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Hiroyuki ARAI, Kiyotaka NISHIKAWA, Keizo INOUE, Yoshinori NOZAWA, Shos ...
1987 Volume 101 Issue 5 Pages
1059-1067
Published: 1987
Released on J-STAGE: June 30, 2008
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We studied the metabolism of phospholipids exogenously added to cultures of the protozoan,
Tetrahymena pyriformis. Tetrahymena cells were found to metabolize the extracellular phospholipids and the fatty acyl chains of the latter were accumulated predominantly as a form of triacylglycerol in the cells. This metabolism was considered to be initiated
via endocytosis of phospholipid vesicles, as judged from the following facts: 1) Cytochalasin B, an inhibitor of endocytosis, suppressed the metabolism almost completely. 2) Phospholipid vesicles were incorporated into a phagosome-like structure in
Tetrahymena cells, as observed under an electron microscope. When phospholipids doubly labeled with
14C and
3H at the glycerol moiety and fatty acyl chain, respectively, were incubated with
Tetrahvinena cells, the glycerol moiety and fatty acyl chain at the
sn-2-position of the exogenous phospholipids were incorporated into the cellular triacylglycerol fraction in a 1 to 1 ratio. Monoacylglycerol acyltransferase activity was detected in the microsomal fraction of
Tetrahymena cells. From these results, together with those of our previous study on lysosomal phospholipid hydrolysis in
Tetrahymena (
J. Biochem.
99, 125-133 (1986)), it is suggested that the extracellular phospholipids which were taken up by the cells
via endocytosis were hydrolyzed through the action of lysosomal phospholipases A
1 and C, and also that one of the products,
sn-2-monoacylglycerol, served as an acyl acceptor for the synthesis of triacylglycerol
via the microsomal “monoacylglycerol pathway.”
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Norio MUTO, Makoto YAMAMOTO, Satoru TANI
1987 Volume 101 Issue 5 Pages
1069-1075
Published: 1987
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Two types of acid proteases, cathepsin D and cathepsin E-like enzyme, from rat gastric mucosa and spleen were compared in their biochemical and immunochemical properties. The enzymes were partially purified by employing the same chromatographic procedures and they showed a single proteolytically active band in polyacrylamide gel electrophoresis. Two low molecular weight enzymes, cathepsins D, from both tissues showed the same molecular weight and the same sensitivities to various inhibitors, but slightly different electrophoretic mobilities. The rabbit antiserum raised against gastric mucosa cathepsin D precipitated both enzymes. On the other hand, high molecular weight enzymes, gastric mucosa cathepsin D-like acid proteinase and spleen cathepsin E-like acid proteinase, were similar to each other as judged by their chromatographic profiles, electrophoretic mobilities, and high stabilities in urea solution. Furthermore, the antiserum specific to gastric mucosa cathepsin D-like acid proteinase inhibited both enzyme activities in a similar manner. However, the antiserum specific to one type of enzyme did not react with the other type. These results indicate that: (a) gastric mucosa cathepsin D is immunologically identical with spleen cathepsin D; (b) gastric mucosa cathepsin D-like acid proteinase has biochemical and immunological properties quite similar to spleen cathepsin E-like enzyme; (c) these two types of acid proteases are quite different proteins existing in the individual tissues.
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Ikuo WADA, Shigeki ETO, Masaru HIMENO, Keitaro KATO
1987 Volume 101 Issue 5 Pages
1077-1085
Published: 1987
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5'-Nucleotidase was found in purified rat liver tritosomes. When tritosomes were subfractionated into the membrane and soluble contents fractions, 73% of the total 5'-nucleotidase activity was found in the membrane fraction and 24% in the soluble contents fraction. Immunoblotting using specific polyclonal antibodies against the rat liver plasma membrane 5'-nucleotidase showed that the mobilities on SDS-polyacrylamide gel electrophoresis of both 5'-nucleotidases in the membrane and contents fractions were identical to that of the enzyme in the plasma membranes (
Mr=72, 000). 5'-Nueleotidases in the membrane and contents fractions were sensitive to neuraminidase and converted into a form that was 4 kDa smaller after digestion, as observed in the case of plasma membrane enzyme. 5'-Nucleotidases, both from the membrane and contents fractions, were purified using immunoaffinity chromatography, and the isoelectric points, heat stability, and oligomeric structure of the purified enzymes were compared. Isoelectric focusing and the heat stability test indicated the resemblance of the soluble enzyme to the membrane-bound enzyme. However, the membrane-bound enzyme aggregated in the absence of Triton X-100, whereas the soluble enzyme behaved as a dimer. The topography of 5'-nucleotidase in the tritosomal membranes was studied using antibodies against 5'-nucleotidase and neuraminidase treatment. The inhibition of 5'-nucleotidase activity by the antibodies and neuraminidase-susceptibility of 5'-nucleotidase were not observed in the intact tritosomal fraction until the tritosomes had been disrupted by osmotic shock. These results show that the active sites and the oligosaccharide chains of 5'-nucleotidase are located on the inside surface of the tritosomal membranes.
