Holoenzymes of cAMP-dependent protein kinase containing the neural form of type I regulatory subunit have an increased sensitivity to cyclic nucleotides.

摘要: Specific isoforms of the cAMP-dependent protein kinase are preferentially expressed within discrete neuronal regions in mouse brain (Cadd and McKnight (1989) Neuron 3, 71-79) suggesting that these subunits might have different functional properties. We used recombinant techniques to express purify type I regulatory subunits, RI alpha beta, catalytic C then reconstituted holoenzymes with various combinations R subunits. The ability form inactive be activated presence cyclic nucleotides was examined. Holoenzymes containing beta had essentially same activation properties exhibited by holoenzymes. However, neural RI, led formation a holoenzyme which at 3-7-fold lower concentration compared alpha. Expression central nervous system may provide mechanism for increasing sensitivity what would otherwise subthreshold levels stimulation. Two mutant forms were constructed converted sequence position 98 (RI Ala) or positions 99 Ala/Ile). These sequences part pseudosubstrate site thought interact subunit. Wild combined vitro purified bovine half maximal constants (Ka) determined nucleotides. Ala Ala/Ile gave Ka values higher than wild double shifting toward value about 30%. results suggest amino acid differences account some, but not all, increased beta.