Sepharose 4B Column Chromatography Research Articles

Cytochrome P-450 in culture forms of Trypanosoma cruzi

1. Trypanosoma cruzi epimastigote and trypomastigote forms contain microsomal peptides in the 40–60,000 mol. wt region, some of which are heme-staining-positive and are induced by phenobarbital, as indicated by SDS-gel electrophoresis and by double-labeling experiments. 2. Epimastigotes show induced peptides of mol. wt 56,000, 52,500, 49,000, 44,000, 42,000 and 40,500 whereas only one peptide (52,500 mol. wt) is increased in trypomastigotes. 3. Fractionation of microsomes derived from epimastigotes by octylamine Sepharose-4B column chromatography reveals the presence of two heme peptides with mol. wt of 55,800 and 56,600. 4. The pooled fraction has a typical cytochrome P-450 CO-difference spectrum and appears to correspond to a high spin form. 5. The demonstration of the existence of this family of hemoproteins in T. cruzi further supports the idea that resistance to chemotherapeutic agents is due to active metabolism. 6. The active metabolism, however, may not be similar in the various developmental forms of this organism since differences exist in the patterns of induction of heme-positive microsomal peptides.

Age-related changes in chemical composition and physical properties of mucus glycoproteins from rat small intestine.

Mucus glycoproteins from newborn and adult rat small intestine were radiolabelled in vivo with Na2 35SO4 and isolated from mucosal homogenates by using Sepharose 4B column chromatography followed by CsCl-density-gradient centrifugation. Non-covalently bound proteins, lipids and nucleic acids were not detected in the purified glycoproteins. Amino acid, carbohydrate and sulphate compositions were similar to chemical compositions reported for other intestinal mucus glycoproteins, as were sedimentation properties. There were, however, important differences in the chemical and physical characteristics of the mucus glycoproteins from newborn and adult animals. The buoyant density in CsCl was higher for the glycoproteins from newborn rats (1.55 g/ml versus 1.47 g/ml). On sodium dodecyl sulphate/polyacrylamide/agarose-gel electrophoresis, the glycoprotein from newborn rats had a greater mobility than the adult-rat sample. Although both preparations had similar general amino acid compositions, variations were observed for individual amino acids. The total protein content was greater in the glycoprotein from newborn animals (27%, w/w, versus 18%, w/w). The molar ratio of carbohydrate to protein was less in the newborn, primarily owing to a decreased fucose and N-acetylgalactosamine content. Comparison of the molar ratio of fucose and sialic acid to galactose for both glycoproteins demonstrated a reciprocal relationship similar to that described by Dische [(1963) Ann. N.Y. Acad. Sci. 106, 259-270]. The sulphate content was greater in the glycoprotein from newborn rats (5.5%, w/w, versus 0.9%, w/w). Both had similar sedimentation coefficients in a dissociative solvent. These results suggest an age-related difference in the types of mucus glycoproteins synthesized by small intestine.

Characterisation of small unilamellar vesicles produced in unsonicated phosphatidic acid and phosphatidylcholine-phosphatidic acid dispersions by pH adjustment

As the pH of phosphatidic acid and mixtures of phosphatidylcholine and phosphatidic acid dispersed in water is raised vesicles are formed. These vesicles are stable when the pH is returned to neutrality. Dispersions of phosphatidic acid transiently raised to pH 10–11 have been resolved by Sepharose 4B column chromatography into large structures ( > 100 nm ), which elute at the void volume, and small vesicles ( < 60 nm ), which elute at 0.44–0.48 column volumes (Hauser, H. and Gains, N. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 1683–1687). A more detailed characterisation of these vesicles has now been made by including 22Na +, 35SO 4 2− and the fluorochromes, pyranine and harmaline, in the aqueous dispersion before the pH was raised. The encapsulated and unencapsulated ions were then separated, with the vesicles, by Sepharose 4B column chromatography. The material eluting in the void volume consists mainly of unilamellar vesicles with an encapsulated water phase of approx. 15 l per mole of phospholipid. The small vesicles formed from phosphatidic acid encapsulate approx. 0.5 l/mol. Their size is increased by increasing the concentration of sodium chloride in the initial dispersion medium and decreased by both increasing the maximum pH to which the dispersion is raised and increasing the rate of the pH change. Rapidly (in about 1 s) raising the pH to 9 or above results in more than 70% formation of small vesicles. This percentage is decreased in the presence of sodium chloride and increased by both increasing the maximum pH to which the dispersion is raised and increasing the rate of the pH change. The sodium salt of dilauroylphosphatidic acid does not form small vesicles (less than 60 nm) when the pH is rapidly raised to 11, unless the sodium ions have been removed by acid washing. The half-time of efflux of 22Na + and 35SO 4 2− from these vesicles is of the order of days.

