited phosphoad- phosphate ions were strongly inhibitory at concentrations enylylating activity with the preferential modification of p40 usually applied in buffer solutions. Divalent cations like (or a closely related acceptor). In the EAT cells, however, Mg2", Ca2+, and especially Mn2+ were also potent inhibitors most activity was associated with the cytosol. In this case, (Fig. 5). No marked influence on phosphoadenylylation of the principal acceptor appeared to be slightly larger than p40 p40 was seen with EDTA or with sulfhydryl-blocking agents (cf. Fig. 5). like iodoacetate or mercury salts. The apparent Km for Preliminary data indicate that extracts from submitochon- P-ADP-Rib in liver microsomes or EAT cytosol was close to drial particles (inner membranes) from rat liver also exhibit 2 x 10-4 M. P-AMP transfer to endogenous polypeptides when incubated Chemical Stability of the Bond Linking P-AMP to the with NADP or P-ADP-Rib. However, the mitochondrial Microsomal Acceptor. The linkage of the microsomal conju- system differed significantly from the microsomal reaction, gate(s) appears to be relatively labile at neutral pH as shown leading to the modification ofmultiple acceptor polypeptides. by a 30-min incubation of resuspended acid-precipitated Furthermore, in this system, Mn2+ ions intensified labeling reaction products at pH 7 (in Hepes buffer) and 37°C that led and changed the pattern of modified proteins. Experiments to a loss of about 20% of the adenine residues. At pH 1 none performed with NADP labeled in the NMN moiety showed were lost. Increasing the pH to 13 induced a complete release that the mitochondrial system also catalyzed the transfer of of P-AMP equivalents. No acceleration of breakdown was P-AMP rather than a modification of P-ADP-Rib residues. seen with 3 M NH20H, alkaline phosphatase, phosphodies- terase, or RNase at neutral pH. The release effected by proteinase K rather reflects degradation to acid-soluble phosphoadenylylated oligopeptides than a direct effect on the 118 linkage of the modifying group. Although these data appear - 66 to be compatible with a phosphodiester bond not accessible }NR 1. - 45 Table Different sensitivities towards various treatments of - .~ phosphoadenylyltransfer from NADP versus P-ADP-Rib G= 36 P-AMP transfer, cpm incorporation relative to --29 controls Treatment [3H]NADP [3H]P-ADP-Rib I None 100 100 FiG. 5. Modification ofpolypeptides in microsomal and cytosolic INH at 20 mM 11 80 preparations from different precursors. Protein-stained gel (lanes G 56°C, 10 min 21 70 and H) and autoradiogram ofNaDodSO4 gel electropherogram (lanes 95°C, 5 min <0.1 <0.1 A-F). Lanes A-E and G: Triton X-114-extracted microsomes (500 ,g NaOH at 100 mM, 0°C, 5 min 5 1 of protein). Lanes F and H: EAT cell cytosol (300 Ag of protein). EAT cell cytosol was incubated with P-[3H]ADP-Rib, and micro- Triton X-114-extracted microsomes (600 jg of protein) were somes were incubated with [3H]NAD (lane A), with [3H]NADP (lane treated as indicated followed by incubation (2 hr, 37°C; ± INH) in the B), or with P-[3H]ADP-Rib (lanes C-E), plus 10 mM Mg2+ (lane C), presence of 100 uM labeled precursor (about 2 x 106 cpm). or plus 10 mM Mn2+ (lane D) for 2 hr with 200 ,uM precursor in Incorporation into p40 was analyzed. INH, isonicotinic acid Tris/acetate, pH 6.8. Molecular size markers in kDa (lane I) are hydrazide. indicated. Downloaded by guest on September 28, 2021 Biochemistry: Hilz et al. Proc. Natl. Acad. Sci. USA 83 (1986) 6271 DISCUSSION an essential coenzyme, these NADP glycohydrolases pro- vide the ultimate substrate for P-AMP transferase(s), the When exploiting the group transfer potential of NADP as specificity of which precludes direct use of NADP or opposed to NAD, incorporation of adenine equivalents NADPH for the modification reaction. Glycohydrolase ac- together with 2'-phosphate groups into microsomal and tivity could thus become a rate-limiting factor in phospho- mitochondrial preparations first suggested the presence of adenylylation. 2'-phosphoADP-ribosyltransferase activity. However, incu- While the cellular NADP concentration in liver appears to bation with NADP species labeled in various parts of the be well above 200 ,uM (cf. ref. 17), steady state levels of molecule revealed that these transferases made use of the P-ADP-Rib may be much lower. However, phosphoadenyl- energy-rich bond in the pyrophosphate group ofNADP rather ylation ofp40 proceeds even at 3 ,uM substrate concentration than of the bond linking P-ADP-Rib to the quaternary with remarkable efficiency (about 10% of that at the Km nitrogen of the pyridine ring (cf. Fig. 1). To our knowledge, concentration). Furthermore, NADP glycohydrolase and this type of covalent modification has not been described phosphoadenylyltransferase may form a complex thus chan- before. It differs not only from enzymatic ADP-ribosylation neling the glycohydrolase product P-ADP-Rib directly to the reactions (cf. ref. 13), but also is distinct from nonenzymatic transferase. modification reactions, in