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Nobuo MIKI, Retsu MIURA, Yoshihiro MIYAKE
1987 Volume 101 Issue 5 Pages
1087-1094
Published: 1987
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Cytochrome P-450 which catalyzes the 7α-hydroxylation of cholesterol was purified from liver microsomes of untreated rabbits. The minimum molecular weight of the cytochrome P-450 was estimated to be 48, 000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The preparation contained 7 nmol of cytochrome permg of protein. The oxidized form of the P-450 showed absorption maxima at 568, 535, and 417 nm, which are characteristic of a low spin hemoprotein, while the reduced form showed maxima at 545 and 413 nm. The carbon monoxide complex of the reduced form showed maxima at 550 and 447 nm. The cholesterol 7α hydroxylase system of untreated rabbit liver microsomes was reconstituted with the purified P-450, NADPH-cytochrome P-450 reductase, and cytochrome
b5. The P-450 catalyzed the 7α-hydroxylation of cholesterol 500 times more efficiently than the starting microsomes. The reconstituted hydroxylase system showed a substantial salt dependency. In the presence of cytochrome
b5 the activity was maximum at 0.4M KCl (4.55 nmol product formed/mg of protein per min), whereas in the absence of cytochrome
b5 the activity was marginal (0.65 nmol product formed/mg of protein per min) and inhibited by KCl. Thus, cytochrome
b5 stimulated the hydroxylase activity by one order of magnitude. These results indicate that cytochrome
b5 is an essential component of the cholesterol 7α-hydroxylase system of untreated rabbit liver microsomes.
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Eric H. HOLMES, Sen-itiroh HAKOMORI
1987 Volume 101 Issue 5 Pages
1095-1105
Published: 1987
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The enzyme GDPFuc: GM
1 α1→2fucosyltransferase, induced by chemical carcinogens in precancerous rat liver as well as rat hepatoma cells, was found previously to be membrane bound, and was inactivated by various detergents, while the activities of many other transferases are generally enhanced by detergents (Holmes, E. H. & Hakomori, S. (1983)
J. Biol. Chem.
258, 3706-3717). The effects of phospholipids and detergents on rat hepatoma H 35 cells, the conditions of solubilization and subsequent affinity chromatography of the enzyme, and a possible association of phospholipids with the enzyme have been studied with the following major results: i) The α1→2 fucosyltransferase activity in Golgi membrane was diminished on treatment of membranes with phospholipase A
1 or phospholipase C. ii) The enzyme activity was stimulated 7-fold in the presence of cardiolipin or phosphatidylglycerol (and 3-fold by phosphatidylethanolamine) but not other phospholipids. The stimulatory effect of phosphatidylglycerol was eliminated when a variety of ionic or non-ionic detergents were added to the reaction mixture, with the exception of the cationic detergent G-3634-A, which provided a 10-fold total stimulation in the presence of phosphatidylglycerol. The kinetic analysis indicated that addition of phosphatidylglycerol has a negligible effect on apparent
Km values but increases the
Vmax of the enzyme 5- to 6- fold. iii) The enzyme activity was solubilized by the dialyzable detergent CHAPSO without inhibition of the enzyme activity, and the solubilized enzyme in the presence of 0.4% CHAPSO is partially purified by chromatography on GDP-hexanolamine-Sepharose. Removal of CHAPSO from the affinity purified enzyme by dialysis resulted in a 66% loss of the original activity, which was restored by addition of phosphatidylglycerol. iv) Chromatography of the affinity-purified enzyme with
3H-labeled phosphatidylglycerol on a Biogel
A0.5 column indicated an association of the enzyme with the phospholipid that occurred only in the absence of detergent. These results suggest that phospholipid has a direct effect on the enzyme and that the inhibitory effect of detergents can be ascribable to disturbing interaction between phospholipids and the enzyme. A possible role of specific phospholipids on
in vivo transferase activity for glycolipids is discussed.
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Kojiro TAKAHASHI, Mitsuhiro SHIMIDZU, Heisaburo SHINDO, Tomoyuki KAWAM ...
1987 Volume 101 Issue 5 Pages
1107-1114
Published: 1987
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The dephosphorylation of phospho-amino acids with alkaline phosphatase (AlPase) from calf intestine or
Escherichia coli and the phosphorylation of bovine serum albumin (BSA) with epidermal growth factor (EGF) receptor kinase from human A 431 epidermoid carcinoma cells were investigated by
31P NMR spectroscopy. The initial rates of the dephosphorylation of phospho-tyrosine (P-Tyr) and phosphoserine (P-Ser) with AlPase were essentially the same in the one-substrate system. In the two-substrate system (P-Tyr plus P-Ser), however, the ratio of the initial rate for P-Tyr
vs. P-Ser was 2.4 to 4.5 depending on the buffer and pH conditions employed. This substantiates for the first time the specificity of AlPases to P-Tyrover P-Ser at the free amino acid level. In the stationary phase of the overall process, the dephosphorylation of P-Ser became slow compared to that of P-Tyr in the one-substrate system. The decrease in the rate for P-Ser was further pronounced in the two-substrate system. For this remarkable effect, the rephosphorylation of serine was responsible, as demonstrated in the reaction mixture containing serine, P
1, and AlPase. BSA phosphorylated by EGF receptor kinase exhibited sharp
31P resonances around O ppm at neutral pH, far distant from the peak positions (4.9 ppm) of histone H 1 phosphorylated by cAMP-dependent protein kinase. These NMR data are direct evidence that BSA was phosphorylated exclusively at the tyrosyl residues, whereas the phosphorylation of histone H 1 was at the seryl residues.
