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FIGURE 2-31 Evolutionary relationship of P450 genes. The vertical scale describes evolutionary time in millions of years. The Roman numerals refer to the standardized nomenclature employed for P450 gene families (see Nebert et al., 1987). The question mark suggests the probable existence of other mitochondrial families of P450 such as those required for vitamin D metabolism and bile acid hydroxylation (see Table 2-6). Adapted from Figure 7 of Miller, W. (1988). Molecular biology of steroid hormone biosynthesis. Endocr. Rev. 9, 295-318.

(see reaction 2 of Table 2-6 and Figure 2-18). This transformation is mediated by one enzyme located in the endoplasmic reticulum that carries out the two steps. The same enzyme is apparently involved in the adrenals, ovaries, testes, and placenta.

The 3/3-hydroxysteroid dehydrogenase A5,A4-isomerase has an obligatory cofactor NAD+ and catalyzes the reactions shown in Figure 2-32. The enzyme has been cloned and expressed; evidence indicates the expression of multiple forms of the 3/3-hydroxy dehydrogenase / A5, A4-isomerase.

2. 5a-Reductase

The conversion of testosterone (15) to dihydrotes-tosterone (86) (see Figure 2-22 and reaction 14 of Table 2-6) is mediated by the 5a-reductase. 5a-Reductase is an NADPH-dependent non-P450 enzyme that is present in the nuclear membrane of some target cells for testosterone, e.g., genital skin and hair follicles.

3. 17-Ketosteroid Reductase

17-Ketosteroid reductase is a non-P450 enzyme present in the endoplasmic reticulum; it requires NADPH (see reaction 9 of Table 2-6 and Figures 2-22 and 2-23). The catalyzed reactions of the 17-ketosteroid

FIGURE 2-32 Reaction catalyzed by the 3/3-hydroxysteroid dehydrogenase, A5,A4-isomerase.

FIGURE 2-32 Reaction catalyzed by the 3/3-hydroxysteroid dehydrogenase, A5,A4-isomerase.

reductase are readily reversible. It is present in the testes, ovaries, and placenta.

4. lip-Steroid Dehydrogenase

The enzyme 11/3-steroid dehydrogenase (see reaction 17 in Table 2-6 and Figure 2-21) is responsible for the NADPH-dependent reduction of the 11/3-hydroxyl on Cortisol (13) to yield cortisone (93). This enzyme is present in kidney, liver, brain, and lung. One significant contribution of this enzyme in the kidneys is to protect the aldosterone receptor from inappropriate exposure to Cortisol, which is an effective ligand. Cortisone, in contrast, is a weak ligand for the aldosterone receptor (see Chapter 15).

5. Steroid Acute Regulatory Protein (StAR)

The first step in classical steroid hormone biosynthesis involves the conversion of cholesterol into pregnenolone by the cholesterol side chain cleavage enzyme P450scc. Although the side chain cleavage reaction is known to be the rate-limiting step for adrenal and gonadal steroid hormones, it is not the catalytic process of the enzyme that is rate limiting but, instead, the delivery of the substrate cholesterol from the cytoplasm across the outer mitochondrial membrane to the inner mitochondrial membrane site of the P450scc, which has been found to be the rate-limiting step.

A specific cholesterol transport protein, known as steroid acute regulatory protein (StAR), has been implicated as being an essential mediator of cholesterol delivery and the consequent activation of P450scc (see Figure 2-33). StAR is biosynthesized as a 285-amino

StAR

StAR

FIGURE 2-33 Proposed model for the role of the steroid acute regulatory (StAR) protein in cholesterol side chain cleavage. StAR is believed to facilitate the transfer of the hydrophobic cholesterol molecule across both the outer mitochondrial membrane and the inner membrane space to the inner mitochondrial membrane, which is the location of the P450scc where metabolism to pregnenolone occurs. [Adapted from Waterman, M. R. (1955). A Rising StAR: An essential role in cholesterol transport. Science 267, 1780-1781.]

FIGURE 2-33 Proposed model for the role of the steroid acute regulatory (StAR) protein in cholesterol side chain cleavage. StAR is believed to facilitate the transfer of the hydrophobic cholesterol molecule across both the outer mitochondrial membrane and the inner membrane space to the inner mitochondrial membrane, which is the location of the P450scc where metabolism to pregnenolone occurs. [Adapted from Waterman, M. R. (1955). A Rising StAR: An essential role in cholesterol transport. Science 267, 1780-1781.]

acid protein with a mitochondrial targeting sequence of 25 amino acid residues, which is cleaved from the NH2-terminus after it is transported into the mitochondria.

The most convincing evidence for the essential nature of StAR was obtained from molecular evaluation of the defect associated with the disease known as lipoid congenital adrenal hyperplasia (CAH). (See

Chapter 10 for a discussion of the autosomal recessive diseases that result from a blockade in several of the steps of steroid biosynthesis.) In lipoid congenital adrenal hyperplasia, which is characterized by a deficiency of both adrenal and gonadal steroid hormones, there is a mutation in the StAR gene, so that the StAR protein is nonfunctional due to premature stop codons. Lipoid CAH is the only steroid hormone synthesis disorder that is not a result of a mutation in the gene for a steroidogenic enzyme.

