Early labeled heme synthesis in normal rats and rats with iron deficiency anemia.

Heme synthesis from [2-14C]glycine was studied in liver and red blood cells. In normal rats liver contained two early [14C] heme peaks maximal at 1 and 4.5 h, followed by a long plateau of heme labeling. These phases were present in both microsomes and mitochondria. Cycloheximide suppressed formation of the first but not the second heme component. All phases of hepatic heme labeling were reduced in iron-deficient rats, with better preservation ofthe microsomal fraction. In iron-deficient rats responding to iron therapy, the first peak merged with an enlarged and premature second component; the increase was most marked in mitochondria. Thus, labeled heme metabolism was less perturbed in microsomes than mitochondria in both of these conditions. Peripheral blood also contained a [14C] heme peak at 1 h in all experimental groups. This was highest with the increased eythroid response observed in iron-treated rats. The first heme peak, present in both hepatic and erythroid cells, may represent a pool of free or unassigned heme. The later heme component may reflect formation of hemoproteins, which could be related directly or in directly to the initial, rapid turnover heme component.     

Cytochrome P-450 heme and the regulation of hepatic heme oxygenase activity

The degradation of cytochrome P-450 heme in the liver has been studied by a new approach. In rats, hepatic heme was labeled by administration of a tracer pulse of [5-¹⁴C]δ-aminolevulinic acid (ALA), and its degradation was analyzed in terms of labeled carbon monoxide (¹⁴CO) excretion, which is a specific degradation product of the labeled heme. Within minutes after administration of [5-¹⁴C]ALA, 14CO was detectable and increased after 2 h to an “early peak,” reflecting the elimination of labeled heme from a rapidly turning over pool in the liver. Beyond the early peak, the rate of ¹⁴CO production decreased in a log-linear manner, consistent with the degradation of heme in stable hepatic hemoproteins. From the rate at which ¹⁴CO production declined during this phase, from the predominant labeling of cytochrome P-450 heme by the administered [5-¹⁴C]ALA and from the known turnover characteristics of this hemoprotein in the liver, it could be inferred that production of ¹⁴CO—between 16 and 30 h after administration of labeled ALA—largely reflected degradation of cytochrome P-450 heme. This approach, which permits serial measurements in a single animal, was used to study the effect on cytochrome P-450 heme of administered heme or endotoxin, both of which are potent stimulators of hepatic heme oxygenase activity. Both of these substances caused marked acceleration of the degradation of cytochrome P-450 heme, the effect occurring over the same dose range as that for stimulation of hepatic heme oxygenase. The findings suggest that stimulation of this enzyme activity in the liver is closely related to the rate of degradation of cytochrome P-450 heme.     

Lung surfactant synthesis: a Ca++-dependent microsomal phospholipase A2 in the lung.

We present the first direct evidence for a highly active, Ca⁺⁺-dependent phospholipase A₂ in the microsomal fraction of rat lung homogenate. Several previously reported studies from other laboratories strongly implicate this enzyme as a key metabolic step in the biosynthesis of dipalmitoyl lecithin, the primary component of pulmonary surfactant. In the present study, stoichiometric amounts of [³H]lysophosphatidylethanolamine and [¹⁴C]fatty acid were released during incubation of 1-[9, 10-³H]palmitoyl-2-sn-[1′-¹⁴C]linoleoyl phosphatidylethanolamine with the lung microsomal fraction. Marker enzyme measurements showed that the microsomal activity cannot be due to contamination with mitochondria, which also show phospholipase A₂ in both lung and liver. In contrast, liver microsomes show predominantly a phospholipase A₁ activity.     

Heme transport from rat liver mitochondria to the microsomes invitro

When rat liver mitochondria labeled with [⁵⁹Fe]heme were incubated with microsomes in the presence of cytosol, about 16 % of the heme in mitochondria was transported to microsomes during a 1 hr-incubation period. In the absence of cytosol, little heme was transported. DEAE-Sephadex column chromatography of the cytosol partially purified by pH 5.1 treatment and ammonium sulfate precipitation (45–65%) revealed that there were at least two proteins with a releasing activity from mitochondria via heme transport.     

Adriamycin treatment increases 3-hydroxy-3-methylglutaryl CoA reductase and isoprene biosynthesis in the rat liver

Treatment of rats with Adriamycin caused an increase in the incorporation into hepatic cholesterol of [1-¹⁴C] acetate, but not of [2-¹⁴C] mevalonate. The step affected was found to be 3-hydroxy-3-methylglutaryl CoA reductase whose activity in the liver microsomes increased in Adriamycin-treated animals, but was inhibited when the drug was added in the assay medium. Also, the concentration of ubiquinone in the liver and of cholesterol in the plasma increased.     

