Topological disposition of the sequences -QRKIVE- and -KETYY in native (Na sup + + K sup + )-ATPase

Journal Article · Tue Mar 06 00:00:00 EST 1990 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00461a007· OSTI ID:6828743

Bayer, R ^[1]

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1. Univ. of California, San Diego, La Jolla (USA)

The dispositions with respect to the plane of the membrane of lysine-905 in the internal sequence -EQRKIVE- and of lysine-1012 in the carboxy-terminal sequence -RRPGGWVEKETYY of the {alpha}-polypeptide of sodium and potassium ion activated adenosinetriphosphatase have been determined. These lysines are found in peptides released from the intact {alpha}-polypeptide by the extracellular protease from Staphylococcus aureus strain V8 and by trypsin, respectively. Synthetic peptides containing terminal sequences of these were used to prepare polyclonal antibodies, which were then used to prepare immunoadsorbents directed against the respective peptides. Sealed, right-side-out membrane vesicles containing native (Na{sup +} + K{sup +})-ATPase were labeled with pyridoxal phosphate and sodium ({sup 3}H)borohydride in the absence or presence of saponin. The labeled {alpha}-polypeptide was isolated from these vesicles and digested with appropriate proteases. The incorporation of radioactivity into the peptides binding to the immunoadsorbent directed against the sequence pyrERXIVE increased 3-fold int the presence of saponin as a result of the increased accessibility of this portion of the protein to the reagent when the vesicles were breached by saponin; hence, this sequence is located on the cytoplasmic face of the membrane. It was inferred that the carboxy-terminal sequence -KETYY is on the extracytoplasmic face since the incorporation of radioactivity into peptides binding to the immunoadsorbent directed against the sequence -ETYY did not change when the vesicles were breached with saponin.

• OSTI ID: 6828743
• Journal Information: Biochemistry; (USA), Vol. 29:9; ISSN 0006-2960
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
ATP-ASE
MOLECULAR STRUCTURE
LYSINE
LABELLING
POLYPEPTIDES
BIOSYNTHESIS
ELECTROPHORESIS
STAPHYLOCOCCUS
TRITIUM COMPOUNDS
ACID ANHYDRASES
AMINO ACIDS
BACTERIA
CARBOXYLIC ACIDS
ENZYMES
HYDROGEN COMPOUNDS
HYDROLASES
MICROORGANISMS
ORGANIC ACIDS
ORGANIC COMPOUNDS
PEPTIDES
PHOSPHOHYDROLASES
PROTEINS
SYNTHESIS
550201* - Biochemistry- Tracer Techniques