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Volume 88 (1980)
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Volume 88, Issue 5
Displaying 1-42 of 42 articles from this issue
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VI. Polysaccharide-Induced Aggregation of Multilamellar Liposomes of Egg LecithinJunzo SUNAMOTO, Kiyoshi IWAMOTO, Hiroki KONDO, Seiji SHINKAI1980 Volume 88 Issue 5 Pages 1219-1226
Published: November 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSInduced aggregation of multilamellar liposomes of egg lecithin with several polysaccharides, such as pullulan, dextran, hydroxyethylstarch, and amylopectin, has been investigated in conjunction with structural characteristics of polysaccharides, incubation temperature, ionic strength, and surface charge of liposomes. Aggregation of liposomes as a function of time was followed by monitoring turbidity of the liposome suspension at 600 nm. The inverse relationship between the minimal concentration and molecular weight of polysaccharides for the induced aggregation of liposomes was observed for all the polysaccharides employed. Polysaccharides having larger excluded volume more easily bind to liposomes, leading to the acceleration of the aggregation of liposomes. Hence, the order of efficiency of polysaccharides for the promotion of liposome aggregation was pullulan>dextran>hydroxyethyl-starch_??_amylopectin. Increasing the surface charge of liposomes brought about a decrease in aggregation irrespective of the sign. Even when negatively charged liposomes were incubated with the cationic DEAE-dextran, the liposome aggregation was depressed. These results including salt effect and temperature dependency of the induced aggregation of liposomes supported a plausible mechanism for the reaction, the polysaccharide-bridging mechanism, which has been provided for the polysaccharide-promoted agglutination of human erythrocytes.View full abstractDownload PDF (594K) -
Keiichi KAWAI, Yoshitomo EGUCHI1980 Volume 88 Issue 5 Pages 1227-1231
Published: November 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBlue dextran or Cibacron Blue F3GA has been shown to inhibit yeast phosphoglycerate kinase [EC 2. 7. 2. 3] competitively with respect to ATP (Thompson et al. (1975) Proc. Natl. Acad. Sci. U. S. 72, 663-667; Beissner and Rudolph (1979) J. Biol. Chem. 254, 6273-6277). However, we have found that phosphoglycerate kinase of Lactobacillus plantarum was inhibited by Cibacron Blue F3GA, the blue chromophore of blue dextran, noncompetitively with respect to ATP, but competitively with respect to 3-phosphoglycerate. Further inhibition studies with Cibacron Blue F3GA suggest that one molecule of the dye was bound per molecule of phosphoglycerate kinase at a saturated level of either substrate, but two molecules of the dye were bound per molecule of the kinase with an unsaturated level of either substrate used as a fixed substrate. Furthermore, phosphoglycerate mutase [EC 2. 7. 5. 3] of Leuconostoc dextranicum was also inhibited by Cibacron Blue F3GA competitively with respect to 3-phosphoglycerate and noncompetitively with respect to 2, 3-bisphosphoglycerate. These results suggest that the 3-phosphoglycerate-binding site on both phosphoglycerate kinase and phosphoglycerate mutase can interact with Cibacron Blue F3GA.View full abstractDownload PDF (366K) -
Functional and Structural StudiesAnna GIULIANI, Donatella MAFFI, Maria Pia CAPPABIANCA, Leonardo TENTOR ...1980 Volume 88 Issue 5 Pages 1233-1237
Published: November 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHb Legnano (α2 141 (HC3) Arg→Leu β2) is an abnormal hemoglobin, for which preliminary structural and functional studies demonstrated an amino acid substitution (Arg→Leu) in the α-C-terminus. This substitution modifies the functional properties observed in whole blood as well as in red blood cells, as reported in this paper.
On the basis of its pI, previously determined by analytical isoelectric focusing (IEF), Hb Legnano was purified on a preparative IEF slab. The purified fraction was subjected to functional ultracentrifugal studies under various conditions of pH and salt concentration.
The findings are compared with those for Hb Suresnes (α2 141 (HC3) Arg→His β2) and Hb-CPB, a normal hemoglobin in which the C-terminal α 141 Arg has been cleaved by carboxypeptidase B.
Hb Legnano, like the other hemoglobins considered, shows an increased P50, a decreased Hill's “n” values and a decreased Bohr effect that are partially restored in presence of organic phosphates.
The presence of inorganic ions decreases the Bohr effect and enhances the dissociation into dimers, as observed in Hb-CPB. The dissociation of hemoglobin in carboxy form in ultracentrifugal studies and the different slope of Hill's “n” value as a function of pH are presumably due to presence of Leu, which probably modifies the stereochemistry of the variant.View full abstractDownload PDF (1271K) -
Yoshitaka GOTO, Jun SHIMIZU, Ryoiti SHUKUYA1980 Volume 88 Issue 5 Pages 1239-1249
Published: November 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPhosphoenolpyruvate carboxykinase from bullfrog liver mitochondria has been purified to electrophoretical and immunological homogeneity by an improved method using hydrophobic chromatography on Sepharose-hexane-GMP and affinity chromatography on phosphocellulose. The molecular weight was determined to be 70, 000 by SDS-gel electrophoresis, 65, 000 by Sephadex G-100 gel filtration and 71, 000 by glycerol gradient centrifugation. The isoelectric point was determined to be 6.2, differing from that of the cytosol enzyme. The rabbit IgG fraction against the mitochondrial PEP carboxykinase precipitated not only the mitochondrial but also the cytosol enzyme. The dissociation constant of the nucleotide-enzyme complex was determined to be 3 μM for GTP, 8.5 μM for GDP, and 171 μM for GMP. The affinity of GTP for the enzyme was reduced in the presence of phosphoenolpyruvate or Mn2+, whereas that of GDP was not changed. GMP inhibited the enzyme competitively with GDP for the phosphoenolpyruvate carboxylation and competitively with GTP for the exchange reaction between [14C]HCO3- and oxaloacetate. The purified enzyme was found to have a cysteine residue which reacted with iodoacetamide to form inactive enzyme. Guanine nucleotides or IDP and Mn2+ at a lower concentration prevented the inactivation by iodoacetamide of the enzyme in a competitive manner. Binding of guanine nucleotide to the enzyme and the relation of the sulfhydryl group to the nucleotide binding are discussed.View full abstractDownload PDF (1389K) -
Hidenori HAYASHI, Sigehiro MORITA1980 Volume 88 Issue 5 Pages 1251-1258
Published: November 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSLight harvesting (LH) bacteriochlorophyll (Bchl) protein complexes were isolated from chromatophores of Chromatium vinosum, by the combination of detergent solubilization, sodium dodecylsulfate (SDS) polyacrylamide gel electrophoresis (PAGE) and hydroxyapatite chromatography. Addition of the absorption spectra of these complexes reproduced the absorption spectrum of chromatophores from which these complexes were derived. This result led to the conclusion that these isolated complexes retained the near-infrared absorption spectra which these complexes showed as they existed in chromatophores. Two kinds of spectrally different chromatophores were obtained under different culture conditions. One of them contained two kinds of LH Bchl protein complexes; B 890 containing and B 850-B 800 containing complexes, and the other contained, in addition, B 820-B 800 containing complex. B 890 containing complexes from the two types of chromatophores were spectrally similar, whereas B 850-B 800 containing complexes were not the same with respect to the location of absorption maxima and the content of B 850. It was shown that the variation of the nearinfrared absorption spectra of the chromatophores is due to not only the variation of the ratio of complexes but also the variation of the absorption spectrum of a particular complex.View full abstractDownload PDF (623K) -
Takao KODAMA, Nagomi KUREBAYASHI, Yasuo OGAWA1980 Volume 88 Issue 5 Pages 1259-1265
Published: November 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. Measurements were made of heat production and proton release during ATP hydrolysis by fragmented sarcoplasmic reticulum (FSR) prepared from bullfrog and rabbit skeletal muscle. A microcalorimeter with an improved time resolution was used (Kodma, T. & Woledge, R. C. (1979) J. Biol. Chem. 254, 6382-6386). The FSR was incubated under ATP-limiting conditions in a medium containing 100mM KCl, 4mM MgCl2, 0.1-0.3mM CaCl2, 60 μM P1, P5-di(adenosine-5')pentaphosphate, and 20mM MOPS-KOH (pH 7.1) at 8 and 20°C.
