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Volume 112 (1992)
- Issue 6 Pages 725-
- Issue 5 Pages 573-
- Issue 4 Pages 427-
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- Issue 2 Pages 173-
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Volume 112, Issue 2
Displaying 1-22 of 22 articles from this issue
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Masahiro Sakurai, Ko Onodera, Hideaki Moriyama, Osamu Matsumoto, Nobuo ...1992 Volume 112 Issue 2 Pages 173-174
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA new type of chimeric 3-isopropylmalate dehydrogenase (2T2M6T) was produced by expressing the fused gene of Bacillus subtilis and Thermus thermophilus. The enzyme shows heat stability intermediate between those of the parents. The crystal of the enzyme belongs to the space group of P3221, with cell dimensions of a=b=78.9 Å and c=158.9 Å. Two thermostable mutants of the chimeric enzyme were prepared by site-directed mutagenesis and then crystallized.View full abstractDownload PDF (169K) -
Masazumi Tada, Saori Takahashi, Motoshige Miyano, Yoshihiro Miyake1992 Volume 112 Issue 2 Pages 175-182
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRat gene for renin-binding protein (RnBP) was shown to be expressed in the kidney, adrenal gland, brain, lung, spleen, ovary, testis, and heart. On sodium depletion and captopril administration, the rat showed a marked increase in the adrenal RnBP mRNA level and a slight decrease in the kidney RnBP mRNA level. In two-kidney, one clip hypertensive rats, the RnBP mRNA levels of the clipped and contralateral kidneys were unchanged and also its adrenal mRNA level was maintained at the control level. The recombinant rat RnBP was synthesized in Escherichia coli cells and purified to apparent homogeneity. The RnBP existed as a homodimer and formed a heterodimer with rat renin to inhibit renin activity extensively. Intravenous injection of the RnBP into rats resulted in a rapid and strong inhibition of plasma renin activity, which persisted at least for 2h. These results suggest that the expression of RnBP gene in the kidney and adrenal gland is regulated independently, and the function of RnBP is related to electrolyte homeostasis, probably through the interaction with renin.View full abstractDownload PDF (3041K) -
Mina Itakura, Takayoshi Iio1992 Volume 112 Issue 2 Pages 183-191
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCa2+ binding to calmodulin triggers conformational change of the protein which induces exposure of hydrophobic surfaces. Melittin has been believed to bind to Ca2+-bound calmodulin through the exposed hydrophobic surfaces. However, tryptophan fluorescence measurements and gel chromatography experiments with the melittin-calmodulin system revealed that melittin bound to calmodulin at zero salt concentration even in the absence of Ca2+; addition of salt removed melittin from Ca2+-free calmodulin. This means not only the hydrophobic interaction but also the electrostatic interaction contributes to the melittincalmodulin binding. The fluorescence stopped-flow studies of the dissociation reaction of melittin-calmodulin complex revealed that Ca2+ removal from the complex induced a conformational change of calmodulin, resulting in reduction of the hydrophobic interaction between melittin and calmodulin, but the electrostatic interaction kept melittin still bound to calmodulin for a subsecond lag period, after which melittin dissociated from calmodulin. The fluorescence stopped-flow experiments on the dissociation reaction of complex of melittin and tryptic fragment(s) of calmodulin revealed that the lag period of the melittin dissociation reaction was attributable to the interaction between the C-terminal half of calmodulin and the C-terminal region of melittin.View full abstractDownload PDF (1056K) -
Hiroshi Yokota, Akira Yuasa, Ryo Sato1992 Volume 112 Issue 2 Pages 192-196
