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Lactate dehydrogenase from rabbit muscle

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Lactate dehydrogenase from rabbit muscle is an enzyme that catalyzes the interconversion of lactate and pyruvate. It is a widely used reagent in biochemistry and clinical diagnostics.

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Lab products found in correlation

Prism 8, by GraphPad (2 mentions) Uv transparent 96 well plate, by Corning (2 mentions) Pyruvate kinase from rabbit muscle, by Merck Group (2 mentions) Spark multimode microplate reader, by Tecan (2 mentions)

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Lactate dehydrogenase (112 citations) Lactate dehydrogenase from (2 citations) L-lactate dehydrogenase from rabbit muscles (2 citations)

3 protocols using lactate dehydrogenase from rabbit muscle

1

Sperm Lactate Production Assay

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Sperm lactate production was measured with a spectrophotometric assay that monitors the accumulation of NADH during the oxidation of lactate to pyruvate by lactate dehydrogenase (Pesce et al. 1975 (link); Danshina et al. 2001 (link)). Sperm were incubated for 2 hr using our standard conditions for capacitation, except that lactate and pyruvate were omitted from the HTF medium. Duplicate aliquots were removed at 0 and 2 hr to measure lactate accumulation in the medium. After sperm was removed by centrifugation, samples were added to the assay buffer (pH 9.0) containing 1 mM NAD+ and 10 U/mL lactate dehydrogenase from rabbit muscle (Sigma-Aldrich, St. Louis, MO) and incubated for 2 hr at 25°. The concentration of lactate in the sample was proportional to the increase in absorbance at 340 nm, as NAD+ was reduced to NADH. Sperm in each sample were counted and lactate production (μmole/108 sperm) during the 2-hr incubation period was calculated.
Odet F., Pan W., Bell T.A., Goodson S.G., Stevans A.M., Yun Z., Aylor D.L., Kao C.Y., McMillan L., de Villena F.P, & O’Brien D.A. (2015). The Founder Strains of the Collaborative Cross Express a Complex Combination of Advantageous and Deleterious Traits for Male Reproduction. G3: Genes|Genomes|Genetics, 5(12), 2671-2683.
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2

Kinetic Analysis of Nucleoside Kinase Enzymes

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Purified enzyme was subjected to kinetic analysis using an ATPase-coupled assay that monitors the reduction of NADH to NAD+ via decrease in absorbance at 340 nm.32 (link),41 ,42 Each reaction contained 20 mM HEPES (pH 7.2), 100 mM KCl, 2 mM MgCl2, 50–300 μM cytidine or uridine for UCK2, 50–8000 μM cytidine or uridine for UCK1, 100 μM – 10 mM 2’AzCd or 2’AzUd for UCK2, 1–40 mM 2’AzCd or 2’AzUd, 1 mM ATP, 250 μM NADH, 250 μM phosphoenolpyruvate, 0.5 U lactate dehydrogenase from rabbit muscle (Sigma Aldrich), 0.5 U pyruvate kinase from rabbit muscle (Sigma Aldrich), and 100 nM of purified UCK1 or UCK2 enzyme. Continuous measurements were taken at 340 nm in 150 μL fill volumes in 96-well UV-transparent plates (Corning), using a Spark Multimode microplate reader (Tecan). Kinetic constants were obtained through a nonlinear fit of the data using the Michaelis-Menten equation with Prism 8 GraphPad software.
Nainar S., Cuthbert B., Lim N.M., England W.E., Ke K., Sophal K., Quechol R., Mobley D.L., Goulding C.W, & Spitale R.C. (2020). An Optimized Chemical-Genetic Method for Cell-Specific Metabolic Labeling of RNA. Nature methods, 17(3), 311-318.
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3

Kinetic Analysis of Nucleoside Kinase Enzymes

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Purified enzyme was subjected to kinetic analysis using an ATPase-coupled assay that monitors the reduction of NADH to NAD+ via decrease in absorbance at 340 nm.32 (link),41 ,42 Each reaction contained 20 mM HEPES (pH 7.2), 100 mM KCl, 2 mM MgCl2, 50–300 μM cytidine or uridine for UCK2, 50–8000 μM cytidine or uridine for UCK1, 100 μM – 10 mM 2’AzCd or 2’AzUd for UCK2, 1–40 mM 2’AzCd or 2’AzUd, 1 mM ATP, 250 μM NADH, 250 μM phosphoenolpyruvate, 0.5 U lactate dehydrogenase from rabbit muscle (Sigma Aldrich), 0.5 U pyruvate kinase from rabbit muscle (Sigma Aldrich), and 100 nM of purified UCK1 or UCK2 enzyme. Continuous measurements were taken at 340 nm in 150 μL fill volumes in 96-well UV-transparent plates (Corning), using a Spark Multimode microplate reader (Tecan). Kinetic constants were obtained through a nonlinear fit of the data using the Michaelis-Menten equation with Prism 8 GraphPad software.
Nainar S., Cuthbert B., Lim N.M., England W.E., Ke K., Sophal K., Quechol R., Mobley D.L., Goulding C.W, & Spitale R.C. (2020). An Optimized Chemical-Genetic Method for Cell-Specific Metabolic Labeling of RNA. Nature methods, 17(3), 311-318.
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