Mg Of Microsomal Protein Research Articles

Microsomal drug metabolism in acute and chronic galactosamine induced hepatic injuries.

To determine the microsomal functions of the injured liver, female rats were given D-galactosamine (GalN) acutely (400 mg/kg body weight, i.p.) or chronically (250 mg/kg body weight for 7 months, daily, i.p.). In the acute study, GalN administration resulted in a five-fold increase in hepatic triglyceride content. Microsomal protein and phospholipid concentrations were not affected. However microsomal cytochrome P450 content and aminopyrine demethylase and also aniline hydroxylase activities were significantly decreased whether expressed per mg of microsomal protein or per gram of liver. Cytochrome b5 content was significantly increased. By contrast, chronic administration of GalN produced a marked hepatomegaly and an extensive hepatic fibrosis with nodular formation without triglyceride accumulation. Electron-microscopically, there was apparent proliferation of mitochondria, and smooth endoplasmic reticulum showed focally accentuated hypertrophy without liposomes in the dilated tubules. Although microsomal protein concentration was significantly decreased, cytochrome P450 content was increased significantly when expressed per mg of microsomal protein or per unit of body weight. The aminopyrine demethylase activity was significantly enhanced only when expressed per unit of body weight. In both acute and chronic studies, microsomal glucose-6-phosphatase activity was significantly reduced. These data suggest that in acute liver injury, there is an impairment of hepatic microsomal drug metabolizing activity, whereas in chronic liver injury, there is an apparent enhancement of the activity possibly due to an enlarged hepatic mass even in the presence of a decreased drug metabolizing capacity per unit of the injured liver.

Extraction and assay of small quantities of free fatty acids produced by hydrolysis of microsomal membranes

It has been shown that the Dole procedure extracts as much free fatty acids from rat liver microsomes as does homogenization in chloroform-methanol, 2:1. If microsomal phospholipids in the heptane phase from the Dole procedure are removed with silicic acid, microsomes give a background in the 63Ni assay for free fatty acids equivalent to about 10 nmol of fatty acid/mg of microsomal protein. Only about 2% of this background is due to endogenous free fatty acids. Thus, use of the Dole extraction procedure, “clean-up” of the hexane phase with silicic acid, and the 63Ni assay allow determination of the hydrolysis of 5% or less of microsomal phospholipids using only small amounts of material (0.5–1.0 mg of microsomal protein). This procedure was found to be useful in following the breakdown of microsomal phospholipids by phospholipase A 2.

