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Abstract
Two-dimensional gel electrophoresis was used to monitor proteins synthesized in unstimulated control and in adrenocorticotropic hormone (ACTH)- or cAMP-stimulated rat adrenal cells. Four proteins, which have similar proteolytic peptide maps, have been identified. The two found primarily in unstimulated cells are referred to as pb and pa, where pb is the protein with more basic isoelectric point. Similarly, proteins ib and ia were detected only in stimulated cells. The synthesis of pb occurs only in unstimulated cells and that of ib only in stimulated cells. Protein ib accumulates with the same lag time, rate, and stimulant dose response as the increase in steroid hormone synthesis. Pulse-chase studies showed that protein ib is not produced from pb by a post-translational modification. Proteins pb and ib thus seem identical with proteins p and i previously identified in rat adrenal cortex and corpus luteum (Krueger, R.J., and Orme-Johnson, N. R. (1983) J. Biol. Chem. 258, 10159-10167, and Pon, L.A., and Orme-Johnson, N.R. (1986) J. Biol. Chem. 261, 6594-6599). The acidic forms, pa and ia, appear after a longer lag time and are produced at a slower rate than the basic forms. Pulse-chase studies showed that the disappearance of the basic form of each protein occurs concurrently with the appearance of the corresponding acidic form. Addition of [32P]orthophosphate to stimulated adrenal cells allowed direct demonstration that proteins ib and ia are phosphorylated. Moreover, alkaline phosphatase treatment of [35S]methionine-labeled, cAMP-stimulated adrenal cells caused a large decrease in the amounts of ib and ia and the appearance of proteins with the same two-dimensional electrophoretic mobilities as pb and pa. These observations suggest that protein ib may mediate stimulation of steroidogenesis, be produced by an ACTH- or cAMP-dependent, cotranslational phosphorylation of protein pb, and be lost by a cycloheximide-insensitive, post-translational conversion to ia.
Highlights
Acute andinadren~orticotropic hormone (ACTH) Regulation of Adrenal Corticosteroid~ i o s ~ n t ~ e s i s
Protein ib accumulates with the same lag time, rate, and stimulant dose response as the increase in steroid hormone synthesis
Pulse-chase studies showed that protein ibis not produced from pb by a p~t-translational modi~cation Proteins pb and ibthus seem identical with proteinsp and i previously identified in rat adrenal cortex and synthesis depends on ongoing translation (13-17) but does not require transcription (14,18)
Summary
Eitherthese phosphorylations occurred revealed that exogenous methionine is in rapid equilibrium with a much longer lag time than thatof acute stimulationof with the translation-directed amino acid pools It is steroidogenesis, or they were insensitive to inhibitors of pro- possible to obtain accurate quantitation of protein synthesis tein synthesis. Koroscil and Gallant (28) the present studies, rat adrenal cell suspensions were stimuobserved ACTH-dependent phosphorylation of several pro- lated with ACTH or cAMP prior to addition of radiolabel. Teins, including three with apparent molecular weights be- Under these conditions, where synthesis of proteins unique tween 20,000 and 30,000 These protein modifications corre- to the unstimulated state was repressed before [35S]methiolated with the rateof steroidogenesis in time andwith respect nine was added, significant changes were detected in the to ACTH dose. In ACTH-stimulated cells, two forms of protein i (i. and ib), i.e
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