In the presence of ATP and a cytosolic factor, cholera toxin fragment A1 catalyzes the transfer of ADP-ribose from NAD to a number of soluble and membrane-bound proteins of the pigeon erythrocyte. Evidence is presented that suggests that the most readily modified membrane protein (M, 42,000 The activation of adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1.] by cholera toxin involves the interaction of some part of the cyclase system exposed on the inner surface of the plasma membrane with NAD, a nucleoside triphosphate, and a cytoplasmic protein (1-4) and is conveniently studied in lysed pigeon erythrocytes. The likelihood that the NAD might function as an adenosine diphosphoribose donor in a toxincatalyzed reaction of the type NAD+ + protein T ADP-ribosyl protein + nicotinamide + H+ was increased by the demonstrations that the active A1 fragment of cholera toxin catalyzes the slow hydrolysis of NAD to ADP-ribose and nicotinamide (5) and the transfer of ADP-ribose to arginine and related compounds (6) and to itself (C. King, personal communication; ref. 7).If the postulated acceptor were adenylate cyclase or an associated membrane protein, it should be possible to demonstrate a toxin-dependent transfer of ADP-ribose from radioactive NAD to a small number of sites on pigeon erythrocyte ghosts. There is, however, a large toxin-independent incorporation of ADP-ribose which was difficult to decrease without losing the toxin response. We now report the resolution of this problem and the demonstration of several toxin-specific ADP-ribose acceptors, principally a 42,000 Mr membrane protein. METHODSThe medium used throughout consisted of 0.13 M NaCl, 0.01% sodium azide, Trasylol (aprotinin: FBA Pharmaceuticals) at 2 kallikrein inactivator units/ml, 10 mM N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (Hepes), and NaOH to give pH 7.3 at 370. Washed and purified pigeon erythrocytes suspended in an equal volume of this medium were lysed by rapid freezing and then thawing. The lysate was centrifuged at 10,000 X g for 5 min to separate ghosts and cytosol. The cytosol was mixed with well-washed pig brain NAD glycohydrolase (Sigma) at a ratio of 1 ml/10 mg, agitated at 370 for 30 min, and then centrifuged to remove the enzyme. The ghosts were washed three times in 20 vol of medium. There are about 8 X 109 ghosts per ml (packed).Portions of lysate reconstituted from 1 part washed ghosts and 2 parts NAD-depleted cytosol fraction were incubated for 30 min at 250 or 370 with 10 mM thymidine, 5

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When equimolar amounts of purified homologous A- and B-chains from abrin or ricin are mixed and dialyzed to remove 2-mercaptoethanol, the disulfide bridge between the chains reforms and fully toxic molecules are reconstituted. In a similar manner, when heterologous chains are mixed and dialyzed, the toxic hybrid molecules abrin A/ricin B or ricin A/abrin B are formed in good yield. Stable hybrid molecules were not formed between the two A-chains or the two B-chains. Whereas rabbit anti-abrin and anti-ricin sera each neutralizes only its homologous toxin, neither antitoxin can distinguish between the hybrid toxins. On the other hand, antibodies specifically directed against determinants on A- or B-chains readily distinguish between the two hybrids. Neutralization by antisera of the toxic effects of abrin, ricin, and their hybrids on protein synthesis in HeLa cell cultures has been studied quantitatively. The results are discussed in relation to other interspecies hybrid molecules and in relation to the mode of action of certain glycoprotein tropic hormones.