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The neurosteroid pregnenolone sulfate (PREGS), which is synthesized in glial cells, plays a significant role in learning and memory performance. The aim of this study was to investigate the regulation of expression of the steroid sulfotransferase SULT2B1a, which catalyzes the conversion of pregnenolone to PREGS, using the rat C6 glioma cell line. Rat C6 glioma cells expressed the SULT2B1a isoform, which sulfonates pregnenolone, but, neither the SULT2B1b isoform, which catalyzes cholesterol, nor the prototypical steroid sulfotransferase SULT2A1 were expressed in these cells. Increasing concentrations of l-glutamic acid in the presence of cyclothiazide, which prevents AMPA receptor desensitization, attenuated SULT2B1a mRNA expression; however, neither NMDA nor kainic acid had a significant effect. Exposure to the synthetic glutamate analogue alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) in the presence of cyclothiazide also inhibited SULT2B1a expression. Attenuation of SULT2B1a expression by L-glutamic acid was reversed by the selective AMPA/kainate receptor antagonist 2,3-dioxo-6-nitro-7-sulfamoylbenzo(f)quinoxaline (NBQX), and partially reversed by the specific neuronal nitric oxide synthase (NOS) inhibitor 7-nitroindazole (7-NI). Induction of inducible NOS by TNF-alpha in combination with lipopolysaccharide (LPS) dramatically attenuated SULT2B1a expression; this was partially reversed by the specific inducible NOS inhibitor N(6)-(1-iminoethyl)-L-lysine hydrochloride (L-NIL). Furthermore, exposure to exogenous NO donors inhibited SULT2B1a mRNA expression, and exposure to sodium nitroprusside, LPS/TNF-alpha and L-glutamic acid in combination with cyclothiazide increased the production of nitrite, a stable degradation product of NO. These findings suggest that expression of SULT2B1a, which catalyzes PREGS production, is inhibited by activation of excitatory amino acid receptors of the AMPA subtype, via facilitation of intracellular NO signaling.

Oxysterols constitute a class of cholesterol derivatives that exhibit broad biological effects ranging from cytotoxicity to regulation of nuclear receptors. The role of oxysterols such as 7-ketocholesterol (7-KC) in the development of retinal macular degeneration and atheromatous lesions is of particular interest, but little is known of their metabolic fate. We establish that the steroid/sterol sulfotransferase SULT2B1b, known to efficiently sulfonate cholesterol, also effectively sulfonates a variety of oxysterols, including 7-KC. The cytotoxic effect of 7-KC on 293T cells was attenuated when these cells, which do not express SULT2B1b, were transfected with SULT2B1b cDNA. Importantly, protection from 7-KC-induced loss of cell viability with transfection correlated with the synthesis of SULT2B1b protein and the production of the 7-KC sulfoconjugate (7-KCS). Moreover, when 7-KCS was added to the culture medium of 293T cells in amounts equimolar to 7-KC, no loss of cell viability occurred. Additionally, MCF-7 cells, which highly express SULT2B1b, were significantly more resistant to the cytotoxic effect of 7-KC. We extended the range of oxysterol substrates for SULT2B1b to include 7alpha/7beta-hydroxycholesterol and 5alpha,6alpha/5beta,6beta-epoxycholesterol as well as the 7alpha-hydroperoxide derivative of cholesterol. Thus, SULT2B1b, by acting on a variety of oxysterols, offers a potential pathway for modulating in vivo the injurious effects of these compounds.

Sterol 27-hydroxylase (CYP27A1) is a mitochondrial P-450 enzyme with broad substrate specificity for C27 sterols including 7-ketocholesterol (7kCh). CYP27A1 is widely expressed in human tissues but has not been previously demonstrated in the retina. In this study, we examined the expression and localization of CYP27A1 in the monkey retina where it localized mainly to the photoreceptor inner segments. CYP27A1 was also observed in Müller cells with faint immuno staining detected in the RPE and choriocapillaris. We also determined that the 27-hydroxylation of 7-ketocholesterol (27OH7kCh) rendered it non-toxic to cultured RPE cells. Moreover, the 27OH7kCh when mixed with 7-ketocholesterol significantly reduced the toxicity of 7-ketocholesterol. These data, when taken in context of the known functions of CYP27A1 imply that expression in the retina serves to modify the biological activity of oxidized sterols that are either transported or generated locally by photo-oxidation. Published by Elsevier Ltd.

cDNAs for rat SULT2B1 steroid/sterol sulfotransferase isoforms were cloned, and the encoded proteins overexpressed, purified and characterized. The rat SULT2B1a isoform avidly sulfonates pregnenolone but poorly utilizes cholesterol as a substrate, whereas cholesterol is more efficiently sulfonated than pregnenolone by the SULT2B1b isoform; on the other hand, neither isoform sulfonates dehydroepiandrosterone to any significant degree. Real-time PCR revealed that SULT2B1a was only expressed in brain and testis, whereas SULT2B1b was mainly expressed in skin, intestine and kidney. The SULT2B1 gene is unique among steroid/sterol sulfotransferase genes in that it encodes for two isoforms as a result of an alternative exon I. Interestingly, whereas the orthologous human and mouse SULT2B1 gene structures are identical, the rat SULT2B1 gene structure diverges. Similar to human and mouse SULT2B1 genes the rat SULT2B1 gene consists of an alternative exon I; however, as a result of exonic rearrangement, the genic locations of exons IA and IB are reversed in the rat gene. Where exon IA is located downstream of exon IB in the human and mouse SULT2B1 genes, in the rat SULT2B1 gene exon IA is located upstream of exon IB. Furthermore, unlike the case with human and mouse SULT2B1 genes where differential splicing is necessitated since a portion of exon IA is fused with exon IB to complete the SULT2B1b mRNA, this step is not required with the rat gene.

The steroid/sterol sulfotransferase gene (SULT2B1) encodes for two isozymes of which one (SULT2B1b) sulfonates cholesterol and is selectively expressed in skin. The human SULT2B1 gene contains neither a TATAAA nor a CCAAT motif upstream of the coding region for SULT2B1b; however, this area is GC-rich. Of five Sp1 elements identified two had regulatory activity utilizing immortalized human keratinocytes: one element is located above the ostensible transcription initiation site, whereas the other is located within the 5′-untranslated region of the SULT2B1b mRNA. Sp1 and Sp2 transcription factors identified by supershift analyses induced reporter gene activity, an effect markedly augmented by histone deacetylase inhibition.

Cholesterol sulfate is a highly amphipathic molecule that is present in a relatively high concentration in the epidermis of human skin, particularly in the granular layer. The physiologic significance of this finding, however, is not well-understood. Therefore, we examined expression of the gene encoding for the enzyme that sulfonates cholesterol (SULT2B1b). Of the three enzymes known to sulfonate steroids/sterols, only the SULT2B1b isozyme was detected in cultures of normal human epidermal keratinocytes (NHEK) in response to Ca 2+ -induced terminal differentiation as well as by normal human epidermal tissue. Immunocytochemical analysis of normal skin as well as specific skin disorders was carried out. In normal skin, the expression of SULT2B1b was localized to the granular layer of the epidermis similar to that of filaggrin, an acknowledged late marker of differentiation and in contrast to that of involucrin, an early marker of terminal differentiation, which was expressed throughout the suprabasal region. The confinement of SULT2B1b to the granular layer coincides with this being the area with the highest cholesterol sulfate content suggesting that the physiologic action of cholesterol sulfate is likely carried out in this region of the living epidermis. Additionally, 88% of cholesterol sulfate in NHEK was membrane-associated further suggesting a cellular location for cholesterol sulfate action.

