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Volume 100, Issue 1
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Sumihiro HASE, Satoshi KOYAMA, Hiromi DAIYASU, Hiroshi TAKEMOTO, Sabur ...1986 Volume 100 Issue 1 Pages 1-10
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn asparagine-linked sugar chain of a protease inhibitor from barbados pride (Caesalpinia pulcherrima Sw.) was liberated by hydrazinolysis. After N-acetylation, the reducing end residue of this carbohydrate unit was coupled with 2-aminopyridine and the pyridylamino (PA-) derivative was purified by gel-filtration and reversed-phase HPLC. The structure of the resulting PA-sugar chain was determined mainly by stepwise exoglycosidase digestions and 500 MHz 1H-NMR spectroscopy and proved to be as follows:
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Tetsu HOZUMI1986 Volume 100 Issue 1 Pages 11-19
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSLimited subtilisin digestion of myosin subfragment 1 (S-1) was carried out, varying the enzyme : substrate weight ratio from 1:200 to 1:10, and changes in structure, and in the MgATPase activities of S-1 and acto-S-i after proteolysis, were followed. When the starting material-tryptically-cleaved S-1 (27kDa-50kDa-20kDa) (“split S-1”)-was subjected to further subtilisin digestion, it was found that with increasing enzyme concentration, the 50 kDa fragment degraded into an 18kDa fragment via a 33kDa peptide (50→33→18kDa), which was not cross-linked with F-actin. On the other hand, the 27 and 20kDa fragments were rather stable at lower subtilisin concentrations and started to degrade only at higher subtilisin concentrations. These degradations lowered the MgATPase activities of S-1 and acto-S-1. The losses of MgATPase activities of S-1 and of acto-S-1 were mianly due to the degradations of the 27 and 20kDa fragments, respectively. Addition of EDTA did not affect the subtilisin cleavage pattern of split S-1 but the breakdown of the 50kDa fragment was extremely depressed, suggesting that some conformational change of the 50 kDa fragment is induced by the binding of divalent cation. The binding of MgADP to split S-1 accelerated the degradation of the 27kDa fragment and produced a new cut in the 27kDa fragment (27→20kDa), resulting in a further loss of the S-1 MgATPase activity. Although the cleavage pattern of split S-1 was not affected by F-actin, the effect induced by MgADP was abolished when split S-1 was a ternary complex with actin and MgADP. These results suggest that the intersite communication system is still effective, even in split S-1. Ca2+ or Mg2+ accelerates only the further degradation of the 50kDa fragment, suggesting that interaction with divalent cation induces a conformational change in this fragment.View full abstractDownload PDF (2574K) -
Koichi MIZUSAKI, Yuji SUGAHARA, Hideaki TSUNEMATSU, Satoru MAKISUMI1986 Volume 100 Issue 1 Pages 21-25
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn order to study the conformation of the side chain of lysine substrates bound to the active center of trypsin, two lysine analogs, cis- and trans-2, 6-diamino-4-hexenoic acids (4, 5-dehydrolysines) were synthesized and kinetic parameters for the hydrolysis of benzoyl methyl esters and phenylthiazolones of these analogs by this enzyme were compared with those of the corresponding lysine derivatives. The derivatives of cis-4, 5-dehydrolysine were hydrolyzed much more slowly than those of lysine, owing largely to the small kcat values for the former. On the other hand, the derivatives of the trans-isomer were hydrolyzed at about the same rates as those of lysine and the values of both Km and kcat of the former are also similar to those of the latter. These results indicate that the conformation of the side chain of the lysine derivatives hydrolyzed by trypsin is such that the β-and ε-carbons are in a trans-like conformation, as suggested by X-ray crystallographic studies of inhibitor-trypsin complex.View full abstractDownload PDF (380K) -
James O. OCHANDA, Bunei SYUTO, Iwao OHISHI, Masaharu NAIKI, Shuichiro ...1986 Volume 100 Issue 1 Pages 27-33
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe binding characteristics of Clostridium botulinum neurotoxins of types B, C1, and F to gangliosides was studied by thin layer chromatography plate and microtiter plate methods at low (10mM NaCI in 10mM Tris-HCI buffer, pH 7.2) or high (150mM NaCI in 10mM Tris-HCI buffer, pH 7.2) ionic strengths and at 0 or 37°C. The three types of toxins bound exclusively to three kinds of gangliosides, GD1a, GD1b, and GT1b, in both the thin layer chromatography plate and the microtiter plate methods. Type C1, toxin bound to the three gangliosides under all the conditions, while type B and F toxins bound only at low ionic strength and 37°C. At low ionic strength, the binding kinetics for the three toxins was monophasic in Scatchard plots, and the association constants obtained in the microtiter plate system were 2-4×108M-1. In contrast, the binding kinetics of type C1 toxin in high ionic strength was biphasic in the Scatchard plot, and two association constants were obtained in the microtiter plate system. The heavy chain facilitated the binding of the toxin to the gangliosides.
