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Volume 109 (1991)
- Issue 6 Pages 799-
- Issue 5 Pages 667-
- Issue 4 Pages 499-
- Issue 3 Pages 371-
- Issue 2 Pages 199-
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Volume 109, Issue 4
Displaying 1-27 of 27 articles from this issue
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Takayoshi Iio, Mina Itakura, Sho Takahashi, Seiji Sawada1991 Volume 109 Issue 4 Pages 499-502
Published: April 01, 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA molecular rotor, 9-(dicyanovinyl)julolidine (DCVJ), is a fluorescent dye whose intra-molecular rotation determines its fluorescence yield [Kung, C. E. & Reed, J. K. (1989) Biochemistry 28, 6678-6686]. DCVJ binds to bovine brain calmodulin and emits strong fluorescence. In fluorescence titration experiments, the dissociation constant and the number of binding sites were determined to be 20±10 μM and 0.7 ± 0.5 in the presence of Ca2+, and 22±10 μM and 0.6±0.5 in the absence of Ca2+, respectively. The fluorescence intensity of bound DCVJ increased 10-fold in the presence of Ca2+ compared to that in the absence of Ca2+. The fluorescence titration curve of DCVJ-calmodulin showed a transition at pCa 6.5. Over the same Ca2+ range, a decrease in molecular ellipticity at 222 nm and an increase in tyrosine fluorescence of calmodulin were observed. These results mean that the conformational change of calmodulin due to the Ca2+ binding induces the microenviron-mental change of the DCVJ binding site from the flexible to the rigid state, resulting in inhibition of the intramolecular rotation of DCVJ and an increase in its fluorescence.View full abstractDownload PDF (373K) -
Minoru Suzuki, Tamio Yamakawa, Akemi Suzuki1991 Volume 109 Issue 4 Pages 503-506
Published: April 01, 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA micro method involving high-performance liquid chromatography-fast atom bombard-ment mass spectrometry (IIPLC/FAB/ MS) has been developed for the sensitive structural characterization of neutral glycosphingolipids and monosialogangliosides. The method involves a micro silica gel column (0.3 mm i.d. × 100 mm) and a micro HPLC apparatus working at a flow rate of 6 μ1/min. All injected materials can be structurally characterized by mass spectrometry without the splitting or wasting of materials, which was not possible with our previous method [M. Suzuki et al. (1990) J. Biochem. 108, 92-98]. A mixture containing 160 ng each of five neutral glycosphingolipids (GlcC, er, LacCer, Gb3Cer, Gb4Cer, and IV3 αGalNAc-Gb4Cer) and a mixture containing 160 ng each of three monosialoganglio-sides [GM3(NeuAc), GM2(NeuAc), and GM1(NeuAc)] were injected into the micro HPLC with programmed elution with isopropanol-n-hexane-water with or without ammonium hydroxide. Each glycosphingolipid was separated by mass chromatography and the obtained mass spectra were suitable for structural characterization. Thus, the characterization of glycosphingolipids was achieved with small amounts of materials, 160 ng each, and in mixtures.View full abstractDownload PDF (1613K) -
Kiichi Imamura, Ziyuan Wang, Kikuno Murayama-Oda, Hee-Kyoung Kim, Take ...1991 Volume 109 Issue 4 Pages 507-513
Published: April 01, 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn this work deviation of liver metabolism by cytokines, especially recombinant human interleukin 1-α (rhIL1-α), was investigated. Administration of rhIL1- α or recombinant human tumor necrosis factor (rhTNF/cachectin) to normal mice resulted in rapid, dose-dependent induction of high liver ornithine decarboxylase (ODC) activity. The effects of these cytokines on liver ODC were not indirect effects mediated by eicosanoids. The induction of liver ODC by rhIL1- α was at least partly a direct effect on hepatocytes, and was due to increase in de novo synthesis of the enzyme protein after increase in ODC mRNA. No specific protein was required for increase in the level of ODC-mRNA. On ILl treatment, actinomycin D caused superinduction of liver ODC, which was at least partly due to increased stability of the ODC enzyme, because actinomycin D doubled the apparent half-life (from 50 to 95 min). Daily administration of 2 × 103U of rhILl-α to mice for 3 days also caused decrease in the level of the differentiated type of pyruvate kinase isozyme (PK-L) and marked increase in that of the prototype isozyme (PK-M2) in the liver, but did not cause significant change in the isozyme patterns of the kidney, thymus, and spleen. RhIL1-α also induced hypertrophy of the spleen. These results indicate that rhIL1-α causes metabolic deviation of the liver similar to that in tumor-bearing hosts.View full abstractDownload PDF (1919K) -
Keiichi Takagaki, Toshiya Nakamura, Atsushi Kon, Shinri Tamura, Masahi ...1991 Volume 109 Issue 4 Pages 514-519