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe topological disposition of a form of UDP-glucuronyltransferase (called GT-1) in rat liver microsomes was examined. Concanavalin A-Sepharose failed to bind microsomal vesicles even though GT-1 has sugar chains of “high mannose” type, indicating that mannose-containing sugar chains of microsomal glycoproteins including GT-1 are not exposed to the outer surface of microsomal vesicles. Polyclonal antibodies raised against purified GT-1 could bind to microsomal vesicles, indicating that at least part of the GT-1 polypeptide chain is extruded to the outside of the microsomal membrane. Intact microsomal vesicles were digested with carboxypeptidase Y and then subjected to immunoblot analysis using the anti-GT-1 antibodies. It was thus found that the digestion resulted in cleavage of a C-terminal, 2-kDa fragment, leaving a 52-kDa fragment of GT-1 still tightly bound to the membrane. From these results, it is concluded that GT-1 is a transmembrane protein, which extrudes its C-terminal end (at least 2 kDa) to the outside of the membrane, whereas most of its polypeptide chain together with the sugar chains are located on the luminal side of the membrane.View full abstractDownload PDF (3067K) -
Michiki Kasai, Takashi Kawasaki, Kouki Yamamoto1992 Volume 112 Issue 2 Pages 197-203
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPermeation of neutral molecules as well as Ca2+ through the Ca2+ channel in sarcoplasmic reticulum vesicles has been studied by the tracer and/or by the light scattering methods. In the absence of KCl, the Ca2+ channel was found not to be able to pass neutral molecules such as glucose, xylose, and glycine under the condition that the channel was open, although the channel could pass Ca2+. On the other hand, submolar concentrations of KCl made the channel become permeable to neutral molecules as well as Ca2+. Since the effect of KCl could be replaced by NaCl and KNO3, but not by sucrose and glucose, this effect of KCl is considered to be due to ionic strength and not to osmotic pressure. These results suggest that low ionic strength transforms the Ca2+ channel protein in such a manner as to block the permeation of neutral molecules without modifying the gating mechanism of the channel.View full abstractDownload PDF (809K) -
Yoshitaka Ueda, Shuichi Kojima, Kouhei Tsumoto, Shigeki Takeda, Kin-ic ...1992 Volume 112 Issue 2 Pages 204-211
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA proteinaceous protease inhibitor was isolated from the culture broth of Streptomyces lividans 66 by a series of purification steps (salting out by ammonium sulfate, ion-exchange chromatography on DEAE-cellulose, hydrophobic chromatography on Phenyl-Sepharose, and gel-filtration on Sephacryl S-200), and was named S. lividans protease inhibitor (SLPI). The purified SLPI existed in a dimeric form consisting of two identical subunits, each of which was composed of 107 amino acids. SLPI exhibited strong inhibitory activity toward subtilisin BPN'. These features were similar to those of protein protease inhibitors produced by other Streptomyces (SSI family inhibitor). In addition, SLPI was capable of inhibiting trypsin with an inhibitor constant (K1) of about 10-9M. The primary structure of SLPI and location of two disulfide bridges were homologous to those of the other serine protease inhibitors of Streptomyces. The reactive site of SLPI was found to be Arg67-Glu68 from the sequence analysis of cleaved SLPI which was produced by acidification of subtilisin-SLPI complex. An Arg residue at the P1 site was consistent with the trypsininhibitory property of SLPI. Sequence comparison with other members of the SSI family revealed that amino acid replacements in SLPI were mainly localized on the surface of the SLPI molecule, and many of the amino acid residues in β-sheets and hydrophobic core were well conserved.View full abstractDownload PDF (1916K) -
Jun-ichi Nezu, Kiyoto Motojima, Hiro-omi Tamura, Shoji Ohkuma1992 Volume 112 Issue 2 Pages 212-219
