Comparison of protein phosphorylation patterns produced in adrenal cells by activation of cAMP-dependent protein kinase and Ca-dependent protein kinase.

Bovine adrenal fasciculata cells, exposed to either ACTH or AII, synthesize glucocorticoids at an enhanced rate. It is generally accepted that the signaling pathways triggered by these two peptides are not identical. ACTH presumably acts via a cAMP-dependent protein kinase (PKA) and AII, via a calcium-dependent protein kinase. We have found that either peptide hormone stimulates synthesis of a mitochondrial phosphoprotein pp37, leading to accumulation of its proteolytically processed products pp30 and pp29. On the basis of a number of criteria, this 37 kDa protein is the bovine homolog of the 37 kDa protein that we have characterized in rodent steroidogenic tissue (Epstein L. F. and Orme-Johnson N. R.: J. Biol. Chem 266 (1991) 19,739-19,745). Further, bovine pp37 is phosphorylated when PKA or protein kinase C (PKC) is activated directly by (Bu)2 cAMP or PMA, respectively. These studies indicate that either pp37 is a common substrate for PKA and PKC in these cells or there is a common downstream kinase, which is activated by exposure to either ACTH or AII. Rat adrenal glomerulosa cells, exposed to either ACTH or AII, show an enhanced rate of mineralocorticoid synthesis. As for bovine fasciculata cells, it is thought that the signaling pathway triggered by ACTH differs from that triggered by AII. As we found for bovine fasciculata, pp37 is phosphorylated when the rat cells are exposed to either peptide hormone. However, in contrast to the finding for bovine fasciculata, while exposure of the rat glomerulosa cells to (Bu)2cAMP does cause the synthesis of pp37, exposure of the cells to PMA does not. Taken together, these findings provide further evidence that the subcellular signaling events, triggered by the action of AII on bovine adrenal fasciculata and rat adrenal glomerulosa cells, differ. Further, the fact, that pp37 is phosphorylated only when the rate of steroidogenesis is enhanced, reaffirms its potential involvement in the signaling pathway that causes stimulation of steroid hormone biosynthesis.

Acute action of luteinizing hormone on mouse Leydig cells: accumulation of mitochondrial phosphoproteins and stimulation of testosterone synthesis.

Upon stimulation of Leydig cells with luteinizing hormone (LH) or dibutyryl-3',5'-cyclic AMP (Bt2cAMP) at 37 degrees C, two mitochondrial phosphoproteins accumulate with the same stimulant dose response as the increased rate of testosterone synthesis. The proteins pp32 and pp30 have apparent isoelectric points of 6.6 and 6.5 and molecular weights of approximately 32 30 kDa respectively, as determined by two-dimensional polyacrylamide gel electrophoresis. These two phosphoproteins are not detected in mouse adipose or liver cells nor in the total testicular cell population, of which Leydig cells constitute a small percentage. However, both proteins are also observed in mouse adrenal cells stimulated by ACTH or Bt2cAMP. The appearance of pp32 and pp30 is prevented by inhibitors of cytosolic protein translation, indicating that only newly synthesized protein is available as a substrate for phosphorylation. Proteolytic peptide mapping indicates that both of these mouse Leydig and adrenal proteins have structural similarity to pp30 (formerly denoted as ib), the 30 kDa mitochondrial phosphoprotein that we have observed previously in peptide hormone or Bt2cAMP-stimulated rat adrenal cortex (Pon, L.A., Hartigan, J.A. and Orme-Johnson, N.R. (1986) J. Biol. Chem. 261, 13309-13316; Alberta, J.A., Epstein, L.F., Pon, L.A. and Orme-Johnson, N.R. (1989) J. Biol. Chem. 264, 2368-2372) and rat corpus luteum cells (Pon, L.A. and Orme-Johnson, N.R. (1986) J. Biol. Chem. 261, 6694-6599). Since pp32 is a larger mitochondrial protein of similar primary structure to pp30, it is a potential precursor of this protein. Finally, the detection of the mitochondrial phosphoprotein pp30 in a third steroidogenic tissue type and a third species provides further correlative evidence that the production of pp30 may be an integral part of the subcellular mechanism by which peptide hormones stimulate steroid hormone biosynthesis.

