Cytochrome b5 Augments the 17,20-Lyase Activity of Human P450c17 without Direct Electron Transfer*

Richard J. AuchusDagger §, Tim C. LeeDagger , and Walter L. MillerDagger par **

From the Departments of Dagger  Pediatrics and § Internal Medicine and the par  Metabolic Research Unit, University of California, San Francisco, California 94143-0978

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

In the biosynthesis of steroid hormones, P450c17 is the single enzyme that catalyzes both the 17alpha -hydroxylation of 21-carbon steroids and the 17,20-lyase activity that cleaves the C17-C20 bond to produce C19 sex steroids. Cytochrome b5 augments the 17,20-lyase activity of cytochrome P450c17 in vitro, but this has not been demonstrated in membranes, and the mechanism of this action is unknown. We expressed human P450c17, human P450-oxidoreductase (OR), and/or human cytochrome b5 in Saccharomyces cerevisiae and analyzed the 17alpha -hydroxylase and 17,20-lyase activities of the resulting yeast microsomes. Yeast expressing only P450c17 have 17alpha -hydroxylase and trace 17,20-lyase activities toward both Delta 4 and Delta 5 steroids. Coexpression of human OR with P450c17 increases the Vmax of both the 17alpha -hydroxylase and 17,20-lyase reactions 5-fold; coexpression of human b5 with P450c17 also increases the Vmax of the 17,20-lyase reactions but not of the 17alpha -hydroxylase reactions. Simultaneous expression of human b5 with P450c17 and OR, or addition of purified human b5 to microsomes from yeast coexpressing human P450c17 and OR, further increases the Vmax of the 17,20-lyase reaction without altering 17alpha -hydroxylase activity. Genetically engineered yeast and mixing experiments demonstrate that OR is both necessary and sufficient for microsomal 17,20-lyase activity. Addition of purified human holo-b5, apo-b5, or cytochrome c to microsomes containing both human P450c17 and OR demonstrate that the stimulatory action of b5 does not require electron transfer from b5 to P450c17. These data suggest that human b5 acts principally as an allosteric effector that interacts primarily with the P450c17·OR complex to stimulate 17,20-lyase activity.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Among the many chemical transformations catalyzed by cytochrome P450 enzymes, steroid hormone hydroxylations, and cleavages are of particular interest because of their mechanistic complexities and essential roles in physiology (1). P450c17 catalyzes both 17alpha -hydroxylase and 17,20-lyase activities (2) (for review see Ref. 3) and also has a modest degree of 16alpha -hydroxylase activity (4). In human beings, the 17alpha -hydroxylase reaction leads to the glucocorticoid, cortisol, and the subsequent 17,20-lyase reaction leads to precursors of sex steroids. As the sole pathway leading to biosynthesis of circulating sex steroids, the regulation of this 17,20-lyase activity is central to understanding the developmental regulation of dehydroepiandrosterone sulfate (DHEA)1 with adrenarche and aging, and to the pathogenesis of the polycystic ovary syndrome (3). The 17,20-lyase activity, involving the oxidative cleavage of a carbon-carbon bond, is regulated in a tissue-specific and developmentally programmed manner by factors such as the abundance of the electron donor flavoprotein P450-oxidoreductase (OR) (5, 6), the co-existence of 3beta -hydroxysteroid dehydrogenase and P450c21 (7), and post-translational modification of P450c17 (8).

To perform catalysis, P450c17, like all other microsomal P450 oxygenases, must receive two electrons from NADPH via OR. Cytochrome b5 has also been implicated as a component of the 17,20-lyase reaction, as b5 augments 17,20-lyase activity and occasionally 17alpha -hydroxylase activity of P450c17 in reconstituted systems (9, 10); however, our laboratory could not confirm this effect in transfected monkey kidney COS-1 cells (5). Inconsistencies in the animal species of P450c17, OR, and b5 used in previous studies preclude extrapolation of the available biochemical data to human adrenal and gonadal physiology; furthermore, the mechanism(s) of these reported b5-mediated increases in 17,20-lyase activity remain unknown.

Among the various systems developed to study mammalian cytochromes P450, transfection of genetically modified yeast cells provides the opportunity to study the activities of a cytochrome P450 in the presence of various combinations of electron transfer proteins in the native microsomal environment (11). To clarify the function of cytochrome b5 in 17,20-lyase activity, we systematically varied the abundance of putative electron transfer proteins in yeast microsomes containing human P450c17. We find that human, but not yeast cytochrome b5 can selectively augment the rate of the 17,20-lyase reaction by more than 10-fold. However, this augmentation requires OR and occurs without electron transfer to or from cytochrome b5.

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Yeast Strains and Expression Vectors-- Wild type yeast strains W303A (Y150WT) (leu2-3, 112; his3-11, 15; trp1-1; ade2-1; ura3-1; mat a) and W303B (JC104) (trp1-1; ura3-1; ade2-1; can1-100; mat alpha ) were generous gifts of Drs. Gregory Petsko and Ira Herskowitz. Engineered yeast strains, W(B), W(hR), and W(BDelta ), generated by targeted disruption of the yeast CPR1 or YCY b5 loci (11-14) and the yeast expression vectors V10 and V60 (11) were generous gifts of Dr. Denis Pompon (CNRS, Gif-sur-Yvette, France). Human P450c17 cDNA (15) was PCR amplified with Pfu polymerase (Stratagene, La Jolla, CA) using primers c17-S-1 and c17-AS-1 (Table I) and pECE-c17 (16) as template. The resulting PCR product was digested with BamHI and EcoRI, facilitating directional cloning into complementary ends of BglII-EcoRI digested V10 vector, destroying the BglII site and placing the P450c17 cDNA under the control of the constitutive pgk promoter, producing vector V10-c17. V60 was modified by disruption of the ura3 gene by digestion with NcoI, blunting of the staggered ends, and religation, yielding vector pYeSF1. Using the unique BamHI site at the 5' end of the pgk promoter in V10, a BamHI-EcoRI fragment of the V10-c17 plasmid was cloned into BamHI-EcoRI digested pYeSF1 to provide ade2 complementation to the pgk-regulated P450c17 (vector pYeSF2-c17). Vector cDE2, used to generate doubly transformed yeast with either V10-c17 or pYeSF2-c17, was a generous gift from Dr. Ira Herskowitz (17). Human P450-oxidoreductase cDNA was PCR amplified from pECE-OR (5) using primers OR-S-1 and OR-AS-1 (Table I). The 5'-PCR primer contained two silent base pair changes from the wild type sequence to remove hairpin structures surrounding the translation initiation codon (12) which can inhibit transcription and translation in yeast (18). Cytochrome b5 cDNA was generated by reverse transcription of total human testicular RNA using random hexamers followed by PCR amplification with primers b5-S-1 and b5-AS-1 (Table I) based on the human b5 cDNA sequence (19). The human b5 and OR cDNAs were then cloned into the EcoRI site of cDE2 vector under control of the constitutive adc1 promoter with a trp1 selectable marker. The accuracy and orientation of all constructions were confirmed by DNA sequencing.