Published: April 01, 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHuman skin fibroblasts were incubated in the presence of a fluorogenic xyloside, 4-methylumbelliferyl β-D-xyloside. Three fluorogenic components were isolated and purified from the culture medium by gel permeation high-performance liquid chromatography. Their structures were then characterized by enzymatic digestion, fast-atom-bombardment mass spectrometry, gas-liquid chromatography, and electrophoresis on cellulose acetate membrane. The results showed that one of the components was a mixture of dermatan sulfate (70%) and chondroitin sulfate (30%), bearing the 4-methylumbelliferone at the reducing termini, and having an average molecular weight of 9, 200. The others had the structures galactosyl-galactosyl-xylosyl-4-methylumbelliferone and galactosyl-xylosy1- 4-methylumbelliferone, respectively, representing the linkage region between the glycos-aminoglycan chains and core protein, except that 4-methylumbelliferone replaced the amino acid. Moreover, it was demonstrated that these oligosaccharides were intermediates of glycosaminoglycan synthesis, not depolymerized products.View full abstractDownload PDF (1346K) -
Kingo Takiguchi1991 Volume 109 Issue 4 Pages 520-527
Published: April 01, 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSuzuki et al. [Biochemistry 28, 6513-6518 (1989)] have shown that, when F-actin is mixed with inert high polymer, a large number of actin filaments closely align in parallel with overlaps to form a long and thick bundle. The bundle may be designated non-polar, as the constituent filaments are random in polarity (Suzuki et al. 1989). I prepared non-polar bundles of F-actin using methylcellulose (MC) as the high polymer, exposed them to heavy meromyosin (11MM) in the presence of ATP under a light microscope, and followed their morphological changes in the continuous presence of MC. It was found that bundles several tens of micrometers long contracted to about one-third the initial length, while becoming thicker, in half a minute after exposure to HMM. Subsequently, each bundle was split longitudinally into several bundles in a stepwise manner, while the newly formed ones remained associated together at one of the two ends. The product, an aster-like assembly of actin bundles, was morphologically quiescent; that is, individual bundles never contracted upon second exposure to HMM and ATP, although they were still longer than the F-actin used. Bundles in this state consisted of filaments with parallel polarity as examined by electron microscopy. This implies that non-polar bundles were transformed into assemblies of polar bundles with ATP hydrolysis by HMM. Importantly, myosin subfragment-1 caused neither contraction nor transformation. These results are interpreted as follows. In the presence of ATP, the two-headed 11MM molecule was able to cross-bridge antiparallel actin filaments, as well as parallel ones. In the antiparallel case, HMM induced sliding forces between the two filaments, while, in the parallel case, HMM traveled along the pair directionally, producing resistance against external sliding forces acting upon the two filaments.View full abstractDownload PDF (4802K) -
Tatsuya Sakai, Shohei Yamashina, Shun-ichi Ohnishi1991 Volume 109 Issue 4 Pages 528-533
Published: April 01, 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe fluorescence of FL cells after endocytosis of rhodamine-labeled transferrin initially appeared as a dispersed punctate pattern over the whole cell and then accumulated in the cytocenter on further incubation. In nocodazole-treated cells, the punctate fluorescence appeared along the cell edges, and stayed there on further incubation but did not accumulate in the cytocenter. The localization of transferrin was examined at the electron microscopic level with horseradish peroxidase (HRP)-labeled transferrin. Nocodazole did not affect endosome formation but affected the distribution of the endosomes. Several types of endosomes (tubular, small spherical, and microvesicular endosomes) were observed in nocodazoletreated cells, as in control cells. The endosomes were in the Golgi area of the cytocenter and also in peripheral cytoplasm in control cells. In contrast, the endosomes were only in the periplasm, along the cell edges, in nocodazole-treated cells. The uptake and release of HRP-transferrin and the release of ferric ion into the cytoplasm in nocodazole-treated cells followed in the same time-course as those in control cells. The release of transferrin was the exponential with a half-time of 12 min. The activation energy of a rate-limiting step in the recycling was 5.5 kcal•mol-1 at around 37°C and increased to 29 kcal•mol-1 below 25°C. These results indicated that microtubule-dependent endosome transport was faster than the overall recycling process and was independent of the return event of transferrin to the plasma membrane.View full abstractDownload PDF (3650K) -
Ryoji Masui, Sadao Wakabayashi, Hiroshi Matsubara, Youssef Hatefi1991 Volume 109 Issue 4 Pages 534-543
