Formation of Tightly Bound Forms of Annexins V and VI in Rabbit Skeletal Muscle Membranes Isolated in the Presence of Barium Ions

K. S. Krasavchenko, E. I. Akimova, and V. I. Melgunov^*

Department of Molecular Biology, School of Biology, Lomonosov Moscow State University, Moscow, 119899 Russia; fax: (7-095) 939-3181; E-mail: vim@protein.bio.msu.su

^* To whom correspondence should be addressed.

Received March 17, 1999; Revision received April 20, 1999

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After isolation of rabbit skeletal muscle membranes in the presence of Ba²⁺ or Ca²⁺, significant portions of annexin V and VI tightly bind to membranes and become inaccessible for chelating agents. Tightly bound annexin VI is virtually completely solubilized only after treatment with a buffer supplemented both with EGTA and detergent. The portion of tightly bound annexin V cannot be removed even by extraction with buffer containing both EGTA and detergent. In some cases, tightly bound annexin V or VI is detected even in the control (not treated with cations) membranes, thus indicating the possible formation of tight annexin--membrane complexes in situ. The addition of exogenous cations seems to promote only the accumulation of tightly bound annexins within the cell. After temperature-induced phase separation, annexin V and VI bound to the membranes isolated in the presence of Ba²⁺ or Ca²⁺ remains mainly in the aqueous phase, similarly to annexins isolated from the control membranes. Neither annexin partitions into the detergent-enriched phase. This indicates the absence of hydrophobicity change in comparison with the standard EGTA-soluble annexins.

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KEY WORDS: annexins, Ca²⁺-dependent phospholipid-binding proteins, membrane binding, detergents, chelating agents, calcium, barium

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