These results indicate that different types of botulinum toxins bind to the gangliosides under different optimal conditions and that gangliosides may not be the common receptor for all types of botulinum toxins. The gangliosides may bind to type C1 toxin together with other potential receptor (s) on synaptosomal membranes.View full abstractDownload PDF (1194K) -
Yoshiaki BANDO, Eiki KOMINAMI, Nobuhiko KATUNUMA1986 Volume 100 Issue 1 Pages 35-42
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCathepsin L was purified to apparent homogeneity from rat kidney. The molecular weight of the enzyme was estimated to be 30, 000, but part of the enzyme was found to consist of two polypeptide chains of Mr 25, 000 and 5, 000. Antibody against rat kidney cathepsin L did not cross-react with rat cathepsin B or H and detected only cathepsin L in crude rat tissue preparations on immunoblotted sheets. The concentrations of cathepsin L in various rat tissues and peripheral blood cells of rats were determined by a sensitive immunoassay, in which the minimum detectable amount of cathepsin L was 20pg/assay. The concentration of cathepsin L was found to be highest in the kidneys, where it was more than 3 times higher than in the liver, spleen, lungs, and brain. Nervous tissues, especially the cerebellar cortex, also contained fairly high concentrations of cathepsin L, but the heart, skeletal muscle, and gastrointestinal tract contained low concentrations, as did peripheral blood cells. The cathepsin L content of macrophages was 20% of that of cathepsin B. The concentrations of cathepsin L in lymphocytes, neutrophils, and erythrocytes were 10%, 20%, and less than 0.2%, respectively, of those in resident macrophages.View full abstractDownload PDF (1502K) -
Naoko SAKIHAMA, Masateru SHIN, Hiroko TODA1986 Volume 100 Issue 1 Pages 43-47
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHydrophobic chromatography on a TSK-gel Phenyl-5PW column separated highly purified spinach ferredoxin into two distinct molecular species in their native forms. The two ferredoxins showed almost the same absorption spectra in spite of a difference in amino acid composition. Both ferredoxins were active in the NADPH-cytochrome c reducing system, and no significant difference was observed between their activities. The new separation method was also applied to ferredoxins highly purified from wheat plants and barley. Interestingly, all ferredoxin preparations so far examined contained two molecular species of ferredoxin.View full abstractDownload PDF (324K) -
Shoji WAKASUGI, Shuichiro MAEDA, Kazunori SHIMADA1986 Volume 100 Issue 1 Pages 49-58
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe cloned a genomic DNA fragment which covers the entire sequence of the mouse prealbumin gene and then studied the structure. The coding regions are separated into four exons by three introns, and these numbers, the sizes of the exons and the relative sites of the exon-intron junctions are all in complete agreement with those determined for the human gene. The sequences of four exons can be aligned perfectly with that of the previously determined mouse prealbumin cDNA. In addition to the exon regions, we found two highly conserved DNA regions between the mouse and human prealbumin genes, one in the 5'-flanking region of the gene and the other in the 3' end region of the first intron. These DNA regions contain several consensus glucocorticoid receptor-binding site sequences, and the latter also contains an enhancer sequence present in the immunoglobulin kappa-chain joining-constant, intron. RNA hybridizing to the mouse prealbumin cDNA was detected in the extracts from liver, brain, and kidney, but was not detected in testes, spleen, or heart. Little change was caused in the level of prealbumin mRNA in the liver by administration of dexamethasone to mice.View full abstractDownload PDF (2205K) -
Kazushi TANABE, Chikako SATO, Takaaki KOBAYASHI, Taijo TAKAHASHI1986 Volume 100 Issue 1 Pages 59-65