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA cDNA (T3-L) encoding the 16 kDa subunit of vacuolar H+-ATPase was cloned from a cDNA library of rat liver. A polypeptide of 155 amino acids with a molecular mass of 15, 807 Da (pI=9.5) having four hydrophobic stretches was predicted. T3-L polypeptide was 92% and 100% identical with the 16 kDa proteolipid of bovine chromaffin granule and that of mouse, respectively. Antisera raised against the NH2-terminal of the T3-L polypeptide reacted positively with the membrane ghosts of rat liver tritosomes and the partially purified H+-ATPase thereof. Western blotting of subcellular fractions with the antisera showed high abundance of 16 kDa protein in the lysosomes, although a significant amount was also detected in the Golgi apparatus. Western blotting of rat tissues revealed high levels of 16 kDa proteolipid in the brain and the kidney. Northern blots with T3-L similarly showed considerably high expression of T3-L mRNA in the brain and the kidney. Southern hybridization of rat genomic DNA with T3-L showed at most three distinct bands, regardless of the stringency of hybridization and whether hybridization was performed with its subfragments. This suggests the possibility of multiple (at least three) homologous/identical genes encoding 16 kDa proteolipid. The possible presence and significance of isoforms of 16 kDa proteolipid in rats are discussed.View full abstractDownload PDF (3472K) -
Akihiko Kimura, Tomoji Uda, Shoichi Nakashima, Haruhiko Ikeda, Seiji Y ...1992 Volume 112 Issue 2 Pages 220-223
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMonoclonal antibodies (P3-9H, P3-1F, P3-2H, P3-4A, and P3-4C) to human erythrocyte band 3 were produced using human erythrocyte membranes as the immunogen. All epitopes defined by these antibodies were found on the amino-terminal cytoplasmic domain of erythrocyte band 3. The antibodies crossreacted variously with erythrocyte band 3 of primates (chimpanzee, orangutan, Rhesus monkey, Japanese monkey, spider monkey, and capuchin monkey) in enzyme-linked immunosorbent assay. P3-9H did not crossreact with erythrocyte band 3 of any primate examined; P3-1F crossreacted only with that of chimpanzee; P3-2H crossreacted with erythrocyte band 3 of chimpanzee, spider monkey, and capuchin monkey; and P3-4A and P3-4C crossreacted with erythrocyte band 3 of all primates examined. These results suggest that evolutional changes in primates are accumulated in the amino-terminal cytoplasmic domain of band 3 and that species-specific epitopes exist on this domain.View full abstractDownload PDF (1643K) -
Takahiro Tsujita, Hiromichi Okuda1992 Volume 112 Issue 2 Pages 224-228
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe activity of phospholipase C from Clostridium perfringens on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) as a monolayer at an air/water interface was examined. With a pure POPC monolayer, sharp cut-off of the enzyme activity was observed on increase in surface pressure. However, this cut-off disappeared on addition of a 0.3 molar fraction of 1, 2-dioleoylglycerol (1, 2-DO) to the monolayer. An abrupt change in the enzyme activity was observed with molar fractions of between 0.2 and 0.3 1, 2-DO in the POPC monolayer at an initial surface pressure of 35 mN/m. For examination of the effect of 1, 2-DO on the phospholipase C activity, the quantity of [125I]phospholipase C adsorbed to the surface was determined. The enzyme was found to be adsorbed nonspecifically to all lipid films except that of POPC only. The adsorption of enzyme was not affected by the presence or absence of Ca2+ and Zn2+. The rate constant for enzyme adsorption to a 1, 2-DO film was 4.5 times that for its adsorption to a POPC film. The adsorption decreased linearly with increase in the surface concentration of POPC, and increased with increase in the surface concentration of 1, 2-DO. These data suggest that 1, 2-DO (a reaction product) regulates the interaction of phospholipase C with films containing substrate and may also regulate the enzyme activity.View full abstractDownload PDF (646K) -
Yi-Ping Huang, Katsuya Seguro, Masao Motoki, Katsuhisa Tawada1992 Volume 112 Issue 2 Pages 229-234
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe action on muscle proteins of microbial transglutaminase (MTGase), which catalyzes the formation of a “zero-length” covalent cross-link between glutamine and lysine residues in peptides, was studied in order to define a basis for future application of MTGase cross-linking to the study of muscle protein interaction. We examined the cross-linking of skeletal muscle myosin, myosin subfragments, actin, and myofibrils by treatment with MTGase and the possible side-effects of the cross-linking on the enzymic activity of myosin, and found that the rod portions of myosin in myosin filaments were quickly cross-linked to each other by the action of MTGase, but myosin subfragment 1 was not cross-linked to actin. The MgATPase activities at 0.5M KCl of myosin, heavy meromyosin, subfragment 1, and subfragment 1-actin were not significantly affected by the MTGase reaction. A very small fraction of the head portion of heavy meromyosin was cross-linked to actin in their rigor complexes by MTGase, and the ATPase activity at 0.5M KCl of the cross-linked heavy meromyosin-actin complexes was slightly enhanced.View full abstractDownload PDF (3262K) -
