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Cytochrome P450s, proteins involved in metabolism of sterols, steroids, and a variety of foreign compounds, have been grouped into families based on amino acid sequence. We have identified a microsomal cytochrome P450 in guinea pig adrenal immunochemically related to P450c,d (P450I), induced in rat liver by methylcholanthrene. The inner zone localization, male predominance, and suppression by ACTH of this protein correspond to the ability of the adrenal microsomes to metabolize ethylmorphine. Its immunoreactivity, localization, and regulation distinguish it from P450(17) alpha (P450XVII) and P450(21) (P450XXI), known microsomal steroid hydroxylases. To examine whether other cytochrome P450s homologous to those in liver might be present in the guinea pig adrenal, microsomes were reacted with antibodies to hepatic P450s from families II and III. Each probe detected proteins in microsomes, but not in mitochondria, which were in the lower mol wt range of cytochrome P450s (47-50K). The immunoreactivity of all was diminished in animals treated with spironolactone, a compound which destroys cytochrome P450s in the adrenal, but not in liver. All were present in both outer and inner zones and in both males and females. None was suppressed by ACTH in the inner zone. Thus, only the previously described 52K protein reactive with anti-P450I corresponds in both distribution and ACTH response to the capacity for xenobiotic metabolism in guinea pig adrenal microsomes. On the other hand, unlike the 52K protein, the newly discovered proteins related to P450s II and III were all suppressed in the outer zone following dexamethasone treatment, suggesting that they might be related to dexamethasone-suppressible functions, such as metabolism of sterols and steroids.

A rapid gas chromatographic method for the determination of medium-chain and long-chain free fatty acids (C14:0 to C24:0 fatty acids) from different biological specimens is presented. After a rapid one-step transesterification method in methanol-acetyl chloride (50:1, v/v), fatty acid methyl esters were extracted into n-hexane and analysed on a 15-m Durabond-Wax column within a 12-min chromatographic run. The detection limit is 500 pg per injection.

The drug metabolizing enzyme activities, the vitamin A content and the fatty acid composition in the endoplasmic reticulum membrane were studied in rat liver after a single injection of the polychlorobiphenyls (PCBs) 3,3',4,4'-tetrachlorobiphenyl [(3,4)2Cl] or 2,2',4,4',5,5'-hexachloro-biphenyl [(2,4,5)2Cl], 300 mumol/kg each. The microsomal vitamin A level was markedly lowered 3 days after treatment with (3,4)2Cl, a coplanar type inducer of cytochrome P-450. A marked increase in microsomal AHH and UDPGT activities occurred within 3 days after injection of (3,4)2Cl whereas (2,4,5,)2Cl treatment enhanced APDM activity only. Arachidonic, stearic and linoleic acid microsomal contents were enhanced by the two congeners. (3,4)2Cl caused the proportion of docosahexaenoic acid to decrease. No highly significant correlation was found between the vitamin A content and lipid components in the microsomal membrane. However, the vitamin A level was inversely related to the activities of drug metabolizing enzymes induced by coplanar compounds (cytochrome P-450 towards benzo[a]pyrene and UDP glucuronosyl transferase towards 4-nitrophenol).

Phenobarbital treatment induces an isozyme(s) of liver microsomal cytochrome P450 susceptible to CCl4 and enhances the latter's lethality. We have now studied phenobarbital's effect on the specificity of phosphatidyl fatty acid changes in rat liver microsomes. Male Sprague-Dawley rats were pretreated with three daily ip doses of phenobarbital (50 mg/kg) or saline and then orally dosed with CCl4 (2.5 ml/kg). Liver microsomes were prepared 7.5 to 180 min after CCl4 treatment, the lipid fraction was extracted, diene conjugate content was determined, and phospholipids were separated by HPLC for fatty acid content determination. Protein, phospholipid, and phosphatidyl fatty acid residue loss occurred early (7.5 to 30 min) and in some cases later (60 to 180 min) in both pretreated groups, suggesting that two phases of CCl4-mediated injury occurred. Phenobarbital pretreatment accelerated the CCl4-induced formation of diene conjugates in the microsomal lipids. In studies on the separated phospholipids, phenobarbital alone altered microsomal fatty acid content, primarily decreasing arachidonic acid in favor of linoleate, particularly in phosphatidylserine. During the early phase of CCl4 injury, phenobarbital pretreatment shifted the major loss of arachidonic acid from phosphatidylserine to phosphatidylethanolamine. During the later phase, arachidonic acid loss was still prominent, but the most extensive CCl4-induced changes in fatty acids occurred in the neutral lipid fraction, regardless of pretreatment. These changes included loss of neutral lipid linoleic and docosahexanoic acids associated with an increase in palmitic acid. These data demonstrate that phenobarbital pretreatment is associated with a shift in the predominant phospholipid locus from phosphatidylserine to phosphatidylethanolamine for the early CCl4-induced fatty acid changes in rat liver microsomes.

