Cyclic AMP-dependent protein kinase in rat mammary tissue: expression of catalytic and regulatory subunits throughout pregnancy and lactation

Abstract

‘Expressed’ and ‘total’ activities of cyclic AMP-dependent protein kinase (PK-A) were measured in extracts of rat mammary tissue sampled throughout pregnancy and lactation. Expression of the genes encoding the catalytic subunit (C-subunit) isoforms C alpha and C beta was examined by Northern blotting, as a function of mammary development, to determine relative levels of their respective mRNAs. The content of C-subunit protein (all isoforms) was estimated immunochemically and related to levels of C-subunit catalytic activity and of mRNAs. It was found that C-subunit isoform mRNAs are expressed co-ordinately during mammary development and that a marked decline in expression, per cell, at around parturition is paralleled by a fall in ‘total’ PK-A activity. The ‘expressed’ activity of PK-A activity underwent characteristic changes throughout pregnancy and lactation, reaching a peak late in pregnancy. The PK-A activity ratio reached a peak in early lactation. C-subunit protein mass closely parallel ‘total’ PK-A activity throughout pregnancy and lactation, thereby demonstrating the constancy of C-subunit specific catalytic activity during these developmental events. Regulatory subunits (R-subunits) were probed with the photoaffinity label 8-azido-[32P]cAMP. The abundance of R-II as a proportion of total R-subunit increased throughout pregnancy and lactation, and quantitative analysis of the photoaffinity labelling suggested inconstancy in the ratio of R:C subunits, with highest values occurring in late pregnancy/early lactation.