Published: April 01, 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMitochondrial NADH: ubiquinone oxidoreductase (complex I) is the most complicated system in the respiratory chain. It consists of many subunits, some of which hold iron-sulfur clusters, but structural information is still limited. The amino acid sequences of two 13 kDa polypeptides, 13 kDa-A and 13 kDa-B polypeptides, of ironsulfur protein fraction (IP) of bovine heart mitochondrial complex I were determined by a combination of protease digestion, Edman degradation, and carboxypeptidase digestion. The 13 kDa-A polypeptide was composed of 96 amino acids with a molecular weight of 10, 536. The 13 kDa-B polypeptide consisted of 114 amino acids and had an acetylated amino terminus. The molecular weight of this protein was calculated to be 13, 130 including the acetyl group. These proteins had no obvious sequence similarity to other known proteins. The partial amino acid sequence of 30 kDa-B polypeptide of IP was also determined to reveal a characteristic arrangement of cysteine residues that could be involved in iron-sulfur cluster formation.View full abstractDownload PDF (823K) -
Yoshihiko Kitajima, Sachiko Matsuhashi, Hiromi Nishida, Yozo Takasaki, ...1991 Volume 109 Issue 4 Pages 544-550
Published: April 01, 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThree monoclonal antibodies (MAbs1A2, 3C5, and 4C2) for human aldolase A [EC 4.1.2.13] were established. MAbs1A2, 3C5, and 4C2 were shown to belong to subclasses IgM, IgG1, and IgG2a, respectively. None of the MAbs inhibits aldolase A activity. Their epitopes were mapped in detail on the molecule by examining the reactivities of the MAbs to chimeric proteins between aldolases A and B [Kitajima et al. (1990) J. Biol. Chem. 265, 17493-17498] in ELISA and to the CNBr-cleaved fragments of aldolase A in immuno-blotting. MAbs1A2 and 3C5 reacted with sites located within amino acid residues 306-363 at the C-terminal region of the enzyme. MAb4C2 recognized an epitope of the enzyme present within amino acid residues 34-108 at the N-terminal region. In a competitive binding assay, MAbs1A2 and 3C5 competed with each other for binding to the antigen and also interfered with the binding of MAb4C2, whereas MAb4C2 failed to inhibit the binding of MAbs1A2 and 3C5 to the antigen. MAb3C5 showed a species-specificity in the reaction with the antigen; it reacted with human and rabbit aldolase A with similar reactivity but not at all with the rat and mouse enzymes, which differ from the human and rabbit enzymes in two amino acid residues at positions 328 and 348. Reactivities of MAbs to aldolase A were further examined with engineered enzymes containing an amino acid substitution. The epitopes for MAbs3C5 and 4C2 were shown to be highly sensitive to amino acid substitution, substrate-binding, or thermal treatment of the aldolase A molecule, whereas the epitope for MAb1A2 remained unchanged under these circumstances, indicating that MAbs4C2 and 3C5 would be useful tools for detecting conformational changes on the aldolase A molecule.View full abstractDownload PDF (3499K) -
Koji Kobayashi, Mikiharu Yoshida, Yasuhiko Shinoda, Michio Yazawa, Koi ...1991 Volume 109 Issue 4 Pages 551-558
Published: April 01, 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA monoclonal antibody (IM7) toward scallop testis calmodulin and another one (PBE2) toward wheat germ calmodulin were produced. Ca2+ was required for IM7 to react with scallop calmodulin. IM7 reacted with the C-terminal region (Asp78-Lys148) of the calmodulin. As observed on competitive ELISA, IM7 reacted with chicken calmodulin, but not with Euglena gracilis or wheat calmodulin, troponin C, myosin light chains, or parvalbumin. It is assumed that the cluster of Thr143, Thr146, and Ser147 in the C-terminal region acts as the antigenic site. IM7 (and Fab of IM7) inhibited the activities of myosin light chain kinase and cAMP-phosphodiesterase. PBE2 reacted with wheat germ calmodulin irrespective of the presence or absence of Ca2+, the antigenic site being in the N-terminal region (Alal-Met37). It reacted with wheat and spinach calmodulins, but not with scallop, chicken, or Euglena calmodulin, troponin C, myosin light chains, or parvalbumin. PBE2 had no effect on the activities of myosin light chain kinase and cAMP-phosphodiesterase.View full abstractDownload PDF (1189K) -
Hiroshi Saito, Tsunehisa Araiso, Haruhisa Shirahama, Tomiyasu Koyama1991 Volume 109 Issue 4 Pages 559-565
Published: April 01, 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe motion of the head group of phospholipid molecules in the bilayer structure was investigated by a picosecond fluorescence anisotropy technique using a newly synthesized fluorescent phospholipid, dipalmitoyl-L-α-phosphati dyl-(3-p-methoxyphenyl) umbelliferone (DPPU). In this phospholipid, a coumarin derivative is attached covalently to the phosphate moiety. The motion of the acyl chain of the phospholipid was also investigated by the same method using 1-palmitoy1-2-(3-diphenylhexatrieny1)-propanoyl-L-α-phos-phatidylcholine (DPHpPC). From fluorescence anisotropy decay the wobbling diffusion rate (Dw) of DPPU and DPHpPC in DPPC vesicles at 45°C was calculated to be 2.7×109s-1 and 5.1×107 s-1 using the wobbling-in-cone-model. The range of the motion was calculated as the cone angle (θc), which is half of the angle of the cone in which the fluorophore can diffuse. The cone angle of the coumarin skeleton of DPPU in DPPC vesicles at 45°C was 64°, which was larger than that of the DPH skeleton of DPHpPC, 40°. These results indicate that the motion of the head group is much faster and wider than that of the acyl chain. When