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Ken-ichi NAGAI, Ineo ISHIZUKA
1987 Volume 101 Issue 5 Pages
1115-1127
Published: 1987
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Some properties of the enzyme activity that catalyzes the transfer of
N-acetylgalactosamine from UDP-
N-acetylgalactosamine to exogenous lactosylceramide-II
3-sulfate (SM3) and
N-acetylneuraminosyllactosylceramide (GM3) were studied using the enzyme preparation solubilized from the 100, 000×
g pellet of 6-day-old rat brain. The products from SM3 and GM3 were identified as gangliotriaosylceramide-II
3sulfate (SM2) and
N-acetylneuraminosylgangliotriaosylceramide (GM2), respectively, by TLC-autoradiography. Optimal conditions for both activities were similar: pH (Hepes-NaOH), 7.0-7.5; detergent (heptylthioglucoside), 0.64% and Mn
2+, 5-10 mM. The concentrations of the detergent optimal for both enzyme activities were also examined at various concentrations of the acceptors. The lower the amounts of acceptors, the less the amounts of detergent that were required, and
vice versa, for the maximum activities. The acceptor-saturation curve for SM2 synthesis was triphasic, exhibiting a sigmoidal region at lower concentrations, a hyperbolic region and finally a descending region. For GM2 synthesis, the curve was biphasic without the descending region. The donor-saturation curves were classical hyperbolic ones for both syntheses. The
Km values calculated for SM3 and GM3 were 0.37 and 0.19 mM, respectively, when the data corresponding to the hyperbolic regions were used for the double-reciprocal plots. The
Km values for UDP-
N-acetylgalactosamine in the SM2- and GM2-synthesis were 82 and 26 μM, respectively. SM3 and GM3 were the best acceptors for this enzyme preparation. From the results of the acceptor competition study, it was suggested that the two synthetic reactions are catalyzed by a single enzyme.
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Yoshio IMAI
1987 Volume 101 Issue 5 Pages
1129-1139
Published: 1987
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cDNA complementary to mRNA coding for a minor form of cytochrome P-450 from phenobarbital-treated rabbit liver (pHP3) was isolated using cDNA for the major phenobarbital-inducible cytochrome P-450 of rat liver as a probe in the first screening of a cDNA library. The nucleotide sequence of pHP3 was determined and contained a continuous reading frame encoding 490 amino acids. The deduced amino acid sequence of pHP3 protein exhibited about 50% homology with the major cytochrome P-450 from phenobarbital-treated rabbit liver, while the homology was as high as 80% between two minor cytochrome forms, pHP2 and pHP3. Two expression plasmids, pAHF3 and pAH
ΔN3, were constructed by insertion of pHP3 fragment between yeast alcohol dehydrogenase I (ADH1) promoter and terminator regions. pAHF3 contained the entire coding sequence of pHP3, but nucleotide sequences for the N-terminal region of pHP3 protein (from the 2 nd to the 23 rd amino acid) were deleted in pAH
ΔN3. On introduction of the constructed plasmids into
Saccharomyces cerevisiae AH22 cells, the absorption spectrum of cytochrome P-450 was detected in the microsomal fraction from the transformed cells carrying pAHF3. On the other hand, cytochrome P-450 could not be detected spectrophotometrically in any subcellular fractions from the yeast cells carrying pAH
ΔN3, although the transcript of pHP3 insert was detected in RNA blot analysis. These results suggest that the N-terminal region of pHP3 protein plays an important role in accumulation of the newly synthesized pHP3 protein in yeast cells. Cytochrome P-450 (pHP3) was solubilized from microsomal membranes of the transformed yeast cells and purified partially on an aminooctyl Sepharose column (specific content, about 6 nmol per mg of protein). In the oxidized state the cytochrome preparation exhibited an absorption spectrum characteristic of a low-spin ferric cytochrome P-450. The reduced CO complex of the cytochrome showed a Soret absorption maximum at 450 nm. The monooxygenase activity of cytochrome P-450 (pHP3) was examined in a reconstituted system containing the cytochrome preparation and NADPH-cytochrome P-450 reductase. Cytochrome P-450 (pHP3) catalyzed
N-demethylation of benzphetamine and aminopyrine and denitrification of lnitropropane. Addition of cytochrome
b5 to the reconstituted system resulted in stimulation of the
N-demethylation activities but inhibition of the denitrification activity. Neither 7-ethoxycoumarin
O-deethylation activity nor acetanilide
phydroxylation activity was detected, either in the presence or absence of cytochrome
b5. This substrate specificity closely resembles that of P-450, which has been purified from liver microsomes of drug-untreated rabbits.