Rats were treated with 10, 200 or 400 mg/kg brotizolam (Lendormin) for 4 weeks, then liver microsomes were prepared and the in vitro transformation of several model substances studied. Furthermore, after similar treatment of rats, the metabolite pattern in the plasma was studied using [14C]brotizolam as a marker. Finally the same investigations were performed after pretreating the rats with the enzyme inducers, phenobarbital or 3-methylchol-anthrene, for 3 days instead of brotizolam. The amount of microsomal protein in the rat liver was increased after all 3 doses of brotizolam, the liver weight after the highest dose only. Activity of the flavoenzyme NADPH cytochrome-c reductase was the only enzyme activity increased after 200 and 400 mg/kg brotizolam, whereas cytochrome P-450 content decreased after 400 mg/kg brotizolam. Activities of the mixed-function oxidases studied were not changed at all. Marked changes after brotizolam administration were seen in the metabolite pattern. The higher doses led to reduced amounts of both of the very polar metabolites. Simultaneously metabolite We 964 (= brotizolam hydroxylated at the methyl group) and the unchanged brotizolam increased several-fold. Treatment of rats with phenobarbital or 3-methylcholanthrene showed the typical but different changes in enzyme activities. The metabolite pattern of brotizolam, however, was not changed. From the results it is concluded that a 4-week treatment of rats with up to 400 mg/kg brotizolam causes no induction of mixed-function oxidases in the liver. The changes of the metabolite pattern described can be discussed as an effect of liver enzyme saturation.

4-Hydroxytetrahydroisoquinoline (4OH-TIQ) was detected as a metabolite of a possible parkinsonism-inducing substance, tetrahydroisoquinoline (TIQ), in rat liver microsomes and rat urine. Urinary excretion of 4OH-TIQ was significantly reduced in female DA rat, an animal model of a poor debrisoquine metabolizer. The female DA rat also showed significantly higher brain accumulation of TIQ. These results suggest that the metabolic detoxication process is depressed and TIQ accumulation in the brain is enhanced in a poor debrisoquine metabolizer, which may be one possible explanation for poor debrisoquine metabolizers being susceptible to Parkinson's disease.

Positive structural identification of the PBN-trichloromethyl spin adduct in vitro was accomplished with the use of high pressure liquid chromatography and/or gas chromatography coupled with mass spectrometry. Both thin layer and liquid chromatography were used to separate a complex mixture of compounds from rat liver extracts treated with CCl4 in vitro and in vivo. Deuterated PBN's (PBN-d9; tert-butyl deuteration, or PBN-d14; both phenyl and tert-butyl deuteration) were also used to aid in the mass spectral analysis of spin adducts from liver extracts of CCl4 exposed rat livers, since the tert-butyl group fragment ion. C4D9+ (m/z = 66) is always present for PBN and PBN spin adducts. In addition, the masses of the ion peaks increase by the amount of deuteration, i.e. an increase of 9 for PBN-d9 or PBN-d14 in comparison to normally synthesized PBN.

Microsomes were prepared from livers of malignant hyperthermia susceptible (MHS) or resistant (MHR) pigs. On incubation with the spin trap alpha-(4-pyridyl-l-oxide)-N-tert-butylnitrone (4-POBN), the microsomes from MHS pigs produced a characteristic electron spin resonance (ESR) signal at a greater rate than those from MHR pigs. Increased formation in the incubations of thiobarbituric acid reactive substances (TBARS) by the microsomes of the MHS pigs indicated an enhanced susceptibility to free radical-mediated lipid peroxidation. These results provide further evidence that MHS pigs have an antioxidant abnormality which may contribute to the fatal MH response. However the nature of the abnormality is unclear. The enhanced formation of unstable free radicals and indices of lipid peroxidation was not due to decreased vitamin E concentration or glutathione peroxidase activity in the microsomes. Furthermore, fatty acid profiles were similar in microsomes from MHS and MHR pigs indicating similar amounts of potential substrate for TBARS formation.

