|Reinhardt, Timothy - Tim|
Submitted to: Iowa State University Biochemistry Biophysics and Molecular Biology Symposi
Publication Type: Abstract Only
Publication Acceptance Date: 11/5/1999
Publication Date: N/A
Citation: N/A Interpretive Summary:
Technical Abstract: We investigated the expression of the bovine secretory pathway Ca2+ ATPase (bSPCA), and plasma membrane Ca2+ ATPase isoforms (PMCA1 and PMCA2) in bovine mammary tissue during the periparturient period. Mammary tissue samples from 15 cows were collected during -21, -7, 0, +7, and +14 days of calving. Competitive RT-PCR was used to quantitate specific mRNA expression. The bSPCA mRNA is expressed in large amounts 1 wk before lactation and its expression is highly correlated with the degree of hypocalcemia observed in cows at parturition. Mammary tissue PMCA1, mRNA expression paralleled the observed increase in the RNA/DNA ratio. PMCA2 expression increased postpartum to approx. 4X that observed prepartum. Mammary tissue microsomal membranes and milk fat membranes were assayed for Ca2+ ATPase protein by Western blotting using antibodies, 5F10, NR4, and 220 which recognize all PMCAs, PMCA4, and PMCA2. There were 3 immunoreactive bands with molecular mass around 130-140 KD. However, less apparent splice form heterogeneity was detected in the milk fat membrane. Immunohistochemistry was also performed in an attempt to define the subcellular localization of these proteins in lactating rat mammary tissue. We utilized antibodies 227, which recognizes rSPCA, 5F10 and 220. The 5F10 antibody, which recognizes all PMCAs, detected antigens in both the apical and baso-lateral membrane of mammary alveolar epithelium. However, the PMCA2 was localized only in the apical membrane of the epithelium as detected by antibody 220 and the SPCA was localized in the vesicular membrane of secretory vesicles. The knowledge gained from this study will allow us to further investigate the role of Ca2+ ATPases in mammary gland calcium homeostasis during the periparturient period.