Akihiko Kuniyasu, Kozo Oka, Tomoko Ide-Yamada, Yasumaru Hatanaka, Teru ...1992 Volume 112 Issue 2 Pages 235-242
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCa2+-channel was purified 230-fold from digitonin extracts of the porcine cardiac sarcolemmal membranes by means of a four-step procedure. Two antibodies, a site-directed antibody against the sequence 1691-1707 of the rabbit cardiac α1 subunit (anti-CCP5) and a monoclonal antibody directed to rabbit skeletal muscle α2δ subunit-complex (MCC-1), effectively immunoprecipitated the 125I-labeled cardiac Ca2+-channel complex in 0.2% digitonin. SDS-PAGE analysis of the immunoprecipitates under reducing conditions revealed that the cardiac channel is mainly composed of two large polypeptides of 190 and 150 kDa, and five smaller polypeptides of 60, 55, 35, 30, and 25 kDa. An additional polypeptide of either 79 or 55 kDa is crosslinked with the 190 kDa component to form 250-270 kDa (_??_270 kDa) to the extent of 15-20% through disulfide bond(s). The 190 kDa component (α1) is responsible for photoaffinity labeling with [3H]diazipine, since minor photolabeled _??_270 kDa was converged to the major labeled 190 kDa component when electrophoresed under reducing conditions. The 150 kDa component (α2) was derived by reduction of disulfide bonds from another 190 kDa component of glycopolypeptide which was separated from the channel complex in 1% Triton X-100 and capable of binding to WGA-Sepharose. The four smaller components of 60, 35, 30, and 25 kDa were not covalently associated with the large components through disulfide bonds, whereas the 55 kDa polypeptide was suggested to be a mixture of two kinds of peptides with respect to the disulfide bond: one was crosslinked with α1 through disulfide linkage and the other was not covalently associated with any other component. None of the polypeptides in the purified preparation or the sarcolemmal membranes which were phosphorylated by the cAMP-dependent protein kinase, was immunoprecipitated by either of the antibodies.View full abstractDownload PDF (5153K) -
Masao Sakaguchi, Naomi Hachiya, Katsuyoshi Mihara, Tsuneo Omura1992 Volume 112 Issue 2 Pages 243-248
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMitochondrial porin is a major integral membrane protein of the outer membrane. To assess the stop-transfer sequence in the yeast porin molecule (P), we constructed the following chimeric proteins. (i) The signal sequence of interleukin 2, a secretory protein, was fused to the amino-terminus of porin (SP). (ii) The matrix targeting presequence of cytochrome c oxidase subunit IV was fused to the amino-terminus of porin (CP). (iii) The amino-terminal segment consisting of 42 amino acid residues of “70 kDa protein” of yeast mitochondria, a major membrane protein of the outer membrane, was introduced into the middle portion of interleukin 2 (IL70). These chimeric proteins were expressed with an in vitro transcription-translation system and their integration into microsomal membrane or mitochondrial membranes was examined. When the proteins were synthesized in vitro with wheat germ cell-free system in the presence of rough microsomal membrane (RM), SP was completely translocated across the membrane, processed by the signal peptidase, and glycosylated. The translocation of IL70 molecule across RM was stopped at the introduced amino-terminal segment of 70 kDa protein. The authentic porin did not interact with the microsomal membrane. To assess the interaction with mitochondria, porin and CP were synthesized with the reticulocyte lysate system and subjected to posttranslational import reaction with isolated rat liver mitochondria. The authentic porin was integrated into the outer membrane in an alkali-resistant fashion. CP was imported into the mitochondria and its presequence was cleaved by the processing protease in the matrix. Upon alkali extraction, the processed form of CP, the authentic porin portion, was extracted into the supernatant, indicating that it was imported into mitochondria but not integrated into the membrane. These results demonstrate that the primary structure of porin does not contain any sequence which interrupts the protein translocation across the ER (endoplasmic reticulum) membrane and mitochondrial membranes.View full abstractDownload PDF (2037K) -
