BIOCHEMICAL-IDENTIFICATION OF TRANSMEMBRANE SEGMENTS OF THE CA2-ATPASE OF SARCOPLASMIC-RETICULUM()

The transmembrane segments of sarcoplasmic reticulum Ca2+-ATPase were determined by trypsinization of cytoplasmic side out intact sarcoplasm ic reticulum vesicles. The membrane portion of tryptic digest comprisi ng the transmembrane fragments, joined by the intravesicular segments, was separated by sodium dodecyl sulfate-polyacrylamide gel electropho resis after labeling with fluorescein 5-maleimide in the presence of s odium dodecyl sulfate. In this way, seven fluorescent bands of tryptic fragments below 11 kDa were observed which were derived from 4 pairs of membrane spanning segments and one hydrophobic sequence at the C-te rminal end. Two peptides of 10.8 and 10.6 kDa had the identical N-term inal sequence beginning at Glu(826), representing the transmembrane se gments M7 and M8 and their connecting loop. A band at 8.1 kDa containe d one peptide beginning at Tyr(36) (M1/loop/M2). A 7.7-kDa peptide sta rting at Leu(253) (M3/loop/M4) and a 7.3-kDa peptide beginning at Ala( 752) (M5/loop/M6) were also observed. A band at 6.7 kDa contained two peptides, one beginning at Ser(48) (M1/loop/M2) and another beginning at Tyr(763) (M5/loop/M6). In addition, a 4-kDa peptide beginning at Me t(925) was observed. The size of this peptide did not allow for a comp lete pair of transmembrane segments, but this peptide could have been derived from trypsinolysis between the last pair of membrane spanning segments. These data therefore provide biochemical evidence for at lea st 8 transmembrane segments and perhaps two more at the C-terminal end of the enzyme.