Role of cytochrome P450 in the oxidation of glycerol by reconstituted systems and microsomes.

Glycerol can be oxidized by rat liver microsomes to formaldehyde in a reaction that requires the production of reactive oxygen intermediates. Studies with inhibitors, antibodies, and reconstituted systems with purified cytochrome P4502E1 were carried out to evaluate whether P450 was required for glycerol oxidation. A purified system containing phospholipid, NADPH-cytochrome P450 reductase, P4502E1, and NADPH oxidized glycerol to formaldehyde. Formaldehyde production was dependent on NADPH, reductase, and P450, but not phospholipid. Formaldehyde production was inhibited by substrates and ligands for P4502E1, as well as by anti-pyrazole P4502E1 IgG. The oxidation of glycerol by the reconstituted system was sensitive to catalase, desferrioxamine, and EDTA but not to superoxide dismutase or mannitol, indicating a role for H2O2 plus non-heme iron, but not superoxide or hydroxyl radical in the overall glycerol oxidation pathway. The requirement for reactive oxygen intermediates for glycerol oxidation is in contrast to the oxidation of typical substrates for P450. In microsomes from pyrazole-treated, but not phenobarbital-treated rats, glycerol oxidation was inhibited by anti-pyrazole P450 IgG, anti-hamster ethanol-induced P450 IgG, and monoclonal antibody to ethanol-induced P450, although to a lesser extent than inhibition of dimethylnitrosamine oxidation. Anti-rabbit P4503a IgG did not inhibit glycerol oxidation at concentrations that inhibited oxidation of dimethylnitrosamine. Inhibition of glycerol oxidation by antibodies and by aminotriazole and miconazole was closely associated with inhibition of H2O2 production. These results indicate that P450 is required for glycerol oxidation to formaldehyde; however, glycerol is not a direct substrate for oxidation to formaldehyde by P450 but is a substrate for an oxidant derived from interaction of iron with H2O2 generated by cytochrome P450.     

Rapid hydrolysis of diacylglycerol formed during phosphatidate phosphatase assay by lipase activities in rat liver cytosol and microsomes

Side reactions which may affect the determination of phosphatidate phosphatase activity were investigated in rat liver cytosol and microsomes. Incubation of these subcellular fractions with either 14C-labeled phosphatidate bound to microsomal membranes (PAmb) or that coemulsified with microsomal lipids resulted in rapid formation of water-soluble products, most of which were identified as glycerol, in addition to diacylglycerol. Neither lysophosphatidate nor glycerol 3-phosphate accumulated under any of the conditions used and only a minute amount of activity catalyzing hydrolysis of glycerol 3-phosphate could be detected in cytosol and microsomes, suggesting that glycerol was not formed by the deacylation of phosphatidate to glycerol 3-phosphate and subsequent dephosphorylation. On the other hand, pretreatment of cytosol or microsomes with diisopropylfluorophosphate abolished the formation of water-soluble products, indicating that glycerol was formed from diacylglycerol, the product of the phosphatidate phosphatase reaction, by lipase-type activities. Rapid deacylation of diacylglycerol by these subcellular fractions was also observed with an emulsion of phosphatidate, which has been purified from the total lipid extract of PAmb as substrate. The rate of hydrolysis of diacylglycerol was maximum when the concentration of diacylglycerol was less than 20 microM with either cytosol or microsomes. The present results suggest that it is essential to characterize the reaction products before employing specific assay conditions for phosphatidate phosphatase. At least under the conditions we tested, reliable measurement of the enzyme activity in rat liver cytosol and microsomes can be achieved only by determining the release of Pi or that of water-soluble activity from 32P-labeled phosphatidate.     