                              
View this table:
[in this window]
[in a new window]
 
Table I
PCR primers
Restriction sites (BamHI or EcoRI) are underlined, and ATG start codons are in bold type. Silent base pair changes to eliminate hairpin loop formation in OR-S-1 are underlined and in bold.

Yeast Transformation and Growth-- Yeast were transformed using 700 µl of 40% polyethylene glycol 3350, 0.1 M lithium acetate, 10 mM Tris-HCl (pH 8), 1 mM EDTA to transform 106 yeast in 100 µl of 0.1 M lithium acetate, 10 mM Tris-HCl (pH 8), 1 mM EDTA with 1-2 µg of plasmid DNA and 50 µg of denatured herring sperm carrier DNA (20). Cells were washed in 100 µl of 1 M sorbitol before final resuspension in 100 µl of 10 mM Tris-HCl (pH 8), 1 mM EDTA and plating onto selective media. All transformations introduced two plasmids simultaneously: the first, expressing P450c17, was either V10-c17 or pYeSF2-c17; the second was cDE2, containing no cDNA insert or the cDNA for either human OR or b5. Thus, P450c17 expression was always under the control of the constitutive pgk promotor, and all yeast producing different combinations of electron donor proteins were grown in the same culture medium. For microsome preparations, transformed yeast were cultured in minimal SD media containing 20 g/liter D-glucose or D-galactose, 1.7 g/liter yeast nitrogen base without amino acids or ammonium sulfate (Difco, Detroit, MI), 5 g/liter ammonium sulfate, and supplemented with the requisite combination of 10 mg/liter leucine, 15 mg/liter adenine, and 10 mg/liter histidine (11).

Microsome Preparation and Characterization-- Yeast cells harvested at a density of 4.5-6 × 107 cells/ml were disrupted by manual breakage with glass beads (450-600 micron) for 5 min (11). The breakage was stopped at 1-min intervals, and cells were iced for 30 s; 3 µl of 0.5 M ethanolic phenylmethylsulfonyl fluoride was added after the first minute of breakage. For a typical 300-ml culture, crude extracts and beads were washed twice with 5-7 ml of 50 mM Tris-HCl (pH 8), 1 mM EDTA, 0.4 M sorbitol, and the cellular debris was collected by centrifugation at 4 °C twice for 10 min at 14,000 × g. Microsomes were pelleted by centrifugation at 4 °C for 45 min at 100,000 × g and were resuspended in 50 mM Tris-HCl (pH 8), 1 mM EDTA, 20% glycerol at 5-20 µg/µl total protein. Preparations were homogenized by shearing microsomes through a 27-gauge needle 10 times and were kept frozen at -70 °C until needed. Human adrenal microsomes were prepared from excess surgical tissue as described (8).

Microsomal proteins were quantitated colorimetrically. Immunoblotting on polyvinylidene difluoride membranes (Millipore, Bedford, MA) was performed with rabbit antiserum to human P450c17 (5) or to human OR (generously provided by Prof. C. Roland Wolf, Imperial Cancer Institute, Dundee, United Kingdom) using secondary antibody-peroxidase conjugate and ECL reagents (Amersham, Arlington Heights, IL) and with goat antiserum to human b5 (Oxford Biomedical, Rochester Hills, MI) using secondary antibody-peroxidase conjugate (Santa Cruz Biotechnology, Santa Cruz, CA) and ECL reagents. Microsomal P450 and cytochrome b5 contents were measured spectroscopically (21) using either a Cary 3E or a Shimazdu UV160U spectrophotometer. P450 oxidoreductase activity was measured as described (22).

P450c17 Enzyme Assay-- Microsomes were assayed under initial rate kinetics by preincubation in 50 mM potassium phosphate buffer (pH 7.4) with 0.5-5 µM steroid (added in 4 µl of ethanol) in 200 µl total volume at 37 °C for 2 min before the addition of 1 mM NADPH to start the reaction. Each reaction contained either 20,000 cpm of [3H]pregnenolone, [3H]17alpha -hydroxypregnenolone (NEN Life Science Products Inc., Boston, MA), or [3H]17alpha -hydroxyprogesterone (Amersham) or 10,000 cpm of [14C]progesterone (NEN Life Science Products). Steroids were extracted with 400 µl of ethyl acetate/isooctane (1:1), concentrated under nitrogen, separated by thin layer chromatography (Whatman PE SIL G/UV silica gel plates, Maidstone, Kent, UK) using 3:1 chloroform/ethyl acetate, and quantitated as described (23). Purified recombinant human cytochrome b5 (Pan Vera, Madison, WI), apo-human cytochrome b5, or horse heart cytochrome c (Sigma) were included in incubations as indicated. Apo-cytochrome b5 was prepared from the Pan Vera holo-cytochrome b5 as described (24), and absent electron transfer properties of the resulting material was confirmed by difference spectroscopy. Kinetic behavior was approximated as a Michaelis-Menten system for data analysis, and all error bars shown represent standard deviations.

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Yeast Transfection and Microsome Characterization-- The capacity of b5 to increase the 17,20-lyase activity of P450c17 has been shown by several laboratories using purified, reconstituted protein systems (9, 25), but this phenomenon has not been observed in intact cell and microsome preparations (5). The development of "humanized" yeast strains (12) that express both P450c17 and selected electron donor proteins has enabled us to dissect this problem without detergent solubilization of individual components.

