Abstract:
:Because of the conflicting conclusions that have been reached regarding the location of the two putative membrane-spanning segments from cysteine 911 through isoleucine 929 and from isoleucine 946 through cysteine 964 in the alpha subunit of native ovine Na+/K(+)-transporting ATPase, the disposition of lysine 943 with respect to the plane of the lipid bilayer was investigated. Sealed, right-side-out vesicles were modified with pyridoxal phosphate and Na[3H]BH4 in the presence and absence of saponin, a reagent that creates holes in the membranes. Modified alpha polypeptide was isolated, and digested with trypsin and chymotrypsin to release the desired peptides, QQGMK and QQGMK([3H]pyr)NK (where [3H]pyr designates the modification on lysine 943). These peptides, after cyclization of their amino-terminal glutamines, were isolated with an immunoadsorbent specific for the amino-terminal sequence pyroglutamyl-QGM-followed by high-pressure liquid chromatography on a C-18 reverse phase column. Comparisons were made of the extent of incorporation of radioactivity into lysine 943 between sealed vesicles and sealed vesicles pretreated with saponin. An increase in incorporation into lysine 943 of 5-fold to 18-fold was seen in vesicles pretreated with saponin prior to the modification with pyridoxal phosphate. This increase in incorporation is consistent with a cytoplasmic location for lysine 943. This conclusion places the residues on the carboxy-terminal side of the putative membrane-spanning segment from cysteine 911 through isoleucine 929 and the amino-terminal side of the putative membrane-spanning segment from isoleucine 946 through cysteine 964 in the ovine alpha subunit on the cytoplasmic side of the membrane.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Anderberg SJdoi
10.1021/bi00029a027subject
Has Abstractpub_date
1995-07-25 00:00:00pages
9508-16issue
29eissn
0006-2960issn
1520-4995journal_volume
34pub_type
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