Preparation of a human lung purified plasma membrane fraction: confirmation by enzyme markers, electron microscopy, and histamine H1 receptor binding.
A simple and rapid method of isolating plasma membranes from human peripheral lung tissue is described. The method involves homogenization of tissue in 0.25 M sucrose-buffered medium followed by differential and sucrose density gradient centrifugation. Enzymatic and morphological characterization of the plasma membrane fraction revealed minimal contamination by nonplasma membrane fragments. The isolated plasma membranes showed an 18-fold purification of 5'-nucleotidase activity compared to the original homogenate. Electron-microscopic studies of the plasma membrane fraction revealed the presence of small membrane vesicles having a trilaminar membrane structure. To further examine the purity of the plasma membrane preparation, the binding of the H1 receptor antagonist, 3H pyrilamine, to the plasma membrane-enriched fraction was compared to the binding to crude membrane preparations. Both the plasma membrane-enriched fraction and the crude membrane preparation had similar Kd's for the histamine antagonist, but the plasma membrane-enriched fraction had a threefold greater binding capacity, reflecting the relative enrichment of plasma membranes of the preparation. Thus, a method has been developed for the isolation of plasma membranes from human peripheral lung which should provide material for a variety of biochemical and pharmacological studies.
D015720:5'-Nucleotidase; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D003584:Cytological Techniques; D003576:Electron Transport Complex IV / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008168:Lung / Q000201:enzymology / Q000378:metabolism / Q000648:ultrastructure*; D008565:Membrane Proteins / Q000378:metabolism; D008854:Microscopy, Electron; D009245:NADH Dehydrogenase / Q000378:metabolism; D009708:Nucleotidases / Q000378:metabolism; D011969:Receptors, Histamine H1 / Q000378:metabolism; D013347:Subcellular Fractions / Q000648:ultrastructure
D008565:Membrane Proteins; D011969:Receptors, Histamine H1; D007770:L-Lactate Dehydrogenase; D009245:NADH Dehydrogenase; D003576:Electron Transport Complex IV; D009708:Nucleotidases; D015720:5'-Nucleotidase