To study the effects of human OR and b5 on human P450c17 activities in yeast microsomes, parental yeast strain W303B was doubly transfected with vector V10 expressing human P450c17 and with vector cDE2 expressing either the cDNA for human OR or b5 (or empty vector). Microsomes from these transfectants were characterized and used for kinetic studies; microsomes were also prepared from transfections using the same vectors with the cDNA inserts exchanged. Both sets of transfections were also performed using yeast strain W303A; abbreviated kinetic experiments using these W303A-derived microsome preparations yielded qualitatively similar results as did microsomes and spheroplasts prepared from the W303B transfectants; thus, W303B doubly transfected with V10-c17 plus cDE2 (for expressing an electron donor) was used in all subsequent experiments.

P450 content, total OR (cytochrome c reductase activity), and total cytochrome b5 content were similar among the three microsome preparations from co-transfected W303B yeast (Table II). Essentially all of the P450 is from P450c17, whereas cytochrome c reductase activity and total cytochrome b5 content were similar in all transfectants, indicating that endogenous yeast OR and b5 are the predominant electron transfer proteins in these microsomes. Comparable expression of P450c17 was demonstrated in all samples by Western blotting, and human OR and b5 were detected only in samples from yeast containing their respective cDNAs, as expected (Fig. 1).

                              
View this table:
[in this window]
[in a new window]
 
Table II
Total P450 content, cytochrome c reductase activity, and cytochrome b5 content in yeast microsomes


View larger version (101K):
[in this window]
[in a new window]
 
Fig. 1.   Immunoblot of yeast microsomal proteins. Denatured microsomal proteins (2-5 µg) from yeast transfected with the various vectors as labeled at the top of the figure were electrophoresed through a 10% SDS-polyacrylamide gel, blotted to polyvinylidene difluoride, and probed with antisera to P450c17 and OR. Expression of human P450c17 (c17) is comparable in all samples, but human OR is expressed only in yeast containing the OR expression vector. Immunoblotting with antisera to rat or rabbit b5 showed aggregation and cross-reactivity (not shown). Dash (-) indicates use of empty vector.

Kinetics-- To determine how the presence of human OR and/or b5 alters the activities of human P450c17 in yeast microsomes, we measured apparent Km and Vmax values for both 17alpha -hydroxylase and 17,20-lyase reactions for Delta 5 and Delta 4 substrates (Table III). Lineweaver-Burk plots (Fig. 2) show that yeast transfected with human P450c17 alone perform both 17alpha -hydroxylase and 17,20-lyase reactions despite the absence of human electron transfer proteins, indicating that the endogenous yeast OR can couple with human P450c17, as has been shown for bovine P450c17 (26, 27). In this system, however, 17,20-lyase activity is very low but not absent, as reported for bovine P450c17 (26). Co-expression of human OR substantially increases both activities. Using the Delta 5 steroid substrates pregnenolone and 17alpha -hydroxypregnenolone, co-expression of human OR raises the Vmax for both the 17alpha -hydroxylase and 17,20-lyase reactions 5-fold. In yeast microsomes, the apparent Km for both pregnenolone and 17alpha -hydroxypregnenolone is about 1 µM, and co-expression of human OR lowers the Km to below 0.5 µM, suggesting that the association of human P450c17 and OR may increase the affinity of P450c17 for Delta 5 substrates. The presence of OR also alters the orientation of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in the substrate-binding pocket of P450 2D6 (28), suggesting that OR may participate in substrate discrimination by P450 enzymes. DHEA formed by microsomes containing both human P450c17 and OR is metabolized further to a more polar compound, possibly 16alpha -hydroxy-DHEA, the major DHEA metabolite of a bovine P450c17/rat OR fusion protein (29). Human OR markedly stimulated both activities without a significant change in total cytochrome c reductase activity, indicating that yeast OR is an inefficient electron donor for P450c17 and does not significantly interfere with catalysis in the presence of human OR.

                              
View this table:
[in this window]
[in a new window]
 
Table III
Kinetic constants
Apparent Km and Vmax values were obtained from linear regression analysis of the data in Fig. 2.


View larger version (12K):
[in this window]
[in a new window]
 
Fig. 2.   Lineweaver-Burk plots of 17alpha -hydroxylase and 17,20-lyase activities. Lines were derived from least-squares fit to data points (r2 > 0.92 for all lines). The apparent Km and Vmax values obtained from these data are shown in Table III. Microsomes were prepared from W303B yeast co-transfected with V10-c17 and the empty cDE2 vector (squares), V10-c17 and cDE2-OR (circles), or V10-c17 and cDE2-b5 (triangles). Panel A, incubations with Delta 5 pregnenolone. Panel B, incubations with Delta 5 17alpha -hydroxypregnenolone. Panel C, incubations with Delta 4 progesterone. Panel D, incubations with Delta 4 17alpha - hydroxyprogesterone.

When Delta 4 substrates are used, human P450c17 efficiently catalyzed the conversion of progesterone to 17alpha -hydroxyprogesterone, but the conversion of 17alpha -hydroxyprogesterone to androstenedione was much less efficient than the corresponding conversion of 17alpha -hydroxypregnenolone to DHEA (Table III). The slow turnover of 17alpha -hydroxyprogesterone by human P450c17 explains why circulating androgens in humans derive principally from the isomerization and reduction of DHEA rather than by cleavage of 17alpha -hydroxyprogesterone to androstenedione, the predominant pathway in rodents. Guinea pig P450c17, for example, preferentially converts progesterone to androstenedione, some of which is sequentially metabolized without dissociation of the intermediate 17alpha -hydroxyprogesterone from the active site (30, 31). Co-expression of human OR similarly increases the Vmax of both activities toward Delta 4 steroids but without a significant change in apparent Km values (Table III). A second, more polar product, presumably 16alpha -hydroxyprogesterone (4, 5, 32), constitutes ~20-25% of the products when Delta 4-progesterone is the substrate with all microsomes tested.

Co-expression of human b5 with human P450c17 increases Vmax 10-fold for the 17,20-lyase reaction but not for the 17alpha -hydroxylase reaction with both Delta 5 and Delta 4 substrates and does not change the apparent Km for any substrate tested (Table III). Although human OR improves the catalytic efficiency of P450c17 in yeast microsomes, both by lowering the Km of Delta 5 substrates and increasing the Vmax for all reactions, the sole effect of human b5 is to augment the Vmax for the 17,20-lyase reactions. Our results generally agree with those obtained with reconstituted recombinant human P450c17 and rat OR, except that rat b5 approximately doubles the rate of hydroxylation of Delta 5-pregnenolone but not of Delta 4-progesterone (25). Differences in species of origin of the OR and b5 used may explain some differences in the results obtained in the two systems, as well as subtle differences in the activities of microsomal and detergent-solubilized proteins.

