Tryptic digest of the alpha subunit of lamb kidney (Na+ + K+)-ATPase.
The Mr approximately equal to 100 000 alpha subunit was prepared from highly purified lamb kidney (Na+ + K+)-ATPase. Its N-terminal sequence is Gly-Arg-Asx-Lys-Tyr-Glu. The alpha subunit was S-carboxymethylated, succinylated, and cleaved at its 40 arginine residues with trypsin. Four major, well-differentiated peptide fractions (A to D) were obtained by chromatography of the digest on a Sephadex G-50 column. Fraction A eluted at the void volume of the column and contained aggregated, very hydrophobic peptides, possibly from regions of alpha that are buried within the membrane lipid bilayer in the native enzyme. Fractions B to D, which together accounted for about 75% of the total protein, contained water-soluble peptides. To test the feasibility of using antibodies to identify and purify specific peptides of alpha subunit, studies were carried out using antibodies to native (Na+ + K+)-ATPase. Carboxymethylation and succinylation did not significantly decrease total antibody binding to alpha subunit, although the affinity of the anti-(Na+ + K+)-ATPase antibodies for alpha subunit was reduced by about 50%. The tryptic peptides of alpha subunit also retain significant immunochemical reactivity. Fractions A, B and C (but not D) of the digest all bind antibodies. To characterize further the tryptic digest, 16 peptides from fraction D were isolated and sequence studies on these were carried out.
D000595:Amino Acid Sequence; D000818:Animals; D007679:Kidney Medulla / Q000201:enzymology*; D046911:Macromolecular Substances; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D012756:Sheep; D000254:Sodium-Potassium-Exchanging ATPase; D014357:Trypsin
D046911:Macromolecular Substances; D010446:Peptide Fragments; D014357:Trypsin; D000254:Sodium-Potassium-Exchanging ATPase