Practical 1 Activity Of Lactate

D Naughtonpage 323012012ls2050 Practical 1activity Of Lactate Deh

D Naughtonpage 323012012ls2050 Practical 1activity Of Lactate Deh

D. Naughton 23/01/2012 LS2050 Practical 1 Activity of Lactate Dehydrogenase in Mammalian Tissues Lactate dehydrogenase (LDH) catalyzes the reversible reduction of pyruvate to lactate using NADH as a coenzyme. The activity of LDH can be measured by continuously monitoring the decrease in absorbance at 340 nm, using pyruvate as the substrate. Varying the pyruvate concentration in the presence of excess NADH allows for the determination of the enzyme's kinetic parameters with respect to pyruvate. This practical involves preparing reaction mixtures with different pyruvate concentrations, measuring the rate of NADH oxidation spectrophotometrically, and analyzing the data to determine Km and Vmax values for LDH from different mammalian tissues, such as liver, heart, and muscle.

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The reaction catalyzed by lactate dehydrogenase (LDH) involves the conversion of pyruvate to lactate, with concomitant oxidation of NADH to NAD+. The chemical equation can be represented as:

CH3–CO–COOH + NADH + H+ → CH3–CHOH–COOH + NAD+

In this reversible reaction, the enzyme facilitates the reduction of pyruvate to lactate while oxidizing NADH to NAD+. The molecular structures include pyruvate, a keto acid with a triple-bonded oxygen on the carbonyl carbon, and lactate, a hydroxy acid with an additional hydroxyl group. The enzyme's activity in the laboratory is monitored spectrophotometrically by measuring the decrease in absorbance at 340 nm, which corresponds to the oxidation of NADH. Because NADH absorbs strongly at this wavelength, whereas NAD+ does not, the reduction in absorbance over time provides a direct measure of enzyme activity.

To analyze enzyme kinetics, reaction rates are measured at varying substrate (pyruvate) concentrations while keeping NADH in excess to ensure the reaction is primarily dependent on pyruvate concentration. The initial rate of NADH oxidation (expressed as A340 per minute) is calculated from the absorbance change, then converted into molar quantities of NADH oxidized, facilitating the determination of kinetic parameters like Km (Michaelis constant) and Vmax (maximum velocity).

The preparation of reaction mixtures involves setting up seven tubes, each containing phosphate buffer, NADH, cytosol extract from different tissues, and varying amounts of pyruvate. After equilibrating the tubes at 37°C, the reaction is initiated by adding pyruvate, and the change in absorbance at 340 nm is recorded over time. The data collected allows for the plotting of Michaelis-Menten curves and their linear transformations, such as Lineweaver-Burk plots, to extract kinetic parameters.

Calculations involve converting observed absorbance changes into nmol NADH oxidized per minute per mg tissue, taking into account molar extinction coefficients, reaction volume, and tissue concentration in the cytosol preparations. The Km and Vmax values derived reflect the enzyme's affinity and capacity within each tissue type. Comparing these values reveals differences in enzyme activity, possibly related to tissue-specific isoforms or metabolic requirements.

For tissue-specific isozyme characterization, techniques such as electrophoresis, immunohistochemistry, and isozyme-specific activity assays can be employed. These approaches can distinguish between different LDH isoforms (e.g., LDH-1, LDH-5) based on mobility differences or antigenic properties, providing insights into the metabolic roles of LDH variants in various tissues. Understanding isozyme distribution is crucial for elucidating tissue-specific metabolic adaptations and pathological states involving lactate metabolism.

References

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