The gene for human hydroxysteroid sulfotransferase (SULT2B1) encodes two peptides, SULT2B1a and SULT2B1b, that differ only at their amino termini. SULT2B1b has a predilection for cholesterol but is also capable of sulfonating pregnenolone, whereas SULT2B1a preferentially sulfonates pregnenolone and only minimally sulfonates cholesterol. We have determined the crystal structure of SULT2B1a and SULT2B1b bound to the substrate donor product 3′-phosphoadenosine 5′-phosphate at 2.9 and 2.4 A, respectively, as well as SULT2B1b in the presence of the acceptor substrate pregnenolone at 2.3 A. These structures reveal a different catalytic binding orientation for the substrate from a previously determined structure of hydroxysteroid sulfotransferase (SULT2A1) binding dehydroepiandrosterone. In addition, the amino-terminal helix comprising residues Asp19 to Lys26, which determines the specificity difference between the SULT2B1 isoforms, becomes ordered upon pregnenolone binding, covering the substrate binding pocket.

The sulfonation of endogenous molecules is a pervasive biological phenomenon that is not always easily understood, and although it is increasingly recognized as a function of fundamental importance, there remain areas in which significant cognizance is still lacking or at most minimal. This is particularly true in the field of endocrinology, in which the sulfoconjugation of hormones is a widespread occurrence that is only partially, if at all, appreciated. In the realm of steroid/sterol sulfoconjugation, the discovery of a novel gene that utilizes an alternative exon 1 to encode for two sulfotransferase isoforms, one of which sulfonates cholesterol and the other pregnenolone, has been an important advance. This is significant because cholesterol sulfate plays a crucial role in physiological systems such as keratinocyte differentiation and development of the skin barrier, and pregnenolone sulfate is now acknowledged as an important neurosteroid. The sulfonation of thyroglobulin and thyroid hormones has been extensively investigated and, although this transformation is better understood, there remain areas of incomplete comprehension. The sulfonation of catecholamines is a prevalent modification that has been extensively studied but, unfortunately, remains poorly understood. The sulfonation of pituitary glycoprotein hormones, especially LH and TSH, does not affect binding to their cognate receptors; however, sulfonation does play an important role in their plasma clearance, which indirectly has a significant effect on biological activity. On the other hand, the sulfonation of distinct neuroendocrine peptides does have a profound influence on receptor binding and, thus, a direct effect on biological activity. The sulfonation of specific extracellular structures plays an essential role in the binding and signaling of a large family of extracellular growth factors. In summary, sulfonation is a ubiquitous posttranslational modification of hormones and extracellular components that can lead to dramatic structural changes in affected molecules, the biological significance of which is now beginning to be appreciated.

As a result of an alternative exon 1, the gene for human hydroxysteroid sulfotransferase (SULTB1) encodes for two peptides differing only at their amino termini. The SULT2B1b isoform preferentially sulfonates cholesterol. Conversely, the SULT2B1a isoform avidly sulfonates pregnenolone but not cholesterol. The outstanding structural feature that distinguishes the SULT2B1 isoforms from the prototypical SULT2A1 isozyme is the presence of extended amino- and carboxyl-terminal ends in the former. Investigating the functional significance of this unique characteristic reveals that removal of 53 amino acids from the relatively long carboxyl-terminal end that is common to both SULT2B1 isoforms has no effect on the catalytic activity of either isoform. On the other hand, removal of 23 amino acids from the amino-terminal end that is unique to SULT2B1b results in loss of cholesterol sulfotransferase activity, whereas removal of 8 amino acids from the amino-terminal end that is unique to SULT2B1a has no effect on pregnenolone sulfotransferase activity. Deletion analysis along with site-directed mutagenesis of SULT2B1b reveal that the amino acid segment 19-23 residues from the amino terminus and particularly isoleucines at positions 21 and 23 are crucial for cholesterol catalysis. In the gene for SULT2B1, exon 1B encodes for only the unique amino-terminal region of SULT2B1b; however, exon 1A encodes for the unique amino-terminal end of SULT2B1a plus an additional 48 amino acids. Thus, if the gene for SULT2B1 employs exon 1B, cholesterol sulfotransferase is synthesized, whereas if exon 1A is used, pregnenolone sulfotransferase is produced.

Sulphonation, a fundamental process essential for normal growth and development, requires the sulphonate donor molecule 3′-phosphoadenosine 5′-phosphosulphate (PAPS), which is produced from ATP and inorganic sulphate by the bifunctional enzyme PAPS synthase. In humans, two genes encode isoenzymes that are 77% identical at the amino acid level, and alternative splicing creates two subtypes of PAPS synthase 2. The question as to whether distinctions in amino acid composition are reflected in differences in activity has been examined. The specific activity of the PAPS synthase 2 subtypes is 10- to 15-fold higher than that for PAPS synthase 1. The greater catalytic efficiency of the PAPS synthase 2 subtypes is demonstrated further by the 3- to 6-fold higher kcat/Km ratios for ATP and inorganic sulphate as compared with the ratios for PAPS synthase 1. In humans, PAPS synthase 1 is expressed ubiquitously, and is the dominant isoform in most tissues, whereas expression of the PAPS synthase 2 subtypes is variable and tissue-specific. It is noteworthy that, similar to other human tissues, PAPS synthase 1 also appears to be the dominant isoform expressed in cartilage. The latter finding initially created a conundrum, since there is a specific human dwarfing disorder that is known to be caused by a mutation in the PAPS synthase 2 gene. This apparent enigma would seem to be resolved by examination of cartilage from guinea-pigs as an animal model. Similar to humans, cartilage from mature animals predominantly expresses PAPS synthase 1. In contrast, expression of PAPS synthase 1 is relatively low in the cartilage of immature guinea-pigs, including the growth plate of long bones, whereas PAPS synthase 2 is the highly expressed isoenzyme.

Sulphonation is a fundamental process that is essential for normal growth and development as well as maintenance of the internal milieu. The universal sulphonate donor molecule essential for all sulphoconjugation reactions is adenosine 3′-phosphate 5′-phosphosulphate (PAPS), which is produced from ATP and inorganic sulphate by the action of bifunctional PAPS synthase. There are two isozymes encoded by genes located on chromosome 4 (PAPS synthase 1) and chromosome 10 (PAPS synthase 2). The promoter for PAPS synthase 2 contains neither a TATAAA nor a CCAAT box, although a consensus initiator motif is present. Three human cell lines were used to examine promoter activity after transfection with various lengths of the 5′-flanking region of the PAPS synthase 2 gene fused to a reporter gene. Proximal promoter activity was located between bp −84 and bp −124 upstream of the purported transcription start site. This region contains two GC/GT boxes that are essential for full promoter activity, as indicated by deletion analysis and supported further by mutagenesis. A nuclear extract of SW13 cells, which highly express PAPS synthase 2, contained proteins that bound to probes possessing promoter-specific GC/GT boxes. Furthermore, the presence of specificity protein (Sp) 1, Sp2 and Sp3 proteins in the nuclear extract was confirmed by supershift analysis. Co-transfection experiments using SL2 cells yielded additional support for the involvement of Sp1 in transcriptional regulation of the PAPS synthase 2 gene; the involvement of Sp2 and/or Sp3 remains to be clarified further.