2. In the presence of 6 μM A 23187, which abolishes the Ca uptake by FSR without impairing its ATPase activity, the results with frog and rabbit FSR were very similar at each temperature. The heats of ATP hydrolysis (ΔHATP) thus obtained were -25.2±1.5 and -26.8±0.9 kJ•mol-1 at 8 and 20°C, respectively, after correction for the heat (-18.7 kJ•mol-1) due to the interaction of MOPS with 0.89±0.01 mol of H+ released per mol of ATP hydrolyzed. The ΔHATP at pH 8.0 determined at 8 and 20°C under conditions otherwise the same as above were -18.5±1.8 and -19.7±1.3 kJ•mol-1, respectively. These values are in good agreement with those calculated according to the method of Alberty (J. Biol. Chem. 244, 3290-3302 (1969)).
3. When the ATP hydrolysis was coupled to the Ca uptake, some protons (0.26-0.30 mol for rabbit FSR; 0.16-0.25 mol for frog FSR) were released in addition to those derived from the ATP hydrolysis itself, but the heat produced was not much different from that of the ATP hydrolysis uncoupled from the Ca uptake. Using values for the stoichiometry of Ca uptake and ATP hydrolysis determined in separate experiments, the heat of Ca uptake was estimated. The values for rabbit FSR were +13.1 and +5.6 kJ•mol-1 at 8 and 20°C, respectively. Corresponding values for frog FSR were +4.8 and +3.0 kJ•mol-1 It can be concluded that the ATP-driven Ca uptake by FSR is an endothermic process accompanied by the release of a fraction of a proton in exchange for a Ca ion sequestered.View full abstractDownload PDF (577K) -
I. Physical Properties of Lipid Bilayers in Whole CellsAtsushi HYONO, Shigeko KURIYAMA, Hisako HARA, Ikuya YANO, Masamiki MAS ...1980 Volume 88 Issue 5 Pages 1267-1274
Published: November 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe lipid membrane properties of a moderately halophilic gram-negative bacterium, Pseudomonas halosaccharolytica ATCC 29423, were studied by the use of stearate spin labels, 5 NS, 12 NS, and 16 NS, changing the temperature of ESR measurement from 15 to 50°C. The order parameter and the rotational correlation time of the spin labels incorporated into intact cell membranes of this bacterium grown at various temperatures in media containing different NaCl concentrations were calculated. The activation energy of rotational microviscosity was obtained from Andrade plots.
At low growth temperature and low NaCl concentration in the medium, extractable lipids of this bacterium contained comparatively large amounts of unsaturated fatty acids, but as the growth temperature and NaCl concentration in the medium increased, the contents of saturated and cyclopropanoic fatty acids increased to more than half of the total fatty acids. 5 NS gave the highest order parameters for the intact cells of this bacterium, while 12 NS gave lower and 16 NS gave the lowest results. The order parameters of 5 NS, 12 NS, and 16 NS were completely separated, and all order parameters decreased gradually as the measuring temperature was increased. In contrast, the rotational correlation times of the intact cells with 12 NS were as large as those with 5 NS, while those with 16 NS were distinctly smaller. Increasing NaCl concentrations in the growth medium caused an increase of the rotational correlation times, that is, stiffened the lipid bilayers. The Andrade plot for 16 NS was approximately a straight line, whereas 5 NS and 12 NS gave two straight lines crossing at a temperature near the growth temperature, indicating phase transition from solid to liquid. The microviscosity activation energies were 5-10 kcal/mol in the liquid phase and 15-25 kcal/mol in the solid phase.View full abstractDownload PDF (498K) -
II. Effect of Extreme Growth Conditions on Liposome PropertiesHisako HARA, Atsushi HYONO, Shigeko KURIYAMA, Ikuya YANO, Masamiki MAS ...1980 Volume 88 Issue 5 Pages 1275-1282
Published: November 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSLipid preparations from the cells of a moderately halophilic bacterium, Pseudomonas halosaccharolytica grown under the two extreme conditions of high temperature-high NaCl concentration and low temperature-low NaCl concentration showed distinctively different profiles in phospholipid and fatty acid composition. Cells grown at 40°C in medium containing 3.5M NaCl had high concentrations of saturated and C19 cyclopropanoic fatty acids (about 50 per cent of the total), whereas cells grown at 20°C in medium containing 0.5M NaCl had decreased concentrations of these fatty acids with increased concentrations of the corresponding unsaturated fatty acids. The phospholipid composition was also affected by the culture conditions; cells grown at 40°C in 3.5M NaCl had large amounts of acidic phospholipids, whereas those grown at 20°C in 0.5M NaCl had small amounts. ESR studies on liposomes prepared from lipids of cells grown under the two conditions showed characteristic profiles for correlation times and order parameters of three spin labels of stearic acid derivatives similar to those of membranes of whole cells of this bacterium. ESR studies showed that the physical properties of the liposomes from the total extractable lipids and isolated phosphatidylglycerol from the cells were completely different from those of synthetic dioleoyl-phosphatidylglycerol. Liposomes of the lipids extracted from cells grown at 40°C in 3.5M NaCl showed change in rotational viscosity on altering the NaCl concentration to 0.5M, whereas liposomes of lipids extracted from cells grown at 20°C in 0.5M NaCl did not show change in rotational viscosity on increasing the NaCl concentration to 3.5M.View full abstractDownload PDF (509K) -
Panagiotis PANTAZIS, Masao SAKAMOTO1980 Volume 88 Issue 5 Pages 1283-1289
