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F Fukai

Publications and source records attributed to F Fukai.

At least 37 records · Page 2Linked to original sources
PubMed1989

Protection of glutathione S-transferase from bilirubin inhibition.

Inhibition of the enzyme activity of glutathione S-transferase (GST) by a physiological concentration of bilirubin was studied using various substrates. When rat liver cytosol was used as an unfractionated GST, its GSH-conjugation activity toward 1-chloro-2,4-dinitrobenzene was decreased to one-half by bilirubin, while the activity toward 1,2-dichloro-4-nitrobenzene, p-nitrobenzyl chloride, or 1,2-epoxy-(p-nitrophenoxy)propane and also the non-selenium dependent GSH-peroxidase activity toward cumene hydroperoxide (CHPx activity) were hardly affected under the same conditions. In contrast, bilirubin inhibited each of the purified GST isozymes and no remarkable difference in bilirubin inhibition was observed with any of the substrates tested. From the chromatographic analysis of the cytosol incubated with [3H]bilirubin, it was found that a major part of the added bilirubin binds to subunit 1 (Ya) of GST isozyme, leaving not only the conjugation activity derived from 3-4 type GST but also the CHPx activity of subunit 2 (Yc) quantitatively intact. The bilirubin inhibition of both the conjugation activity of GST 3-4 and the CHPx activity of GST 2-2 was prevented almost completely by addition of a 3-fold molar excess of GST 1-1. From these results, it was assumed that the enzyme activities of both 3-4 type GSTs and subunit 2 (Yc) were protected from the inhibitory action of bilirubin by the scavenger effect of subunit 1 (Ya).

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PubMed1989

Multiple classes of binding sites for palmitic acid on the fatty acid-binding protein molecule.

Binding characteristics of fatty acid-binding protein (FABP) toward palmitic acid were studied. On the analysis of the interaction between FABP and [3H]palmitic acid over a wide range of concentrations of the fatty acid, at least three saturation plateaux were observed. By Scatchard-plot analysis, it appeared that FABP possesses three classes of binding sites for palmitic acid with different affinities [Kd1 = 1 x 10(-6) M (N = 1), Kd2 = 4 x 10(-6) M (N = 2), Kd3 = 2 x 10(-5) M (N = 10)]. Results of both sedimentation analyses and chromatofocusing of FABPs labeled with various concentrations of [3H]palmitic acid suggested that the FABP used was homogeneous. These results indicate that several classes of binding sites for palmitic acid with different affinities are present on the FABP molecule.

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PubMed1988

Characterization of fibronectin metabolites in normal rat urine.

Fibronectin fragments showing immunoreactivity against an anti-rat plasma fibronectin antibody were detected in normal rat urine. Untreated urine showed immunoreactivity and this reactivity could not be dialyzed. These rat urinary fibronectin-related substances (RUF) were adsorbed on heparin-immobilized gel but hardly at all on gelatin-immobilized gel. They were separated into three fractions by DEAE-Sephadex ion exchange chromatography and each fraction was further purified by heparin-Sepharose affinity chromatography. The partially purified preparations, RUF-1, RUF-2 and RUF-3, were analyzed by the sandwich-type enzyme-linked immunosorbent assay (ELISA) method, and it was found their maximum reactivities were about 30%, 45% and 20% that of the intact plasma fibronectin, respectively. Plasma fibronectin was digested with elastase and then analyzed by the ELISA method to elucidate the patterns shown by the fragments. The fragments showed almost the same patterns on ELISA analysis, and the maximum immunoreactivity decreased in parallel with the degradation. On SDS-polyacrylamide gel electrophoresis, the RUF preparations were found to be composed of multiple forms of molecules, Mr 15,000-100,000, which reacted with the antibody. These results suggest that various forms of fibronectin metabolites are excreted in normal rat urine.

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PubMed1988

Novel immunochemical characteristics of glutathione S-transferase isozymes of rat liver cytosol.