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMicrotubules were purified from porcine brain by two cycles of temperature-dependent assembly and disassembly, then microtubule associated proteins, MAP-1, MAP-2, and tau, were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Two-dimensional tryptic peptide maps of radioiodinated polypeptides were compared with each other by means of mixed sample experiments, and the following results were obtained. 1) Subspecies of MAP-1 (355-345 and 325kDa) showed about 33% homology in the tryptic peptide maps. 2) Structural homology of MAP-1 and MAP-2 was very low; only 3 out of 40 peptide spots of MAP-2 were identical with those of MAP-1-C. 3) Subspecies of tau proteins (65 and 60kDa) were very closely related. 4) Structural similarity between MAP-2 and tau was very low. 5) MAP-1 from porcine brain and rat brain showed very high structural homology.View full abstractDownload PDF (2338K) -
Yukie SUMA, Keiko SHIMIZU, Kinji TSUKADA1986 Volume 100 Issue 1 Pages 67-75
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSS-Adenosylmethionine synthetase exists in at least two distinct forms, α- and β-forms, in adult liver. The β-form was purified to homogeneity from the soluble fraction of rat liver with a yield of about 10%. An antiserum directed against the purified β-form from rat liver was prepared by injecting the purified enzyme into a rabbit. Ouchterlony double diffusion analysis and immunochemical titrations revealed that the isozymes, α- and β-forms, are identical. Thus, the α-form was isolated from rat liver as a single protein using immunoaffinity chromatography against the β-form. The molecular weights of the β- and α-forms were determined to be 48, 000 each by sodium dodecyl sulfate disc gel electrophoresis, and about 100, 000, and 200, 000, respectively, by Sephacryl S-200 gel filtration. These results indicate that the β-form consisted of two subunits of 48, 000 daltons and the α-form of four subunits of 48, 000 daltons. The sedimentation coefficient was calculated to be 5.5S for the β-form and 8.OS for the α-form.View full abstractDownload PDF (1330K) -
Kirk L. PARKIN, Herbert O. HULTIN1986 Volume 100 Issue 1 Pages 77-86
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA crude microsomal fraction isolated from red hake (Urophycis chuss) muscle demethylated trimethylamine-N-oxide (TMAO). Two cofactor systems were capable of stimulating activity; the system of NADH and FMN required anaerobic conditions while the other system, composed of iron and cysteine and/or ascorbate functioned in the presence or absence of oxygen. The components of each cofactor system functioned synergistically and kinetic parameters were established for each. Of several amine compounds common to fish muscle, TMAO was the only substrate demethylated by the microsomes. Activity was inhibited by iodoacetamide, potassium cyanide, and sodium azide under certain conditions, but not by carbon monoxide. An enzymic nature of the reaction was demonstrated by the properties of heat lability, sensitivity to protease treatment, the requirement of microsomes for TMAO demethylation and by the exhibition of typical hyperbolic kinetics with respect to substrate (TMAO). Moreover, TMAO demethylation by the microsomes was 3 to 4 orders of magnitude faster than the non-enzymic reaction and the reaction was specific for dimethylamine (DMA) as product. It appears the two cofactor systems may share a common catalytic unit in the process of TMAO demethylation.View full abstractDownload PDF (856K) -
Kirk L. PARKIN, Herbert O. HULTIN1986 Volume 100 Issue 1 Pages 87-97
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDetergent treatments were examined for their efficacy in purifying trimethylamine-N-oxide (TMAO) demethylase activity from fish muscle microsomes. Tritons X-100 and X-45, deoxycholate, Brijs, Tweens 20, 65, and 80, and SDS were generally ineffective in solubilizing demethylase activity from this membrane fraction, at concentrations up to 10mg detergent per mg protein. In all of these cases, specific activity became enriched in the particulate fraction obtained post-treatment. Highest fold-purification was achieved by using 10mg SDS per mg protein in 5mM his-tidine, pH 7.0 at 10-14°C. Activity was relatively stable to the presence of SDS at this level, and with this treatment, TMAO demethylase activity became purified in the resultant particulate fraction 28- and 58-fold for activity stimulated by ascorbate-iron-cysteine and FMN-NADH, respectively. The presence of urea or 2-mercaptoethanol, or sonication of the SDS-microsome suspension during purification resulted in significant losses of recovered activity.