Sulfonation, which is essential for normal growth, development and maintenance of the internal milieu, requires the universal sulfonate donor molecule 3′-phosphoadenosine 5′-phosphosulfate (PAPS) produced from ATP and inorganic sulfate by two bifunctional PAPS synthase isozymes. The gene for PAPS synthase 1 containing neither a TATA nor a CCAAT box was found to be under the influence of the Sp1 family of transcription factors. Multiple GC/GT boxes are present in the proximal promoter region and deletion analysis implicated their involvement in transcription, a finding supported by mutational analysis of specific GC/GT boxes. Nuclear extract of SW13 cells, which highly express PAPS synthase 1, contains proteins that bind to probes possessing specific GC/GT boxes; furthermore, the presence of Sp1, Sp2, and Sp3 proteins in nuclear extracts was confirmed by supershift analysis. Cotransfection experiments using SL2 cells yielded additional support for the involvement of Sp1 in transcriptional regulation of the PAPS synthase 1 gene; the involvement of Sp2 and/or Sp3 is presently unclear.

Cholesterol sulfate is abundant in the human epidermis and is a putative natural ligand for retinoic acid receptor-related orphan receptor alpha (RORα). Although direct binding of cholesterol sulfate is expected to activate RORα, cholesterol sulfate can also induce RORα expression and increase RORα target gene expression. The purpose of this study was to determine whether cholesterol sulfate induces profilaggrin expression, a precursor of the barrier protein filaggrin in the epidermis, through activation of RORα by directly binding to RORα, or through increased RORα expression. Immunohistochemical and polymerase chain reaction (PCR) analyses showed that RORα was expressed in normal human epidermal keratinocytes (NHEKs) and that its expression increased during keratinocyte differentiation in parallel with that of profilaggrin and cholesterol sulfotransferase, which catalyzes the synthesis of cholesterol sulfate. Exogenous cholesterol sulfate significantly increased both RORα and profilaggrin expression in NHEKs, whereas no effect on profilaggrin expression was observed in cells in which RORα was knocked down with small interfering RNA (siRNA). Additionally, a luciferase reporter gene assay revealed that exogenous RORα dose-dependently increased the activity of the profilaggrin gene promoter even in the absence of cholesterol sulfate, and that this response involves activator protein-1. In conclusion, the results of this study indicate that cholesterol sulfate induces filaggrin expression through increased RORα expression. Further studies are required to fully elucidate the mechanisms involved.

Estrogen sulfotransferase (EST) activity expressed by Chinese hamster ovary (CHO)-K1 cells stably transfected with a plasmid containing a guinea pig EST complementary DNA insert was subjected to biochemical characterization, and the EST protein was further examined by nondenaturing isoelectric focusing and immunoblot analysis. CHO-K1 cells transfected with the same plasmid without the EST complementary DNA insert as well as untransfected CHO-K1 cells did not demonstrate either EST activity or the presence of an immunologically related protein. The EST expressed by the stably transfected CHO-K1 cells was found to manifest Michaelis-Menten kinetics and would use only estrogenic steroids as substrates, whereas other forms of steroids, such as pregnenolone, dehydroepiandrosterone, cortisol, and testosterone, were not acted on. When 17 beta-estradiol was used as a substrate, sulfonation occurred exclusively at the 3 position; 17-sulfonate was not formed. Thus, the expressed EST acted selectively on the 3-hydroxyl group of phenolic steroids. The apparent Km values for estrone, 17 beta-estradiol, and estriol were 60, 70, and 40 nM, respectively. The maximum velocity (Vmax) determinations for estrone and 17 beta-estradiol were equivalent, whereas the Vmax for estriol was reduced by 33%. Of the three estrogens, only 17 beta-estradiol caused substrate inhibition at a high concentration. Steroid sulfonation requires 3'-phosphoadenosine-5'-phosphosulfate (PAPS) as the active sulfonate donor, and the Km value for PAPS was 1.2 microM. In steroid sulfotransferase reactions, two products are formed: the sulfonated steroid product and the desulfonated cofactor, 3'-phosphoadenosine-5'-phosphate (PAP). The sulfonation of 17 beta-estradiol was inhibited by PAP in a dose-dependent manner. In addition, the Km for PAPS was increased by PAP, whereas the Vmax was unaffected, indicating competitive inhibition (Ki, approximately 0.52 microM). The EST protein expressed by the CHO-K1 cell stable transfectants demonstrated a mol wt of 34 kilodaltons, as determined by sodium dodecyl sulfate-gel electrophoresis. Additionally, when the expressed EST protein was subjected to isoelectric focusing, it was found to consist of multiple charge isoforms. These findings are comparable to what has been previously reported for native guinea pig adrenocortical EST. Furthermore, the charge isoform pattern that was demonstrated for the expressed EST was similar to the pattern observed for the native protein.

A novel stereoselective hydroxysteroid sulfotransferase (HST) that acts on neutral steroids having the 3-hydroxyl group in the alpha orientation but not on steroids where the 3-hydroxyl group is oriented in the beta position has been cloned and expressed. Primary screening of a guinea pig adrenal cDNA library was performed by colony hybridization using an oligonucleotide probe based on a highly homologous region among known steroid sulfotransferases. Selected clones consisted of overlapping sequences but were incomplete. The rapid amplification of cDNA ends procedure was used to construct a full-length guinea pig HST cDNA. The guinea pig HST cDNA transiently transfected into Chinese hamster ovary K1 cells expressed a protein identical in size to that of purified guinea pig HST specific for 3 alpha-hydroxylated neutral steroids that was recently reported (Driscoll, W. J., Martin, B. M., Chen, H.-C., and Strott, C. A. (1993) J. Biol. Chem. 268, 23496-23503). The expressed HST likewise exhibited sulfotransferase activity that was directed specifically toward steroid substrates containing a 3-hydroxyl group in the alpha orientation; on the other hand, steroids with a 3 beta-hydroxyl group were not sulfonated by the expressed HST. Thus, the cloned HST cDNA clearly coded for a steroid sulfotransferase with chiral specificity for 3 alpha-hydroxylated neutral steroids and was, therefore, given the designation of guinea pig 3 alpha-hydroxysteroid sulfotransferase (gp3 alpha-HST). The full-length gp3 alpha-HST cDNA consisted of 1182 base pairs and coded for a protein containing 287 amino acids. The deduced amino acid sequence of the protein shares 65/79, 65/80, and 62/76% identity/similarity with rat, mouse, and human HST, respectively. Northern blot analysis of guinea pig tissues revealed no apparent gender differences in either mRNA species or distribution; a single 1.4-kilobase HST mRNA species was present in adrenal and liver tissue. Interestingly, the adrenal mRNA content was considerably more abundant than that found in the liver. Evidence for 3 alpha-HST mRNA was not detected in kidney, heart, lung, muscle, spleen, or uterus.

