All issues
Volume 114 (1993)
- Issue 6 Pages 761-
- Issue 5 Pages 623-
- Issue 4 Pages 449-
- Issue 3 Pages 299-
- Issue 2 Pages 163-
- Issue 1 Pages 1-
Volume 114, Issue 4
Displaying 1-31 of 31 articles from this issue
- |<
- <
- 1
- >
- >|
-
Takashi Nakaoka, Naoya Kojima, Toshiro Hamamoto, Nobuyuki Kurosawa, Yo ...1993 Volume 114 Issue 4 Pages 449-452
Published: October 01, 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA calcium-independent phosphatidylserine specific binding protein detected on liposome blotting analysis was purified from rat brain and revealed to be identical to myristoylated, alanine-rich C kinase substrate (MARCKS). MARCKS specifically binds to phosphatidyl-serine but not phosphatidylcholine. The binding of MARCKS to phosphatidylserine was abolished on protein kinase C-dependent phosphorylation. Since bacterially expressed MARCKS also specifically binds to phosphatidylserine, myristoylation of the N-terminal glycine seems not to be essential for the binding of MARCKS to phosphatidylserine. These data suggest that phosphatidylserine is a membranous target molecule of MARCKS.View full abstractDownload PDF (2154K) -
Bang-Jian Gong, Katsuhide Mabuchi, Katsuhito Takahashi, Bernardo Nadal ...1993 Volume 114 Issue 4 Pages 453-456
Published: October 01, 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCalponin is a thin filament-associated protein that is implicated in the regulation and maintenance of smooth muscle contraction. Molecular cloning of chicken gizzard calponin indicated the presence of two isoforms, α and β, the expression of the α-isoform being uniformly more abundant in various smooth muscle tissues [Takahashi, K. & Nadal-Ginard, B. (1991) J. Biol. Chem. 266, 13284-13288]. For the long-range goal of understanding of the structure and function of calponin, we have started bacterial expression and site-directed mutagenesis of α calponin. The amino acid composition and N-terminal sequence of the recombinant α calponin were found to be identical to those deduced from its nucleotide sequence. Recombinant α calponin is capable of binding to calmodulin, troponin C, tropomyosin, and actin, and of inhibiting skeletal muscle acto-subfragment-1 ATPase activity. A mutant α calponin with α replacement in the putative inhibitory region (residues 146-171) has impaired ability to inhibit the acto-subfragment-1 ATPase activity, suggesting that this region of calponin may be involved in the modulation of the actin-myosin interactions.View full abstractDownload PDF (1454K) -
Masaru Tanokura, Hiroshi Sasaki, Tomonari Muramatsu, So Iwata, Toru Ha ...1993 Volume 114 Issue 4 Pages 457-458
Published: October 01, 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSProteinase A from Aspergillus niger var. macrosporus is a non-pepsin-type acid proteinase, whose catalytic residues and mechanism remain to be elucidated. A new form of proteinase A crystals more suitable for crystallography than that obtained previously was prepared from an ammonium sulfate solution at pH 3.5 by the hanging-drop vapor diffusion method. The space group of the crystals was P212121 with unit cell dimensions of α=69.75±0.06 Å, b=87.55±0.05 Å, and c=60.83±0.04 Å. On the assumption of two enzyme molecules per asymmetric unit, the calculated volume to unit protein mass ratio (Vm) was 2.08 Å3/Da. By assuming the specific volume to be 0.74cm3/g, the solvent content (Vsol) was estimated to be 41%, i.e., much larger than that of the crystal form obtained previously at pH 2.0 (Vsol=26%). Diffraction data were collected up to a resolution higher than 1.6 Å, using the Weissenberg camera for macromolecular crystallography with synchrotron radiation.View full abstractDownload PDF (873K) -
Hatsue Waki, Ai Masuzawa, Kazuo Kon, Susumu Ando1993 Volume 114 Issue 4 Pages 459-462
Published: October 01, 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn O-acetylated trisialoganglioside that is converted to GT 2 on mild base treatment was found in cod brain. This alkali-labile ganglioside was isolated using high-performance liquid chromatography, and its chemical structure was characterized. This novel ganglio-side was identified as a GT 2 derivative having an acetyl group at the C-9 position of the external sialic acid. Its chemical structure is as follows:
II3 (9-O-Ac-NeuAc 2-SNeuAc 2-8 NeuAc 2-3)-Gg3Cer.