The cytochrome P-450 isoforms have been studied in liver microsomes of some fish species from Lake Baikal. Using the inhibitory analysis of microsomal monooxygenase activities carried out by the specific polyclonal antibodies it has been shown that 3-methylcholanthrene, beta-naphthoflavone and arochlor 1254 induce isoforms immunologically related to cytochrome P-488c but not to the rat cytochrome P-450b in fish liver microsomes. The immunologic identity in isoforms of fish and rat cytochromes induced by methylcholanthrene has not been revealed. A possibility to use the method of the inhibitory analysis of fish microsomal activities by specific antibodies to the rat cytochrome P-450 isoforms for biomonitoring and biotesting of polycyclic hydrocarbons and polychlorinated biphenyls in aquatic systems is discussed.

Male Sprague-Dawley rats were maintained for 31 days on a liquid diet containing 36% of calories as ethanol. Pair-fed controls were administered a similar diet, but with maltose-dextrin isocalorically substituted for ethanol. A phospholipid analysis has been carried out in liver microsomes and mitochondria isolated from the two groups of animals. The phospholipid phosphorus/protein ratio was not significantly different in the organelles of the ethanol-fed animals as compared to the same organelles of liquid diet controls, which indicates that ethanol feeding did not influence the total phospholipid content of microsomes and mitochondria. The phospholipid distribution within organelles was not changed, except for a significant increase in the phosphatidylinositol content of microsomes from ethanol-fed animals. The fatty acid compositions of both microsomal and mitochondrial phospholipids were significantly altered by ethanol feeding. In microsomes from ethanol-fed rats, palmitic acid levels were lowered in the total phospholipid fraction, phosphatidylcholine and phosphatidylethanolamine; oleic acid levels were elevated in microsomal phosphatidylethanolamine. In mitochondria from ethanol-fed animals, palmitic and arachidonic acid were lowered in phosphatidylcholine and phosphatidylethanolamine. Oleic and linoleic acid were elevated in the same phospholipids. In contrast, linoleic acid levels in cardiolipin were depressed significantly. These alterations in the fatty acid composition are suggestive of ethanol-induced changes in fatty acid desaturation activities.

GH receptors (GHRs) have been shown by affinity cross-linking to be present in late pregnant mouse liver microsomes in three forms with cross-linked mol wts of 125,000, 62,000, and 56,000. The two lower mol wt forms of the receptor were partially purified by bovine GH-affinity chromatography of 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate-solubilized extracts of late pregnant mouse hepatic microsomes. The GHRs were identified from the partially purified receptor preparation and isolated by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isolated GHRs had mol wts of 40,700 and 37,500, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. Enzymatic cleavage of N-linked glycosylation from the isolated GHRs reduced their apparent mol wts to 33,600 and 30,900, respectively. Sixteen of the amino-terminal 17 amino acid residues of the two isolated receptors were sequenced and determined to be identical. One amino acid residue in each of the proteins, at position 14, could not be identified. Rabbit polyclonal antiserum was produced against the isolated GHRs. The resulting antiserum precipitated the isolated 40,700 and 37,500 mol wt proteins as well as cross-linked mouse GHRs (including the high mol wt form of the receptor). However, the antiserum did not inhibit the binding of mouse GH to either membrane bound or 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate-solubilized GHRs.

The present study was designed to determine if Golgi fractions from fetal rat liver contain glucagon receptors and to characterize the properties of such receptors. Purification patterns of liver plasma membranes and Golgi fractions from fetal and adult rats were similar, as verified by morphological and biochemical approaches. Glucagon binding was greater in plasma membranes of adult than fetal rats, while in Golgi fractions glucagon binding was similar in both groups. The modifications in in glucagon binding reflect changes in glucagon receptors. Glucagon association and glucagon receptor inactivation by liver membranes were similar in the two groups of animals, while glucagon degradation was lower in fetal than in adult rats.

Carbohydrate-containing structures in rat liver rough microsomes (RM) were localized and characterized using iodinated lectins of defined specificity. Binding of [125I]Con A increased six- to sevenfold in the presence of low DOC (0.04--0.05%) which opens the vesicles and allows the penetration of the lectins. On the other hand, binding of [125I]WGA and [125I]RCA increased only slightly when the microsomal vesicles were opened by DOC. Sites available in the intact microsomal fraction had an affinity for [125I]Con A 14 times higher than sites for lectin binding which were exposed by the detergent treatment. Lectin-binding sites in RM were also localized electron microscopically with lectins covalently bound to biotin, which, in turn, were visualized after their reaction with ferritin-avidin (F-Av) markers. Using this method, it was demonstrated that in untreated RM samples, binding sites for lectins are not present on the cytoplasmic face of the microsomal vesicles, even after removal of ribosomes by treatment with high salt buffer and puromycin, but are located on smooth membranes which contaminate the rough microsomal fraction. Combining this technique with procedures which render the interior of the microsomal vesicles accessible to lectins and remove luminal proteins, it was found that RM membranes contain binding sites for Con A and for Lens culinaris agglutinin (LCA) located exclusively on the cisternal face of the membrane. No sites for WGA, RCA, soybean (SBA) and Lotus tetragonobulus (LTA) agglutinins were detected on either the cytoplasmic or the luminal faces of the rough microsomes. These observations demonstrate that: (a) sugar moieties of microsomal glycoproteins are exposed only on the luminal surface of the membranes and (b) microsomal membrane glycoproteins have incomplete carbohydrate chains without the characteristic terminal trisaccharides N-acetylglucosamine comes from galactose comes from sialic acid or fucose present in most glycoproteins secreted by the liver. The orientation and composition of the carbohydrate chains in microsomal glycoproteins indicate that the passage of these glycoproteins through the Golgi apparatus, followed by their return to the endoplasmic reticulum, is not required for their biogenesis and insertion into the endoplasmic reticulum (ER) membrane.

