Various types of preparation used to study biochemical processes in the metabolic pathways. If we arranged in their decreasing order of complexity first of all we kept studies at the whole animal level. Which can include removal of organ (e.g. hepatectomy), alteration of diet (e.g. fasting — feeding) administration of a toxin, administration of a drug (e.g. phenobarbital), use of an animal with a specific disease and use of sophisticated techniques such as MMR spectroscopy and position emission tomograph, second of all we kept studies with micro-organisms such as bacteria, virus, yeast moulds and bacteriophages, which used in several areas of biochemistry and genetics. Third, we kept studies with perfused organs by analyzing the arterial blood entering into an organ and the venous blood tissue slices and at last we kept sub-cellular fractions.
Now we take studies with tissue slices in detail. In the techniques of tissue slices, the tissue is dissected out of the animal and frozen rapidly in order to keep the tissue alive and also make slices of the tissue. The frozen tissue is sliced in a special instrument known as cryostate. The frozen slices of the tissue are brought to room temperature (thawing) and are incubated in the presence of oxygen (aerobic incubation) with various substrates. For example, to study the activity of succinate dehydrogenase (a enzymes involve in TCA cycle), succinate is used as a substrate in the phosphate buffer medium along with co-enzyme NAD.
If the tissue slices contain active succinate dehydrogenase, it converts the substrate succinate into fumarate. At the end of the incubation use find decreasing concentration of fumarate. This technique is also used to study metabolic pathways in plant tissue. For example, leaf tissue are used in studying the effect of phyto-hormone cytokinin on the phenomenon of aging in plants. The effect of various organic materials on the enzyme system and the effect of change in the substrate concentration an enzyme kinetics are also studies by this techniques. You will find that as the substrate declines and that of the product rises.
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