Teichoic acid synthesis in Bacillus stearothermophilus

1. Particulate enzyme preparations obtained from Bacillus stearothermophilus B65 by digestion with lysozyme were shown to catalyse teichoic acid synthesis. With CDP-glycerol as sole substrate the preparations synthesized 1,3-poly(glycerol phosphate). It was characterized by alkaline hydrolysis, by glucosylation to the alkali-stable 2-glucosyl-1,3-poly(glycerol phosphate) with excess of UDP-glucose and a Bacillus subtilis Marburg enzyme system, by degradation of this latter product with 60%HF and periodate oxidation of the resulting glucosylglycerol. The specificity of the B. subtilis system previously reported (Glaser & Burger, 1964), was confirmed in the present work. 2. Pulse-labelling experiments, followed by periodate oxidation of the product and isolation of formaldehyde from the glycerol terminus of the polymer, showed that the B. stearothermophilus enzyme system transferred glycerol phosphate units to the glycerol end of the chain. The transfer reaction was irreversible. It was not determined if these poly(glycerol phosphate) chains were synthesized de novo, but it was shown that the newly synthesized oligomers were bound to much larger molecules. 3. When the B. stearothermophilus enzyme system was supplied with both CDP-glycerol and UDP-glucose, 1-glucosyl-2,3-poly(glycerol phosphate) was synthesized in addition to the 1,3-isomer. The former polymer was characterized by acid and alkaline hydrolysis, degradation with HF and periodate oxidation of the resulting glucosylglycerol, and periodate oxidation of the intact polymer followed by mild acid hydrolysis. This latter procedure removed the glucose substituents without disrupting the poly(glycerol phosphate) chain. 4. The poly(glycerol phosphate) isomers were distinguished by glucosylation with the B. subtilis enzymes and alkaline hydrolysis, the 2,3-isomer remaining alkali-labile. The proportion of 2,3-poly(glycerol phosphate) in the product increased with increasing amounts of UDP-glucose in the incubation mixture, but the total glycerol phosphate incorporated into products remained constant. It is suggested that the synthetic pathways of the two poly(glycerol phosphate) species may share a rate-limiting step.     

Oxidation of pyrazole by reconstituted systems containing cytochrome P-450 IIE1

Rat liver microsomes oxidize pyrazole to 4-hydroxypyrazole and this oxidation is increased in microsomes isolated from rats treated with inducers of cytochrome P-450 IIE1, such as pyrazole or ethanol. A reconstituted system containing the P-450 IIE1, purified from pyrazole-treated rats, oxidized pyrazole to 4-hydroxypyrazole in a time- and P-450-dependent manner. Oxidation of pyrazole was dependent on the concentration of pyrazole over the range of 0.15 mM to 1.0 mM. In isolated microsomes, glycerol inhibited pyrazole oxidation by about 50% under concentration conditions which occur in the reconstituted system; hence, the values for pyrazole oxidation by the reconstituted systems are underestimated because of the presence of glycerol. Oxidation of pyrazole was inhibited by competitive substrates for P-450 IIE1, such as 4-methylpyrazole, aniline and ethanol, as well as by an antibody raised against the pyrazole-induced P-450 IIE1. Thus, pyrazole is an effective substrate for oxidation by purified P-450 IIE1, extending the substrate specificity of this isozyme to potent inhibitors of alcohol dehydrogenase.     

Oxidative demethylation of t-butyl alcohol by rat liver microsomes

Tertiary butyl alcohol has often been used experimentally as a “non-metabolizable” alcohol. In this report, evidence is presented that t-butanol serves as a substrate for rat liver microsomes and that it is oxidatively demethylated to yield formaldehyde. The apparent K_m for t-butanol is 30 mM while V_max is about 5.5 nmol per min per mg microsomal protein. Formaldehyde production is stimulated by azide, which prevents destruction of H₂O₂ by catalase. Hydroxyl radical scavenging agents, such as benzoate, mannitol, and 2-keto-4-thiomethylbutyrate, suppress formaldehyde production. Therefore, the microsomal reaction pathway appears to involve the interaction of t-butanol with hydroxyl radicals generated from H₂O₂ by the microsomes. Formaldehyde is also produced when t-butanol is incubated with model hydroxyl radical-generating systems such as the iron-EDTA-stimulated oxidation of xanthine by xanthine oxidase or the iron-EDTA-catalyzed autoxidation of ascorbate. These results indicate that t-butanol cannot be used to distinguish metabolically-linked from non-metabolically-linked actions of ethanol.     

An apolipoprotein preferentially enriched in cholesteryl ester-rich very low density lipoproteins

Phosphatidyl glycerol is present in lamellar bodies and in the material obtained by alveolar wash representing 12.3 and 11.5%, respectively, of the total phospholipid phosphorus. Lung microsomes catalyze the formation of phosphatidyl glycerol from the known precursors, L-glycerol 3-phosphate and CDP-diglyceride. The rate of [¹⁴C]L-glycerol 3-phosphate incorporation into phosphatidyl glycerol was 30% higher in microsomes as compared to mitochondria. The addition of mercuric chloride inhibited the synthesis of phosphatidyl glycerol, and stimulated the incorporation into another as yet incompletely identified lipid. After pulse labeling of microsomal phosphatidyl glycerol $\text{in vitro}$, further incubation of microsomes with lamellar bodies or alveolar wash resulted in nearly quantitative appearance of label in surfactant.     