Nature of considerable variability of hexobarbital sleeping time and drug metabolism efficiency within a single strain of rats were investigated. Wistar or Sprague-Dawley rats with shorter than average hexobarbital sleeping time had also higher rates of in vitro hepatic microsomal metabolism of hexobarbital, aminopyrine, aniline and benzene, higher liver weight, microsomal protein content and P-450 level, and faster hexobarbital blood level decline (but similar volumes of distribution) after intraperitoneal hexobarbital sodium than those with relatively longer hexobarbital sleeping time, but awakened with the same hexobarbital blood level. The differences were maintained throughout the life of rats and inherited in their offspring. It indicated a possible genetic control of hexobarbital sleeping time and efficiency of drug metabolisms with apparent differences in selection response for Type I and Type II substrates (hexobarbital and aminopyrine vs aniline): it might indicate different heredity mechanism for these types of substrates. Stronger hexobarbital narcotic effect in females was associated with the rate of hexobarbital metabolism, but also with higher brain sensitivity. Hexobarbital sleeping time pattern indicated more general pattern of drug metabolism (better for Type I substrates) and success of selection of rats for different efficiency of drug metabolism (up to 8-fold differences in F5 generation) suggested considerable genetic non-homogeneity of two common strains of laboratory rats.

The lipid compositions of homogenates and microsomal fractions derived from surgical samples of highly differentiated human hepatoma, morphologically normal regions outside the tumours and from normal livers were analysed. A few enzyme activities were also assayed. Hepatoma microsomes demonstrated considerably lowered levels of cytochromes P-450 and b5. Hepatoma homogenates exhibited increased levels of cholesterol, normal amounts of dolichyl-P and slightly lowered levels of total phospholipid. The levels of dolichol, dolichol ester and ubiquinone in hepatoma homogenates were prominently decreased. In tumour microsomes the levels of cholesterol and dolichyl phosphate were increased considerably while the levels of phospholipid and dolichol were lowered. The phospholipid composition of tumour homogenates was roughly similar to that of control tissue. In tumour microsomes the relative amounts of phosphatidylserine and phosphatidylinositol were about 30% decreased, whereas the major phospholipids showed minor increases in amount. The rate and pattern of incorporation of [3H]glycerol into individual phospholipids in liver slices from control and hepatoma tissue did not differ to any larger extent. The fatty acid composition of tumour homogenates exhibited minor differences in comparison to the control with the greatest changes in the sphingomyelin fraction. In hepatoma microsomes the fatty acid compositions of the major phospholipids were altered moderately, with evident decreases in the relative amounts of the long-chain polyunsaturated fatty acids. In hepatoma homogenates the fatty acid composition of dolichol esters differed only slightly from the control pattern. These results indicate that the major disturbance in the lipid metabolism of highly differentiated hepatomas is localized to the mevalonate pathway, thus affecting mainly the levels of cholesterol, dolichol and ubiquinone.

Changes in lipid composition and function of subcellular organelles have been described in transplanted and primary tumours. We examine here the fatty acid composition of individual phospholipids (PL) in hyperplastic nodules and primary hepatoma induced by diethylnitrosamine (DEN), compared to that of normal liver and of transplantable Yoshida AH-130 hepatoma. Phosphatidylcholine and phosphatidylethanolamine fatty acid composition in mitochondria and microsomes from primary hepatoma were markedly different from normal liver; C18:0/C18:1 ratio was lower and the ratio between monosaturated and polyunsaturated fatty acids was higher. Linoleic acid content of mitochondrial cardiolipin, usually very high in normal rat liver, was notably lower in primary hepatoma. Cholesterol/phospholipid ratio in both microsomes and mitochondria from DEN-induced hepatoma was higher than in normal liver. Hyperplastic nodules showed no changes in cholesterol content whereas modifications in fatty acid composition were already observable. These modifications of membrane structure may be related to the functional changes found in nodular cells. Changes in fatty acid composition of membrane phospholipids, occurring in both primary hepatoma and preneoplastic nodules, might be one of the causes for decreased rate of lipid peroxidation peculiar to these tissues.