Metabolism of cyclophosphamide by purified cytochrome P-450 from microsomes of phenobarbital-treated rats

Incubation of [³H]-sidechain-labeled and [¹⁴C]-C(4)-ring-labeled cyclophosphamide (CPA) with purified cytochrome P-450 from liver microsomes of rats treated with phenobarbital resulted in the production of a major metabolite that contained both labels, was unaffected by diazomethane, possessed high polarity, was identical in TLC and HPLC behavior to a synthetic standard, didechlorodihydroxy-CPA, and was converted to CPA and bis(2-chloroethyl)amine by thionyl choloride. These results indicate that phenobarbital-inducible cytochrome P-450 is able to dechlorinate CPA and may account, in part, for the inability of phenobarbital to enhance the therapeutic activity and toxicity of this important anticancer and immunosuppressive agent.     

N-methyl oxidation in liver mitochondria of triiodothyronine-treated and thyroidectomized rats

The levels of sarcosine dehydrogenase and acid-nonextractable flavin in the inner matrix of mitochondria of rat liver are decreased in animals treated with triiodothyronine and are elevated in the mitochondria obtained from thyroidectomized animals. Administration of triiodothyronine does not affect the electron-transfer flavoprotein associated with the sarcosine dehydrogenase or the relative amounts of soluble and membrane-bound proteins of the mitochondria. In phosphate-washed mitochondria from either the controls or the triiodothyronine-treated rats, the O₂ uptake equals the total of the [¹⁴C]formaldehyde and [β-¹⁴C]serine isolated as reaction products of the sarcosine-[¹⁴C]methyl group. In contrast to its restraint of sarcosine or choline oxidation in preparations capable of oxidative phosphorylation, ADP does not inhibit the oxidation of these substrates in mitochondria of rats given triiodothyronine.     

Studies on the interaction of furan with hepatic cytochrome P-450

In vitro incubation of rat liver micro-somes with [¹⁴C]-furan in the presence of NADPH resulted in the covalent incorporation of furan-derived radioactivity in microsomal protein. Compared to microsomes from untreated rats a two- to threefold increase in binding was observed with microsomes from phenobarbital-treated rats and a four- to five-fold increase was observed with microsomes from rats pretreated with imidazole or pyrazole. Covalent binding was reduced with microsomes from rats pretreated with β-naphthoflavone. Chemicals containing an amine group (semicarbazide), those in which the amine group is blocked but have a free thiol group (N-acetylcysteine), and those which have both an amine and a thiol group (glutathione) effectively blocked binding of [¹⁴C]-furan to microsomal protein. A decrease in cytochrome P-450 (P-450) content and decreases in the activities of P-450-dependent aniline hydroxylase, 7-ethoxycoumarin-O-deethylase (BCD), and 7-ethoxyresorufin-O-deethylase (ERD) was observed 24 hours after a single oral administration of 8 or 25 mg/kg of furan, suggesting that the reactive intermediate formed during P-450 catalyzed metabolism could be binding with nucleophilic groups within the P-450. In vitro studies indicated a significant decrease in the activity of aniline hydroxylase in pyrazole microsomes and BCD in phenobarbital microsomes without any significant change in the CO-binding spectrum of P-450 or in the total microsomal heme content, suggesting that furan inhibits the P-450s induced by PB and pyrazole. An almost equal distribution of furan-derived radioactivity in the heme and protein fractions of the CO-binding particles after In vitro treatment of microsomes with furan suggests binding of furan metabolites with heme and apoprotein of P-450, and, probably, due to this interaction, furan is acting as a suicide inhibitor of P-450.     