This study demonstrated the presence of 9-O-acetyl GT 2 in fish brain for the first time.View full abstractDownload PDF (1799K) -
Ken Kim, Yuji Homma, Yoshihide Ikeuchi, Atsushi Suzuki1993 Volume 114 Issue 4 Pages 463-467
Published: October 01, 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe factors affecting the conversion of α-connectin to β-connectin induced by pressurization of muscle were investigated over a pressure range from 100 to 400 MPa by using SDS-PAGE and immunoblot analysis. When muscles were exposed to high pressures, the conversion of α-connectin to β-connectin was the most pronounced at a pressure of 300 MPa, and the appearance of 1, 200-kDa peptide accompanied by conversion of α- to β-connectin was observed. Connectin was relatively resistant to degradation under a pressure of 400 MPa. The degradative products of β-connectin reactive with mAb 2D4 were not observed. The effect of high pressure on connectin in isolated myofibrils was similar to that on connectin in muscle. Addition of leupeptin and E-64 to the isolated myofibrils resulted in the prevention of the degradation of connectin at each stage of the pressurization. The ability of calcium-activated protease (calpain) to hydrolyze connectin from α to β gradually declined with increasing pressure. The results indicate that calpain is respon-sible for the pressure-induced conversion of α- to β-connectin. The rate of this conversion is probably regulated by the pressure-dependent structural change of α-connectin and inactivation of calpain.View full abstractDownload PDF (3831K) -
Mizue Morioka, Haiime Ishikawa1993 Volume 114 Issue 4 Pages 468-472
Published: October 01, 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSymbionin, a homologue of Escherichia coli GroEL, which functions as a molecular chaperone in the aphid endosymbiont, exists as a double-doughnut structure, consisting of two rings of seven 63-kDa subunits. Symbionin had a more labile oligomeric structure than GroEL and completely disassembled into monomeric (1-mer) components in 3M urea. The urea-dissociated symbionin self-assembled into 14-mer symbionin in a Mg-ATP dependent manner. When 1-mer symbionin was incubated in the presence of Mg-ATP, its reassembly proceeded hyperbolically with time. The yield of reassembled symbionin increased in response to increase in the initial concentration of 1-mer symbionin. The reassembled symbionin not only had the same molecular mass as native symbionin but also exhibited the same ATPase and phosphotransferase activity, suggesting that the correct three-dimensional structure was restored in vitro. While the self-assembly of symbionin was markedly stimulated by the presence of reassembled symbionin, it was not affected by the presence of native symbionin, which may suggest that native symbionin contains component (s) inhibitory to its chaperoning activity. As in the self-assembly of GroEL, that of symbionin was stimulated by the presence of GroES.View full abstractDownload PDF (535K) -
Yuji Sugita, Yasuko Nakano, Eiichi Oda, Keiichi Noda, Takashi Tobe, Na ...1993 Volume 114 Issue 4 Pages 473-477
Published: October 01, 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMACIF (CD 59) is a glycosyl-phosphatidylinositol (GPI)-anchored membrane glycoprotein which inhibits the formation of membrane attack complex of human complement. MACIF prepared from human erythrocyte membranes was digested with pronase. When the digest was subjected to two-phase partition with butanol and 0.1 N HCl, the carboxyl-terminal peptide was recovered in the butanol phase because of the attachment of the highly hydrophobic GPI. The amino acid sequence of the peptide was determined to be Asn72 at its amino-terminus and up to Glu76, while the presence of Asn77 was ambiguous. To allow unequivocal determination of the carboxyl-terminus, a soluble form of MACIF was prepared from human urine on a large scale. The carboxyl-terminal peptide from the soluble form was prepared by tryptic digestion followed by reversed-phase HPLC. The sequence and composition of the peptide unequivocally revealed Asn77 as the carboxyl-terminus. The pattern of disulfide bonds of MACIF was also determined with the membrane form as well as the soluble form. Cystine-containing peptides were prepared by chymo-tryptic and tryptic digestion, purified by HPLC, and their amino acid sequences were determined. The results indicated that disulfide bonds were formed at Cys3-Cys26, Cys6-Cysl3, Cys19-Cys39, Cys45-Cys63 (or 64), and Cys63 (or 64)-Cys69.View full abstractDownload PDF (516K) -
Taketoshi Uzawa, Nobuko Hamasaki, Tairo Oshima1993 Volume 114 Issue 4 Pages 478-486