Two physically distinct hydroxysteroid sulfotransferases (HSTs) that demonstrate substrate specificity with respect to the orientation of the ring A 3-hydroxyl group have been isolated from the guinea pig adrenal gland. Nondenaturing liquid-phase isoelectric focusing permitted the separation of the activities, and reverse phase high performance liquid chromatography was used to purify the two proteins to homogeneity. The 3 beta-HST had an apparent molecular mass of 33 kDa and utilized pregnenolone, 17-hydroxypregnenolone, and dehydroepiandrosterone as substrates. The 3 alpha-HST was slightly smaller at 32 kDa and utilized allopregnanolone and androsterone as substrates. The proteins were further distinguished by isoelectric point, immunoreactivity, and tryptic peptide mapping. Peptides isolated from both guinea pig HSTs demonstrated significant amino acid sequence homology (approximately 65% identity) to rat liver HST; however, available sequence data from the two proteins did not yield differences that might account for their stereospecific substrate selectivity. This paper represents the first definitive report demonstrating the existence of discrete HSTs that exhibit substrate specificity based on the stereochemistry of the 3-hydroxyl group.

This report describes for the first time the cellular and subcellular localization of estrogen sulfotransferase (EST) as well as the subcellular localization of hydroxysteroid sulfotransferase (HST) in the mammalian adrenal cortex. A 34-kilodalton EST and two HSTs with 3 alpha- and 3 beta-hydroxysteroid substrate specificities (32 and 33 kilodaltons, respectively) were previously purified from guinea pig adrenal cortex and characterized. Western blots were used to establish that two antisera generated against EST were highly specific for EST, whereas three antisera generated against the HSTs were highly specific for the HSTs, but did not distinguish between the 3 alpha- and 3 beta HSTs. Light and electron microscopic immunoperoxidase labeling with these antisera revealed that the sulfotransferases were expressed only within the ACTH-responsive layers of the guinea pig adrenal cortex, with EST localized to zona fasciculata and zona reticularis cells, and the HSTs confined to the zona reticularis. No labeling was detected in the zona glomerulosa or adrenal medulla. EST was concentrated in cell nuclei; sparse labeling was distributed throughout the cytoplasm. HST labeling was intense in smooth endoplasmic reticulum of zona reticularis cells, but was absent from nuclei. Ovoid inclusions about 1-4 microns in diameter, with no limiting membrane, were observed in zona reticularis cells; these inclusions were strongly labeled for both EST and HSTs. No gender-specific differences in distribution or labeling intensity were apparent. The high concentration of EST immunoreactivity in nuclei suggests that EST may play a role in modulating the ability of active estrogens to regulate gene expression in ACTH-responsive cells. The distribution of HST labeling suggests that sulfonation of adrenocortical 3-hydroxysteroids takes place largely within smooth endoplasmic reticulum in the zona reticularis in adult guinea pigs.

Complementary DNA for the guinea pig adrenocortical estrogen sulfotransferase (EST) has been cloned and expressed. Oligonucleotides, based on amino acid sequences of the purified 34-kilodalton protein, were synthesized and used to generate a specific probe by polymerase chain reaction for screening a guinea pig adrenal cDNA library. The polymerase chain reaction rapid amplification of cDNA ends procedure was employed to obtain the 3' and 5' cDNA ends, and a full-length cDNA was constructed. The cloned cDNA consists of 1192 base pairs and encodes a protein of 296 amino acids with a calculated molecular mass of 35,161 daltons. A computer search of the protein data banks revealed significant homology with several sulfotransferases: 71% with bovine placental estrogen sulfotransferase, 52% with rat liver phenol sulfotransferase, 35% with rat liver hydroxysteroid sulfotransferase, and 36% with rat liver senescence marker protein 2. The EST cDNA was inserted into the pcDNA I eukaryotic expression vector and transfected into COS-7 cells. The successful expression of EST cDNA in COS-7 cells was ascertained by Western blot analysis using antibody generated against the protein used to obtain the original amino acid sequence. Additionally, the expressed protein was clearly functional. Only after transfection with EST cDNA was there detectable estradiol sulfotransferase activity in COS-7 cell cytosol. The expressed EST had a single pI of 6.4, whereas native guinea pig adrenocortical EST exhibits four primary charge isoforms. The majority of adrenocortical EST activity focuses as a broad bimodal band in the pH range of 6.6-6.2; additionally, three other discrete immunocross-reactive isoforms are present with pIs of 5.5, 5.4, and 5.2. Antibodies generated against each individual isoform cross-react with all the other isoforms and with the expressed protein. These isoforms were previously reported to be isomers of a pregnenolone-binding protein; however it is now evident that the isoforms and antibodies raised against them are EST specific. Under high stringency hybridization conditions, EST mRNA was only detected in the adrenal gland, where two mRNA species of 1.4 and 1.8 kilobases were evident; when low stringency conditions were used, a faint 1.4-kilobase band was also detected in the liver. Primer extension analysis revealed that the multiple mRNAs do not arise from differential transcription initiation sites, and genomic Southern blot analysis indicated that the multiple mRNAs arise from a single gene.

This paper presents data identifying adenosine 3',5'-diphosphate (3',5'-ADP) as the small heat-stable factor essential for the active steroid binding complex of the adrenocortical pregnenolone-binding protein (PBP). Factor activity obtained from the boiled supernatant of partially purified PBP was isolated by high performance liquid chromatography using weak anion-exchange and hydrophobic (C18) chromatography sequentially. The purified material retained characteristic factor activity and presented a UV spectrum identical to that for authentic 3',5'-ADP. Mass spectroscopic analysis of the isolated factor revealed an M-H ion of appropriate mass (m/z = 426) and a decomposition pattern for the M-H ion that was consistent with the structure of 3',5'-ADP. The studies presented here demonstrate that authentic 3',5'-ADP can categorically substitute for factor prepared from the soluble fraction of the guinea pig adrenal. Specifically, 3',5'-ADP potentiated ligand binding of partially purified native PBP and restored binding capacity to alkaline phosphatase-inactivated PBP in a dose-dependent manner. As is the case for adrenocortical factor activity, these effects were negated by pretreating the 3',5'-ADP with calf intestinal alkaline phosphatase. Other nucleotides similarly tested, including ADP isomers, were ineffective as factor substitutes. The sulfated form of 3',5'-ADP (i.e. 3'-phosphoadenosine 5'-phosphosulfate) demonstrated some potential for restoring binding capacity to phosphatase-inactivated PBP; however, this compound was clearly inhibitory rather than stimulatory for native PBP activity. Taken collectively, the data overwhelmingly demonstrate that 3',5'-ADP is in fact the molecule required by the PBP for high affinity steroid binding complex formation. It is not yet known whether 3',5'-ADP acts allosterically or contributes directly to the structure of the steroid binding site.

Nuclei of the guinea pig adrenal cortex contain a protein that specifically binds progesterone and that, biochemically, is clearly distinct from the classical progesterone receptor. The adrenocortical nuclear progesterone-binding protein has now been purified more than 2000-fold by steroid-affinity chromatography with a 75% yield. The purified protein preparation demonstrated three major bands on sodium dodecyl sulfate-polyacrylamide gel of 79K, 74K, and 50K. To determine which of the three might represent the progesterone-binding protein, steroid photoaffinity labeling was performed which resulted in the specific and exclusive labeling of a 50K band. Thus, the adrenocortical nuclear progesterone-binding protein appears to be distinct from the classical progesterone receptor not only biochemically, but also on the basis of molecular size. To test whether the adrenocortical nuclear progesterone-binding protein can be hormonally stimulated, guinea pigs were treated with ACTH. The chronic administration of ACTH caused a 4- to 6-fold increase in the specific progesterone binding capacity without a change in the binding affinity. There appeared to be no significant difference in nuclear progesterone binding between the zona fasciculata and zona reticularis. This finding suggests a mediating role for the progesterone-binding protein in ACTH action. In addition, the nuclear progesterone-binding protein bound to nonspecific DNA sequences, further suggesting a possible transcriptional regulatory role.