Induction of hepatic heme oxygenase activity by bromobenzene

Hepatic heme oxygenase, an enzyme which converts heme to carbon monoxide and bile pigment in vitro, is inducible by heme but also by large “toxic” doses of such nonheme substances as hormones, endotoxin, and heavy metal ions. When we gave rats a single hepatotoxic dose of allyl alcohol, ethionine, acetaminophen, furosemide, or endotoxin, hepatic heme oxygenase activity rose modestly (two- to fivefold) after 20 h. In contrast, administration of bromobenzene (5 mmol/kg) induced heme oxygenase in the liver an average of 15-fold after 20 h but was without effect on the enzyme in the kidney or spleen. The change in heme oxygenase was accompanied by a loss in cytochrome P-450 concentration and, in rats labeled with 5-δ-amino[¹⁴C]levulinic acid, an increased rate of degradation of hepatic [¹⁴C]heme to ¹⁴CO. Induction of heme oxygenase by bromobenzene was blocked by cycloheximide, an inhibitor of protein synthesis, but not by actinomycin D, an inhibitor of RNA synthesis. This suggests that bromobenzene stimulates de novo enzyme synthesis at the step of translation. Subtoxic doses of bromobenzene (less than 1 mmol/kg) gave proportionately greater induction of heme oxygenase. Furthermore, induction of the enzyme remained unaffected when bromobenzene hepatotoxicity was blocked by pretreatment of rats with SKF-525A, 3-methylcholanthrene, or cysteine (which supplements liver sulfhydryl content), or when hepatotoxicity was enhanced by pretreatment with phenobarbital or with diethylmaleate (which depletes hepatic glutathione). These data suggest that with induction of heme oxygenase by bromobenzene, neither liver cell necrosis nor alteration in hepatic sulfhydryl metabolism is indispensible. The latter characteristic differs from induction of the enzyme by metal ions in which depletion of sulfhydryl-containing constituents has been thought to be essential. We conclude that bromobenzene is a novel inducer of heme oxygenase activity in the liver, differing from other nonheme substances in potency and specificity for the liver, and in utilizing mechanism(s) which require neither production of hepatotoxicity, depletion of hepatic glutathione, nor sensitivity to actinomycin D.     

Exogenous heme restores in vivo functional capacity of hepatic cytochrome P-450 destroyed by allylisopropylacetamide.

Administration of allylisopropylacetamide (AIA) produces a dose-related destruction of the heme moiety of the phenobarbital-induced subspecies of hepatic cytochrome P-450. This results in delayed plasma disappearance of the inactivating agent as determined after injection of [¹⁴C]AIA. In phenobarbital-pretreated rats, infusion of heme reversed this AIA-mediated impairment of the plasma disappearance of [¹⁴C]AIA. In the absence of phenobarbital pretreatment, cytochrome P-450 destruction by AIA was minimal and heme infusion failed to enhance plasma disappearance of [¹⁴C]AIA. Since exogenously administered heme is incorporated into hepatic cytochrome P-450 $\text{in vivo}$, these observations suggest that the infused heme restored the functional capacity of the phenobarbital-induced mixed function oxidase system by substituting for the prosthetic heme moiety destroyed by AIA. Heme infusion is a potentially useful therapeutic modality for enhancing drug biotransformation after intoxication with compounds that inactivate cytochrome P-450.     

The oxidation of choline by liver slices and mitochondria during liver development in the rat

Betaine is the major oxidation product of [Me-¹⁴C] choline produced by rat liver slices. Liver slices from adult rats rapidly oxidize [Me-¹⁴C] choline to betaine and the bulk of the betaine produced is recovered in the incubation medium. Considerably more choline is oxidized to betaine than is phosphorylated to phosphorylcholine. The rate of phosphorylation of choline appears to be independent of the rate of choline oxidation. Liver slices from fetal and young rats oxidize choline to betaine at a lower rate than adult liver slices.The ability of mitochondria to oxidize [Me-¹⁴C] choline to betaine aldehyde and betaine is considerably lower in fetal liver than in adult liver. The major product with both fetal and adult mitochondria is betaine aldehyde. Choline oxidation by mitochondria begins to increase 1 day prior to birth and increases progressively to adult levels by 18 days. The developmental pattern for choline oxidation is similar to the pattern for succinic dehydrogenase activity.     

Role of endogenous and exogenous cholesterol in liver as the precursor for bile acids in rats

There is considerable evidence suggesting that compartmentalized functional pools of cholesterol in the liver contribute differently to the formation of bile acids as the precursor. The present paper deals with the incorporation of [1-¹⁴C]acetate and of [1,2-³H]cholesterol carried on lipoproteins (LDL and HDL) into biliary bile acids in perfused rat livers and bile-fistula rats. The results showed that endogenous cholesterol synthesized newly from [1-¹⁴C]acetate in the liver was incorporated into both cholic acid and chenodeoxycholic acid in a similar way, while exogenous lipoprotein-[1,2-³H]cholesterol delivered to hepatocytes from hepatic circulation was incorporated into chenodeoxycholic acid at a higher rate.     

