Regulation of rat hepatic 3β-hydroxysterol Δ7-reductase: substrate specificity, competitive and non-competitive inhibition, and phosphorylation/dephosphorylation
1998; Elsevier BV; Volume: 39; Issue: 12 Linguagem: Inglês
10.1016/s0022-2275(20)33327-7
ISSN1539-7262
AutoresSarah Shefer, G Salen, Akira Honda, A K Batta, L. B. Nguyen, G S Tint, Yiannis A. Ioannou, Robert J. Desnick,
Tópico(s)Lipid metabolism and biosynthesis
ResumoThe mechanism for the catalytic reduction of the double bond at C-7,8 in 7-dehydrocholesterol by 3β-hydroxysterol Δ7-reductase was investigated by testing structurally related sterols as substrates and potential inhibitors. The hepatic smooth endoplasmic reticulum was identified as the site of enzyme activity. All putative substrates contained 27 carbons, but differed from 7-dehydrocholesterol by the addition of either an ethyl substituent at C-24 (7-dehydrositosterol), a double bond at C-22 with a methyl substituent at C-24 (ergosterol), epimerization of the hydroxyl from the 3β- to 3α-configuration (7-dehydroepicholesterol), or a saturated double bond at C-5,6 (lathosterol). Two non-steroidal compounds that inhibit 3β-hydroxysterol Δ7-reductase in vivo (AY 9944 and BM 15.766) were also tested. Ergosterol, 7-dehydrositosterol, and 7-dehydroepicholesterol were reduced at C-7,8 to form brassicasterol, sitosterol, and epicholesterol, respectively, but 75% less efficiently than 7-dehydrocholesterol. Increasing concentrations of these sterols competitively inhibited 3β-hydroxysterol Δ7-reductase activity. The double bond at C-7,8 in lathosterol was not reduced. AY 9944 and BM 15.766 inhibited 3β-hydroxysterol Δ7-reductase activity non-competitively. 3β-Hydroxysterol-Δ7-reductase activity declined after microsomes were exposed to alkaline phosphatase, and enzyme activity was increased by phosphorylation with Mg2+, and ATP. These results demonstrate that the reduction of the double bond at C-7,8 requires binding of the enzyme protein with the B-ring of the sterol substrate that contains a double bond at C-5,6. The reaction is hindered by substituents located on the apolar side-chain and epimerization of the hydroxyl group in ring A to a 3α-configuration. 3β-Hydroxysterol Δ7-reductase exists in two forms: an active phosphorylated form and an inactive dephosphorylated form.—Shefer, S., G. Salen, A. Honda, A. K. Batta, L. B. Nguyen, G. S. Tint, Y. A. Ioannou, and R. Desnick. Regulation of rat hepatic 3β-hydroxysterol Δ7-reductase: substrate specificity, competitive and non-competitive inhibition, and phosphorylation/dephosphorylation. J. Lipid Res. 1998. 39: 2471–2476. The mechanism for the catalytic reduction of the double bond at C-7,8 in 7-dehydrocholesterol by 3β-hydroxysterol Δ7-reductase was investigated by testing structurally related sterols as substrates and potential inhibitors. The hepatic smooth endoplasmic reticulum was identified as the site of enzyme activity. All putative substrates contained 27 carbons, but differed from 7-dehydrocholesterol by the addition of either an ethyl substituent at C-24 (7-dehydrositosterol), a double bond at C-22 with a methyl substituent at C-24 (ergosterol), epimerization of the hydroxyl from the 3β- to 3α-configuration (7-dehydroepicholesterol), or a saturated double bond at C-5,6 (lathosterol). Two non-steroidal compounds that inhibit 3β-hydroxysterol Δ7-reductase in vivo (AY 9944 and BM 15.766) were also tested. Ergosterol, 7-dehydrositosterol, and 7-dehydroepicholesterol were reduced at C-7,8 to form brassicasterol, sitosterol, and epicholesterol, respectively, but 75% less efficiently than 7-dehydrocholesterol. Increasing concentrations of these sterols competitively inhibited 3β-hydroxysterol Δ7-reductase activity. The double bond at C-7,8 in lathosterol was not reduced. AY 9944 and BM 15.766 inhibited 3β-hydroxysterol Δ7-reductase activity non-competitively. 