Regulation of steroid hormone biosynthesis. Identification of precursors of a phosphoprotein targeted to the mitochondrion in stimulated rat adrenal cortex cells.

Two short-lived precursor proteins, pp37 and pp32, of the mitochondrial phosphoprotein pp30 (formerly denoted as ib) have been detected in Bt2cAMP-stimulated rat adrenal cortex cells, incubated at 25 degrees C or with 1,10-ortho-phenanthroline at 37 degrees C. Subsequently, these two precursor proteins were also identified in cells incubated at 37 degrees C, where they are present only at low levels due to their short half-life. pp30 is produced in several steroidogenic tissues in response to trophic hormone or second messenger analogue. pp37 and pp32 are also phosphoproteins located in the mitochondrion that are produced in response to cAMP analogue and give rise to proteolytic peptide maps similar to that of pp30. As for pp30, inhibition of cytosolic translation prevents the production of pp37 and pp32. The larger precursor protein pp37 has an apparent molecular mass of 37 kDa, an isoelectric point of approximately 7.1, and a half-life at 37 degrees C of 3-4 min. Pulse-chase studies indicate that this protein is processed into the smaller protein, pp32, which has an apparent molecular mass of 32 kDa, an isoelectric point of approximately 6.4, and a half-life at 37 degrees C of 3-4 min. This latter protein is the immediate precursor of pp30. Since ortho-phenanthroline inhibits the mitochondrial processing protease, while the lower incubation temperature slows both protein import and protease processing, the experimental conditions necessary to detect these proteins are consistent with pp37 being a precursor protein that contains two cleavable presequences and is imported into the mitochondrion. The sequential removal of these sequences produces the mature protein pp30.

Characterization of the adrenal 11 beta-hydroxylase in inbred salt-sensitive and resistant rats.

Rats have been bred for susceptibility and resistance to the hypertensive effect of dietary salt (S/JR & R/JR). S/JR have an abnormal adrenal steroid 11 beta,18-hydroxylase activity resulting in increased production of 18-OH-DOC. S/JR also produce increased quantities of 19-nor-DOC, which may be related, since the 11 beta,18-hydroxylase also catalyzes the 19-hydroxylation of DOC, a pivotal step in 19-nor-DOC biosynthesis. The purpose of the present studies was to further characterize the mutant S/JR adrenal steroid 11 beta,18-hydroxylase. Preliminary studies are also presented on assessing the renal 19-desmolase, the last step in 19-nor-DOC biosynthesis. Adrenal glands were harvested from R/JR and S/JR and prepared for incubation studies, protein immunoblotting, and RNA analysis. Kidneys from Sprague-Dawley rats were also used for isolated renal perfusion studies. Both S/JR and R/JR strains had a single immunostaining band for 11 beta,18-hydroxylase at 51,000 molecular weight which were equal in intensity. Both strains had a single RNA transcript at 4.3 kilobases which hybridized with equal intensity to the bovine cDNA (pB11-9). The Km for 11 beta- and 18-hydroxylation was identical within strains but was different between strains. The Km for 19-hydroxylation was different between S/JR and R/JR, and was much greater than 11 beta- and 18-hydroxylation in both strains. This suggests that the catalytic site for 19-hydroxylation is different than that for 11 beta- and 18-hydroxylation and that the S/JR enzyme binds the substrate with higher affinity than the R/JR enzyme. In the isolated perfusion studies the rat kidneys converted 80% of 19-oxo-DOC to either 19-oic-DOC or 19-nor-DOC. These data demonstrate that the difference in S/JR enzyme activity is probably due to a point mutation in the enzyme or to a change in a regulatory protein.

Inhibition of steroidogenesis in rat adrenal cortex cells by a threonine analogue.