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Nobuko MINAGAWA, Akio YOSHIMOTO
1987 Volume 101 Issue 5 Pages
1141-1146
Published: 1987
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Cyanide-resistant respiration was induced in the yeast,
Hansenula anornala in the presence of cyanide or antimycin A, which blocks the electron transport after ubiquinone. The
de novo protein synthesis in cytosol and oxygen were deduced to be involved in this induction process. The period required for the induction varied during the growth stage, suggesting that involvement of additional physiological factor (s) in this induction process. The organism could multiply in the presence of antimycin A by developing cyanide-resistant respiration despite a decreased growth rate.
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Minoru TAMURA, Toshitsugu YUBISUI, Masazumi TAKESHITA, Shun-ichiro KAW ...
1987 Volume 101 Issue 5 Pages
1147-1159
Published: 1987
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NADH-cytochrome
b5 reductases purified from bovine erythrocytes and from bovine brain and liver microsomes solubilized with lysosomal protease were subjected to structural analysis by using HPLC mapping, amino acid analysis of the resulting peptides, and NH
2-terminal sequence analysis of apoproteins. HPLC maps of the tryptic peptides derived from these enzymes were very similar to each other, and amino acid analysis of the HPLC-separated peptides indicated that the structures of these enzymes are identical except for the NH
2-terminal region. The NH
2-terminal sequence of the brain enzyme determined by automated Edman degradation was as follows:
NH
2-Phe-Gln-Arg-Ser-Thr-Pro-Ala-Ile-Thr-Leu-Glu-Asn-Pro-Asp-Ile-Lys-Tyr-Pro-Leu-Arg-Leu-Ile-Asp-Lys-Glu-Val-Ile-
This sequence is identical to that of liver enzyme except that the liver enzyme started at the 3 rd Arg or 4 th Ser. The NH
2-terminal amino acid residue of the soluble erythrocyte enzyme was not detected by automated Edman degradation. The sequence analysis of a tryptic peptide from the erythrocyte enzyme indicated that Leu is present before the NH
2-terminal Phe of the brain enzyme. The recently reported sequence of the apparently identical protein (Ozols
et al. (1985)
J. Biol. Chem.
260, 11953-11961) differs in two amino acid assignments from our sequence.
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Akira HIRAGA, Shinri TAMURA, Kunimi KIKUCHI, Shigeru TSUIKI
1987 Volume 101 Issue 5 Pages
1161-1168
Published: 1987
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To gain more insight into the nature of the substrate specificity of protein phosphatases, four forms of glycogen synthase D were used as substrates for previously characterized protein phosphatases, IA, IB, and II, from rat liver cytosol. The phosphatase activity was measured as the conversion of glycogen synthase D to synthase I. While glycogen synthase isolated from rat liver as the D-form was activated mainly by phosphatase IA, rabbit skeletal muscle glycogen synthase previously phosphorylated
in vitro by cyclic AMP-dependent protein kinase or phosphorylase kinase was activated efficiently by phosphatases IA, IB, and II. Glycogen synthase isolated from rabbit skeletal muscle as the D-form, however, was a poor substrate for all three phosphatases. These results suggest that the phosphorylation state as well as the primary structure of synthase D markedly affects the rate of its activation by individual protein phosphatases. A protein phosphatase released from rat liver particulate glycogen, on the other hand, activated all forms of synthase D used here readily and at about the same rate.
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Tohru NAKANO, Ayako TERAWAKI, Hitoshi ARITA
1987 Volume 101 Issue 5 Pages
1169-1180
Published: 1987
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A stable thromboxane A
2 (TXA
2) mimetic, U46619, induced a shape change of rat platelets, but did not induce phosphoinositide breakdown, aggregation or secretion. However, when U46619 was added to platelets which had been previously stimulated with collagen in the presence of indomethacin, all biological responses were induced about 1 min after the occurrence of shape change. Furthermore, two phases of phosphorylation of myosin light chain (MLC) were observed under the same conditions, one coinciding with shape change and the other with aggregation. Similar two-phase Ca
2+ mobilization has been observed using aequorin (Nakano, T., Terawaki, A., & Arita, H. (1986)
J. Bioehein.
99, 1285-1288). From these results, collagen-induced signal transduction is considered to be composed of three stages. The first stage is the initial TXA
2 generation. The second stage involves inositol trisphosphate-independent first-phase Ca
2+ mobilization and the first-phase MLC phosphorylation by the action of TXA
2 alone, leading to the shape change of platelets. The third stage is initiated by an abrupt phosphoinositide breakdown via the synergistic action of TXA
2 and occupation of the collagen receptor, and the resulting inositol trisphosphate may induce the second-phase Ca
2+ mobilization to produce the second-phase MLC phosphorylation together with aggregation and secretion.
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Fred VAN LEUVEN, Peter MARYNEN, Jean-Jacques CASSIMAN, Herman VAN DEN ...