This partially purified fraction represented about 1% of the orginal microsomal protein and SDS-PAGE revealed the presence of several protein components. The partially purified demethylase could utilize the same two cofactor systems as the native microsomes. It displayed a curvilinear dependence on iron for activity and a sigmoidal response for cysteine. Utilization of NADH, FMN, and ascorbate differed for the purified fraction as compared to the microsomes. Substrate inhibition by TMAO was observed for the partially purified preparation, whereas saturation kinetics were previously noted for microsomal activity. Using the most conservative estimates, the partially purified preparation retained catalytic properties such that demethylation was 2 to 4 orders of magnitude faster than that for the non-enzymic reaction.View full abstractDownload PDF (1770K) -
Tetsuya OIDA1986 Volume 100 Issue 1 Pages 99-113
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe interactions of human serum albumin (HSA) with a number of ligands (mostly drugs) were examined by proton nuclear magnetic resonance spectroscopy. Ligand presence-absence difference spectra of HSA solutions were measured. Nonspecifically bound drugs such as tiaramide showed difference spectrum patterns which were similar to the spectra of the drugs themselves but were broadened as to the line-widths of signals. Thus, the difference spectra of these drugs reflect only the changes in the surroundings of the drug molecules, that is, between the bound and free states. In contrast, specifically bound drugs like ibuprofen and warfarin showed difference spectra in which signals from the HSA molecule only were observed. Furthermore, according to the characteristic peaks in these difference spectrum patterns, specifically bound drugs may be classified into several groups; the drugs in the first group bind to the ibuprofen binding site, those in the second group to the warfarin binding site, and those in the third group to sites other than the warfarin and ibuprofen sites. These findings suggest that the specific binding of drugs to HSA brings about a conformational change of this protein which is specifically correlated to the binding site.View full abstractDownload PDF (1040K) -
Ikuo MATSUI, Kunio OISHI1986 Volume 100 Issue 1 Pages 115-121
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSL-Fucose, D-mannose-specific lectin (SFL 100-2) particles produced by Streptomyces no. 100-2 were labeled with N-succinimidyl-[2, 3-3H]propionate to investigate quantitatively their binding properties to human erythrocytes. The labeling did not influence the physical properties or the hemagglutinating activity of the lectin particles. The binding studies suggested that two kinds of receptor sites were present on the erythrocytes. Association constants (Ka's) of the lectin particles to the receptor sites and the numbers of the receptor sites (n) on human 0 erythrocytes were calculated to be 4.60×108 M-1 and 3.17×104/cell for high-affinity receptor sites, and 7.5×107 M-1 and 1.33×105/cell for low-affinity ones.
The inhibition constants (K1's) for L-fucose, p-nitrophenyl (PNP)-β-L-fucoside, D-mannose, and PNP-α-D-mannoside were calculated to be 1.20×103, 1.82×103, 1.82×102, and 2.40×102 M-1, respectively. The numbers of carbohydrate-binding sites (m) on the lectin particles were estimated to be 2.82, 2.18, 2.19, and 2.21 for L-fucose, PNP-β-L-fucoside, D-mannose, and PNP-α-D-mannoside, respectively, suggesting that SFL 100-2 has two carbohydrate-binding sites per particle.View full abstractDownload PDF (506K) -
Miho TAKAHASHI, Tsuneko UCHIDA1986 Volume 100 Issue 1 Pages 123-131
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSNB8 DNA ligase from an extract of Thermus thermophilus HB8 could catalyze bluntend ligation in the presence of high concentration of polyethylene glycols (PEG) or in the presence of polyamines. In the presence of high molecular weight PEG 20, 000, 6, 000, or 1, 000 (8-28%), the enzyme catalyzed blunt-end intermolecular joining to yield linear oligomers, but no circular DNA forms. But in the presence of low molecular PEG 400, 200 (8-80%), or the monomer, ethylene glycol (16-80%), the circular forms were also detected by intramolecular ligation. In the presence of polyamines, the blunt-end ligation products were linear oligomers and the optimum concentrations were as follows: caldopentamine (0.05mM), thermine (0.1-0.2mM), spermine (0.2mM), thermospermine (0.4mM), and sperminediol (0.75mM). Spermidine and putrescine were less capable of producing oligomers. PEG and polyamines elevated the ligation temperature by HB8 DNA ligase. The optimum temperature of blunt-end ligation was about 65°C.View full abstractDownload PDF (3956K) -
Yasutada IMAMURA, Masao KAWAKITA1986 Volume 100 Issue 1 Pages 133-141
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSarcoplasmic reticulum membranes were treated with trypsin under conditions leading to accumulation of B and three other fragments a little smaller than A1, namely A1a, A1b, and C (Mr 27, 000-28, 000) (Saito, K. et al. (1984) J. Biochem. 95, 1297-1304), and enzymatic properties of trypsin-digested ATPase were investigated. The tryptic cleavage pattern of SR membranes in the presence of 1M glycerol and 5mM CaCl2 at 35°C was qualitatively similar to that obtained in the presence of Ca2+ alone. However, considerably more Al-derived fragments, A1a and Alb, which are stabilized by the binding of Ca2+ to the enzyme, were accumulated. The sample digested under this condition for 60min was mainly composed of A1b and B, and was designated as Alb+B complex. ATPase activity was lost in parallel with the formation of Ala and A1b. On the other hand, E-P forming activity was still retained by A1b+B complex. E-P formation with this complex was strictly dependent on the presence of Ca2+ ions at micromolar concentration. This indicates that Ca2+ binding site is well conserved in this complex. E-P formed with Alb+B complex was ADP-sensitive (E1-P), and was not further decomposed, since the transition from E1-P to E2-P was blocked.View full abstractDownload PDF (1999K) -
Yuji SUGITA, Takaharu NEGORO, Toshio MATSUDA, Terufumi SAKAMOTO, Motow ...1986 Volume 100 Issue 1 Pages 143-150
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDAF (decay-accelerating factor) is one of the integral membrane proteins of erythrocytes, and is considered to play an important role in the regulation of complement activation.
