The 97-kd α-actinin-like protein in chromaffin granule membranes from adrenal medulla: evidence for localization on the cytoplasmic surface and for binding to actin filaments

Abstract

α-Actinin, a protein of the contractile apparatus known to be associated with the chromaffin granule membrane, has been partially purified and localized by one-dimensional gel electrophoresis by electrophoretic transfer onto nitrocellulose sheets and detection with antiserum raised against α-actinin purified from bovine skeletal red muscle. The α-actinin-like protein from granule membrane has a molecular weight of M r = 97 kD , close to the molecular weight of skeletal muscle α-actinin. and is antigenically related to skeletal muscle α-actinin. Chromaffin granule membrane proteins were separated using two-dimensional gel electrophoresis; electrophoretic maps revealed a high degree of complexity because of the large number of protein species and because of the microheterogeneity of some of these components. In addition, certain components did not focus well and were poorly separated. Nevertheless, the 97-kD α-actinin-like protein and actin were localized on two-dimensional gel electrophoregrams of chromaffin granule membrane proteins. They appeared as minor components, showing great variability depending on media used for preparing granules. Isolation of granules in classical sucrose gradient steps (0.32 M sucrose; 1.6 M sucrose, both buffered with HEPES) resulted in low recovery of the α-actinin-like protein because it detached from the membrane in low ionic strength buffers. Isolation of granules in buffered isotonic KCl media led to an enrichment of the α-actinin-like protein. When granules were prepared in buffered sucrose media containing 2 mM Mg 2+, there was a marked increase of 97-kD α-actinin-like protein. Labelling experiments with Na[ 125I]lactoperoxidase were carried out on intact granules and broken membranes; in both experiments, the α-actinin-like protein was not found to be labelled, while actin was radio-iodinated with no increase of the radioactivity when comparing intact granule experiment with broken membrane experiment. Mild digestion of intact granules with Streptomyces griseus pronase led to the disappearance of all protein species with a molecular weight above 70 kD and of some unidentified components with molecular weight below 70 kD. The 97-kD α-actinin-like protein and actin were digested. We concluded that α-actinin-like protein and actin are attached to the vesicle membrane facing towards the cytoplasmic side. In addition, the α-actinin-like protein is probably a peripheral membraneassociated protein rather than an integral membrane protein. Chromaffin granule membranes are known to stimulate actin polymerization and to bind actin filaments. Anti-α-actinin antiserum and monospecific anti-α-actinin Fab fragments were found to inhibit by 50% [ 3H]actin binding to granule membranes. Inhibition was dependent on the amount of antibody present in the incubation medium. Membranes isolated from intact granules which have previously been incubated with pronase in conditions to digest completely α-actinin-like protein as judged from two-dimensional gel electrophoresis, were characterized by a lower (50%) actin binding capacity that we attributed to pronase-resistant binding sites to G-actin. All these data showed that α-actinin-like protein plays a role in the binding of actin filaments to the chromaffin granule membrane, either stabilizing actin nuclei present in this membrane and/or cross-linking actin filaments emerging from the membrane.