Change in the metabolism of 7,12-dimethylbenz(a)anthracene under the influence of vitamin A]

The in vitro metabolism of 7,12-dimethylbenz(a)anthracene in its incubation with the liver microsomes, the liver and mammary gland homogenates of rats, kept on vitamin A-enriched diet, was studied. Vitamin A inhibited the formation of lipophilic metabolites and increased the generation of water-soluble metabolites. The amount of lipophilic metabolites extracted from the microsomes and the liver and mammary gland homogenates were decreased by a factor of 2.2 and 5, respectively. The amount of unmetabolized DMBA in the liver microsomes was the same in control and experimental animals.     

Quantitative analysis of the ring-hydroxylated metabolites of 2-fluorenylacetamide by gas-liquid chromatography

An analytical procedure is described for quantitative estimation of the C₅- and C₇-hydroxylated metabolites of the carcinogenic amide, 2-fluorenylacetamide. After incubation of 2-fluorenylacetamide in the presence of rat or hamster liver microsomes and a NADPH-generating system, these metabolites were extracted and derivatized with heptafluorobutyrylimidazole. Subsequent analysis by gas-liquid chromatography with electron-capture detection was sensitive enough for determination of the kinetic parameters of these hydroxylation reactions. The effects of pretreatment with 3-methylcholanthrene or 2,3-benzanthracene on these parameters were also studied.     

Metabolic activation of N-thiazol-2-yl benzamide as glucokinase activators: Impacts of glutathione trapping on covalent binding

Glucokinase activators (GKAs) are currently under investigation as potential antidiabetic agents by many pharmaceutical companies. Most of GKAs reported previously possess N-aminothiazol-2-yl amide moiety in their structures because the aminothiazole moiety interacts with glucokinase (GK) and shows strong GK activation. During the development of N-aminothiazol-2-yl amide derivatives, we identified a bioactivation and metabolic liability of 2-aminothizole substructure of GKA 3 by assessing covalent binding, metabolites in liver microsomes and glutathione (GSH) trap assay.     

Stereoselective hydroxylation at the aliphatic carbons of 7,8- and 9,10-dihydrobenzo[a]pyrenes by rat liver microsomes

Optically active 7-hydroxy-7,8-dihydrobenzo[a]pyrene and 8-hydroxy-7,8-dihydrobenzo[a]pyrene were identified as two of the major metabolites formed by incubation of 7,8-dihydrobenzo[a]pyrene with rat liver microsomes. Optically active 9-hydroxy-9,10-dihydrobenzo[a]pyrene and 10-hydroxy-9,10-dihydrobenzo[a]pyrene were similarly identified as two of the minor metabolites of 9,10-dihydrobenzo[a]pyrene. The formation of these metabolites was abolished either by prior treatment of liver microsomes with carbon monoxide or the absence of NADPH, but was not inhibited by an epoxide hydrolase inhibitor. The results indicate that the aliphatic carbons of dihydro polycyclic aromatic hydrocarbons may undergo stereoselective hydroxylation reactions catalyzed by the cytochrome P-450 system of rat liver microsomes.     

