Edward E. Penhoet

[15] Fructose-diphosphate aldolase, pyruvate kinase, and pyridine nucleotide-linked activities after electrophoresis

Publisher Summary This chapter describes the fructose-diphosphate aldolase, pyruvate kinase, and pyridine nucleotide-linked activities after electrophoresis. For the detection of aldolase by tetrazolium dye reduction, proteins in the sample with differing electrophoretic mobilities are resolved by electrophoresis on cellulose polyacetate strips. Bands of aldolase activities may then be detected by placing the strip in contact with an agar film containing the assay reagents. The aldolase on the strip comes in contact with its substrate fructose 1,6-diphosphate and catalyzes its cleavage to 3-phosphoglyceraldehyde and dihydroxyacetone phosphate. Bands of aldolase activity are detected by placing the strips, containing the electrophoresed sample on the surface of an agar plate. For the detection by UV absorbance, pyruvate kinase activity is detected by NADH oxidation produced by coupling the enzyme with lactate dehydrogenase. Aldolase may be detected either by NADH oxidation or NAD reduction by coupling with triosephosphate isomerase and α -glycerophosphate dehydrogenase or glyceraldehyde-3-phosphate dehydrogenase, respectively. The enzymes are first resolved by electrophoresis on cellulose polyacetate strips, then the strips are applied to a thin agar film containing the assay reagents. Aldolase may be detected by the above method, both by coupling to NAD reduction through glyeeraldehyde-3-phosphate dehydrogenase (NAD method) and by coupling to NADH oxidation through triosephosphate isomerase and α-glycerophosphate dehydrogenase (NADH method). The detection method for NAD and NADH consists of an agar film prepared as described for pyruvate kinase.