GO:0004769 steroid Delta-isomerase activity: Mechanism, Genes and Research Methods
Research-grade guide for scientists and biopharma professionals
Key Takeaways
• GO:0004769 steroid Delta-isomerase activity catalyzes the conversion of a 3-oxo-delta(5)-steroid to a 3-oxo-delta(4)-steroid, a key step in steroid hormone biosynthesis.
• The reaction is essential for the production of all classes of steroid hormones, including androgens, estrogens, glucocorticoids, and mineralocorticoids.
• In humans, this activity is carried out by bifunctional enzymes such as 3-beta-hydroxysteroid dehydrogenase/delta-5-delta-4 isomerase (3-beta-HSD) and by glutathione transferase A3-3 (GSTA3-3).
• The catalytic mechanism involves a conserved tyrosine and aspartate residue that act as a general acid and base, respectively, to isomerize the double bond.
• Dysregulation of steroid Delta-isomerase activity is implicated in endocrine disorders, skin pathologies, and hormone-dependent cancers.
• CRISPR-based knockout, point mutation, and knock-in models are powerful tools to dissect the precise roles of isomerase enzymes in health and disease.
Description
Steroid Delta-isomerase activity (GO:0004769) is a fundamental enzymatic function in steroid metabolism, responsible for the isomerization of the double bond in 3-oxo-delta(5)-steroids to the delta(4) position. This reaction is a critical step in the biosynthesis of all steroid hormones, as it converts intermediates such as pregnenolone and dehydroepiandrosterone into their active delta(4) forms, which are precursors for androgens, estrogens, and corticosteroids. The enzyme is widely distributed in mammals, from human placenta and skin to bovine adrenal glands, and its activity is essential for normal endocrine function. Researchers study this activity to understand hormone biosynthesis, metabolic disorders, and the mechanisms of bifunctional enzymes that catalyze multiple reactions. The importance of steroid Delta-isomerase activity extends to clinical areas such as endocrinology, dermatology, and oncology, where altered enzyme levels can contribute to disease pathogenesis.
steroid Delta-isomerase activity At A Glance
| GO ID | GO:0004769 |
|---|---|
| GO term | steroid Delta-isomerase activity |
| Ontology | molecular_function |
| Synonym | 3-oxosteroid delta5-delta4-isomerase activity; delta5-3-ketosteroid isomerase activity; hydroxysteroid isomerase activity; steroid isomerase activity |
| Major function | Catalyzes the isomerization of 3-oxo-delta(5)-steroids to 3-oxo-delta(4)-steroids, a key step in steroid hormone biosynthesis |
| Reaction | a 3-oxo-delta(5)-steroid = a 3-oxo-delta(4)-steroid |
| EC number | 5.3.3.1 |
| Found in | Mammals, including human placenta, skin, adrenal glands, and bovine adrenal glands |
| Associated enzymes | 3-beta-hydroxysteroid dehydrogenase/delta-5-delta-4 isomerase, glutathione transferase A3-3 |
What Is GO:0004769?
According to the Gene Ontology, steroid Delta-isomerase activity (GO:0004769) is defined as the catalysis of the reaction: a 3-oxo-delta(5)-steroid = a 3-oxo-delta(4)-steroid. In simpler terms, it is an enzyme activity that moves a double bond from the delta(5) position to the delta(4) position in a steroid molecule that already has a ketone group at the 3-position. This isomerization is a prerequisite for the subsequent actions of other steroidogenic enzymes, and it is often associated with 3-beta-hydroxysteroid dehydrogenase activity in bifunctional proteins.
Why Is steroid Delta-isomerase activity Important in Cell Biology?
Steroid Delta-isomerase activity is indispensable for the production of all biologically active steroid hormones. Without this isomerization, the delta(5) intermediates cannot be converted into the delta(4) steroids that serve as substrates for downstream enzymes, leading to a block in hormone synthesis. This activity is therefore central to endocrine physiology, and its dysregulation has been linked to disorders such as adrenal hyperplasia, skin diseases, and hormone-dependent cancers. Moreover, the bifunctional nature of some isomerases, such as 3-beta-HSD, makes them attractive targets for understanding enzyme evolution and for developing therapeutic inhibitors.
• Essential for the biosynthesis of androgens, estrogens, glucocorticoids, and mineralocorticoids.
