Biochemical characterization of recombinant mouse amelogenins: protein quantitation, proton absorption, and relative affinity for enamel crystals.
Four recombinant mouse amelogenins, which varied by the presence or absence of the exon 4 encoded segment as well as the carboxyl-terminus were heterologously expressed and purified from bacteria. The rM193 and rM179 contain the carboxyl-terminus, whereas the rM180 and rM166 do not. The rM193 and rM180 contain the polypeptide segment encoded by exon 4 of the amelogenin gene. A precisely weighed sample of purified rM179 was quantified by Lowry, Bicinchoninic Acid and Bradford assays. It was determined that these protein quantification methods characteristically under or overestimate the amount of amelogenin. The calculated correction factors were: Lowry (x 1.35), BCA (x 1.96), and Bradford (x 0.78). Recombinant mouse amelogenin (rM179) was characterized with respect to its hydrogen ion binding properties. The protein absorbs 11.9 +/- 1.7 protons during a pH change from 8.0 to 5.0, suggesting that amelogenins buffer the enamel fluid in vivo. Crystal binding experiments were performed using rM193, rM180, rM179 and rM166. The carboxyl-terminus enhanced the binding of amelogenin to enamel crystals while the exon 4 encoded segment did not appreciably affect crystal binding.
Ryu
O H
OH
University of Texas School of Dentistry, Health Science Center at San Antonio, Department of Pediatric Dentistry, 78284-7888, USA.
Hu
C C
CC
Simmer
J P
JP
D000042:Absorption; D053523:Amelogenin; D000818:Animals; D003460:Crystallization; D003743:Dental Enamel / Q000378:metabolism*; D003746:Dental Enamel Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D051379:Mice; D011485:Protein Binding; D011522:Protons; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013552:Swine
D053523:Amelogenin; C507200:Amelx protein, mouse; D003746:Dental Enamel Proteins; D011522:Protons; D011993:Recombinant Fusion Proteins