Reevaluation of the immunochemical relationships among the individual glutathione S-transferase (GST) isozymes (GSTs 1-1, 1-2, 2-2, 3-3, 3-4, 4-4, and GSTs with isoelectric points of 7.5 and 6.8) of rat liver cytosol was performed utilizing the immunoblot technique. As a result, we found that the respective isozymes of two isozyme classes of rat liver cytosol might possess a common epitope(s) which has been undetected by the Ouchterlony double-diffusion method. The assumption was further supported by the results of the effects of Fab' prepared from some anti-GST antibodies on the enzymatic activity of GSTs.

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PubMed1987

A novel role of fatty acid-binding protein as a vehicle of retinoids.

Intracellular transport and storage of retinoids were shown to be conducted by fatty acid-binding protein (FABP). When rat liver cytosol was gel filtrated, retinyl palmitate-binding activity was mainly eluted in the fraction with a Mr. of around 14,000, in which both FABP and cellular retinol-binding protein (CRBP) co-existed. From the binding analysis of purified FABP and CRBP to retinyl palmitate, FABP was found to have a relatively high affinity (Kd = 1.4 X 10(-6) M) to retinyl palmitate, while binding of retinyl palmitate to CRBP was scarcely detectable. By using anti-FABP serum, it was shown that FABP was distributed in organs relating to absorption and storage of retinoids, such as jejunum, ileum, and liver. In liver, the protein was localized in the parenchymal cells and with particularly high concentration in the perisinusoidal cells, probably fat-storing cells.

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PubMed1985

Effect of molybdate on the molecular organization of the estrogen receptor system of porcine uterus.

Molybdate was shown to have complex effects in modulating the molecular organization of the constituents of the estrogen receptor (ER) system of porcine uterus. We showed previously the presence of one basic ER molecule (vero-ER) (sedimentation coefficient, 4.5S; Stokes radius, 44 A) and ER-binding factors (ERBFs) ["8S" ER-forming factor ("8S" ER-FF), (component A) X (component B)6; "6S" ER-FF, (component B)6; "5S" ER-FF, component A] in the porcine uterus [Fukai, F. & Murayama, A. (1981) J. Biochem. 95, 1697-1704]. Molybdate regulates the specific interaction of vero-ER with ERBFs in a complex way. The apparent Kd value (6.7 X 10(-10) M) of vero-ER with "8S" ER-FF in the presence of molybdate (30 mM) was decreased remarkably as compared with that (2.7 X 10(-9) M) in the absence of molybdate. In contrast, the apparent Kd value (3.7 X 10(-9) M) of vero-ER with "5S" ER-FF observed in the presence of molybdate (30 mM) was increased over ten-fold as compared with that in the absence of molybdate. Meanwhile, the affinity (Kd, 5 X 10(-9) M) of vero-ER for "6S" ER-FF was scarcely influenced by molybdate. These results reveal the mechanism by which molybdate selectively stabilizes "8S" ER. Molybdate further affected the molecular constitution of ERBFs. The dissociation of "8S" ER-FF into component A and component B, which takes place under hypertonic (0.4 M KCl) conditions at higher temperature (25 degrees C), was suppressed almost completely by molybdate (30 mM).(ABSTRACT TRUNCATED AT 250 WORDS)

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PubMed1984

Association and dissociation of estrogen receptor with estrogen receptor-binding factors is regulated by Mg2+.

It was recently shown that uterine estrogen receptor (ER) is translocated from the cytoplasm into the nucleus in the form of vero-ER X E (basic ER molecule bound with estradiol), and the translocation is inhibited by the specific binding of vero-ER X E with the cytoplasmic protein factors designated as ER-binding factors (ERBFs) ["5S" ER-forming factor ("5S" ER-FF), "6S" ER-FF, "8S" ER-FF] [Murayama, A. & Fukai, F. (1983) J. Biochem. 94, 511]. It was found that the specific interaction of vero-ER X E with the ERBFs is regulated by Mg2+ at physiological concentrations. The apparent Kd values of vero-ER X E for the ERBFs ["5S" ER-FF, 2.7 X 10(-9) M; "6S" ER-FF, 6.0 X 10(-8) M; "8S" ER-FF, 3.5 X 10(-8) M] observed in the presence of 1 mM Mg2+ were all increased over 10 times as compared with in the absence of Mg2+. The inhibitory effects of the ERBFs on the nuclear translocation of vero-ER X E were reduced by Mg2+.