• Defects in 3-beta-HSD/isomerase can cause endocrine disorders and adrenal hyperplasia.
• Altered activity in skin may contribute to acne and other dermatological conditions.
• The enzyme is a target for active-site-directed inhibitors, with potential for drug development.
• Bifunctional catalysis by a single protein provides a model for understanding enzyme promiscuity.
• Membrane lipid environment can modulate isomerase activity, linking it to cellular signaling.
• Glutathione transferase A3-3 exhibits high steroid isomerase activity, suggesting a role in steroidogenesis outside classical endocrine tissues.
• The catalytic mechanism involving D38 and Y14 residues is a paradigm for general acid-base catalysis.
• CRISPR screens can identify novel regulators of steroid isomerase activity in disease models.
• Understanding this activity aids in the design of hormone therapies and contraceptives.
What Happens During steroid Delta-isomerase activity?
Substrate Binding and Orientation
In simple terms: The enzyme grabs the steroid molecule and positions it perfectly for the chemical reaction.
The first step in the isomerization reaction is the binding of the 3-oxo-delta(5)-steroid substrate to the active site of the enzyme. In human 3-beta-HSD/isomerase, the substrate binds in a hydrophobic pocket that positions the delta(5) double bond near the catalytic residues. In glutathione transferase A3-3, specific active-site residues govern high steroid isomerase activity by orienting the substrate for efficient catalysis. The binding is often facilitated by the membrane lipid environment, as shown for bovine adrenal 3-oxo-delta(5)-steroid isomerase.
Catalytic Isomerization
In simple terms: A base and an acid work together to move the double bond from one position to another.
The isomerization proceeds through a general acid-base mechanism. In the well-studied bacterial and mammalian enzymes, a conserved aspartate residue (e.g., D38 in the bacterial enzyme) acts as a base to abstract a proton from the steroid, while a tyrosine residue (e.g., Y14) acts as an acid to donate a proton to the opposite face, resulting in the migration of the double bond from delta(5) to delta(4). In the D38A mutant, the enzyme recruits aspartate-99 as an alternative base, demonstrating the flexibility of the active site. This step is rate-limiting and is highly dependent on the precise geometry of the substrate-enzyme complex.
Product Release and Enzyme Turnover
In simple terms: The newly formed hormone precursor is released, and the enzyme is ready to do it again.
After isomerization, the 3-oxo-delta(4)-steroid product is released from the active site. The enzyme can then bind another substrate molecule. The turnover rate can be influenced by the membrane environment, as shown by studies on bovine adrenal isomerase, where changes in lipid composition affected activity. In bifunctional enzymes like 3-beta-HSD/isomerase, the isomerase activity is coordinated with the dehydrogenase activity, allowing efficient channeling of intermediates.
Regulation of Enzyme Levels
In simple terms: The amount of enzyme in the cell can go up or down depending on hormonal signals.
The expression and activity of steroid Delta-isomerase can be regulated by hormones such as adrenocorticotropin (ACTH) and corticosterone. In rat adrenal glands, ACTH treatment increased 3-beta-HSD/isomerase expression and activity, while corticosterone had suppressive effects. This regulation ensures that steroid hormone production meets physiological demands. Additionally, the enzyme's activity can be modulated by post-translational modifications and interactions with other proteins, although these mechanisms are less well characterized.
Key Genes Involved in GO:0004769 steroid Delta-isomerase activity
The following genes and proteins are directly associated with steroid Delta-isomerase activity, either as catalytic subunits or as bifunctional enzymes that harbor this activity.