The purification of DAF has been impeded by the difficulty in removing glycophorin. We devised an effective method for removing glycophorin. Through the limited trypsinization of stromata prior to the extraction of DAF, glycophorin was readily digested so that the DAF could be purified free of glycophorin by DEAE-Sephacel and Bio-Gel A 0.5m chromatographies.
On SDS-PAGE, DAF from trypsinized stromata showed the same mobility as that from native stromata: its molecular weight was estimated to be about 70kDa. Amino acid analysis of DAF showed high contents of serine and glutamic acid. The amino-terminal sequence of DAF prepared by the present method, determined for the 29 residues, did not show significant homology with that of glycophorin.View full abstractDownload PDF (2440K) -
Issei MABUCHI, Yoshiaki NONOMURA1986 Volume 100 Issue 1 Pages 151-156
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAcrosomal actin bundles were isolated from the sperm of horseshoe crabs from four different sources, three from Asia and one from North America, and their protein constituents and structures were compared. The bundle from the American Limulus polyphemus sperm was composed of actin and two associated proteins of MW 95, 000 and MW 52, 000, as reported previously (Tilney, L.G. (1975) J. Cell Biol. 64, 289-310). However, those from the three Asian species (Tachypleus tridentatus, T. gigas, and Carcinoscorpius rotundicauda) were composed only of actin and the protein of MW 95, 000. Electron microscopic and optical diffraction studies indicated that both the helical structures and the interfilament spacing of the actin filaments composing the acrosomal bundle were indistinguishable among the four species. These results suggest that the MW 95, 000 protein crosslinks actin filaments in the bundle. Moreover, they support the idea that Limulus and the three Asian species have evolved independently from a common ancestor.View full abstractDownload PDF (1553K) -
Satoshi SAIGO1986 Volume 100 Issue 1 Pages 157-165
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo elucidate the relationship between local and gross conformational changes, three types of conformational changes, i.e., alkaline isomerization, ligand replacement, and guanidine hydrochloride (GuHCI) denaturation, of a set of homologous and modified cytochromes c were investigated by spectroscopic methods. Cytochromes c examined include the horse, tuna, Candida, monomeric Saccharomyces, and disulfide-linked dimeric Saccharornyces proteins. Correlations were found between the apparent pK (pKa) for the isomerization and the equilibrium constants for the binding of imidazole and azide to the heme iron: the lower the pKa value, the higher the binding constant. This is explained by the fact that the isomerization and ligand replacement involve similar conformational changes localized around the sixth coordination position of the heme. A good correlation was also observed between the susceptibilities to local and gross conformational changes: the lower the pKa value for the isomerization, the lower the GuHCl concentration for the midpoint of the denaturation. Thermodynamic analysis suggests that this correlation is not due to the involvement of similar conformational changes in the two processes. Cooperative stabilization of the tertiary structure is proposed to interpret this correlation.View full abstractDownload PDF (657K) -
Kimi WATANABE, Atsuhiko OOHIRA, Isao URAMOTO, Tsuyoshi TOTSUKA1986 Volume 100 Issue 1 Pages 167-173
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSGlycosaminoglycans were isolated from the skeletal muscle of either normal or dystrophic mice aged from 3 to 18 weeks. The glycosaminoglycan content of the normal muscle, based on the tissue weight, decreased slightly during the period from 3 to 10 weeks, and remained almost unchanged after 10 weeks. The major glycosaminoglycan in normal muscle was hyaluronate, the relative amount of which increased slightly (from 70% to 80%) with age. Both dermatan sulfate and heparan sulfate were also obtained. The relative amounts of these sulfated glycosamino-glycans tended to decrease with age. On the other hand, the glycosaminoglycan content of the dystrophic muscle was higher than that of normal muscle even at 3 weeks. The proportion of hyaluronate was almost constant (about 65%) throughout the age range examined. The relative amount of dermatan sulfate increased from 20% to 30% with a compensatory decrease in the amount of heparan sulfate. Further, the incorporation of [35S]sulfate into glycosaminoglycans by the dystrophic muscle was reduced to about 60% of the normal. These differences in glycosamino-glycan composition and [35S]sulfate incorporation between the normal and the dystrophic muscles may be related to the progressive muscular dysfunction seen in this disease.View full abstractDownload PDF (1175K) -
Satoru YAMAMOTO, Emi KUSUNOSE, Schuichiro MATSUBARA, Kosuke ICHIHARA, ...1986 Volume 100 Issue 1 Pages 175-181
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe microsomes of placenta and uterus from pregnant rabbits have been found to catalyze the ω-hydroxylation of PGE1, PGE2, PGF2α, and PGA1, as well as the ω-and (ω-1)-hydroxylation of palmitate and myristate in the presence of NADPH. These activities were greatly inhibited by carbon monoxide, indicating the involve-ment of cytochrome P-450. The apparent Km for PGE1 was 2.38μM and 2.1μM with the placental and uterus microsomes, respectively.