Phosphatidate Phosphohydrolase and the Regulation of Glycerolipid Biosynthesis

Phosphatidate is an intermediate in the biosynthesis of diacylglycerol, triacylglycerol, phosphatidylethanolamine, phosphatidylcholine and CDP-diacylglycerol. In addition, phosphatidate may be subject to degradation by particulate phospholipases. The factors regulating the disposition of phosphatidate have interested numerous investigators. We have studied the reactions of phosphatidate metabolism in several tissues and under various environmental conditions. Phosphatidate phosphohydrolase activity is found primarily in the microsomal and supernatant fractions of liver, adipose tissue and intestine. Presumably the physiologically important substrate for this reaction is the particulate-bound phosphatidate and methods for the preparation of this substrate have been developed (Fallon et al., 1975). Radioactively labelled phosphatidate bound to microsomal particles is prepared from sn-[1 ,3-14C]glycerol 3-phosphate. A wide range of microsomal phosphatidate concentrations may be prepared by this technique, although the usual microsomal concentration is approx. 5 nmol/mg of microsomal protein. This substrate has been used in most of our studies of phosphatidate phosphohydrolase, CDP-diacylglycerol formation and phospholipase A. There are several lines of evidence which support an important role for phosphatidate phosphohydrolase in the regulation of neutral-lipid formation by liver or adipose-tissue particulate preparations. (1) The reaction rate of phosphatidate phosphohydrolase under optimum conditions in vitro is the lowest in the overall synthetic pathway in liver microsomal fractions (Lamb & Fallon, 1974a). The rate of phosphohydrolase activity was determined as 0.6-0.8 nmol/min per mg of microsomal protein when radiolabelled particulate-bound phosphatidate was used as substrate. The overall rate of glycerolipid formation by these microsomal preparations was approx. l.Onmol/min per mg of microsomal protein. The optimum rate of microsomal sn-glycerol 3-phosphate acyltransferase was 5-7-fold higher. In the presence of added rat liver supernatant fraction the rate of microsomal phosphatidate phosphohydrolase is increased 5-20-fold. However, at ratios of microsomal protein to supernatant protein estimated for the intact cell, the increase in phosphohydrolase activity is much more modest. (2) Induction of increased liver triacylglycerol formation by administration of a high-fructose diet is accompanied by an equivalent rise in the activity of the phosphohydrolase, as shown in Table 1. These changes in lipid metabolism occur simultaneously over a 10-day period (Lamb & Fallon, 19746). Further, the phosphatidate content of liver microsomal preparation is diminished by approx. 15 %, and diacylglycerol increased by approx. 90 % under these dietary conditions. (3) The increase in liver triacylglycerol content after partial hepatectomy is accompanied by a corresponding increase in phosphatidate phosphohydrolase activity (Mangiapane et al., 1973). (4) Several drugs which cause substantial inhibition of phosphatidate phosphohydrolase activity in vitro also result in inhibition of neutral-lipid formation when administered to the intact animal (Brindley & Bowley, 1975; Lamb et al., 1977). A series of 1,3-bis(substituted phenoxy)-2-propanones inhibited microsomal phosphatidate phosphohydrolase activity as measured in vitro. The inhibition ranged from 8 to 92% total activity. In animals fed on these agents in laboratory chow the incorporation of [14C]glycerol into hepatic triacylglycerol was decreased by 3-75 %. Agents which caused a 50 % or greater decrease in phosphatidate phosphohydrolase activity in vitro also lowered

STUDIES ON THE LIVER ENLARGEMENT INDUCED BY DIETARY ADMINISTRATION OF DI-(2-ETHYLHEXYL) PHTHALATE (DEHP).

Di-(2-ethylhexyl) phthalate (DEHP) was mixed into diet and given to Wistar male rats for 16 days. A significant and dose-dependent increase in the relative liver weight (RLW) was observed in the rats fed DEHP at dietary levels greater than 0.1%. The RLW increased progressively with the duration of the treatment, reaching its maximun in two weeks. Biochemical analysis of the principal hepatic components has shown that the increase in RLW induced by DEHP was due to an increase in the total amounts of protein, water, lipid, and nucleic acids. The increase in protein was most marked and was due mainly to the increase in non-collagen protein. The total amounts of glycogen and collagen did not change. However, most of these components were found to be unchanged or reduced in their contents per liver weight, except that of protein. The increase in RLW is caused equally by the increase in cell number and in the cell volume, the nuclear DNA content being unaffected. The content of cytochrome P-450 in microsomes increased in the rats of both sex fed a 0.5% DEHP-diet for 16 days. However, the activities per mg of microsomal protein or per g of liver of aminopyrine N-demethylase and aniline hydroxylase decreased in male rats but increased in female rats. Total activities of these enzymes increased markedly in both sex of animals. Glucose 6-phosphatase, acid phosphatase and cytochrome c oxidase were reduced significantly in their activities per liver weight but glucose 6-phosphate dehydrogenase was unchanged. The significance of the liver enlargement induced by DEHP is discussed in relation to the physiological response of liver and to possible pathological changes of liver.

Energy-dependent calcium transport in endoplasmic reticulum of adipocytes.