Comparative study of lipid transfer between cell organelles using lipid-transfer proteins in vitro

The transfer of de novo synthesized lipids from microsomes to lipid non-synthesizing membranes was studied in vivo and in vitro from the ratios of specific radioactivities of [14C]cholesterol, [14C] and [32P]phosphatidylcholine and [32P]phosphatidylethanolamine in the nuclei and mitochondria to that in microsomes. The radioactivity of lipids transferred from microsomes to mitochondria and nuclei was identical both in vitro and in vivo and when the lipid-exchange protein of the 105 000 g supernatant was used. Acceleration of lipid metabolism in the liver of gamma-irradiated rats was concomitant with the increase in the rate of labeled cholesterol transfer cation to liver cell nuclei and mitochondria, but remained unchanged in in vitro studies involving lipid-exchange protein. The reduction of phosphatidylethanolamine transfer to the nuclei in vitro and in vivo diminished in the same way. The existence in the cell of mechanisms of transfer of de novo synthesized cholesterol other than lipid-exchange protein is postulated.     

N-Hydroxylation of arylamides by the rat and guinea pig: Evidence for substrate specificity and participation of cytochrome P1-450

The hypothesis that N-hydroxylation of arylamides is essential for carcinogenicity was examined in vivo and in vitro with N-2-fluorenylacetamide, a potent carcinogen, and with N-3-fluorenylacetamide, an isomer with marginal carcinogenicity. About 10–20% of 2-[9-¹⁴C]fluorenylacetamide administered intraperitoneally to the rat was excreted in the bile as the N-hydroxy-2-[9-¹⁴C]-derivative, whereas <0.1% of 3-[G-³H]fluorenylacetamide was found as the N-hydroxy metabolite in bile and urine. N-Hydroxylation of the 2- isomer by hepatic microsomes of untreated or 3-methylcholanthrene-treated rats was 40 to 50-fold greater than that of the 3- isomer. The role of cytochromes P-450 and P₁-450 in N-hydroxylation of arylamides by rat liver microsomes was shown by inhibition of the reaction with carbon monoxide and cobaltous chloride. Interaction of the arylamides with cytochrome P₁-450 was also demonstrated by binding spectra obtained on addition on 2- and 3-fluorenylacetamide to hepatic chromosomes of methylcholanthrene-treated rats. There appeared to be no correlation between the magnitude of the spectra and the extent of N-hydroxylation. N-Hydroxylation of the 2- isomer by hepatic microsomes of the guinea pig, a species resistant to the carcinogenecity of this compound, was markedly less than N-hydroxylation by rat liver microsomes, even though, as judged by the appearance of the binding spectra, both 2- and 3- isomers were bound by cytochrome P₁-450 of guinea pig-liver microsomes. The results are in agreement with the view that the microsomal N-hydroxylation of arylamides parallels their carcinogenicity.     

Incorporation of heme to microsomal cytochrome P-450 in the absence of protein biosynthesis

Determination of the heme and protein portions of phenobarbital (PB)-inducible and 3-methylcholanthrene inducible forms of cytochrome P-450, P-450(PB-1), and P-450(MC-1), in the liver microsomes of drug-treated animals indicated the presence of 20-30% of apo-cytochrome P-450 in both cases. Inhibition of protein synthesis by cycloheximide injection to the rats did not significantly inhibit the incorporation of delta-amino[14C]levulinic acid (ALA) into the heme of P-450(PB-1) or P-450(MC-1) in the liver, indicating that the heme incorporation into microsomal cytochrome P-450 is not tightly coupled with the synthesis of the apo-cytochrome. When heme-labeled cytosol prepared from [14C]ALA-injected rats was incubated with non-radioactive microsomes in vitro, a significant amount of labeled heme was incorporated into microsomal P-450(PB-1), whereas the incorporation into P-450(MC-1) was much less. The in vitro transfer of heme from cytosol to microsome-bound cytochrome P-450 was stimulated by the addition of an NADPH-generating system to the incubation mixtures, and inhibited when the microsomes were solubilized with sodium cholate and Emulgen-913. Although the in vitro incubation of heme-labeled microsomes with non-radioactive cytosol resulted in some release of labeled heme from the microsomes, no reversible transfer of heme between cytochrome P-450 molecules bound to separate microsomal vesicles was detected when heme-labeled microsomes were incubated with non-radioactive microsomes in the presence and absence of cytosol.     

Vitamin K dependent formation of gamma-carboxyglutamate residues in tumor microsomes

Vitamin K stimulated the incorporation of ¹⁴C into proteins when microsomes from melanoma, mammary gland, mast cell and lymphoma tumors were incubated with Na₂¹⁴CO₃. The ¹⁴C label in the [¹⁴C] proteins was identified as [¹⁴C] γ-carboxyglutamate (Gla), which is formed by carboxylation of glutamic acid residues. Carboxylation in tumor microsomes ranged from 2 to 19% of the carboxylation in normal liver microsomes per mg of microsomal protein. Carboxylation in microsomes was completely blocked by 10 μM Warfarin. SDS-polyacrylamide gel analysis of the melanoma [¹⁴C] Gla protein(s) revealed one major peak of ¹⁴C with an apparent MW of less than 6,000.     