cholesterol was added to the DPPC vesicles, the range of motion of the acyl chain decreased, but that of the head group increased. These facts show that cholesterol restricts the motion of the acyl chain but enhances that of the head group in the phospholipid bilayer.View full abstractDownload PDF (733K) -
Ikuo Matsui, Kazuhiko Ishikawa, Eriko Matsui, Sachio Miyairi, Sakuzo F ...1991 Volume 109 Issue 4 Pages 566-569
Published: April 01, 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe kinetic parameters (kcat/Km) and the cleaved-bond distributions for the hydrolysis of linear maltooligosaccharides Gn (3_??_n_??_9) by Saccharomycopsis α-amylase (Sfamy) secreted from Saccharomyces cerevisiae were determined at pH 5.25 and 25°C. The subsite affinities of Sfamy were also evaluated from these data. The subsite structure of Sfamy is characteristic of the active site of an endo-cleavage type enzyme, consisting of internal repulsive sites with the catalytic residues and external attractive sites. Moreover, the pKa values of the catalytic residues were calculated from the pH dependence plot of the kinetic parameter (kcat/Km). The amino acid residues which contribute to the subsite affinities and the catalytic activity of Sfamy are proposed and compared with those of Taka-amylase A.View full abstractDownload PDF (393K) -
Katsura Inoue, Seiki Kuramitsu, Akihiro Okamoto, Ken Hirotsu, Taiichi ...1991 Volume 109 Issue 4 Pages 570-576
Published: April 01, 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTyr225 in the active site of Escherichia coli aspartate aminotransferase (AspAT) was replaced by phenylalanine or arginine by site-directed mutagenesis. X-ray crystallo-graphic analysis of Y225F AspAT showed that the benzene ring of Phe225 was situated at the same position as the phenol ring of Tyr225 in wild-type AspAT. The mutations resulted in a great decrease in the rate of the transamination reaction, suggesting that Tyr225 is important for efficient catalysis. The kinetic analysis of half-transamination reactions of Y225F AspAT with four substrates (aspartate, glutamate, oxalacetate, and 2-oxoglutarate) and some analogues (2-methylaspartate, succinate, and glutarate) revealed a considerable increase in the affinities for all these compounds. In contrast, affinity for the amino acid substrates was decreased by mutation to arginine, but affinities for the keto acid substrates and the two dicarboxylates (succinate and glutarate) were increased. The electrostatic interaction between O(3') of the coenzyme [pyridoxal 5'-phosphate (PLP)] and the residue at position 225 affected the pKa value of the Schiff base, which is formed between the E-amino group of Lys258 and the aldehyde group of PLP; based on the spectrophotometric titration the pKa values were determined to be 6.8 for wild-type AspAT, 8.5 for Y225F AspAT, and 6.1 for Y225R AspAT in the absence of substrate. The absorption spectra of the three AspATs were almost identical in the acidic pH region, but the spectrum of Y225F AspAT differed from that of wild-type or Y225R AspAT in the alkaline pH region. These results suggest that the hydroxyl group of Tyr225 (wild-type AspAT) or the guanidinium group of Arg225 (Y225R AspAT) interacts with O(3') of PLP in the un-protonated Schiff base, and that these interactions are lost when the Schiff base is protonated.View full abstractDownload PDF (777K) -
Hiroyuki Kataoka, Norihisa Sakiyama, Masami Makita1991 Volume 109 Issue 4 Pages 577-580
Published: April 01, 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThis study was carried out to investigate the distribution and contents of O-phosphoserine (P-Ser), O-phosphothreonine (P-Thr), and O-phosphotyrosine (P-Tyr) as their free forms in animal tissues. After extraction of a tissue sample with trichloroacetic acid, these O-phosphoamino acids were converted into their N-isobutoxycarbonyl trimethyl ester derivatives and then quantitated by GLC, with flame photometric detection, on a DB-1701 capillary column. Using this method, nanogram levels of O-phosphoamino acids in tissue samples could be accurately and precisely determined without any interference by coexisting substances. Free P-Ser and P-Thr were widely found in animal tissues, pancreas, spleen, stomach, kidney, liver, and lung containing considerable amounts of these O-phosphoamino acids. On the other hand, free P-Tyr was not detected in any of the tissues investigated in this study.View full abstractDownload PDF (409K) -
Tadayuki Hatta, Teruko Wakabayashi, Eiji Itagaki1991 Volume 109 Issue 4 Pages 581-586