Published: October 01, 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSEffects of novel, naturally occurring polyamines on protein synthesis catalyzed by Thermus thermophilus cell-free extract were investigated. The results revealed the physiological importance of a branched quaternary polyamine, tetrakis (3-aminopropyl) ammonium, in thermophile protein biosynthesis. Longer polyamines than triamine supported the polypeptide synthesis at high temperature, though both the activity and the optimum temperature varied depending on polyamines added. The highest activity was found when tetrakis (3-aminopropyl) ammonium and a tetraamine were simultaneously present. The optimum temperature of the reaction supported by the combination of the branched polyamine and spermine was the highest and in accord with the optimum temperature of the bacterial growth. These results suggested an essential role of the quaternary amine in protein synthesis in vivo. This amine effectively stabilized the ternary complex between ribosomes, the messenger, and phenylalanyl-tRNA, and this stabilization may account, at least in part, for its action on the present reaction. In contrast, another branched polyamine, tris (3-aminopropyl) amine supported the activity only moderately even in the presence of another polyamine, though the tris amine stabilized the ternary complex as effectively as the quaternary amine. This result suggests the presence of another essential site for polyamine action in the thermophile polypeptide synthesis, in addition to the stabilization of the ternary complex. The effects of polyamines on MS2 RNA directed reaction resembled those on poly (U) directed polypeptide synthesis, indicating that polyamines are essential in protein biosynthesis directed by natural messengers in vivo. The quaternary amine inhibited the aminoacylation of tRNAPphe, and the inhibition was canceled by the addition of another polyamine. When phenylalanyl-tRNA instead of free phenylalanine was added to the reaction mixture in order to investigate the effect of polyamines on polypeptide formation, single addition of tetrakis (3-aminopropyl) ammonium was enough for the highest activity, and the synergistic effect disappeared. The results indicate that the role of spermine in the synergism is to relieve the inhibition of aminoacylation caused by the quaternary amine.View full abstractDownload PDF (934K) -
Kazunori Anzai, Masako Masumi, Koh Kawasaki, Yutaka Kirino1993 Volume 114 Issue 4 Pages 487-491
Published: October 01, 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA novel positively charged planar bilayer membrane was formed from a mixture containing 20% cationic lipid, 1, 2-dioleoyloxy-3-(trimethylammonio) propane, and neutral phos-pholipid mixture of 56% phosphatidylethanolamine and 24% egg phosphatidylcholine. The basic properties of the bilayer were essentially the same as those previously reported for neutral and negatively charged lipid bilayers. Using the positively charged bilayer in addition to neutral and negatively charged bilayers, the effects of charge of the planar bilayers upon vesicle-planar membrane fusion were investigated by measuring the fusion, to the bilayers, of liposomes containing nystatin-ergosterol channels and carrying a net negative charge. The tendency for fusion was evaluated in terms of the time elapsed before the first fusion event (denoted fusion time). In the absence of calcium ions, a fusion time of about 1min was measured with the positively charged planar bilayers, and about 5 and over 15min with the neutral and negatively charged planar bilayers, respectively. These results indicated that the vesicle-planar membrane fusion without calcium ions is greatly enhanced by the presence of cationic lipids in the planar bilayers, and suggested the usefulness of cationic lipid bilayers.View full abstractDownload PDF (551K) -
Sun-Hee Park, Kazutaka Kano, Yousuke Seyama1993 Volume 114 Issue 4 Pages 492-497
Published: October 01, 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSUsing a primary culture system of guinea pig Harderian gland cells, we investigated the metabolism of a unique lipid: 1-alkyl-2, 3-diacylglycerol containing methyl-branched fatty acids. The cells were obtained by collagenase digestion, and cells with lipid-droplets were collected by two-step centrifugation. We cultured these cells, and examined their lipid and fatty acid compositions. The de novo synthesis of lipids in these cells was studied as to the incorporation of [1 (2)-14C] acetate and [U-14C]glucose. The major lipid proved to be 1-alkyl-2, 3-diacylglycerol, as in tissue, and it contained a large amount of methylbranched fatty acids specific to this gland. The incorporation of [14C] acetate and [14C] glucose into 1-alkyl-2, 3-diacylglycerol in the cultured cells amounted to 79.7 and 88.2% of the total incorporation into the lipid fraction, respectively. The incorporation of [14C] acetate into fatty acids in the cultured cells was detected for the chain lengths of C 14 to C 25. The activities of glycerol-3-phosphate dehydrogenase in the cultured cells and Harderian gland were lower than that in adipose tissue. These results confirm that cultured cells reflect the lipid metabolism originating in the Harderian gland and show that this culture system can serve as one part of the armamentarium for further study of this unique lipid metabolism.View full abstractDownload PDF (1342K) -