THE SYNTHESIS AND TURNOVER OF RAT LIVER PEROXISOMES : IV. Biochemical Pathway of Catalase Synthesis

Early events in the biosynthesis of liver catalase were studied on female rats receiving [³H]leucine or [³H]δ-aminolevulinic acid or a mixture of [³H]leucine with [¹⁴C]δ-aminolevulinic acid by intraportal injection. Catalase antigen was selectively separated from homogenates by immunoprecipitation, both without and after partial purification of the enzyme. Label from both precursors appeared first in immunoprecipitable material which was lost upon purification of catalase; the label subsequently became associated with material indistinguishable from catalase. Kinetic analysis of the results indicates that the nonpurifiable material identified by early labeling consists of two distinct biosynthetic intermediates, the first lacking heme and representing about 1.6% of the total catalase content or 13 µg/g liver, the second containing heme and representing about 0.5% of the total catalase content or 4 µg/g liver. The first intermediate migrates at the same rate as catalase upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and therefore has a monomeric molecular weight of about 60,000.     

Lecithin biosynthesis in liver mitochondrial fractions

The pretreatment of rat liver mitochondrial fractions with phospholipase C preparations enhanced the incorporation of cytidine diphospho-[¹⁴C]-choline into phospholipids several-fold. Similar pretreatment of the microsomal fraction produced a similar stimulation. When the extent of microsomal contamination in the mitochondria was determined, and increments of pretreated microsomes were added to the mitochondria, the incorporation values extrapolated to zero for zero microsomal contamination. It was concluded that lecithin biosynthesis from endogenous diglycerides in the mitochondrial fractions could be ascribed to contaminating microsomes.     

Apparent pyridine nucleotide synthesis in mitochondria: An artifact of NMN and NAD glycohydrolase activity?

Intact and Triton disrupted mitochondria incorporate [¹⁴C]nicotinamide into [¹⁴C]NMN and [¹⁴C]NAD. Dialyzed Triton extracts lose this activity. The ability to form [¹⁴C]NMN is restored by addition of a fraction of boiled mitochondrial extract or of NMN. PRPP and ATP are not required for [¹⁴C]NMN formation. The specific activity of [¹⁴C]NMN formation decreases with serial washing of mitochondria while that of an outer membrane enzyme (kynurenine-3-monooxygenase) remains about constant. These finding suggest that the previously reported synthesis of NMN and NAD by mitochondria may be due to exchange reactions catalyzed by active glycohydrolase(s) in contaminating microsomes.     

The metabolism of cholesterol in the presence of liver mitochondria from normal and thyroxine-treated rats

1. [26-(14)C]- and [4-(14)C]-Cholesterol were incubated with liver mitochondria from normal and thyroxine-treated rats, and the radioactivity was measured in the carbon dioxide evolved during the incubation, in a butanol extract of the incubation mixture and in a volatile fraction containing substances of low molecular weight derived from the side chain of cholesterol. The butanol extract was separated by paper chromatography into three radioactive fractions, one of which contained the steroids more polar than cholesterol. 2. The butanol extract from incubations with [4-(14)C]cholesterol contained a radioactive substance moving with the same R(F) as cholic acid on thin-layer chromatography. 3. After incubation with [26-(14)C]-cholesterol, 60-80% of the radioactivity extracted by steam-distillation of the incubation mixture at acid pH was recovered as [(14)C]propionic acid. 4. In the presence of [26-(14)C]cholesterol, mitochondria from thyroxine-treated rats produced more radioactivity in carbon dioxide and in the volatile fraction, and less radioactivity in the fraction containing the polar steroids, than did mitochondria from normal rats. In the presence of [4-(14)C]cholesterol, mitochondria from thyroxine-treated rats produced the same amount of radioactivity in the polar steroids as did normal mitochondria. 5. Thyroxine treatment had no effect on the capacity of the mitochondria to oxidize propionate to carbon dioxide. 6. These results are best explained by supposing that thyroxine stimulates a rate-limiting reaction leading to the cleavage of the side chain of cholesterol, but has little or no influence on the hydroxylations of the ring system or on the oxidation of the C(3) fragment removed from the side chain.