Adrenal Cortex Adrenodoxin

The purification procedure has been published elsewhere. Two flow sheets of the purification are as follows  

A conventional purification procedure for adrenodoxin has been explored as follows  

The yield of adrenodoxin from one kg of pig adrenal gland is approximately 10.0 mg of protein. An alternative method has been reported by Omura et al. (45, 46). 

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In mitochondria (Fig. lb), the electron acceptor protein is also a flavoprotein termed NADPH-adrenodoxin reductase (MW 50 kDa) because it was discovered in the adrenal cortex and because it donates its electrons not directly to the P450 but to the smaller redox protein adrenodoxin (MW 12.5 kDa). The two iron-sulphur clusters of this protein serve as electron shuttle between the flavoprotein and the mitochondrial P450. 

Adrenodoxin. Adrenodoxin is the only iron-sulfur protein which has been isolated from mammals. This protein from mitochondria of bovine adrenal cortex was purified almost simultaneously by Kimura and Suzuki (32) and Omura et al. (33). It has a molecular weight of 12,638 (34) and the oxidized form of the protein shows maximal absorbances at 415 and 453 nm. Adrenodoxin acts as an electron carrier protein in the enzyme system required for steroid hydroxylation in adrenal mitochondria. In this system, electron transfer is involved with three proteins cytochrome P. gQ, adrenodoxin and a flavoprotein. Reduced NADP gives an electron to Tne flavoprotein which passes the electron to adrenodoxin. Finally, reduced adrenodoxin transfers the electron to cytochrome Pas shown in Fig. 3. The mechanism of cytochrome P cq interaction with steroid, oxygen and adrenodoxin in mixed-function oxidase of adrenal cortex mitochondria has been reviewed by Estabrook et al. (35). 

FIGURE 21-47 Side-chain cleavage in the synthesis of steroid hormones. Cytochrome P-450 acts as electron carrier in this mixed-function oxidase system that oxidizes adjacent carbons. The process also requires the electron-transferring proteins adrenodoxin and adrenodoxin reductase. This system for cleaving side chains is found in mitochondria of the adrenal cortex, where active steroid production occurs. Pregnenolone is the precursor of all other steroid hormones (see Fig. 21-46). 

Figure 17.8 Typical hydroxylation reaction involving cytochrome P450. Ad, adrenodoxin. The substrate is a steroid hormone biosynthesis intermediate. The location of the reaction is in the mitochondria of adrenal cortex. (Reproduced by permission from Bezkorovainy A. Biochemistry of Nonheme Iron. New York Plenum Press, 1980, p. 380.)...

Animal "Adrenodoxin (adrenal cortex, testis, ovary)... 

The purification procedure for the pig testis non-heme iron protein has been published elsewhere (28, 43). The procedure is similar to Procedure B . The isolation of testis mitochondrial fraction, Procedure A , is not recommended due to the low yield of the mitochondria and loss of the non-heme iron protein. The yield of the non-heme iron protein from testis was very much lower than the yield of adrenodoxin from adrenal cortex. This may indicate that testis hydroxylation is less active than adrenal hydroxylation. 

Our study on the distribution of electron transferring proteins in animal sources is still in progress. From present knowledge, adrenodoxin can be found in adrenal cortexes from pig, beef, and rat. Further, a similar protein was isolated from pig testis (see II-A-2), and it was also found in the ovary. However, brain, heart, liver, kidney, and pancreas appear to lack adrenodoxin-like protein. If this is correct, the proteins of the ferredoxin family are located solely in the glands which directly act in the biosynthesis of steroid hormones. It is of interest that adrenodoxin-like protein does not participate in the steroid hydroxylation involved in cholesterol and cholic acid biosyntheses. All of these reactions without the participation of adrenodoxin are similar to enzymes responsible for microsomal non-specific hydroxylation, which consist of the following sequence of electron transfer ... 

The protein globule of cytochrome P450 from the adrenal cortex consists of two fragments. The hydrophilic fragment FI has a molecular mass of 27000 and contains a heme and an adrenodoxin-binding site. The hydrophobic fragment of molecular mass 22000 binds the enzyme to the biomemrane. The presence of extensive hydrophobic portions has also been detected in liver cytochrome. 

Cytochromes are used in the conversion of cholesterol to the steroid hormones. Iliese hormones include aldosterone, cortisol, and the sex hormones. Synthesis of aldosterone, for example, occurs in the mitochondria of the adrenal cortex. One of the steps in aidostero ne synthesis is a hydroxylation that is cataly .ed by a complex of adrenodoxin reductase (FAD-containing protein), adrenodoxin (nonheme iron protein), and cytochrome P450 (heme protein). 

The 2 Fe 2S plant type ferredoxins, MW 12,000 dal ton, Em = —430 mV, were first isolated from chloroplast and photosynthetic bacteria. Similar proteins have been purified from the bacteria E. coli (264) and Pseudomonas putida [ putidaredoxin , Em7 = —235 mV, (275)] and from mammalian adrenal cortex mitochondria [ adrenodoxin Em = — 367 mV, 13,100 dalton (165)] among other sources. 

The existence of mitochondrial cytochrome P450 in adrenal cortex was reported originally by Harding et al (1964) and confirmed by subsequent studies. Adrenal cortical mitochondria catalyze a number of NADPH- and molecular-oxygen-dependent hydroxylation reactions that contribute to the biosynthesis of corticosteroids. The enzyme system for 11 jS-hydroxyla-tion has been isolated, and a successful reconstitution of its activity has been achieved by the interaction of three proteins, namely an NADPH-dependent flavoprotein (adrenodoxin reductase), an iron-sulfur protein (adrenodoxin), and the heme protein (cytochrome P450) that serves as the terminal oxidase for the electron transport system from NADPH to oxygen (Wang and... 

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