3β-Hydroxysterol-Δ7-reductase activity declined after microsomes were exposed to alkaline phosphatase, and enzyme activity was increased by phosphorylation with Mg2+, and ATP. These results demonstrate that the reduction of the double bond at C-7,8 requires binding of the enzyme protein with the B-ring of the sterol substrate that contains a double bond at C-5,6. The reaction is hindered by substituents located on the apolar side-chain and epimerization of the hydroxyl group in ring A to a 3α-configuration. 3β-Hydroxysterol Δ7-reductase exists in two forms: an active phosphorylated form and an inactive dephosphorylated form. —Shefer, S., G. Salen, A. Honda, A. K. Batta, L. B. Nguyen, G. S. Tint, Y. A. Ioannou, and R. Desnick. Regulation of rat hepatic 3β-hydroxysterol Δ7-reductase: substrate specificity, competitive and non-competitive inhibition, and phosphorylation/dephosphorylation. J. Lipid Res. 1998. 39: 2471–2476. 3β-Hydroxysterol Δ7-reductase catalyzes the last reaction in the cholesterol biosynthetic pathway, the reduction of the double bond at C-7,8 in 7-dehydrocholesterol to form cholesterol (Fig. 1). This enzyme, also known as 7-dehydrocholesterol Δ7-reductase, is inherited defectively in the Smith-Lemli-Opitz syndrome, a recessive birth defect (1Salen G. Shefer S. Batta A.K. Tint G.S. Xu G. Irons M. Elias E.R. Abnormal cholesterol biosynthesis in the Smith-Lemli-Opitz syndrome.J. Lipid Res. 1996; 37: 1169-1180Google Scholar, 2Shefer S. Salen G. Batta A.K. Honda A. Tint G.S. Irons M. Elias E.R. Chen T.C. Holick M.F. Markedly inhibited 7-dehydrocholesterol Δ7-reductase activity in liver microsomes from Smith-Lemli-Opitz homozygotes.J. Clin. Invest. 1995; 96: 1779-1785Google Scholar). Homozygotes show a characteristic clinical phenotype with abnormal brain development, microcephaly, facial dysmorphism, syndactyly and polydactyly of the fingers and toes, and congenital anomalies of the heart and kidneys. Low plasma and tissue cholesterol levels with the accumulation of the precursor, 7-dehydrocholesterol, and its 8-dehydrocholesterol isomer are prominent biochemical features (3Irons M. Elias E.R. Salen G. Tint G.S. Batta A.K. Defective cholesterol synthesis in Smith-Lemli-Opitz syndrome.Lancet. 1993; 341: 1414Google Scholar, 4Tint G.S. Irons M. Elias E.R. Batta A.K. Frieden R. Chen T.S. Salen G. Defective cholesterol biosynthesis associated with the Smith-Lemli-Opitz syndrome.N. Engl. J. Med. 1994; 333: 107-113Google Scholar, 5Batta A.K. Tint G.S. Shefer S. Abuelo D. Salen G. The identification of 8-dehydrocholesterol (cholest-5,8-dien-3β-ol) in patients with Smith-Lemli-Opitz syndrome.J. Lipid Res. 1995; 36: 705-713Google Scholar). To better understand the enzymatic defect in the Smith-Lemli-Opitz syndrome, where 3β-hydroxysterol Δ7-reductase activity is markedly inhibited (2Shefer S. Salen G. Batta A.K. Honda A. Tint G.S. Irons M. Elias E.R. Chen T.C. Holick M.F. Markedly inhibited 7-dehydrocholesterol Δ7-reductase activity in liver microsomes from Smith-Lemli-Opitz homozygotes.J. Clin. Invest. 