REFOLDING OF THE PRECURSOR AND MATURE FORMS OF MITOCHONDRIAL ASPARTATE-AMINOTRANSFERASE AFTER GUANIDINE-HYDROCHLORIDE DENATURATION

The mitochondrial isozyme of aspartate aminotransferase (mAspAT), a di meric pyridoxal phosphate (PLP)-dependent enzyme, is encoded by the nu clear genome and synthesized in the cytoplasm as a precursor protein ( pmAspAT) containing a 29-residue amino-terminal signal peptide which i s essential for its targeting and import into mitochondria. In the cyt osolic-like environment of rabbit reticulocyte lysate, newly synthesiz ed rat liver pmAspAT has been found to slowly fold and bind PLP (Matti ngly, J. R., Jr., Youssef, J., Iriarte, A. and Martinez-Carrion, M. (1 993) J. Biol. Chem. 268, 3925-3937). On the other hand, isolated mamma lian (pig) mAspAT, when denatured with guanidine hydrochloride, seems unable to refold to a catalytically active state (West, S. M., and Pri ce, N. C. (1990) Biochem. J. 265, 45-50). With the availability of rat liver recombinant precursor and mature forms of mAspAT as homogeneous , stable preparations, an assessment of the influence of the signal pe ptide on the in vitro refolding of this protein can be made. Following unfolding induced by guanidine hydrochloride, we have investigated th e refolding process of this complex, dimeric coenzyme-dependent protei n system by activity, fluorescence, and circular dichroism. Both mAspA T and pmAspAT can be efficiently renatured after rapid dilution of the denaturing agent at low protein concentrations. The equilibrium unfol ding/refolding transitions and the kinetics of folding are protein con centration-independent and identical for both protein forms. Binding o f coenzyme into the active site pocket seems to occur at a late step i n the folding process of both mAspAT and pmAspAT, suggesting that in t hese proteins the coenzyme does not direct the folding of the polypept ide chain. These results indicate that the in vitro refolding of mAspA T is not regulated or influenced by the presence of the amino-terminal signal peptide. On the other hand, in vitro refolding in buffer is si gnificantly faster than the folding of newly synthesized precursor pro tein in reticulocyte lysate examined in our previous report (reference above), pointing at the likely influence of cytosolic factors in modu lating folding in the cell.