Na,K-ATPase subunit isoforms in human reticulocytes: evidence from reverse transcription-PCR for the presence of alpha1, alpha3, beta2, beta3, and gamma.
The objective of this study has been to determine which Na,K-ATPase isoforms are expressed in red blood cells and whether kinetic differences in the uncoupled sodium efflux mode between the human red blood cell Na,K-ATPase and other preparations can be explained by differences in the underlying subunit composition. To this end, human reticulocyte RNA was isolated, reverse transcribed, amplified by PCR and appropriate primers, and sequenced. Primers from highly conserved areas as well as isoform-specific primers were used. The alpha1 and alpha3 isoforms of the alpha subunit, and the beta2 and beta3 isoforms of the beta subunit were found. The complete coding regions of the cDNAs for the reticulocyte subunits were sequenced from overlapping PCR fragments. No difference was found between the reticulocyte isoforms and the ones already known. The fact that we found beta2 but not beta1 in reticulocyte single-stranded cDNA, and beta1 but not beta2 in a leukocyte library indicates that leukocyte contamination of our reticulocyte preparation was negligible. Analysis of a human bone marrow library showed that alpha1, alpha2, and alpha3 as well as all three beta isoforms were present. The extent to which the kinetic properties of uncoupled sodium efflux might depend on different isoform combinations is not yet known.
Stengelin
M K
MK
Department of Cellular and Molecular Physiology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA.
Hoffman
J F
JF
D001483:Base Sequence; D001853:Bone Marrow; D017931:DNA Primers; D018076:DNA, Complementary; D015723:Gene Library; D006412:Hematopoietic Stem Cells / Q000201:enzymology; D006801:Humans; D007527:Isoenzymes / Q000097:blood* / Q000737:chemistry; D007962:Leukocytes / Q000201:enzymology; D046911:Macromolecular Substances; D016133:Polymerase Chain Reaction; D012156:Reticulocytes / Q000201:enzymology*; D000254:Sodium-Potassium-Exchanging ATPase / Q000097:blood* / Q000737:chemistry
D017931:DNA Primers; D018076:DNA, Complementary; D007527:Isoenzymes; D046911:Macromolecular Substances; D000254:Sodium-Potassium-Exchanging ATPase