1995; 96: 1779-1785Google Scholar), we examined the mechanism for the reduction of the double bond at C-7,8 as catalyzed by 3β-hydroxysterol Δ7-reductase. The hepatic subcellular location of the enzyme, substrate specificity, competitive and non-competitive inhibition, and short-term regulation by phosphorylation/dephosphorylation were examined in rat liver. A major aim was to gain insight into the Δ7-reduction mechanism and its regulation to better understand gene mutations that might be responsible for the Smith-Lemli-Opitz syndrome. Male Sprague-Dawley rats (250 g) were fed Purina rodent chow and killed at 10 am at the nadir of the diurnal cycle of cholesterol biosynthesis (6Higgins M. Kawchi T. Rudney H. The mechanism of the diurnal variation of hepatic HMG-CoA reductase activity in the rat.Biochem. Biophys. Res. Commun. 1971; 45: 138-144Google Scholar). Livers were excised and mitochondria, microsomes and the 100,000 g cytosolic fractions were isolated bydifferential ultracentrifigation in the presence of sodium chloride (2Shefer S. Salen G. Batta A.K. Honda A. Tint G.S. Irons M. Elias E.R. Chen T.C. Holick M.F. Markedly inhibited 7-dehydrocholesterol Δ7-reductase activity in liver microsomes from Smith-Lemli-Opitz homozygotes.J. Clin. Invest. 1995; 96: 1779-1785Google Scholar). Protein concentrations were determined by the method of Lowry et al. (7Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. Protein measurement with the Folin phenol reagent.J. Biol. Chem. 1951; 193: 265-275Google Scholar). Cholesterol 7α-hydroxylase activity was assayed in each fraction as a marker of microsomal contamination (8Hansson R. Wikvall K. Purification and properties of cholesterol 7α-hydroxylase.in: Fears R. Sabine J.R. Cholesterol 7α-Hydroxylase (7α-Monooxygenase). CRC Press, Inc., Boca Raton, FL1986: 51-66Google Scholar). Briefly, the assay is performed by measuring the conversion of [14C]cholesterol to 7α-hydroxycholesterol (9Shefer S. Salen G. Batta A.K. Fears R. Sabine J.R. Cholesterol 7α-Hydroxylase (7α-Monooxygenase EC 1.14.13.17) Methods of Assay. CRC Press, Inc., Boca Raton, FL1986: 43-49Google Scholar). 3β-Hydroxysterol Δ7-reductase activity was assayed using the method previously described by Shefer et al. (2Shefer S. Salen G. Batta A.K. Honda A. Tint G.S. Irons M. Elias E.R. Chen T.C. Holick M.F. Markedly inhibited 7-dehydrocholesterol Δ7-reductase activity in liver microsomes from Smith-Lemli-Opitz homozygotes.J. Clin. Invest. 1995; 96: 1779-1785Google Scholar) with minor modifications. Microsomes (0.5 mg of protein) were incubated in a final volume of 300 μl buffer (pH 7.3) containing 100 mm K2HPO4, 1 mm DTT, 30 mm nicotinamide, 0.1 mm EDTA and NADPH generating system: 3.4 mm NADP+, 30 mm glucose-6-phosphate, and 0.3 IU glucose-6-phosphate dehydrogenase. The reaction was initiated by the addition of [3H]7-dehydrocholesterol (30 nmol, 8,000 cpm) solubilized with 15 μl of a 13% solution of β-cyclodextrin (Pharmatec Inc., Alachua, FL). The reaction was stopped after 30 min incubation at 37°C with the addition of 1 ml of 1 N ethanolic NaOH, and the mixture was allowed to stand for 1 h at 37°C. After adding 0.5 ml water, the products were extracted twice with 2 ml n-hexane, separated by argentation thin-layer chromatography, and radioactivity was measured by liquid scintillation spectroscopy (2Shefer S. Salen G. Batta A.K. Honda A. Tint G.S. Irons M. Elias E.R. Chen T.C. Holick M.F. Markedly inhibited 7-dehydrocholesterol Δ7-reductase activity in liver microsomes from Smith-Lemli-Opitz homozygotes.J. Clin. Invest. 