1987 Volume 101 Issue 5 Pages
1181-1189
Published: 1987
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Mice were immunized with the isolated C-terminal heat-induced fragment of human α
2-macroglobulin (α
2M) and the spleens were used to prepare hybridomas. A monoclonal antibody (Mab) designated F17D5 was selected for further characterization. The epitope defined by Mab F17D5 was not expressed on α
2M, on α
2M-methylamine, or on α
2M-proteinase complexes. On the other hand, the antibody reacted avidly with denatured human α
2M and with some types of α
2M from other species, including mouse, on nitrocellulose-immunoblotting. The epitope of Mab F17D5 was mapped to less than 250 residues C-terminal of the internal thiolester of human α
2M. This was based on CNBr fragmentation of the 60 kDa C-terminal heat-fragment and on peptide mapping of α
2M, derivatized at the internal thiolester GLX-residue with
125I-labeled histamine. Murine α
2M was confirmed to contain two types of subunit: a 185 kDa subunit and a combination of 165 kDa and 35 kDa polypeptides. By partial disulfide bond reduction, heat-fragmentation and immunoblotting with Mab F17D5, the structure of murine α
2M was compared to that of human α
2M. The F17D5-epitope was mapped to a 30 kDa heat-induced fragment, which was released by denaturation without reduction. This fragment contained an intrachain disulfide bridge. By analogy with human α
2M, the 35 kDa subunit would be located at the C-terminal end of murine α
2M, disulfide-bonded to the major 165 kDa subunit.
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Takashi MATSUMOTO, Takahisa OHTA, Izumi KUMAGAI, Tairo OSHIMA, Katsuto ...
1987 Volume 101 Issue 5 Pages
1191-1198
Published: 1987
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The efficiency of methylation of tRNA by a thermostable tRNA(guanosine-2') methyltransferase (Gm-methylase) was examined at various temperatures using several species of tRNA isolated from
Escherichia coli, yeast and
Bacillus subtilis, each possessing different thermal properties. The optimal temperature for the methylation reaction was
ca. 20°C lower than the melting temperature of the tRNA in each case. Arrhenius plots of the methylation reactions with various tRNAs gave straight lines below the optimal temperatures in all cases, with similar activation energies of between 10 and 14 kcal/mol. Above the optimal temperatures, the methyl acceptor activity decreased as the incubation temperature was raised to 80°C, at which point the methylase was still active. A correlation was observed between the remaining methyl acceptor activity and the hyperchromicity of tRNA. These results suggest that Gm-methylase recognizes the tertiary structure of tRNA, and it is not the substrate tRNA but the enzyme which is activated by heat.
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Jun UTSUMI, Shojiro YAMAZAKI, Kazuo HOSOI, Shigenobu KIMURA, Keizo HAN ...
1987 Volume 101 Issue 5 Pages
1199-1208
Published: 1987
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Homogeneous
E. coli-derived recombinant human interferon-β (
E. coli-rHuIFN-β) was characterized in order to elucidate its physicochemical properties, as compared with those of fibroblast human interferon-β (fibroblast HuIFN-β). Purified
E. coli-rHuIFN-β and fibroblast HuIFN-β exhibited a single band of
Mr, 19, 000 and 23, 000, respectively, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE). The primary structure of
E. coli-rHuIFN-β was identical to the prediction from the cDNA sequence. Furthermore, both the circular dichroism (CD) spectra and the
1H nuclear magnetic resonance (NMR) spectra of
E. coli-rHuIFN-β and fibroblast HuIFN-β at pH 6.8 were closely similar to each other. On the other hand, on reverse-phase high-performance liquid chromatography (HPLC) using a C 18 column, the retention time of
E. coli-rHuIFN-β was longer than that of fibroblast HuIFN-β. Moreover, although the isoelectric point of
E. coli-rHuIFN-β was pH 8.9, purified fibroblast HuIFN-β exhibited multiple isoelectric points, probably due to heterogeneity of the carbohydrate moiety. These results indicate that the
E. coli-rHuIFN-β polypeptide folds similarly to fibroblast HuIFN-β, and the carbohydrate moiety of natural HuIFN-β has little influence on higher-order structure but does influence the hydrophobic and the electrostatic properties of the molecule.
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Alistair G. C. RENWICK, Tsuguo MIZUOCHI, Naohisa KOCHIBE, Akira KOBATA
1987 Volume 101 Issue 5 Pages
1209-1221
Published: 1987
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The asparagine-linked sugar chains of human follicle-stimulating hormone (hFSH) were liberated as radioactive oligosaccharides from the polypeptide moiety by hydrazinolysis followed by
N-acetylation and NaB
3H
4 reduction. Ninety-five percent of the oligosaccharides were acidic and all were converted to a mixture of neutral oligosaccharides on sialidase treatment. The mixture of neutral oligosaccharides was subjected to sequential immobilized lectin column chromatography on E-PHA-agarose, AAL-Sepharose, and Con A-Sepharose, and six fractions were obtained. The results of sequential exoglycosidase digestion of each oligosaccharide and methylation analysis led us to propose that the asparagine-linked sugar chains of hFSH are a mixture of complex-type bi-, tri-, and tetraantennary sialylated sugar chains with and without a fucose residue linked at the C-6 position of the proximal
N-acetylglucosamine. Some of these sugar chains contain bisecting
N-acetylglucosamine residue.