Activities in the Absence of Yeast OR or Yeast Cytochrome b5-- The experiments described above were performed in the presence of endogenous yeast OR and b5 in the microsome preparations. To determine whether the yeast electron donors influence human P450c17 activities, we expressed human P450c17, with human OR or b5, in engineered yeast strains lacking the endogenous yeast OR or b5 genes. When human P450c17 and OR were coexpressed in yeast strain W(BDelta ), which lacks the yeast homolog of the human b5 gene (13), the resulting microsomes contained 85% of the 17alpha -hydroxylase activity and 73% of the 17,20-lyase activity of microsomes from W303B yeast (Fig. 3A). The 17,20-lyase activity was minimally affected by the absence of yeast b5, demonstrating that OR is both necessary and sufficient to confer both 17alpha -hydroxylase and 17,20-lyase activity to human P450c17 in yeast microsomes.


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 3.   Human P450c17 activities in engineered yeast strains. Panel A, 17alpha -hydroxylase (open bars) and 17,20-lyase (hatched bars) activities in microsomes prepared from yeast strains W(BDelta ), lacking yeast b5, or from W303B yeast using 1 µM steroid. Note the 10-fold difference in the scales of hydroxylase activity (left) and lyase activity (right). Error bars (±S.D.) are too tight to be seen in three of the four bars. Panel B, P450c17 activities in microsomes from W(B) yeast (lacking yeast OR) co-transfected with V10-c17 and empty cDE2 vector or cDE2-OR. Clones were grown in glucose, producing trace (lo) amounts of human b5, or in galactose, inducing high (hi) amounts of b5. Incubations contained 1 µM steroid and 25 or 125 µg of microsomal protein to assay 17alpha -hydroxylase activity (1 h incubation, lanes 1-4) or 17,20-lyase activity (4 h incubation, lanes 5-8), respectively. Panel C, Lineweaver-Burk plot of 17,20-lyase activity in microsomes from W(B) yeast co-transfected with pYeSF2-c17 and cDE2-OR. Microsomes were prepared from the same yeast clone expressing trace human b5 (grown in glucose, squares), or expressing high b5 (grown in galactose, circles). Apparent Km and Vmax values were derived from least-squares fits to the data.

To confirm that OR was required for catalysis, we expressed human P450c17 in strain W(B), in which the endogenous yeast OR locus is replaced by the human b5 cDNA under the control of the inducible Gal10/Cyc1 promoter (12). No 17alpha -hydroxylase or 17,20-lyase activity is present in microsomes prepared from W(B) yeast transfected with human P450c17 and empty cDE2 vector, but both activities are restored by co-transfection of human OR (Fig. 3B). When W(B) yeast, transfected with both human P450c17 and OR, were grown in galactose to induce expression of human b5 as well, the presence of human b5 increased the Vmax of the 17,20-lyase reaction using 17alpha -hydroxypregnenolone from 0.14 min-1 to 1.1 min-1 but did not change the apparent Km (0.3 µM) (Fig. 3, B and C). This induction of human b5 did not significantly change 17alpha -hydroxylase activity, reflected by comparable pregnenolone consumption (Fig. 3B, lanes 3 and 4), but the 17alpha -hydroxypregnenolone formed in the presence of high amounts of human b5 was rapidly converted to DHEA, so that little 17alpha -hydroxypregnenolone accumulated (Fig. 3B, lane 4).

Effect of Exogenous Soluble b5 on 17alpha -Hydroxylase and 17,20-Lyase-- Exogenously added soluble b5 can influence other P450 reactions in yeast microsomes (11); therefore, we added purified human b5 to yeast microsomes containing human P450c17. Although 17alpha -hydroxylase activity against Delta 5-pregnenolone or Delta 4-progesterone was not changed (Fig. 4, A and C), 17,20-lyase activity against 17alpha -hydroxypregnenolone was increased up to 10-fold in microsomes that did not already contain human b5 (Fig. 4B). Purified b5 also increased 17,20-lyase activity toward 17alpha -hydroxyprogesterone, but only about 2-fold (Fig. 4D). These data demonstrate that yeast b5 can neither support nor stimulate human P450c17 activities, as found for other human P450s (12). Furthermore, our results show that the only effect of human b5, either added in solution or coexpressed into microsomes, is to increase the rate of the 17,20-lyase reactions, and that this action of b5 requires the presence of yeast or human OR.


View larger version (36K):
[in this window]
[in a new window]
 
Fig. 4.   Effect of exogenously added human b5 on P450c17 activities. Microsomes were prepared from yeast strain W303B expressing human P450c17 and co-transfected with cDE2 vector expressing no protein, OR, or b5 as indicated. The indicated steroid for each panel (5 µM) was incubated with (+) and without (-) 1 molar equivalent of exogenously added purified human b5 per molar equivalent of P450c17. Incubations contained Delta 5 pregnenolone (panel A), Delta 5 17alpha -hydroxypregnenolone (panel B), Delta 4 progesterone (panel C), and Delta 4 17alpha -hydroxyprogesterone (panel D). Note the different scales in each panel.

The results described above do not exclude a contribution of human b5 as the donor of the second of the two electrons in the P450 catalytic cycle, as has been suggested (33, 34). If b5 functions as the donor of the second electron, b5 should support catalysis by transporting electrons either from a reducing agent (sodium dithionite) or from NADPH-reduced OR to microsomes containing P450c17 that has already been reduced with the first electron. Dithionite, which can provide one electron to either P450c17 or b5, does not support catalysis in microsomes containing both human P450c17 and b5, but dithionite does not abolish catalysis when the second electron is provided to P450c17 from NADPH via OR (Fig. 5A). To confirm this observation, we attempted to reconstitute 17,20-lyase activity by transferring electrons from NADPH to one pool of microsomes containing human OR (and no P450c17), then to soluble human b5 as an electron conduit, and finally to human P450c17 in another pool of microsomes lacking OR. Soluble b5 was first reduced with NADPH by microsomes containing OR (35), and then added to microsomes lacking yeast OR (strain W(B)) but containing human P450c17 alone (lane 1), human P450c17 and OR (lane 2), or human P450c17 and b5 (but no OR, lane 3), all of which had been preincubated with 17alpha -hydroxypregnenolone and dithionite to provide the first electron to P450c17. Microsomes lacking human OR converted only a trace of 17alpha -hydroxypregnenolone to DHEA under these conditions, but microsomes containing both human P450c17 and OR could use the added NADPH to convert substrate to DHEA (Fig. 5B). These results confirm that b5, reduced either by dithionite or OR, cannot provide sufficient electron transfer to P450c17 to support significant 17,20-lyase activity. Therefore, these data suggest that the mechanism by which b5 enhances 17,20-lyase activity does not involve electron transfer.


