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Hi. Im an undergrad medical student currently doing Advance Medical Science. Im interested in determining Na/K-ATPase activity and Ca-ATPase activity in rat lens.
Regarding Na/K-ATPase assay, im using method described in Effects of DL-a-Lipoic Acid on Peripheral Nerve Conduction, Blood Flow, Energy Metabolism, and Oxidative Stress in Experimental Diabetic Neuropathy by Martin J. Stevens, Irina Obrosova, Xianghui Cao, Carol Van Huysen, and Douglas A. Greene.
The question is, how to convert amount of the 2 enzymes (pyruvate kinase and lactate dehydrogenase) from microgram into concentration or activity unit?
" Aliquots of homogenate were assayed enzymatically for total ATPase activity in 1 ml reaction mixture containing 100 mmol/l NaCl,
10 mmol/l KCl, 2.5 mmol/l MgCl 2 , 1 mmol/l Tris-ATP, 1 mmol/l phosphoenolpyruvate,30 mmol/l imidazole-HCl buffer, pH 7.3, 0.15 mmol/l NADH,
50 µg lactate dehydrogenase, and 30 µg pyruvate kinase (40). After an initialstabilization period, activity was monitored spectrophotometrically at 340 nm for at least 15 min."
here is an attachment of my flowchart regarding this assay.
