We have investigated the orientation of isolated fragments of Halobacterium halobium purple membrane (PM) adsorbed to poly-L-lysine-treated glass (PL-glass), by quanitative electron microscopy. Three lines of evidence support the conclusion that the cytoplasmic side of the membrane is preferentially absorbed. First, monolayer freeze-fracture reveals nonrandom orientation; more fracture faces (89%) are particulate than smooth. Second, the amount of each membrane surface present can be assayed using polycationic ferritin; 90% of all adsorbed membrane fragments are labeled. Third, it is possible to distinguish two surfaces, "cracked" (the extracellular surface) and "pitted" (the cytoplasmic surface) , in slowly air-dried, platinum-carbon-shadowed membranes. When applied under standard conditions, more than 80% appear cracked. Selection for the cytoplasmic by the cationic substrate suggests that the isolated PM, buffered at pH 7.4 and in the light, has a higher negative charge on its cytoplasmic surface than on its extracellular surface. Nevertheless, cationic ferritin (CF) preferentially adsorbs to the extracellular surface. Orientation provides a striking example of biomembrane surface asymmetry as well as the means to examine the chemical reactivity and physical properties of surfaces of a purified, nonvesicular membrane fragment.

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