View larger version (38K):
[in this window]
[in a new window]
 
Fig. 5.   Reconstitution of human P450c17 activities using sodium dithionite and cytochrome b5. Panel A, 17alpha -hydroxylase and 17,20-lyase activities in microsomes from W(B) yeast (lacking yeast OR) co-transfected with pYeSF2-c17 and cDE2-b5 (0.1 mg of protein containing 9 pmol of P450 and 42 pmol of b5). Microsomes were incubated with either 1 µM pregnenolone (lanes 1 and 3) or 1 µM 17alpha -hydroxypregnenolone (lanes 2 and 4) and a saturating portion of solid sodium dithionite (21). Incubations for lanes and 4 also contained NADPH and additional microsomes (16 µg of protein) containing human P450c17 (1 pmol) and OR. Panel B, 17,20-lyase activity in microsomes containing 5 pmol of human P450c17 alone (lane 1), with human OR (lane 2), or with human b5 (lane 3). Microsomes were preincubated with 1 µM 17alpha -hydroxypregnenolone and solid sodium dithionite in 100 µl, and the reactions were started by adding an equal volume of a second incubation containing soluble human b5 (50 pmol) plus microsomes containing human OR but no P450c17 (50 µg of protein, cytochrome c reductase activity, 104 ± 15 nmol/min/mg protein) and NADPH (2 mM) to reduce the soluble b5. Migrations of Delta 5 steroids are indicated (S, 17alpha -hydroxypregnenolone standard).

How Does Human Cytochrome b5 Augment 17,20-Lyase Activity?-- To explore the mechanism by which b5 increases 17,20-lyase activity, we assayed the 17,20-lyase activity of microsomes containing constant, high amounts of P450c17 and OR and varying amounts of b5. A sharp increase in 17,20-lyase activity was observed when the molar ratio of b5 to P450c17 approached 1:1 (Fig. 6A). Activity reached a maximum at ratios of b5 to P450c17 between 10:1 and 30:1; however, further addition of human b5 progressively inhibited 17,20-lyase activity in both yeast and human adrenal microsomes. If human b5 was acting as the preferred electron donor, 17,20-lyase activity should saturate and remain constant rather than fall at high b5/P450c17 ratios. Similarly, when we examined the influence of b5 on the 17,20-lyase activity of microsomes containing very small amounts of human OR and no yeast OR (strain W(hR) transfected with P450c17 grown to high density in glucose), maximal stimulation occurred at a b5/P450c17 ratio between 1:1 and 3:1, and higher ratios were again inhibitory (Fig. 6A). Thus, the influence of human b5 changes dramatically as the abundance of human OR and the b5/P450c17 ratio are varied. These data suggest that b5 does not function as an electron donor, but instead exerts some other action, perhaps facilitating electron transfer from OR to P450c17 or improving coupling efficiency, as has been suggested for other P450 reactions stimulated by b5 (24, 36).


View larger version (13K):
[in this window]
[in a new window]
 
Fig. 6.   Activation and inhibition of 17,20-lyase activity by cytochromes b5 and c. Conversion of 0.5 µM 17alpha -hydroxypregnenolone to DHEA by human adrenal microsomes (triangles), yeast microsomes with high amounts of human OR (squares), or yeast microsomes with low amounts of human OR (circles) plus the indicated molar ratios of human holo-b5 (panel A), human apo-b5 (panel B), or horse heart cytochrome c (panel C). Activity is expressed as the percent of conversion by the microsomes alone. The yeast microsomes were prepared from strain W(hR) either co-transfected with pYeSF2-c17 plus cDE2-OR and grown in galactose, yielding the microsomes with high amounts of human OR, or co-transfected with pYeSF2-c17 plus empty cDE2 vector and grown in glucose, yielding the microsomes with low amounts of human OR (cytochrome c reductase activities of 223 and 12 nmol/min/mg protein, respectively).

Inhibition of enzymatic activity at high b5/P450c17 ratios, a phenomenon also observed in guinea pig adrenal microsomes (10), could result from a second, inhibitory b5-binding site on P450c17 or from a competition between b5 and P450c17 for electrons from limiting amounts of OR. Cytochrome c, which is also a substrate for reduction by OR (37), also inhibits 17,20-lyase activity at molar ratios above 10:1, the same molar ratios at which b5 becomes inhibitory (Fig. 6C). Inhibition by equivalent molar ratios of cytochrome c to P450c17 is consistent with b5 competing with P450c17 for reduction when OR is limiting, but "reverse" electron transfer from P450c17 to b5 (38) may also contribute to the inhibition observed at higher b5/P450c17 ratios. These data suggest that electron transfer from OR to b5 is actually detrimental to 17,20-lyase activity. Therefore, we determined whether human apo-b5, which lacks the heme and hence cannot participate in electron transfer, modulates 17,20-lyase activity differently than human holo-b5. In microsomes containing either low or high amounts of human OR, molar ratios of apo-b5 to P450c17 between 1:1 and 10:1 augment 17,20-lyase activity (Fig. 6B). Unlike the data with holo-b5, the stimulatory effect of apo-b5 remains constant rather than falling at higher b5/P450c17 ratios. These results exclude direct electron transfer from b5 as the principal means by which b5 augments 17,20-lyase activity and suggest that b5 exerts a saturable, allosteric effect on the P450c17·OR complex.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

The 17,20-lyase/17alpha -hydroxylase ratio in the human adrenal rises dramatically with the onset of adrenarche at age 8-10, reaches maximal values at age 25-35, and then falls progressively with aging (39); as these phenomena occur only in human beings and great apes (40), their study is difficult. These selective, physiologic, developmentally programmed changes in human adrenal 17,20-lyase activity imply regulatory mechanisms beyond transcription of P450c17 or OR (3, 8). Most P450 enzymes catalyze multiple reactions, but the ratio of their activities remains fixed. The developmentally and possibly hormonally programmed changes in the ratio of 17,20-lyase to 17alpha -hydroxylase activities of human P450c17 provide a unique system for studying the differential regulation of two reactions catalyzed by a single P450 enzyme.