Published: November 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSChromatin was prepared from calf thymus by three different methods differing in the ionic composition of the solutions used. The contents of histones and nonhistones per DNA unit were dependent on the preparative method utilized. Variations in protein content were analyzed by SDS-polyacrylamide gel electrophoresis. Although the amount of all histones varied with the isolation method, the abundance of H 1 histone in chromatin was the most sensitive to the presence of magnesium ions during the isolation procedure. The variation in protein content affects the thermal denaturation profiles of chromatin. The amount of low-temperature melting chromatin is most sensitive to the preparation method used. Additionally, a very high-temperature denaturation step, designated δ, is detected for chromatin prepared in the absence of magnesium.View full abstractDownload PDF (994K) -
Yasunaga KAMEYAMA, Kazuo OHKI, Yoshinori NOZAWA1980 Volume 88 Issue 5 Pages 1291-1303
Published: November 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThermally induced phase separation was observed to occur in microsomal membranes of the ciliate Tetrahymena pyriformis, using the technique of freeze-fracture electron microscopy. In the present study, we attempted to fractionate the phase-separated membranes which were produced by chilling cells by sucrose density gradient centrifugation. When Tetrahymena was grown in the presence of palmitic acid, cells rapidly incorporated the fatty acid into their phospholipids. The resulting endoplasmic reticulum containing a high level of palmitic acid was more susceptible to thermotropic phase separation. Despite the profound alterations in the fatty acid composition, the cells retained normal growth rate, appearance and cell motility. Smooth microsomes isolated from palmitic acid-supplemented Tetrahymena cells were sonicated and then fractionated into three major subfractions. Fraction-I with lower buoyant density was rich in phospholipids and saturated fatty acids, while Fraction-III with higher density was rather rich in proteins and contained more unsaturated fatty acids in the phospholipids. A significant change was also observed in the polar head composition of phospholipids in these fractions. ESR analysis demonstrated that the extracted lipids from Fraction-III were more fluid than those from Fraction-I. In addition, the motion of the spin probe in the native membranes was more restricted than in extracted lipids. These results indicate that the lipid phase separation causes “squeezing out” of the membrane proteins from the less fluid to the fluid areas. Furthermore, we examined the temperature dependence of the activities of glucose-6-phosphatase and palmitoyl CoA desaturase.View full abstractDownload PDF (1937K) -
Akira MURAYAMA, Fumio FUKAI, Tadashi YAMAMOTO1980 Volume 88 Issue 5 Pages 1305-1315
Published: November 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe cytoplasmic progesterone receptor (PR) system of hen oviduct was shown to be similar to the cytoplasmic estrogen receptor (ER) system of cow uterus described previously. A molecule (sedimentation coefficient, 4.5 S; Stokes radius, 45 A; molecular weight, 84, 000) designated as native “4 S” PR was the sole native component which bound specifically with progesterone. Native “4 S” PR was easily modified by a cytoplasmic enzyme to give a partially proteolyzed PR (sedimentation coefficient, 4.5 S; Stokes radius, 34 A; molecular weight, 63, 000) designated as modified “4 S” PR. Modified “4 S” PR dissociated in the presence of 0.4M NaSCN to give a fragment (sedimentation coefficient, 2.9 S; Stokes radius, 22 A; molecular weight, 26, 000), which reassociated into modified “4 S” PR when NaSCN was removed. The presence of a cytoplasmic protein component (Stokes radius, 50 A; molecular weight, 140, 000) designated as “8 S” PR-forming factor [(“8 S” PR)-FF], which specifically binds with native “4 S” PR to form “8 S” PR (sedimentation coefficient of 8.0 S) was indicated. (“8 S” PR)-FF was dissociated in the presence of 0.4M KCl into subunits designated as component A (Stokes radius, 36 A; molecular weight, 56, 000) and component B (Stokes radius, 17.5 A; molecular weight, 13, 000). Component A bound with native “4 S” PR to give “5 S” PR (sedimentation coefficient, 5.3 S), and was further designated as “5 S” PR-forming factor [(“5 S” PR)-FF]. Component B formed a hexamer (Stokes radius, 46 A; molecular weight, 78, 000) under hypotonic (low salt) conditions, which bound with native “4 S” PR to give “6 S” PR (sedimentation coefficient, 6.3 S). The hexamer of component B was designated as “6 S” PR-forming factor [(“6 S” PR)-FF]. (“8 S” PR)-FF was estimated to be a 1:1 complex of (“5 S” PR)-FF and (“6 S” PR)-FF. “4 S” PR was dissociated from “8 S” PR, “6 S” PR, and “5 S” PR in hypertonic (0.4M KCl) buffer. (“5 S” PR)-FF, (“6 S” PR)-FF, and (“8 S” PR)-FF were designated as progesterone receptor binding factors (PRBFs).View full abstractDownload PDF (757K) -
Mutumi MURAMATU, Seiichiro MORI, Yutaka MATSUZAWA, Yoshiko HORIGUCHI, ...1980 Volume 88 Issue 5 Pages 1317-1329
Published: November 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA urinary trypsin inhibitor, UTI68, was purified from the urine of healthy men by the following procedures: treatment of the urine with Celite; treatment on a QAE-Sephadex A-25 column at 33.8 m_??_•cm-1; chromatography of UTI on a QAE-Sephadex A-25 column; gel filtration on a Sephadex G-100 column.
The molecular weight of the purified UTI was estimated as 68, 000, and so the UTI was named UTI68. The purified UTI68 was completely free from uropepsinogen (Upg). Since the recovery of UTI68 amounted to 40 to 50 per cent of the total UTI in urine, UTI68 was concluded to be the main UTI in human urine. Another UTI with a molecular weight of 25, 000 was named UTI25. However, too little of UTI25 was obtained to permit its further examination. The purified UTI68 appeared homogeneous on polyacrylamide gel electrophoresis. UTI68 strongly inhibited the esterolytic and caseinolytic activities of trypsin, and one molecule of UTI68 inhibited four molecules of bovine trypsin. UTI68 also markedly inhibited the esterolytic activity of bovine chymotrypsin, but its inhibitory effect on the caseinolytic activity of chymotrypsin was very low (less than 40 per cent).
The results of amino acid analysis of UTI68 showed high values of aspartic acid, glutamic acid, and glycine. The content of half-cystine was also high. UTI68 contained 10 per cent of neutral sugar.
