Jack Kyte

The (Na+ + K+) adenosine triphosphatase was obtained from canine renal medulla and partially purified. A highly active, membrane-bound enzyme was prepared from the microsomal fraction of a homogenate. The enzyme was then transferred from this particulate state to the supernatant phase by treatment with sodium deoxycholate and, in this form, it was further purified by gel filtration. The final preparation had a high specific activity which was constant over a significant region of the elution profile. When this material was examined by electrophoresis in a sodium dodecyl sulfate solvent, more than 90% of the protein was accounted for by two polypeptide chains which were present in approximately equimolar amounts. The ratio of these two polypeptides remained constant in those fractions from the gel filtration which had the highest specific activity. The protein traveled as a single component in an acetic acid-urea electrophoresis system. These results suggest that a specific complex responsible for the (Na+ + K+) adenosine triphosphatase activity has been isolated.

Abstract The (Na+ + K+)-dependent adenosine triphosphatase from canine renal medulla is composed of lipids and of two polypeptide chains. The larger one has a molecular weight of 135,000, NH2-terminal glycine, and has a high content of non-polar amino acids. The smaller polypeptide chain has NH2-terminal alanine and it is a sialoglycoprotein. The two polypeptides are present in a mass ratio of 1.7:1 for the ratio of large to small chain. This mass ratio probably corresponds to a molar ratio of one large chain to two small chains. The two chains are close to one another in the purified enzyme because they can be covalently crosslinked by dimethyl suberimidate. Finally, the large polypeptide chain, which has all of the properties of a membrane-bound enzyme, is soluble in simple aqueous solvents in the absence of detergents and lipids, although it no longer has enzymatic activity.

The distribution of (Na+ + K+) ATPase over the plasma membranes of the proximal convoluted tubule from canine renal cortex has been determined. Ultrathin frozen sections of this tissue were stained with rabbit antibodies to this enzyme and ferritin-conjugated goat antirabbit gamma-globulin. It is demonstrated that high concentrations of this enzyme uniformly line the intercellular spaces of this epithelium. The consequences of this observation are discussed in terms of the low resistant tight junctions of these tubules and the isotonic fluid transport which they support. Furthermore, antibodies to (Na+ + K+) ATPase recognize an antigen on the luminal surfaces of the tubules within the brush border. It is proposed that the enzyme is present in this region of the plasma membrane as well, although at much lower concentration. To further substantiate this conclusion, a brush border fraction has been purified from rabbit kidney and been shown to contain significant (Na+ + K+) ATPase. These results contradict earlier conclusions about the location of (Na+ + K+) ATPase in this tissue.