We have investigated the ability of amino acid analogues of serine and threonine to inhibit the increase in steroidogenesis elicited by addition of ACTH or cAMP in cells isolated from the rat adrenal cortex. We have found that the serine analogues, D, L-isoserine, alpha-methyl-D, L-serine and L-homoserine, are almost totally ineffective in inhibiting this process but that the threonine analogue, D, L-beta-hydroxynorvaline, at a concentration of 300 microM inhibits stimulated steroid hormone biosynthesis by ca 95%, while inhibiting overall protein synthesis by only ca 40%. This inhibition was found to occur in a dose-dependent manner and to be reversible by a stoichiometric concentration of threonine. These studies suggest that beta-hydroxynorvaline is functioning as a threonine analogue in our experimental system. Both the onset of inhibition by analogue and reversal of this inhibition by the natural amino acid occurred rapidly, without detectable lag. Since results obtained using cAMP as stimulant parallel those obtained using ACTH, the inhibitory effect of the analogue seems to occur subsequent to the synthesis of cAMP. Additionally, the analogue does not inhibit the conversion of pregnenolone to corticosterone, suggesting the site of action of analogue occurs prior to the synthesis of pregnenolone from cholesterol. Thus, the analogue may be exerting its effect on a protein that is synthesized subsequent to ACTH addition and is important in the acute phase of stimulated steroid hormone biosynthesis. Further, since ACTH action on adrenal cortex cells causes the activation of protein kinase A, which phosphorylates serine and threonine residues, it is possible that the effect of the analogue is to prevent the phosphorylation of a newly-synthesized protein.

Distinctive properties of adrenal cortex mitochondria.

The mitochondria in cells that synthesize steroid hormones not only have enzymes not present in mitochondria of non-steroidogenic cells but also have unique mechanisms for regulating the steroid substrate availability for certain of these enzymes. We have considered in detail the cytochrome P-450scc system that is located in the inner mitochondrial membrane and that catalyzes the initial and rate-determining step in the steroid hormone biosynthetic pathway. The flux through this pathway is regulated both by the levels of these catalysts themselves and by the availability of the substrate cholesterol for conversion to pregnenolone. These two levels of regulation occur in different time frames but are both controlled externally by the action of tissue-specific peptide hormone. We have used the adrenal cortex fasciculata cells as our paradigmatic cell type. The overall picture seems closely similar for mitochondria in other such steroidogenic cells when analogous data are available. Thus, in adrenal cortex fasciculata cells ACTH triggers several long-term (trophic) and short-term (acute) effects upon and within mitochondria that influence the initial and rate-determining step in the steroid hormone biosynthetic pathway. The only second messenger for both effects characterized thus far is cAMP. An increase in membrane-associated cAMP rapidly activates cAMP-dependent protein kinase, which in turn phosphorylates several cellular proteins, e.g., cholesterol ester hydrolase (vide supra). The trophic action, i.e., that produced by exposure of the cells to increased levels of ACTH or cAMP for a prolonged period (minutes to hours), increases the amounts of the steroid hormone synthesizing proteins in the mitochondria by increasing the transcription of the relevant nuclear genes. This latter process is not needed for the acute increase in the rate of steroid hormone biosynthesis. Whether induction of steroidogenic enzymes requires activation of a kinase has not been determined. However, the postulated SHIP proteins provide a mechanism by which cAMP levels and protein synthesis itself may regulate this induction. Mitochondria in steroidogenic tissues exert control over this process by their ability to recognize, import and process correctly the nuclear encoded precursors of the steroidogenic enzymes. Whether control at this level is ultimately dictated by nuclear or mitochondrial gene products or by an interplay between them is still unknown.(ABSTRACT TRUNCATED AT 400 WORDS)

19-Hydroxylase inhibition of adrenal mitochondrial P450 11 beta/18/19-hydroxylase by a suicide inhibitor.

