Sodium- and potassium-activated adenosine triphosphatase of the nasal salt gland of the duck (Anas platyrhynchos). Purification, characterization, and NH2-terminal amino acid sequence of the phosphorylating polypeptide.
Sodium- and potassium-activated adenosine triphosphatase (NaK-ATPase) was purified from nasal salt glands of the duck (Anas platyrhynchos). Enzyme of specific activity 2,000 to 2,300 mumol of Pi/mg/hour was routinely obtained by sodium dodecyl sulfate treatment of a microsomal fraction of gland homogenate in the presence of 3 mM ATP followed by pelleting of the enzyme through a sucrose density gradient. Purified NaK-ATPase was stable for over 3 months at -20 degree. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration chromatography purified NaK-ATPase was shown to contain two polypeptide chains of molecular weight 94,000 and 60,000, the smaller of which was a glycoprotein. Purified enzyme of activity 2,300 mumol of Pi/mg/hour bound 3,600 pmol of ouabain/mg of enzyme protein. Reaction with [gamma-32P]ATP in the presence of Mg2+ and Na+ gave 7,025 pmol of acyl phosphate/mg of enzyme protein. The turnover number calculated from phosphorylation data was 5,460 min-1. Amino acid analysis of the polypeptide components of duck salt gland enzyme after separation by gel filtration chromatography in sodium dodecyl sulfate demonstrated strong compositional homology with highly purified NaK-ATPase preparations from other organs and species. The NH2-terminal amino acid of the 94,000-dalton component was glycine and of the 60,000-dalton component, alanine. With a combination of manual sequencing and automated Edman degradation, the NH2-terminal amino acid sequence of the 94,00-dalton catalytic subunit was found to be Gly-Arg-Asn-Lys-Tyr-Glu-Thr-Thr-Ala-()-Ser-Glu.
D000251:Adenosine Triphosphatases / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D004372:Ducks; D004789:Enzyme Activation / Q000187:drug effects; D008297:Male; D008861:Microsomes / Q000201:enzymology; D009666:Nose; D010042:Ouabain / Q000494:pharmacology; D011188:Potassium / Q000494:pharmacology; D011485:Protein Binding; D012491:Salt Gland / Q000201:enzymology*; D012964:Sodium / Q000494:pharmacology; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology
D000596:Amino Acids; D012967:Sodium Dodecyl Sulfate; D010042:Ouabain; D012964:Sodium; D000251:Adenosine Triphosphatases; D011188:Potassium