Solubilization and partial characterization of alkylglycerol monooxygenase from rat liver microsomes.

The enzyme alkylglycerol monooxygenase was solubilized with 2% of Triton X-100 and partially purified approximately to 83-fold with a 36% yield after a procedure which included 6-aminohexyl-Sepharose and DEAE-cellulose column chromatography, followed by a sucrose density gradient centrifugation. The molecular weight of the native form was estimated to be approximately 400000 as judged by Sepharose 6B column chromatography in the presence of Triton X-100. The enzyme had a pH optimum near 8.5 and a Km of 0.66 mM for 1-O-hexadecylglycerol. The microsomal alkylglycerol monooxygenase was stimulated by Triton X-100, but did not affect kinetically the initial velocity except to maintain it for a much longer period of time. The purified enzyme required absolutely both molecular oxygen and reduced pteridine as an electron donor. Furthermore, reduced glutathione and phospholipids were necessary for expression of full enzyme activity. N-Ethylmaleimide and p-chloromercuribenzoate significantly inhibited the enzyme activity, suggesting that alkylglycerol monooxygenase is a -SH enzyme.

A Poly(U)-Binding Factor Stimulating EF-1 Activity in the Wheat-Germ Soluble Fraction

A poly(U)-binding activity is present in the high-speed supernatant fraction of embryo homogenates from wheat seeds. The factor responsible for such activity was found to have a stimulatory effect on the elongation factor 1 (EF-1). It copurifies with EF-1L, the lighter form of EF-1, through Sephadex G-200, DEAE-cellulose, hydroxyapatite and poly(U)-Sepharose 4B column chromatography. The two factors could be separated only through a heating step which destroyed EF-1 activity whilst leaving most of the poly(U)-binding activity unaltered.

Enzyme-linked immunosorbent assay for detection of Pseudomonas aeruginosa Lipopolysaccharides.

A double-antibody sandwich method of enzyme-linked immunosorbent assay was developed to detect lipopolysaccharides (LPS) from the eight most prevalent Pseudomonas aeruginosa serotypes (O1, O2,5,16, O3, O4, O6, O9, O10, and O11). Immunoglobulin M fractions from rabbit antisera were used as the coating antibody and as the antibody to be conjugated to an enzyme. When two fractions of LPS (I and II) obtained by Sepharose 2B column chromatography were assayed, LPS II showed 10 to 100 times more activity than LPS I; the detection level of LPS II was 0.1 ng/ml. When LPS in purified preparations or in culture filtrates was examined with both homologous and heterologous antibody systems, the same specificity pattern was demonstrated, suggesting that, in crude filtrates, antigens other than LPS do not interfere in the assay. The method described can be used to detect LPS in biological fluids.

The fate of an N-formylated chemotactic peptide in stimulated human granulocytes. Subcellular fractionation studies.