which free ADP-Rib orP-ADP-Rib So far, neither the function of the acceptor polypeptide(s) formed acid-stable adducts with mitochondrial polypeptides nor the consequences of their modification are known. (9, 14, 15) and with glucose-6-phosphate dehydrogenase (16), However, the occurrence of this reaction in various higher respectively. While these reactions also proceeded in heat- , in dividing cells and resting tissues, in tumors, inactivated preparations (14), significant impairment of p40 and in terminally differentiated cells suggest an important modification by P-ADP-Rib was seen already after short function in cellular regulation. exposure ofmicrosomes to 560C, and complete elimination of the reaction was achieved by heating to 960C for 5 min. Short The authors thank Kunihiro Ueda (Kyoto) who sent H.H. (as a treatment with cold alkali or cold acid also destroyed the birthday present) ['4C]NAD labeled in the nicotinamide-proximal transfer, as expected for an enzymatic reaction. Together ribose, thus providing the first tool to unravel the covalent modifi- with the reaction optimum at pH 6.8, the rather specific cation reation. We are also deeply indebted to Mike Jacobson (Fort inhibition by P-ADP-Rib analogs and by certain divalent ions Worth) whose stimulating discussions promoted this work and who as well as the high substrate specificity, these data strongly supported us with another generous gift of NMN-ribose-labeled point to the enzymatic nature of the phosphoadenylylation NAD. Our thanks go also to Renate Pforte who helped edit the paper. reaction. This work was supported by the Deutsche Forschungsgemeinschaft. It is interesting to note that the microsomal and cytosolic 1. Lynen, F. & Reichert, E. (1951) Z. Angew. Chem. 63, 290. system preferentially modified a single polypeptide (p40) 2. Hilz, H. & Lipmann, F. (1955) Proc. Natl. Acad. Sci. USA 41, while in mitochondria several proteins became phosphoad- 880-890. enylylated. The pattern of mitochondrial polypeptides car- 3. Zatman, L. J., Kaplan, N. 0. & Colowick, S. P. (1953) J. Biol. rying P-AMP residues could be markedly changed by the Chem. 200, 197-212. presence of Mn2+ ions. 4. Moss, J. & Vaughan, M. (1982) in ADP-Ribosylation Reac- Without isolation ofmodified p40, no reliable data as to the tions, eds. Hayaishi, 0. & Ueda, K. (Academic, New York), stoichiometry of the reaction can be given. Preliminary data, pp. 637-645. however, indicate that more than 15% of the p40 were 5. Shapiro, B. M., Kingdon, H. S. & Stadtman, E. R. (1967) modified under that not Proc. Nati. Acad. Sci. USA 58, 642-647. conditions did lead to saturation. 6. Wulif, K., Mecke, D. & Holzer, H. (1967) Biochem. Biophys. Basically, phosphoadenylylation bears some similarity to Res. Commun. 28, 740-745. adenylylation reactions first described by Stadtman and 7. Lehmann, I. R. (1974) Science 186, 790-797. co-workers (5) and by Holzer and co-workers (6) as a 8. Nolde, S. & Hilz, H. (1972) Hoppe-Seylers Z. Physiol. Chem. mechanism to modulate activity in 353, 505-513. . In this case, however, ATP is used as a 9. Kun, E., Kirsten, E. & Piper, W. (1979) Methods Enzymol. 55, substrate providing the activated form of adenylate. There is 115-118. also a certain similarity to the bacterial DNA ligase reaction, 10. Bordier, C. (1981) J. Biol. Chem. 256, 1604-1607. where transfer of AMP to the active center of the enzyme 11. Adamietz, P., Wielckens, K., Bredehorst, R., Lengyel, H. & Hilz, H. (1981) Biochem. Biophys. Res. Commun. 101, 96-103. appears to be an intermediate step in the overall reaction 12. McCreanor, G. M. & Bender, D. A. (1983) Biochim. Biophys. occurring with NAD as a substrate (7). Acta 759, 222-228. Modification of eukaryotic proteins by phosphoadenylyla- 13. Hilz, H. & Stone, P. R. (1976) Rev. Physiol. Biochem. tion seems to be closely linked to the metabolism of NADP, Pharmacol. 76, 1-58. because the true substrate P-ADP-Rib presumably can only 14. Hilz, H., Koch, R., Fanick, W., Klapproth, K. & Adamietz, P. be provided via NADP. The modification process, therefore, (1984) Proc. Natl. Acad. Sci. USA 81, 3929-3933. might depend not only on the activity of the phosphoadenyl- 15. Hilz, H., Koch, R., Kreimeyer, A., Adamietz, P. & Jacobson, yltransferase, but also on the cellular concentration ofNADP M. K. (1985) in ADP-Ribosylation of Proteins, eds. Althaus, as well as on the ratio since NADPH is F. R., Hilz, H. & Shall, S. (Springer, Heidelberg), pp. NADP/NADPH quite 518-526. resistant to NADP glycohydrolase. 16. Skala, H., Vibert, M., Kahn, A. & Dreyfus, J. C. (1979) Phosphoadenylylation of proteins may also allow us to Biochem. Biophys. Res. Commun. 89, 988-996. attribute a new function to the that hydrolyze 17. Sies, H. (1982) in Metabolic Compartmentation, ed. Sies, H. NADP at the glycosidic bond linking P-ADP-Rib to the N1 of (Academic, New York), pp. 205-231. the nicotinamide moiety. Beyond a mere catabolic action on 18. Laemmli, U. K. (1970) Nature (London) 227, 680-685. 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