Progesterone, which is normally produced in the endoplasmic reticulum, was found to be rapidly degraded in the cytosolic fraction of the guinea pig adrenal cortex in vitro. Assuming this finding reflects what happens in vivo raises a question as to the source of progesterone for interacting with a nuclear progesterone-binding protein (P4-BP) that exists in this model system. It was subsequently found that pregnenolone, which in contrast to progesterone is relatively stable in the cytosol, was converted to progesterone by endogenous nuclear 3 beta-ol dehydrogenase. It was also determined that the nuclear-derived progesterone specifically bound to the nuclear P4-BP which is distinct from the classical progesterone receptor. The guinea pig adrenocortical cytosol contains a specific pregnenolone-binding protein (P5-BP) that could be virtue of its pregnenolone binding activity regulate the conversion of pregnenolone to progesterone in the nuclear compartment and thereby reduce the binding of progesterone to the nuclear P4-BP. A partially purified P5-BP preparation markedly inhibited the nuclear conversion of pregnenolone to progesterone and reduced the binding of progesterone to the nuclear P4-BP (P5-BP did not directly inhibit binding of progesterone to the nuclear P4-BP). The ability of P5-BP to inhibit the conversion of pregnenolone to progesterone was destroyed by heat and alkaline phosphatase treatment. The binding of pregnenolone to the P5-BP, as previously reported, is regulated by phosphorylation/dephosphorylation, and alkaline phosphatase-treated P5-BP loses the ability to bind pregnenolone; this process can be reversed by a cytosolic kinase. This provides a mechanism for controlled release of bound steroid. These results suggest that P5-BP regulates the nuclear conversion of pregnenolone to progesterone and thus the binding of progesterone to the nuclear P4-BP.

The pregnenolone-binding protein (PBP) in guinea pig adrenocortical cytosol is inactivated (converted to a nonsteroid-binding form) by incubation with calf intestinal alkaline phosphatase at pH 9. Previously bound pregnenolone does not prevent this inactivation, and dephosphorylation causes dissociation of bound ligand from the protein. Cytosolic PBP, partially purified PBP, and highly purified PBP are equally susceptible to alkaline phosphatase-mediated inactivation. No change in apparent molecular weight or immunoreactivity is evident by Western blot analysis. Loss of pregnenolone-binding capacity of cytosolic PBP (but not partially purified PBP) could be reversed by inhibiting the phosphatase, lowering the pH to approximately 7, and adding ATP to the incubation. Reactivation is absolutely and specifically dependent upon ATP, which restores binding capacity in a concentration-dependent manner. Other nucleoside triphosphates, including the nonhydrolyzable ATP analogue adenosine 5'-(beta, gamma-imido)triphosphate, as well as cAMP and cGMP are ineffectual as cofactors for reactivation. These data strongly implicate a cytosolic kinase which is apparently inactivated or separated from PBP during purification. Preliminary investigations indicate that the reactivating kinase is not cAMP-dependent, but may have a requirement for calcium and/or calmodulin. The identification of phosphorylation/dephosphorylation as the regulatory mechanism for steroid binding should prove pivital in elucidating the functional role of PBP.

Background— Cholesterol sulfate, the most important sterol sulfate in the human circulation, has emerged as a multifaceted molecule. Among its many demonstrated regulatory actions is its ability to influence blood clotting and fibrinolysis. Additionally, cholesterol sulfate is a constituent of human platelets, where it has been shown to support platelet aggregation. Methods and Results— We have documented the presence of the enzyme (SULT2B1b) that sulfonates cholesterol in human platelets and examined the influence of plasma lipoproteins on the expression and activity of this enzyme. SULT2B1b mRNA was detected by reverse transcription-polymerase chain reaction and found to be the only steroid/sterol sulfotransferase expressed in these discoid anucleate particles. Using real-time polymerase chain reaction for quantification, we found that the level of SULT2B1b mRNA in platelets was maintained at 4°C but substantially diminished over a period of 4 hours at 37°C. The loss of SULT2B1b mRNA, however, was markedly reduced in the presence of HDL but not LDL. The stabilizing influence of HDL was attributable specifically to its apolipoprotein (apo) A-I component, whereas apoA-II and apoE were without effect. Importantly, there was a direct correlation between platelet SULT2B1b mRNA and protein levels in the presence or absence of lipoprotein that was reflected in enzymatic activity and cholesterol sulfate production. Conclusions— Human platelets selectively express SULT2B1b, the physiological cholesterol sulfotransferase. Furthermore, the stability of SULT2B1b mRNA and protein in platelets maintained at 37°C is subject to regulation by the apoA-I component of HDL.

The gene for human hydroxysteroid sulfotransferase (SULT2B1) encodes two peptides, SULT2B1a and SULT2B1b, that differ only at their amino termini. SULT2B1b has a predilection for cholesterol but is also capable of sulfonating pregnenolone, whereas SULT2B1a preferentially sulfonates pregnenolone and only minimally sulfonates cholesterol. We have determined the crystal structure of SULT2B1a and SULT2B1b bound to the substrate donor product 3'-phosphoadenosine 5'-phosphate at 2.9 and 2.4 A, respectively, as well as SULT2B1b in the presence of the acceptor substrate pregnenolone at 2.3 A. These structures reveal a different catalytic binding orientation for the substrate from a previously determined structure of hydroxysteroid sulfotransferase (SULT2A1) binding dehydroepiandrosterone. In addition, the amino-terminal helix comprising residues Asp19 to Lys26, which determines the specificity difference between the SULT2B1 isoforms, becomes ordered upon pregnenolone binding, covering the substrate binding pocket.

Cholesterol sulfate is quantitatively the most important known sterol sulfate in human plasma, where it is present in a concentration that overlaps that of the other abundant circulating steroid sulfate, dehydroepiandrosterone (DHEA) sulfate. Although these sulfolipids have similar production and metabolic clearance rates, they arise from distinct sources and are metabolized by different pathways. While the function of DHEA sulfate remains an enigma, cholesterol sulfate has emerged as an important regulatory molecule. Cholesterol sulfate is a component of cell membranes where it has a stabilizing role, e.g., protecting erythrocytes from osmotic lysis and regulating sperm capacitation. It is present in platelet membranes where it supports platelet adhesion. Cholesterol sulfate can regulate the activity of serine proteases, e.g., those involved in blood clotting, fibrinolysis, and epidermal cell adhesion. As a result of its ability to regulate the activity of selective protein kinase C isoforms and modulate the specificity of phosphatidylinositol 3-kinase, cholesterol sulfate is involved in signal transduction. Cholesterol sulfate functions in keratinocyte differentiation, inducing genes that encode for key components involved in development of the barrier. The accumulating evidence demonstrating a regulatory function for cholesterol sulfate appears solid; the challenge now is to work out the molecular mechanisms whereby this interesting molecule carries out its various roles.