Experiments were performed to examine how human granulocytes process the chemotactic peptide N-formyl-Met-Leu-Phe after stimulation by the same peptide. Purified human granulocytes were stimulated with 50 nM N-formyl-Met-Leu-[3H]Phe at 37 degrees C for various times, washed, lysed by N2 cavitation, and fractionated by isopycnic sucrose density gradient sedimentation. The major subcellular fractions identified were plasma membrane, Golgi, granules, endoplasmic reticulum, and mitochondria. After 1 min of stimulation, radioactivity was found only in the plasma membrane (sedimentable) and cytosol (soluble) fraction. At 5, 10, and 25 min, radioactivity also appeared in a sedimentable, low density fraction (25-28% sucrose) enriched in galactosyl transferase activity and containing Golgi structures. The accumulation in the sedimentable fractions was maximal after 5 min but continued to increase linearly in the cytosol fraction. Incorporation of radioactivity into cells or membrane and soluble fractions was 60 to 85% specific and was inhibited if incubation with N-formyl-Met-Leu-[3H]Phe was performed at 4 degrees C. 80-90% of the radiolabel in the plasma membrane or Golgi-containing fractions remained sedimentable despite freeze thawing or sonication. Solubilization of these fractions in Triton X-100 followed by Sepharose 4B column chromatography revealed that the radiolabel eluted in the void volume. Our results are consistent with internalization which proceeds by passage of an occupied receptor in a high affinity, supramolecular complex from the plasma membrane to the Golgi followed by accumulation of peptide in the cytosol.

Zearalenone reductase from rat liver.

Zearalenone is known to be reduced by rat liver preparations to a more active estrogenic metabolite, alpha-zearalenol. To elucidate the enzymatic feature of zearalenone reductase, we worked out a simple assay procedure for this enzyme using [3H]-zearalenone and thin layer chromatography (TLC). The NAD(P)H-dependent zearalenone reductase localized in the microsomes was most active at pH 4-4.5, while the reductase localized in the cytosol was active at neutral pH. The former enzyme reduced zearalenone only to alpha-zearalenol and the latter reduced both alpha-zearalenol and beta-zearalenol. The microsomal enzyme was solubilized with Triton X-100 and purified by DEAE-Sephadex, hydroxyapatite and Sepharose 4B column chromatographies. The partially purified enzyme showed the following properties. a) The molecular weight of the enzyme was estimated to be about 230,000 by Sepharose 4B chromatography. b) The apparent Km value of the enzyme was 1.0 X 10(-5) M for zearalenone at an NADPH concentration of 1 mM. c) The enzyme activity was inhibited by a high concentration of KCl but not by 1 mM Co2+, Mn2+, Zn2+, EDTA, or 20 mM N-ethylmaleimide.

Effect of several hormones on cyclic 3',5'-nucleotide phosphodiesterase in rat kidneys

Kidney function is regulated by several hormones which act through adenylate cyclase-cyclic AMP system. The present study was undertaken to investigate cyclic AMP- and cyclic GMP-phosphodiesterase (cAMP-PDE and cGMP-PDE respectively) activities in the rat kidney, and also the effect of several hormones affecting the kidney function on these enzyme activities in vitro. Rat kidneys were separated into cortex and medulla. These were homogenized in 50 mM Tris-HCl buffer, pH 7.5, containing 0.32 M sucrose and fractionated by centrifugation. PDE activity was measured in all fractions, using the two-step assay system. A low substrate concentration (0.5 microM) was used, unless otherwise stated. Substantial activity was present in all of the fractions and most of the activity existed in the soluble fraction (105000 X g supernatant). Cyclic GMP-PDE activity was dominant in both cortex and medulla. The rat kidney contained two forms of cAMP-PDE, one of which had a Km of 2.0 X 10(-4) M and another which had a low Km of 2.5 X 10(-5) M, and one form of cGMP-PDE with a Km of 2.5 X 10(-5) M. These cAMP-PDE and cGMP-PDE were purified by Sepharose-6B column chromatography. Cyclic AMP-PDE activity was found in a broad area associated with two peaks and cGMP-PDE activity had one peak corresponding to the same peak as the high molecular weight cAMP-PDE. Calmodulin was eluted after the peak of cGMP-PDE activity. Both cAMP-PDE and cGMP-PDE activities were inhibited by calcium ion at a concentration of more than 5.0 X 10(-4) M. Cyclic GMP-PDE activity was not activated by calmodulin in the presence of enough calcium ion. The effect of 1 alpha, 25(OH)2 Vit D3, parathyroid hormone (PTH), antidiuretic hormone (ADH), calcitonin (CT), angiotensin II, and trichlormethiazide on the partially purified cAMP-PDE and cGMP-PDE activities were examined. 1 alpha, 25(OH)2 Vit D3 activated cAMP-PDE activity and did not affect cGMP-PDE activity. The concentrations of 1 alpha, 25(OH)2 Vit D3 producing 50% activation of cAMP-PDE activity were 5.0 X 10(-11) M (cortex) and 6.7 X 10(-10) M (medulla). CT and ADH inhibited both cAMP-PDE activities. The concentrations of CT producing 50% inhibition of cAMP-PDE activity were 4.0 X 10(-5) M (cortex) and 3.3 X 10(-7) M (medulla), and those of cGMP-PDE activity were 1.0 X 10(-5) M (cortex) and 1.0 X 10(-4) M (medulla). Concerning ADH, the concentrations required for 50% inhibition of cAMP-PDE activity were 5.3 X 10(-6) M (cortex) and about 1.0 X 10(-3) M (medulla), and those of cGMP-PDE activity were 5.3 X 10(-3) M (cortex) and 5.3 X 10(-8) M (medulla).(ABSTRACT TRUNCATED AT 400 WORDS)