The relative participation of liver microsomal amine oxidase and cytochrome P-450 in N-demethylation reactions.

N-Demethylation of benzphetamine and p-chloro-N-methylaniline measured in the presence and absence of specific antibodies to NADPH-cytochrome c (P-450) reductase demonstrates that part of the formaldehyde formed from the sec-N-methylamine arises from non-cytochrome P-450-dependent oxidation catalyzed by pig, hamster, and rat liver microsomes. The additional formaldehyde formed can be inhibited by adding methimazole, a non-formaldehyde-producing substrate specific for the microsomal mixed-function amine oxidase, to the reaction media. Purified amine oxidase catalyzes the oxidation of sec-N-methylamines to sec-N-methylhydroxylamines that, upon oxidation and hydrolysis, yield formaldehyde. Approximately 65, 40, and 15% of total formaldehyde is formed by this route during oxidation of p-chloro-N-methylaniline catalyzed by pig, hamster, and rat liver microsomes, respectively.     

In vitro metabolism of 3-t-butyl-4-hydroxyanisole and its irreversible binding to proteins

A method for the preparation of methyl-labelled 3-t-butyl-4-hydroxyanisole (BHA) is described. Metabolism of [14C]BHA using four different enzyme systems (liver microsomes + NADPH; liver microsomes + cumene hydroperoxide (CHP); sheep seminal vesicle (SSV) microsomes (as a source of prostaglandin synthetase) + arachidonic acid (AA); horseradish peroxidase (HRP) + hydrogen peroxide) was investigated. In all systems, BHA was oxidized to a variety of products including formaldehyde, a dimer di-BHA, polar and water soluble metabolites as well as a reactive intermediate(s) that binds irreversibly to proteins. With liver microsomes and NADPH, phenobarbital (PB) induction gave increased yields of all products while 3-methylcholanthrene (MC) induction specifically increased protein binding but decreased other metabolite formation. BHA addition effectively discharged the activated oxygen complex of cytochrome P-450 (liver microsomes) as well as Comp. I and Comp. II of HRP suggesting that it is a good one electron peroxidase donor. BHA addition also increased the net rate of NADPH oxidation in the presence of liver microsomes suggesting uncoupling. It is proposed that in all system investigated BHA is oxidized predominantly via a one electron oxidation process to yield first the BHA free radical which then dimerizes, forms more products or binds to proteins.     

The vanadate-stimulated oxidation of NAD(P)H by biomembranes is a superoxide-initiated free radical chain reaction

Rat liver microsomes catalyze a vanadate-stimulated oxidation of NAD(P)H, which is augmented by paraquat and suppressed by superoxide dismutase, but not by catalase. NADPH oxidation was a linear function of the concentration of microsomes in the absence of vanadate, but was a saturating function in the presence of vanadate. Microsomes did not catalyze a vanadate-stimulated oxidation of reduced nicotinamide mononucleotide (NMNH), but gained this ability when NADPH was also present. When the concentration of NMNH was much greater than that of NADPH a minimal average chain length could be calculated from 1/2 the ratio of NMNH oxidized per NADPH added. The term chain length, as used here, signifies the number of molecules of NMNH oxidized per initiating event. Chain length could be increased by increasing [vanadate] and [NMNH] and by decreasing pH. Chain lengths in excess of 30 could easily be achieved. The Km for NADPH, arrived at from saturation of its ability to trigger NMNH oxidation by microsomes in the presence of vanadate, was 1.5 microM. Microsomes or the outer mitochondrial membrane was able to catalyze the vanadate-stimulated oxidation of NADH or NADPH but only the oxidation of NADPH was accelerated by paraquat. The inner mitochondrial membrane was able to cause the vanadate-stimulated oxidation of NAD(P)H and in this case paraquat stimulated the oxidation of both pyridine coenzymes. Our results indicate that vanadate stimulation of NAD(P)H oxidation by biomembranes is a consequence of vanadate stimulation of NAD(P)H or NMNH oxidation by O-2, rather than being due to the existence of vanadate-stimulated NAD(P)H oxidases or dehydrogenases.     

