Binding of estrogen and progesterone-BSA conjugates to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the effects of the free steroids on GAPDHenzyme activity: physiological implications

In this study rat brain solubilized plasmalenuna-microsomal fractions (B-P3 ) or cytosolic fractions were applied to P-3-BSA (progesterone linked to BS A at C-3 position) and E-6-BSA (17 beta -estradiol linked to BSA at C-6 pos ition) affinity columns. It is interesting that a 37 kDa protein was retain ed by both columns which was identified as glyceraldehyde-3-phosphate dehyd rogenase (GAPDH) by N-terminal sequencing. The 37 kDa protein (GAPDH) was n ot retained by either a control BSA conjugated affinity column or a cortico sterone-BSA affinity column. E-6-BSA bound to GAPDH with higher binding aff inity than P-3-BSA or T-3-BSA (testosterone linked to BSA at C-3 position) affinity columns. In addition, the binding of 17 beta -E-6-BSA to GAPDH was impeded by free estrogen (17 beta -estradiol) completely. Binding studies of E-6-BSA and P-3-BSA to commercial GAPDH from rabbit skeletal muscle usin g radiolabeled ligand binding assays revealed that P-3-BSA had 10X lower GA PDH binding affinity than E-6-BSA. Nest, the effects of estrogen and proges terone on GAPDH activity were studied. Rapid and significant increases in V -max and changes in K-m were observed by the addition of 10 nM estradiol, w hereas 100 nM progesterone decreased only V-max significantly. Testosterone , corticosterone, 17 alpha -estradiol, and diethylstilbestrol did not affec t the enzyme activity. The results indicate that GAPDH is a target site for 17 beta -estradiol and progesterone and suggest possible roles in the regu lation of cellular metabolism rind synaptic remodeling in which GAPDH has b een reported to be involved. (C) 2001 Elsevier Science Inc. All rights rese rved.