A pulmonary cytochrome P-450 was purified from lung microsomes of 3-methylcholanthrene (MC)-treated rats by immunoaffinity chromatography using a monoclonal antibody to MC-induced rat liver cytochrome P-450. The isolated pulmonary cytochrome P-450 was MC-inducible and had an apparent molecular weight of 57 kD on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight, as well as the NH2-terminal sequence of the first nine amino acids of the pulmonary cytochrome P-450, was identical to that of an epitopically related MC-induced rat liver cytochrome P-450. In addition, partial proteolysis of both cytochromes P-450 yielded indistinguishable peptide patterns on SDS-Page. Treatment of rats with MC, therefore, induces a pulmonary cytochrome P-450 which is structurally identical to the MC-induced hepatic enzyme by several criteria.

The effect of M7310U, a new non-steroidal analgesic anti-inflammatory agent, on liver microsomal drug-metabolizing enzymes was investigated. Rats were treated orally with M73101 (100, 200, 500 mg/kg), henylbutazone (PZ, 200 mg/kg), aminopyrine (AM, 100 mg/kg) or phenobarbital sodium (PB, 100 mg/kg) once daily for 2 weeks and then were observed for 2 weeks during which treatment was not given. On treatment with M73101, PZ, AM and PB, the liver enlarged but was restored to normal 1 week after the last administration. The rate of increase in the case of M73101 was lower than that seen with the reference compounds. M73101 markedly increased the content of microsomal protein, cytochrome P-450 or b5 and NADPH cytochrome C reductase, aniline hydroxylase and AM demethylase activity, but these increments returned to the normal level 1 week after the last administration. The serum concentration of M73101 after repeated administration (200 mg/kg, p.o.) for 1 week was lower than that after a single administration. Furthermore, M73101 increased Vmax for both aniline hydroxylase and AM demethylase, whereas it increased Km only for aniline hydroxylase. M73101 did not enhance the lipid peroxidation. Our observations suggest that the enlargement of rat liver seen with M73101 was due to the induction of drug-metabolizing enzymes and that this agent can probably be classified as a phenobarbital-type inducer.

Primary products of lipid peroxidation in liver tissue (diene conjugates) were extracted using various systems of solvents. In the system heptane-isopropanol 1:1 the diene conjugates were mainly extracted by the alcohol. The procedure for estimation of the tissue diene conjugates involved extraction with heptane-isopropanol, purification and spectrophotometric measurement of the extinction in the alcohol fraction.

The nuclear pore complex is a prominent structural component of the nuclear envelope that appears to regulate nucleoplasmic molecular movement. Up to now, none of its polypeptides have been defined. To identify possible pore complex proteins, we fractionated rat liver nuclear envelopes and microsomal membranes with strong protein perturbants into peripheral and intrinsic membrane proteins, and compared these fractions on SDS gels. From this analysis, we identified a prominent 190-kilodalton intrinsic membrane polypeptide that occurs specifically in nuclear envelopes. Lectin binding studies indicate that this polypeptide (gp 190) is the major nuclear envelope glycoprotein. Upon treatment of nuclear envelopes with Triton X-100, gp 190 remains associated with a protein substructure of the nuclear envelope consisting of pore complexes and nuclear lamina. We prepared monospecific antibodies to gp 190 for immunocytochemical localization. Immunofluorescence staining of tissue culture cells suggests that gp 190 occurs exclusively in the nucleus during interphase. This polypeptide becomes dispersed throughout the cell in mitotic prophase when the nuclear envelope is disassembled, and subsequently returns to the nuclear surfaces during telophase when the nuclear envelope is reconstructed. Immunoferritin labeling of Triton-treated rat liver nuclei demonstrates that gp 190 occurs exclusively in the nuclear pore complex, in the regions of the cytoplasmic (and possibly nucleoplasmic) pore complex annuli. A polypeptide that cross-reacts with gp 190 is present in diverse vertebrate species, as shown by antibody labeling of nitrocellulose SDS gel transfers. On the basis of its biochemical characteristics, we suggest that gp 190 may be involved in anchoring the pore complex to nuclear envelope membranes.