| Gene | Major Role | Research Relevance |
|---|---|---|
| HSD3B1 | Encodes 3-beta-hydroxysteroid dehydrogenase/delta-5-delta-4 isomerase type I, a bifunctional enzyme with isomerase activity | Studied for its role in placental and peripheral steroidogenesis; target for endocrine disorders |
| HSD3B2 | Encodes 3-beta-hydroxysteroid dehydrogenase/delta-5-delta-4 isomerase type II, primarily in adrenal glands and gonads | Mutations cause congenital adrenal hyperplasia; key for cortisol and aldosterone synthesis |
| GSTA3 | Encodes glutathione transferase A3-3, which exhibits high steroid isomerase activity | Investigated for its non-classical role in steroidogenesis and detoxification |
| GSTA1 | Glutathione transferase A1-1, with lower steroid isomerase activity | Used as a comparison to understand active-site determinants of isomerase activity |
| GSTA2 | Glutathione transferase A2-2, with minimal steroid isomerase activity | Serves as a negative control in studies of GSTA3-3 |
| SRD5A1 | 5-alpha-reductase type 1, acts downstream of delta(4) steroids | Studied in context of androgen metabolism; not directly isomerase but related pathway |
| SRD5A2 | 5-alpha-reductase type 2, converts testosterone to dihydrotestosterone | Relevant for understanding androgen action after isomerization |
| CYP17A1 | 17-alpha-hydroxylase/17,20-lyase, acts on delta(4) steroids | Downstream enzyme that uses products of isomerase reaction |
| CYP19A1 | Aromatase, converts androgens to estrogens | Uses delta(4) androgens produced after isomerization |
| STAR | Steroidogenic acute regulatory protein, transports cholesterol to mitochondria | Upstream of isomerase in steroidogenesis; often co-studied |
| CYP11A1 | Cholesterol side-chain cleavage enzyme, produces pregnenolone | Generates the delta(5) substrate for isomerase |
| HSD17B1 | 17-beta-hydroxysteroid dehydrogenase type 1 | Acts on steroids after isomerization; studied in breast cancer |
| HSD17B2 | 17-beta-hydroxysteroid dehydrogenase type 2 | Inactivates steroids; balance with isomerase activity |
| NR5A1 | Steroidogenic factor 1, transcription factor regulating steroidogenic genes | Regulates expression of HSD3B genes |
| POR | P450 oxidoreductase, supports CYP enzyme activities | Indirectly affects isomerase substrate supply |
| FDX1 | Ferredoxin 1, electron transfer in mitochondria | Supports CYP11A1, upstream of isomerase |
| FDXR | Ferredoxin reductase | Part of mitochondrial steroidogenic machinery |
| CYP21A2 | 21-hydroxylase, acts after isomerization in cortisol pathway | Deficiency causes congenital adrenal hyperplasia |
How Is steroid Delta-isomerase activity Regulated?
Steroid Delta-isomerase activity is regulated at multiple levels. Hormonal signals such as adrenocorticotropin (ACTH) can upregulate the expression of 3-beta-HSD/isomerase in the adrenal gland, while corticosterone can suppress it. The activity is also influenced by the membrane lipid environment, as demonstrated for bovine adrenal 3-oxo-delta(5)-steroid isomerase, where changes in phospholipid composition altered catalytic efficiency. Additionally, the bifunctional nature of 3-beta-HSD/isomerase suggests that conformational changes may regulate the switch between dehydrogenase and isomerase activities. Post-translational modifications and protein-protein interactions may further modulate activity, though these mechanisms require further investigation.
steroid Delta-isomerase activity and Human Disease
| Gene | Disease / Biology | Potential Experimental Model |
|---|---|---|
| HSD3B2 | Congenital adrenal hyperplasia | Knockout mouse or patient-derived iPSCs with point mutations |
| HSD3B1 | Placental steroidogenesis, breast cancer | Knockdown in breast cancer cell lines; overexpression in MCF-7 |
| GSTA3 | Steroidogenesis in peripheral tissues, cancer | CRISPR knockout in HEK293 or cancer cell lines |
| HSD3B1/2 | Skin disorders (acne) | 3D skin models with CRISPR knockout of HSD3B1 |
| HSD3B2 | Adrenal insufficiency | Adrenal cell lines with point mutations (e.g., D38A equivalent) |
Endocrine Disorders
Deficiencies in 3-beta-HSD/isomerase, the enzyme responsible for steroid Delta-isomerase activity in humans, lead to congenital adrenal hyperplasia, a group of disorders characterized by impaired cortisol and aldosterone synthesis and excessive androgen production. This can cause ambiguous genitalia in females, salt-wasting crises, and precocious puberty. The regulation of this enzyme by ACTH and corticosterone is critical for maintaining hormonal balance, and disruptions in this axis can exacerbate disease severity.
Skin Pathologies
Steroid Delta-isomerase activity is present in human skin, where it contributes to local steroidogenesis. Altered activity of 3-beta-HSD/isomerase in skin has been implicated in acne and other dermatological conditions, as androgens produced locally can stimulate sebum production. Inhibitors of this activity are being explored as potential treatments for androgen-dependent skin disorders.