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Takamitsu HORI, Harumi OKUYAMA
1987 Volume 101 Issue 5 Pages
1223-1231
Published: 1987
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The oleate (
Δ9-18:1)/
cis-vaccenate (
Δ11-18:1) ratios in phospholipids increased in the order of normal liver, host liver and hepatoma in rats. The amount of oleate increased in phospholipids and decreased in triacylglycerol in the same order, where as the distributions of
cis-vaccenate among the major lipid classes were relatively unchanged among the three kinds of cells. Biochemical bases for these differences were sought by characterizing the microsomal elongation system and by analyzing the elongation and desaturation products
in vitro. Kinetic parameters for the elongations of palmitoyl-CoA and palmitoleoyl-CoA did not account for the observed differences in the oleate/
cis-vaccenate ratios in these cells. However, the oleate/
cis-vaccenate ratios varied depending on the availability of substrates, NADH, NADPH, and malonyl-CoA, in the elongation and desaturation of palmitoyl-CoA. Based on the results, it is proposed that differences in the concentrations of substrates for the elongation and desaturation systems might account at least in part for the differences in the oleate/
cis-vaccenate ratios among the three kinds of cells.
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Motonobu YOSHIDA
1987 Volume 101 Issue 5 Pages
1233-1245
Published: 1987
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Antisera against purified contact site A glycoprotein, with an apparent molecular weight of 80×10
3 (80 kDa), from
Dictyostelium discoideum were raised by using Freund's adjuvant (antiserum-A) and by using Alu-Gel-S (antiserum-B) as immunoadjuvants. They were converted into Fab fragments for the cell agglutination assay. Fab fragments of antiserum-B inhibited only EDTA-stable cell contact, whereas Fab fragments of antiserum-A (Fab-A) inhibited EDTA-sensitive cell contact as well as EDTA-stable cell contact. We prepared several cell types in order to identify target antigens for the adhesion-blocking Fab-A in EDTA-sensitive cell contact or EDTA-stable cell contact. One of these cell types produced contact site A without
N-glycosidically-linked carbohydrate chains. It is known that contact site A contains two kinds of
N-glycosidically-linked carbohydrate chains (carbohydrates I and II, Yoshida, M., Stadler, J., Bertholdt, G., and Gerisch, G. (1984)
EMBO J.
3, 2653-2670). When growth-phase cells were treated with tunicamycin (TM) at a final concentration of 2 μg/ml in nutrient medium (TM-pretreated cells), the cells produced contact site A without
N-glycosidically-linked carbohydrate chains (53 kDa) at the normal developmental stage. These cells lacked EDTA- sensitive cell contact as well as EDTA-stable cell contact. The neutralization of the adhesion-blocking Fab-A was done by using particulate fractions from each cell type. The blocking activity in EDTA-stable cell contact was neutralized by the cell type with carbohydrate II. Taking these results into consideration, EDTA-stable cell contact may be formed by the interaction between protein moieties of contact site A and carbohydrate II. Concerning EDTA-sensitive cell contact, the blocking activity was neutralized by each cell type irrespective of TM treatment. This suggests that
O-glycosidically-linked carbohydrate chains play a role in EDTA-sensitive cell contact. Moreover, the biological activity in EDTA-sensitive cell contact of TM-pretreated cells suggests that
N-glycosidically-linked carbohydrate chains may also be involved in this contact.
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Yoshihiro SATO, Tomoko MURAI, Taiko ODA, Hazime SAITÔ, Masahiko ...
1987 Volume 101 Issue 5 Pages
1247-1252
Published: 1987
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Dienestrol, meso-hexestrol, and
dl-hexestrol, synthetic nonsteroidal estrogens, were shown to be inhibitors of microtubule assembly
in vitro using microtubule proteins isolated from porcine brains. The order of activity of the synthetic estrogens as inhibitors of microtubule assembly is: dienestrol>diethylstilbestrol>meso-hexestrol>
dl-hexestrol>isodienestrol. The activity of dienestrol as an inhibitor was of the same order as that of (+)-griseofulvin, as determined by turbidity measurement. Electron microscopic observation revealed that twisted ribbon structures are formed from microtubule proteins in the presence of some synthetic estrogens (dienestrol, meso-hexestrol, and
dl-hexestrol).
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Masayuki MORITA, Susumu TSUNASAWA, Yoshinobu SUGINO
1987 Volume 101 Issue 5 Pages
1253-1264
Published: 1987
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The Tn3 transposase accumulated to about 4% of total cell protein in a minicellproducing
Escherichia coli strain harboring a transposase overproducer plasmid. This accumulation of the transposase seems to be due to four factors: i) derepression of transcription resulting from inactivation of the repressor gene (
tnpR); ii) efficient translation caused by a mutation within the Shine-Dalgarno (SD) sequence, iii) the dosage effect of the increased plasmid copy number resulting from deletion of the
rom gene of the plasmid; and iv) use of a minicell-producing strain as the host. The Tn3 transposase was purified by a procedure involving five steps; i) sonication, ii) precipitation of protein by adding polyethyleneimine to the sonic supernatant, followed by extraction of transposase fraction with a buffer containing ammonium sulfate, iii) ammonium sulfate precipitation, iv) gel filtration through Sephacryl S-300, and v) phosphocellulose and DNA-cellulose chromatography. Milligram quantities of pure transposase can be obtained from one gram of wet cells. A small fraction of the accumulated transposase had a blocked amino-terminus and was eluted separately from the normal protein in the chromatography.