Published: April 01, 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe inducible 3-keto-5α-steroid-Δ4-dehydrogenase of Nocardia corallina was purified to homogeneity using affinity chromatography on 19-nortestosterone-17-acetoxyaminoethyl Sepharose 4B. SDS-polyacrylamide gel electrophoresis, gel filtration and spectral analysis of flavin suggest that the purified dehydrogenase is a monomeric protein of Mr 60, 000 containing one flavin. It has a typical absorption spectrum of flavoprotein with maxima at 457, 375, and 277 nm. The values shifted to 470 and 395 nm on binding of 19-nortestosterone. The enzyme catalyzed the dehydrogenation of 3-keto-5α -steroid at the 4- and 5-position, e.g. the conversion of 5 α -androst-1 -ene-3, 17-dione to 1, 4-androstadiene-3, 17-dione with the reduction of phenazine methosulfate. The substrate 3-ketosteroid has essentially the 5α-configuration. The enzyme did not reduce potassium ferricyanide but did reduce cytochrome c at a moderate rate, and exhibited only a weak steroid oxidase activity. Stereochemical study demonstrated that the enzyme abstracts the 4β, 5α-hydrogens of the substrate as a hydrogen ion through a protein-based reaction and as a hydride ion by transfer to FAD, respectively. The enzyme oxidizes a wide variety of 3-keto-5α-steroids but not 3β-hydroxysteroid. The dehydrogenase also catalyzed steroid transhydrogenation between 3-keto-5α -steroid and 3-keto-1, 4-diene-steroid. The properties of this enzyme are compared with those of 3-keto-steroid-Δ1-dehydrogenase.View full abstractDownload PDF (644K) -
Suguru Kawato, Ikuo Ashikawa, Tadashi Iwase, Ei-ichi Hara1991 Volume 109 Issue 4 Pages 587-593
Published: April 01, 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSEffect of drug-induction on the rotation of cytochrome P-450 and on lipid fluidity in rat
liver microsomes was examined. Rotational diffusion of cytochrome P-450 was examined by observing the decay of absorption anisotropy, r(t), after photolysis of the heme-CO complex by a vertically polarized laser flash. Analysis of r(t) was based on a “rotation-about-membrane normal” model. Microsomal lipid fluidity was measured by observing fluorescence anisotropy of DPH incorporated in the lipid bilayer. The absorption anisotropy decayed within 2 ms to a time-independent value. Rotational diffusion of cytochrome P-450 was dependent on the drug-induction with PB, MC, and PCB when compared with non-induced CON-microsomes. The observed values for the normalized time-independent anisotropy r(∞)/r(0) are r(∞)/ r(0) =0.41 (CON-microsomes), 0.54 (PB-microsomes), 0.52 (MC-microsomes), and 0.57 (PCB-microsomes). The average rotational relaxation time φ =580-690 μs was almost unchanged over all microsomes presently examined. A significantly high value of r(∞)/ r(0)=0.41-0.57 implies the co-existence of mobile and immobile populations of cytochrome P-450. Based on the assumption that the heme tilts about 55° from the membrane plane for all species of P-450s besides P-450PB, 59% (CON-microsomes), 46% (PB-microsomes), 48% (MC-microsomes), and 43% (PCB-microsomes), respectively, of the cytochrome P-450 in microsomes is calculated to be mobile. Upon drug-induction the microsomal membrane was fluidized to some extent as judged by the steady-state fluorescence anisotropy of 0.156 for CON-microsomes and 0.139-0.148 for drug-induced microsomes. The present observations imply that the drug-inductions with PB, MC, and PCB cause an immobilization of cytochrome P-450 molecules which might be due to increased protein-protein interactions of the drug-induced P-450 molecules, because no rigidification in the lipid due to the induction was observed.View full abstractDownload PDF (706K) -
Yoshihiro Ohta, Kazutoshi Yanagibashi, Takayuki Hara, Masahiro Kawamur ...1991 Volume 109 Issue 4 Pages 594-599
Published: April 01, 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe rotational diffusion of cytochrome P-450 in submitochondrial particles (SMP) of bovine adrenocortical mitochondria was measured by detecting the decay of absorption anisotropy, r(t), after photolysis of the heme•CO complex by a vertically polarized laser flash. Analysis of r(t) was based on a “rotation-about-membrane normal” model. The measurements were used to investigate the effect of KCI on intermolecular interactions involving cytochrome P-450 and to investigate the interactions of cytochrome P-450 with other redox partners. The rotational diffusion of cytochrome P-450 was significantly dependent on KC1 concentration. When the KC1 concentration was increased from 0 to 1, 000 mM, the mobile population of cytochrome P-450 was increased from 33 to 82%. After removing the KCI, the mobile population of cytochrome P-450 returned to the original 33%. These results suggest that nonspecific protein aggregates are dissociated by the presence of KC1, possibly due to the change in electrostatic interactions, resulting in mobilization of cytochrome P-450. SMP were observed to be nearly free from adrenodoxin and adrenodoxin reductase. The addition of adrenodoxin to SMP increased the mobile population of cyto-chrome P-450 from 35 to 54%. Further addition of adrenodoxin reductase to SMP containing adrenodoxin immobilized cytochrome P-450 by 6%. The addition of only adrenodoxin reductase to SMP, however, did not immobilize cytochrome P-450. The present results are consistent with our previous observations [Ohta, Y., Mitani, F., Ishimura, Y., Yanagibashi, K., Kawamura, M., & Kawato, S. (1990) J. Biochem. 107, 97-104] that cholesterol-bearing P-4505scc forms a transient ternary association with adrenodoxin and adrenodoxin reductase.View full abstractDownload PDF (735K) -
