Complex formation of EDTA-extractable proteins from calf lens fiber membranes with calcium and acidic phospholipids

Abstract

The nature of the membrane components, which are involved in the calcium-dependent binding of EDTA-extractable proteins (EEP) to calf lens fiber membranes, has been investigated. Association experiments of radioiodinated EEP with several lens membrane preparations by means of the gel-overlay technique have shown that only phospholipids have the ability to bind EEP in the presence of calcium. The water-soluble crystallins, cytoskeletal proteins and membrane proteins of lens cells lack EEP-binding properties in this technique. Binding studies of EEP with sonicated vesicles of separate phospholipids revealed that acidic calcium-binding phospholipids, i.e. phosphatidylserine (PS), phosphatidylethanolamine (PE) and phosphatidylinositol (PI) are effective in binding provided that calcium is present. Phosphatidylcholine (PC) and sphingomyelin (SM) do not bind EEP in the presence of calcium. The results of association and binding studies indicate that the EEP-lipid binding is purely electrostatic and is accomplished very likely by coupling of the anionic groups of EEP and phospholipids via calcium ions. The interaction of EEP with (isolated) lens fiber membranes is resistant to the non-ionic detergent Triton X-100 in the presence of calcium. This result confirms the earlier idea that the EEP-membrane binding is predominantly or even exclusively electrostatic. It is suggested that calcium-binding phospholipids, rather than proteins, function as binding sites for EEP in this hydrophilic calcium-dependent binding of EEP to lens fiber membranes.