Inhibition of the low-Km mitochondrial aldehyde dehydrogenase by diethyl maleate and phorone in vivo and in vitro. Implications for formaldehyde metabolism.

Formaldehyde can be oxidized primarily by two different enzymes, the low-Km mitochondrial aldehyde dehydrogenase and the cytosolic GSH-dependent formaldehyde dehydrogenase. Experiments were carried out to evaluate the effects of diethyl maleate or phorone, agents that deplete GSH from the liver, on the oxidation of formaldehyde. The addition of diethyl maleate or phorone to intact mitochondria or to disrupted mitochondrial fractions produced inhibition of formaldehyde oxidation. The kinetics of inhibition of the low-Km mitochondrial aldehyde dehydrogenase were mixed. Mitochondria isolated from rats treated in vivo with diethyl maleate or phorone had a decreased capacity to oxidize either formaldehyde or acetaldehyde. The activity of the low-Km, but not the high-Km, mitochondrial aldehyde dehydrogenase was also inhibited. The production of CO2 plus formate from 0.2 mM-[14C]formaldehyde by isolated hepatocytes was only slightly inhibited (15-30%) by incubation with diethyl maleate or addition of cyanamide, suggesting oxidation primarily via formaldehyde dehydrogenase. However, the production of CO2 plus formate was increased 2.5-fold when the concentration of [14C]formaldehyde was raised to 1 mM. This increase in product formation at higher formaldehyde concentrations was much more sensitive to inhibition by diethyl maleate or cyanamide, suggesting an important contribution by mitochondrial aldehyde dehydrogenase. Thus diethyl maleate and phorone, besides depleting GSH, can also serve as effective inhibitors in vivo or in vitro of the low-Km mitochondrial aldehyde dehydrogenase. Inhibition of formaldehyde oxidation by these agents could be due to impairment of both enzyme systems known to be capable of oxidizing formaldehyde. It would appear that a critical amount of GSH, e.g. 90%, must be depleted before the activity of formaldehyde dehydrogenase becomes impaired.     

Further evidence for the existence of different diacylglycerol pools of the phosphatidylcholine synthesis in microsomes

Endogenous diacylglycerol and diacylglycerol, synthesized in vitro by glycerol 3-phosphate acylation, are not mixed and represent different substrate pools for the biosynthesis of phosphatidylcholine in microsomes of rat muscle, liver and lung. Freshly isolated lung microsomes contain 12-18 nmol diacylglycerol per mg protein, and incubation with CDPcholine showed a biphasic curve for the synthesis of phosphatidylcholine as lung microsomes enriched in diacylglycerol through the glycerol phosphate pathway. With respect to the synthesis of phosphatidylcholine, a part of this endogenous diacylglycerol (0.4-0.8 nmol/mg) was comparable with diacylglycerol de novo formed in vitro by glycerol 3-phosphate acylation. An increase in the relative proportion of de novo-formed diacylglycerol in the total amount of diacylglycerol caused an increase in phosphatidylcholine synthesis by nearly the same factor. The apparent Km of the de novo-formed diacylglycerol substrate for the choline phosphotransferase was 10-times higher than the pool size of this diacylglycerol substrate in freshly isolated lung microsomes. The results supported the idea that the availability of this substrate type may be rte limiting for the de novo synthesis of phosphatidylcholine. As shown by use of the proteolytic technique measuring the mannose-6-phosphatase as lumenal control activity, the phosphatidylcholine synthesis from de novo-formed diacylglycerol and endogenous as well as exogenous diacylglycerol seems to be located on the cytoplasmic leaflet of the microsomal vesicles isolated from rat lung.     

Regulation by noradrenaline of the mitochondrial and microsomal forms of glycerol phosphate acyltransferase in rat adipocytes.