Metabolism of benzo(a)pyrene by duck liver microsomes

The metabolism of benzo(a)pyrene [BP], a model carcinogenic PAH, by hepatic microsomes of two duck species, mallard (Anas platyrhynchos) and common merganser (Mergus merganser americanus) collected from chemically-contaminated and relatively non-contaminated areas was investigated. The rate of metabolism of BP by liver microsomes of common merganser and mallard collected from polluted areas (2,650 +/- 310 and 2,200 +/- 310 pmol/min per mg microsomal protein, respectively) was significantly higher than that obtained with liver microsomes of the two species collected from non-polluted areas (334 +/- 33 and 231 +/- 30 pmol/min per mg microsomal protein, respectively). The level of cytochrome P-450 1A1 was significantly higher in the liver microsomes of both duck species from the polluted areas as compared to the ducks from the non-polluted areas. The major BP metabolites, including BP-9, 10-diol, BP-4, 5-diol, BP-7, 8-diol, BP-1, 6-dione, BP-3, 6-dione, BP-6, 12-dione, 9-hydroxy-BP and 3-hydroxy-BP, formed by liver microsomes of both duck species from polluted and non-polluted areas, were qualitatively similar. However, the patterns of these metabolites were considerably different from each other. Liver microsomes of ducks from the polluted areas produced a higher proportion of benzo-ring dihydrodiols than the liver microsomes of ducks from the non-polluted areas, which converted a greater proportion of BP to BP-phenols. The predominant enantiomer of BP-7,8-diol formed by hepatic microsomes of the two duck species had an (-)R,R absolute stereochemistry. The data suggest that duck and rat liver microsomal enzymes have different regioselectivity but similar stereoselectivity in the metabolism of BP.     

Metabolism of benzo(a)pyrene by microsomes from tissues of pregnant and fetal hamsters

Pretreatment of hamsters with benzo (a) pyrene (BaP) greatly increased the $\text{in vitro}$ metabolism of BaP by lung microsomes from pregnant hamsters, and had less effect on the metabolism of BaP by liver microsomes. The production of various metabolites of BaP by lung microsomes was increased to different extents: 3-hydroxy-BaP (3-OH-BaP) was one of the major metabolites; the metabolic yields of 9, 10-dihydrodihydroxy-BaP (9, 10-diol) and 7,8-diol were increased more than that of the 4,5-diol. In the case of liver microsomes, only the yields of 9,10-diol and 7,8-diol were increased over the control levels. The presence of cyclohexene oxide in the incubation mixtures decreased the production of the diols. Basal-level enzyme activities in placental, fetal liver, and fetal skin microsomes in metabolizing BaP were very low. Pretreatment of pregnant hamsters with BaP induced BaP-metabolizing enzymes in fetal tissue 2–3 fold.     

Inhibition of steroid 5 alpha-reductase and its effects on testosterone hydroxylation by rat liver microsomal cytochrome P-450

It has been shown previously that liver microsomal steroid 5 alpha-reductase activity increases with age in female but not male rats, which coincides with a female-specific, age-dependent decline in the cytochrome P-450-dependent oxidation of testosterone to 1 beta-, 2 alpha-, 2 beta-, 6 alpha-, 6 beta-, 7 alpha-, 15 beta-, 16 alpha-, 16 beta-, and 18-hydroxytestosterone and androstenedione. To determine whether the increase in steroid 5 alpha-reductase activity is responsible for the decrease in testosterone oxidation, we have examined the effects of the steroid 5 alpha-reductase inhibitor, 4-MA (17 beta-N,N-diethylcarbamoyl-4-methyl-4-aza-5 alpha-androstan-3-one), on the pathways of testosterone oxidation catalyzed by rat liver microsomes. We have also determined which hydroxytestosterone metabolites are substrates for steroid 5 alpha-reductase. At concentrations of 0.1 to 10 microM, 4-MA completely inhibited steroid 5 alpha-reductase activity without inhibiting the pathways of testosterone oxidation catalyzed by liver microsomes from rats of different age and sex, and from rats induced with phenobarbital or pregnenolone-16 alpha-carbonitrile. 4-MA (10 microM) had little or no effect on the oxidation of testosterone catalyzed by liver microsomes from mature male rats (which have low steroid 5 alpha-reductase activity). In contrast, the hydroxylated testosterone metabolites formed by liver microsomes from mature female rats (which have high steroid 5 alpha-reductase activity) accumulated to a much greater extent in the presence of 4-MA. Evidence is presented that 4-MA increases the accumulation of hydroxytestosterones by two mechanisms. First, 4-MA inhibited the 5 alpha-reduction of those metabolites (such as 6 beta-hydroxytestosterone) that were found to be excellent substrates for steroid 5 alpha-reductase. In the absence of 4-MA, these metabolites eventually disappeared from incubations containing liver microsomes from mature female rats. Second, 4-MA inhibited the formation of 5 alpha-dihydrotestosterone, which otherwise competed with testosterone for oxidation by cytochrome P-450. This second mechanism explains why 4-MA increased the accumulation of metabolites (such as 7 alpha-hydroxytestosterone) that were found to be poor substrates for steroid 5 alpha-reductase. Despite its marked effect on the accumulation of hydroxylated testosterone metabolites, 4-MA had no effect on their initial rate of formation by liver microsomes from either male or female rats.(ABSTRACT TRUNCATED AT 400 WORDS)     

