Determination of subunit molecular weights of canine renal (Na+,K+)-ATPase by low-angle laser light scattering coupled with high performance gel chromatography in the presence of sodium dodecyl sulfate.

Abstract

Subunit molecular weights of the (Na+,K+)-ATPase of canine renal outer medulla were estimated in the presence of sodium dodecyl sulfate (SDS) by the measuring system consisted of components connected in the following sequence: a TSK-gel G3000 SW column, a UV spectrophotometer, a low-angle laser light scattering photometer, and a differential refractometer. Polypeptide molecular weights of the alpha- and beta-subunits were determined to be 117,900 and 39,400, respectively. The measurement required the extinction coefficient at 280 nm of the sample polypeptide in addition to the outputs from the three detectors. The extinction coefficients at 280 nm of the alpha- and beta-subunits were determined to be 0.931 and 1.41 ml X mg-1 X cm-1, respectively by the quantitative amino acid analysis. The above procedure seems to be most appropriate to determine uniquely the composition of subunits molecular weights of an oligomeric membrane protein.