Incubation of rat adipocytes with 1 microM-noradrenaline caused a decrease in both the N-ethylmaleimide-sensitive (microsomal) and N-ethylmaleimide-insensitive (mitochondrial) glycerol phosphate acyltransferase activities measured in homogenates from freeze-stopped cells. The effects of noradrenaline on glycerol phosphate acyltransferase activity were apparent over a wide range of concentrations of glycerol phosphate and palmitoyl-CoA. The effect of noradrenaline was reversed within cells by the subsequent addition of insulin or propranolol. Inclusion of albumin in homogenization buffers abolished the effect of noradrenaline on the N-ethylmaleimide-sensitive activity. The effect of noradrenaline on the N-ethylmaleimide-insensitive (mitochondrial) activity was, however, not abolished by inclusion of albumin in buffers for preparation of homogenates from freeze-stopped cells. Inclusion of fluoride in homogenization buffers did not alter the observed effect of noradrenaline. The inactivating effect of noradrenaline persisted through the subcellular fractionation procedures used to isolate adipocyte microsomes (microsomal fractions). The effect of noradrenaline on mitochondrial glycerol phosphate acyltransferase did not persist through subcellular fractionation. Noradrenaline treatment of cells significantly decreased the Vmax. of glycerol phosphate acyltransferase in isolated microsomes without changing the activity of NADPH-cytochrome c reductase. Glycerol phosphate acyltransferase activity in microsomes from noradrenaline-treated cells is unstable, being rapidly lost on incubation at 30 degrees C. Bivalent metal ions (Mg2+, Ca2+) or post-microsomal supernatant protected against this inactivation. Glycerol phosphate acyltransferase activity in microsomes from noradrenaline-treated cells could not be re-activated by incubation with either alkaline phosphatase or phosphoprotein phosphatase-1. Addition of cyclic AMP-dependent protein kinase catalytic subunits to adipocyte microsomes incubated with [gamma-32P]ATP considerably increased the incorporation of 32P into microsomal protein, but did not cause inactivation of glycerol phosphate acyltransferase. These findings provide no support for the proposal that inactivation of adipocyte microsomal glycerol phosphate acyltransferase by noradrenaline is through a phosphorylation type of covalent modification.     

The role of cytochrome P-450 in the hydroperoxide-catalyzed oxidation of alcohols by rat-liver microsomes.

The organic hydroperoxide cumene hydroperoxide is capable of oxidizing ethanol to acetaldehyde in the presence of either catalase, purified cytochrome P-450 or rat liver microsomes. Other hemoproteins like horseradish peroxidase, cytochrome c or hemoglobin were ineffective. In addition to ethanol, higher alcohols like 1-propanol, 1-butanol and 1-pentanol are also oxidized to their corresponding aldehydes to a lesser extent. Other organic hydroxyperoxides will replace cumene hydroperoxide in oxidizing ethanol but less effectively. The cumene-hydroperoxide-dependent ethanol oxidation in microsomes was inhibited partially by cytochrome P-450 inhibitors but was unaffected by catalase inhibitors. Phenobarbital pretreatment of rats increased the specific activity of the cumene-hydroperoxide-dependent ethanol oxidation per mg of microsomes about seven-fold. The evidence suggests that cytochrome P-450 rather than catalase is the enzyme responsible for hydroperoxide-dependent ethanol oxidation. However, when H2O2 is used in place of cumene hydroperoxide, the microsomal ethanol oxidation closely resembles the catalase system.     

Reactions of direct formaldehyde oxidation to CO2 are non-essential for energy supply of yeast methylotrophic growth

Mutants of the methylotrophic yeast Hansenula polymorpha deficient in NAD-dependent formaldehyde or formate dehydrogenases have been isolated. They were more sensitive for exogenous methanol but retained the ability for methylotrophic growth. In the medium with methanol the growth yields of the mutant 356–83 deficient in formaldehyde dehydrogenase and of the wild-type strain were identical (0.34 g cells/g methanol) under chemostat cultivation. These results indicate that enzymes of direct formaldehyde oxidation are not indispensable for methylotrophic growth. At the same time inhibition of tricarboxylic acid cycle has resulted in suppression of growth in the media with multicarbon nonfermentable substrates such as glycerol, succinate, ethanol and dihydroxyacetone as well as with methanol, but not with glucose. In the experiments with the wild-type strain H. polymorpha it has been shown that citrate and dihydroxyacetone inhibit the radioactivity incorporation from ¹⁴C-methanol into CO₂. All obtained data indicate that for the dissimilation of methanol and the supplying of energy for methylotrophic growth, the functioning of tricarboxylic acid cycle reactions as oppossed to those of direct formaldehyde oxidation is essential.     

