Modification of human placental alkaline phosphatase by periodate-oxidized 1,N6-ethenoadenosine monophosphate.
Oxidation of 1,N6-ethenoadenosine monophosphate (epsilon AMP) with periodate cleaved the cis-diol of the ribose ring and resulted in the formation of a dialdehyde derivative (epsilon AMP-dial). At room temperature epsilon AMP-dial was unstable and underwent beta-elimination to give 4',5'-anhydro-1,N6-ethenoadenosine dialdehyde acetal (A epsilon Ado-dial). These nucleotide analogues were found to inactivate human placental alkaline phosphatase in a time- and concentration-dependent manner. epsilon AMP-dial was shown to be an affinity label for the enzyme on the basis of the following criteria. (a) Kinetics of the enzyme activity loss over a wide range of epsilon AMP-dial concentration showed a saturating phenomenon. Removal of the phosphate group made the reagent (A epsilon Ado-dial) become a general chemical modifying reagent. (b) The artificial substrate p-nitrophenyl phosphate gave substantial protection of the enzyme against inactivation. (c) epsilon AMP-dial was a substrate and a partial mixed-type inhibitor for the enzyme. Results of the inhibition and protection studies indicated that the reagent and substrate could combine with the enzyme simultaneously. Besides the phosphate-binding domain, an induced hydrophobic region is proposed for the substrate-binding site for human placental alkaline phosphatase.
Chang
G G
GG
Department of Biochemistry, National Defense Medical Center, Taipei, Taiwan, Republic of China.
Shiao
M S
MS
Lee
K R
KR
Wu
J J
JJ
D000249:Adenosine Monophosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D000469:Alkaline Phosphatase / Q000302:isolation & purification / Q000378:metabolism*; D005260:Female; D006801:Humans; D007700:Kinetics; D008433:Mathematics; D008962:Models, Theoretical; D010084:Oxidation-Reduction; D010504:Periodic Acid; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D011485:Protein Binding
D010504:Periodic Acid; C030368:etheno-AMP; D000249:Adenosine Monophosphate; D000469:Alkaline Phosphatase