Purification of mixed-function amine oxidase from rat liver microsomes

To clarify the metabolism of carcinogenic aminoazo dyes in target tissues, mixed function amine oxidase (MFAO) was purified from rat liver. The MFAO was solubilized from microsomes with Triton X in the presence of 20% glycerol and 1 mM EDTA and purified successively with DEAE Sepharose CL-6B, 2′,5′-ADP Sepharose 4B and Hydroxyapatite column chromatography. The purified enzyme yielded a single protein band on sodium dodecyl sulfate (SDS)-poly-acrylamide gel electrophoresis. The apparent molecular weight was about 59,000. When dimethylaniline (DMA) was used as a substrate, the specific activity of the enzyme fortified with NADPH was about 430 nmol DMA N-oxide formed/mg protein/min with a yield of about 15%. N-Demethylation of dimethyl-aminoazobenzene (DAB) with the enzyme proceeded only when iron was added to the reaction system.

Generality and diversity of winged bean (Psophocarpus tetragonolobus) protein in eight various lines.

Protein components from eight lines of winged bean (Psophocarpus tetragonolobus) seeds which were originally introduced from Papua New Guinea, Indonesia, Nigeria, and Ishigaki, and cultivated in Okinawa and Fukuoka, were investigated. Two major peaks which had sedimentation coefficients, s20, of about 2.5S and about 6.5S (6.0 to 6.6 for the 8 lines), and no larger component were observed in all specimens with more than 90% extraction. Electrophoretic profiles of the “6.5S” component(s) which was separated with Sepharose 6B column chromatography showed a main broad band and a few minor bands which seemed to be essentially similar among the eight lines of winged bean. Thus the “6.5S” protein surely could be regarded as the common storage protein in winged bean seeds. The subunit structure of the “6.5S” component(s) in SDS solution consisted of four major bands. The “2.5S” components were mixtures and combinations of various proteins which were distinctly different from one selection to another.

Monoclonal antibodies against ethacrynic acid high-sensitive Mg-ATPase.

Ethacrynic acid (EA) high-sensitive Mg-ATPase activity is an anion sensitive, non-mitochondrial Mg-ATPase activity observed in the brain (Biochem. Pharmacol. 31, 877 and 4077, 1982). In the present study, we prepared monoclonal antibodies against EA high-sensitive Mg-ATPase for use in the purification of the enzyme and examination of its localization. BALB/c mouse was immunized with crude microsomal fractions from rat brain and boostered 4-7 weeks later. Antibody titer was measured by inhibition assay of EA high-sensitive Mg-ATPase activity. Hybridomas were obtained and cloned by the method of Köhler and Milstein. These selected hybridomas were injected i.p. to BALB/c mice, and the ascites were collected. Both of the hybridomas were positively stained by antimouse γ2b. or anti-mouse λ rabbit serum and FITC antirabbit immunoglobulins. IgG fractions of ascites were prepared by (NH4)2SO4. fractionation or by DEAE cellulose or protein A Sepharose 4B column chromatography. The antibodies inhibited EA high-sensitive Mg-ATPase activity without significant effects on activities of EA low-sensitive Mg-ATPase or Na, K-ATPase. These results suggest that monoclonal antibodies (IgG2b,λ) were obtained against the catalytic site of EA high-sensitive Mg-ATPase.

