Study of microsomal mixed function oxidative demethylation and deformylation of 4-methyl and 4-hydroxymethylene sterols.

Oxidative deformylation of 4-hydroxy[“Clmethylene-5cycholest-7-en-d-one catalyzed by rat liver microsomes has been studied using both NADH and NADPH as a source of reducing equivalents. Microsomes treated with a nonionic detergent, Triton WR-1339, will catalyze oxidative deformylation in the presence of NADPH. Both NADH and NADPH will serve as a source of reducing equivalents, however, if the microsomes are treated further with deoxycholate at a concentration of 10 mg/ml. Using deoxycholatetreated microsomes, the oxidase-catalyzing oxidative deformylation of 4-hydroxy[14Clmethylene-5cu-cholest-7-en-3-one has been compared with methyl sterol oxidase which participates in oxidative demethylation of 4,4-dimethyl-5cY-[30,3114Clcholest-7-en-3p-ol. Oxidative metabolism of both substrates was inhibited in a similar manner upon heating the microsomes at 47°C or including dithiothreitol or CNin the reaction vessel. Cytochrome c, however, caused marked inhibition (40%) of methyl sterol oxidase when NADH was used. Deformylation was virtually unaffected at concentrations of cytochrome c as high as 10 pM. Furthermore, methyl sterol oxidase was inhibited noncompetitively by 4hydroxymethylene-5a-cholest-7-en-3-one. Finally, if Tritontreated microsomes are treated with deoxycholate at a higher concentration (20 mglml) both oxidative deformylation activity and methyl sterol oxidase activity appear in the 105,000 x g supernatant fraction. However, specific activity of the oxidase catalyzing oxidative deformylation decreases significantly, whereas the specific activity of methyl sterol oxidase increases. The ratio between the two activities changes by as much as 5-fold.

Oxidative deformylation of 4-hydroxy["Clmethylene-5cycholest-7-en-d-one catalyzed by rat liver microsomes has been studied using both NADH and NADPH as a source of reducing equivalents.
Oxidative metabolism of both substrates was inhibited in a similar manner upon heating the microsomes at 47°C or including dithiothreitol or CN-in the reaction vessel. Cytochrome c, however, caused marked inhibition (40%) of methyl sterol oxidase when NADH was used. Deformylation was virtually unaffected at concentrations of cytochrome c as high as 10 pM. Furthermore, methyl sterol oxidase was inhibited noncompetitively by 4hydroxymethylene-5a-cholest-7-en-3-one.
Finally, if Tritontreated microsomes are treated with deoxycholate at a higher concentration (20 mglml) both oxidative deformylation activity and methyl sterol oxidase activity appear in the 105,000 x g supernatant fraction. However, specific activity of the oxidase catalyzing oxidative deformylation decreases significantly, whereas the specific activity of methyl sterol oxidase increases. The ratio between the two activities changes by as much as 5-fold.
During oxidative demethylation of 4-methyl sterols by rat liver to form cholesterol a microsomal, mixed function oxidase, methyl sterol oxidase, is functional and requires either NADH or NADPH and uses molecular oxygen as the oxidizing agent (l-7). The oxidase system is proposed to catalyze three sequential reactions in which a 4-methyl group is oxidized first to an alcohol, next to an aldehyde, and finally to a carboxylic acid * This research was supported from the Memphis State University Faculty Research Funds. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ To whom reprint requests should be sent.
Since NADH also can serve as a source of reducing equivalents for methyl sterol oxidase, we have compared oxidative demethylation of 4,4-dimethyl-5a-I30.31-"Clcholest-7-en-3P-ol and oxidative deformylation of 4-hydroxy[14Clmethylene-5a-cholest-7-en-3-one in the presence of NADH. In attempting to establish a system that would allow this, we have observed evidence that oxidative demethylation is different from oxidative deformylation. for 20 min at 10,000 x g. The supernatant fraction was centrifuged for 1 h at 105,000 x g and the microsomal pellet frozen in liquid nitrogen and stored at -28". Frozen microsomes were thawed and treated with Triton WR-1339 as described previously (1). Triton-treated microsomes were isolated by centrifugation at 105,000 x g for 1 h. Triton-treated microsomes were suspended in 0.1 M Tris buffer, pH 7.1, at 25", containing 10% glycerol (v/v), and treated with deoxycholate dissolved in 1 ml of water.
The amount of deoxycholate added, based on the volume of microsomal suspension, was either 10 mg/ml or 20 mgiml depending upon the experiment.
The volume of Tris buffer used was equal to l/4 the volume of the supernatant resulting from the initial centrifugation at 10,000 x g. This volume ratio was used for all microsomal suspensions.
The deoxycholate-treated microsomes were left at room temperature for 20 min and passed through a Sephadex G-25 column (3 x 13 cm) of medium pore size equilibrated with water. The eluted colored band of protein was collected at 0". From 8 ml of microsomes, 30 to 35 ml of eluant were collected.
The -This assay for oxidative demethylation of 4,4-dimethyl-5a-[30,31-'4Clcholest-7-en-3/3-ol was conducted essentially as previously described (1). The first aerobic step of the assay was conducted at 37" for 10 min using either a NADH-or NADPH-generating system with microsomes and substrate in a final volume of 1.0 ml as described for deformylation.
The enzymatic process was terminated by heating at steam bath temperature for 5 min. The flasks were cooled on ice, capped, and flushed 1 min at 37" with nitrogen.
Fresh Triton-treated microsomes (0.1 ml) and a NAD+-generating system in 0.4 ml of buffer were then added and the reaction flasks incubated anaerobically for 10 min. 1 mg of glucose oxidase and 15 mg of glucose were included in each flask to maintain anaerobic conditions. The reaction was terminated as described above for collection of '*CO,. Generating systems in the assay flask were prepared as follows: NADH (1 mM NADH, 10 mM P-hydroxybutyrate, and 0.25 unit of fi-hydroxybutyrate dehydrogenase), NADPH (1 rn~ NADPH, 10 rn~ isocitrate, and 0.4 unit of isocitrate dehydrogenase), and NAD+ (0.67 mM NAD+, 6.7 mM pyruvate, and 5 units of lactate dehydrogenase).
Preparation of 4.Hydroxyl "CJmethylene-5a-cholest-7-en-3-one and 4,4-dimethyl-5~u-/30,31-~~Clcholest-7-en-3~-ol   (Table I). Hence, deformylation of 4hydroxy 1 "CJmethylene-50c-cholest-7-en-3-one was similar to demethylation of 4-methyl sterols in that both NADH and NADPH could contribute reducing equivalents to the oxidative process (1). Since 4-hydroxyl "Clmethylene-50-cholest-7en-3-one serves as a model substrate for the methyl sterol oxidase when NADPH is used (81, we investigated the comparable role using NADH. Deformylation of4-Hydroxycyl '~~lmethylenr~-5Lu-c~holrst-7-en-3-onr Compared to Demethylation of 4.4-Dimethyl-5cu-130.31. "Cl~holrst-7-en-3Bol -4-Hydroxyl 14Clmethylene-5~-cholest-7-en-3-one was incubated aerobically with deoxycholatetreated microsomes and NADH. 14C0, release was measured. 4,4-dimethyl-5a-130,31-'4Clcholest-7-en-3~-ol was incubated likewise using the two-step assay to measure 14C0, release. Dithiothreitol, cyanide, heat, and cytochrome c were tested for inhibitory effects upon the oxidative metabolism of these two substrates (Fig. 3). Parallel results were obtained using dithiothreitol, heat, and cyanide. However, when cytochrome c was used, oxidative demethylation was considerably more sensitive to inhibition than was oxidative deformylation. Demethylation was inhibited to 60% of control by 10 pM cytochrome c whereas deformylation was unaffected. The inhibition due to cytochrome c was investigated using normal microsomes and Triton-treated microsomes as well to assure that deoxycholate treatment had not produced an artificial system not indicative of the true microsomal oxidase activity. Using low levels of NADH (10 FM) so as to observe 14C0, Results are the average of two experiments release from 4-hydroxyl '"C]methylene-5rY-cholest-7-en-3-one, a similar response was observed with microsomes and Tritontreated microsomes.' Oxidative demethylation of 4,4-dimethyl-501-130,31-'4C]cholest-7-en-3p-ol was inhibited much more markedly than oxidative deformylation of 4hydroxyl '*Clmethylene-5cu-cholest-7-en-3-one when NADH was used as a cofactor regardless of the condition of the microsomes. Also, under conditions in which oxidative demethylation was inhibited 80%, the second step of the twostep assay was unaffected.' Thus, the reactions in the first step catalyzed by methyl sterol oxidase are the ones affected by cytochrome (2. It has been previously shown that deformylation and demethylation are inhibited to the same extent by cytochrome ( when NADPH serves as the source of reducing equivalents (8). These studies were done using Triton-treated microsomes and have been verified in our laboratory as well. A similar response was also observed using NADPH and deoxycholatetreated microsomes.' Thus, deformylation and demethylation can be distinguished as two dissimilar processes regarding inhibition by cytochrome c only when NADH is used regardless of the state of the microsomes.

