The first five amino acids of the catalytic alpha 1-subunit predicted from its cDNA are not found in purified mammalian Na(+)-K(+)-ATPase, suggesting co- or posttranslational cleavage. To facilitate evaluation of amino-terminal structure and the cleavage process, we developed a site-directed antibody (anti-VGR) specific for the first nine residues of nascent alpha 1 from rat. In immunoblots of polypeptides generated by in vitro translation, anti-VGR detected a prominent band with a mobility appropriate for the alpha 1-subunit (100 kDa). Immunoblots of total protein from various rat organs, however, revealed no significant binding, implying that virtually all the alpha 1-subunit expressed in vivo was modified. We also assessed amino-terminal structure in various heterologous expression systems. Binding of anti-VGR was observed in Escherichia coli transformed with a vector containing an alpha 1/troponin fusion protein and in insect cells infected with baculovirus containing full-length alpha 1 or alpha 1T. This suggests that modification of the introduced alpha 1 in these expression systems was absent or different from that in mammals. In contrast, green monkey kidney cells (COS-1) transfected with alpha 1 did not reveal significant binding of the antibody, indicating that the introduced isoform was processed appropriately. These results demonstrate that the structure of the alpha 1-subunit's amino terminus differs among various expression systems. The results further imply that efficient co- or posttranslational processing of nascent alpha 1 is conserved among various organs within the rat, yet the required modification enzymes are not present in distant phyla.

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By CHARLES HUGH CLARKE and FRANCIS FRANCIS. ALTHOUGH Tiemann and Friedlander (Ber., 1881, 14, 1968) were unable to prepare a-amino-a-phenylacetamide in a pure state from its hydrochloride, we have found the base is stable, and may be easily obtained from mandelonitrile. The action of alcoholic ammonia on the aldehydecyanohydrins results in the formation of the a-amino-nitriles, but if the reaction is carried out in the presence of potassium hydroxide, partial saponification of the nitrile group takes place ; and, if benzaldehydecyanohydrin is used, a-amino-a-phenylacetamide is formed, thus : C,H,*CH(OH)*CN + NHT,-+ C , H , * C H (~I I ,) * C O * N~~~. The isolation of the base from the other products of the reaction is difficult, but if the decGmposition is carried out in the presence of benzaldehyde, the well crystalline Z, enzylidene derivative can be readily obtained to the extent of about 65 per cent. of the cyanohydrin taken, and a-amino-a-phenylacetamide is best prepared from it by treating it with phenylhydrazine, when the separation of the resulting benza.1dehydephenylEydrazone from the bas? is easily carried out, and a quantitative yield obtained. a-Amino-a-phenylacetamide is characterised by the readiness with which i t condenses with aromatic aldehydes, forming products of the type C,II,*CH(N:CIIIR)*C0.NH2. A similar reaction takcs place with acetoacetic ester and ethylacetoacetic ester, but not with the diethyl derivative. Greater interest lies in the ease with which the five-membered ring, phenylhydantoin, can be formed from the base. The carbethoxy-compound gives an 85 per cent. yield of phenylhydantoin when treated with alcoholic potassium hydroxide : The ease with which tlie base and its carbethoxy-derivative can Lo prepared xcnders this the best method for the preparation of this substance. Corresponding experiments with a-amino-a-p-methoxyphenylacetamide showed that this also lent itself readily to the formation of p-metlboayph e n y lhytlunt oin. As compared with the ease with which five-membered rings uf the above type can be formed is the difficulty experienced in the preparation of any six-membered heterocyclic derivative,

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