Abstract
Serine: pyruvate/alanine: glyoxylate aminotransferase in the liver is a class IV aminotransferase. The present study was undertaken to characterize the pyridoxal 5'-phosphate (PLP) binding to a recombinant rat serine: pyruvate/alanine: glyoxylate aminotransferase (SPT10), which is a homodimer of 44.4 kDa subunits. Purified SPT10 exhibited absorption maxima at _??_330nm in addition to a 278nm protein peak and a _??_420nm peak of PLP bound via Schiff base, and contained 0.56-0.69 mol of PLP per mol of subunit. Apo-SPT10 without measurable bound PLP did not exhibit the absorbance at _??_420nm, but still showed the _??_330nm peak. Upon reconstitution, 0.73-0.79 mol of PLP per mol of subunit was bound to apo-SPT10 with an apparent Kd of _??_0.1μM, resulting in a holo-SPT10 preparation whose specific activity and A_??_420/A_??_330 absorbance ratio were higher than those of the original SPT10. On SDS/PAGE of BrCN-cleavage peptides of NaBH4-reduced SPT10 22-23 kDa fragments migrated as a pair of bands. On amino acid sequencing, the _??_22 and _??_23 kDa pair gave the same sequence, except that Lys was released only from the _??_22 kDa band material in the cycle corresponding to Lys209. NaB3H4-treated SPT10 also migrated as a pair of 44-45 kDa bands and 3H was incorporated only into the _??_45 kDa band. It appears that SPT10 has the capacity to bind 1 mol of PLP to Lys209 of every subunit, but usually binds less PLP in a Schiff base structure, probably due to the presence of a 330nm-absorbing chromophore.