Tomohiro Mega, Sumihiro Hase1991 Volume 109 Issue 4 Pages 600-603
Published: April 01, 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe used high performance liquid chromatography to determine the concentrations of free ligands after equilibrium dialysis to examine lectin-sugar interactions. The binding of p-nitrophenyl 1-thio-α-mannoside, p-nitrophenyl N-acetyl-1-thio-β-glucosaminide, and the pyridylamino derivative of Man6G1cNAc2 to concanavalin A and Triticum vulgaris lectin was examined. The binding constant, Ka, and the concentration of total binding sites, [L]t were calculated from the trace amounts of sugars and lectins using the equation, [S]/ [LS] =1/Ka [L]t+ [ S]/[L]t, where [S] is the concentration of free ligand and [LS] the concentration of bound ligand.View full abstractDownload PDF (340K) -
Hiroko Hama, Takashi Kayahara, Masaaki Tsuda, Tomofusa Tsuchiya1991 Volume 109 Issue 4 Pages 604-608
Published: April 01, 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have reported that a major cause of growth inhibition of Escherichia coli by L-serine is its inhibition of homoserine dehydrogenase I (HDH I), which is involved in the biosyntheses of threonine and isoleucine [Hama, H., Sumita, Y., Kakutani, Y., Tsuda, M., & Tsuchiya, T. (1990) Biochem. Biophys. Res. Commun. 168, 1211-1216]. However, Patte et al. reported that L-serine does not inhibit HDH I [Patte, J.-C., Truffa-Bachi, P., & Cohen, G. N. (1966) Biochim. Biophys. Acta 128, 426-439]. In studies on the reason for these discrepant results, we found that the concentration of K+ and the pH in the assay mixture strongly influenced the inhibitory effect of L-serine. L-Serine strongly inhibited the HDH I activities in both the forward and reverse reactions between aspartate semialdehyde and homoserine at a physiological K+ concentration (100 to 200mM) and physiological pH (7.5) for E. coli cells. On the other hand, two well-known inhibitors of HDH I, L-threonine and L-cysteine, strongly inhibited the activity regardless of the K+ concentration and pH.View full abstractDownload PDF (583K) -
Tomoko Ohkusa, Jaw Jou Kang, Magotoshi Morii, Noriaki Ikemoto1991 Volume 109 Issue 4 Pages 609-615
Published: April 01, 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHeavy sarcoplasmic reticulum vesicles were labeled with the thiol-reacting fluorescent probe N-(7-dimethylamino-4-methyl-4-coumarinyl)maleimide (DACM), and the DACM-labeled foot protein moiety was purified. The fluorescence intensity of the DACM attached to the foot protein decreased by the addition of low (activating) concentrations of ryanodine, while it increased at higher (inhibitory) concentrations, suggesting that the lower fluorescence represents the active state of the foot protein, while the higher fluores-cence, its inactive state. Under conditions that induce Ca2+ release from SR (Ca2+ jump, addition of Ca2+ release inducing reagents such as caffeine and polylysine), the fluorescence intensity of the protein-attached DACM decreased rapidly (e. g. k_??_70 s-1 under optimum conditions). The initial rate of Ca2+ release from the DACM-labeled SR showed a close correlation with the amplitude of the fluorescence change of the foot protein-attached DACM under variety of conditions; e. g. in the presence of Ca2+, polylysine, ATP, and ruthenium red, etc. The fluorescence change of the foot protein was much faster than Ca2+ release from SR under a variety of conditions of Ca2+ release. We propose that the binding of release triggering reagents to the foot protein induces a rapid conformational change, which in turn regulates Ca2+ release.View full abstractDownload PDF (1156K) -
Young Jae Kim, Shoji Mizushima, Hajime Tokuda1991 Volume 109 Issue 4 Pages 616-621
Published: April 01, 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSGeneration of membrane potential (inside-positive) and ΔpH (inside-acidic) at two kinds of NADH:quinone oxidoreductase segments, the Na+-motive segment and another segment, of Vibrio alginolyticus was examined by monitoring the quenching of fluorescence of oxonol V and that of quinacrine, respectively, with inside-out membrane vesicles. Transient generation of membrane potential at the segment occurred when ubiquinone-1 was added in the presence of KCN and NADH. The membrane potential was resistant to a proton conductor, carbonylcyanide m-chlorophenylhydrazone, indicating that the membrane potential was generated specifically at the Na+-motive segment. On the other hand, neither membrane potential nor ΔpH was generated at another segment. The Na+-motive segment did not generate ΔpH, indicating that only Na+ is extruded at this segment. Furthermore, generation of membrane potential and ΔpH at the NADH:quinone oxidoreductase segment of V. anguillarum was examined by using the fluorescence quenching technique. This segment of the bacterium was also found to generate Δψ by the extrusion of Na+ but not H+. These results revealed that the fluorescence quenching technique is useful for the rapid identification and characterization of the respiratory segment involved in Na+ translocation.View full abstractDownload PDF (571K) -
