Surface‐replica cytochemistry as a tool for studying cell‐surface enzyme activity: Membrane ecto‐ATPase localization in liver cell cultures

Abstract

AbstractA stain‐replica technique is described for cytochemical examination of ecto‐adenosine triphosphatase (ATPase) activity over the membrane surface of monolayer cell cultures. Rat liver epithelial cells grown on a plastic substrate were fixed in glutaraldehyde, incubated in situ in an ATPase‐lead reaction medium, ethanol‐dehydrated and air‐dried. The cell surface of the monolayer cultures was replicated with plasma polymerization of hydrocarbon gas in the negative phase of glow discharge. X‐ray microprobe analysis confirmed the site‐specific deposition of lead phosphate in the polymer‐replica films. The cytochemical localization of lead was mirrored in the replicas of epithelial cells, demonstrating that ATPase activity was expressed along the apical margins of cell‐to‐cell contacts. Little or no activity was present over the remainder of the smooth‐surface membranes. In transformed epithelial cells, there were abundant reaction products over the microvilli and intercellular boundaries. These observations were consistent with biochemical data on the liver epithelial cells in culture and suggested the potential of surface‐replica cytochemistry.