Pyruvate Kinase Enzyme Activity Assay

The lactate dehydrogenase (LDH) coupled enzyme assay was used to measure pyruvate kinase (PK) enzyme activity (15 (link)). The assay was carried out in the presence of mouse retinal lysate containing an enzyme buffer mixture (50 mM Tris–HCl [pH 7.4], 100 mM KCl, 5 mM MgCl₂, 1 mM ADP, 0.5 mM PEP, and 0.2 mM NADH [reduced form of NAD⁺]) and 8 U of LDH with a reaction volume of 1.0 ml. The PK activity was measured spectrophotometrically by monitoring the reduction in the absorbance at 340 nm from the oxidation of NADH.

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Rajala A., Bhat M.A., Teel K., Gopinadhan Nair G.K., Purcell L, & Rajala R.V. (2023). The function of lactate dehydrogenase A in retinal neurons: implications to retinal degenerative diseases. PNAS Nexus, 2(3), pgad038.

Publication 2023

Assay Buffer Enzyme Enzyme activity Enzyme assay Lactate dehydrogenase Mgcl2 Mouse Nadh Pyruvate kinase Retinal Tris

Corresponding Organization :

Other organizations : Dean McGee Eye Institute, University of Oklahoma Health Sciences Center

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Pyruvate kinase (PK) enzyme activity

control variables

Enzyme buffer mixture (50 mM Tris–HCl [pH 7.4], 100 mM KCl, 5 mM MgCl2, 1 mM ADP, 0.5 mM PEP, and 0.2 mM NADH [reduced form of NAD+])
8 U of lactate dehydrogenase (LDH)
Reaction volume of 1.0 ml

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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