Cholesterol and phospholipid content, and phospholipid composition (sphingomyelin, phosphatidylcholine, phosphatidylserine, phosphatidylinositol, phosphatidylethaolamine) were assayed in rat liver microsomes during regeneration, foetal development and pregnancy. Cholesterol was assayed using Liebermann-Buchard reagent; the phospholipid extract was separated by thin-layer chromatography. While in pregnancy no changes were observed, during foetal development and liver regeneration there was a significative decrease of cholesterol/phospholipid ratio, and of phosphatidylcholine content. Moreover, in developing liver microsomes, there is also a significative increase of sphingomyelin and phosphatidylserine + phosphatidylinositol.

Biotin clearance, its distribution in liver and liver fractions after intravenous administration of 5 muCi/100 g body weight (21.55 microgram) of biotin-14COOH in normal and biotin-deficient rats are reported. In the biotin deficient animal there is a more rapid disappearance of the labeled biotin from the blood stream. Biotin-14COOH incorporation in the liver of the deficient rat is more rapid and larger than the incorporation in normal rat liver. Almost all the biotin recovered from liver homogenate is found in the mitochondria and in the pH 5.2 cytosol fraction; whereas in the microsomes only a very small amount is present. The intracellular distribution of biotin is in agreement with its known metabolic roles.

(1) A method was devised for the subfractionation of normal rat liver and for the subfractionation of the mitochondrial fraction into inner and outer membrane fractions. The purity of the fractions was assessed using marker enzyme measurements. (2) alpha-Tocopherol was measured in all the fractions by a sensitive HPLC technique. Profiles of phospholipid fatty acids were also determined by gas-liquid chromatography in all the fractions, and these values were calculated in terms of the percentage of each fatty acid in the total fatty acid of the fraction, as well as of the mass of each fatty acid per mumol of phospholipid phosphorus. Tocopherol values were expressed as the mass of tocopherol per g wet liver and per mumol of phospholipid phosphorus. (3) The results show that the mitochondrial and microsomal fractions were the major tocopherol-containing fractions, and both the inner and outer mitochondrial fractions contained substantial amounts of alpha-tocopherol. (4) The mitochondrial and microsomal fractions also had the highest levels of polyunsaturated fatty acids (PUFA) in their phospholipid fraction, especially 20:4 and 22:6, which were particularly localised in the inner mitochondrial membrane fraction. The high inner mitochondrial and microsomal PUFA levels were particularly apparent when the sum of all the unsaturated fatty acids with three or more double bonds was calculated. (5) Calculation of molar ratios of of some phospholipid fatty acids to alpha-tocopherol gave values of the order of several thousand to one. (6) It is concluded that the protective effect of each molecule of alpha-tocopherol must be exerted towards a large number of molecules of membrane unsaturated fatty acids simultaneously. This perhaps implies specific localisation of tocopherol in regions of membrane particularly liable to attack, such as might be expected to occur close to respiratory enzymes that can donate electrons to molecular oxygen.

Treatment of rats with carbon tetrachloride (CCl4) resulted in early reproducible losses of either one or two specific polypeptides (depending on the inducing agent with which the animals had been treated) in the molecular weight range of the multiple forms of cytochrome P-450. The loss was correlated with a decrease in total cytochrome P-450 content in the microsome. The results of this study and those in the accompanying report indicate that CCl4 was metabolized by a specific form of cytochrome P-450 (52,000 daltons), which was rapidly destroyed in the process. The early loss of this peptide occurred simultaneously with the previously demonstrated production of highly reactive trichloromethyl radicals (CCl3). This polypeptide, which was shown to disappear from liver microsomes following treatment of rats with CCl4 was demonstrated in the accompanying report to be the form of cytochrome P-450 specifically required for production of the highly reactive trichloromethyl radical in a reconstituted monooxygenase system.

Signal recognition particle (SRP) and SRP receptor are known to be essential components of the cellular machinery that targets nascent secretory proteins to the endoplasmic reticulum (ER) membrane. Here we report that the SRP receptor contains, in addition to the previously identified and sequenced 69-kD polypeptide (alpha-subunit, SR alpha), a 30-kD beta-subunit (SR beta). When SRP receptor was purified by SRP-Sepharose affinity chromatography, we observed the co-purification of two other ER membrane proteins. Both proteins are approximately 30 kD in size and are immunologically distinct from each other, as well as from SR alpha and SRP proteins. One of the 30-kD proteins (SR beta) forms a tight complex with SR alpha in detergent solution that is stable to high salt and can be immunoprecipitated with antibodies to either SR alpha or SR beta. Both subunits are present in the ER membrane in equimolar amounts and co-fractionate in constant stoichiometry when rough and smooth liver microsomes are separated on sucrose gradients. We therefore conclude that SR beta is an integral component of SRP receptor. The presence of SR beta was previously masked by proteolytic breakdown products of SR alpha observed by others and by the presence of another 30-kD ER membrane protein (mp30) which co-purifies with SR alpha. Mp30 binds to SRP-Sepharose directly and is present in the ER membrane in several-fold molar excess of SR alpha and SR beta. The affinity of mp30 for SRP suggests that it may serve a yet unknown function in protein translocation.