1995; 96: 1779-1785Google Scholar). Incubations were performed with the following substrates: ergosterol, 7-dehydrositosterol, 7-dehydroepicholesterol, or lathosterol substituting for 7-dehydrocholesterol (Fig. 2) using the same cofactors and conditions as described in the previous section. Each substrate was solubilized with 15 μl of 13% solution of β-cyclodextrin. The reaction was stopped by adding 1 ml of 1 N ethanolic NaOH, and after extraction with n-hexane as described above, trimethylsilyl-ether derivatives were formed. Quantitation was carried out by gas–liquid chromatography–mass spectrometry with selected-ion monitoring (SIM) using a Hewlett-Packard model 5988 mass spectrometer. A nonpolar CP-Sil 5CB (25 m × 0.25 mm ID) capillary column (Chrompack, Raritan, NJ) was used with a flow-rate of helium carrier gas of 1.0 ml/min. The column oven temperature was programmed to increase from 100°C to 265°C at 35°C/min, after a 2-min delay from the start time. The mass spectral resolution was about 1000. The multiple ion detector was focused on m/z 363 for ergosterol, m/z 380 for brassicasterol, m/z 325 for 7-dehydroepicholesterol, m/z 329 for epicholesterol, m/z 484 for 7-dehydrositosterol, m/z 486 for sitosterol, m/z 458 for lathosterol, and m/z 445 for cholestanol (Fig. 2). The inhibitory effects of ergosterol, 7-dehydrositosterol, 7-dehydroepicholesterol, and lathosterol on hepatic 3β-hydroxysterol Δ7-reductase activities were determined by measuring catalytic activities in rat hepatic microsomal fractions in the presence of increasing concentrations of the inhibitors. Microsomes (0.5 mg of protein) were incubated in a final volume of 300 μl buffer (pH 7.3) containing an NADPH generating system and increasing concentrations of the test compounds (ergosterol, 7-dehydrositosterol, 7-dehydroepicholesterol, and lathosterol) solubilized with 15 μl of a 13% solution of β-cyclodextrin. The reaction was initiated by the addition of [3H]7-dehydrocholesterol (30 nmol, 8,000 cpm) and incubated for 30 min at 37°C. The reaction was stopped and the product [3H]cholesterol was extracted and quantitated as described above in the 3β-hydroxysterol Δ7-reductase assay. To determine the mechanism of inhibition, rat hepatic microsomes were assayed for 3β-hydroxysterol Δ7-reductase activities after incubations with increasing concentrations of the 3H-labeled 7-dehydrocholesterol substrate in the presence of 100 and 300 μm inhibitors, and the Lineweaver-Burk double reciprocal plots were analyzed. AY 9944 [1,4-bis(2-dichlorobenzylaminomethyl) cyclohexane] and BM 15.766 [4-(2-[1-(4-chlorocinnamyl) piperazin-4-yl] ethyl) benzoic acid] (Fig. 3) were solubilized in a 13% solution of β-cyclodextrin and added to the incubation mixture described above. The effect of increasing concentrations of the non-steroidal compounds on the activity of 3β-hydroxysterol Δ7-reductase was evaluated, and the mechanism of inhibition was defined by the Dixon plot, of 1/V versus the increasing concentrations of the inhibitors. Hepatic microsomes were prepared by differential ultracentrifugation with 50 mm NaCl or 50 mm NaF (10Nguyen L.B. Shefer S. Salen G. Chiang J.Y.L. Patel M. Cholesterol 7α-hydroxylase activities from human and rat liver are modulated in vitro post translationally by phosphorylation/dephosphorylation.Hepatology. 