Large-scale preparation of sodium-potassium ATPase from kidney outer medulla.

Abstract

Large amounts of Na,K-ATPase are needed for studies involving protein chemistry. Preparation of Na,K-ATPase from kidney by the widely used, rapid procedure of Jørgensen (Biochim Biophys Acta 356:36-52, 1974; Methods Enzymol 156:29-43, 1988) includes labor-intensive dissection of tissue from the outer medulla and centrifugation into a step gradient of sucrose solution. In a large-scale modification presented here, tissue was dissected with a surgical instrument, a rongeur, and centrifugation was simply a five times repeated differential centrifugation. The procedure took seven days and 68 person-hours of work. The yield of activity from 26 kg of whole kidneys was 6600 units (micromol Pi/min) in one preparation. The amount of protein was 240 mg and the specific activity was 28 micromol Pi/min per mg protein. There is a significant saving of labor to obtain a product with a specific activity similar to that commonly obtained. The microsomal fraction may be useful for preparing other membrane proteins from the outer medulla.