19-Nor-deoxycorticosterone (19-nor-DOC) is a mineralocorticoid that is increased in some forms of experimental and human hypertension. The pivotal step in 19-nor-DOC biosynthesis is adrenal P450 19-hydroxylase, but this enzyme has not been clearly distinguished from P450 11 beta/18-hydroxylase. This study attempted to specifically inhibit adrenal 19-hydroxylation of deoxycorticosterone (DOC) using a suicide aromatase inhibitor, 19-acetylenic androstenedione (19-AA). Purified bovine P450 11 beta/18/19-hydroxylase was incubated with excess substrate DOC, adrenodoxin, and adrenodoxin reductase in the presence of increasing doses of the inhibitor, 19AA. 11 beta-, 18-, and 19-hydroxylation were measured by quantification of corticosterone, 18-OH-DOC, and 19-OH-DOC respectively. Measurements of these products demonstrated that 11 beta- and 18-hydroxylation was not inhibited whereas 19-hydroxylation was inhibited as manifested by decreased 19-OH-DOC formation (p less than .05). The IC50 of 19-AA was approximately 10(-12) M. The specific inhibition of 19-hydroxylation suggests that the 19-hydroxylase may be an enzyme distinct from the P450 11 beta/18-hydroxylase. This further suggests that 19-nor-DOC biosynthesis may be under independent regulation and may be amendable to specific in vivo inhibition.

Mitochondrial localization of a phosphoprotein that rapidly accumulates in adrenal cortex cells exposed to adrenocorticotropic hormone or to cAMP.

We have reported previously that a phosphoprotein, ib, is present in adrenal cortex, corpus luteum, and Leydig cells stimulated with either tissue-specific peptide hormone or with cAMP. The accumulation of protein ib in each of these cell types has been found to parallel the stimulation of steroid synthesis with respect to both time course and stimulant dose response. Thus, protein ib is a potential mediator in the acute stimulation of steroidogenesis by peptide hormone or cyclic AMP. A second protein, pb, the unphosphorylated form of ib, is synthesized constitutively in unstimulated but not stimulated cells and is not converted post-translationally to ib upon stimulation. Using two-dimensional gel electrophoresis of subcellular fractions isolated from rat adrenal cortex cells labeled with [35S] methionine, we have determined the intracellular localization of proteins p and i. We demonstrate that proteins ib and pb are localized predominantly in the mitochondria and are tightly associated with that organelle. We also find that inhibition of mitochondrial protein synthesis by chloramphenicol affects neither the accumulation of these proteins nor the stimulation of steroidogenesis. Thus, protein pb and its phosphorylated counterpart, ib, are synthesized in the cytosol and transported to the mitochondria, the site of the rate-limiting step in steroid hormone biosynthesis.

Subcellular localization of a protein produced in adrenal cortex cells in response to ACTH.

We have reported previously that a protein, ib, is produced in adrenal cortex and other steroidogenic cells with the same tissue-specific peptide hormone or cAMP dose-response and the same kinetics as the increase in steroid hormone biosynthesis. In this study, we have fractionated adrenal cortex cells into subcellular components and used two-dimensional electrophoresis to characterize the proteins in these fractions. We have demonstrated previously that inhibition of cytosolic translation, e.g. by cycloheximide, prevents the production of protein ib. We also report that the production of this protein is not affected by inhibition of mitochondrial translation by chloramphenicol.

Acute stimulation of corpus luteum cells by gonadotrophin or adenosine 3',5'-monophosphate causes accumulation of a phosphoprotein concurrent with acceleration of steroid synthesis.

A protein (ib), which we have detected previously in peptide hormone or cAMP-stimulated corpus luteum cells, is shown to be a phosphoprotein and to be posttranslationally converted into a more acidic phosphoprotein (ia). Phosphorylation is demonstrated by two types of experiments, both using two-dimensional gel electrophoresis. In the first type, gels from [35S]methionine-labeled solubilized cell extracts are compared to gels from such extracts treated with alkaline phosphatase. This in vitro phosphatase treatment converts protein ib quantitatively into protein pb, which is synthesized in vivo only in unstimulated cells. Similarly, ia is converted into pa, the posttranslational product of pb. The second type of experiment demonstrates 32P label incorporation into proteins with the same electrophoretic mobilities as proteins ib and ia. Limited proteolytic digestion of all four proteins from phosphatase-treated and untreated corpus luteum cells shows that the newly detected acidic products, ia and pa, give rise to cleavage patterns similar to those of ib and pb. Further, these patterns resemble those produced by all four such proteins from the adrenal. These findings suggest that in both stimulated corpus luteum and adrenal, a similar protein ib, which accumulates with kinetics and stimulant dose response paralleling those of steroid hormone biosynthesis, is phosphorylated during its synthesis and is degraded by conversion to another phosphoprotein (ia).