1996; 24: 1468-1474Google Scholar). In the experiments of phosphorylation, microsomes (0.5 mg of protein) prepared with buffer containing NaCl were preincubated for 60 minat 37°C in a final volume of 150 μl. Tris-HC1 buffer (50 mm), pH 7.4, containing 0.3 m sucrose, 1 mm DTT, 1 mm EDTA, 5 mm MgC12, 5 mm ATP. In some experiments, 50 μm cAMP and 20 units of cAMP-dependent protein kinase were also included in the preincubation mixture. For dephosphorylation experiments,microsomes (0.5 mg of protein) prepared with NaF were preincubated for 60 min at 37°C in a final volume of 150 μl, Tris-HC1 buffer (50 mm), pH 7.4, containing 0.3 m sucrose, 1 mm DTT, 1 mm EDTA, and 10 units of E. coli alkaline phosphatase. The reactions were started by the addition of [3H]7-dehydrocholesterol (30 nmol, 8,000 cpm) solubilized with 15 μl of a 13% solution of β-cyclodextrin, and were stopped after 30 min, and [3H]cholesterol was isolated and quantitated as described above. Figure 4 shows 3β-hydroxysterol Δ7-reductase activity in rat liver whole homogenate and three subcellular fractions: microsomes, mitochondria, and 100,000 g cytosolic fraction prepared by differential ultracentrifugation (2Shefer S. Salen G. Batta A.K. Honda A. Tint G.S. Irons M. Elias E.R. Chen T.C. Holick M.F. Markedly inhibited 7-dehydrocholesterol Δ7-reductase activity in liver microsomes from Smith-Lemli-Opitz homozygotes.J. Clin. Invest. 1995; 96: 1779-1785Google Scholar, 11Shefer S. Kren B.T. Salen G. Steer C.J. Nguyen L.B. Chen T.S. Tint G.S. Batta A.K. Regulation of bile acid synthesis by deoxycholic acid in the rat. Different effects in cholesterol 7α-hydroxylase and sterol 27-hydroxylase.Hepatology. 1995; 22: 1215-1221Google Scholar). Proof of purity was based on the recovery of cholesterol 7α-hydroxylase activity in the various fractions. 3β-Hydroxysterol Δ7-reductase was most active in the microsomal fraction. Whole homogenate, which contains about 30% microsomes (12Nguyen L.B. Salen G. Shefer S. Bullock J. Chen T. Tint G.S. Chowdhary I. Lerner S. Deficient ileal HMG-CoA reductase activity in sitosterolemia: sitosterol is not a feedback inhibition of intestinal cholesterol biosynthesis.Metabolism. 1994; 43: 1-5Google Scholar), was also active but the total activity reflected the dilution of the microsomes by other cellular protein. Virtually no activity was detected in the cytosolic fraction, and the mitochondria that were contaminated with 1.8% microsomes (13Biardi L. Sreedhar A. Zokaei A. Vartak N.B. Bozeat R.L. Shackelford J.E. Keller G.A. Krisans S.K. Mevalonate kinase is predominantly localized in peroxisomes and is defective in patients with peroxisome deficiency disorders.J. Biol. Chem. 1998; 269: 1197-1205Google Scholar) showed very low activity. Thus, 3β-hydroxysterol Δ7-reductase is almost exclusively located in the microsomal fraction. In order to gain some insight into the properties of 3β-hydroxysterol Δ7-reductase, we tested the enzyme's substrate specificity, competitive and non-competitive inhibition, and short-term regulation by phosphorylation/dephosphorylation. Figure 2 illustrates the structures of the substrates tested and the products that were formed. Only sterols that have a double bond at C-7,8, but differ in the orientationof the hydroxyl group in ring A (7-dehydroepicholesterol), the absence of the double bond at C-5 in ring B (lathosterol) or the addition of substituents and double bonds in the sidechain (ergosterol, 7-dehydrositosterol), were tested. Ergosterol differs from 7-dehydrocholesterol by having an additional double bond at C-22 and a methyl group at C-24; 7-dehydrositosterol, contains an extra ethyl substituent at C-24; 7-dehydroepicholesterol, has an α-orientation of the hydroxyl group at C-3; and lathosterol contains only a single double bond in ring B, at C-7,8. Figure 5 shows 3β-hydroxysterol Δ7-reductase activity as measured by pmoles of reduced products (Fig. 2) formed per mg microsomal protein per min. The highest activity was observed with 7-dehydrocholesterol as