Department of Medical Biosciences, University of the Western Cape, Bellville, South Africa

Enzyme kinetics, metabolites and protein

For cells to live, ATP is required. This ATP is derived from the breakdown of more complex fuels, such a phosphocreatine, glucose, glycogen and fat (see Fuelling performance in Muscle 101).

Each enzyme of the metabolic pathways, has a specific rate at which it can produce ATP from the above mentioned fuels, known as its activity. However, this rate can be altered acutely from either allosteric activation or inhibition, or interactions with substrates, or activation by other enzymes, or temperature. Additionally, various external stimuli can induce chronic adaptations that can alter this rate, either increasing the enzyme’s activity (e.g. endurance exercise training over 3 months) or decreasing the enzyme’s activity (e.g. bedridden for 3 months).

Our lab uses a multitude of approaches, ranging from spectrophotometric and fluorometric assays by means of kinetic enzyme assays, histochemistry (e.g. NADH stain) or Western blotting. The former assays are very specific to the pathways and enzyme of interest. To illustrate, the graph shows the rate of activity of a particular enzyme related to exercise fatigue resistance – in this case citrate synthase. The slope of each line is calculated, normalised to the muscle weight (or protein concentration) and expressed as µmol/min/g tissue or protein. The slope of the lion is much lower than that of the human, indicating that the human has higher activity for CS, and thus, better endurance capacity than a lion. 

The following enzyme and metabolite assays are routinely performed in our lab, each providing information on the flux capacity through a specific pathway:

☆ 3-hydroxyacetyl Co A dehydrogenase
☆ creatine kinase
☆ citrate synthase
☆ lactate dehydrogenase
☆ phosphofructokinase
☆ phosphorylase

☆ muscle lactate
☆ muscle glycogen
☆ muscle creatine
☆ muscle soluble protein content

Assays for other enzymes in the tissues not listed above, can also be developed on request.

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