Ankyrin, the membrane attachment protein for human erythrocyte spectrin, is tightly linked in a 1:1 molar ratio with band 3 in detergent extracts of spectrin-depleted membranes. Ankyrin-linked band 3, which represents 10--15% of the total band 3, spans the membrane, and is nearly identical to the major band 3 by peptide analysis. Spectrin binds to solubilised ankyrin-linked band 3, but not to free band 3. A portion of band 3 remains firmly associated with detergent-extracted cytoskeletal proteins. It is concluded that a fraction of band 3 is attached to the erythrocyte cytoskeleton through association with ankyrin, which in turn is bound to spectrin.

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Rat liver and isolated hepatocytes contain high levels of a soluble adenylate cyclase stimulator, whereas rat erythrocytes lack this activity. Accordingly, a reconstitution system was developed with adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1] from erythrocyte. ghosts. and the soluble activator from liver cytosol. Pretreatment of erythrocyte ghosts with the cytosolic factor resulted in a 5-to 15-fold activation of adenylate cyclase in the presence or absence of NaF, 5'-guanylyl imidodiphosphate, or isoproterenol and GTP. The sequence of addition of the cytosolic component and the other activators was critical in determining the maximal activity of the enzyme. The cytosolic factor appears to be a heat-labile Mr 105,000 protein, which activates adenylate cyclase in a saturable reaction involving binding of the protein to the erythrocyte ghosts. This molecular interaction was accompanied by stabilization of a labile thiol group that was essential for catalytic activity. The cytosolic component also unmasks latent adenylate cyclase activity in human erythrocyte ghosts and in cytoskeletal preparations from rat erythrocyte ghosts. These observations suggest that the cytosolic activator may also occur as a native, peripheral membrane component of adenylate cyclase systems and may be required for the expression and stabilization of catalytic activity.Soluble components from a variety of organs, cell preparations, and cultured cell lines stimulate particulate adenylate cyclase activity [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1]. The relevant literature has been recently summarized elsewhere (1-5). These presumably cytosolic activators differ in their sensitivity to protease digestion and to heat inactivation, and they frequently exhibit differential effects in the presence of guanyl nucleotides, sodium fluoride, or various hormones (1, 4-13). Maturation-and age-dependent changes in the levels of the soluble activators have been detected in rat reticulocytes (4) and lung (14), respectively.Attempts have been made to separate and purify some of these cytosolic factors. Two active fractions that act synergistically were derived from rat lung supernatants by DEAE-cellulose chromatography (3). Another two factors were separated from rat osteosarcoma cytosol, and their molecular weights were estimated at 55,000 and 29,000 by Sephadex G-100 gel-permeation chromatography (11). A Mr 13,000 heat-stable factor from liver cytosol that potentiates hormonal stimulation of adenylate cyclase in a GTP-like fashion has been purified 1,000-fold (15). The heat-stable and NaF-specific factor from rat brain was purified about 3,000-fold and behaves as a Mr 59,000 monomer (10).A sensitive reconstitution system has been developed here, utilizing rat erythrocyte ghosts as a source of adenylate cyclase and rat liver cytosol as a source of the cytosolic activator. This assay has been employed to characterize the physical parameters of the cytosolic factor and to study the kinetic characteristics and the molec...