Platelet-mediated cardiac ischemia

Although platelets have been associated with angina pectoris, myocardial infarction, and sudden death, the platelet's capacity for induction and propagation of cardiac ischemia remains incompletely defined. We therefore evaluated the effects of platelet activation occurring within the coronary circulation and tested the hypothesis that inhibition of platelet function would prevent platelet-induced cardiac ischemia. Human platelets were isolated from blood obtained from normal donors by Sepharose 2B column chromatography, resuspended in Hepes buffer, and added to the perfusate of a Langendorff rabbit heart (platelet counts > 10,000/μl). Without, and with low dose (10 μM) prostaglandin E 1 (PGE 1), a reversible inhibitor of platelet function, immediate and irreversible global cardiac ischemia, as monitored by NADH fluorescent photography, ensued ( N = 4) following platelet activation with thrombin (0.1 to 1 U/ml). Higher concentrations of PGE 1 (0.1 to 1 m M, N = 2) or aspirin ingestion (1000 mg taken approximately 12, 4, and 1 hr prior to experiment, N = 2) completely prevented this platelet-induced myocardial ischemia. Aspirin, unlike PGE 1, was effective despite its inability to block thrombin-induced platelet aggregation in our in vitro gel-filtered system. We conclude that activation of platelets within the coronary circulation is sufficient for induction of irreversible cardiac ischemia. The efficacy of aspirin, a cyclooxygenase inhibitor, further suggests that the products of arachidonate metabolism (e.g., thromboxanes) have a fundamental role in the genesis of platelet-mediated myocardial ischemia.

Streptokinase-dependent potentiating factor (SK-potentiator) for plasminogen activation from human plasma: its identification as a fibrinogen degradation product.

A factor, named SK-potentiator, which is known to potentiate the activation of plasminogen by streptokinase (SK), was isolated from human plasma. The procedures consisted of column chromatographies on DEAE-Sephadex A-50 and heparin-agarose, followed with gel-filtration on a Sepharose 6B column and affinity chromatography on a lysine-Sepharose 4B column. The isolated SK-potentiator markedly potentiated the rate of activation of plasminogen by streptokinase. However, it did not show a potentiating effect on the activation of plasminogen by urokinase or on the plasmin activity. SK-potentiator showed a similar mobility to that of fibrinogen on both SDS-polyacrylamide gel and agarose gel electrophoresis, and cross-reacted with anti-fibrinogen antiserum. The amino acid composition of SK-potentiator was very close to that of human fibrinogen, although the content of serine and threonine was significantly lower. SK-potentiator showed a single band with a molecular weight of 300,000 on SDS-gel electrophoresis in the absence of 2-mercaptoethanol. In the presence of 2-mercaptoethanol, it showed two major bands with molecular weights of 53,000 and 48,000, respectively, which corresponded to the B beta chain and gamma chain of fibrinogen. To establish further that the isolated SK-potentiator may be one of the fibrinogen degradation products (FDP), human fibrinogen was digested with plasmin and the SK-potentiator activity generated in the course of the digestion was measured. As a result, the SK-potentiator activity was found to initially increase and then decrease with incubation time, suggesting that an early FDP has the ability to potentiate the SK-mediated activation of plasminogen. The early FDP was then isolated by gel-filtration on a column of Sepharose 6B and it was found that the SK-potentiator activity was associated with the early FDP. Moreover, the early FDP showed the same electrophoretic mobility as the isolated SK-potentiator on SDS-gel electrophoresis and their amino acid compositions were quite similar to each other. From these results, the SK-potentiator was identified as the early FDP.