VI. PLASMA TRANSPORT, CATABOLISM, AND EXCRETION OF STEROID HORMONES

A. General Comments

The hormonally active form of most steroids is generally the molecular species released from the endocrine gland and transported systemically to various distal target tissues. A target tissue is defined as one that has stereospecific receptors permitting the accumulation of the steroid in the target tissue against a concentration gradient. This in turn permits generation of the appropriate biological response in that target tissue for the specific steroid in question. Thus, a key determinant of the ability of a target tissue to bind the steroid hormone is the hormone's actual blood concentration. The concentration of a steroid in the plasma at any particular time depends on three factors: (i) the rate at which the steroid is biosynthesized and enters the body pools; (ii) the rate at which the steroid is biologically inactivated by catabolism and removed from body pools; and (iii) the "tightness" of binding of the steroid to its plasma carrier protein.

Previous sections of this chapter have considered in detail the metabolic pathways of production of many steroid hormones; subsequent chapters will discuss in detail the regulation of steroid metabolism. The remaining sections of this chapter will briefly consider the plasma transport proteins for steroids and general

TABLE 2-7 Plasma Transport Proteins for Steroid, Thyroxine, and Retinoid Hormones

Plasma protein Abbreviation Principal steroids bound Molecular weight (x 103)

Vitamin D-binding protein DBP Vitamin D3, 25(OH)D3, l,25(OH)2D3, 50 (glycoprotein)

24,25(OH)2D3

Corticosteroid-binding globulin (transcortin) CBG Glucocorticoids, progesterone 52 (glycoprotein)

Sex hormone-binding globulin SHBG Testosterone, estradiol A dimer, each subunit 42

Thyroxine-binding globulin TBG Thryoxine (T4), triiodothyronine (T3) 63 (glycoprotein)

Retinol-binding protein RBP Retinol 41

FIGURE 2-34 Example of capillary wall fenestration. The existence of fenestrations or windows in the capillary wall may allow plasma steroid transport proteins to exit the circulatory system and approach the outer cell membrane of the target cell for the steroid hormone in question. (A) Plasmalemmal vesicular openings in the P-face (P) of a diaphragm muscle capillary (X60,000). (B) Lightly etched luminal surface from a continuous capillary in the kidney medulla displays small depressions containing a central particle similar to surface views of muscle capillaries (X 80,000). (C) Diagram representing the possible processes of exit or entry of hormones. (D) A fenestrated diaphragm in the endothelium of an adrenal cortex capillary. Note the eight, dark, wedge-shaped communicatory channels. Adapted with permission from Figures 2 and 6 of Bearer, E., and Orci, L. (1985). Endothelial fenestral diaphragms: A quick-freeze, deep-etch study. /. Cell Biol. 100, 418-428.

FIGURE 2-34 Example of capillary wall fenestration. The existence of fenestrations or windows in the capillary wall may allow plasma steroid transport proteins to exit the circulatory system and approach the outer cell membrane of the target cell for the steroid hormone in question. (A) Plasmalemmal vesicular openings in the P-face (P) of a diaphragm muscle capillary (X60,000). (B) Lightly etched luminal surface from a continuous capillary in the kidney medulla displays small depressions containing a central particle similar to surface views of muscle capillaries (X 80,000). (C) Diagram representing the possible processes of exit or entry of hormones. (D) A fenestrated diaphragm in the endothelium of an adrenal cortex capillary. Note the eight, dark, wedge-shaped communicatory channels. Adapted with permission from Figures 2 and 6 of Bearer, E., and Orci, L. (1985). Endothelial fenestral diaphragms: A quick-freeze, deep-etch study. /. Cell Biol. 100, 418-428.

pathways of steroid hormone inactivation and excretion.

B. Plasma Transport of Steroid and Thyroid Hormones

The steroid hormones are transported from their sites of biosynthesis to their target organs through the blood compartment. Due to the intrinsic low water solubility of all the steroid hormones, their transport is effected by a family of plasma transport proteins (see Table 2-7). Each plasma transport protein has a specific ligand-binding domain that allows the high-affinity binding of their cognate hormones. The exception is aldosterone, which is believed to circulate as the free steroid in the plasma compartment. Although all five plasma transport proteins are synthesized in the liver, they have no amino acid sequence homology. Also, there is no apparent sequence homology between the ligand-binding domain of the plasma transport protein and either the nuclear-cytosol receptor's ligand-binding domain or the substrate-bindiing domain of the P450 enzyme that generated the steroid.

In the plasma compartment, the steroid hormones move through the circulatory system bound to their transport protein. However, an important issue con cerns the details of the mode of delivery of steroid hormones to their target cells. Since the "free" form of the steroid hormone is believed to be the form of steroid movement across the outer cell membrane of a target cell, it had been postulated that the steroid ligand bound to a plasma transport protein dissociates and diffuses first through the capillary wall and then through the outer wall membrane of target cells. However, as illustrated in Figure 2-34, it is apparent that the endothelial wall of capillaries contains fenestrations. Thus, it is conceivable that the plasma steroid transport protein could exit the capillary bed through a fenestration and move immediately adjacent ("dock?") to the outer cell membrane of the appropriate target cell for the steroid hormone in question.

Each plasma transport protein binds its ligand(s) with high affinity, the Kd values fall in the range of (5-500) X 10"8 M. In general, for any given hormone, the Kd of the hormone for its target organ receptor is 10-100 X tighter than for its plasma transport protein.

C. Measurements of Rates of Secretion and Metabolic Clearance

The plasma concentration of a steroid is determined by the balance between biosynthesis and bioinactiva-tion. With the ready availability of high specific activity

TABLE 2-8 Mean Secretion Rates, Plasma Concentrations, and Metabolic Clearance Rates (MCR) of Various Steroids"

Plasma

Secretion rate concentration MCR (liters

Steroid Men Women Men Women Men Women

Cortisol

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