The endoplasmic reticulum from isolated rat adipocytes has the ability to actively accumulate calcium. The calcium uptake was characterized using the 20,000 X g supernatant (S1 fraction) of total cellular homogenate. Endoplasmic reticulum vesicles isolated from the S1 fraction as a 160,000 X g microsomal pellet prior to testing demonstrated little ability to accumulate calcium. The calcium uptake in the S1 fraction was localized to the endoplasmic reticulum vesicles by morphologic appearance, by the use of selective inhibitors of calcium uptake, and by high speed sedimentation of the accumulated calcium. The uptake was MgATP- and temperature-dependent and was sustained by the oxalate used as the intravesicular trapping agent. Uptake was linear with time for at least 30 min at all calcium concentrations tested (3 to 100 muM) and exhibited a pH optimum of approximately 7.0. The sulfhydryl inhibitor p-chloromercuribenzene sulfonate produced a dose-dependent inhibition of calcium uptake with total inhibition at 0.07 mumol/mg protein. Ruthenium red and sodium azide inhibited less than 5% of the uptake at concentrations (5 muM and 10 mM, respectively) which completely blocked calcium uptake by mitochondria isolated from the same cells. The Km for calcium uptake was 10 muM total calcium which corresponded to approximately 3.6 muM ionized calcium in the assay system. The maximum velocity of the uptake was 5.0 nmol (mg of microsomal protein)-1 (min)-1 at 24 degrees under the assay conditions used and exhibited a Q10 of 1.8. The uptake activity of the endoplasmic reticulum vesicles in the S1 fraction exhibited a marked time- and temperature-dependent lability which might account in part for the lack of uptake in the isolated microsomal fraction. This energy-dependent calcium uptake system would appear to be of physiologic importance to the regulation of intracellular calcium.

The metabolism and binding of catecholamines by the hepatic microsomal mixed-function oxidase of the rat.

Noradrenaline and adrenaline were metabolized by an NADPH- and oxygen-dependent process located within the hepatic microsomal fraction of the rat. Metabolism was inhibited by CO and compound SKF 525A, but not by pargyline, an inhibitor of monoamine oxidase, or by 3,4-dimethoxy-5-hydroxybenzoic acid, an inhibitor of catechol O-methyltransferase. It is concluded that the enzyme system responsible for the metabolism of the catecholamines was the microsomal mixed-function oxidase. The Km for noradrenaline was 2.4 mM and for adrenaline 1.0 mM, and V 15.6 and 3.6 nmol/min per mg of microsomal protein respectively. Both catecholamines bound to the microsomal fraction, producing a type II spectral change, with a Ks for noradrenaline of 0.9 mM and for adrenaline of 1.0 mM, and showed other characteristics of type II compounds by inhibited the reduction of cytochrome P-450 by NADPH and exhibiting an enhanced metabolism in the presence of acetone. The major product of catecholamine metabolism was an as yet unidentified alkali-labile compound, which did not correspond to any of the recognized catecholamine metabolites.

Effect of pregnancy on hepatic microsomal drug metabolism in rabbits and rats.

In Dutch-belted rabbits, pregnancy caused several-fold decrease of in vitro hepatic microsomal aminopyrine, benzphetamine, and hexobarbital biotransformations. In pregnant Sprague-Dawley rats, various kinds of expressing the in vitro rates of hexobarbital biotransformation (per mg of microsomal protein, g of liver, 100 g of body weight) indicated unchanged or slightly elevated microsomal enzyme activity. In vivo, the course of hexobarbital blood levels after i.p. hexobarbital sodium, 100 mg/kg, indicated that the fate of hexobarbital was not primarily determined by the small changes of microsomal enzyme activity but, rather, by changed hexobarbital distribution. Different ways of expressing in vitro rates of aniline biotransformation showed decreased or unchanged enzyme activity during pregnancy and in vivo experiments indicated that these changes did not affect aniline metabolism in living rats. The results pointed out marked species differences in the effect of pregnancy on drug metabolism. Interpretation of in vitro biotransformation data for living animals suggested that with different substrates, microsomal enzyme activity and distribution, respectively, may exert different effects playing either significant or apparently minor role in drug disposition.