Hormone-Dependent Cancers
Many breast and prostate cancers depend on steroid hormones for growth. The isomerase activity of 3-beta-HSD and GSTA3-3 contributes to the production of androgens and estrogens in peripheral tissues, which can fuel tumor growth. Targeting steroid Delta-isomerase activity, either directly or through upstream regulators, is a potential therapeutic strategy for hormone-dependent cancers.
From steroid Delta-isomerase activity-Related Genes to Experimental Models
| Research Question | Suitable Model |
|---|---|
| What is the effect of complete loss of steroid Delta-isomerase activity? | CRISPR knockout of HSD3B2 in adrenal cell lines (e.g., H295R) |
| How does a specific catalytic residue mutation affect isomerase activity? | Point mutation (e.g., D38A) knock-in in HSD3B1 using CRISPR |
| Can we tag the enzyme to study its localization? | Knock-in of fluorescent tag (e.g., GFP) at the endogenous HSD3B1 locus |
| What happens when the enzyme is overexpressed in a non-steroidogenic tissue? | Overexpression of HSD3B1 or GSTA3 in HEK293 cells |
| Which genes regulate steroid Delta-isomerase activity? | CRISPR library screening in steroidogenic cells with a reporter for isomerase activity |
| How does the enzyme interact with other steroidogenic proteins? | Knock-in of proximity labeling tags (e.g., BioID) followed by proteomics |
How to Study the steroid Delta-isomerase activity Process
| Method | What It Measures | Typical Application |
|---|---|---|
| UV spectrophotometric assay | Conversion of delta(5) to delta(4) steroid | Kinetic characterization of purified enzymes |
| Radioactive substrate assay | Enzymatic activity with high sensitivity | Tissue homogenates and cell lysates |
| qRT-PCR | mRNA levels of HSD3B1, HSD3B2, GSTA3 | Expression profiling in disease models |
| Western blot | Protein expression and post-translational modifications | Validation of enzyme levels |
| CRISPR knockout screen | Genes regulating isomerase activity | Identification of novel regulators |
| X-ray crystallography | Three-dimensional structure of enzyme | Active-site mapping and inhibitor design |
| Site-directed mutagenesis | Role of specific amino acids in catalysis | Mechanistic studies |
| Membrane lipid analysis | Effect of lipid environment on activity | Biophysical regulation studies |
Enzymatic Activity Assays
The most direct way to measure steroid Delta-isomerase activity is through spectrophotometric assays that monitor the conversion of a 3-oxo-delta(5)-steroid to its delta(4) isomer, which absorbs UV light at 240-250 nm. This method has been used to characterize the activity of purified enzymes, such as human GSTA3-3 and 3-beta-HSD/isomerase, and to assess the effects of inhibitors. Radioactive substrate assays can also be employed for higher sensitivity.
Gene Expression Analysis
Quantitative RT-PCR and RNA-seq can measure the mRNA levels of genes encoding steroid Delta-isomerase, such as HSD3B1, HSD3B2, and GSTA3, under different physiological conditions or in disease models. This helps determine whether changes in activity are due to altered expression or post-translational regulation. Western blotting with specific antibodies can confirm protein levels.
CRISPR-Based Genetic Screens
Genome-wide CRISPR knockout or activation screens can identify genes that regulate steroid Delta-isomerase activity. For example, a reporter cell line expressing a fluorescent substrate that becomes fluorescent upon isomerization can be used to sort cells with altered activity, followed by sequencing to identify guide RNAs. This approach can uncover novel regulators and pathways.
Structural and Biophysical Methods
X-ray crystallography and NMR spectroscopy have been used to solve the structures of steroid isomerases, revealing the active-site architecture and catalytic residues. Site-directed mutagenesis combined with kinetic studies can dissect the roles of individual amino acids, as demonstrated for the D38A mutant of 3-oxo-delta(5)-steroid isomerase. These methods provide mechanistic insights that can guide drug design.
