The high frequency of P/Q-type calcium-channel antibodies found in patients with Lambert-Eaton syndrome implies that antibodies of this specificity have a role in the presynaptic pathophysiology of this disorder.

The U1 snRNP-specific 70K protein is one of the few snRNP proteins from higher eukaryotic cells that is phosphorylated in vivo (1,2). Immunoaffinity purified spliceosomal snRNPs (U1, U2, U5, and U4/U6) were tested for their ability to phosphorylate in vitro the U1-specific 70K protein. An snRNP-associated kinase activity which phosphorylates all U1-70K isoelectric variants was identified. Like its in vivo counterpart, this snRNP-associated enzyme phosphorylates solely serine residues of the 70K protein, preferentially utilizing ATP as a phosphodonor. Tryptic phosphopeptide analysis revealed an overlapping set of at least four radiolabeled peptides in the in vivo and in vitro phosphorylated protein, suggesting that the snRNP-associated serine kinase is responsible, at least in part, for the 70K protein phosphorylation observed in vivo. Chymotryptic digestion of in vitro, 32P-labeled 70K protein and in vitro phosphorylation studies with a synthetic peptide, indicated that the multiple 70K phosphorylation sites are limited to a highly charged, C-terminal domain of the protein. In vitro phosphorylation studies with the splicing factor ASF/SF2 and several deletion mutants demonstrated that, similar to the U1-70K protein, the snRNP-associated serine kinase phosphorylates the carboxy terminal RS-rich domain of ASF/SF2. A potential general role for this enzyme in the phosphorylation of splicing factors and its consequences for pre-mRNA splicing regulation are discussed.

The proteins of the major human snRNPs U1, U2, U4/U6 and U5 were characterised by two-dimensional electrophoresis, with isoelectric focussing in the first dimension and SDS-polyacrylamide gel electrophoresis in the second. With the exception of protein F, which exhibits an acidic pl value (pl = 3.3), the snRNP proteins are basic. Post-translational modification was found among the proteins associated specifically with the U1 and U2 particles. The most complex modification pattern was observed for the U1-specific 70K protein. This was found in at least 13 isoelectric variants, with pl values ranging from 6.7 to 8.7; these variants differed also in molecular weight. All of the 70K variants are phosphorylated in the cell. Thin-layer analysis of their tryptic phosphopeptides revealed that the 70K variants have four major phosphopeptides in common, in addition to which at least four additional serine residues are phosphorylated to different extents. The comparative phosphopeptide analysis shows that differential phosphorylation alone is not sufficient to explain the occurrence of the many isoelectric variants of 70K, so that the final charge of the 70K variants is determined both by phosphorylation and by other, as yet unidentified posttranslational modifications. By two-dimensional separation of snRNP proteins obtained from mouse Ehrlich ascites tumour cells, it was shown that the pattern of pl values of the mouse proteins was almost identical with the corresponding pattern for human proteins. Even the complex modification patterns of the 70K protein are identical in mouse and man, indicating that the presence in the cell of so many variants of this protein may have functional importance. The major difference between murine and human snRNP proteins is the absence of protein B' from mouse snRNPs. This suggests that the homologous protein B may be able to carry out the task of protein B'.

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Biochemical properties of the ␣ 1 subunits of class A brain calcium channels (␣ 1A ) were examined in adult rat brain membrane fractions using a site-directed antipeptide antibody (anti-CNA3) specific for ␣ 1A . Anti-CNA3 specifically immunoprecipitated high affinity receptor sites for -conotoxin MVIIC (K d ϳ100 pM), but not receptor sites for the dihydropyridine isradipine or for -conotoxin GVIA. In immunoblotting and immunoprecipitation experiments, anti-CNA3 recognized at least two distinct immunoreactive ␣ 1A polypeptides, a major form with an apparent molecular mass of 190 kDa and a minor, full-length form with an apparent molecular mass of 220 kDa. The 220-and 190-kDa ␣ 1A polypeptides were also specifically recognized by both anti-BI-Nt and anti-BI-1-Ct antibodies, which are directed against the NH 2 -and COOH-terminal ends of ␣ 1A predicted from cDNA sequence, respectively. These data indicate that the predicted NH 2 and COOH termini are present in both size forms and therefore that these isoforms of ␣ 1A are created by alternative RNA splicing rather than post-translational proteolytic processing of the NH 2 or COOH termini. The 220-kDa form was phosphorylated preferentially by cAMP-dependent protein kinase, whereas protein kinase C and cGMP-dependent protein kinase preferentially phosphorylated the 190-kDa form. Our results identify at least two distinct ␣ 1A subunits with different molecular mass, demonstrate that they may result from alternative mRNA splicing, and suggest that they may be differentially regulated by protein phosphorylation.