Purification of a pregnenolone-binding protein in the soluble fraction of the guinea-pig adrenal cortex: Differentiation from pregnenolone sulfotransferase

Strott, Charles A.; Goff, A.K.; Lyons, Curtis D.

doi:10.1016/0022-4731(83)90070-5

Cited by 23 publications

(7 citation statements)

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“…However, EST activity was markedly low in the adrenal glands. Guinea pig adrenal cortical EST (gpEST) was purified and cloned as a pregnenolone binding protein by Oeda et al (39) and Strott et al (40). gpEST is also known to specifically bind to E2 with a relatively high affinity.…”

Section: Discussionmentioning

confidence: 99%

Systemic Distribution of Steroid Sulfatase and Estrogen Sulfotransferase in Human Adult and Fetal Tissues

Miki

Nakata

Suzuki

et al. 2002

The Journal of Clinical Endocrinology &Amp; Metabolism

154

125

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Estrogens play a key role in various target tissues. Enzymes involved in the biosynthesis and metabolism of these sex steroids also regulate estrogenic actions in these tissues. Estrone sulfate (E1S) is a major circulating plasma estrogen that is converted into the biologically active estrogen, estrone (E1), by steroid sulfatase (STS). E1 is also sulfated and reverted into E1S by estrogen sulfotransferase (EST). These two enzymes have recently been shown to play important roles in the in situ estrogen actions of various sex steroid-dependent human tumors. However, the distribution of STS and EST in normal adult and fetal human tissues remains largely unknown. Therefore, in this study, in addition to examining the tissue distribution of both STS and EST mRNA in human adult and fetal tissues using RT followed by quantitative PCR, we studied the activity of these enzymes using 3 H-labeled E1/E1S as substrates in the homogenates of various human adult tissues. We also examined the localization of STS and EST protein in human adult and fetal tissues using immunohistochemistry, and that of EST mRNA in the adult kidney using laser dissection microscopy and PCR. STS mRNA, enzyme activity, and immunoreactivity were either absent or detected at very low levels in all adult and fetal tissues examined in this study. EST mRNA expression, however, was detected in all of the tissues examined, except for adult spleen and pancreas. EST enzyme activities were consistent with those of mRNA expression in the great majority of the tissues examined. Marked EST immunoreactivity was detected in hepatocytes, adrenal gland (adult, zona fasciculate to the reticularis; fetus, fetal zone), and epithelial cells of the gastrointestinal tract, smooth muscle cells of the tunica media in aorta, Leydig cells of the testis, and syncytiotrophoblast of the placenta. Patterns of EST immunolocalization were similar between adult and fetal human tissues, but EST immunoreactivity was detected in the urinary tubules of adult kidney, whereas in the fetal kidney, it was localized in the interstitial cells surrounding the urinary tubules. In the adult kidney, the presence of EST mRNA was also confirmed in the cells of urinary tubules using laser dissection microscopy and RT-PCR.Although the number of human tissues available for examination in this study was limited, our results suggest that between the enzymes involved in estrogen activation or inactivation, EST and not STS is the more widely expressed enzyme in various peripheral tissues in humans. We speculate that EST may play an important role in protecting peripheral tissues from possible excessive estrogenic effects. (J Clin Endocrinol Metab 87: 5760 -5768, 2002)

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Section: Discussionmentioning

confidence: 99%

Systemic Distribution of Steroid Sulfatase and Estrogen Sulfotransferase in Human Adult and Fetal Tissues

Miki

Nakata

Suzuki

et al. 2002

The Journal of Clinical Endocrinology &Amp; Metabolism

154

125

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“…This site is also considered to be the major control point of zona glomerulosa biosynthesis (Aguilera & Catt, 1979;Kramer, Gallant & Brownie, 1979), although in addition angiotensin II, ACTH and K + have all been shown to affect the 'late' pathway directly, causing small but significant increases in the conversion of corticosterone to aldosterone (Williams, McDonnel, Tait & Tait, 1972;Aguilera & Catt, 1979). Evidence for further mechanisms of control of output has recently increased, indicating both functional sequestration of product within the gland (Whitehouse & Vinson, 1971; Vinson & Whitehouse, \973a,b;Inaba & Kamata, 1974;Strott, 1977;Holzbauer, 1981; Sibley, Whitehouse, Vinson, Goddard & McCredie, 1981;Nussdorfer & Mazzochi, 1982) and an active process for release of product (Rubin, Sheid, McCauley & Laychock, 1974;Gemmel, Laychock & Rubin, 1977;Nussdorfer, Mazzochi & Meneghelli, 1978;Nussdorfer, 1980). This laboratory has consistently put forward the view that these processes, sequestration and active release of preformed product, together form a specific control of steroid output (Whitehouse & Vinson, 1972;Vinson, Whitehouse, Goddard & Sibley, 1979).…”