A novel mouse hydroxysteroid sulfotransferase cDNA has been cloned, and organization of its gene structure has been determined. The new mouse sulfotransferase, SULT2B1a, and its closely related isoform, SULT2B1b, are derived from a single SULT2B1 gene as a result of an alternative exon I and differential splicing. Thus, the only structural distinction between the two SULT2B1 isoforms is at their amino-terminal ends. Importantly, in contrast to the prototypical mouse hydroxysteroid sulfotransferase SULT2A1, the SULT2B1 isoforms have a predilection for cholesterol. Real-time RT-PCR reveals that the SULT2B1a isoform is most abundantly expressed in the brain and spinal cord, whereas SULT2B1b and SULT2A1 are weakly, if at all, expressed in the central nervous system. On the other hand, the SULT2B1b isoform is the most prominent hydroxysteroid sulfotransferase expressed in skin, whereas SULT2A1 is strikingly expressed in the liver. The substrate specificities and differential expression patterns of the three SULT2 isozymes strongly suggest that they have distinct biologic roles to play. Of further interest, the mouse SULT2B1 and SULT2A1 genes are differentially expressed during embryonic development, with the former being expressed at all stages from E8.5-E19, whereas the latter is not expressed until E19. It is speculated that, during embryonic development, SULT2B1b is required for production of cholesterol sulfate essential for normal skin development, whereas SULT2B1a produces pregnenolone sulfate, an essential neurosteroid during development of the central nervous system.

In humans, the biotransformation of cholesterol and its hydroxylated metabolites (oxysterols) by sulfonation is a fundamental process of great importance. Nevertheless, the sulfotransferase enzyme(s) that carries out this function has never been clearly identified. Cholesterol is a relatively poor substrate for the previously cloned hydroxysteroid sulfotransferase (HST), i.e. dehydroepiandrosterone (DHEA) sulfotransferase (HST1). Recently, cloning of a single human gene that encodes for two proteins related to HST1 was reported. These newly cloned sulfotransferases (HST2a and HST2b), while exhibiting sequence similarity to other members of the soluble sulfotransferase superfamily, also contain unique structural features. This latter aspect prompted an examination of their substrate specificity for comparison with HST1. Thus, HST1, HST2a, and HST2b were overexpressed as fusion proteins and purified. Furthermore, a novel procedure for the isolation of cholesterol and oxysterol sulfonates was developed that was used in association with HPLC to resolve specific sterol sulfonates. HST1 preferentially sulfonated DHEA and, to a lesser extent, oxysterols; whereas cholesterol was a negligible substrate. The reverse, however, was the case for the HST2 isoforms, particularly HST2b, which preferentially sulfonated cholesterol and oxysterols, in contrast to DHEA, which served as a poor substrate for this enzyme. RT-PCR analysis revealed distinct patterns of HST1, HST2a, and HST2b expression. It was particularly notable that both HST2 isoforms, but not HST1, were expressed in skin, a tissue where cholesterol sulfonation plays an important role in normal development of the skin barrier. In conclusion, substrate specificity and tissue distribution studies strongly suggest that HST2a and HST2b, in contrast to HST1, represent normal human cholesterol and oxysterol sulfotransferases. Furthermore, this study represents the first example of the sulfonation of oxysterols by a specific human HST.

During the course of isolating, characterizing, and cloning estrogen and 3-hydroxysteroid sulfotransferases from the guinea pig adrenal gland, it was noted that cytosolic preparations from this tissue would also sulfonate testosterone. Therefore, we set out to isolate and clone the enzyme that performs this reaction. Testosterone sulfotransferase (TST) was isolated from the guinea pig adrenal by using the standard procedures of ion exchange, affinity, and high-performance liquid chromatography. When purified, TST was examined by liquid-phase nondenaturing isoelectric focusing, it was found that the TST activity profile completely overlapped with the activity profile of the 3alpha-hydroxysteroid sulfotransferase (3alphaHST) isoform, but not the 3beta-hydroxysteroid sulfotransferase (3betaHST) isoform. This finding was further investigated by overexpressing the cDNAs for 3alphaHST and 3betaHST in Escherichia coli and examining the expressed proteins for TST activity. This experiment confirmed that 3alphaHST does indeed function as a TST. In addition, 3alphaHST was also found to sulfonate estradiol but not estrone, a finding that further suggested that 3alphaHST may function as a general 17beta-hydroxysteroid sulfotransferase.

The 3-hydroxysteroid sulfotransferases that have been isolated and cloned from humans and rodents appear to have broad substrate specificities. In the guinea pig, however, two 3-hydroxysteroid sulfotransferases have been isolated that function according to an innate stereospecificity: the alpha-isoform acts on steroids with a 3-hydroxyl group oriented in the alpha position, whereas the beta-isoform acts on steroids where the 3-hydroxyl group is in a beta orientation. To examine the structural basis for this remarkable stereoselectivity, chimeras of the two enzymes, which are 87% identical, were constructed. A chimera consisting of the NH(2)-terminal 91 amino acids of the alpha-isoform and the COOH-terminal 196 amino acids of the beta-isoform displayed activity similar to that of the alpha-isoform. Site-selected mutagenesis of this 3alpha/beta-hydroxysteroid sulfotransferase chimera involving the 12 amino acid differences that exist between the two isoforms within the 91 amino acid NH(2)-terminal region revealed that the amino acid residue at position 51 plays a fundamental role in determining the stereospecificity exhibited by the alpha- and beta-isoforms, i.e. if residue 51 is an asparagine, alpha activity predominates, whereas if an isoleucine is in that position, beta activity prevails.

3'-Phosphoadenosine-5'-phosphosulfate (PAPS) synthase is a bifunctional protein consisting of an NH2-terminal APS kinase and a COOH-terminal ATP sulfurylase. Both catalytic activities require ATP; the APS kinase domain involves cleavage of the beta-gamma phosphodiester bond of ATP, whereas the ATP sulfurylase domain involves cleavage of the alpha-beta phosphodiester bond of ATP. Previous mutational studies have suggested that beta-gamma phosphodiesterase activity involves a highly conserved NTP-binding P-loop motif located in the adenosine-5'-phosphosulfate kinase domain of PAPS synthases. Sequence alignment analysis of PAPS synthases and the superfamily of TagD-related nucleotidylyltransferases revealed the presence of a highly conserved HXGH motif in the ATP sulfurylase domain of PAPS synthases, a motif implicated in the alpha-beta phosphodiesterase activity of cytidylyltransferases. Thus, site-selected mutagenesis of the HXGH motif in the ATP sulfurylase domain of human PAPS synthase (amino acids 425-428) was performed to examine this possibility. Either H425A or H428A mutation produced an inactive enzyme. In contrast, a N426K mutation resulted in increased enzymatic activity. A G427A single mutant resulted in only a modest 30% reduction in catalytic activity, whereas a G427A/H428A double mutant produced an inactive enzyme. These results suggest an important role for the HXGH histidines in the ATP sulfurylase activity of bifunctional PAPS synthase and support the hypothesis that the highly conserved HXGH motif found in the ATP sulfurylase domain of PAPS synthases is involved in ATP binding and alpha-beta phosphodiesterase activity.

Inhibition of protein synthesis blocks ACTH-stimulated steroidogenesis. Researchers have proposed that a "regulatory" protein functions at an intramitochondrial site to promote the translocation of cholesterol substrate from the outer to the inner membrane where the rate-limiting P-450 scc enzyme complex is located. To date three different proteins have been put forth as a "regulatory" protein. These are structurally distinct, yet appear to perform the same in vitro function; they are widely distributed, and are normally involved in disparate activities unrelated to steroidogenesis. Thus, it is difficult to understand how a physiological role for the three proteins in ACTH-stimulated steroidogenesis might exist.