Cytochrome P-450 has been solubilized with 1% cholate from the placental microsomes, and partially purified by chromatography on 6-amino-n-hexyl Sepharose 4B, DEAE-Sephadex A-50 and hydroxylapatite columns. The partially purified cytochrome P-450 efficiently catalyzed the ω-hydroxylation of various prostaglandins such as PGE1, PGE2, PGF2α, PGD2, and PGA1 in a reconstituted system containing NADPH-cytochrome P-450 reductase, cytochrome b5, and phosphatidylcholine. The reconstituted system also hydroxylated palmitate and myristate at the ω- and (ω-1)-position, but could not hydroxylate laurate. These catalytic properties resemble those of a new form of cytochrome P-450 highly purified from the lung microsomes of progesterone-treated rabbits (Yamamoto, S., Kusunose, E., Ogita, K., Kaku, M., Ichihara, K., and Kusunose, M. (1984) J. Biochem. 96, 593-603). This type of cytochrome P-450, viz., cytochrome P-450 with high prostaglandin ω-hy-droxylase activity may play a role in the regulation of prostaglandin levels in pregnancy.View full abstractDownload PDF (585K) -
Yoko KASAI, Mitsushi INOMATA, Masami HAYASHI, Kazutomo IMAHORI, Seiich ...1986 Volume 100 Issue 1 Pages 183-190
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFifteen hybridomas secreting antibodies against calcium-activated neutral protease (CANP), especially those for rabbit muscle mCANP with low calcium sensitivity, have been produced by the cell fusion technique. Eight of the monoclonal antibodies belong to the class IgG1, one to the class IgG2a, and six to the class IgG2b. The antibodies from these clones were characterized with regard to their relative binding affinities to the large subunits (80K) and the small subunits (30K) of mCANP as well as μCANP, which is another type of CANP with high calcium sensitivity. Fourteen antibodies bound only to the 80K subunit of mCANP and one antibody bound to the 80K subunit of both mCANP and μCANP. These antibodies recognized rat mCANP but not chicken CANP, with the exception of one antibody. Examination of the effects of these antibodies on the enzyme activity of mCANP showed that six antibodies partially inhibited the enzyme activity and the others were noninhibitory. These monoclonal antibodies should be useful for analyzing the fine structure of CAN-Ps and the mechanism of the activation of mCANP, and also for determining the intracellular localization of mCANP.View full abstractDownload PDF (1998K) -
Youichi KAWANO, Naotaka HAMASAKI1986 Volume 100 Issue 1 Pages 191-199
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA hydrophobic 5, 300-dalton peptide was isolated from the 38, 000-dalton domain of Band 3 by sodium dodecyl sulfate polyacrylamide gel electrophoresis and reversedphase high-performance liquid chromatography. The peptide was affinity labeled with pyridoxal phosphate and sodium [3H]borohydride when erythrocytes were incubated in vitro. The peptide was not labeled with these agents when cells were incubated in the presence of a specific inhibitor of anion transport, suggesting that the peptide contains at least a part of the active center for the anion transport system in the cell membrane.