Measurement of Rat Liver Microsomal 3-Hydroxy-3-methylglutaryl-Coenzyme A Reductase Activity, Partially Purified or in Microsomal Suspensions

mevalonate formed by t.1.c. (Shapiro et af., 1969), g.1.c. (Hamprecht & Lynen, 1970) or electrophoresis (Berndt & Gaumert, 1971). The method is reliable and sensitive, but slow because of the analytical step needed to separate the radioactive product from the radioactive substrate. However, for various reasons several other theoretically possible assay methods cannot be used. The rat liver enzyme shows a diurnal variation in activity (Hamprecht et al., 1969; Shapiro REdwards G Gregoryetaf., 1972),achieving at the daily maximum a specific activity, when assayed as a microsomal suspension, of about 1.0, and at the daily minimum of about 0.1. Specific enzyme activity is defined as units/mg of protein, where one unit of activity converts 1 nmol of 3-hydroxy-3-methylglutaryl-CoA to mevalonate in 1 min. No sufficiently sensitive chemical or enzymic test for mevalonate is known, so that direct estimation of the mevalonate formed is not practicable. The oxidation of NADPH during the 3-hydroxy-3-methylglutaryl-CoA reductase reaction cannot be used, because microsomal suspensions oxidize NADPH even when no other substrate is added (Gillette et af., 1957; Das et af., 1968). According to Das etaf. (1968) the initial rate of this NADPH oxidation is in the order of 4nmol/min per mg of microsomal protein; similar rates have been observed in this laboratory, and there is no known way of selectively inhibiting this ‘NADPH oxidase’ activity. Assay of rat liver microsomal 3-hydroxy-3-methylglutaryl-CoA reductase by measuring the CoASH released does not seem to have been attempted, although the enzyme in pigeon liver microsomal fractions has been assayed in this way by using Ellman’s reagent (Ellman, 1959) to estimate the thiol group of CoASH (Hulcher & Oleson, 1973). We have assayed the reductase in rat liver microsomal fraction by using 2-oxoglutarate dehydrogenase (Garland et al., 1965) to measure the CoASH released. For 3-hydroxy-3-methylglutaryl-CoA reductase assay microsomal fraction was incubated as described previously (Gregory et al., 1972) except that the 3-hydroxy-3methylglutaryl-CoA was not radioactively labelled. After deproteinization with HCI a sample of the reaction mixture was assayed fluorimetrically for CoASH essentially as

Irreversible protein binding of [ 14C]imipramine with rat and human liver microsomes

After incubation of [ 14C]imipramine with rat liver microsomcs up to 0–7 mole/mg was irreversibly bound per mg of microsomal protein. If albumin was added to the microsomal incubations [ 14C]imiprámine was also irreversibly bound to this protein. The irreversible binding of imipramine to protein was determined by exhaustive solvent extraction and charcoal adsorption, and measurement of the remaining 14C-radioactivity in the protein. The binding reaction was dependent on oxygen, NADPH, microsomal protein content and substrate concentration. It was inhibited by CO and SKF 525-A. Pretreatment of rats with phenobarbital did not increase the amount of imipramine irreversibly bound to protein. Glutathione and other cysteine derivatives diminished the binding, whereas incubation with the epoxide hydrase inhibitor trichloropropene oxide resulted in an increase of imipramine irreversibly bound to protein. The results favour the concept that irreversible protein binding of imipramine is catalyzed by a cytochrome P-450-dependent hydroxylation via an epoxidation step. Irreversible protein binding of imipramine was also detectable with three samples of human liver microsomes.