Since purified UTI68 was completely free from Upg, it remained intact on incubation for 30min at pH 2.0 and 37°C. However, readdition of Upg, which was separated from UTI68 during the purification procedures described above, and incubation of the mixture at pH 2.0 and 37°C, resulted in the modification of UTI68 to another UTI. The conversion of UTI68 to another UTI by uropepsin was rapid, and further incubation resulted in a gradual decrease of UTI68 with production of two UTI's, but finally both UTI activities disappeared.
After incubation of UTI68 and Upg for 15min at pH 2.0, the two reaction products and the remaining UTI68 were separated by QAE-Sephadex A-25 column chromatography, and identified by polyacrylamide gel electrophoresis. The results suggested that UTI68 was cleaved by uropepsin to form two types of UTI, both with a molecular weight of 30, 000.View full abstractDownload PDF (972K) -
Kazuko OHGI, Masachika IRIE1980 Volume 88 Issue 5 Pages 1331-1339
Published: November 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1) The inactivation of a RNase from Aspergillus saitoi (RNase Ms) was studied to obtain information on its active site.
2) Inactivation of RNase Ms by iodoacetamide was greater at an alkaline pH, and was protected more by 2', (3')-AMP than by 2', (3')-GMP.
3) Analysis of the hydrolysis products with 6 N HCl and alkaline treatment of carboxamido-methylated RNase Ms showed that the sites of reaction were one carboxyl group and one histidine residue.
4) Since the incorporation of a carboxamidomethyl group into carboxylic acid was not protected by 2', (3')-AMP, it was concluded that the formation of N1-carboxamidomethyl-histidine was responsible for the loss of enzymatic activity of RNase Ms.View full abstractDownload PDF (636K) -
XI. Site of Synthesis and Segregation by Stripped ER MembranesTakashi TOBE, Tokuhiko HIGASHI1980 Volume 88 Issue 5 Pages 1341-1347
Published: November 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe reinvestigated the site of synthesis of rat liver catalase, and it has been reconfirmed that catalase is synthesized not only by free polysomes but also by membrane-bound polysomes. Considerable amounts of nascent catalase on rough microsomes were released from the membrane into the medium upon incubation with puromycin, not transported directly into the intracisternal cavity of microsomes. On the other hand, catalase newly synthesized in vitro was shown to be segregated by stripped rat liver microsomal membranes in a state resistant to proteolysis. Since this segregation occurred without coupled protein synthesis, catalase appears to be transported by a mechanism different from co-translational transfer. A hypothesis is presented regarding the mechanism of intracellular transport of liver catalase.View full abstractDownload PDF (594K) -
Atsushi IKAI1980 Volume 88 Issue 5 Pages 1349-1357
Published: November 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe interaction of human serum low density lipoprotein with a non-ionic detergent, Tween 80, was investigated by sedimentation velocity measurements and Sepharose CL-4 B gel chromatography. The properties of Tween 80-extracted apoprotein (apo B) were studied by sedimentation velocity and sedimentation equilibrium measurements, gel chromatography, electron microscopy and chemical cross-linking.
Tween 80, at low concentrations, increased the sedimentation coefficient of LDL in 3% NaCl to a plateau value. At the same time, the effective Stokes radius of LDL expressed as the partition coefficient in gel chromatography became larger. The effect was reversed in part after protease treatment. A further increase of Tween 80 concentration caused the all-or-none dissociation of the entire mass of apo B from LDL. The remaining lipid core of LDL was not destroyed by detergent at 25°C, but it was at 35°C.
The Tween 80-extracted apo B was purified by gel chromatography and its molecular weight and partial specific volume were determined by sedimentation equilibrium analysis in H2O and D2O buffers. It formed a complex of 1, 200, 000 molecular weight with Tween 80, with ƒ/ƒ0=2.2. The molecular weight of the protein part of the complex was estimated to be about 500, 000, corresponding to the total apo B in an LDL molecule. This result was confirmed by chemical cross-linking of apo B in Tween 80. Under an electron microscope the apo B-Tween 80 complex appeared as a flexible string, a little less than 1, 000 A in length and 50 to 60 A in width.View full abstractDownload PDF (2123K) -
Atsushi IKAI, Yutaka YANAGITA1980 Volume 88 Issue 5 Pages 1359-1364
Published: November 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSChemical cross-linking with dimethylsuberimidate was applied to the study of apo B interactions in human serum low density lipoprotein (LDL). Since apo B is known to have a mass of 500, 000 to 600, 000 daltons in non-ionic detergent solutions (i.e., a dimer of apo B based on the molecular weight of 260, 000 for a single peptide of apo B), the possibility was investigated that the cross-linking reagent might be used to identify the interacting site of apo B in LDL. Although extensive cross-linking was observed in our experiment most of the reaction took place not between apo B's in one LDL (intramolecular) but between apo B's of different LDL's (intermolecular). Only a small fraction of cross-linked apo B dimer was formed intramolecularly. We interpret the results as indicating that the exposed or hydrophilic regions of LDL are scattered on the surface of LDL and that the protein-protein interaction site is not accessible to dimethylsuberimidate, probably being hidden under the surface layer of LDL.View full abstractDownload PDF (429K) -
I. Simultaneous Binding of Three Sodium and Two Potassium or Rubidium Ions to the EnzymeMotonori YAMAGUCHI, Yuji TONOMURA1980 Volume 88 Issue 5 Pages 1365-1375
Published: November 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe previously measured the amounts of Na+ and K+ ions bound to the Na+, K+-dependent ATPase [EC 3. 6. 1. 3] purified from porcine kidney by a modified membrane filtration method [(1979) J. Biochem. 86, 509-523]. In this study, we improved the method for measuring the amount of the active site and measured the amount of Rb+ ions (a K+ congener) bound to the ATPase as well as those of Na+ and K+ ions to get more accurate information on the K+-and Na+-binding sites. The following results were obtained.
Two kinds of cation-binding sites were found to exist on the ATPase molecule. One was the Na+-binding sites (3 mol per mol of active site). Na+ ions were bound to the sites cooperatively (Hill coefficient, 2.5-3), and the apparent dissociation constant was 0.20-0.32mM. Three moles of Na+ ions bound to the sites was displaced by 1 mol of K+ ions bound to the ATPase (ØK, 24 μM).
The other was the K+-binding sites (2 mol per mol of active site). Two moles of K+, Rb+, or Na+ ions was bound to the sites cooperatively (Hill coefficient, 1.5-2), and their apparent dissociation constants were 0.044, 0.024, and 2.2mM, respectively.
We measured the amounts of Na+ and Rb+ ions bound to the ATPase in the presence of 0.8mM NaCl and 0.13mM RbCl, and obtained unequivocal evidence for the simultaneous binding of 3 mol of Na+ ions and 2 mol of Rb+ ions per mol of active site of the ATPase.View full abstractDownload PDF (735K) -
II. Acceleration of Transition from a K+-Bound Form to a Na+-Bound Form by Binding of ATP to a Regulatory Site of the EnzymeMotonori YAMAGUCHI, Yuji TONOMURA1980 Volume 88 Issue 5 Pages 1377-1385
Published: November 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo kinds of ATP binding sites were found to exist on the ATPase molecule. One was the catalytic site (1 mol/mol phosphorylation site) and its apparent dissociation constant for ATP was about 1 μM. The other was the regulatory site (s) and its apparent dissociation constant for ATP was equal to or higher than about 0.2mM. The affinities of both sites for AMPPNP were three times lower than those for ATP.