Hepatic metabolism of ergot alkaloids in beef cattle by cytochrome P450

This study was conducted to investigate the involvement of cytochrome P450 3A (CYP3A) in the metabolism of ergotamine in beef liver microsomes. When incubated with liver microsomes, ergotamine was hydroxylated to metabolites M1 and M2. Similarly, its isomer was hydroxylated to M1-Iso and M2-Iso (8-hydroxy-derivatives). Further incubation resulted in a second hydroxylation of M1 and M2 to metabolites M3 and M4 (8,9-dihydroxy derivatives). Maximum formation of metabolites was reached after 20 min, and ergotamine and its isomer were almost totally metabolized after 60 min of incubation. The formation of these metabolites was completely dependent on the presence of NADPH or the NADPH generating system and was also dependent on microsome concentration. Ergotamine was converted at a rate of 2 nM/microgram microsome/min when incubated with bovine liver microsomes to produce a metabolite profile (M1, M2, M1-Iso and M2-Iso) similar to the metabolites produced (2.2 nM/microgram/min) when ergotamine was incubated with liver microsomes of dexamethasone treated rats. This work provides information on the modification of ergotamine in bovine liver microsomes by CYP3A, which is of importance in understanding the detoxification and the clearance of ergotamine and other ergot alkaloids by bovine.     

Metabolism of carbosulfan. I. Species differences in the in vitro biotransformation by mammalian hepatic microsomes including human

The in vitro metabolism of carbosulfan, a widely used carbamate insecticide, by hepatic microsomes from human, rat, mouse, dog, rabbit, minipig, and monkey was studied. Altogether eight (8) phase I metabolites were detected by LC–MS; phase II metabolites were not found in human homogenates fortified with appropriate cofactors. The primary metabolic pathways were the initial oxidation of sulfur to carbosulfan sulfinamide (‘sulfur oxidation pathway’) and the cleavage of the nitrogen sulfur bond (N–S) to give carbofuran and dibutylamine (‘carbofuran pathway’). Carbofuran was further hydroxylated to 3-hydroxycarbofuran and/or 7-phenolcarbofuran, which were further oxidized to 3-ketocarbofuran or 3-hydroxy-7-phenolcarbofuran, respectively, and finally to 3-keto-7-phenolcarbofuran. 3-Hydroxycarbofuran was the main metabolite in all species, but otherwise there were some qualitative interspecies differences in carbofuran pathway metabolites. Only rabbit liver microsomes were able to metabolize carbofuran via hydroxylation to 7-phenolcarbofuran. Carbofuran was not detected in dog liver microsomes due to rapid further metabolism. In general, liver microsomes from all seven species produced more toxic products (carbofuran, 3-hydroxy-carbofuran, 3-ketocarbofuran) more rapidly than a detoxification product (carbosulfan sulfinamide). Differences in intrinsic hepatic clearances (CL_int) between the lowest and highest species were moderate; 2-fold for the carbofuran pathway, 2.7-fold for carbosulfan sulfinamide and 6.2-fold for dibutylamine. Our studies, although restricted to in vitro metabolic data from human and animal hepatic preparations, provide valuable quantitative carbosulfan-specific data for risk assessment, which suggest that interspecies differences, for carbosulfan active chemical moiety, in toxicokinetics are within the standard applied factor for species extrapolation in toxicokinetics. These results will be valuable in further defining the risks associated with exposure to carbosulfan.     