Section: Introductionmentioning

confidence: 93%

Serine proteases selectively control the output of 18-hydroxycorticosterone and aldosterone in stimulated zona glomerulosa tissue of the rat adrenal

Raven¹,

McAuley²,

Vinson³

1983

Journal of Endocrinology

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The finding that incubation of rat adrenal capsules (largely zona glomerulosa) with trypsin reproducibly releases aldosterone and 18-hydroxycorticosterone (18-OH-B) from tightly protein-bound tissue stores leads to the hypothesis that the secretion of these steroids may be under the control of endogenous proteases. Rat adrenal capsule whole tissue and collagenase-dispersed cells were incubated under conditions of stimulation by (1-24)ACTH (10(-7) mol/l), potassium (8.4 X 10(-3) mol/l) or dibutyryl cyclic AMP (dbcAMP) (10(-4) mol/l) with the addition in some flasks of one of the following protease inhibitors at the appropriate concentration for their known actions: N alpha-p-tosyl-L-arginine methyl ester (TAME; 10(-2) mol/l), 2-nitro-4-carboxyphenyl-N,N'-diphenylcarbamate (NCDC; 2 X 10(-6) mol/l), N alpha-benzoyl-L-arginine (BA; 10(-2) mol/l), p-nitrophenyl-N alpha-benzyloxycarbonyl-L-lysinate (CBZ-NL; 2 X 10(-6) mol/l) and soybean trypsin inhibitor (STI; 1 mg/ml). The (1-24)ACTH-stimulated steroid output in dispersed cells was not affected by NCDC, BA or CBZ-NL. However, all of the inhibitors except STI produced selective effects on aldosterone and 18-OH-B production by whole capsule tissue under certain conditions. Thus TAME and NCDC significantly inhibited the dbcAMP-stimulated production of these two steroids (aldosterone values decreased from 328 +/- 35 to 128 +/- 15 and 157 +/- 32 ng/gland pair respectively) and furthermore NCDC elicited the same effect in potassium- or ACTH-stimulated whole tissue (e.g. in K+-stimulated tissue aldosterone decreased from 79 +/- 15 to 40 +/- 7 ng/gland pair). The reverse effect was shown by BA and CBZ-NL in potassium-stimulated whole tissue, and yields of aldosterone and 18-OH-B were significantly enhanced (aldosterone increased from 79 +/- 15 ng/pair to 151 +/- 14 ng in the presence of BA). The high molecular weight inhibitor STI had no effect on potassium-stimulated whole tissue, but enhanced the yield of extractable aldosterone from 9.7 +/- 1.7 to 16.9 +/- 2.3 ng/pair when added to incubations of a cytosol preparation. The results suggest that endogenous proteases in rat adrenal whole capsule tissue play specific roles in the control of aldosterone and 18-OH-B secretion. Some (type 1) whose action is mimicked by trypsin, are inhibited by TAME and NCDC and appear to be involved in the release of these two steroids from their tight (apparently covalent) binding to protein.(ABSTRACT TRUNCATED AT 400 WORDS)

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“…The PAGE multiphasic buffer system designated no. 2365 (13) consisted of chloride, sulfate, asparagine, and N-(j3-hydroxyethyl)morpholine; the PAGE procedure was performed as described (9). For some experiments [3H]pregnenolone (NEN) was added to the gel solution before casting to give a baseline of =1000 cpm per 1-mm slice of gel.…”