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Tetsuro YOSHIMURA, Shigenori MAEZAWA, Keelung HONG
1987 Volume 101 Issue 5 Pages
1265-1272
Published: 1987
Released on J-STAGE: June 30, 2008
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Clathrin induces fusion of phospholipid membranes containing phosphatidylserine when the pH of the medium is reduced below about 6. The hydrophobicity of clathrin in the membrane fusion-inducible pH region was examined. In the presence of clathrin, the fluorescence maximum of 1-anilinonaphthalene-8-sulfonate was shifted to shorter wavelengths and its fluorescence intensity increased at pH values below about 6. Steep increase of the fluorescence intensity of the fluorescent probe,
N-(1-anilinonaphthyl-4) maleimide covatently bound to clathrin was observed in a similar pH region. The efficiency of resonance energy transfer from tryptophan to anilinonaphthyl residues in the clathrin molecule showed similar pH dependency. When Triton X-114 solutions containing clathrin were subjected to phase separation by raising the temperature from 0° to 30°C at different pH values, clathrin remained in the aqueous phase above pH 6, whereas the protein was partitioned between the aqueous and detergent phases at pH 5-6, and was present only in the detergent phase below pH 5. The effective hydrophobicity of clathrin determined by the fluorescence method using
cis-parinaric acid also increased at pH values below 6. Moreover, clathrin was aggregated by lowering the pH below 6. These results show that exposure of hydrophobic domains of clathrin through conformational change occurs in its membrane fusion-inducible pH region, suggesting the crucial role of protein hydrophobicity in the initiation of membrane fusion.
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Masao HIRAIWA, Yutaka UDA, Masatoyo NISHIZAWA, Tadashi MIYATAKE
1987 Volume 101 Issue 5 Pages
1273-1279
Published: 1987
Released on J-STAGE: June 30, 2008
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A sialidase [EC 3. 2. 1. 18] has been partially purified from human placenta by means of procedures comprising Con A-Sepharose adsorption, ammonium sulfate precipitation, sucrose density gradient centrifugation, and high-pressure liquid chromatography on a Shim pack Diol 300 column. On high-pressure liquid chromatography, most of the β-galactosidase that comigrated with the sialidase on sucrose density gradient centrifugation was removed. The sialidase was purified 3, 600-fold from the preparation obtained by Con A-Sepharose adsorption. The enzyme liberated the sialic acid residues from (α2-3) and (α2-6) sialyllactose, colomic acid, fetuin, and transferrin, but not from bovine submaxillary mucin. The enzyme also hydrolyzed gangliosides G
M3, G
D1a, and G
D1b in the presence of sodium cholate as a detergent, but G
M1 and G
M2 were less susceptible to the enzyme. The optimum pHs for 4-methylumbelliferyl-
N-acetylneuraminate, sialyllactose, fetuin, and G
M3 lay between 4.0 and 5.0.
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Yoshimasa SAITO, Yoshinori ISHII, Mineo NIWA, Ikuo UEDA
1987 Volume 101 Issue 5 Pages
1281-1288
Published: 1987
Released on J-STAGE: June 30, 2008
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Direct expression of a growth-promoting peptide hormone, somatomedin C/insulinlike growth factor I, which is quite difficult due to the instability of somatomedin C itself in
Escherichia coli, has been achieved by the use of a two-cistron system. Assuming that basic somatomedin C might be stabilized by forming a complex with an acidic polypeptide, we constructed synthetic genes consisting of two cistrons; an acidic 93-amino-acid polypeptide was coded in the first cistron followed by a synthetic somatomedin C gene in the second cistron. The chain termination codon for the first polypeptide overlapped the initiation codon for the second polypeptide in the intercistronic region, as occurs in the polycistronic
E. coli tryptophan operon, whose products are associated in multi-subunit enzyme complexes. In the expression of the resulting genetic system, recombinant somatomedin C associated with the acidic polypeptide was accumulated to high levels in the cells. After treatment of the product with acetic acid to dissociate the two components, the recombinant somatomedin C was isolated in a yield of 4.0mg from a liter of culture broth at
A600=1.6. It was determined to be Met-somatomedin C by chymotryptic mapping as well as amino-terminal analysis.