Florian Schifferer, Wolfgang Cordroch, Heinz Beitinger, Dietmar Mö ...1991 Volume 109 Issue 4 Pages 622-626
Published: April 01, 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effect of a negatively charged mono-sialoglyco-sphingolipid (GM1-ganglioside) on the molecular organization and on physicochemical properties of lipid/peptide (valinomycin) systems was investigated in monolayers at the air/water interface. At a high molar fraction of GM1, the surface pressure/area isotherms of the two-component films of the system GM1/valinomycin and the isotherm of the pure ganglioside monolayer are identical concerning the space requirement of the molecules and thereby the packing of the monolayer. Using space-filling molecular models, a simple calculation gives the theoretical amount of 4.5 ganglioside molecules associated with one molecule of the depsipeptide valinomycin. The average surface potential indicates, that valinomycin, interacting with the polar head group of GM1, becomes partly embedded within the lipid interface. For GMl/eicosanol and valinomycin/eicosanol mixtures, the agreement between theory and experimental data strongly supports the model of ideal mixing without any molecular interactions between the different components. The results suggest the formation of a ganglioside/valinomycin complex with simultaneous alteration of the surface potential and molecular structure of the single components.View full abstractDownload PDF (1319K) -
Tomohito Kakegawa, Yan Guo, Yuko Chiba, Toshiko Miyazaki, Mutsumi Naka ...1991 Volume 109 Issue 4 Pages 627-631
Published: April 01, 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe functions of acetylpolyamines were examined with respect to stimulation of protein synthesis and cell growth. Unlike polyamines, acetylpolyamines could not lower the optimal Mg2+ concentration of protein synthesis, and the degree of stimulation of protein synthesis by acetylpolyamines was small. The addition of N1-acetylspermine did not stimulate cell growth of a polyamine- requiring mutant of Escherichia coli MA261, although acetylspermine was accumulated in the cells. Acetylspermine did not interfere with polyamine stimulation of protein synthesis and cell growth of E. coli MA261. The binding of acetylpolyamines to RNA was very weak, and the binding of polyamines to RNA was not disturbed significantly by the presence of acetylpolyamines. When the growth of E. coli MA261 was stimulated by addition of polyamines, significant amounts of acetylpoly-amines were also formed in the cells. These results suggest that acetylation of polyamines, together with polyamine excretion, may regulate the intracellular level of the parent polyamines when excess amounts of polyamines accumulate intracellularly.View full abstractDownload PDF (484K) -
Masahiro Maeda, Susumu Satoh, Shingo Suzuki, Mineo Niwa, Nobuyuki Itoh ...1991 Volume 109 Issue 4 Pages 632-637
Published: April 01, 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe cloned cDNA for batroxobin has been expressed in E. coli. Batroxobin could only be obtained as intracellular aggregates of fusion proteins, fused with a small peptide. To obtain the mature batroxobin, the recognition sequence for thrombin was inserted between the peptide and the mature batroxobin. This fusion protein accumulated in an insoluble form and could easily be purified. After site-specific cleavage of the fusion protein with thrombin, recombinant batroxobin was isolated by preparative electrophoresis. Batroxobin with enzymatic activity was obtained by the refolding of recombinant batroxobin.View full abstractDownload PDF (1846K) -
Yujiro Higashi, Takako Hiromasa, Ayako Tanae, Tetsuo Miki, Jun Nakura, ...1991 Volume 109 Issue 4 Pages 638-644
Published: April 01, 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRecent observations have suggested that the pathological mutations in human P-450(C21) deficiency are generated through gene conversion-like events between the functional gene [P-450(21)B] and the pseudogene [P-450(C21)A]. To address this point more extensively, we investigated the effects of the base changes in the A pseudogene on the P-450(21) activity by using the COS cell expression system. In addition to the defective mutations found previously in the pseudogene, four single base changes with amino acid substitutions of Pro(30), Ile(172), Val(282), or Arg(356) were further identified as causing complete [Arg(356)] or partial [Pro(30), Ile(172), and Val(282)] inactivation of P-450(C21). Blot hybridization analysis of patient DNAs using oligonucleotide probes specific for these mutations revealed that the splicing mutation in the 2nd intron was distributed most frequently in both simple-virilizing and salt-wasting forms. The mutation Ile(172) seemed to be frequent in patients with the less severe simple-virilizing form, whereas the mutation Arg(356), together with other most serious mutations reported previously, was preferen-tially associated with salt-wasting, the most severe form of the disease. In combination with the present results of the effects of various mutations on the P-50(C21) activity, a survey of the distribution of the various mutations in the patient genomes so far reported suggests that the heterogeneous clinical symptoms of this genetic disease are somehow related to the degree of attenuation of the activities of the mutated gene products.View full abstractDownload PDF (1100K) -
