Characterization of recombinant RI beta and evaluation of the presence of RI beta protein in rat brain and testicular extracts.
Based upon recent reports that the mRNA from the regulatory (R) RI beta subunit of cAMP-dependent protein kinase (PKA) was expressed in testicular extracts, we determined whether testicular extracts exhibited RI beta protein. To accomplish this goal, we initially determined the fundamental labeling and ionic characteristics of recombinant RI beta. Recombinant RI beta 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 RI alpha. As predicted by its amino acid sequence homology to RI alpha, recombinant RI beta was not phosphorylated by PKA but was labeled specifically with 8-azido-adenosine 3':5'-[32P]monophosphate (8-N3[32P]cAMP). Additionally, RI antisera reacted equally with RI alpha (47 kDa) and recombinant RI beta (53 kDa). However, recombinant RI beta exhibited an unexpectedly basic pI of 6.65-6.85. By using a pH gradient for isoelectric focussing that allowed for clear focussing of 8-N3[32P]cAMP-labeled recombinant RI beta, 8-N3[32P]cAMP-labeled RI beta was readily detected by two-dimensional gel electrophoresis in rat brain particulate extracts and exhibited a pI equivalent to that of recombinant RI beta. The 53-kDa RI beta was undetectable either by its immunoreactivity or upon photoaffinity labeling with 8-N3[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-N3[32P]cAMP-labeled RI beta 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 pI of RI beta 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 RI beta mRNA and protein.
Department of Cell and Molecular Biology, Northwestern University Medical School, Chicago, IL 60611.
D A
DA
DeManno
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V
Jackiw
E
E
Brooks
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Hunzicker-Dunn
D000818:Animals; D001345:Autoradiography; D001665:Binding Sites; D001921:Brain / Q000201:enzymology*; D000242:Cyclic AMP / Q000378:metabolism; D015180:Electrophoresis, Gel, Two-Dimensional; D005260:Female; D006863:Hydrogen-Ion Concentration; D008297:Male; D011494:Protein Kinases / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000737:chemistry; D013737:Testis / Q000201:enzymology*
D011994:Recombinant Proteins; D000242:Cyclic AMP; D011494:Protein Kinases