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unknown enzyme concentration


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#1 Sarah Diyana

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Posted 30 September 2012 - 10:17 PM

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.

#2 Sarah Diyana

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Posted 30 September 2012 - 10:35 PM

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."

#3 mdfenko

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Posted 01 October 2012 - 05:45 AM

your enzyme concentration, in the assay, would be 50ug/ml ldh and 30ug/ml pk. you can get the units from the spec sheet that comes with the enzyme (or you can go to the supplier's website and look up the specs for the lot#).

but, i'm not sure that that is what you meant to ask, is it?
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