An augmentation of the 17,20-lyase activity of P450c17 by b5 has been observed in vitro (9, 25) but was not seen in transfected COS-1 cells (5), possibly because the endogenous b5 in those cells was sufficient to stimulate 17,20-lyase activity maximally. Thus, it has not been clear how or if b5 regulates human P450c17 activities in vivo. The use of microsomes from yeast engineered to express human P450c17, OR, or b5 from inducible promoters permits the quantitative manipulation of each protein in a membrane environment that should simulate events in vivo. This permits greater experimental flexibility than the use of bicistronic plasmids (41), fusion proteins (29), or viral vectors (42), and obviates concerns about the relevance of data from detergent-solubilized systems to in vivo systems.

Titration experiments with purified human holo-b5, apo-b5, and cytochrome c showed that the stimulatory effect of b5 on 17,20-lyase activity is not mediated by electron transfer from b5 and suggest that b5 exerts an allosteric effect on the P450c17·OR complex. This proposed mechanism could explain three observations from other laboratories. First, b5 facilitates electron transfer from OR to P450 3A4 only when all three proteins are premixed before adding NADPH and substrate, but not when b5 is premixed with P450 3A4 and added to OR, NADPH, and substrate in stop-flow experiments (24). These data suggested that the stimulatory action of b5 on testosterone 6beta -hydroxylation by P450 3A4 was an allosteric effect and was not mediated by an action of b5 as an alternate electron donor (24). Second, b5 is a more potent stimulator of 17,20-lyase activity when the abundance of OR is low, and this stimulation is quite sensitive to small changes in these low amounts of OR (10). Our results corroborate these studies and suggest that b5 interacts primarily with the P450c17·OR complex and not with P450c17 alone. Third, the redox-active core 1 segment of porcine b5 alone cannot augment the 17,20-lyase activity of human P450c17 (43), consistent with our findings that electron transfer from human b5 is not required to stimulate 17,20-lyase activity.

Three conclusions about human physiology emerge from our analysis of the kinetics of human P450c17. First, human androgen biosynthesis proceeds predominantly through the pathway 17alpha -hydroxypregnenolone right-arrow DHEA right-arrow androstenedione, rather than through the pathway 17alpha -hydroxypregnenolone right-arrow 17alpha -hydroxyprogesterone right-arrow androstenedione. The pathway via DHEA predominates because the apparent Km for Delta 4 17alpha -hydroxyprogesterone is about 10-fold higher and its Vmax is one-tenth as fast as the corresponding values for Delta 5 17alpha -hydroxypregnenolone. Thus, the catalytic efficiency Vmax/Km for the 17,20-lyase reaction is nearly 100-fold greater for Delta 5 17alpha -hydroxypregnenolone than for Delta 4 17alpha -hydroxyprogesterone. Second, significant androgen biosynthesis via the Delta 4 pathway can only occur in the presence of very high Delta 4 17alpha -hydroxyprogesterone concentrations, as found in untreated patients with 21-hydroxylase deficiency (44). Third, considerable microsomal 17,20-lyase activity is found even in the complete absence of b5; therefore, b5 deficiency cannot cause a syndrome of complete 17,20-lyase deficiency (23) as has been suggested (45).

The structural nature of the interaction of P450c17 with OR is not known, but the x-ray crystal structures of rat OR (46) and P450-BMP (47) provide useful clues. The redox-partner binding site for P450-BMP, a Type II (microsomal) P450, comprises the surface surrounding a depression in the "proximal" face of the protein that extends down to the face of the heme opposite the substrate-binding pocket (47, 48). This crevasse is lined on one side with positively charged residues from the J' and K helices (in P450-BMP, lysines 325, 328, and 331) which appear to participate in electrostatic pairing with negatively charged residues in OR. Molecular modeling shows that human P450c17 has a similarly located crevasse of positively charged residues that interact with redox partners (23). The electron-donating FMN moiety of rat OR also lies at the base of a concave cleft formed by the butterfly-shaped apposition of the FMN and FAD domains (46). However, the FMN domain joins the remainder of the protein via a disordered, flexible hinge that must flex about 90° for the FMN moiety to extend out from the concave cleft of OR (46) to approach the concave redox-partner binding site of P450c17 (23).

Because b5 normally participates in redox reactions such as methemoglobin reduction (49) and stearyl-CoA desaturation (50) and can serve as an alternate electron donor in some other P450 reactions (51), our demonstration that b5 serves a role as an allosteric facilitator of electron transfer from OR to P450c17 was unexpected. The binding of redox partners to P450c17 must transmit subtle changes to the substrate-binding pocket, as evidenced by the lower Km values for Delta 5 substrates in the presence of human OR (Table III) and by analogy to the altered regiospecificity of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine oxygenation by P450 2D6 in the presence of OR (28). Furthermore, the oxidative scission of the C17-C20 bond of 17alpha -hydroxypregnenolone appears to impose much more stringent constraints on the active-site topology of P450c17 than does the 17alpha -hydroxylase reaction. Therefore, we propose that b5 optimizes the geometry of the P450c17·OR complex for the more sensitive 17,20-lyase reaction perhaps by forming a ternary complex (Fig. 7). The structural core 2 domain of b5 may be the region that stimulates 17,20-lyase activity, as core 2 adopts a similar conformation in the NMR structures of both isolated holo-b5 (52) and apo-b5 (53), whereas the heme-binding core 1 domain is disordered in apo-b5 (53). Furthermore, core 2 retains its overall topology during molecular dynamics simulations of apo-b5, while core 1 loses secondary structure and exhibits conformational mobility (54).