The affinity of the ATPase for ATP was reduced by the addition of KCl, but unaffected by the addition of NaCl. As thermodynamically expected, the affinity of the Na+-binding sites for Na+ ions was almost completely unaffected by the addition of ATP, which markedly decreased that of the K+-binding sites for K+ and Rb+ ions.
In the absence of KCl, Na+ ions were bound very rapidly to the Na+-binding sites [(1979) J. Biochem. 86, 509-523]. However, Na+ ions were bound very slowly to the enzyme preincubated with 50 μM KCl, and the Na+ binding was markedly accelerated by the addition of ATP or AMPPNP at concentrations much higher than several μM. On the other hand, in the presence of 50 μM KCl, 1 mol of ATP was bound to the catalytic site with the same dissociation constant as that in the absence of KCl, and another 1 mol of ATP bound with a dissociation constant of about 0.1mM. Therefore, we concluded that the Na+ binding to the enzymein a K+ form is markedly accelerated by the binding of ATP to the regulatory site.View full abstractDownload PDF (603K) -
III. Marked Changes in Affinities for Monovalent Cations Induced by Formation of an ADP-Insensitive but not an ADP-Sensitive PhosphoenzymeMotonori YAMAGUCHI, Yuji TONOMURA1980 Volume 88 Issue 5 Pages 1387-1397
Published: November 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe measured the amounts of Rb+ ions (a K+ congener) as well as Na+ and K+ ions bound to the ATPase during the ATPase reaction at pH 7.5 and 0°C. The affinity of the Na+-binding sites for three Na+ ions decreased markedly but that of the K+-binding sites for two K+ or Rb+ ions increased markedly upon formation of an ADP-insensitive phosphorylated intermediate. Furthermore, the present experiment did not give any indication of a change in the Hill coefficient of 2, and showed an increase in the affinity of the K+-binding sites for Rb+ ions of about 28 times upon the formation of an ADP-insensitive EP. The enzyme state with a high affinity for Rb+ was maintained after the disappearance of EP. When the ATPase was treated with N-ethylmaleimide (NEM), almost all the EP formed was ADP-sensitive. The formation of an ADP-sensitive EP with the NEM-treated enzyme induced no change in the affinities of the ATPase for Na+ and Rb+ ions.View full abstractDownload PDF (687K) -
Hitoshi ARITA, Jun'ichi KAWANAMI1980 Volume 88 Issue 5 Pages 1399-1406
Published: November 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSome analogs of phenyl 6-halogeno-6-deoxy-β-D-glucopyranosides have been found to be inhibitors of glucose uptake by Rhesus monkey kidney cells (LLCMK2 cells). The structures of the glycone and aglycone parts were both found to contribute to the inhibitory activity. Introduction of alkyl groups into the phenyl residue caused appreciable enhancement of the inhibition. p-(sec-Butyl)phenyl-6-chloro-6-deoxy-β-D-glucopyranoside, which is the most potent inhibitor in this series, showed competitive and reversible inhibition. As a result of this study, we prepared p-azidopheny1-6-chloro-6-deoxy-β-D-glucopyranoside as a possible compound for photoaffinity labeling of the glucose uptake site in animal cells.View full abstractDownload PDF (553K) -
Makoto TSUBOKAWA, Hiroshi KAWAUCHI, Choh Hao LI1980 Volume 88 Issue 5 Pages 1407-1412
Published: November 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA highly purified somatotropin (growth hormone) has been isolated from fin whale (Balaenoptera physalus) pituitary glands in a yield of 1, 014mg/kg of wet weight tissue. The preparation was shown to be homogenous by isoelectric focusing on polyacrylamide with growthpromoting potency comparable to that of ovine growth hormone by the rat tibia test. The molecular weight was found to be 20, 000 daltons by SDS gel electrophoresis and 40, 000 daltons by exclusion chromatography on Sephadex G-100 at pH 8.2. The amino acid composition and α-helix content of fin whale somatotropin were similar to those of human, bovine, and ovine hormones. The circular dichroism in the side chain region, however, differed from those of the mammalian hormones. The NH2, -terminal amino acid sequence was nearly identical with those of the bovine and ovine hormones as determined by the fluorescein isothiocyanate method.View full abstractDownload PDF (805K) -
Kenji SUGIYAMA, Toshihiro EMORI, Sumi NAGASE1980 Volume 88 Issue 5 Pages 1413-1417
Published: November 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSNagase analbuminemic rats (NAR) are deficient in serum albumin. Extravascular regions of the tissues of NAR were tested for rat serum albumin by the immunofluorescence staining technique. Albumin was not detectable in any of the tissues of NAR tested (liver, kidney, muscle, skin, and small intestine), whereas it was clearly demonstrated in all these tissues of normal rats. Furthermore, extracts of NAR tissues, including the liver, contained no albumin detectable by the single radial immunodiffusion method. These findings indicate that albumin is not present in either the serum or extravascular regions of tissues of NAR, and suggest that the deficiency of serum albumin in NAR is not due to impaired albumin secretion from the liver or alteration of the tissue distribution of albumin in certain tissues, but to the absence of its synthesis in the liver.View full abstractDownload PDF (1570K) -
Shuzo YAMAGATA1980 Volume 88 Issue 5 Pages 1419-1423
Published: November 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSStudies with crude preparations obtained from a cysteine auxotroph of Saccharomyces cerevisiae showed that O-acetylserine sulfhydrylase could be separated from O-acetylhomoserine sulfhydrylase by chromatography on a DEAE-cellulose column and centrifugation in a sucrose density gradient. On the basis of sedimentation distance, the molecular weights of these enzymes were calculated to be about 99, 000 and 182, 000, respectively. The former did not react with the amino acid substrate of the latter, and vice versa. The wild-type strain was also demonstrated to possess O-acetylserine sulfhydrylase (molecular weight: about 96, 000), in addition to a large amount of O-acetylserine-O-acetylhomoserine sulfhydrylase (Yamagata et al. (1974) J. Biochem. 75, 1221).View full abstractDownload PDF (373K) -
Nobuyuki YAMAMOTO, Michiki KASAI1980 Volume 88 Issue 5 Pages 1425-1435
Published: November 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe Donnan potential in sarcoplasmic reticulum vesicles prepared from rabbit skeletal muscle was measured by using a fluorescent cyanine dye, diS-C3-(5) (3, 3'-dipropylthiadicarbocyanine iodide). The Donnan potential is caused by the existence of negative fixed charges inside the vesicles. On average, sarcoplasmic reticulum vesicles have internal negative fixed charges of 420 nmol/mg protein at pH 6.8. The fixed charges are mainly due to low affinity Ca2+ binding proteins. The Donnan potential in sarcoplasmic reticulum vesicles was about -72 mV in 5mM K-MES buffer (pH 6.8). When Ca2+ was accumulated into sarcoplasmic reticulum vesicles with ATP, the concentration of negative fixed charges decreased from 140mM to 95mM due to Ca2+ binding to the negative fixed charges, and sarcoplasmic reticulum vesicles were depolarized by about 10 mV. It was found that there are two types of sarcoplasmic reticulum vesicles, one type (60%) having many negative fixed charges inside the vesicles and the other (40%) having few fixed charges.View full abstractDownload PDF (841K) -
Toshiaki HIRATSUKA, Koki UCHIDA1980 Volume 88 Issue 5 Pages 1437-1448
Published: November 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo mol of N-methyl-2-anilino-6-naphthalenesulfonyl (Mns) groups was preferentially incorporated into pig cardiac myosin in the absence of divalent metal ions, 1 mol rapidly and 1 mol slowly. In the presence of divalent metal ions, I mol was rapidly incorporated but subsequent incorporation was strongly suppressed. No substantial effect of incorporation of Mns groups in the presence or absence of divalent metal ions on the Ca2+- and K+-ATPase activities of myosin was found. However, the fluorescence spectra due to attached Mns groups were different in the two cases.