Evidence for the involvement of a labile protein in stimulation of adrenal steroidogenesis under conditions not inhibitory to protein synthesis.

Evidence is presented to support the hypothesis that synthesis of a labile protein is required for stimulation of steroidogenesis in rat adrenocortical cells. Amino acids L-canavanine and L-S-aminoethylcysteine, at concentrations as high as 5 mM, each inhibited steroidogenesis to a much greater extent than they inhibited protein synthesis. S-Aminoethylcysteine caused a 50% decrease in the stimulated rate of corticosterone production under conditions where incorporation of [35S]methionine into protein was unchanged. Both amino acids block stimulation of steroid synthesis at a step subsequent to the formation of cAMP and before the synthesis of progesterone. The onset of this effect, after the addition of the amino acids, on corticosterone production is quite rapid. These results provide support, that is not dependent on inhibition of protein synthesis, for the hypothesis that a labile protein mediates stimulation of steroidogenesis. Reversal of canavanine and S-aminoethylcysteine inhibition of steroidogenesis by arginine and lysine, respectively, suggests that the inhibitors are functioning as amino acid analogs. S-Aminoethylcysteine inhibits the incorporation of [3H]lysine into protein as well as inhibits steroidogenesis; further, [3H]S-aminoethylcysteine is incorporated into protein that is nonstimulatory. These results suggest that lysine residues play an essential role in the function of the labile protein or that the labile protein contains a large number of lysine residues.

Acute ACTH regulation of adrenal corticosteroid biosynthesis. Rapid accumulation of a phosphoprotein.

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.

Acute stimulation of steroidogenesis in corpus luteum and adrenal cortex by peptide hormones. Rapid induction of a similar protein in both tissues.

Two-dimensional electrophoresis was used to detect a protein (ic) synthesized in rat corpus luteum cells in response to acute stimulation by human chorionic gonadotropin or dibutyryl cyclic AMP. This induced protein ic is isoelectric at pH 6.5 (isoelectric focusing) and has an apparent molecular weight of 28,000 (sodium dodecyl sulfate electrophoresis). The human chorionic gonadotropin or dibutyryl cyclic AMP dose response and time course of synthesis of the protein parallel those of progesterone synthesis in stimulated luteal cells. Additionally, cycloheximide, which inhibits the increase in progesterone formation caused by human chorionic gonadotropin or cAMP, also inhibits the synthesis of ic. Proteolytic polypeptide mapping suggests that ic has a very similar primary structure to another protein (pc), which has the same molecular weight as ic, differs from ic in pI, and is synthesized only in unstimulated cells. These polypeptide maps also demonstrate the close similarity of pc and ic to two proteins p and i, synthesized in control and in adrenocorticotropic hormone-stimulated rat adrenal cortex cells, respectively (Krueger, R. J. and Orme-Johnson, N. R. (1983) J. Biol. Chem. 258, 10159-10167). In both adrenal cortex and corpus luteum, binding of a tissue-specific polypeptide hormone acts via cAMP to cause increased steroidogenesis and induction of the synthesis of protein i (ic), with the same time course and hormone dose dependence. Also in both tissues, inhibition of protein synthesis at the level of translation (e.g. by cycloheximide addition) causes inhibition of i (ic) synthesis and of stimulated steroid production. This close correlation between the two different tissues in conditions which cause induction of the synthesis of these proteins suggests that the proteins may be common intermediaries in the control by polypeptide hormones of steroidogenesis in endocrine tissues.

