In vitro reassociation of EDTA-extractable proteins with calf lens fiber membranes

Nature and site of membrane binding of the EDTA-extractable proteins (EEP) from calf lens fiber membranes have been studied. Reassociation of EEP to EEP-free lens fiber membranes only occurs by means of calcium, not by magnesium ions. This EEP-membrane binding is not hindered by the cytoskeleton. The proportional distribution of the EEP protein components is not altered by reattachment of EEP to the membrane. Calcium appears to be a limiting factor in the reassociation of EEP with the membrane. The total amount of reassociated EEP increases with increasing calcium concentration and may largely exceed the quantity of naturally occurring EEP in lens fiber membranes. In addition, the latter EEP-containing membranes are able to bind an additional amount of EEP in the presence of calcium. These results indicate that most of the EEP-binding sites in lens fiber membranes are not occupied by EEP. Trypsin- or Staphylococcus aureus V8 protease-treated fiber membranes retain the capacity to bind EEP in the presence of calcium. This result indicates that the small polypeptide fragment of the main intrinsic protein (MIP), which is accessible to proteolytic attack, very likely is not the membrane attachment point for EEP. It is suggested that phospholipids rather than membrane proteins are involved in the calcium-dependent binding of EEP to calf lens fiber membranes.