Ca 2+-binding proteins in rat synaptic fractions surveyed by the 45Ca 2+ overlay method

Abstract

Ca 2+-binding proteins in the synaptic and subsynaptic fractions (P 2, synaptosome, synaptic plasma membrane, and postsynaptic density [PSD]-enriched fractions) and soluble fraction of rat brain were surveyed by a 45Ca 2+ overlay method. The PSD-enriched fraction from cerebral cortex contained two major Ca 2+-binding proteins (55,000 M r and 19,000 M r) and a distinct group (in 140,000 M r region)m and two minor ones (66,000 M rand16,000 M r); and the fraction from cerebellum contained two (55,000 M rand19,000 M r). The proteins with 55,000 M rand19,000 M r were identified as tubulin and calmodulin, respectively, and present in all the fractions investigated. The Ca 2+-binding proteins of 140,000 M r region were found only in the PSD-enriched fraction isolated from cerebral cortex: neither the PSD-enriched fraction isolated from cerebellum nor other subcellular fractions prepared from cerebral cortex and cerebellum contained the proteins. The 140,000 M r Ca 2+-binding proteins were the substrates for the Ca 2+/calmodulin-dependent protein kinase II associated with PSD, and no change in the Ca 2+-binding was detected by the 45Ca 2+ overlay method after phosphorylation of the proteins by the protein kinase. The 16,000 M r Ca 2+-binding protein might be the β-subunit of calcineurin. Calretinin and calbindin-D 28k were also detected as Ca 2+-binding proteins in the soluble fractions of both cerebral cortex and cerebellum.