A quantitative analysis of G-actin binding proteins and the G-actin pool in developing chick brain

The large G-actin pool in individual actively motile cells has been shown t o be maintained primarily by the actin sequestering protein thymosin beta f our (T beta(4)). It is not clear whether T beta(4) or an isoform also plays a primary role in neural tissue containing highly motile axonal growth con es. To address this question we have made a definitive analysis of the rela tive contributions of all the known G-actin sequestering proteins: T beta(4 ), T beta(10), profilin, and phosphorylated (inactive) and unphosphorylated (potentially active) forms of both ADF and cofilin, in relation to the G-a ctin pool in developing chick brain at embryonic days 13 and 17. From our m easurements we estimate the intracellular concentration of G-actin as 30-37 mu M and of T beta(4) as 50-60 mu M in an 'average' brain cell in embryoni c chick brain. No other beta thymosin isoforms were detected in these brain extracts. The ratio of soluble, unphosphorylated ADF to T beta(4) is only 1:7 at 13 embryonic days, but increases to 1:4 at 17 days. Profilin and cof ilin concentrations are an order of magnitude lower than T beta(4). Combini ng the contributions of T beta(4), unphosphorylated ADF and unphosphorylate d cofilin, we estimate a mean G-actin critical concentration of similar to 0.45 mu M and similar to 0.2 mu M, respectively, in day 13 and day 17 embry onic brain extracts, suggest-ing a significant developmental decrease. We c onclude that (a) T beta(4) is the major actin sequestering protein in embry onic chick brain and the only beta thymosin isoform present; (b) ADF may pl ay a significant developmental role, as its concentration changes significa ntly with age; (c) the known G-actin binding proteins can adequately accoun t for the G-actin pool in embryonic chick brain. (C) 1999 Elsevier Science B.V. All rights reserved.