The aim of this proposal is to purify the factor or factors present in the plasma membranes of senescent human diploid fibroblasts that inhibit DNA synthesis in young fibroblasts. The literature contains a number of methods for isolation and purification of plasma membranes from quiescent and senescent mammalian cells. Some of these procedures are suitable for large scale work. The purified membranes would be treated with agents that dissociate peripheral proteins. Activities of interest might be releasable, or intrinsic to the membrane, or due to an intrinsic factor acting together with a releasable co-factor. If the activity remains membrane bound, agents such as octyl glucoside supplemented with guanidine- HCL, Triton C-100, or CHAPS will be tried as solubilizing agents and to maintain the activity in a soluble state. Stability difficulties will be attached in various ways, including the use of reducing agents, heavy metal chelators, high concentrations of glycerol, etc. Membrane associated inhibitory activity has been reported to be trypsin sensitive, so appropriate protease inhibitors will be used if necessary. Fractions will be recombined in the event that the activity is due to multiple factors. It is anticipated that many different kinds of fractionation procedures under a variety of conditions of pH, ionic strength, etc., will be explored. Properties of the purified material will be compared with the inhibitory factor purified from quiescent young fibroblasts. The mechanism of action of the purified fractions will be explored.