substrate. The activity was more than 4-times higher than for ergosterol, 7-dehydrositosterol, or 7-dehydroepicholesterol as substrates. There was virtually no reduction of the double bond at C-7,8 when lathosterol was used as a substrate. Note that lathosterol is the only substrate tested that does not have a double bond at C-5,6. Thus, a double bond at C-5,6 in ring B of the substrate is essential for the reduction of the double bond at C-7,8. In addition, substitutions on the side-chain and reversal of the orientation of the hydroxyl group at C-3 reduced the catalytic efficiency of the 3β-hydroxysterol Δ7-reductase. Figure 6 shows the effects of ergosterol, 7-dehydrositosterol, and 7-dehydroepicholesterol on the activity of 3β-hydroxysterol Δ7-reductase. In these experiments, incubations were carried out with increasing amounts of unlabeled ergosterol, 7-dehydrositosterol, or 7-dehydroepicholesterol solublized in β-cyclodextrin added to 0.5 mg of rat hepatic microsomal protein. [3H]7-dehydrocholesterol was the substrate and [3H]cholesterol was the product measured. The results show that ergosterol and 7-dehydroepicholesterol are more potent inhibitors than 7-dehydrositosterol. Ergosterol and 7-dehydroepicholesterol in a concentrationof 400 μm inhibited 3β-hydroxysterol Δ7-reductase activity about 30%, whereas 400 μm of 7-dehydrositosterol inhibited the same enzyme by only 17%. In separate experiments, 200 μm and 400 μm unlabeled lathosterol were tested and did not inhibit 3β-hydroxysterol Δ7-reductase activity. Figure 7 shows 3β-hydroxysterol Δ7-reductase activities with increasing concentrations of [1, 2-3H]7-dehydrocholesterol substrate, in the absence and presence of 200 or 400 μm concentrations of unlabeled ergosterol. The Lineweaver-Burk double reciprocal plots (1/V vs. 1/S) all intersect at thesame point on the ordinate, which indicates competitive inhibition of 3β-hydroxysterol Δ7-reductase activity by ergosterol. Similar results were obtained with either 7-dehydrositosterol or 7-dehydroepicholesterol but not lathosterol. These three structurally similar sterols that undergo reduction of the double bond at C-7,8 competitively inhibit the reduction of 7-dehydrocholesterol by binding at the enzyme active site. Figure 3 illustrates the structures of AY 9944 and BM 15.766, two additional inhibitors of 3β-hydroxysterol Δ7-reductase activity. These non-steroidal compounds are structurally and chemically unrelated to 7-dehydrocholesterol and show different physical properties, but are powerful inhibitors of 3β-hydroxysterol Δ7-reductase in vivo and produce congenital anomalies similar to the Smith-Lemli-Opitz syndrome when fed to pregnant rats. (14Lindstrom-Olsson B. Differences in mechanisms of modulation between rat liver cholesterol 7α-hydroxylase and HMG-CoA reductase.FEBS Lett. 1985; 189: 124-128Google Scholar, 15Honda M. Tint G.S. Honda A. Batta A.K. Chen T.S. Shefer S. Salen G. Measurement of 3β-hydroxysteroid Δ7-reductase activity in cultured skin fibroblasts utilizing ergosterol as a substrate: a new method for the diagnosis of the Smith-Lemli-Opitz syndrome.J. Lipid Res. 1996; 37: 2433-2438Google Scholar). Both are more powerful inhibitors than the competitive inhibitors, ergosterol, 7-dehydrositosterol, and 7-dehydroepicholesterol [AY 9944 is 500 times more potent than BM 15.766; (I50 = 5 × 10−8 vs. 1 × 10−6, respectively)]. These concentrations are several orders of magnitude lower than the concentrations of the competitive inhibitors that produced only up to 30% inhibition of 3β-hydroxysterol Δ7-reductase activity. To