Extensive pronase digestion of labeled myosin indicated that the Mns groups were attached predominantly to lysyl residues, regardless of the labeling conditions. Peptide mapping of the labeled myosin digested with subtilisin, pepsin or trypsin uniformly showed the selective incorporation of an Mns group into essentially one species of peptide. However, the peptide labeled in the absence of divalent metal ions was clearly different from that labeled in their presence.
The present results confirm that pig cardiac myosin heavy chains contain two distinct lysyl residues, which are both accessible to labeling with Mns groups only when divalent metal ions are absent. The results also suggest that conformational changes occur around these residues when divalent metal ions are added.View full abstractDownload PDF (2259K) -
A Study on the Molecular Evolution of 5 S Ribosomal RNA from a Living FossilHiroyuki KOMIYA, Nobuyoshi SHIMIZU, Makoto KAWAKAMI, Shosuke TAKEMURA1980 Volume 88 Issue 5 Pages 1449-1456
Published: November 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBy chromatography on columns of DEAE-Sephadex A-50 and Sephadex G-100, and electrophoresis on polyacrylamide gel, 5 S rRNA was purified from a low-molecular-weight RNA fraction extracted from the total tissues of Lingula anatina. Complete digests of the 5 S rRNA with RNase T1 [EC 3. 1. 4. 8] and pancreatic RNase [EC 3. 1. 4. 22] were sequenced by conventional column chromatography procedures. The nucleotide sequence of this RNA was determined mainly by a chemical method for sequencing the RNA 3' end-labeled with 32P (1), with the complement of the oligonucleotide catalog obtained by the complete RNase digestions of the RNA. By comparing the sequences of several invertebrate, vertebrate, and Chlorella 5 S rRNAs, a phylogenic tree of the rRNAs was constructed and the time of divergence of Lingula was estimated.View full abstractDownload PDF (1911K) -
Akira MURAYAMA, Fumio FUKAI, Tadashi YAMAMOTO1980 Volume 88 Issue 5 Pages 1457-1466
Published: November 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAnalysis of estrogen receptor-binding factors (ERBFs) under isotonic (0.15M KCl) conditions showed the presence of a factor designated as “7 S” estrogen receptor-forming factor [(“7 S” ER)-FF], which binds with native “4 S” estrogen receptor (ER) to form “7 S” ER, besides the previously described component B and its hexamer [“6 S” ER-forming factor, (“6 S” ER)-FF]. Under hypertonic (0.4 tit KCl) conditions at lower temperature (4°C), (“7 S” ER)-FF and the monomer of component B were present. Under hypertonic conditions at higher temperature (25°C), ERBF existed as component A [“5 S” ER-forming factor, (“5 S” ER)-FF] and component B. (“7 S” ER)-FF was estimated to be a 1:2 complex of component A and component B. Under isotonic conditions, approximately 30% of the cytoplasmic ER was “4 S” form, and the rest was “6 S” and “7 S” forms. “5 S” ER, “6 S” ER, “7 S” ER, and “8 S” ER were reconstituted from partially purified native “4 S” ER and the corresponding ERBFs. “5S” ER was estimated to be a 1:1 complex of native “4 S” ER and (“5 S” ER)-FF. “6 S” ER was estimated to be a 1:1 complex of native “4 S” ER and (“6 S” ER)-FF. “7 S” ER was estimated to be a 1:1 complex of native “4 S” ER and (“7 S” ER)-FF. “8 S” ER was estimated to be a 1:1 complex of native “4 S” ER and the “8 S” ER-forming factor [(“8 S” ER)-FF] previously described, in which component A is bound to “4 S” ER not directly, but via component B. A schematic molecular model of the in vitro dissociation and assembly of the constituents of the cytoplasmic ER system of cow uterus is presented.View full abstractDownload PDF (592K) -
III. Difference Spectra of Their Cellular and Subcellular FractionsKatsuko KAKINUMA, Teruhide YAMAGUCHI, Kohkichi SHIMADA, Nobuhiro SATO1980 Volume 88 Issue 5 Pages 1467-1474
Published: November 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe respiratory pigments of rabbit polymorphonuclear leukocytes (PMN) and alveolar macrophages (A-MØ) were compared in a spectrophotometric study conducted at liquid nitrogen temperature. In the difference spectra of the KCN-treated minus the untreated states and of the reduced minus the oxidized states, the cellular and granular fractions of PMN showed sharp absorption maxima identical with the absorption bands of myeloperoxidase (MPO). Even though A-MØ revealed some guaiacol-peroxidation activity, the difference spectra of cellular and subcellular A-MØ fractions did not show an MPO spectrum. The enzyme catalyzing the guaiacol peroxidation by A-MØ appears to be an enzyme other than the MPO found in PMN. Despite the absence of the MPO spectrum, the difference spectra of A-MØ granules when measured with KCN, dithionite, and succinate showed characteristic absorption bands of mitochondrial cytochromes, in contrast to the difference spectra of PMN, which showed far smaller cytochrome absorption bands. The results obtained suggest that the phagocytic metabolism of rabbit A-MO depends on mitochondrial respiration for its energy supply.View full abstractDownload PDF (599K) -
Hiro'omi TAMURA, Kazuhisa SEKIMIZU, Masuo OBINATA, Shunji NATORI1980 Volume 88 Issue 5 Pages 1475-1480
Published: November 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe amount of S-II, a protein specifically stimulating RNA polymerase II, was measured in various mouse organs by a micro complement fixation assay. The amount was almost the same in brain, liver, kidney, spleen, and Ehrlich ascites tumor cells on the basis of DNA, being about 3.8 μg/mg DNA, which corresponds to 3.6×105 molecules/cell. However, the amount of S-II decreased greatly during erythro-differentiation of Friend leukemia cells and no S-II was detected in matured erythrocytes.View full abstractDownload PDF (434K) -
Akihiko KAWAGUCHI, Shigetoshi TSUBOTANI, Yousuke SEYAMA, Tamio YAMAKAW ...1980 Volume 88 Issue 5 Pages 1481-1486