Acute cAMP stimulation in Leydig cells: rapid accumulation of a protein similar to that detected in adrenal cortex and corpus luteum.

Addition of cAMP (as the dibutyryl compound) to a primary culture of mouse Leydig cells caused the accumulation of a protein, i(b), with the same dependence on cAMP concentration as the increase in testosterone synthesis. Stimulation of both protein i(b) and testosterone production were inhibited by cycloheximide. Additionally, cAMP caused repression of synthesis of another protein, p(b), with the same approximate molecular weight (28,000 daltons) as i(b), but more basic isoelectric point. This behavior resembles an event which has been documented in the adrenal cortex (Krueger, R.J., and Orme-Johnson, N.R., J. Biol. Chem., 258, 10159-10167, 1983) and corpus luteum (Pon, L.A., and Orme-Johnson, N.R., J. Biol. Chem., 261, 6594-6599, 1986). The discovery of these proteins in a third steroid-producing cell type and the close correlation between conditions causing increased steroid synthesis and increased i(b) production is further indication that protein i(b) may be an intermediary in peptide hormone or cAMP control of steroid hormone biosynthesis.

Protein synthesis requirement for acute ACTH stimulation of adrenal corticosteroidogenesis.

The rapid, cAMP mediated increase produced by ACTH in adrenal corticosteroidogenesis depends also on the rapid synthesis of protein. This obligatory involvement of protein synthesis has been established in several laboratories mainly on the basis of studies demonstrating a parallelism, under a variety of experimental conditions, of the capacity of adrenal cells to synthesize protein with their ability to increase steroid production in response to ACTH or cAMP. More recent correlative studies have disclosed the existence of two proteins, denoted as p and i. Protein i appears only in ACTH or cAMP stimulated cells and with the same time course and ACTH dose response as the increase in corticosteroid synthesis. Protein p, which closely resembles i in molecular weight and primary structure but differs in pI, is synthesized only in unstimulated cells. Neither the function of these two proteins nor the exact nature of the interplay of their synthesis on regulation has been established, although it seems reasonably certain that i is stimulatory rather than p inhibitory. The protein glycosylation inhibitor tunicamycin was found to inhibit the ACTH produced increase in steroidogenesis and also to inhibit the synthesis of protein i under conditions which did not inhibit overall protein synthesis. Therefore it seems probable that i is an N-glycosylated form of protein p and that adrenal cells are unresponsive to acute ACTH action if they are incapable of synthesizing glycosylated proteins specifically.

Characterization of 3-iron ferredoxins by means of the linear electric field effect in EPR.

We describe a method for the differentiation of 3-iron from 2-iron and 4-iron Fe/S proteins based on consideration of both the magnetic field dependence of shifts in g induced by an externally applied electric field (LEFE) and the continuous wave EPR spectra properties. The magnetic field dependence and the magnitude of the LEFE for 3-iron ferredoxins are similar to those for 4-iron ferredoxins but differ considerably from those for 2-iron ferredoxins or for high potential iron proteins. Furthermore, as 3-iron ferredoxins and high potential iron proteins are EPR-active when oxidized while 2-iron and 4-iron ferredoxins are only EPR-active when reduced, the differentiation among all of them can be made on the basis of both continuous wave EPR and LEFE properties, but not by each individually.

Oxidation state dependence of proton exchange near the iron-sulfur centers in ferredoxins and high-potential iron-sulfur proteins.

For both the [2Fe-2S] and the [4Fe-4S] ferredoxins, dialysis against 2H2O prior to single electron reduction leads to the appearance of a deuterium modulation pattern in the electron spin echo decay envelope indicative of deuteron-proton exchange very near the paramagnetic center. In contrast, if the ferredoxin is exposed to 2H2O after its reduction in H2O, far less deuterium exchange near the metal center takes place. Thus, proton exchange with solvent is in part dependent on the redox state of the protein. For high potential iron-sulfur proteins, this type of proton-deuteron exchange near the metal center does not occur unless the protein is partially unfolded in dimethylsulfoxide in 2H2O.

