Refolding of the precursor and mature forms of mitochondrial aspartate aminotransferase after guanidine hydrochloride denaturation (original) (raw)

Journal of Biological Chemistry, 1993

Abstract

The mitochondrial isozyme of aspartate aminotransferase (mAspAT), a dimeric pyridoxal phosphate (PLP)-dependent enzyme, is encoded by the nuclear genome and synthesized in the cytoplasm as a precursor protein (pmAspAT) containing a 29-residue amino-terminal signal peptide which is essential for its targeting and import into mitochondria. In the cytosolic-like environment of rabbit reticulocyte lysate, newly synthesized rat liver pmAspAT has been found to slowly fold and bind PLP (Mattingly, J. R., Jr., Youssef, J., Iriarte, A. and Martinez-Carrion, M. (1993) J. Biol. Chem. 268, 3925-3937). On the other hand, isolated mammalian (pig) mAspAT, when denatured with guanidine hydrochloride, seems unable to refold to a catalytically active state (West, S. M., and Price, 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 peptide on the in vitro refolding of this protein can be made. Following unfolding induced by guanidine hydrochloride, we have investigated the refolding process of this complex, dimeric coenzyme-dependent protein system by activity, fluorescence, and circular dichroism. Both mAspAT and pmAspAT can be efficiently renatured after rapid dilution of the denaturing agent at low protein concentrations. The equilibrium unfolding/refolding transitions and the kinetics of folding are protein concentration-independent and identical for both protein forms. Binding of coenzyme into the active site pocket seems to occur at a late step in the folding process of both mAspAT and pmAspAT, suggesting that in these proteins the coenzyme does not direct the folding of the polypeptide chain. These results indicate that the in vitro refolding of mAspAT is not regulated or influenced by the presence of the amino-terminal signal peptide. On the other hand, in vitro refolding in buffer is significantly faster than the folding of newly synthesized precursor protein in reticulocyte lysate examined in our previous report (reference above), pointing at the likely influence of cytosolic factors in modulating folding in the cell.

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