[A method of selective isolation of tryptophan- and cysteine-containing peptides by covalent chromatography. Analysis of the topography of the Na+,K+-ATPase alpha-subunit].
A procedure for highly selective isolation of tryptophan- and cysteine-containing peptides from protein hydrolysates has been developed on the basis of covalent chromatography. It includes incorporation of a thiol group into the tryptophan residues by sequential treatment of peptides with 2-nitrophenylsulfenyl chloride and beta-mercaptoethanol followed by immobilization on the corresponding supports via thiol-disulfide exchange. The technique is applicable to the analysis of the hydrolysate of the Na+, K+-ATPase alpha-subunit obtained by limited trypsinolysis of the membrane-bound enzyme. Fifteen tryptophan- and cysteine-containing tryptic peptides, which comprise the protein portions exposed outside the membrane, have been isolated in addition to those previously identified. This structural information allows unequivocal determination of boundaries of transmembrane segments of the alpha-subunit in the spatial model earlier proposed.
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine; D006868:Hydrolysis; D007668:Kidney / Q000201:enzymology; D046911:Macromolecular Substances; D010449:Peptide Mapping; D010455:Peptides / Q000302:isolation & purification*; D000254:Sodium-Potassium-Exchanging ATPase / Q000302:isolation & purification*; D013438:Sulfhydryl Compounds; D013552:Swine; D014357:Trypsin; D014364:Tryptophan
D046911:Macromolecular Substances; D010455:Peptides; D013438:Sulfhydryl Compounds; D014364:Tryptophan; D014357:Trypsin; D000254:Sodium-Potassium-Exchanging ATPase; D003545:Cysteine
Metod selektivnogo vydeleniia triptofan- i tsisteinsoderzhashchikh peptidov kovalentnoĭ khromatografieĭ. Analiz topografii alpha-sub''edinitsy Na+,K+-ATP-azy.