View larger version (40K):
[in this window]
[in a new window]
 
Fig. 7.   Proposed function of cytochrome b5. I, NADPH donates two electrons to the FAD domain of OR (touching the microsomal membrane), which then pass to the FMN moiety. II, the FMN domain of OR, which is connected to the FAD domain by a connecting domain and a hinge region (H) must rotate about 90° (counterclockwise in the figure) to dock with the redox-partner binding site of P450c17. The interaction of P450c17 and OR is adequate to support 17alpha -hydroxyation, but this complex rarely adopts the geometry required to catalyze the 17,20-lyase reaction. III, the presence of either holo-b5 or apo-b5 favors the interaction of OR and P450c17 in an orientation that satisfies the more stringent conformational restrictions required by the 17,20-lyase reaction, facilitating productive electron transfer from OR to P450c17 and subsequent catalysis. The precise site(s) of action of b5 remain unknown.

A role for b5 as an allosteric effector protein is consistent with our observation that serine phosphorylation of P450c17 selectively increases 17,20-lyase activity (8) and that mutations of arginine residues in the redox-partner binding site of human P450c17 cause isolated 17,20-lyase deficiency (23). The precise orientation of OR in the electron-donor docking region of P450c17 required to assemble the active oxygenating complex for the 17,20-lyase reaction is impaired by mutation of this surface and enhanced by b5 or apo-b5. Phosphorylation of P450c17 probably favors assembly of productive complexes so that electron transfer is more rapid and coupling efficiency is higher; however, the exact mechanism by which phosphorylated serine residues enhance 17,20-lyase activity is not yet known. A more detailed understanding of these complexes is essential for understanding the regulation of 17,20-lyase activity; this in turn may permit development of agents to inhibit this activity, which will aid in the treatment of sex steroid-dependent malignancies and disorders of androgen excess.

    ACKNOWLEDGEMENTS

We thank Dr. Denis Pompon for yeast strains W(B), W(hR), and W(BDelta ) and for yeast vectors V10 and V60; Drs. Gregory Petsko and Ira Herskowitz for yeast strains W303A and W303B and for vector pYcDE2; Dr. C. Roland Wolf for antiserum to human OR; and Drs. Phillipe Urban and Gilles Truan for valuable discussions.

    FOOTNOTES

* This work was supported in part by the National Cooperative Program for Infertility Research, University of California, San Francisco, Grant U54-HD34449 (to W. L. M.), National Institutes of Health Grants DK37922 and DK42154 (to W. L. M.), and Clinical Investigator Award DK02387 (to R. J. A.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Postdoctoral fellow of the Howard Hughes Medical Institute during the initial stages of this work.

** To whom correspondence should be addressed: Dept. of Pediatrics, Bldg. MR-IV, Box 0978, University of California, San Francisco, San Francisco, CA 94143-0978. Tel.: 415-476-2598; Fax: 415-476-6286.