Metabolism of [4-14C]estrone in hamster and rat hepatic and renal microsomes: species-, sex- and age-specific differences.

The metabolism of [4-14C]estrone (E1) was examined in liver and kidney microsomes of adult castrated male and ovariectomized female hamsters and rats and in neonatal and immature hamster renal microsomes. In castrated male hamster liver microsomes, E1 was metabolized extensively to six major metabolites; 15 beta-hydroxyestrone, 7 alpha-hydroxyestrone, 6 alpha-hydroxyestrone, 6 beta-hydroxyestrone, 2-hydroxyestrone, and delta(9,11)-dehydroestrone, and a nonpolar fraction. Six minor metabolites of E1 were also detected. In contrast, kidney microsomes derived from castrated male hamsters metabolized E1 to mainly 17 beta-estradiol, 2- and 4-hydroxyestrone, 6 alpha-hydroxyestrone, 6 beta-hydroxyestrone and one monohydroxyestradiol metabolite. However, 16 alpha-hydroxyestrone was not detected. A variable, but low amount of estriol was also found. Interestingly, the quantity of 2-hydroxyestrone found in kidney microsomes of the hamster represented 26% of the total amount of metabolites formed, whereas in liver microsomes, only 9% of the overall metabolism resulted in the formation of 2-hydroxyestrone. The ability of kidney microsomes of female ovariectomized hamsters and two different rat strains to metabolize E1 was 5.9- and 9.4-fold lower, respectively, compared to renal microsomes of male castrated hamsters. The onset of oxidative metabolism in newborn hamster kidneys during development was also assessed. The results indicate that the oxidative metabolism of [14C]E1 in renal microsomes of newborn hamsters was 20-fold less than in kidney microsomes of adult hamsters. While catechol E1 metabolites were essentially negligible in hamster kidneys of these ages, it was evident that the conversion of E1 to estradiol via 17 beta-hydroxysteroid dehydrogenase resembles levels seen in the adult animals. Between the age of one and two months, the male hamster kidney exhibited the capacity to metabolize E1 at levels seen in fully mature adult hamsters.     

In vitro oxidative metabolism study of (-)-rhazinilam

Metabolism studies were conducted in order to investigate the reasons for the in vivo lack of activity of (-)-rhazinilam 1, an original poison of the mitotic spindle. Bioconversion by Beauveria bassiana strains, rat and human liver microsomes allowed the identification of metabolites 2, 3, and 4 oxidized in positions 3 and 5 of rhazinilam. Further experiments indicated that CYP2B6 was the main CYP responsible for the oxidation of 1 by human liver microsomes. All isolated metabolites were markedly less active than rhazinilam in vitro, which might explain its in vivo inactivity.     

Oxidation of carbon tetrachloride,bromotrichloromethane, and carbon tetrabromide by rat liver microsomes to electrophilic halogens

In order to determine whether CCl₄, CBrCl₃, CBr₄ or CHCl₃ undergo oxidative metabolism to electrophilic halogens by liver microsomes, they were incubated with liver microsomes from phenobartital pretreated rats in the presence of NADPH and 2,6-dimethylphenol. The analysis of the reaction mixtures by capillary gas chromatography mass spectrometry revealed that 4-chloro-2,6-dimethylphenol was a metabolite of CCl₄ and CBrCl₃ whereas 4-bromo-2,6-dimethylphenol was a metabolite of CBr₄. The formation of the metabolites was significantly decreased when the reactions were conducted with heat denatured microsomes, in the absence of NADPH or under an atmosphere of N₂. These results indicate that the chlorines of CBrCl₃ and CCl₄ and the bromines of CBr₄ are oxidatively metabolized by rat liver microsomes to electrophilic and potentially toxic metabolites.     