How CRISPR Can Be Used to Study GO:0004769 steroid Delta-isomerase activity
Knockout
CRISPR knockout of HSD3B2 or HSD3B1 can completely abolish steroid Delta-isomerase activity in cell models, allowing researchers to study the consequences of enzyme loss on steroid hormone production and cellular physiology. For example, knockout of HSD3B2 in adrenal H295R cells would block cortisol synthesis, mimicking congenital adrenal hyperplasia. Such models are valuable for testing potential therapies and understanding compensatory mechanisms.
Point Mutation
Introducing specific point mutations, such as the D38A substitution in the catalytic site, via CRISPR knock-in can dissect the contribution of individual residues to isomerase activity. This approach preserves the rest of the protein structure and allows comparison of mutant and wild-type enzymes in a physiological context. It is particularly useful for studying bifunctional enzymes where one activity is selectively impaired.
Knock-in
Knock-in of epitope tags (e.g., FLAG, HA) or fluorescent proteins (e.g., GFP) at the endogenous HSD3B1 or GSTA3 locus enables real-time tracking of enzyme localization and interaction partners without overexpression artifacts. This can reveal dynamic changes in enzyme distribution during steroidogenesis or in response to hormonal signals.
Overexpression
CRISPR activation (CRISPRa) or traditional cDNA overexpression can increase steroid Delta-isomerase activity in cell lines that normally have low levels. Overexpressing HSD3B1 or GSTA3 in HEK293 cells can create a gain-of-function model to study the effects of excess isomerase activity on steroid metabolism and cell proliferation. This is useful for identifying downstream targets and potential oncogenic roles.
How EDITGENE Supports steroid Delta-isomerase activity Research
Researchers studying steroid Delta-isomerase activity-related genes often need to determine whether a candidate gene is causally involved in hormone biosynthesis, disease progression, or drug response. EDITGENE provides a comprehensive suite of CRISPR-based services to create precisely engineered cell models, enabling functional validation of genes associated with GO:0004769.
Contact EDITGENE today to design your custom CRISPR model for steroid Delta-isomerase activity research.
Related Products
| Product name | Cat.No. | Species | Gene ID | |
|---|---|---|---|---|
| GSTA1 Knockout HEK293 Cell Line | EDJ-KQ2570 | Human | 2938 | Details Get a Quote |
| GSTA3 Knockout HEK293 Cell Line | EDJ-KQ4804 | Human | 2940 | Details Get a Quote |
| HSD3B2 Knockout HEK293 Cell Line | EDJ-KQ4948 | Human | 3284 | Details Get a Quote |
| HSD3B1 Knockout HEK293 Cell Line | EDJ-KQ50365 | Human | 3283 | Details Get a Quote |
| EBP Knockout HEK293 Cell Line | EDJ-KQ51000 | Human | 10682 | Details Get a Quote |
| GSTA1 Knockout HeLa Cell Line | EDJ-KQ53448 | Human | 2938 | Details Get a Quote |
| GSTA3 Knockout HeLa Cell Line | EDJ-KQ53450 | Human | 2940 | Details Get a Quote |
| HSD3B1 Knockout HeLa Cell Line | EDJ-KQ53574 | Human | 3283 | Details Get a Quote |
| HSD3B2 Knockout HeLa Cell Line | EDJ-KQ53575 | Human | 3284 | Details Get a Quote |
| EBP Knockout HeLa Cell Line | EDJ-KQ55461 | Human | 10682 | Details Get a Quote |
| GSTA1 Knockout A-549 Cell Line | EDJ-KQ61922 | Human | 2938 | Details Get a Quote |
| GSTA3 Knockout A-549 Cell Line | EDJ-KQ61924 | Human | 2940 | Details Get a Quote |
| HSD3B1 Knockout A-549 Cell Line | EDJ-KQ62040 | Human | 3283 | Details Get a Quote |
| HSD3B2 Knockout A-549 Cell Line | EDJ-KQ62041 | Human | 3284 | Details Get a Quote |
| EBP Knockout A-549 Cell Line | EDJ-KQ63946 | Human | 10682 | Details Get a Quote |
Displaying Records 1 To 15 Of 20 Records
Frequently Asked Questions About steroid Delta-isomerase activity
What is steroid Delta-isomerase activity?
Steroid Delta-isomerase activity (GO:0004769) is an enzymatic activity that catalyzes the conversion of a 3-oxo-delta(5)-steroid to a 3-oxo-delta(4)-steroid, a key step in steroid hormone biosynthesis.