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PubMed1984

Action of calpain on the basic estrogen receptor molecule of porcine uterus.

Basic estrogen receptor (ER) molecule (vero-ER) of the cytosol of porcine uterus was purified 1,200-fold after successive chromatographies on phenyl-Sepharose, hydroxylapatite, and DEAE-cellulose, followed by Sephadex G-150 gel filtration. The purified vero-ER was completely free from endogenous protease and ER-binding factor. The action of Ca2+-dependent cysteine proteinase (calpain) on vero-ER was studied by utilizing the purified receptor and calpains from porcine uterus (endogenous calpain), porcine kidney, and human erythrocytes. Proteolysis of vero-ER was followed by monitoring the disappearance of the binding capability of vero-ER with "8S" ER-forming factor. Vero-ER was proteolyzed by both the endogenous and the exogenous calpains in the presence of Ca2+. The calpains did not attack vero-ER in the absence of Ca2+. The results indicated the absolute requirement by calpain for Ca2+ for the limited hydrolysis of vero-ER. Uterine cytosol was shown to contain, in parallel with calpain, a protease which does not require Ca2+ for the limited proteolysis of vero-ER. The strongly hydrophobic domain of vero-ER, recently shown to be indispensable for the nuclear translocation of vero-ER (Murayama, A. & Fukai, F. (1983) FEBS Lett. 158, 255), was preferentially destroyed by both the Ca2+-requiring and -nonrequiring enzymes. It was assumed that calpain might intervene in the estrogen action by diminishing irreversibly the amount of the cytoplasmic ER capable of translocating into the nucleus.

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PubMed1983

The strong hydrophobic domain of the activated estrogen receptor of porcine uterus.

Basic estrogen receptor (ER) molecule (vero-ER) of porcine uterus, which was previously shown to be the activated ER necessary to translocate from the cytoplasm into the nucleus, possesses a strongly hydrophobic nature. The strong hydrophobicity of vero-ER was concealed through binding with ER-binding factors (ERBFs). Vero-ER lost its strong hydrophobicity and its capability to bind with ERBFs after limited proteolysis by endogenous protease. The strong hydrophobic domain of vero-ER, indispensable for the nuclear translocation, was assumed to be located near the binding site with ERBFs.

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PubMed1983

Studies on the molecular mechanism of the translocation of estrogen receptor from the cytoplasm into the nucleus.

A detailed study of the molecular mechanism of the translocation of estrogen receptor (ER) from the cytoplasm into the nucleus was undertaken in an in vitro system of porcine uterus. The capabilities of vero-ER . E (basic ER molecular bound with estradiol) (sedimentation coefficient 4.5S; Stokes radius 44 A) and the complexes ["5S" ER . E, (vero-ER . E) . (component A); "6S" ER . E, (vero-ER . E) . (component B)6; "8S" ER . E, (vero-ER . E) . (component B)6 . (component A)] with ER-binding factors (ERBFs) to translocate into the isolated nuclei were estimated by subtracting the amounts of ER adsorbed by the nuclear envelopes from those of ER bound to the whole nuclei. The results strongly supported our previous assumption that vero-ER . E translocates into the nuclei, and the complexes with ERBFs do not. The results suggested also that the binding site of vero-ER to ERBFs is required to be unoccupied in the process of the translocation of ER from the cytoplasm into the nucleus. The presence of a cytoplasmic factor (component C) which binds specifically with "5S" ER . E under low salt conditions was indicated. The complex, ("5S" ER . E) . (component C), was shown to possess relatively high affinity towards nuclear envelopes, but not to translocate into the nuclei.(ABSTRACT TRUNCATED AT 250 WORDS)

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PubMed1982

Inhibition of the translocation of estrogen receptor from the cytoplasm into the nucleus by estrogen receptor-binding factors.