Jean-Luc Fausser, Ernst Ungewickell, Jean Victor Ruch, Hervé Le ...1993 Volume 114 Issue 4 Pages 498-503
Published: October 01, 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo further document the interaction of vinculin with the clathrin heavy chain (CHC) which was observed by using gel overlay, co-sedimentation experiments were performed and attempts were made to localize the domains involved on both molecules. The binding properties of proteolytic fragments of vinculin were investigated after cleavage with V 8 protease. Neither the isolated globular domain, nor the C-terminal rod domain were able to interact with the CHC. Either the interaction involved the portion of vinculin which links these two domains, or the region of vinculin mediating the interaction was present on one of the two major fragments, but the cleavage itself resulted in conformational changes which abolished the binding. The first hypothesis could be ruled out using α-chymotrypsin generated fragments of vinculin, suggesting that the native conformation of vinculin might play an important role. Proteolytic cleavage of CHC with trypsin demonstrated that the interaction with vinculin is mediated by the proximal or distal segment of the CHC. Presence of clathrin light chain (CLC) associated with the CHC did not affect its interaction with vinculin. Vinculin did not interact with the CLC.View full abstractDownload PDF (2971K) -
Hiroshi Kitagawa, Hiroshi Nakada, Shigeyuki Fukui, Ikuo Funakoshi, Tos ...1993 Volume 114 Issue 4 Pages 504-508
Published: October 01, 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo novel oligosaccharides with the sialyl-Lea structure were isolated from human milk using a monoclonal antibody, MSW 113. These oligosaccharides were purified by affinity chromatography on a column of the immobilized monoclonal antibody and by high-per-formance liquid chromatography, and structurally characterized by a combination of 600-MHz 1H NMR spectroscopy and fast atom bombardment mass spectrometry. The structural studies indicated the structures of these oligosaccharides to be:_??_View full abstractDownload PDF (933K) -
Shuntaro Hara, Yohsuke Imai, Makoto Murakami, Hiroshi Mori, Katsuhiko ...1993 Volume 114 Issue 4 Pages 509-512
Published: October 01, 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIntrapleural injection of zymosan into rats induces acute inflammation characterized by plasma leakage and cellular influx. The level of type II phospholipase A2 (PLA2) increased in the pleural fluid as well as in exudating leukocytes after the injection of zymosan. Rather low PLA2 activity was found in cell lysates, though treatment of such lysates at low pH increased the PLA2 activity drastically. The appearance of “acid-extracted” PLA2 activity in leukocytes preceded that of the extracellular enzyme activity, suggesting that pleural leukocytes might be one of the origins of the extracellular enzyme. Treatment of exudating pleural leukocytes with purified rat type II PLA2 elicited the production of prostaglandin E2, but not platelet-activating factor appreciably. These findings indicate that type II PLA2 might play a role in the progression of inflammation through the production of eicosanoids in the present inflammation model.View full abstractDownload PDF (453K) -
Yoshimasa Miyake, Mieko Shinomura, Toshiko Ito, Kazuhiko Yamamoto, Kos ...1993 Volume 114 Issue 4 Pages 513-521
Published: October 01, 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSα-Macroglobulin and murinoglobulin were purified to homogeneity from Syrian hamster plasma and their properties were compared with those of their respective homologs from other mammals. The trypsin-inhibiting capacity of hamster murinoglobulin was much weaker than those of rat and mouse murinoglobulins. Hamster α-macroglobulin was cleaved by trypsin at a number of sites whereas the human homolog was split essentially only in a “bait” region into two fragments of similar size. Hamster α-macroglobulin treated with methylamine differed from that treated with trypsin in the electrophoretic mobility, intensity of fluorescence induced by binding of bis (8-anilino-l-naphthalenesulfonate), and plasma clearance pattern, whereas virtually no difference was observed between the human homologs treated in the same manner. The reaction of hamster α-macroglobulin with methylamine, as measured by the generation of thiol groups and the decrease in trypsin-protein amidase activity, was much slower than that of the human