define the mechanism of enzyme inhibition, we have analyzed the activity (1/V) of 3β-hydroxysterol Δ7-reductase with increasing concentrations of the inhibitor in the presence of 10, 20, and 40 μm concentrations of the substrate (Fig. 8, Dixon plots). The two compounds (AY 9944 and BM 15.766) showed similar patterns of inhibition as illustrated for AY 9944. All three lines intersected at the same point on the abcissa which indicated that AY 9944 is a non-competitive inhibitor. Similar results were obtained for BM 15.766. In other words, these inhibitors bind either to the free enzyme or to the enzyme–substrate complex at a site on the enzyme, other than the active site. Table 1 summarizes the results of experiments to determine whether 3β-hydroxysterol Δ7-reductase activity is regulated by phosphorylation/dephosphorylation. Preparation of microsomes in the presence of 50 mm NaF, which is known to inhibit endogenous phosphatases, led to a significant increase (+30%, P < 0.005) in the activity. Preincubation of microsomes with 5 mm MgC12 and 5 mM ATP significantly increased the activity (+150%, P < 0.005) compared with 5 mm MgC12 alone. Mg-ATP was enough to activate the enzyme and the addition of 50 μM cAMP and 20 units of cAMP-dependent protein kinase to the preincubation mixture did not lead to more activation. In contrast, preincubation of microsomes with 10 units of alkaline phosphatase significantly reduced the activity (−13%, P < 0.05) compared with 10 units of boiled alkaline phosphatase. The addition of more than 10 units of alkaline phosphatase did not cause greater inhibition. These results indicate that 3β-hydroxysterol Δ7-reductase is inhibited by dephosphorylation and that enzyme activity can be increased by phosphorylation. Thus, 3β-hydroxysterol Δ7-reductase is short-term regulated by phosphorylation/dephosphorylation so that dephosphorylation inactivates the enzyme and phosphorylation increases its activity.TABLE 1Effects of NaF and alkaline phosphatase on 3β-hydroxysterol Δ7-reductase activitiesAddition During PreincubationMicrosomes PreparationaMicrosomes were prepared with 50 mM NaCl or 50 mM NaF.nbNumber of rats used for assay.3β-Hydroxysterol Δ7-Reductase Activitypmol/min/mg%NoneNaCl5756 ± 322100NoneNaF5922 ± 361130 ± 7cP < 0.005, significantly different from microsomes treated with NaCl.MgCl2 (5 mm)NaCl4526 ± 118100McCl2 (5 mm) + ATP (5 mm)NaCl41216 ± 218250 ± 34dP < 0.005, significantly different from MgCl2 (5 mM).Boiled alkaline phosphatase (10 units)NaF5805 ± 201100Alkaline phosphatase (10 units)NaF5715 ± 19287 ± 5eP < 0.05, significantly different from boiled alkaline phosphatase (10 units).Values given as mean ± SEM.a Microsomes were prepared with 50 mM NaCl or 50 mM NaF.b Number of rats used for assay.c P < 0.005, significantly different from microsomes treated with NaCl.d P < 0.005, significantly different from MgCl2 (5 mM).e P < 0.05, significantly different from boiled alkaline phosphatase (10 units). Open table in a new tab Values given as mean ± SEM. The results of this investigation demonstrate specific structural requirements for the sterol substrate to bind to the enzyme protein for the reduction of the double bond at C-7,8. The reaction is catalyzed by the enzyme, 3β-hydroxysterol Δ7-reductase that is located in the microsomes. When the substrate, 7-dehydrocholesterol, is modified by adding substituents or a double bond to the apolar side-chain or epimerizing the 3β-hydroxy to a 3α-configuration in ring A, Δ7-reductase activity is hindered (Fig. 5). However, the most specific