S-one upon Methyl Sterol Oxidase
-Since the inhibitory effect of cytochrome c upon oxidative demethylation is clearly different from that upon oxidative deformylation, the possibility presented itself that two different oxidases might be involved. If so, the two substrates might not be binding to the same oxidase. The hydroxymethylene substrate, however, has been reported to be a competitive inhibitor of oxidative demethylation (8). Accordingly, 4,4-dimethyl-5~-130,31-'4C]cholest-7-en-3p-01 was incubated aerobically with deoxycholate-treated microsomes and either NADPH or NADH in the presence of 4hydroxymethylene-5cu-cholest-7-en-3-one which was not radioactively labeled. Formation of l"CO, was measured by the twostep assay as described previously. As seen in Fig. 4, oxidative demethylation was inhibited in a noncompetitive manner.
Selective Extraction-To this point we have been able to demonstrate that oxidative deformylation of 4-hydroxy-1 14CJmethylene-5a-cholest-7-en-3-one is functionally different from oxidative demethylation.
If two oxidases are present, extraction procedures might affect the two oxidases differently. This was investigated by extracting Triton-treated microsomes with a higher concentration of deoxycholate (20 mg/ml of microsomes) as described earlier.
Both oxidase activities were found in the supernatant fraction from centrifugation at 105,000 Triton-treated microsomes were treated with deoxycholate (10 mg/ml).
For methyl sterol oxidase assays, 4,4-dimethyl&x-130,31-YYcholest-7-en-3/3-o1 was varied from 10 to 80 pM. Isolation of microsomes and assay procedures are described under "Experimental Procedures." Results are the average of three experiments. to only 75% of control. The differences between the two oxidase systems were also seen when oxidative demethylation was assayed in the presence of 4-hydroxymethylene-5a-cholest-7-en-3-one (Fig. 4) has not yet been investigated so our data cannot be interpreted in terms of known electron transport components.
The data, however, for cytochrome c inhibition (Fig. 3) imply that in the presence of cytochrome c a rate-limiting step for introduction of reducing equivalents from NADH must be different for demethylation and deformylation.
If it is assumed that the rate of demethylation and deformylation is dependent upon the rate of introduction of reducing equivalents, then the