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7-07 ANNEXIN VI-BINDING PROTEINS IN RAT BRAIN. TATSUO WATANABE. MAKOTOmu AND KENJI SOBUE. Deoartment of Neurochemistrv and Neuronharmacologv, Osaka Unive>&

-Medical School. Yamadaoka 2-2, Suita. Osaka 565. Japan

Annexin VI (~68) is a member of a new class of proleins which bind negatively charged phospholipids in a Ca2+-dependent manner. ~68 is abundant in mammalian brain, but its function has been unclear. To elucidate its function, it is of primary importance to identify proteins which interact with ~68. We established the identification method for the p68 binding proteins in the rat brain particulate (Pl) fraction, using ‘blot binding technique’ in which proteins were electrophoretically separated. transferred LO a nitrocellulose sheet, and incubated with I2%labeIled ~68. 125I-p68 bound to a number of proteins including Mr 280.3OOK, 230K, 190K. IOO- 130K, 8OK. 45K and 30K in Ca2+/phosphatidyIserine (PS)-dependent manners. Of these, the protein with Mr 230K was identified to be a membrane skeletal protein, calspectin (brain spectrin). ‘W-p68 specifically bound to p- subunit of calspectin but not to a-subunit. lrsl-p68 also hound to native calspectin Caz+/PS-dependently, which was confirmed by ‘dot-blot binding technique’. The binding of 1Wp68 was saturable and could be replaced by excess cold ~68. These results indicate that ~68 could interact with the membrane skeleton in a Car+-dependent manner. The techniques used in this study SCCII~ useful to identify proteins which interact with ~68, and finally to elucidate physiological roles of ~68.

7-08 INTERACTION OF ANNEXIN VI WITH A SYNAPTIC VESICLE PROTEIN, SYNAPSIV I. MAKOTO INUI, TATSUO WATANABE. MASAHIDE IGA AND KENJI SOBUE,

DeDartment of Neurochemistry and Neuropharmacology. Osaka Universitv Medical School, Yamadaoka2_ 2, Suita. Osaka 565. Japan

Annexins are a new class of Ca2+/phospholipid binding proteins. Annexin VI (~68) has been identified in mammalian brains, although its function has not been known. We have undertaken a study

aimed at identifying the molecular targets of ~68, using the blot binding technique with 12-51-~68. WC found a group of proteins in the particulate (P2) fraction from rat brain, which bound 1251-~68 in C$+- and phosphatidytserine (PS)-dependent manners. Of the 125I-p68 binding proteins found in P2, a protein with Mr 80K was enriched in the synaptic vesicle fraction, and was identified to be synapsin I. 12iI-p68 bound to the purified synapsin I, Caz+/PS-dependently. Synapsin I is thought to link synaptic vesicles to actin filaments in nerve terminals, and to regulate the neurotransmitter release by determining the availability of synaptic vesicles for exocytosis. The centrifugation assay with F-actin revealed that p68 inhibited the interaction between synapsin I and F-actin, Ca2+/PSdependently. These results suggest that p68 may play a role in the process of the neurolransmitler release by interacting with synapsin I.

7-09 SEPTAL KINDLING INCREASES THE IN SlTUPHOSPHORYLATION STATE OF SYNAPSIN I. YOKO YAMAGATA’. KUNIHIKO OBATA’. PAUL GREENGARD2 ANDANDREW,

lLab.2Lab. of Mol. and Cell. Neurosci The Rockefeller Universitv, 1230 York Avenue. New York. NY 10021, U.S.A.

Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) has been implicated in the regulation of neurotransmitter release, in the induction of long-term potentiation and in the process of learning. In order to investigate a possible presynaptic role of CaM kinase II in synaptic plasticity, the effect of septal kindling on the phosphorylation state of synapsin I m situ was studied by immunoblot analyses using phosphorylation state-specific antibodies to synapsin I Phosphorylation of synapsin I at site 3 (one of two CaM kinase II-dependent sites) was significantly increased in kindled ratS compared to paired controls in samples of homogenates from hippocampus and cerebral cortex, but not from cerebellum. A slight increase in phospho- rylation of synapsin I at site 1 (the CAMP- and CaM kinase I-dependent site) was also observed in cerebral cortex. The relative increase in phosphorylation at site 3 was higher than that at site 1, both in hippocampus and in cerebral cortex. The total synapsin I level was unchanged. The observation that kindling is accompanied by an increase in the phosphorylation state of synapsin I at site 3 suggests that increased activity of CaM kinase II in presynaptic termini could result in an increased probability of neurotransmitter release. This presynaptic mechanism may be involved in synaptic plasticity in general.