Abstract
Based upon recent reports that the mRNA for the regulatory (R) RIβ subunit of cAMP-dependent protein kinase (PKA) was expressed in testicular extracts, we determined whether testicular extracts exhibited RIβ protein. To accomplish this goal, we initially determined the fundamental labeling and ionic characteristics of recombinant RIβ. Recombinant RIβ eluted from DEAE-cellulose with a salt concentration (of 0.075 M) equivalent to its elution position from soluble mouse brain extracts with catalytic subunit-free RIα. As predicted by its amino acid sequence homology to RIα, recombinant RIβ was not phosphorylated by PKA but was labeled specifically with 8-azido-adenosine 3′:5′-[ 32P]monophosphate (8-N 3[ 32P]cAMP). Additionally, RI antisera reacted equally with RIα (47 kDa) and recombinant RIβ (53 kDa). However, recombinant RIβ exhibited an unexpectedly basic p I of 6.65–6.85. By using a pH gradient for isoelectric focussing that allowed for clear focussing of 8-N 3[ 32P]cAMP-labeled recombinant RIβ, 8-N 3[ 32P]cAMP-labeled RIβ was readily detected by two-dimensional gel electrophoresis in rat brain particulate extracts and exhibited a p I equivalent to that of recombinant RIβ. The 53-kDa RIβ was undetectable either by its immunoreactivity or upon photoaffinity labeling with 8-N 3[ 32P]cAMP by one or two-dimensional gel electrophoresis in soluble or particulate extracts of testes of 14-day-old, 45-day-old, or adult rats or in epididymal sperm. However, 8-N 3[ 32P]cAMP-labeled RIβ was detected, albeit in very small levels, by two-dimensional electrophoresis upon separation of PKAs in testes of 14-day-old rats by DEAE-cellulose chromatography but was absent in equivalent extracts from adult rat testes. These results demonstrate that the unexpectedly basic p I of RIβ allows for its clear separation by two-dimensional electrophoresis from the RII proteins and therefore allows for its unambiguous identification. Further studies, however, are required to resolve the basis for the apparent disparity in testis RIβ mRNA and protein.
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