The universal sulfonate donor, 3′-phosphoadenosine 5′-phosphosulfate (PAPS), is synthesized by the concerted action of ATP sulfurylase and adenosine 5′-phosphosulfate (APS) kinase, which in animals are fused into a bifunctional protein. The cDNA for human PAPS synthase (hPAPSS) along with polymerase chain reaction products corresponding to several NH2- and COOH-terminal fragments were cloned and expressed in COS-1 cells. A 1–268-amino acid fragment expressed APS kinase activity, whereas a 220–623 fragment evinced ATP sulfurylase activity. The 1–268 fragment and full-length hPAPSS (1–623) exhibited hyperbolic responses against APS substrate with equivalent K m values (0.6 and 0.4 μm, respectively). The 1–268 fragment demonstrated Michaelis-Menten kinetics against ATP as substrate (K m 0.26 mm); however, full-length hPAPSS exhibited a sigmoidal response (apparent K m1.5 mm) suggesting cooperative binding. Catalytic efficiency (V max/K m) of the 1–268 fragment was 64-fold higher than full-length hPAPSS for ATP. The kinetic data suggest that the COOH-terminal domain of hPAPSS exerts a regulatory role over APS kinase activity located in the NH2-terminal domain of this bifunctional protein. In addition, the 1–268 fragment and full-length hPAPSS were overexpressed in Escherichia coli and column purified. Purified full-length hPAPSS, in contrast to the COS-1 cell-expressed cDNA construct, exhibited a hyperbolic response curve against ATP suggesting that hPAPSS is perhaps modified in vivo.

A guinea pig adrenal hydroxysteroid sulfotransferase (gpHST2) has been cloned that is distinct from guinea pig hydroxysteroid sulfotransferase that stereoselectively acts on 3 alpha-hydroxylated neutral steroids (gp3 alpha HST, redesignated gpHST1). The deduced amino acid sequences for gpHST1 and gpHST2 are 86% identical; however, whereas gpHST1 selectively acts on 3 alpha-hydroxylated steroids, gpHST2 demonstrates a clear preference (but not exclusive specificity) for 3 beta-hydroxylated steroids suggesting that gpHST2 is similar to a previously reported guinea pig hydroxysteroid sulfotransferase that selectively acts on 3 beta-hydroxylated neutral steroids (gp3 beta HST). Additionally, gpHST2 (33K) is the same size as gp3 beta HST and larger than gpHST1 (32K), contains amino acid sequences identical to peptides obtained from gp3 beta HST and cross-reacts with antibodies raised against purified gp3 beta HST. Nonetheless, gpHST2 can sulfonate both 3 alpha- and 3 beta-hydroxylated neutral steroids, suggesting that either gp3 beta HST does not have the exquisite stereoselectivity previously indicated or this subfamily of hydroxysteroid sulfotransferases is larger than originally thought.

Point mutations were selectively introduced into a cDNA for guinea pig estrogen sulfotransferase (gpEST); each construct was then expressed in Chinese hamster ovary K1 cells. The molecular site chosen for study is a conserved GXXGXXK sequence that resembles the P-loop-type nucleotide-binding motif for ATP- and GTP-binding proteins and is located near the C terminus of all steroid and phenol(aryl) sulfotransferases for which the primary structures are known. Preliminary experiments demonstrated that the GXXGXXK motif is essential for binding the activated sulfonate donor 3'-phosphoadenosine 5'-phosphosulfate (PAPS). The present study was undertaken to ascertain the relative importance of each individual residue of the motif. While the mutation of a single motif residue had little effect on the interaction between gpEST and PAPS as determined by kinetic analysis and photoaffinity labeling, the mutation of any two residues in concert resulted in an approximate 10-fold increase in the Km for PAPS and reduced photoaffinity labeling. The mutation of all three motif residues resulted in an inactive enzyme and complete loss of photoaffinity labeling. Interestingly, several mutants also displayed a striking effect on the Km for the steroid substrate; double mutants, again, demonstrated greater perturbations (8- to 28-fold increase) than did single mutants. Unexpectedly, whereas the mutation of nonmotif residues had a negligible effect on the Km for PAPS, a marked increase in the Km for the estrogen substrate ( > 30-fold) was noted. On the basis of these findings, it is concluded that the sequence GISGDWKN within the C-terminal domain of gpEST represents a critical component of the active site.

Cytosol prepared from Chinese hamster ovary (CHO)-K1 cells transfected with guinea pig estrogen sulfotransferase (EST) cDNA demonstrated high affinity binding activity for pregnenolone as well as 17 beta-estradiol but failed to bind dehydroepiandrosterone or testosterone. In contrast, cytosol prepared from nontransfected CHO-K1 cells did not demonstrate steroid binding activity. Additionally, the binding activity for pregnenolone and 17 beta-estradiol was dependent on the presence of the cofactor adenosine-3',5'-diphosphate. Pregnenolone and 17 beta-estradiol effectively competed with each other for binding. On the other hand, pregnenolone, which was not sulfonated, did not inhibit the sulfonation of 17 beta-estradiol by expressed EST.

Estrogen sulfotransferase (EST) purified from the guinea pig (gp) adrenal gland consists of multiple charge isoforms with isoelectric points (pls) ranging from 6.5 to 5.2. Four individual isoforms were isolated for use in antibody production, as well as for tryptic fragmentation analysis. The multiple charge isoforms manifested a high degree of relatedness as evidence by complete immunocross-reactivity. This relatedness was further demonstrated by peptide mapping analysis, which revealed essentially identical elution profiles for three of the isoforms, whereas the fourth most acidic isoform, although similar to the other three isoforms, demonstrated some differences. To further explore the nature of the charge isoforms, native gpEST and EST expressed by CHO-K1 cells transfected with the gpEST cDNA were subjected to additional biochemical characterization. An interesting finding was that, in addition to EST activity, gpEST bound estradiol with a high affinity [dissociation constant (Kd) 10 nM]; furthermore, the binding activity was absolutely dependent on the cofactor, adenosine-3',5'-diphosphate (3',5'-ADP). An unexpected and novel finding was that only the most basic pl 6.5 isoform was catalytically active, while estradiol-binding activity was associated with a more acidic isoform (pl 5.5-5.2); however, it has not as yet been possible to definitively assign the binding activity to a specific isoform.(ABSTRACT TRUNCATED AT 250 WORDS)

The steroid-binding capacity of the adrenocortical pregnenolone-binding protein (PBP) is effectively destroyed by extreme temperature (boiling water for 2-5 min); however, the boiled preparation contains a factor that potentiates ligand binding when readded to native PBP. Treatment of the boiled fraction with calf intestinal alkaline phosphatase at pH 9 reverses the stimulatory effect on PBP activity. Additionally, if native PBP is first incubated with alkaline phosphatase, which converts it to a nonbinding form, activity can be fully restored in a dose-dependent manner by the addition of the boiled preparation. The factor (itself devoid of binding capacity) can also be generated by exposing native PBP to acidic conditions (pH 4). The molecule is small (mol wt, less than 2000), as judged by Sephadex G-25 gel filtration and equilibrium dialysis. It is not retained on Concanavalin-A-Sepharose and is not extractable with a variety of organic solvents. The factor remains active after lyophilization and has a net negative charge at pH 7.4 (determined by DEAE-cellulose chromatography). While the binding capacity of native PBP is destroyed by a variety of proteases, the heat-stable factor is unaffected by similar treatment. Additionally, factor activity is not susceptible to RNase, DNase, or lipase digestion. Thus, the protein moiety of the PBP has an absolute requirement for a distinct phosphorylated heat-stable factor for expression of ligand-binding activity, and it may be through this factor that binding activity is regulated. It is not yet known whether the factor is acting allosterically or actually functions as part of the steroid-binding site.

