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Abstract:
The subcellular distribution of adenyl cyclase was investigated in small intestinal epithelial cells. Enterocytes were isolated, disrupted and the resulting membranes fractionated by differential and sucrose gradient centrifugation. Separation of luminal (brush border) and contra-luminal (basolateral) plasma membrane was achieved on a discontinuous sucrose gradient.
The activity of adenyl cyclase was followed during fractionation in relation to other enzymes, notably those considered as markers for luminal and contraluminal plasma membrane. The luminal membrane was identified by the membrane-bound enzymes sucrase and alkaline phosphatase and the basolateral region by (Na+ + K+)-ATPase. Enrichment of the former two enzymes in purified luminal plasma membrane was 8-fold over cells and that of (Na+ + K+)-ATPase in purified basolateral plasma membranes was 13-fold. F- -activated adenyl cyclase co-purified with (Na+ + K+)-ATPase, suggesting a common localization on the plasma membrane. The distribution of K+ -stimulated phosphatase and 5′-nucleotidase also followed (Na+ + K+)-ATPase during fractionation.