Metabolism of polycyclic hydrocarbons by a highly active aryl hydrocarbon hydroxylase system in the liver of a trout species

In sharp contrast to earlier beliefs, some fish (trout) liver-microsomes are highly capable of metabolizing benzo(α)pyrene. The hydroxylating system is a typical monooxygenase system in many respects when compared with the mammalian system, needing oxygen and NADPH for full activity. The trout liver-microsomes convert benzo(α)pyrene to dihydrodiols and hydroxymetabolites at a rate 5 to 10 times higher than the male rat liver-microsomes, when measured per mg of microsomal protein. The trout liver-microsomes metabolize benzo(α)pyrene 15 to 30 times as fast as the male rat liver-microsomes if the activity is measured per unit of cytochrome P-450 and NADPH-cytochrome c reductase.

Biochemical effects of 3,5-diethoxycarbonyl-1, 4-dihydrocollidine in mouse liver

3,5-Diethoxycarbonyl-1,4-dihydrocollidine (DDC) is a porphyrinogenic agent and is a powerful inducer of δ-aminolaevulinate synthetase, the first and rate-limiting enzyme of the haem-biosynthetic pathway, in mouse liver. However, DDC strikingly inhibits mitochondrial as well as microsomal haem synthesis by depressing the activity of ferrochelatase in vivo. The drug on repeated administration to female mice has been found to elicit hypertrophic effects in the liver microsomes initially, but the effects observed at later stages denote either hyperplasia or increase in polyploidal cells. The microsomal protein concentration shows a striking decrease with repeated doses of the drug. The rate of microsomal protein synthesis in vivo as well as in vitro shows an increase with two injections of DDC but decreases considerably with repeated administration of the drug. The activities of NADPH-cytochrome creductase and ribonuclease are not affected in the liver microsomes of drug-treated animals when expressed per mg of microsomal protein. DDC has also been found to cause degradation of microsomal haem, which is primarily responsible for the decrease in cytochrome P-450 content. The drug also leads to a decrease in mitochondrial cytochrome c levels due to inhibition of haem synthesis and also due to degradation of mitochondrial haem at later stages. The biochemical effects of the drug are compared and discussed with those reported for allylisopropylacetamide and phenobarbital.

The Binding of Reduced Nicotinamide Adenine Dinucleotide-Cytochrome b5 Reductase to Hepatic Microsomes

Abstract Incubation of microsomes with the amphipathic form of NADH-cytochrome b5 reductase results in the binding of a maximum of 7.2 ± 0.5 nmoles of reductase per mg of microsomal protein representing a 100-fold increase over endogenous enzyme levels. Interactions with the phospholipid portion of the membrane are involved in this binding and extra bound and endogenous reductase are apparently functionally identical. The binding of reductase is similar to the binding of cytochrome b5 to microsomes and the model for the microsomal membrane formulated on the basis of cytochrome b5 binding is therefore extended to include reductase. This model, involving random distribution and translational diffusion of these proteins on the surface of the membrane, is supported by experiments showing that extra bound reductase and cytochrome b5 interact catalytically and exclude each other from the microsomal membrane.

The hydroxylation of D-amphetamine by liver microsomes of the male rat

This paper describes the conditions under which the hydroxylation of amphetamine to p-hydroxyamphetamine can he studied in isolated rat liver microsomes. The reaction depends on the concentration of NADP. The optimal pH is 6.9–7.0. As Michaelis parameters 1.5 × 10 −4 M ( K m ) and 2.16 nmoles (mg of microsomal protein) −1 (10min) −1 ( V max) were calculated. The production of p-hydroxyamphetamine is not restricted to the microsomes but a smaller biotransformation of amphetamine was also found in mitochondria.

Cytochrome P-450-linked monooxygenase system and drug-induced spectral interactions in human liver microsomes

