Upon stimulation of Leydig cells with luteinizing hormone (LH) or dibutyryl-3',5'-cyclic AMP (Bt2cAMP) at 37°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 and 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.

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.

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.

19-Nor-deoxycorticosterone (19nor-DOC) is a mineralocorticoid that is increased in some forms of experimental and human hypertension. The pivotal step in 19nor-DOC biosynthesis is adrenal P450 19-hydroxylase, but this enzyme has not been clearly distinguished from P450 11β/18-hydroxylase. This study attempted to specifically inhibit adrenal 19-hydroxylation of deoxycorticosterone (DOC) using a suicide aromatase inhibitor, 19-acetylenie androstenedione (19-AA). Purified bovine P450 11β/18/19-hydroxylase was incubated with excess substrate DOC, adrenodoxin, and adrenodoxin reductase in the presence of increasing doses of the inhibitor, 19AA. 11β-, 18-, and 19-hydroxylation were measured by quantification of corticosterone, 18OH-DOC, and 19-OH-DOC respectively. Measurements of these products demonstrated that 11β- and l8-hydroxylation was not inhibited whereas 19-hydroxylation was inhibited as manifested by decreased 19-OH-DOC formation (p <.05). The IC50 of 19-AA was approximately 10-12 M. The specific inhibition of 19hydroxylation suggests that the 19-hydroxylase may be an enzyme distinct from the P450 11β/18-hydroxylase. This further suggests that 19-nor-DOC biosynthesis may be under independent regulation and may be amenable to specific in vivo inhibition.

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.

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.

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).

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.

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.

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.

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.

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).

We describe a method for the differentiation of 3iron 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.

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.

A cytochrome P-450 (P-450,), active in the removal of the side chain from cholesterol to form pregnenolone, was isolated from bovine adrenocortical mitochondria in a cholesterol-containing (P-450a,,(CH)) and a steroidfree (P-45OeC,(SF)) form with a specific heme content of 9 2 1 nmol/mg of protein.Oxidized P-45OaCc(CH) forms a 1:l complex with oxidized adrenodoxin, as monitored by an increase in the amount of high spin heme as well as a decrease in the reduction potential of the minority low spin heme species upon adrenodoxin binding.Data are presented which make unlikely the presence of a tightly bound ternary complex between adrenodoxin reductase, adrenodoxin, and P-45OS,,(CH), at least when all three are oxidized; the stoichiometry determined is 1:2:1, implying either a quaternary complex or two binary complexes.Reduction potentials were measured for both P-45OS,(CH) and P-45OS,,(SF) as isolated and as complexed to steroid substrate, intermediates, and product of the cholesterol desmolase reaction.P-450,,(SF) is predominantly low spin with a reduction potential of -412 a 2 mV for this low spin form.The addition of cholesterol causes P-45OS,,(SF) to become predominantly high spin with a reduction potential of -305 1 mV for this high spin form; both properties are the same as for the protein isolated with cholesterol bound to it, i.e.P-450,,(CH).The reduction potentials of P-450a,,(CH) bound to any two sequential intermediates in the side chain cleavage reaction are the same within experimental error.However, there. is a slight but significant increase (10 to 30 mV) in reduction potential of the P-45OS,, complex with the final intermediate 20,22-dihydroxycholesterol over that of the P-450,,, complex with the substrate cholesterol.The reduction potential of the P-450,,, complex with the product pregnenolone is -30 mV more negative than that of the P-450.,,complex with substrate.

EPR spectroscopic and chemical analyses of spinach nitrite reductase show that the enzyme contains one reducible iron-sulfur center, and one site for binding either cyanide or nitrite, per siroheme. The heme is nearly all in the high spin ferric state in the enzyme as isolated. The extinction coefficient of the enzyme has been revised to E386 = 7.6 X 10(4) cm-1 (M heme)-1. The iron-sulfur center is reduced with difficulty by agents such as reduced methyl viologen (equilibrated with 1 atm of H2 at pH 7.7 in the presence of hydrogenase) or dithionite. Complexation of the enzyme with CO (a known ligand for nitrite reductase heme) markedly increases the reducibility of the iron-sulfur center. New chemical analyses and reinterpretation of previous data show that the enzyme contains 6 mol of iron and 4 mol of acid-labile S2-/mol of siroheme. The EPR spectrum of reduced nitrite reductase in 80% dimethyl sulfoxide establishes clearly that the enzyme contains a tetranuclear iron-sulfur (Fe4S4) center. The ferriheme and Fe4S4 centers are reduced at similar rates (k = 3 to 4 s-1) by dithionite. The dithionite-reduced Fe4S4 center is rapidly (k = 100 s-1) reoxidized by nitrite. These results indicate a role for the Fe4S4 center in catalysis.

This chapter describes the detection and quantitation of free cytochrome P-450 and cytochrome P-450 complexes by electron paramagnetic resonance (EPR) spectroscopy. Cytochromes P-450 in the oxidized state exhibit EPR spectra at low temperature, both in the free form and when bound to substrates, intermediates and products, or certain inhibitors. The EPR spectra fall into two categories, those arising from a high-spin heme species with absorption maxima at approximately g = 8, 4, and 1.8 and those arising from a low-spin heme species (S = 1/2) with absorption maxima at approximately g = 2.4, 2.25, and 1.9; which form is observed depends on the state of ligation of the cytochrome. EPR spectra of high-spin P-450 are not generally observed above 20°K. This species is not easily saturable as the low-spin form, and powers in the mW region may often be used. There is an additional problem related to the zerofield splitting in the case of high-spin heme as opposed to low-spin heme.