Characterization of the placental brush border membrane Na+/H+ exchanger: identification of thiol-dependent transitions in apparent molecular size.
We examined the protein and mRNA encoding the amiloride-sensitive Na+/H+ exchanger from human placenta. Reverse transcriptase PCR of human placental RNA and a human choriocarcinoma cell line showed that the message for the amiloride-sensitive Na+/H+ exchanger from human placenta. Reverse transcriptase PCR of human placental RNA and a human choriocarcinoma cell line showed that the message for the amiloride-sensitive Na+/H+ exchanger is present in the placenta and its derived cell line. Northern blot analysis showed only one species of Na+/H+ exchanger mRNA, of about 5 kb in size. To examine the Na+/H+ exchanger protein two different affinity-purified antibodies were produced against the C-terminal cytoplasmic region of the Na+/H+ exchanger. The antibodies both identified a 105 kDa protein in human placental brush border membrane vesicles. Under non-reducing conditions the amount of 105 kDa protein was greatly decreased, while a 205 kDa protein became apparent. This is probably a dimer of the 105 kDa protein. The monomer-to-dimer transition was dependent on the concentration of beta-mercaptoethanol. The results show that the amiloride-sensitive Na+/H+ exchanger is relatively abundant in human placenta and that it can exist as a larger 205 kDa protein linked by disulphide bonds.
Department of Biochemistry, Faculty of Medicine, University of Alberta, Edmonton, Canada.
L
L
Fliegel
R S
RS
Haworth
J R
JR
Dyck
D000584:Amiloride / Q000494:pharmacology; D000917:Antibody Formation; D001483:Base Sequence; D015152:Blotting, Northern; D002352:Carrier Proteins / Q000032:analysis* / Q000187:drug effects / Q000235:genetics / Q000276:immunology; D003001:Cloning, Molecular; D004277:DNA, Single-Stranded; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D008623:Mercaptoethanol / Q000494:pharmacology; D008871:Microvilli / Q000737:chemistry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000276:immunology; D016133:Polymerase Chain Reaction; D011247:Pregnancy; D011257:Pregnancy Proteins / Q000032:analysis* / Q000187:drug effects / Q000235:genetics / Q000276:immunology; D012333:RNA, Messenger / Q000235:genetics; D017923:Sodium-Hydrogen Exchangers; D014407:Tumor Cells, Cultured
D002352:Carrier Proteins; D004277:DNA, Single-Stranded; D010446:Peptide Fragments; D011257:Pregnancy Proteins; D012333:RNA, Messenger; D017923:Sodium-Hydrogen Exchangers; D008623:Mercaptoethanol; D000584:Amiloride