The Preparation of a Human Lung Purified Plasma Membrane Fraction
Plekes, Julie Kathryn
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In order to study the various plasma membrane receptors associated with lung allergy, it was necessary to develop and prepare a plasma membrane-enriched fraction from human peripheral lung. The procedure begins with the homogenization of the lung tissue, followed by a series of centrifugations. MgCl2 was added to the final supernantant (S4) to a lmM concentration and was layered onto a Sucrose gradient. After centrifugation, 4 distinct fractions were collected and analyzed for 5’-Nucleotidase (SNT), LDH, cytochrome C oxidase (CO), and cytochrome C reductase (CR) activities as markers for plasma membrane, cytosol, mitochondria, and microsomes, respectively. The results of 4 different preparations are expressed as units of enzyme activity/mg protein and reflect an 18-fold increase in the specific activity of SNT in the plasma membrane fract,ion. The plasma membrane fraction (PMF) contains membrane vesicles by electron microscopy. The PMF will be analyzed for the presence of adrenergic and histamine receptors by radioligand binding studies.