Phosphofructokinase from Ascaris suum. Purification and properties.

A rapid and efficient procedure has been developed to purify phosphofructokinase from the muscle of the parasitic roundworm, Ascaris suum. The procedure can be accomplished in 1 day with a 420-fold purification and a 60% yield. The enzyme was shown to be homogeneous by two-dimensional electrophoresis, Sepharose 6B column chromatography, and high performance liquid chromatography utilizing a size exclusion column. The subunit molecular weight of the enzyme was found to be 95,000 by electrophoresis in the presence of sodium dodecyl sulfate. In solutions of low ionic strength, the native enzyme aggregated to species of higher molecular weight than did the rabbit muscle phosphofructokinase. In the presence of 0.2 M (NH4)2SO4, the minimum native molecular weight was determined to be 398,000 by high performance liquid chromatography and Sepharose 6B column chromatography. Therefore, the enzyme appears to be a tetramer with identical or near-identical subunits. The apparent isoelectric point of the enzyme was shown to be 7.3 to 7.4 by both column and gel isoelectric focusing. Amino acid analysis revealed a lower number of the aromatic residues Phe, Tyr, and Trp than in the rabbit muscle enzyme and this is in agreement with the lower extinction coefficient of E1%280 nm = 6.5. Analysis of the purified enzyme revealed 7.4 +/- 0.6 mol of phosphate/mol of enzyme.

Transglutaminase activity during the differentiation of macrophages

Summary Transglutaminase (R-glutaminyl-peptide:amine γ-glutamyltransferase, E.C. 2,3,2,13) activity during the differentiation of murine leukemia cell lines (M1 cells) was investigated. Ml cells contained a significant transglutaminase activity of the tissue type. During the course of differentiation into mature macrophage-like M1 + cells induced with a protein inducer, the enzymatic activity was stimulated more than ten times as much as in the original undifferentiated Ml − cells. A remarkable enhancement of enzymatic activity was also observed when lipopolysaccharide was utilized as an inducer of differentiation. The enzymatic activity of undifferentiated M1 − cells was eluted at the region of M.W. ca. 80,000 as a single and symmetrical peak on Sepharose 4B column chromatography. By contrast, most of the activity in differentiated Ml + cells was eluted at the void volume under the same condition, though some activities were eluted at the same region as in Ml − cells. These data suggest that most of transglutaminase activity exists in the form of a high-molecular-weight complex with some cellular components in differentiated cells. The possible physiological significance of the enzyme in macrophage functions was discussed.

A simple reliable method for producing electron dense markers of uniform size for use in immunoelectron microscopy

A method is presented for the preparation of electron dense markers of defined size for use in immunoelectron microscopy. The electron dense markers were formed by the covalent binding of rabbit IgG and horse spleen ferritin mediated by glutaraldehyde. We demonstrated that the conjugate size, i.e. the number of ferritin grains per unit conjugate, was directly related to the time of incubation af 4°C in a 1000 molar excess of glutaraldehyde. Conjugates were separated on the basis of molecular size by Sepharose 4B column chromatography. Three different sized conjugates were used to delineate pseudomonas toxin receptors on the surface of mouse LM fibroblasts; similar numbers of receptors were observed with all conjugates.

Biosynthesis of myristate in an aphid: Involvement of a specific acylthioesterase