Comparative metabolism of benzo[f]quinoline by liver microsomes from brown bullheads and rats

1. Liver microsomes from rats were considerably more active in metabolizing benzo[f]quinoline (B f Q) than those from brown bullheads (Ictalurus nebulosus). 2. The main B f Q metabolites formed by both rat and brown bullhead liver microsomes were qualitatively similar and included B f Q-7,8-dihydrodiol, B f Q-9,10-dihydrodiol, B f Q-N-oxide, 7-hydroxy B f Q, and 9-hydroxy B f Q. 3. The liver microsomes from control brown bullheads and rats metabolized B f Q primarily at the 7,8-and 9,10-positions, respectively, whereas in the case of microsomes from 3-methylcholanthrene (3-MC)-treated rats or brown bullheads, the major site of metabolic attack was the 7,8-position. 4. A 3-MC-type of cytochrome P-450 appears to be primarily responsible for the oxidation of B f Q by control brown bullhead liver microsomes, whereas a phenobarbital-inducible type of cytochrome P-450 seems to be involved in the metabolism of B f Q by control rat liver microsomes.     

An in vitro reporter gene assay method incorporating metabolic activation with human and rat S9 or liver microsomes

A metabolic activation system with an S9 fraction or liver microsomes was applied to a reporter gene assay in vitro for the screening of estrogenicity of chemicals. The endpoint (luciferase) was luciferase induction in cells transfected with a reporter plasmid containing an estrogen-responsive element linked to the luciferase gene. Compounds were applied to the reporter gene assay system after pretreatment or simultaneous treatment with an S9 fraction or liver microsomes. Both trans-stilbene and methoxychlor themselves showed no or little estrogenicity, but when they were treated with an S9 fraction or liver microsomes, they demonstrated strong effects, indicating their metabolites to be estrogenic. When four pyrethroid insecticides were subjected to this assay system, however, they showed no estrogenicity even with liver microsome or S9 mix treatment.     

Potent bivalent inhibition of human tryptase-beta by a synthetic inhibitor

Human tryptase-beta (HTbeta) is a unique serine protease exhibiting a frame-like tetramer structure with four active sites directed toward a central pore. Potent inhibition of HTbeta has been attained using CRA-2059. This compound has two phenylguanidinium head groups connected via a linker capable of spanning between two active sites. The properties of the CRA-2059:HTbeta interaction were defined in this study. Tight-binding reversible inhibition was observed with an inhibition constant (Ki) of 620 pM, an association rate constant of 7x10(7) M(-1) s(-1) and a relatively slow dissociation rate constant of 0.04 s(-1). Bivalent inhibition was demonstrated by displacement of p-aminobenzamidine from the primary specificity pocket with a stoichiometry, [CRA-2059]0/[HTbeta]0, of 0.5. The potency of the bivalent interaction was illustrated by CRA-2059 inhibition of HTbeta, 24% or 53% inhibited by pre-incubation with an irreversible inhibitor. Two interactions were observed consistent with mono- and bi-valent binding; the Ki value for bivalent inhibition was at least 10(4)-fold lower than that for monovalent inhibition. Comparison of the affinities of CRA-2059 and phenylguanidine for HTbeta finds an approximate doubling of the free energy change upon bivalent binding. This doubling suggests that the linker portion minimally hinders the binding of CRA-2059 to HTbeta. The potency of CRA-2059 is thus attributable to effective bivalent binding.     