The molecular mechanism of the translocation of estrogen receptor (ER) from the cytoplasm into the nucleus was studied in an in vitro system taking into account the specific interactions of the basic constituents of the uterine ER-system which had been reported previously (Murayama, A. et al. (1980) J. Biochem. 88, 1457). It was found that ER is translocated from the cytoplasm into the nucleus in the form of vero-ER . estradiol (basic ER-unit bound with estradiol; sedimentation coefficient, 4.5S; Stokes radius, 44 A; Kd for estradiol, approximately 10(-10) M) dissociated from coexisting estrogen receptor-binding factors (ERBFs). ERBFs inhibited the translocation of vero-ER into nuclei through binding with it. The apparent equilibrium dissociation constants (Kd) of ERBFs for vero-ER were estimated by Scatchard analysis to be 2.6 X 10(-10) M ["5S" ER-forming factor ("5S" ER-FF)], 3.0 X 10(-9) M ("6S" ER-FF), and 2.5 X 10(-9) M ("8S" ER-FF), respectively. The inhibitory effects of ERBFs on the binding of vero-ER to the nucleus paralleled their association constants.

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PubMed1981

Cytoplasmic estrogen receptor system of gilt uterus. Partial purification of component B labeled with [14C]iodoacetic acid.

Analysis of the cytoplasmic estrogen receptor (ER) system of gilt uterus by protecting ER from proteolysis showed that the ER system of gilt uterus is similar to that of cow uterus described previously. There was only one native unit molecule [native "4S" ER (sedimentation coefficient, 4.5S; Stokes radius, 45 A; molecular weight, 82,000)] with specific affinity towards estradiol. This molecule was further designated as vero-ER. "8S" ER-forming factor [("8S"ER)-FF] (Stokes radius, 51 A) was separated from the cytosol under hypotonic (low salt) conditions, and this was further dissociated into component A ["5S" ER-forming factor, ("5S"ER)-FF] (Stokes radius, 37 A) and component B (Stokes radius, 18.5 A) in the presence of sodium thiocyanate. Vero-ER was proteolyzed in the absence of Ca2+ ion by a cytoplasmic protease into modified "4S" ER (sedimentation coefficient, 4.5S; Stokes radius, 35 A; molecular weight, 65,000) which was further designated as secto-ER. The constituents of the cytoplasmic ER system of cow and gilt uteri cross-reacted with each other to undergo similar molecular assembly as in their own systems. When analyzed for various uterine specimens, fluctuation of ("8S"ER)-FF-level (1-50 unit/g tissue) was more remarkable than that of ER-level (3-6 x (10(-12) mol/g tissue). Component B labeled with [14C]iodoacetic acid was purified over 1,500-fold. Mixture of vero-ER and component B labeled with [14C]iodoacetic acid formed "6S' ER with 14C-activities under hypotonic conditions. This clearly excluded the possibility that component B is a catalyzer of self-association (dimerization) of vero-ER.

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PubMed1980

Estrogen receptor of cow uterus. I. Characterization of native and proteolyzed "4S" estrogen receptors.