Male ICR mice were subcutaneously injected with either aqueous Ag (1.5 or 5.0 mg/kg) or Cd (1.5 or 2.5 mg/kg) for 2 consecutive days. Body fluids and livers were collected 24 hr after the second dose. In the hepatic supernatant, Ag and Cd were recovered at 2 and 36-46% of the total dose, respectively. Ag-metallothionein (MT), which is associated with Ag, Cu, and Zn, and Cd-MT, which is associated with Cd, Cu, and Zn, were induced in the liver by the injection of Ag and Cd, respectively. The supernatant Ag and Cd existed in the MT fraction at 34-61 and 97% levels, respectively. Cu concentration in the hepatic supernatant was increased by the Ag and Cd injections. The increased Cu was due to the appearance of Ag-MT and Cd-MT, respectively. Microsomal concentrations of Cu increased in the Cd groups, but decreased in the Ag groups. Serum ceruloplasmin (Cp) activity was remarkably increased by the injection of Cd, but severely decreased by the injection of Ag. These opposing changes in Cp activity induced by Cd and Ag may be due not to the sequestering of Cu in MT, but to the alteration of microsomal Cu concentration and/or the difference in affinity of the induction metals to MT. Hepatic Fe concentration was increased by the Ag injection, but was decreased by the Cd injection. These changes may not be related to induction of MT, but to Cp synthesis in the liver.

Microsomal glucokinase is solubilized by incubation in the presence of several metabolites. After solubilization of the enzymes, the membranes present free sites for specific binding of glucokinase, therefore, they can be purified by affinity chromatography on Sepharose--ATP-glucokinase. This method yields membranous vesicles which contain, in addition to glucokinase, uridylyl-transferase, phosphoglucomutase, sialyl-transferase and adenylate cyclase. Galactosyl-transferase, glucose-6-phosphatase and NADPH cytochrome c reductase are absent. It appears that functionally related enzyme from UDP-glucose biosynthesis are aggregated onto specific patches of the membrane, most likely from Golgi apparatus.

Toxic substances are generated by microsomes undergoing NADPH-dependent lipid peroxidation. In the present experiments the nature of the haemolytic material extracted from peroxidized microsomes is further examined. Suspensions of peroxidized rat liver microsomes were extracted with lipid solvents and the haemolytic material obtained subjected to gel exclusion chromatography. The haemolytic activity eluted as a single broad peak with an average size of 720 daltons. There was no evidence of haemolytic compounds less than 500 daltons. On a silicic acid column the activity eluted with the phospholipid. In both separations the presence of haemolytic activity correlated well with the peroxide content.

A comparative characteristic of the liver main cellular organoids is given for sheep of different age and origin. Crossing of different types of sheep breeds (Krazakh fine-wool and Lincoln) results in an increase in the amount of nucleic acids and proteins in nuclei and microsomes and does not influence other cellular structures of cross-breeds.

The fatty acid patterns of rat liver mitochondrial and microsomal phospholipids were analyzed from term fetuses, 1 and 4 days old, and adult rats. The main fatty acids of phosphatidylethanolamine and -choline were stearic and palmitic acids, although the patterns differed slightly. The fatty acid composition of corresponding phospholipids in mitochondria and microsomes was similar. The fatty acid pattern of cardiolipin was dominated by linoleic acid. The most consistent feature of the developmental changes in the fatty acid patterns of all phospholipids studied was a decrease in the relative amount of monounsaturated fatty acids. The percentages of saturated fatty acids in phosphatidylethanolamine and -choline increased during neonatal development. It is suggested that the high levels of fetal monounsaturated fatty acids were due to low availability of polyunsaturated fatty acids.