A radioimmunoassay for 17-OH-pregnenolone was developed and applied to the study of this steroid in human plasma. Steroids that cross-react with the antiserum could be readily separated from 17-OH-pregnenolone; therefore the assay was highly specific. In normal subjects plasma 17-OH-pregnenolone is mainly of adrenal origin. It was largely suppressible with dexamethasone, readily responsive to ACTH and followed a diurnal rhythm similar to that of plasma fluorogenic corticosteroids with which there was a significant correlation (p < 0.001). Testicular secretion of 17-OH-pregnenolone could be inferred from the observation that while dexamethasone suppressed plasma concentrations incompletely combined treatments with dexamethasone and methyltestosterone caused the steroid to disappear completely. In contrast, women receiving dexamethasone alone had similar levels of 17-OH-pregnenolone to women receiving dexamethasone plus estrogen. Ovarian secretion of 17-OH-pregnenolone was suggested by the finding ...

The role of cytoplasmic microtubules in the process of steroidogenesis was examined in enzyme-dispersed and mechanically dispersed adrenocortical cells obtained from adult male rats. These cells were maintained in culture for periods of 2 h to 2 days and tested with various antimicrotubular drugs as well as ACTH and N6,O2-dibutyryl adenosine 3',5'-monophosphoric acid [(Bu)2cAMP]. The drugs tested, viz. colchicine, vinblastine, and nocodazole, were found to stimulate steroid synthesis in a dose-dependent fashion; the maximal degree of stimulation was found to be the same as that obtained with either ACTH or (Bu)2cAMP. When a maximal concentration of an antimicrotubular drug was mixed with a maximal concentration of either (Bu)2cAMP or ACTH, no additive effect on steroidogenesis was observed. The mixture of maximal concentrations of two antimicrotubular drugs produced a steroidogenic response that was less than when either drug was used alone. However, when submaximal concentrations of two antimicrotubular drugs were mixed, a synergistic response was noted. These antimicrotubular drugs are known to interfere with microtubular function by binding to specific sites on the tubulin protomer and inhibiting normal polymerization. Lumicolchicine, the photoinactivated form of colchicine, does not bind to tubulin and did not stimulate steroid synthesis. These studies support the notion that microtubules can influence the process of steroidogenesis.

The guinea pig adrenal cortex consists of a steroidogenic ACTH-responsive outer zone and an ACTH-unresponsive inner zone. It has been suggested that calmodulin plays an important role in ACTH-stimulated steroidogenesis. Thus, in an effort to examine the calmodulin 'system' in the guinea pig adrenal cortex model, Ca2+-dependent binding of calmodulin to proteins in subcellular fractions of the outer and inner zones was examined by the [125I]iodocalmodulin overlay technique and compared to similar studies utilizing pancreas, brain and liver tissue. Although the general pattern of calmodulin-binding proteins was similar for the two adrenocortical zones, quantitatively there was a striking difference with greater binding in the outer zone; this was particularly noteworthy for the mitochondrial fraction. The two most prominent calmodulin-binding proteins isolated from cytosol by calmodulin-Sepharose column chromatography had Mr of 60,000 and 47,000. The size of these two proteins suggested the presence of Ca2+/calmodulin-dependent protein kinase II. Western blot analysis, however, failed to demonstrate calmodulin kinase II in either zone, although it was clearly detectable in brain cytosol. The 60 K calmodulin-binding protein in the adrenal cortex also suggested the presence of the calmodulin-binding A subunit of the Ca2+/calmodulin-stimulated protein phosphatase, calcineurin. Western blot analysis did reveal the presence of calcineurin in the outer adrenocortical zone; it was not detectable, however, in the inner adrenocortical zone. The relation between the striking zonal differential for calmodulin-binding proteins and the zonal differential in ACTH-stimulated steroidogenesis in the guinea pig adrenal cortex will require further investigation.

The in vitro metabolism of progesterone was studied using the chick oviduct, a known target tissue for progesterone action. Tissue was pulsed with 3H-progesterone at 0 C and then incubated at 37 C for varying periods of time. Following incubation, the tissue was homogenized and subjected to differential centrifugation. Cytosol and nuclear protein preparations were analyzed by density gradient centrifugation which separated specific and nonspecific progesterone-binding proteins. The pattern of progesterone metabolites associated with specific binding proteins clearly differed from the patterns associated with nonspecific binding proteins, those metabolites that were not bound to protein, and those found in the incubation medium, the latter three patterns being essentially the same. The pattern of progesterone metabolites associated with oviduct cytosol proteins from diethylstilbestrol-stimulated immature chicks was in marked contrast to the pattern found when unstimulated chicks were used. The principal me...

A patient with pituitary-dependent hypercortisolism, unresponsive to resection of nodules in the anterior lobe, is described. Histochemical stains of the nodules showed multiple, focal, cellular expansions of the fibrovascular stroma. Transitions between normal and expanded adenohypophysial acini were present. Immunoperoxidase stains for ACTH and other pituitary hormones revealed that these multiple foci contained an excess of ACTH-positive cells. Less than 10% of the cells in these foci were negative for ACTH and positive for other hormones. Serial sections showed that these foci of predominantly ACTH-producing acini were not connected. Clinical, morphological, and immunohistochemical data indicated that ACTH-cell hyperplasia caused Crushing's disease in this patient. Pathologic study of individual cases should concentrate on determining whether hyperplasia or adenoma exist at the time of surgical exploration of the pituitary gland, since this determination is important to proper treatment. Tentative criteria to recognize ACTH-cell hyperplasia are: 1. Multiple foci of ACTH laden cells. 2. A minor subpopulation of cells of alternate hormone series. 3. Expansion without destruction of acini in the adenohypophysis.

Pregnenolone-binding activity isolated from the cytosol of the guinea pig adrenal cortex appears to correspond to a Mr 34,000 protein when examined by SDS-polyacrylamide gel electrophoresis during different stages of purification. To verify this finding the Mr 34,000 protein band was eluted from the SDS gel and used to generate a polyclonal antibody. Immobilized anti 34,000 IgG on protein A-Sepharose was found to extract pregnenolone-binding activity from solution in contrast to pre-immune IgG and an antibody raised against a Mr 30,000 protein isolated simultaneously. In addition, protein eluted from the protein A-anti 34,000 IgG complex exhibited the expected molecular weight of 34,000 when examined on an SDS gel. These results, thus, confirm that the pregnenolone-binding protein is indeed a protein of Mr 34,000.

Steroidogenesis is not stimulated by ACTH in the inner zone of the guinea pig adrenal cortex; adenylate cyclase is normally stimulated. To further explore the lack of a steroidogenic response to ACTH in the inner zone, cAMP-dependent protein kinase activity and protein phosphorylation were examined in the outer and inner adrenocortical zones. To summarize: total cAMP-dependent protein kinase activity was 40% higher in the outer zone than in the inner zone; of the total cAMP-dependent protein kinase activity, cytosol contained 80% for the outer and 70% for the inner zone. In both zones only the type II isozyme was present. Qualitative and quantitative differences in protein phosphorylation were noted for the two zones.