The estrogen receptor (ER) was separated from the cytoplasmic components of the cow uterus through successive gel filtrations of the cytosol in the presence of antipain, a protease inhibitor. The partially purified ER (designated as native "4S" ER) sedimented at 4.5S under both hypotonic (low salt) and hypertonic (0.4 M KCl) conditions. The molecular weight of native "4S" ER was estimated to be approximately 82,000 with a Stokes radius of 44 A. If purification was performed in the absence of antipain, native "4S" ER was very labile to a protease present in the cytoplasm. The modified ER (designated as modified "4S" ER) sedimented at 4.5S like native "4S" ER, but had a smaller stokes radius (35 A) and a smaller molecular weight (65,000). In the presence of 0.4 M NaSCN, native "4S" ER remained intact, but modified "4S" ER dissociated and gave a fragment which retained the estradiol binding site. This fragment sedimented at 2.9S and had a Stokes radius of approximately 24 A (molecular weight, approximately 29,000). In the presence of 0.4 M NaSCN, native "4S" ER remained intact, but modified "4S" ER dissociated and gave a fragment which retained the estradiol binding site. This fragment sedimented at 2.9S and had a Stokes radius of approximately 24 A (molecular weight, approximately 29,000). In the presence of Ca2+ ions and in the absence of antipain, the native ER was modified by the cytoplasmic protease to give ERs sedimenting at 3.8-4.5S. The presence of a cytoplasmic component which specifically binds with native "4S" ER to give "8S" ER under hypotonic conditions was indicated. Modified "4S" ER was unable to bind with this component.

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PubMed1980

Estrogen receptor of cow uterus. II. Characterization of a cytoplasmic factor which binds with native "4S" estrogen receptor to give "8S" estrogen receptor.

Partial purification and characterization of a cytoplasmic component of cow uterus, which specifically binds with native "4S" estrogen receptor (ER) to give "8S" ER was carried out. The component was designated as "8S" ER-forming factor [("8S"ER)-FF]. ("8S"ER)-FF did not bind estradiol by itself. The Stokes radius of ("8S"ER)-FF was estimated to be approximately 51 A. ("8S"ER)-FF sedimented at around 6.9S in sucrose gradient centrifugation under hypotonic conditions. The molecular weight and frictional ratio of ("8S"ER)-FF were calculated to be 145,000 and 1.47, respectively. ("8S"ER)-FF was degraded by trypsin treatment, but was not affected by DNase or RNase. The Stokes radius of "8S" ER was estimated to be approximately 68 A. The molecular weight and frictional ratio of "8S" ER were calculated to be 225,000 and 1.69, respectively. "8S" ER was estimated to be a 1 : 1 complex between native "4S" ER and ("8S"ER)-FF. It was estimated that in the cow uterine cytosol, there was at least a 3-fold molar excess of ("8S"ER)-FF over the amount of "4S" ER. ("8S" ER)-FF inhibited both the Ca2+-dependent and -independent modifications of native "4S" ER by the cytoplasmic protease.

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PubMed1980

Estrogen receptor of cow uterus. III. Molecular constitution of estrogen receptor-binding factors.

"8S" estrogen receptor-forming factor [("8S"ER)-FF], a cytoplasmic factor of cow uterus, which binds specifically with native "4S" estrogen receptor (ER) to give "8S" ER under hypotonic (low salt) conditions was dissociated in the presence of 0.4 M NaSCN into two kinds of subunits designated as component A and component B. Characterization of components A and B was performed. The Stokes radius and molecular weight of component A were estimated to be around 37 A and 58,000, respectively. Those of component B were estimated to be around 18.5 A and 13,700, respectively. These components did not bind with estradiol, and underwent molecular assembly depending on the salt concentration of the medium to give complexes, which, in turn, bound with native "4S" ER to give ERs with higher sedimentation coefficients. Component A [designated as "5S" ER-forming factor, ("5S"ER)-FF] bound with native "4S" ER to give "5S" ER, which was stable under hypertonic (0.4 M KCl) conditions. Under hypotonic conditions, component B formed a hexamer [designated as "6S" ER-forming factor ("6S"ER)-FF], which bound with native "4S" ER to give "6S" ER. "4S" ER was dissociated from "6S" ER under hypertonic conditions. ("8S"ER)-FF was shown to be a 1 : 1 complex of component A and the hexamer of component B. These factors [("5S"ER)-FF, ("6S"ER)-FF and ("8S"ER)-FF] which bind specifically with native "4S" ER to give ERs with higher sedimentation coefficients were designated as estrogen receptor-binding factors (ERBFs).

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