We present three lines of evidence for the existence of an intramicrosomal pool of CMP-NeuNAc in liver slices incubated with 3H-NeuNAc: first, enrichment in the microsomal fraction over the cell homogenate of CMP-3H-NeuNAc relative to CMP-14C-NeuNAc, the latter added as marker for sugar nucleotide trapped between and adsorbed onto vesicles; second, removal from microsomes by isotonic sucrose washing of only half of the CMP-3H-NeuNAc synthesized in vivo under conditions which remove all the CMP-14C-NeuNAc; and third, complete loss of both sugar nucleotides upon treatment of microsomes by hypotonic shock or with low concentrations of detergents. Incubation of CMP-NeuNAc with microsomes in vitro showed that the intact sugar nucleotide was penetrating the vesicles. When the sugar nucleotide is labeled in the sugar moiety, microsomal vesicles accumulate soluble radiolabeled products. This accumulation was saturable and dependent on time, temperature and concentration of sugar nucleotide, and was also inhibited by substrate analogues and by pretreatment of microsomes with proteases. Together, these results provide strong evidence for the existence of a specific transport system for CMP-NeuNAc in microsomes.

The electrochemical behaviour of the electron transfer proteins -- cytochromes b5, c and P-450 was studied by classical polarography and electrolysis with spectrophotometric monitoring. It was shown electrons are directly transferred from the electrode to oxidized cytochromes c and b5. Cytochrome P-450 is not reduced on the electrodes. However, the reduced inactivated from of cytochrome P-420 was detected at high potential values (-1,5 v).

Endoplasmic reticulum (ER)-Golgi relationships in the intracellular transport process of secretory proteins in rat hepatocytes have been studied using lipoprotein particles as a marker for the secretory protein and cytochrome P-450 as a marker enzyme for the ER membranes. Ferritin immunoelectron-microscopic observation revealed that, while almost all the microsomal vesicles derived from ER membranes are heavily labelled with ferritin anti-cytochrome P-450 antibody conjugates, labelling of the small peripheral vesicles containing lipoprotein particles, the stacks of Golgi saccules, especially the outermost saccule which is sometimes fenestrated, condensing vacuoles in the trans-Golgi region and the secretion droplets of lipoprotein were scanty and at the control level. Such a characteristic pattern of labelling was especially evident when these structures were prepared from phenobarbital-treated rats. These findings indicate that the membranes of the small peripheral vesicles do not contain cytochrome P-450 and that the cytochrome is probably not transferred to Golgi saccules in the transport process of lipoprotein from ER to Golgi. It is suggested, therefore, that the small peripheral vesicles are formed by budding of the special regions of ER membrane where microsomal marker proteins such as cytochrome P-450 are excluded and the membrane proteins destined to the Golgi complexes are clustered. It is also shown that lysosomal membranes are not labelled with the anti P-450 antibody conjugates.

A cleavable cross-linking reagent, dithiobis(succinimidyl propionate), DSP, was used to study the topography of the proteins in the endoplasmic reticulum membrane of rat liver. Reaction of untreated (control), phenobarbital- or 3-methylcholanthrene-induced microsomes with 0.5 mM DSP for 30 min at 0 degrees C resulted in the cross-linking of a protein with a molecular weight of about 52 000 to form an apparent dimer. In phenobarbital microsomes, a smaller amount of a 52 000-dalton protein also appeared in a dimer in the absence of DSP if N-ethylmaleimide was not included during homogenization. In phenobarbital and 3-methylcholanthrene microsomes, a 48 000-dalton protein was cross-linked by DSP to a protein of about 57 000. In all three types of microsomes, a protein with an Mr of about 52 000 was also cross-linked to a protein of about 79 000. In phenobarbital and control microsomes, cross-linking resulted in an oligomeric protein of approximate molecular weight 180 000 which contained three proteins, two with Mr of about 52 000 and one about 79 000. Under the cross-linking conditions, little or no denaturation of cytochrome P-450 and NADPH-cytochrome c reductase was observed. The aryl hydrocarbon hydroxylase activity was significantly inhibited by the bifunctional cross-linking reagent, DSP, but not by the monofunctional reagent N-succinimidyl-3-(4-hydroxyphenyl) propionate. However, attempts to regenerate the aryl hydrocarbon hydroxylase activity by cleavage of the disulfide linkage with 2-mercaptoethanol or dithiothreitol were not successful.

Cytochrome b5 from rat liver microsomes (microsomal fractions) was purified in its native form. The procedure described has great capacity, is fast, and the final product is pure as judged from SDS/polyacrylamide-gel electrophoresis. Antibodies to cytochrome b5 are obtained after administration of the antigen inserted into small unilamellar lipid vesicles.

Experiments, carried out on adult and old animals with different specific lifespan (rat, guinea pig, rabbit, and dog), revealed age-related changes in content and activity of liver microsomal oxidation enzymes (cytochrome P-450, b5, aminopyrine demethylase, aniline hydroxylase). The changes become more pronounced with age. This allows to classify these species as chronobiological ones. A negative correlation between the specific lifespan and the level of decrease in activity of aminopyridine demethylase, an integral index of electron transport rate in microsomal chain, in aged animals was established.