Approximately 70% of the total fatty acids of the pea aphid, Acyrthosiphon pisum, is myristate. The fatty acids of the phospholipid fraction consist of over 80% 18-carbon saturated and unsaturated fatty acids, whereas myristate accounts for almost 90% of the fatty acids of the triacylglycerol fraction. In vivo studies showed that [1- 14C]acetate was incorporated into both saturated (primarily myristate) and monounsaturated fatty acids, with the 14-carbon fatty acid incorporated almost exclusively into the triacylglycerol fraction and the 16- and 18-carbon fatty acids preferentially incorporated into the phospholipid fraction. A 105,000 g particle-free supernatant preparation incorporated radiolabel from [ 3H]acetyl-coenyzme A (CoA) primarily into myristate at low malonyl-CoA concentrations, and primarily into 16- and 18-carbon fatty acids at high malonyl-CoA concentrations. Using low malonyl-CoA concentrations, [ 3H]acetyl-CoA was preferentially incorporated into myristate at low ionic stengths, and into 16- and 18-carbon fatty acids at high ionic strengths. Sepharose 6B column chromatography was used to partially purify the fatty acid synthetase (FAS). The partially purified FAS produced 16- and 18-carbon fatty acids exclusively, regardless of malonyl-CoA concentrations or ionic strengths. However, when a separate heat-labile fraction from the Sepharose 6B column was added to the partially purified FAS, it restored the ability of the FAS to produce myristate. Adding larger amounts of this fraction to the partially purified FAS resulted in the formation of a higher percentage of myristate. This fraction contained thioesterase activity, and was partially resolved into two thioesterase activities by Sephadex G-100 column chromatography. The later eluting thioesterase, but not the earlier eluting one, was responsible for chain termination at 14 carbons. This thioesterase readily cleaved 18-, 16-, and 14-carbon acyl-CoA's, but had a much lower specificity for a C 12 CoA. This later eluting thioesterase also restored activity to FAS treated with phenylmethanesulfonyl fluoride. These results suggest the presence of an acylthioesterase in the aphid which releases the growing acyl chain from the FAS when it reaches 14 carbons.

Partial purification and some properties of emodin-O-methyltransferase from (+)-geodin producing strain of Aspergillus terreus.

Emodin-1-O-methyltransferase (E-OMT), an enzyme catalysing methylation of emodin and involved in the biosynthesis of (+)-geodin, was purified upto 89 folds by two-step purification with Blue Sepharose and Sepharose 6B column chromatography. The molecular weight of E-OMT was estimated to be 3.4×105, and Km values for emodin and S-adenosylmethionine (SAM) were 3.3×10-7M and 3.1×10-6M, respectively. Substrate specificity of E-OMT was extremely strict, and the enzyme seems to recognize whole the molecule of substrates.

Alterations of specific forms of cytochrome P-450 in rat liver during acute carbon tetrachloride intoxication

Male rats were pretreated with phenobarbital, 3-methylcholanthrene, or the corresponding vehicles, administered carbon tetrachloride (CCl 4), and sacrificed 3 hr later. Hepatic microsomes were isolated and assayed for cytochrome P-450 content and mixed-function oxidase activity. The residual content of cytochrome P-450 after CCl 4 intoxication was similar in saline and phenobarbital-pretreated animals. Substantially greater amount of cytochrome P-450 remained in animals pretreated with 3-methylcholanthrene and then challenged with CCl 4. In saline-pretreated animals, the dealkylation of 7-ethoxycoumarin, but not benzphetamine or N,N-dimethylaniline, was decreased following CCl 4 exposure. The enhanced dealkylation following phenobarbital, but not following by 3-methylcholanthrene, was also decreased after CCl 4 poisoning. Microsomes were solubilized with sodium cholate; cytochrome P-450 was partially purified by ω-octylamino Sepharose 4B column chromatography; and cytochrome P-450-containing elements were separated by SDS-polyacrylamide gel electrophoresis. A decrease in the staining intensity of a band migrating with a molecular weight of 51,600 was noted following CCl 4 treatment. Furthermore, the phenobarbital-induced component (MW 51,600), but not that appearing after 3-methylcholanthrene induction (MW 57,900), was also diminished. Microsomal proteins separated by SDS-polyacrylamide gel electrophoresis and stained for heme also showed a decrease in staining which was greater in those microsomal proteins from phenobarbital and saline than in 3-methylcholanthrene-induced animals. The data suggest that a specific cytochrome P-450(s) in the saline-treated animals as well as the cytochrome P-450(s) induced by phenobarbital, but not the 3-methylcholanthrene-induced cytochrome P-450, are susceptible to CCl 4-induced destruction.