Yvonne Teunissen, Wÿnand P. M. Geraerts, Harm van Heerikhuizen, R ...1992 Volume 112 Issue 2 Pages 249-252
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe isolated a eDNA encoding a cytochrome P450 from the mollusc Lymnaea stagnalis. The mRNA is 2.1 nucleotides long and contains an open reading frame encoding a protein of 545 amino acids. A conserved heme-binding domain, characteristic of cytochrome P450 proteins, is present in the deduced amino acid sequence. The Lymnaea cytochrome P450 protein shares less than 40% positional identity with any known member of the cytochrome P450 superfamily, and therefore, represents a separate family, which we propose to name CYP10. The CYP10 mRNA is shown to be uniquely and abundantly expressed in the female gonadotropic hormone producing dorsal bodies of L. stagnalis.View full abstractDownload PDF (2992K) -
Purification and Characterization of a Novel Dipeptidyl Carboxypeptidase from a Streptomyces SpeciesShinsuke Miyoshi, Goro Nomura, Masabumi Suzuki, Fumio Fukui, Hideoki T ...1992 Volume 112 Issue 2 Pages 253-257
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn extracellular protease derived from the culture broth of a microorganism, a Streptomyces species, produced Boc-Pro-Pro and diproline from Boc-Pro-Pro-Pro-Pro. The enzyme was purified 726-fold, with a yield of 2.6%, by ammonium sulfate fractionation, ion-exchange chromatography, and gel filtration chromatography. The molecular weight of the enzyme was determined to be 65, 000 by gel filtration and 70, 000 by SDS-PAGE. The enzyme released a C-terminal dipeptide from peptide substrates having a C-terminal proline and a penultimate proline or alanine residue, but did not hydrolyze angiotensin I or bradykinin. When the enzyme hydrolyzed Leu-Pro-Pro-Pro-Pro-Pro, it produced Leu-Pro-Pro-Pro and Pro-Pro before producing Leu-Pro. The enzyme thus seems to be a kind of dipeptidyl carboxypeptidase, its substrate specificity being very different from that of the well known dipeptidyl carboxypeptidases [EC 3. 4. 15. 1] such as the angiotensinconverting enzyme.View full abstractDownload PDF (1834K) -
Takahiro Matsuyama, Kenji Soda, Toshio Fukui, Katsuyuki Tanizawa1992 Volume 112 Issue 2 Pages 258-265
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have constructed an efficient expression plasmid for the leucine dehydrogenase gene previously cloned from Bacillus stearothermophilus. The recombinant enzyme was overproduced in Escherichia coli cells to a level of more than 30% of the total soluble protein upon induction with isopropyl β-D-thiogalactopyranoside. The enzyme could be readily purified to homogeneity by heat treatment and a single step of ion-exchange chromatography. The purified enzyme was inactivated in a time-dependent manner upon incubation with pyridoxal 5'-phosphate (PLP) followed by reduction with sodium borohydride. The inactivation was completely prevented in the copresence of L-leucine and NAD+. Concomitantly with the inactivation, several molecules of PLP were incorporated into each subunit of the hexameric enzyme. Sequence analysis of the fluorescent peptides isolated from a proteolytic digest of the modified protein revealed that Lys80, Lys9l, Lys206, and Lys265 were labeled. Among these residues, Lys8O was predominantly labeled and, in the presence of L-leucine and NAD+, was specifically protected from the labeling. Furthermore, a linear relationship of about 1:1 was observed between the extent of inactivation and the amount of PLP incorporated into Lys80. A slightly active mutant enzyme, in which Lys8O is replaced by Ala, was not inactivated at all by incubation with PLP, showing that the inactivation is correlated with the labeling of only Lys80. Lys8O is conserved in the corresponding regions of all the amino acid dehydrogenase sequences reported to date. These results suggest that Lys80 is located at the active site and plays an important role in the catalytic function of leucine dehydrogenase.View full abstractDownload PDF (2092K) -