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Yukio TANIZAWA, Fumio KISHI, Toshio KANEKO, Atsushi NAKAZAWA
1987 Volume 101 Issue 5 Pages
1289-1296
Published: 1987
Released on J-STAGE: June 30, 2008
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Chicken muscle adenylate kinase was produced in a large amount in
Escherichia coli cells harboring an expression plasmid, pKK-cAKl-1. The plasmid was constructed by placing the cDNA sequence for chicken muscle adenylate kinase after the
tac promoter. After induction by isopropyl-β-D-thiogalactopyranoside, the enzyme protein amounted to about 10% of the bacterial proteins. The enzyme was readily purified in two steps by using phosphocellulose and Sephadex G-100 columns. The apparent molecular weight of the enzyme produced in
E. cola was estimated to be 22, 000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in agreement with the value deduced from the cDNA sequence. Ten amino acids in the NH
2-terminal region were determined, and were identical with the sequence deduced from the cDNA sequence except that the terminal methionine was absent, Michaelis constants for ATP, ADP, and AMP of the enzyme thus synthesized were essentially identical to those determined with the enzyme in crude extracts of chicken skeletal muscle.
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Takanori NAKAMURA, Toshiaki HIRAI, Fuminori TOKUNAGA, Shun-ichiro KAWA ...
1987 Volume 101 Issue 5 Pages
1297-1306
Published: 1987
Released on J-STAGE: June 30, 2008
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A low molecular weight protein protease inhibitor was purified from Japanese horseshoe crab (
Tachypleus tridentatus) hemocytes. It consisted of a single polypeptide with a total of 61 amino acid residues. This protease inhibitor inhibited stoichiometrically the amidase activity of trypsin (
K1=4.60×10
-10M), and also had inhibitory effects on α-chymotrypsin (
K1=5.54α10
-9M), elastase (
K1=7.20α10
-8M), plasmin, and plasma kallikrein. However, it had no effect on
T. tridentatus clotting enzyme and factor _??_, mammalian blood coagulation factors (activated protein C, factor Xa and a-thrombin), papain, and thermolysin. The complete amino acid sequence of this inhibitor was determined and its sequence was compared with those of bovine pancreatic trypsin inhibitor (BPTI) and other Kunitz-type inhibitors. It was found that the amino acid sequence of this inhibitor has a high homology of 47 and 43% with those of sea anemone inhibitor 5-II and BPTI, respectively. Thus, this protease inhibitor appeared to be one of the typical Kunitz-type protease inhibitors.
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Tamio MIZUKAMI, Seiga ITOH
1987 Volume 101 Issue 5 Pages
1307-1310
Published: 1987
Released on J-STAGE: June 30, 2008
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A useful vector, pAGE103, has been developed for the expression of cDNA in animal cells using the simian virus 40 (SV40) expression signals. cDNA could be expressed easily by inserting it into the multiple cloning sites (
HindIII,
SalI/
AccI,
XbaI,
BamHI,
SmaI/
XmaI,
KpnI/
Asp718,
SacI and
EcoRI) of the vector, which are located between the SV40 early promoter and the SV40 early RNA processing signals for splicing and polyadenylation. In addition to the above transcription unit, pAGE103 contains the replication origin of ColE1, and a dual Km
R/G418
R selective gene. Several unique restriction sites are located on the boundaries between the above-mentioned three components of the vector, allowing the easy substitution or insertion of other genetic elements. The human interferon-β gene was inserted into pAGE103 and shown to be expressed transiently in COS-1 cells and stably in several animal cell lines.
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Makio HAYAKAWA, Kazuhiko HORIGOME, Ichiro KUDO, Motowo TOMITA, Shoshic ...
1987 Volume 101 Issue 5 Pages
1311-1314
Published: 1987
Released on J-STAGE: June 30, 2008
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The amino acid composition and partial NH
2-terminal amino acid sequence of the phospholipase A
2 secreted by stimulated rat platelets were determined. The most predominant amino acid in the phospholipase A
2 was cysteine followed by lysine, suggesting that it is a basic one. This finding is consistent with its high affinity to a cation exchange column. The NH
2-terminal 24 amino acids were found to be as follows:
X-Leu-Leu-Glu-Phe-Gly-Gln-Met-Ile-Leu-Phe-Lys-Thr-Gly-Lys-Arg-Ala-Asp-Val-Ser-Tyr-Gly-Phe-Tyr-Gly-The enzyme contains
5Phe,
8Met,
9Ile,
24Tyr, and
25Gly residues, all of which are conserved in the sequenced pancreatic phospholipase A
2. This is the first report of the tentative characterization of a eukaryotic phospholipase A
2, the cellular source of which is known,
i.e., it does not originate from a venom or the pancreas.
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Kenji NISHIMURA, Eiji SATO, Akira NAKAMURA
1987 Volume 101 Issue 5 Pages
1315-1318
Published: 1987
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A fucolipid isolated from Japanese quail intestine was identified as β-galactosyl-1, 4-(α-fucosyl-1, 3-) β-
N-acetylglucosaminyl-1, 3-β-galactosyl-1, 4-β-glucosyl-1, 1-ceramide, a glycolipid which exhibits X-hapten activity. Analysis of the tissue at various embryonic stages showed that the fucolipid is characteristically present at later stages of organogenesis.
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1987 Volume 101 Issue 5 Pages
1319a
Published: 1987
Released on J-STAGE: June 30, 2008
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1987 Volume 101 Issue 5 Pages
1319b
Published: 1987
Released on J-STAGE: June 30, 2008
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1987 Volume 101 Issue 5 Pages
1319c
Published: 1987
Released on J-STAGE: June 30, 2008
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