A high-throughput cell-based toxicity analysis of drug metabolites using flow cytometry

The effects of liver enzymes on drug activities are important considerations in the drug discovery process. Frequently, liver microsomes are used to simulate first-pass metabolism in the liver; however, there are significant disadvantages to the microsome system. As an alternative, a simple cell-based, high-throughput system that allows for examination of metabolite activity is described. Using multiparameter flow cytometry and the low-volume, high-sample format of 96-well plates, it is possible to rapidly evaluate a dose-response curve for metabolites based on variables including initial compound concentrations, hepatocyte cell line metabolic activities, and time. Using HepG2 cells as a surrogate for hepatic metabolism of a potential therapeutic, the impact of metabolites on Jurkat cell death was measured by both propidium iodide dye exclusion and cell cycle analysis. While this system is not proposed to supplant liver microsome studies, this alternative assay provides a highly adaptable, low-cost, and high-throughput measure of drug metabolism.     

Diazepam metabolism by multiple forms of cytochrome P-450 in rat liver microsomes

The synthesis of pharmacologically active diazepam metabolites (oxazepam, 4-hydroxydiazepam, N-demethyldiazepam) in liver microsomes of intact and phenobarbital-, 3-methylcholanthrene- and dexamethasone-induced male and female Wistar rats as well as in a reconstituted system with isolated forms of cytochrome P-450 (P-450a, P-450b, P-450c, P-450d and P-450k according to the Ryan nomenclature) was studied. Marked sex-dependent differences in the rates of diazepam metabolism in liver microsomes of intact and induced animals were revealed. The changes in the spectrum of diazepam metabolites in liver microsomes of induced rats (as compared to control animals) were revealed. In a reconstituted system only phenobarbital-induced cytochromes P-450b and P-450k were found to be active participants of diazepam N-demethylation; none of the isoenzymes tested were shown to be involved in diazepam hydroxylation.     

Reconstitution of testosterone oxidation by purified rat cytochrome P450p (IIIA1)

Cytochrome P450p (IIIA1) has been purified from rat liver microsomes by several investigators, but in all cases the purified protein, in contrast to other P450 enzymes, has not been catalytically active when reconstituted with NADPH-cytochrome P450 reductase and dilauroylphosphatidylcholine. We now report the successful reconstitution of testosterone oxidation by cytochrome P450p, which was purified from liver microsomes from troleandomycin-treated rats. The rate of testosterone oxidation was greatest when purified cytochrome P450p (50 pmol/ml) was reconstituted with a fivefold molar excess of NADPH-cytochrome P450 reductase, an equimolar amount of cytochrome b5, 200 micrograms/ml of a chloroform/methanol extract of microsomal lipid (which could not be substituted with dilauroylphosphatidylcholine), and the nonionic detergent, Emulgen 911 (50 micrograms/ml). Testosterone oxidation by cytochrome P450p was optimal at 200 mM potassium phosphate, pH 7.25. In addition to their final concentration, the order of addition of these components was found to influence the catalytic activity of cytochrome P450p. Under these experimental conditions, purified cytochrome P450p converted testosterone to four major and four minor metabolites at an overall rate of 18 nmol/nmol P450p/min (which is comparable to the rate of testosterone oxidation catalyzed by other purified forms of rat liver cytochrome P450). The four major metabolites were 6 beta-hydroxytestosterone (51%), 2 beta-hydroxytestosterone (18%), 15 beta-hydroxytestosterone (11%) and 6-dehydrotestosterone (10%). The four minor metabolites were 18-hydroxytestosterone (3%), 1 beta-hydroxytestosterone (3%), 16 beta-hydroxytestosterone (2%), and androstenedione (2%). With the exception of 16 beta-hydroxytestosterone and androstenedione, the conversion of testosterone to each of these metabolites was inhibited greater than 85% when liver microsomes from various sources were incubated with rabbit polyclonal antibody against cytochrome P450p. This antibody, which recognized two electrophoretically distinct proteins in liver microsomes from troleandomycin-treated rats, did not inhibit testosterone oxidation by cytochromes P450a, P450b, P450h, or P450m. The catalytic turnover of microsomal cytochrome P450p was estimated from the increase in testosterone oxidation and the apparent increase in cytochrome P450 concentration following treatment of liver microsomes from troleandomycin- or erythromycin-induced rats with potassium ferricyanide (which dissociates the cytochrome P450p-inducer complex). Based on this estimate, the catalytic turnover values for purified, reconstituted cytochrome P450p were 4.2 to 4.6 times greater than the rate catalyzed by microsomal cytochrome P450p.     

