Figure 6. Sequence analysis of [³H]Azicholesterol labeled fragments containing the M4 segment.

Peaks of ³H from the HPLC fractionations of tryptic digests of nAChR subunit fragments (Figure 5) were pooled for sequencing. Panel A, the digest of αV8-10. Fragments were detected beginning at αTyr-401 (□, I_o, 274 pmol; R, 90%) and αSer-388 (◇, I_o, 148 pmol; R, 92.5%) (254,000 cpm loaded on the filter and 25,600 cpm remaining after 30 cycles), with other unidentified peptides present at <10 pmol. Panel B, the digest of βV8-12. The primary sequence began at βAsp-427 (◇, I_o, 86 pmol; R, 94%), with secondary sequences beginning at βLeu-438 (□, I_o, 9 pmol; R, 91%), βLys-216 (βM1, I_o, 9 pmol; R, 94%, not shown), and βMet-249 (βM2, I_o, 4 pmol; R, 92%, not shown) (90,000 cpm loaded, 5,500 cpm remaining after 40 cycles). Panel C, the trypsin digest of γV8-14. Four fragments were present beginning at γVal-446 (□, I_o, 18 pmol; R, 92%), γGlu-429 (◇, I_o, 12 pmol; R, 89%), γThr-276 (γM3, I_o, 28 pmol; R, 90%, not shown), and γLys-218 (γM1, ∼5 pmol) with (53,000 cpm loaded on the filter, 3,300 cpm remaining after 30 cycles). Panel D, the trypsin digest of δV8-11. Peptides were identified beginning at δAsn-437 (□, I_o, 6 pmol; R, 93%), δLeu-456 (◇, I_o, 7 pmol; R, 93%), δLys-224 (δM1, I_o, 12 pmol; R, 94%, not shown), δMet-257 (δM2, I_o, 17 pmol; R, 89%, not shown), and Thr-28 from the β-subunit of the Na⁺/K⁺-ATPase (I_o, 10 pmol; R, 96%, not shown) (90,000 cpm loaded on the filter, 5,500 cpm remaining after 22 cycles). For each sample, ∼83% of each cycle of Edman degradation was analyzed for released ³H (●) and ∼17% for PTH-derivatives (□,◇), with the dotted line corresponding to the fit of the amount of detected PTH-derivatives. The amino acid sequences of the fragments containing the M4 segments are shown above each panel, with the limits of the M4 regions shaded.