requirement for the reduction of the double bond at C-7,8 is the presence of a double bond at C-5,6. Lathosterol which is 5α-dihydrosaturatedand lacks the C-5,6 double bond does not interact with the enzyme protein and its double bond at C-7,8 is not reduced. Increasing concentrations of ergosterol, 7-dehydrositosterol, or 7-dehydroepicholesterol competitively inhibited the conversion of 7-dehydrocholesterol to cholesterol as illustrated in Fig 6. Lineweaver-Burk double reciprocal plots all intersected at the same point on the Y-axis (Fig. 7). In contrast, AY 9944 and BM 15.766 (Fig.3), which are non-competitive inhibitors, reacted with the enzyme protein at a different site so that additional substrate did not displace these inhibitors from the inhibitor–enzyme–substrate complex. As a result, the double bond at C-7,8 was not catalytically reduced by the enzyme. The Dixon plots (Fig. 8) illustrate the kinetics of this inhibition where the plots of increasing inhibitor concentrations all intersect together on the X-axis. An important new observation was the short-term regulation of 3β-hydroxysterol Δ7-reductase activity by phosphorylation/dephosphorylation. Exposure of the microsomes to alkaline phosphatase (dephosphorylation) significantly decreased Δ7-reductase activity. The addition of Mg2+ and ATP (phosphorylation) increased Δ7-reductase activity 2.5-fold. Thus, 3β-hydroxysterol Δ7-reductase activity undergoes short-term regulation similar to cholesterol 7α-hydroxylase (10Nguyen L.B. Shefer S. Salen G. Chiang J.Y.L. Patel M. Cholesterol 7α-hydroxylase activities from human and rat liver are modulated in vitro post translationally by phosphorylation/dephosphorylation.Hepatology. 1996; 24: 1468-1474Google Scholar), but opposite to HMG-CoA reductase, where dephosphorylation activates and phosphorylation inhibits enzyme activity (14Lindstrom-Olsson B. Differences in mechanisms of modulation between rat liver cholesterol 7α-hydroxylase and HMG-CoA reductase.FEBS Lett. 1985; 189: 124-128Google Scholar). Another important implication is that ergosterol, which is chemically more stable than 7-dehydrocholesterol, can be substituted for 7-dehydrocholesterol in the assay to measure the reduction of the double bond at C-7,8. Although enzyme activities are lower with the ergosterol substrate, significant differences in Δ7-reductase activities between homozygotes, heterozygotes, and controls could be demonstrated in fibroblasts (15Honda M. Tint G.S. Honda A. Batta A.K. Chen T.S. Shefer S. Salen G. Measurement of 3β-hydroxysteroid Δ7-reductase activity in cultured skin fibroblasts utilizing ergosterol as a substrate: a new method for the diagnosis of the Smith-Lemli-Opitz syndrome.J. Lipid Res. 1996; 37: 2433-2438Google Scholar). In summary, we have defined the structural requirements necessary to reduce the double bond at C-7,8. A double bond at C-5,6 is an absolute requirement, while substitutions in the side-chain and epimerization of the 3β- to a 3α-hydroxy configuration significantly reduced Δ7-reductase activity. Moreover, 3β-hydroxysterol Δ7-reductase is short-term regulated by dephosphorylation (inhibition) and phosphorylation (activation). This work was supported by a grant from the National Institutes of Health, DK-26756, the Smith-Lemli-Opitz Research Fund, a Grant-in-Aid from the American Heart Association, NJ Affiliate Inc., and the Research Service Veterans Administration, Washington DC. The excellent technical assistance of S. Hauser and B. Rouse is greatly appreciated.
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