Glycerol metabolism in type II pneumocytes isolated from streptozotocin-diabetic rats

Glycerol utilization for phospholipid biosynthesis was examined in type II pneumocytes isolated from normal and streptozocinin-diabetic rats. With glucose in the incubation medium, incorporation of exogenous [1,3-14C]glycerol into disaturated phosphatidylcholine, total phosphatidylcholine (PC), phosphatidylglycerol (PG) and phosphatidylethanolamine (PE) was increased 4-fold in cells from diabetic rats. In the absence of glucose, glycerol incorporation was 5-fold greater than in its presence in cells from normal animals, but was further increased 2.2-fold in cells from diabetic rats. Insulin treatment of diabetic rats returned all incorporation rates to control values. The increased glycerol incorporation rates were not due to differences in either phospholipid turnover or the size of the glycerol 3-phosphate precursor pool. Kinetic analysis of glycerol entry into the acid-soluble cell fraction indicated that glycerol transport occurred largely by simple diffusion, and was not rate limiting for its entry into lipids. Glycerol entry into the total lipid fraction was saturable, reaching a Vmax of 48 pmol/micrograms DNA per h in normal cells and 120 pmol/micrograms DNA per h in cells from diabetic rats, with no change in the Km (0.31 mM). While glycerol oxidation was reduced 23% in cells from diabetic rats in the presence of glucose and by 44% in the absence of glucose, glycerol kinase activity in sonicates of cells from diabetic animals was increased 210% and was reversed by in vivo insulin treatment. These results suggest that glycerol utilization in type II pneumocytes is a hormonally regulated function of both glycerol oxidation and glycerol phosphorylation.     

Glycerol 3-phosphate acylation in microsomes of type II cells isolated from adult rat lung

Glycerol 3-phosphate acylation was studied in type II cells isolated from adult rat lung. The process was found to be largely microsomal. In the microsomes phosphatidic acid is the main product of glycerol 3-phosphate acylation. Glycerol-3-phosphate acyltransferase is rate limiting in the phosphatidic acid formation by the microsomes. Type II cell microsomes incorporate palmitoyl and oleoyl residues into phosphatidic acid at an equal rate if palmitoyl-CoA and oleoyl-CoA are added separately. However, if palmitoyl-CoA and oleoyl-CoA are added as an equimolar mixture the unsaturated fatty acyl moiety is incorporated much faster. Under the latter conditions monoenoic species constitute the most abundant products of glycerol 3-phosphate acylation. The microsomes incorporate both palmitoyl and oleoyl residues readily into both the 1- and 2-position of phosphatidic acid, even when palmitoyl-CoA and oleoyl-CoA are added together. Assuming that both phosphatidic acid phosphatase and cholinephosphotransferase do not discriminate against substrates with an unsaturated acyl moiety at the 1-position and a saturated acyl moiety at the 2-position, the last two observations indicate that a considerable percentage of phosphatidylcholine molecules synthesized de novo may have a saturated fatty acid at the 2-position and an unsaturated fatty acid at the 1-position, and that remodeling at the 1-position may be important for the formation of surfactant dipalmitoylphosphatidylcholine. They also indicate that type II cell microsomes are capable of synthesizing the dipalmitoyl species of phosphatidic acid. However, since there is a preference for the acylation of glycerol 3-phosphate with unsaturated fatty acyl residues, the percentage of dipalmitoyl species in the synthesized phosphatidic acid, and thereby the percentage of dipalmitoyl species in the phosphatidylcholine synthesized de novo, will probably depend on the relative availability of the various acyl-CoA species.     

Microsomal iron-dependent NADPH oxidation: evidence for the involvement of membrane-bound nonheme iron in NADPH oxidation by rat heart microsomes

Rat heart microsomes were found to contain nonheme iron and two lines of evidence suggested that this iron was involved in NADPH oxidation. As first evidence, pretreatment of rats with iron gluconate increased microsomal iron content and NADPH oxidation. As second evidence, treatment of microsomes with nonionic detergent Triton N-101 decreased membrane iron content and NADPH oxidation. Triton N-101-solubilized nonheme iron was nondialyzable and ammonium sulfate-precipitable, indicative of association with protein(s). This protein-bound iron per se did not oxidize NADPH but its addition to detergent-treated microsomes restored very high rates of NADPH oxidation, that were abolished by inhibiting NADPH-cytochrome P450 reductase with p-hydroxymercuribenzoate. Since heart microsomes did not contain cytochrome P450, these results suggested that stimulation of NADPH oxidation was mediated by direct electron transfer from reductase to iron. Purified rat heart ferritin and hemosiderin did not stimulate NADPH oxidation and the stimulation observed with detergent-solubilized microsomal iron was much higher than that observed with EDTA-Fe3+, a very effective electron acceptor for the reductase. This suggested that (i) microsomal iron was different from other intracellular iron-storage proteins, and (ii) microsomal iron was unusually permissive to one-electron transfer from reductase.     