Summary Human liver microsomes isolated from operation biopsy samples from patients with cholelithiasis or a malignant disease, were studied with respect to a drug-oxidizing monooxygenase system. The human liver contained about 30-40 mg of microsomal protein per gram of wet weight and about 50% of that was recovered in the microsomal fraction. The mean levels of cytochrome P-450, NADPH-cytochrome c reductase and cytochrome b5 in the livers of cholelithiatic patients were essentially similar to those in the livers of rat and guinea pig and only slightly lower than those in the rabbit liver. The cytochrome P-450-carbon monoxide difference spectrum exhibited a peak at 450 nm and, in microsomes from several smokers, no peak shift to 448 nm was evident. Studies with ethyl isocyanide as a ligand revealed a typical difference spectrum with a peak ratio at pH 7.4 of 455/430 nm of 0.46 and a pH intercept of at about 7.9. There was no correlation between cigarette smoking and a peak ratio. Drug-induced interactions of type I (SKF 525A, bromobenzene), type II (aniline, n-octylamine, KCN) and reverse type I (phenacetin, n-butanol) variety were detected in liver samples studied. Hexobarbital and aminopyrine gave either type I or reverse type I spectral changes. The spectral dissociation constant for aniline was of the same order of magnitude as in rat liver microsomes. These studies seem to indicate that although qualitative differences between hepatic monooxygenase systems in man and laboratory animals may exist, available evidence suggests that many essential differences are only quantitative in nature.

Effect of ethanol on the amount and enzyme activities of hepatic rough and smooth microsomal membranes

To study the effect of chronic ethanol administration on the hepatic endoplasmic reticulum, rough and smooth microsomal membranes were separated from the liver of female rats pair-fed a nutritionally adequate diet with 36% of total calories either as ethanol or carbohydrate (controls). 6 weeks of ethanol feeding increased protein and phospholipid contents in smooth but not in rough microsomes. Cytochrome P-450 also increased mainly in smooth microsomes. In controls, the activity of the microsomal ethanol-oxidizing system was distributed about equally among the subfractions, whereas after ethanol feeding, it increased mainly in smooth microsomes. Catalase activity after ethanol feeding did not increase; when expressed per mg of microsomal protein, it decreased in smooth microsomes. A reduction of dietary fat content from 35 to 10% of total calories did not influence the effect of ethanol on the hepatic endoplasmic reticulum.

A study on the enzymatic mechanism of guinea-pig hepatic-microsomal bilirubin glucuronyl transferase.

1 Initial velocity studies for the two-substrate system of bilirubin glucuronyl transferase were performed with Triton X-100 as an activator. These studies were performed by varying the concentration of bilirubin in the presence of different concentrations of uridine 5′-diphosphate glucuronic acid (UDP-GlcUA) and gave an intersecting pattern (characteristic of a sequential mechanism) when the data were graphed as double-reciprocal plots. 2 Competitive inhibition was observed for UDP-GlcUA and noncompetitive inhibition for bilirubin when UDP (product) or UMP (dead end) were used as inhibitors. Alternate product inhibition with phenolphthalein glucuronide produced noncompetitive inhibition for both UDP-GlcUA and bilirubin. 3 It was concluded that the reaction under study probably has an ordered mechanism although the iso-Theorell-Chance mechanism is still a possibility. 4 Kinetic constants (mean for 3 animals) for a sequential mechanism yielded 52.3 μM as the apparent Km for bilirubin, 2.3 mM as the apparent Km for UDP-GlcUA and 20.2 nmol bilirubin conjugated per mg of microsomal protein per 30 min as the V for this reaction.

Studies of Prothrombin Biosynthesis in Cell-free Systems: I. COMPARISON OF COAGULATION AND IMMUNOCHEMICAL ASSAYS

Abstract Previous claims that could be detected in subcellular particles of rat liver by coagulation assays have been shown to be based on the artifactual conversion of residual in the reagents to thrombin by lysosomal cathepsin C. The heavy microsome fraction, rich in lysosomes, and prothrombin did not carry out general protein synthesis under the conditions of apparent biosynthesis. The reduced apparent observed in liver microsomes from vitamin K-deficient rats is due to an alteration in the integrity of lysosomes in this deficiency disease. Application of a radiommunoassay for to the detection of in rat liver microsomes before and after incubation showed that the actual content ranged, respectively, from 2 to 10 ng per mg of microsomal protein. The highest amount of detected is only one-tenth of that required to give a reliable coagulation assay.