Section: Methodsmentioning

confidence: 99%

“…But what about pregnenolone, the product of the rate-limiting step? A specific adrenocortical pregnenolone-binding protein (PBP) has been described and partially purified, albeit its precise function is not understood (9,10 2) weighing 700-900 g were used. The animals were anesthetized with CO2 gas and decapitated.…”

mentioning

confidence: 99%

“…The PAGE multiphasic buffer system designated no. 2365 (13) consisted of chloride, sulfate, asparagine, and N-(j3-hydroxyethyl)morpholine; the PAGE procedure was performed as described (9 (14), without urea in the presence of 5% glycerol. The pH gradient was established in 5% acrylamide with a mixture of 3% (vol/vol) Ampholine 5-7 (LKB) and 2% (vol/vol) Bio-Lyte 3/10 (Bio-Rad).…”

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confidence: 99%

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Charge isoforms of the adrenocortical pregnenolone-binding protein: influence of phosphorylation on isoformation and binding activity.

Lee

Driscoll

Strott

1990

Proc. Natl. Acad. Sci. U.S.A.

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Isoelectric focusing of the Mr 34,000 pregnenolone-binding protein (PBP) isolated from the guinea pig adrenal cortex has revealed multiple charge isoforms. Alkaline phosphatase treatment resulted in the disappearance of the pI 5.4 isoform associated with the appearance of pI 5.9 and pI 6.1 isoforms; this alteration in the charge-isoform pattern of the PBP correlated with a loss in pregnenolone-binding activity. This finding appears to be novel for intracellular steroidbinding proteins and has not been demonstrated for steroid receptors, a well-studied group of phosphoproteins. Resolution of the PBP by nondenaturing polyacrylamide gel electrophoresis produced two radioactive peaks of [3Hlpregnenolone in an equilibrium system, while only one peak was present in a nonequilibrium system, suggesting high-and low-binding affinity forms of PBP. Isoelectric focusing of highly purified PBP resolved multiple forms of Mr 34,000 protein with pI values ranging from 6.4 to 5.2. Two of the Mr 34,000 charge isoforms were isolated, and each was used to generate polyclonal antibodies; both antisera were crossreactive against all forms ofMr 34,000 PBP. Western blot analysis revealed that the PBP was present in both the fasciculata and reticularis of the adrenal cortex, though the isoform patterns were not identical for the two zones. Additionally, the pregnenolone-binding activity was =l0-fold greater in the zona reticularis. In vitro alkaline phosphatase treatment of the PBP abolished pregnenolonebinding activity and caused an alteration in the charge-isoform pattern for PBP in the zona reticularis, where pregnenolone binding is high, to resemble the pattern found for the zona fasciculata, where pregnenolone binding is low. The results indicate that phosphorylation/dephosphorylation regulates pregnenolone-binding activity and influences the pattern of the PBP isoformation. The data further suggest that the pI 5.4 isoform may be the active steroid-binding molecule.The rate-limiting step in adrenocortical steroidogenesis is the conversion of cholesterol to pregnenolone (1, 2); however, the precise mechanism by which corticotropin (ACTH) regulates the steroidogenic process is not well understood. There has been an extensive search in recent years for a noncatalytic protein that might play an important regulatory role, presumably by interacting with cholesterol. Three distinct proteins thought to function in such a manner have been described (3-5). The aforementioned proteins are not unique to the adrenal cortex, however, having been found in gonadal as well as nonsteroidogenic tissue (6-8); thus, the cholesterol-interacting proteins appear to play a more universal role in cholesterol metabolism and steroidogenesis. But what about pregnenolone, the product of the rate-limiting step? A specific adrenocortical pregnenolone-binding protein (PBP) has been described and partially purified, albeit its precise function is not understood (9,10 MATERIALS AND METHODSPreparation of Adrenocortical Cytosol. Male guinea pigs (NIH inbred s...

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Purification of a pregnenolone-binding protein in the soluble fraction of the guinea-pig adrenal cortex: Differentiation from pregnenolone sulfotransferase

Cited by 23 publications

References 12 publications

Systemic Distribution of Steroid Sulfatase and Estrogen Sulfotransferase in Human Adult and Fetal Tissues

Systemic Distribution of Steroid Sulfatase and Estrogen Sulfotransferase in Human Adult and Fetal Tissues

Serine proteases selectively control the output of 18-hydroxycorticosterone and aldosterone in stimulated zona glomerulosa tissue of the rat adrenal

Charge isoforms of the adrenocortical pregnenolone-binding protein: influence of phosphorylation on isoformation and binding activity.

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