Structural determinants for alcohol substrates to be oxidized to formaldehyde by rat liver microsomes.

Glycerol can be oxidized to formaldehyde by rat liver microsomes and by cytochrome P450. The ability of other alcohols to be oxidized to formaldehyde was determined to evaluate the structural determinants of the alcohol which eventually lead to this production of formaldehyde. Monohydroxylated alcohols such as 1- or 2-propanol did not produce formaldehyde when incubated with NADPH and microsomes. Geminal diols such as 1,3-propanediol, 1,3-butanediol, or 1,4-butanediol also did not yield formaldehyde. However, vicinal diols such as 1,2-propanediol or 1,2-butanediol produced formaldehyde. With 1,2-propanediol, the residual two-carbon fragment was found to be acetaldehyde, while with 1,2-butanediol, the residual three-carbon fragment was propionaldehyde. Oxidation of 1,2-propanediol to formaldehyde plus acetaldehyde involved interaction with an oxidant derived from H2O2 plus nonheme iron, since production of the two aldehydic products was completely prevented by catalase or glutathione plus glutathione peroxidase and by chelators such as desferrioxamine or EDTA. The oxidant was not superoxide or hydroxyl radical. Product formation was fivefold lower when NADH replaced NADPH, and was inhibited by substrates, ligands, and inhibitors of cytochrome P450. A charged glycol such as alpha-glycerophosphate (but not the geminal beta-glycerophosphate) was readily oxidized to formaldehyde, suggesting that interaction of the glycol with the oxidant was occurring in solution and not in a hydrophobic environment. These results indicate that the carbon-carbon bond between 1,2-glycols can be cleaved by an oxidant derived from microsomal generated H2O2 and reduction of non-heme iron, with the subsequent production of formaldehyde plus an aldehyde with one less carbon than the initial glycol substrate.     

Chemical quantitation of hemoglobin glycosylation: fluorometric detection of formaldehyde released upon periodate oxidation of glycoglobin

A sensitive fluorometric method for the quantitation of hemoglobin glycosylation, based upon periodate oxidation of the carbohydrate moieties present on both the α- and ?-amino groups of globin is described. The formaldehyde product is measured as the fluorescent 3,5-diacetyl-1,4-dihydrolutidine formed from the condensation of formaldehyde with acetylacetone and ammonia.This method is rigorously designed to assay glycosylated hemoglobin levels and to give a direct measure of the number of glycogroups per milligram of hemoglobin. It requires only 1 mg of protein and may also be used to determine the extent of the nonenzmatic glycosylation of other proteins.     

Microsomal electron transport reactions. II. The use of reduced triphosphopyridine nucleotide and-or reduced diphosphopyridine nucleotide for the oxidative N-demethylation of aminopyrine and other drug substrates

The ratio of formaldehyde formed to TPNH oxidized during aminopyrine oxidative demethylation as catalyzed by rabbit liver microsomes was found to be about 0.5. This is less than the expected 1:1 ratio for a mixed function oxidase reaction and may reflect the oxidation of TPNH by other reactions. Similar results were obtained when measuring the oxidative demethylation of codeine and ethylmorphine. In all cases the addition of DPNH significantly increased the yield of formaldehyde formed in the presence of TPNH. The stimulatory effect of DPNH was a linear function of the DPNH concentration added until the initial concentrations of DPNH and TPNH were equal. Increasing the DPNH concentration above a DPNH:TPNH ratio of 1:1 had no further effect upon the final concentration of formaldehyde formed. This observation, as well as the inhibition of DPNH-supported aminopyrine metabolism by TPN⁺, argue against the role of a transhydrogenase mechanism for the DPNH effect. The rate of DPNH oxidation catalyzed by liver microsome was also observed to increase markedly in the presence of TPNH.