Hiroshi Kamitani, Nobuyoshi Esaki, Hidehiko Tanaka, Kenji Soda1991 Volume 109 Issue 4 Pages 645-649
Published: April 01, 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSS-Alkylcysteine α, β-lyase [EC 4. 4. 1. 6] of Pseudomonas putida catalyzes α, β-elimination of L-djenkolate [3, 3'-methylenedithiobis(2-aminopropionic acid)] to produce pyruvate, ammonia, and S-(mercaptomethyl)cysteine initially. Secondly, S-(mercaptomethyl)-cysteine, which was identified in the form of S-(mercaptomethyl)cysteine thiolactone and S-(2-thia-3-carboxypropyl)cysteine in the absence and presence of iodoacetic acid, respec-tively, is decomposed enzymatically to pyruvate, ammonia, and bis(mercapto)methane, or spontaneously to cysteine, formaldehyde, and hydrogen sulfide. Balance studies showed that 1.3 mol each of pyruvate and ammonia and 0.2 mol each of formaldehyde and cysteine were produced with consumption of 1 mol of L-djenkolate. 1, 2, 4, 5-Tetrathiane, 1, 2, 4- trithiolane, 1, 2, 4, 6-tetrathiepane, and 1, 2, 3, 5, 6-pentathiepane, which are derivatives of bis(mercapto)methane, were also produced during the α, β-elimination of L-djenkolate. In addition, a polymer with the general formula of -(CH2S)n- was produced as a white precipitate. When the α, β-elimination of L-djenkolate was carried out in the presence of 20 mM iodoacetic acid, neither formaldehyde, cysteine, hydrogen sulfide, or the polymer were formed. Instead, the S-carboxymethyl derivatives of bis(mercapto)methane and S-(mercaptomethyl)cysteine were produced in addition to pyruvate and ammonia.View full abstractDownload PDF (489K) -
Yukiko Konami, Kazuo Yamamoto, Toshiaki Osawa1991 Volume 109 Issue 4 Pages 650-658
Published: April 01, 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe complete amino acid sequences of the Ulex europeus anti-H(O) lectins I and II were determined by using a protein sequencer. After digestion with endoproteinases Lys-C and Asp-N of the lectins reduced with 2-mercaptoethanol and modified with iodoacetamide, the resulting peptides were purified by reversed-phase high-performance liquid chromatography and subjected to sequence analysis. The complete primary structures of these two Ulex lectins I and II were compared with those of nine lectins already determined, including that of Lotus tetragonolobus anti-H(O) lectin which we have determined. Extensive homologies were found among them.View full abstractDownload PDF (767K) -
Tsutomu Tsuji, Sen-itiroh Hakomori, Toshiaki Osawa1991 Volume 109 Issue 4 Pages 659-665
Published: April 01, 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSGalactoprotein b3 is one of the cell membrane glycoproteins of fibroblasts showing enhanced expression in association with oncogenic transformation. Analysis of cDNA for this glycoprotein from hamster fibroblasts indicated that the glycoprotein is a member of the integrin superfamily [Tsuji, T., Yamamoto, F., Miura, Y., Takio, K., Titani, K., Pawar, S., Osawa, T., & Hakomori, S. (1990) J. Biol. Chem. 265, 7016-7021]. In the present study, we examined the change in the amounts of mRNA for the mouse and human counterparts in fibroblasts after oncogenic transformation by Northern blot analysis using hamster galactoprotein b3 cDNA as a probe. In both human and murine fibroblasts transformed with SV-40, the homologous mRNA to galactoprotein b3 was also found to increase as compared with the progenitor cells. The human homologue of galactoprotein b3 cDNA was cloned from human bladder carcinoma cell line (T24) cDNA library. The cDNA codes for a single polypeptide of which the N-terminal sequence (21 amino acids) is identical with that of human VLA-3 α subunit. Based on this sequence identity and the structural similarities (i.e. the positions of most cysteine residues, the presence of a transmembrane domain near the C-terminus and the presence of metal binding sequences) with other integrins so far cloned, we conclude that human galactoprotein b3 is an integrin α3 subunit. The mature integrin α3 polypeptide was composed of 1, 019 amino acid residues, and the overall structure was quite similar to the hamster counterpart. The integrin α3 subunit is characterized by the presence of a putative cleavage site in its polypeptide chain and the absence of the so-called I-domain which is found in the integrin α2 subunit.View full abstractDownload PDF (1959K)
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