The peptide was eluted from a reversed-phase high-performance liquid chromatography column with a high concentration of acetonitrile (more than 65%), although the elution pattern of the hydrophobic peptide was not as sharp as that of the soluble peptides. However, a satisfactory separation was achieved when this procedure was employed in combination with sodium dodecyl sulfate polyacrylamide gel electrophoresis.View full abstractDownload PDF (3337K) -
Masayasu INOUE, Hideki KOYAMA, Sumi NAGASE, Yoshimasa MORINO1986 Volume 100 Issue 1 Pages 201-206
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo investigate a possible function of plasma albumin in partitioning organic anions into bile and urine, phenolsulfonphthalein (PSP) was administered intravenously and its in vivo fate was studied in normal and analbuminemic mutant rats (NAR). No significant change in the rate of PSP disappearance was observed in bilaterally nephrectomized normal rats. However, biliary excretion of the injected dye increased remarkably in nephrectomized normal rats. Intravenously injected PSP disappeared very rapidly from the circulation of NAR. Thus, the plasma clearance and distribution volume of PSP were significantly larger in NAR than in normal rats. Bilateral nephrectomy also failed to decrease the plasma clearance and distribution volume of the dye in NAR. In striking contrast to the experiments in normal rats, bilateral nephrectomy did not increase the biliary secretion of PSP in NAR. When PSP bound to equimolar albumin was injected into bilaterally nephrectomized NAR, the biliary excretion of PSP increased significantly with concomitant decrease in both plasma clearance and distribution volume of the dye. These results indicate that, in cases of renal transport dysfunction, albumin plays a critical role in hepatic compensatory excretion of PSP, a nephrophilic organic anion, whose molecular weight (MW 354) is close to the threshold value for partitioning a ligand to the eliminatory routes in liver and kidney of a rodent.View full abstractDownload PDF (520K) -
Ikuo KANAZAWA, Kozo HAMAGUCHI1986 Volume 100 Issue 1 Pages 207-212
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThermal unfolding of chicken pancreatic polypeptide at two different concentrations was studied at various pH values. The thermal stability was higher at higher protein concentrations. The transition temperatures at two different protein concentrations changed with pH in parallel and decreased by about 30°C on lowering pH from 5 to 2. The results on the thermal unfolding were analyzed by assuming that the dimerization constant is independent of pH, that the thermal unfolding occurs only after the pancreatic polypeptide dimers dissociated into the monomers, and that one ionizable group participates in the acid unfolding of the monomer. The free energy change for the unfolding of the pancreatic polypeptide monomer was estimated to be 1.4kcal/mol. The unfolding of pancreatic polypeptide by guanidine hydrochloride at pH 6.0 and 25°C was also studied. The stability to guanidine hydrochloride was higher at higher protein concentrations.View full abstractDownload PDF (408K) -
Toshiyuki MIYATA, Hiroaki MATSUMOTO, Masahira HATTORI, Yoshiyuki SAKAK ...1986 Volume 100 Issue 1 Pages 213-220
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe complete cDNA sequence coding for the coagulogen present in the horseshoe crab (Tachypleus tridentatus) hemocytes was determined. Clones carrying cDNA fragments for coagulogen were isolated from a cDNA library of the hemocyte mRNA using synthetic oligodeoxyribonucleotides as probes. The nucleotide sequence analyses of the cloned cDNAs revealed that the hemocyte coagulogen consists of 175 amino acids with 20 amino acids in a presegment, and that there are two types of mRNAs for coagulogen. The two mRNAs exhibited three nucleotide substitutions, two of which were in their protein-coding regions, resulting in two amino acid replacements. Subsequently, two molecular species of coagulogen, named coagulogens type I and type II, were identified by tryptic peptide mapping of the mature proteins isolated from the hemocyte lysate. These results suggest that the two types of coagulogens are first synthesized as preproteins and are incorporated into the granules that are abundantly present in the hemocytes with liberation of the signal peptides.View full abstractDownload PDF (596K) -
Naosada TAKIZAWA, Shigeo HORIE1986 Volume 100 Issue 1 Pages 221-232
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe oscillator strengths of hemoproteins in the light frequency range of 1.11×104 to 3.23×104cm-1 (wavelength range of 900 to 310 nm) were measured by means of computer-assisted spectrophotometry.
1) The obtained values of oscillator strength per molar heme ranged from about 1.4 to 2.2.
2) By comparing the oscillator strength values of the ferric and ferric cyanidebound forms of hemoproteins and also the values of low molecular weight ferric heme complexes, it was found that the oscillator strength was lower for those hemoproteins whose heme was coordinated with strong field ligands. It was also found that the hemoproteins showing a smaller pH-dependent change in the carbon monoxide-difference spectrum had lower oscillator strengths.
3) The following linear relation was observed, with various ligand complexes of bovine methemoglobin, horse metmyoglobin, and ferric horseradish peroxidase, between the oscillator strength (f) determined in the present study and the respective magnetic susceptibility (106•X20°M) values in the literature:
f=A(106•X20°M)+B.
The values of constants A and B in the equation were estimated for horseradish peroxidase, methemoglobin, and metmyoglobin.
4) On varying the temperature in the range of 0 to 40°C, the oscillator strength of the metmyoglobin-azide complex changed in parallel with the change in the spin state.
5) Taking advantage of the fact that fluoride complexes of many hemoproteins show 106•X20°M values close to 14, 500 and also that the values of intersection B are around 86.4% of the respective values of the fluoride complexes of ferric horseradish peroxidase, methemoglobin, and metmyoglobin, an empirical equation was evolved for the calculation of an approximate 106•X20°M value from the f value of a given complex (fobs) and that of the fluoride complex (f F) of a hemoprotein. The approximate magnetic susceptibilities of various ligand complexes of bovine lactoperoxidase could be thus calculated with the equation.