Published: November 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe stereochemical course of dehydrogenation catalyzed by acyl-CoA oxidase was investigated using the enzymes from rat liver peroxisomes and Candida lipolytica. Stearoyl-CoA and nonanoyl-CoA, stereospecifically labeled with deuterium at either C-2 or C-3, were incubated with the enzyme, the products were converted to methyl esters and their deuterium contents were measured by gas chromatography-mass spectrometry. The results suggested that acyl-CoA oxidase-catalyzed dehydrogenation occurred by anti-elimination of the pro-2R and pro-3R hydrogens of acyl-CoA.View full abstractDownload PDF (391K) -
Osamu NUMATA, Tomoyoshi YASUDA, Tamio HIRABAYASHI, Yoshio WATANABE1980 Volume 88 Issue 5 Pages 1487-1498
Published: November 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIsolation and characterization of a fibrous protein component which might be associated with contractile ring of a dividing Tetrahymena cell were attempted by making use of coprecipitation of the protein with rabbit skeletal muscle myosin. The protein was purified to homogeneity by ammonium sulfate fractionation between 40-70% saturation and column chromatography of Sephadex G-200, starting from KCI-extract of Tetrahymena acetone powder. Its molecular weight was calculated to be 38, 000, based on the electrophoretic mobility in sodium dodecyl sulfate (SDS)-polyacrylamide gel, whereas molecular weight of its native state was determined to be 140, 000 by gel filtration on Sephadex G-200, and its sedimentation coeffi-cient was about 9 S as estimated by sucrose density gradient centrifugation. The latter was a particle of 7.7 nm in diameter under an electron microscope and supposed to be a tetramer of the 38, 000-dalton protein. The protein is considered to be a new, unique protein, since it is definitely different from the ubiquitous non-muscle actin in molecular weight, polymerizability in KCl solution and amino acid composition, and it is also different from tropomyosin and tubulin in immunological characteristics and amino acid composition.View full abstractDownload PDF (4101K) -
Osamu NUMATA, Tomoyoshi YASUDA, Tamio HIRABAYASHI, Yoshio WATANABE1980 Volume 88 Issue 5 Pages 1499-1504
Published: November 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAs a step to study the biological function of a new protein, fiber-forming protein-38, 000 (FFP-38), which was previously isolated from Tetrahymena pyriformis, its intracellular localization was investigated by using antiserum specific for FFP-38. Double immunodiffusion tests revealed that the anti-FFP-38 serum reacted with the protein(s) in isolated organelles such as oral apparatus, pellicles and mitochondria, to give rise to a precipitin line confluent with the line formed between the antiserum and the FFP-38.
Furthermore, indirect fluorescent antibody staining showed that fluorescence was intensely localized in the oral apparatus and faintly distributed throughout the cytoplasm. In particular, distinct fluorescence was found along the division furrow of dividing Tetrahymena.View full abstractDownload PDF (2403K) -
Osamu NUMATA, Tomoyoshi YASUDA, Kazuo OHNISHI, Yoshio WATANABE1980 Volume 88 Issue 5 Pages 1505-1514
Published: November 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSUsing a 38, 000-dalton protein (FFP-38) purified from Tetrahymena acetone powder, we have succeeded in the polymerization of this protein into 14-nm filaments. The polymerization was initiated by incubating the purified FFP-38 fraction in a buffer containing 5mM Mes (2-(N-Morpholino)ethanesulfonic acid), 50mM KCl, 1.2mM CaCl2, 0.6mM ATP, pH 6.6, and by shifting the incubation temperature from 0°C to 37°C.
The 14-nm filament is considered to consist of 7-nm globular subunits regularly arranged into 2 start, helical strands with 4 subunits per turn. The subunit may correspond to 9 S tetramer of FFP-38, a native form of FFP-38. Since the subunit arrangement and subunit protein component of this 14-nm filament obviously differ from those of actin filament, 10-nm intermediate filament and microtubule, the 14-nm filament appears to be a newly found intracellular filament.
Concerning the FFP-38 polymerization, some polymorphism appeared: we found ring structures having the diameters of 0.3-3.7 μm and latticed sheet structure, besides typical straight filaments.View full abstractDownload PDF (4352K) -
Sadayuki MATSUDA, Koichi YAGI1980 Volume 88 Issue 5 Pages 1515-1520
Published: November 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe apparent binding constant (KEapp) of EGTA to Ca2+ was determined using a color indicator, murexide, or a Ca2+-selective electrode in the pH range between 6.5 and 7.5, at 5°C and at 20°C. KEapp values obtained at two different temperatures (5°C and 20°C) were essentially the same when measured at the same pH. However, KEapp depended markedly on pH. In the presence of 0.1M KCl and 20mM MOPS-KOH buffer (pH 6.8), KEapp was 6.3×105M-1 The absolute binding constant (K) was calculated as 1010.48 Addition of 10mM MgCl2 did not change the Kpp. The binding of Ca2+ to heavy meromyosin was studied in 0.1M KCl and 20mM MOPS-KOH buffer (pH 7.2), at 5°C. The binding constant and maximum binding number (mol/mol) were obtained as 1.8×106M-1 and 1.4, respectively.View full abstractDownload PDF (353K) -
Mitsuhide NOSHIRO, Nobuhiro HARADA, Tsuneo OMURA1980 Volume 88 Issue 5 Pages 1521-1535
Published: November 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRabbit antibodies were prepared against NADPH-cytochrome P-450 reductase (fpT), NADH-cytochrome b5 reductase (fpD), cytochrome P-450, and cytochrome b5 of rat liver microsomes. The four antibodies were purified by affinity chromatography with antigen-conjugated Sepharose. Monovalent fragments of the antibodies against fpT and cytochrome b5 were also prepared. These antibody preparations were used as specific inhibitors in elucidating the electron pathway from NADPH or NADH to cytochrome P-450 for drug oxidation reactions catalyzed by liver microsomes from normal, phenobarbital (PB)-treated, and 3-methylcho-lanthrene (MC)-treated mice. Drug oxidation activities of the liver microsomes from PB-treated rats were also examined and compared with those of mouse liver microsomes. The drugs used were 7-ethoxycoumarin, benzo(a)pyrene, benzphetamine, and aniline.