The Effect of Age on Tissue Protein Synthesis

Currently available data on the effect of age in tissue protein synthesis and turnover are reviewed and experimental evidence is presented on age changes in amino acid incorporation by rat liver purified microsomes. It has been observed that the purification of ‘crude’ microsomal preparations from contaminating lysosomes by density gradient centrifugation is accompanied by an increase in amino acid incorporatmg activity of the microsomal preparations, no matter whether these are prepared from young or from old animals, and that the amino acid incorporating activity of rat liver purified microsomes prepared from old animals (19–25 months) is lower than that prepared from adult animals (7–11 months) which in turn is lower than that from young animals (2–3 months), no matter whether the activity is expressed per mg of microsomal protein or per mg of microsomal RNA. Also, experimental evidence is presented which is consistent with the presence in liver purified microsomes of heat-labile inhibitor(s) of amino acid incorporation, and with their accumulation (or increase in effect) in the microsomes with the age of the animal. Several major features of the decline with age in tissue protein synthesis are outlined and a number of possible factors or causes, beside the microsomal inhibitor(s), contributing to the decline with age in tissue protein synthesis, are discussed.

Diphenylhydantoin-induced difference spectra with rat-liver microsomes

Addition of diphenylhydantoin (DPH) to rat-liver microsomes caused a type l substrate-induced difference spectrum with a peak at 390 nm and a trough at 425 nm. The magnitude of this substrate-induced change, δA max was relatively small (mean absorbance: 0.027 ± 0.003 S.E., N = 6 per 3 mg of microsomal protein per ml) as compared with that caused by benzphetamine or aniline. The apparent K s for the DPH-inicrosome interaction was low: 27 ± 2 μM(mean ± S.E., N = 6). The δA max of substrate-induced difference spectra caused by addition of DPH to microsomes was proportional to the microsomal protein concentration, but rapidly decayed during storage at 4° (80% reduction in 24 h). Treatment of animals with phenobarbital, DPH, chlordane, or DDT caused a reduction of δA max of the DPH-microsome interaction as compared with that obtained with hepatic microsomes from the control animals. Treatment of animals with 3-methylcholanthrene (3MC) either abolished the δA max of DPH-microsome interaction or caused a reversal to a modified type II response. Addition in vitro of phenobarbital, DPH, chlordane, DDT, or 3MC as modifiers to the microsomes from control animals, reduced or abolished the δA max of substrate-induced difference spectra with DPH as substrate. It may be concluded that DPH despite its low K s is a rather weakly binding substrate which is easily replaced by more strongly binding ligands.

The interaction of a fluorescent probe with rat hepatic microsomes

The interaction of the fluorescent hydrophobic probe 1-anilinonaphthalene-8-sulfonate (ANS) with rat hepatic microsomes was examined. The fluorescence of the dye is enhanced about 250-fold and a 45-nm hypsochromic shift occurs in the peak of the ANS emission spectrum. Pretreatment of the rats with phenobarbital or 3-methylcholanthrene, or the reduction of microsomes with sodium dithionite or NADPH did not affect the intensity of fluorescence per mg of microsomal protein. A nonprotein, nonheme, binding locus, probably a phospholipid, is proposed. A Scatchard plot of the ANS conjugate with hepatic microsomes revealed that there is more than one class of binding sites for ANS on microsomes. Several compounds that have been previously shown to be substrates or inhibitors of rat hepatic microsomal NADPH-dependent mixed-function oxidases, modified ANS-microsome fluorescence without altering markedly the microsomal-ANS binding constants. Increases in fluorescence might reflect structural transitions in microsomes. Warfarin, a substrate, appears to displace ANS competitively from a high-affinity binding site without modifying ANS fluorescence due to interaction at a low-affinity binding site. ANS was not found to be a substrate of the hepatic microsomal NADPH-dependent oxidase system, but was an inhibitor of certain microsomal enzymes when added to incubation mixtures in vitro.