What genes are involved in steroid Delta-isomerase activity?
The main genes are HSD3B1 and HSD3B2, which encode bifunctional 3-beta-hydroxysteroid dehydrogenase/delta-5-delta-4 isomerase enzymes, and GSTA3, which encodes glutathione transferase A3-3 with high isomerase activity.
What is the reaction catalyzed by steroid Delta-isomerase?
The enzyme catalyzes the isomerization of a 3-oxo-delta(5)-steroid to a 3-oxo-delta(4)-steroid, moving the double bond from the delta(5) to the delta(4) position.
Why is steroid Delta-isomerase activity important?
It is essential for the production of all active steroid hormones, including androgens, estrogens, and corticosteroids, and its dysfunction is linked to endocrine disorders and cancers.
How is steroid Delta-isomerase activity regulated?
It is regulated by hormones such as ACTH and corticosterone, which affect enzyme expression, and by the membrane lipid environment.
What diseases are associated with defects in steroid Delta-isomerase activity?
Defects can cause congenital adrenal hyperplasia, skin disorders like acne, and may contribute to hormone-dependent cancers such as breast and prostate cancer.
How can I measure steroid Delta-isomerase activity in the lab?
Common methods include UV spectrophotometric assays monitoring the conversion of delta(5) to delta(4) steroids, radioactive substrate assays, and gene expression analysis.
What model systems are used to study steroid Delta-isomerase activity?
Researchers use cell lines (e.g., H295R, HEK293), knockout mice, and CRISPR-engineered cells to study the enzyme's function and regulation.
Can CRISPR be used to study steroid Delta-isomerase activity?
Yes, CRISPR knockout, point mutation knock-in, and overexpression models allow precise manipulation of genes like HSD3B2 and GSTA3 to study their roles in steroidogenesis.
What are the synonyms for steroid Delta-isomerase activity?
Synonyms include 3-oxosteroid delta5-delta4-isomerase activity, delta5-3-ketosteroid isomerase activity, hydroxysteroid isomerase activity, and steroid isomerase activity.
Conclusion
Steroid Delta-isomerase activity (GO:0004769) is a cornerstone of steroid hormone biosynthesis, catalyzing the essential isomerization of delta(5) steroids to their active delta(4) forms. Its role in endocrine physiology and disease makes it a compelling target for research and therapeutic development. Advances in CRISPR-based genome editing now enable precise interrogation of the genes responsible for this activity, offering new insights into hormone-related disorders and potential treatments.
References
- 1. Johansson AS et al.. 2002. Active-site residues governing high steroid isomerase activity in human glutathione transferase A3-3.. J Biol Chem 277(19):16648-54 PMID: 11872752
- 2. Penning TM et al.. 1981. Inactivation of delta 5-3-oxo steroid isomerase with active-site-directed acetylenic steroids.. Biochem J 193(1):217-27 PMID: 7305923
- 3. Wehrle JP et al.. 1986. Effect of membrane lipid environment on the activity of bovine adrenal 3-oxo-delta 5-steroid isomerase.. Steroids 47(2-3):115-30 PMID: 3564082
- 4. de Launoit Y et al.. 1992. Androgenic 17 beta-hydroxysteroid dehydrogenase activity of expressed rat type I 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase.. Endocrinology 130(1):553-5 PMID: 1309351
- 5. Thomas JL et al.. 1992. Affinity labeling of human placental 3 beta-hydroxy-delta 5-steroid dehydrogenase and steroid delta-isomerase: evidence for bifunctional catalysis by a different conformation of the same protein for each enzyme activity.. Biochemistry 31(24):5522-7 PMID: 1610797
- 6. Tóth I et al.. 1997. Activity and inhibition of 3-beta-hydroxysteroid dehydrogenase/delta-5-4-isomerase in human skin.. Skin Pharmacol 10(3):160-8 PMID: 9287397
- 7. Hénot F et al.. 2000. Catalytic activity of the D38A mutant of 3-oxo-Delta 5-steroid isomerase: recruitment of aspartate-99 as the base.. Biochemistry 39(12):3351-9 PMID: 10727228
- 8. Trudel C et al.. 1991. Regulation of adrenal 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4-isomerase expression and activity by adrenocorticotropin and corticosterone in the rat.. Endocrinology 129(4):2077-84 PMID: 1655393