Acute adrenocorticotropic hormone stimulation of adrenal corticosteroidogenesis. Discovery of a rapidly induced protein.

Two-dimensional electrophoretic techniques were used to identify and characterize a protein that is not produced in quiescent isolated rat adrenal cells but is produced in response to acute stimulation by adrenocorticotropic hormone (ACTH) or dibutyryl cAMP. The molecular weight of this protein is 28,000 (sodium dodecyl sulfate electrophoresis), and its isoelectric point is 6.5 (isoelectric focusing). Mapping of proteolytic peptides suggests that this induced protein (i) is quite similar in primary structure to another protein (p), which is produced only in nonstimulated adrenal cells. The time course of formation of protein i and its ACTH dose response closely parallel the increase of corticosteroid production in stimulated cells. The possibility that protein i is produced in response to increased levels of some steroid of the glucocorticoid pathway is precluded by the observation that inhibition of corticosteroid synthesis by aminoglutethimide does not alter the rate of production of i. Addition of cycloheximide before ACTH, which prevents stimulation of corticosteroidogenesis, also prevents formation of protein i implying that the production of protein i depends on continuing protein synthesis. [35S/32S]Methionine pulse-chase experiments, i.e. addition of excess [32S] methionine and ACTH after prelabeling with [35S]methionine, show that protein i is not produced from pre-existing protein p or other pre-existing proteins even if protein synthesis (and increased steroid production) is not inhibited. These findings exclude post-translational modification as a mechanism for the production of i but are consistent with p and i being related by cotranslational modification. Addition of cycloheximide after stimulation causes the formation of protein i to cease, but the amount of the protein does not decrease with the same kinetics as the return of corticosteroid production to its unstimulated level. [35S/32S]Methionine pulse-chase experiments imply protein i, even under conditions of ongoing ACTH stimulation and protein synthesis, is degraded with approximately the same kinetics as after cycloheximide inhibition. The close concurrence under a wide variety of experimental conditions between the appearance of protein i and the increase in adrenal corticosteroid production, coupled with the fact that the former does not occur as a result of the latter, make protein i a likely candidate for the postulated corticosteroidogenic stimulatory protein (Ferguson, J.J. (1962) Biochim. Biophys. Acta 57, 616-617). The fact that stimulation of steroidogenesis may occur via co-translational modification of a regulatory protein is an intriguing possibility which readily explains both the observed rapid and protein synthesis-dependent stimulation and the lack of dependence of stimulation on transcription (Schulster, D. (1974) Mol. Cell. Endocr. 1, 55-64).

Escherichia coli sulfite reductase hemoprotein subunit. Prosthetic groups, catalytic parameters, and ligand complexes.

Escherichia coli NADPH-sulfite reductase can be dissociated into an oligomeric flavoprotein and a monomeric hemoprotein (HP) subunit in 4 M urea. HP catalyzes stoichiometric 6-electron reductions of SO32- (to S2-) and of NO2-, as well as 2-electron reduction of NH2OH, with reduced methyl viologen (MV+) as reductant. While Vmax values are highest with the nitrogenous substrates, Km for SO32- is 2 to 3 orders of magnitude less than the Km for NO2- or NH2OH. EPR spectroscopic and chemical analyses show that HP contains one siroheme and one Fe4S4 center per polypeptide. The heme is in the high spin Fe3+ state in HP as isolated. Near-quantitative reduction of the Fe4S4 center to a state yielding a g = 1.94 type of EPR spectrum by S2O42- and/or MV+ could be achieved if HP was converted to either the CN- or CO complex or treated with 80% dimethyl sulfoxide. HP binds one SO32- or CN- per peptide. Binding of these ligands, as well as CO, appears to be mutually exclusive and to involve the heme. The heme Fe3+/Fe2+ potential is shifted from -340 mV in the free HP to -155 mV in the HP-CN- complex. The potential of the Fe4S4 center is approximately 70 mV more negative in the CN- as opposed to the CO-ligated HP (-420 mV), a result which indicates the presence of heme-Fe4S4-ligand interaction in the HP complexes.