6) The oscillator strengths of ferrous hemoproteins were also investigated and ligand-dependent regular changes were found.View full abstractDownload PDF (1015K) -
Hidenori YAMADA, Naoko MATSUNAGA, Hideki DOMOTO, Taiji IMOTO1986 Volume 100 Issue 1 Pages 233-241
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDinitrophenylation of hen egg white lysozyme with 2, 4-dinitrofluorobenzene (DNFB) was carried out at pH 7-11 and room temperature in order to examine whether dinitrophenylation could be applied to determine the environments of individual amino groups in lysozyme or not. Lightly dinitrophenylated lysozyme was reduced, S-carboxymethylated and then subjected to reversed-phase high-performance liquid chromatography (RP-HPLC). All tryptic peptides, which contained dinitrophenylated amino groups (one α-amino group, Lys 1(α), and six ε-amino groups, Lys 1(ε), Lys 13, Lys 33, Lys 96, Lys 97, and Lys 116), could be separated and monitored by absorbance measurement at 360 nm on RP-HPLC. The relative reactivities of individual amino groups, determined from the relative peak areas of dinitrophenylated tryptic peptides at 360 run, were found to be sensitive to the reaction pH and to the presence of the trimer of N-acetyl-D-glucosamine or NaCl. It was concluded that dinitrophenylation of a protein with DNFB followed by peptide analysis by RP-HPLC with detection at 360 nm is a good method for probing the environments of individual amino groups in the protein.View full abstractDownload PDF (684K) -
Yasuo TSUNOGAE, Atsuo SUZUKI, Tatsuo SONE, Kazuhiro TAKAHASHI, Isao TA ...1986 Volume 100 Issue 1 Pages 243-246
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCrystallization and preliminary crystallographic study of Bowman-Birk type protease inhibitors, A-I, A-II, and B-I1I from peanut seeds (Arachis hypogaea), and of the A-II+trypsin complex were carried out. A-II, with 70 amino acid residues, crystallizes in a trigonal system, P3121 (or P3221), a=71.8, c=65.9Å, Z=12 or 18. The A-I crystal is isomorphous with that of A-II, indicating that the N-terminal residues are in a disordered state in both crystals. The B-III crystal is monoclinic, C2, a=119.6, b=69.6, c=94.2Å, β=115.1°, Z is about 40. The A-Il+trypsin complex crystallizes in an orthorhombic system, P212121, a=55.5, b=56.0, c=182.1Å, Z=4.View full abstractDownload PDF (1499K) -
Takeo KUMAMOTO, Akio ITO, Tsuneo OMURA1986 Volume 100 Issue 1 Pages 247-254
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAdrenodoxin (Ad) is synthesized as a larger precursor (preAd) by cytoplasmic polysomes and then transported into mitochondria concomitant with its proteolytic processing to the mature form. The protease in bovine adrenal cortex mitochondria, which converts preAd to the mature form, is a metalloprotease in the matrix (Sagara, Y., Ito, A. & Omura, T. (1984) J. Biochem. 96, 1743-1752).
In this study, the protease was purified about 100-fold from the matrix fraction of bovine adrenal cortex mitochondria. The partially purified protease converted not only preAd, but also the precursors of malate dehydrogenase (MDH) and 27 kDa protein (P-27) to the corresponding mature forms. However, it was inactive toward the precursors of P-450(SCC) and of P-450(11β). Since isolated rat liver mitochondria can import and process preAd as efficiently as bovine adrenal cortex mitochondria, we partially purified a preAd-processing protease from rat liver mitochondria and compared its properties with those of the bovine adrenal cortex enzyme. The properties of the rat liver protease were indistinguishable from those of the bovine adrenal cortex enzyme in molecular weight determined from Sephadex G-150 gel filtration, metal requirement and ability to process preMDH and preP-27. The rat liver enzyme was also inactive toward the precursors of P-450(SCC) and P-450(11β).
These results indicate the presence in both adrenal cortex and liver mitochondria of the same type of processing protease, which processes preAd and also the precursors of some other mitochondrial proteins.View full abstractDownload PDF (1269K) -
Masao HIRAIWA, Takayuki SHIRAISHI, Yutaka UDA1986 Volume 100 Issue 1 Pages 255-258
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe multiplicity of bovine liver acid β-galactosidase was investigated. Acid β-galactosidase activity was measured in the presence of glucono-δ-lactone, which inhibited the neutral β-galactosidase activity but not the acid β-galactosidase activity in bovine liver. Three forms of acid, β-galactosidase were separated by Sephadex G-200 gel filtration and the elution pattern of the 4-methylumbelliferyl-β-galactosidase activity coincided with that of the GMl-β-galactosidase activity. These forms were relatively stable under acidic conditions (pH 4.5), but the two high molecular weight forms were inclined to dissociate into the low molecular weight form under neutral conditions (pH 7.0). The three forms of the enzyme showed similar pH-optima and apparent Michaelis constants for GM1 ganglioside.View full abstractDownload PDF (300K)
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