1 The abbreviations used are: DHEA, dehydroepiandrosterone; OR, P450-oxidoreductase; b5, cytochrome b5; V10, vector pYeDP10; V60, vector pYeDP60; cDE2, vector pYcDE-2; PCR, polymerase chain reaction.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Miller, W. L. (1988) Endocr. Rev. 9, 295-318[Medline] [Order article via Infotrieve]
  2. Nakajin, S., and Hall, P. F. (1981) J. Biol. Chem. 256, 3871-3876[Abstract/Free Full Text]
  3. Miller, W. L., Auchus, R. J., and Geller, D. H. (1997) Steroids 62, 135-144
  4. Nakajin, S., Takahashi, M., Shinoda, M., and Hall, P. F. (1985) Biochem. Biophys. Res. Commun. 132, 708-713[Medline] [Order article via Infotrieve]
  5. Lin, D., Black, S. M., Nagahama, Y., and Miller, W. L. (1993) Endocrinology 132, 2498-2506[Abstract]
  6. Yanagibashi, K., and Hall, P. F. (1986) J. Biol. Chem. 261, 8429-8433[Abstract/Free Full Text]
  7. Conley, A. J., and Bird, I. M. (1997) Biol. Reprod. 56, 789-799[Medline] [Order article via Infotrieve]
  8. Zhang, L., Rodriguez, H., Ohno, S., and Miller, W. L. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 10619-10623[Abstract]
  9. Onoda, M., and Hall, P. F. (1982) Biochem. Biophys. Res. Commun. 108, 454-460[Medline] [Order article via Infotrieve]
  10. Kominami, S., Ogawa, N., Morimune, R., Huang, D. Y., Takemori, S. (1992) J. Steroid Biochem. Mol. Biol. 42, 57-64[CrossRef][Medline] [Order article via Infotrieve]
  11. Pompon, D., Louerat, B., Bronine, A., and Urban, P. (1996) Methods Enzymol. 272, 51-64[CrossRef][Medline] [Order article via Infotrieve]
  12. Urban, P., Truan, G., Gautier, J. C., Pompon, D. (1993) Biochem. Soc. Trans. 21, 1028-1034[Medline] [Order article via Infotrieve]
  13. Truan, G., Cullin, C., Reisdorf, P., Urban, P., and Pompon, D. (1993) Gene (Amst.) 125, 49-55[CrossRef][Medline] [Order article via Infotrieve]
  14. Truan, G., Epinat, J. C., Rougeulle, C., Cullin, C., and Pompon, D. (1994) Gene (Amst.) 142, 123-127[CrossRef][Medline] [Order article via Infotrieve]
  15. Chung, B., Picado-Leonard, J., Haniu, M., Bienkowski, M., Hall, P. F., Shivley, J. E., Miller, W. L. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 407-411[Abstract]
  16. Lin, D., Harikrishna, J. A., Moore, C. C. D., Jones, K. L., Miller, W. L. (1991) J. Biol. Chem. 266, 15992-15998[Abstract/Free Full Text]
  17. Hadfield, C., Cashmore, A. M., and Meacock, P. A. (1986) Gene (Amst.) 45, 149-158[CrossRef][Medline] [Order article via Infotrieve]
  18. Baim, S. B., and Sherman, F. (1988) Mol. Cell. Biol. 8, 1591-1601[Medline] [Order article via Infotrieve]
  19. Yoo, M., and Steggles, A. W. (1988) Biochem. Biophys. Res. Commun. 156, 576-580[Medline] [Order article via Infotrieve]
  20. Gietz, D., St. Jean, A., Woods, R. A., Schiestl, R. H. (1992) Nucleic Acids Res. 20, 1425[Medline] [Order article via Infotrieve]
  21. Omura, T., and Sato, R. (1964) J. Biol. Chem. 239, 2370-2378[Free Full Text]
  22. Peyronneau, M. A., Renaud, J. P., Jaouen, M., Urban, P., Cullin, C., Pompon, D., and Mansuy, D. (1993) Eur. J. Biochem. 218, 355-361[Abstract]
  23. Geller, D. H., Auchus, R. J., Mendonça, B. B., Miller, W. L. (1997) Nature Genet. 17, 201-205[Medline] [Order article via Infotrieve]
  24. Yamazaki, H., Johnson, W. W., Ueng, Y. F., Shimada, T., Guengerich, F. P. (1996) J. Biol. Chem. 271, 27438-27444[Abstract/Free Full Text]
  25. Katagiri, M., Kagawa, N., and Waterman, M. R. (1995) Arch. Biochem. Biophys. 317, 343-347[CrossRef][Medline] [Order article via Infotrieve]
  26. Sakaki, T., Shibata, M., Yabusaki, Y., Murakami, H., and Ohkawa, H. (1989) DNA 8, 409-418[Medline] [Order article via Infotrieve]
  27. Barnes, H. J., Jenkins, C. M., and Waterman, M. R. (1994) Arch. Biochem. Biophys. 315, 489-494[CrossRef][Medline] [Order article via Infotrieve]
  28. Modi, S., Gilham, D. E., Sutcliffe, M. J., Lian, L. Y., Primrose, W. U., Wolf, C. R., Roberts, G. C. (1997) Biochemistry 36, 4461-4470[CrossRef][Medline] [Order article via Infotrieve]
  29. Shet, M. S., Fisher, C. W., Arlotto, M. P., Shackleton, C. H. L., Holmans, P. L., Martin-Wixtrom, C. A., Saeki, Y., Estabrook, R. W. (1994) Arch. Biochem. Biophys. 311, 402-417[CrossRef][Medline] [Order article via Infotrieve]
  30. Higuchi, A., Kominami, S., and Takemori, S. (1991) Biochim. Biophys. Acta 1084, 240-246[Medline] [Order article via Infotrieve]
  31. Tagashira, H., Kominami, S., and Takemori, S. (1995) Biochemistry 34, 10939-10945[Medline] [Order article via Infotrieve]
  32. Swart, P., Swart, A. C., Waterman, M. R., Estabrook, R. W., Mason, J. I. (1993) J. Clin. Endocrinol. Metab. 77, 98-102[Abstract]
  33. Hildebrandt, A., and Estabrook, R. W. (1971) Arch. Biochem. Biophys. 143, 66-79[Medline] [Order article via Infotrieve]
  34. Hall, P. F. (1985) Vitam. Horm. 42, 315-368[Medline] [Order article via Infotrieve]
  35. Enoch, H. G., and Strittmatter, P. (1979) J. Biol. Chem. 254, 8976-8981[Medline] [Order article via Infotrieve]
  36. Pompon, D. (1987) Biochemistry 26, 6429-6435[Medline] [Order article via Infotrieve]
  37. Gillette, J. R., Brodie, B. B., and La Du, B. N. (1957) J. Pharmacol. Exp. Ther. 119, 532-541
  38. Pompon, D., and Coon, M. J. (1984) J. Biol. Chem. 259, 15377-15385[Abstract/Free Full Text]
  39. Orentreich, N., Brind, J. L., Rizer, R. L., Vogelman, J. H. (1984) J. Clin. Endocrinol. Metab. 59, 551-555[Abstract]
  40. Cutler, G. B., Glenn, M., Bush, M., Hodgen, G. D., Graham, C. E., Loriaux, D. L. (1978) Endocrinology 103, 2112-2118[Abstract]
  41. Parikh, A., Gillam, E. M. J., and Guengerich, F. P. (1997) Nature Biotech. 15, 346-354
  42. Aoyama, T., Nagata, K., Yamazoe, Y., Kato, R., Matsunaga, E., Gelboin, H. V., Gonzalez, F. J. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 5425-5429[Abstract]
  43. Lee-Robichaud, P., Kaderbhai, M. A., Kaderbhai, N., Wright, J. N., Akhtar, M. (1997) Biochem. J. 321, 857-863[Medline] [Order article via Infotrieve]
  44. Miller, W. L., and Morel, Y. (1989) Annu. Rev. Genet. 23, 371-393[CrossRef][Medline] [Order article via Infotrieve]
  45. Giordano, S. J., Kaftory, A., and Steggles, A. W. (1994) Hum. Genet. 93, 568-570[Medline] [Order article via Infotrieve]
  46. Wang, M., Roberts, D. L., Paschke, R., Shea, T. M., Masters, B. S. S., Kim, J.-J. P. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 8411-8416[Abstract/Free Full Text]
  47. Ravichandran, K. G., Boddupalli, S. S., Hasemann, C. A., Peterson, J. A., Deisenhofer, J. (1993) Science 261, 731-736[Medline] [Order article via Infotrieve]
  48. Hasemann, C. A., Kurumbail, R. G., Boddupalli, S. S., Peterson, J. A., Deisenhofer, J. (1995) Structure 3, 41-62[Medline] [Order article via Infotrieve]
  49. Kuma, F., Prough, R. A., and Masters, B. S. (1976) Arch. Biochem. Biophys. 172, 600-607[Medline] [Order article via Infotrieve]
  50. Oshino, N., Imai, Y., and Sato, R. (1971) J. Biochem. (Tokyo) 69, 155-167[Medline] [Order article via Infotrieve]
  51. Yamazaki, H., Gillam, E. M., Dong, M. S., Johnson, W. W., Guengerich, F. P., Shimada, T. (1997) Arch. Biochem. Biophys. 342, 329-337[CrossRef][Medline] [Order article via Infotrieve]
  52. Muskett, F. W., Kelly, G. P., and Whitford, D. (1996) J. Mol. Biol. 258, 172-189[CrossRef][Medline] [Order article via Infotrieve]
  53. Falzone, C. J., Mayer, M. R., Whiteman, E. L., Moore, C. D., Lecomte, J. T. (1996) Biochemistry 35, 6519-6526[CrossRef][Medline] [Order article via Infotrieve]
  54. Storch, E. M., and Daggett, V. (1996) Biochemistry 35, 11596-11604[CrossRef][Medline] [Order article via Infotrieve]


Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.