Sumihiro Hase1992 Volume 112 Issue 2 Pages 266-268
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPyridylamino (PA) derivatives of sugar chains were converted to 1-amino-1-deoxy derivatives. PA-lactose as a model compound was reduced with hydrogen, then treated with hydrazine. The product obtained was identified as 1-amino-1-deoxylactitol by mass spectrometry and chromatography with 1-amino-1-deoxylactitol as standard. PA-N-acetylglucosamine was converted to 1-amino-1-deoxy-N-acetylglucosaminitol under the same conditions. As an application, Manα1-6 (Manα1-3) Manα1-6 (Manα1-2Manα1-3)-Manβ1-4GlcNAcβ1-4GlcNAc-PA was converted to the 1-amino-1-deoxy derivative, which was further derivatized with fluorescein isothiocyanate or biotin sulfo-N-hydroxysuccinimide ester. Binding of these derivatives to concanavalin A dot-blotted on a nitrocellulose membrane was confirmed by fluorescence and by streptavidin-peroxidase conjugate. This conversion allowed replacement of the PA-group in PA-sugar chains which can be easily purified from glycoconjugates.View full abstractDownload PDF (1322K) -
Satoshi Ogihara, Hiromi Sesaki1992 Volume 112 Issue 2 Pages 269-276
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo 28-kDa calcium- and lipid-binding proteins were isolated from a detergent-insoluble fraction of the Physarum plasmodium. Both proteins have molecular masses of approximately 28 kDa by SDS-PAGE. The protein designated 28K-I has a slightly lower mobility than that designated 28K-II. The purified 28K-I has a dissociation constant of 1.0μM for Ca2+ ions, while the 28K-II has two different dissociation constants: one of 0.32μM and the other of 3.2mM. The 28K-I binds to liposomes at Ca2+ concentrations higher than 1.0μM and has a dissociation constant for lipid of 34μg/ml at 10μM Ca2+. The 28K-II binds to liposomes at concentrations of Ca2+ above the mM range and has a dissociation constant of 36μg/ml for lipid at 2mM Ca2+. There is no evidence of actin-binding activity by either of the 28-kDa (28K) proteins. The 28K proteins crossreacted with an antiserum against chicken brush border calpactin I. The two proteins have quite different phosphorylation levels between a fraction prepared from the cytosolic endoplasm and a fraction prepared from the whole cell. The 28K proteins may play some role in the membrane structure dynamics of the cortical gel layer.View full abstractDownload PDF (4770K) -
Hitomi Fukuda, Nobuko Iritani1992 Volume 112 Issue 2 Pages 277-280
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAfter refeeding a fat-free diet to fasted rats, the time courses of transcriptional rates, mRNA concentrations, and enzyme induction of hepatic acetyl-CoA carboxylase and fatty acid synthase were compared between 1.5- and 18-month-old rats. In the old rats, the levels were mostly 40-70% of those in the young animals. Moreover, the peaks of the levels tended to be delayed in the old animals. The transcriptional rates were increased within only 1h after the refeeding in the young animals, but not until 6h in the old. The mRNA concentrations reached maximum at 16h in the young rats, but at 24h in the old. In the old rats, the incorporation of [3H]leucine into the enzyme proteins was also decreased roughly in proportion to the enzyme induction. The mRNA concentrations in the liver polysomes were roughly proportional to the total mRNAs. Thus, the translational activities did not appear to be altered by aging. It is suggested that the age-dependent decreases of acetyl-CoA carboxylase and fatty acid synthase induction can be mainly ascribed to the transcriptional steps.View full abstractDownload PDF (484K) -
Hideo Kubo, Gang Jung Jiang, Atsushi Irie, Minoru Suzuki, Fuyuhiko Ina ...1992 Volume 112 Issue 2 Pages 281-285
