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


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Klodos
Irena
I
Department of Biophysics, Aarhus University, Aarhus, Denmark.
Esmann
Mikael
M
Post
Robert L
RL
D000818:Animals; D002498:Centrifugation; D004210:Dissection; D007679:Kidney Medulla / Q000201:enzymology*; D008861:Microsomes / Q000201:enzymology; D012967:Sodium Dodecyl Sulfate; D000254:Sodium-Potassium-Exchanging ATPase / Q000302:isolation & purification*; D013501:Surface-Active Agents; D013552:Swine; D017186:Titrimetry
D013501:Surface-Active Agents; D012967:Sodium Dodecyl Sulfate; D000254:Sodium-Potassium-Exchanging ATPase