| pubmed-article:8388803 | pubmed:abstractText | Terminal differentiation of chick lens fiber cells has been previously characterized by the accumulation, acidification via phosphorylation, and increased membrane association of a 49-kDa cytoskeletal protein. In these studies, we examine: (1) the subcellular distribution of the 49-kDa protein with regard to ageing and isoform composition; and (2) potential mechanisms regulating 49-kDa phosphorylation and insolubilization. With conventional Western blotting techniques, the 49-kDa protein is found exclusively in insoluble form within terminally differentiated nuclear fiber cells. Cortical fibers, on the other hand, exhibit a more widespread subcellular distribution of the 49-kDa protein. On two-dimensional gels, cortical 49-kDa isoelectric variants segregate according to their ease of sedimentation. After homogenization in detergent-containing buffers, the major isoform of the 49-kDa protein found in low speed pellets (40,000 g, 20 min) exhibits an acidic pI. The 40,000 g supernate and the high speed pellet (100,000 g, 2 hr) which is sedimented from this supernate are enriched in more basic isoforms of the 49-kDa protein. The 100,000 g supernate overlying the high speed pellet is dominated by the most basic isoform. With in vitro phosphorylation assays, the 49 kDa protein is shown to be a major substrate affected by endogenous cAMP-dependent mechanisms. Both the low and high speed pellets exhibit endogenous cAMP-dependent kinase activity. An inhibitor of cAMP-dependent protein kinase activity is also found in soluble lens fractions. Conversion of the 49-kDa protein into more acidic, phosphorylated isoforms increases its insolubility and ease of sedimentation.(ABSTRACT TRUNCATED AT 250 WORDS) | lld:pubmed |