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA novel fucose-containing neutral glycosphingolipid (GL-5) was purified from the eggs of the sea urchin, Hemicentrotus pulcherrimus. The chemical structure was determined to be Fucα1-3GalNAcβ1-4 (Fucα1-3) GlcNAcβ1-4Glcβ1-1Cer by methylation analysis, partial acid hydrolysis, fast atom bombardment mass spectrometry, and proton nuclear magnetic resonance spectroscopy. The unique characteristics of GL-5 are that: the reducing terminal disaccharide portion is not Galβ1-4Glc but GlcNAcβ1-4Glc; it includes a GalNAcβ1-4GlcNAc sequence and a Fuc-GalNAc linkage; the defucosylated core is a novel trisaccharide chain; and the sugar structure is one of the smallest ever characterized for a difucosylated glycolipid. The major fatty acids were 22:1 and 22h:1, and about 30% of the total acids was 2-hydroxylated. All the long-chain bases were phytosphingosines, of which about 90% was n-t18:0. The similarity of the ceramide moiety to that of glucosylceramide from the same eggs [Kubo, H. et al. (1992) J. Biochem. 111, 726-731] suggests a close biosynthetic relationship between GL-5 and the glucosylceramide.View full abstractDownload PDF (1289K) -
Fuyuhiko Inagaki, Shin-ichi Tate, Hideo Kubo, Motonori Hoshi1992 Volume 112 Issue 2 Pages 286-289
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA novel fucosylglycolipid from the eggs of the sea urchin, Hemicentrotus pulcherrimus, was determined by using two-dimensional NMR methods. Subspectra extraction by the homonuclear Hartmann-Hahn method was useful for identification of the individual sugar components. The homonuclear Hartmann-Hahn and double-quantum-filtered correlated spectra were analyzed to establish the assignments of sugar proton resonances. On the basis of the resonance assignments, the linkages of the individual sugar components were determined to be as follows. Gal_??_Acβ1-4Glc_??_Acβ1-4Glcβ-1Cer This glycolipid contains a novel skeletal structure with the linkages of GalNAcβ1-4GlcNAcβ1-4Glcβ. We also observed that 2-hydroxylation of the fatty acids induced appreciable chemical shift changes of the proton resonances of the phytosphingosine moiety as well as the anomeric proton resonance of the reducing terminal glucose.View full abstractDownload PDF (427K) -
Taketomo Fujiwara, Yoshihiro Fukumori, Tateo Yamanaka1992 Volume 112 Issue 2 Pages 290-298
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA novel type of cytochrome c oxidase was purified to homogeneity from Pseudomonas aeruginosa which was grown aerobically. The purified oxidase contained two molecules of heme a, two atoms of copper, and one molecule of protoheme per molecule. One of the two heme a molecules in the oxidase reacted with carbon monoxide, so that the enzyme was of baa3-type. The oxidase molecule was composed of three subunits with molecular weights of 38, 000, 57, 000, and 82, 000. Although the oxidase oxidized ferrocytochrome c-550 obtained from the bacterial cells grown aerobically, the oxidizing activity was not high. The “resting form” and the “pulsed form” of the oxidase were observed clearly with this enzyme, and the transition from the resting form to the pulsed form was accompanied by a distinct change of the enzymatic activity. The difference in the kinetics of the catalytic reactions between the two forms is discussed.View full abstractDownload PDF (2364K) -
Masaya Imoto, Yoshiko Taniguchi, Kazuo Umezawa1992 Volume 112 Issue 2 Pages 299-302
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSInostamycin, a novel microbial secondary metabolite, inhibited [3H]inositol and 32P1 incorporation into phosphatidylinositol (PtdIns) induced by epidermal growth factor (EGF) in cultured A431 cells, the IC50 being 0.5μg/ml, without inhibiting macromolecular synthesis. The drug inhibited cellular inositol phosphate formation only when it was added at the same time as labeled inositol. It was found to inhibit in vitro CDP-DG: inositol transferase activity of the A431 cell membrane, the IC50 being about 0.02μg/ml. It did not inhibit tyrosine kinase, Ptdlns phospholipase C, or Ptdlns kinase. Therefore, inhibition of Ptdlns turnover by inostamycin must be due to the inhibition of CDP-DG: inositol transferase. Thus, inostamycin is a novel inhibitor of CDP-DG: inositol transferase.View full abstractDownload PDF (1409K)
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