Regulation of arachidonic acid metabolism by cytochrome P-450 in rabbit kidney.

Renal microsomal cytochrome P-450-dependent arachidonic acid metabolism was correlated with the level of cytochrome P-450 in the rabbit kidney. Cobalt, an inducer of haem oxygenase, reduced cytochrome P-450 in both the cortex and medulla in association with a 2-fold decrease in aryl-hydrocarbon hydroxylase, an index of cytochrome P-450 activity, and a similar decrease in the formation of cytochrome P-450-dependent arachidonic acid metabolites by renal microsomes (microsomal fractions). Formation of the latter was absolutely dependent on NADPH addition and was prevented by SKF-525A, an inhibitor of cytochrome P-450-dependent enzymes. Arachidonate metabolites of cortical microsomes were identified by g.c.-m.s. as 20- and 19-hydroxyeicosatetraenoic acid, 11,12-epoxyeicosatrienoic acid and 11,12-dihydroxyeicosatrienoic acid. The profile of arachidonic acid metabolites was the same for the medullary microsomes. Induction of cytochrome P-450 by 3-methylcholanthrene and beta-naphthoflavone increased cytochrome P-450 content and aryl-hydrocarbon hydroxylase activity by 2-fold in the cortex and medulla, and this correlated with a 2-fold increase in arachidonic acid metabolites via the cytochrome P-450 pathway. These changes can also be demonstrated in cells isolated from the medullary segment of the thick ascending limb of the loop of Henle, which previously have been shown to metabolize arachidonic acid specifically via the cytochrome P-450-dependent pathway. The specific activity for the formation of arachidonic acid metabolites by this pathway is higher in the kidney than in the liver, the highest activity being in the outer medulla, namely 7.9 microgram as against 2.5 micrograms of arachidonic acid transformed/30 min per nmol of cytochrome P-450 for microsomes obtained from outer medulla and liver respectively. These findings are consistent with high levels of cytochrome P-450 isoenzyme(s), specific for arachidonic acid metabolism, primarily localized in the outer medulla.     

GLUTAMYL AND ASPARTYL MOIETIES OF CEREBRAL PROTEINS: ENRICHMENT IN MEMBRANE-CONTAINING MICROSOMAL SUBFRACTIONS

Abstract— The total mixed proteins (excluding proteolipids) were isolated from various tissues of the cat and from subcellular fractions of cat cerebral cortex and were compared for contents of total glutamyl and total aspartyl residues and total amide N. The proteins from renal cortex, testis and diaphragm were more acidic (proportionally more glutamyl and aspartyl residues with no increase of amide) than those from cerebral cortex, subcortical white matter and liver. Also the proteins isolated from cerebral cortical microsomes were more acidic than those from highly pure nuclear, mitochondrial and soluble subcellular fractions of cerebral cortex. Hepatic microsomal proteins gave very similar analyses.
Subfractionation of the microsomal preparations from cerebral cortex and liver into deoxycholate-insoluble (ribosomal) and deoxycholate-soluble ('membrane') fractions localized the acidic proteins to the latter, 'membrane' subtraction. The ribosomal proteins isolated from cerebral cortex (and liver) had an amino acid composition very similar to that reported previously for ribosomal proteins in a number of tissues from various species. Upon column chromatography on DEAE-celluIose, the deoxycholate-soluble ('membrane') subtraction of hepatic microsomes yielded a peak (eluted with 0-2 M-phosphate, pH 5-6, plus 0-5 M-NaCl) that contained an exceptionally high proportion (30 per cent) of glutamyl and aspartyl residues. We suggest that such acidic proteins may be characteristic of membranes and may subserve important metabolic functions therein.