The antibody against fpT (anti-fpT IG) strongly inhibited both NADPH-supported and NADH-supported oxidation reactions of all substrates. On the other hand, the antibody against fpD (anti-fpD IG) did not inhibit NADPH-supported reactions but significantly inhibited the NADH-supported oxidation of drugs, except for aniline. The NADH-supported oxidation of 7-ethoxycoumarin, in which the concentration of NADH was lower, was less sensitive to anti-fpT IG and highly sensitive to anti-fpD IG. The dependence of the inhibitory effects of these two antibodies on the concentration of NADH was confirmed; the results suggested that the relative contributions of fpT and fpD to the electron flow from NADH to cytochrome P-450 vary with the concentration of NADH. Judging from the inhibitory effects of antibody, cytochrome b5 participates in both NADH-supported and NADPH-supported drug oxidation reactions, but the extent of its involvement in the reactions varies significantly according to the type of drug and the induced states of the animals. Inhibition studies using fpT-depleted microsomes suggested that the fpD-cytochrome b5 couple can support NADH-dependent drug oxidation at a slow but detectable rate. Since purified fpT and cytochrome P-450 could reconstitute not only NADPH-supported but also NADH-supported oxidation of benzphetamine, the fpD-cytochrome b5 couple and fpT can separately support NADH-dependent drug oxidation reactions, although these two electron pathways seem to cooperate in intact microsomes.View full abstractDownload PDF (1106K) -
Norio HAYASHI, Motoko TERASAWA, Kohei YAMAUCHI, Goro KIKUCHI1980 Volume 88 Issue 5 Pages 1537-1543
Published: November 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe administration of heroin to rats which had been treated with 3, 5-dicarbethoxy-1, 4-dihydrocollidine (DDC) brought about a rapid decrease of the δ-aminolevulinate (ALA) synthase activity in the mitochondria and an appreciable increase of the enzyme activity in the cytosol in the liver. In DDC-treated rats, however, the degree of accumulation of ALA synthase in the liver cytosol resulting from the heroin administration was relatively small and the total ALA synthase activity in the liver was considerably decreased after the heroin administration; these results are in contrast with those for allylisopropylacetamide-treated rats. Further studies by combined use of [3H]leucine and an anti-ALA synthase IgG revealed that incor-poration of [3H]leucine into the mitochondrial ALA synthase was greatly reduced in hemininjected rats and that incorporation of [3H]leucine into total ALA synthase in the liver (the sum of radioactivity incorporated into the mitochondrial enzyme and the cytosolic enzyme) was also decreased to a considerable extent in these rats; this decrease in incorporation of [3H]leucine appeared to occur immediately after the administration of hemin. It was concluded that heroin actually inhibits the translocation of ALA synthase from the cytosol into the mitochondria and that hemin also inhibits de novo synthesis of ALA synthase, possibly at a post-transcriptional level in DDC-treated rats.View full abstractDownload PDF (568K) -
Kunio YONEMASU, Takako SASAKI, Hiroshi SHINKAI1980 Volume 88 Issue 5 Pages 1545-1554
Published: November 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBovine complement subcomponent Clq was purified, in a highly hemolytically active form, by a combination of precipitation with EGTA, ion-exchange chromatography, and gel filtration. Yield ranged from 22 to 28% as protein amounts, and the activity of final preparations was in the range of 2×1013-4×1013 effective molecules/mg. The molecular weight of undissociated Clq was 407, 000, as determined by polyacrylamide gel electrophoresis containing sodium dodecyl sulfate (SDS). Clq was shown to be composed of two non-covalently linked subunits of approximately 46, 000 and 45, 000 molecular weights in a molar ratio of 2:1. On reduction, the higher molecular weight subunit gave two chains having approximate molecular weights of 23, 600 and 22, 200 in equimolar ratio, and the lower molecular weight subunit gave one chain with a molecular weight of approximately 22, 000. Clq contained hydroxyproline, hydroxylysine, a high percentage of glycine and approximately 9% carbohydrate and 14.8% nitrogen. The absorption coefficient (A1%1cm) in 300mM NaCl was found to be 7.3±0.12 at 280nm. From these results, overall molecular structure of bovine Clq looks similar to that of human complement subcomponent Clq.View full abstractDownload PDF (1765K) -
Shoji ODANI, Teruo ONO1980 Volume 88 Issue 5 Pages 1555-1558
Published: November 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA method of exchanging amino acid residue (P1 residue) which determines primarily the inhibitory specificity of a proteinase inhibitor is described. Leu43 (P1 residue) at the chymotrypsin reactive site of Bowman-Birk soybean inhibitor was removed by limited proteolysis of Leu43-Ser44 bond followed by carboxypeptidase digestion. An appropriate amino acid methyl ester was introduced into this position by water-soluble carbodiimide. Derivatives having methyl esters of Gly, Ala, Val, Met, Leu, Phe, Trp, or D-Trp at position 43 were prepared and were shown to restore the activity to various extents. This method provides new approach to study structure-function relationship of proteinase inhibitors and to prepare novel inhibitors.View full abstractDownload PDF (245K) -
Yukio KAWAMURA, Teruyoshi MATOBA, Etsushiro DOI1980 Volume 88 Issue 5 Pages 1559-1561
Published: November 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe two forms of pig kidney cathepsin A (A, L and A, S) were found to be composed of three kinds of polypeptides as common structural subunits with molecular weights of 20, 000, 25, 000, and 55, 000. [32P]-DFP was incorporated into the subunit with a molecular weight of 25, 000. This subunit seems to be a catalytic one, on which active serine is present. A subunit com-position ratio of 1:1:1 in native cathepsin A, S was estimated by its molecular weight and those of the subunits. Native cathepsin A, L also seems to have a subunit composition ratio of 1:1:1. These results suggest a monomer-polymer relationship between both enzymes.View full abstractDownload PDF (702K) -
Takeyori SAHEKI, Atsuko UEDA, Masakazu HOSOYA, Tsunehiko KATSUNUMA, No ...1980 Volume 88 Issue 5 Pages 1563-1566
Published: November 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe urea cycle in the liver of conventional mice was compared with that of germ-free mice. Among the urea cycle enzymes and urea cycle-related substances, only ornithine in the liver of the conventional mice was markedly more abundant than in that of the germ-free mice. The significance of this finding was supported by the comparison of the urea synthesis in the liver slices from these two types of mice.View full abstractDownload PDF (239K) -
1980 Volume 88 Issue 5 Pages 1567a
Published: 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDownload PDF (26K) -
1980 Volume 88 Issue 5 Pages 1567b
Published: 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDownload PDF (26K)
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