homolog. Trypsin in a complex with hamster α-macroglobulin retained its fibrinolytic activity, but this was not the case for human or rabbit α-2-macroglobulin. These results suggest that, compared with the human homolog, hamster α-macroglobulin is more loosely packed in the native state, undergoes conformational change more slowly on treatment with methylamine, and less efficiently hinders the access of proteinaceous substrates to trapped proteinase. The serum concentration of hamster α-macroglobulin was 6.9mg/ml, or about 3-fold higher than that of the human type, and showed little change during the acute-phase reaction. These results account for the fact that pulmonary emphysema can be readily produced in hamsters by intratracheal inhalation of elastase or papain: the inhaled proteinase may be trapped by α-macroglobulin in much greater amounts than by other proteinase inhibitors since the α-macroglobulin concentration is high, and the trapped proteinase may be more potent for destroying the extracellular matrix than that trapped by α-macroglobulins from other species, since hamster α-macroglobulin is much less efficient in hindering the access of proteinaceous substrates. These events may eventually lead to the development of emphysema.View full abstractDownload PDF (2562K) -
Kazuhiko Tokita, Kazuko Hori, Toshitsugu Kurotsu, Masayuki Kanda, Yosh ...1993 Volume 114 Issue 4 Pages 522-527
Published: October 01, 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe mutant gene coding for a proline-activating domain (grs2-pro) was cloned and sequenced from Bacillus brevis Nagano, BII-3 strain, which produces gramicidin S synthetase 2 defective in proline-activation. By comparison of the nucleotide sequence with the wild-type sequence, a single point mutation was found at the 2609th guanine, which was replaced with adenine, resulting in the change of the 870th glycine to glutamic acid. Homology search for the deduced amino acid sequence of grs2-pro gene revealed that the 870th glycine was conserved in adenylate-forming enzymes, and its flanking sequence was highly conserved among the aminoacyl adenylate-forming enzymes, such as antibiotic peptide synthetases: gramicidin S synthetase 1 and 2 (GS1, GS2), tyrocidine synthetase 1 (TS1), and δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACVS); and other aminoacyl adenylation enzymes: α-aminoadipate reductase (LYS2), EntF, and AngR. On the other hand, this flanking sequence was not conserved in the other adenylate-forming enzymes lacking amino acid activation, such as acetyl-CoA synthetase, long-chain acyl-CoA synthetase, luciferase, and 4-coumarate CoA ligase. Single base substitutions at the 870th GGG codon were carried out by oligonucleotide site-directed mutagenesis. Four mutagenized clones were isolated, containing grs2-pro genes which exchange 870-Gly for alanine, valine, arginine, and tryptophan. The translated products from these clones could scarcely catalyze proline-dependent ATP-32PPi exchange reaction. The coil structure of 870-Gly region was lost in the mutants. These results suggest that the 870-Gly residue of grs2-pro protein is essential for aminoacyl-adenylation in the antibiotic peptide synthe-tase family.View full abstractDownload PDF (2637K) -
Cloning of the Rat Aortic Smooth Muscle Na+/Ca+ Exchanger and Tissue-Specific Expression of IsoformsYasuaki Nakasaki, Takahiro Iwamoto, Hironori Hanada, Toshiaki Imagawa, ...1993 Volume 114 Issue 4 Pages 528-534
Published: October 01, 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe amino acid sequences of two isoforms of the rat aortic smooth muscle Na+/Ca2+ exchanger have been deduced by cloning and sequencing the cDNAs. These isoforms are identical in nucleotide sequence except that one has a 23-amino acid insertion at amino acid position 570. They are highly homologous to the canine cardiac exchanger except for the NH2-terminal portion and part of the large central hydrophilic domain (amino acid residues 570-631). They are 902 and 925 (with the insertion) amino acid long with calculated molecular masses of 100, 676 and 103, 200 (with the insertion), respectively, if the NH2-terminal 32-amino acid residues are eliminated as a cleaved signal sequence. Amplification of the variable region (amino acids 570-631) of the exchanger by means of the reverse transcriptase-polymerase chain reaction and DNA sequencing revealed that many isoforms of the exchanger are expressed in different rat tissues. The two clones isolated in this study are the predominant isoforms expressed in aorta, stomach, liver, and kidney. In cardiac and skeletal muscles, another isoform is dominant, which is equivalent to the canine cardiac exchanger. In brain, a third type is predominantly expressed. Alignment of the nucleotide sequences of these isoforms and Southern blot analysis of rat genomic DNA suggested that each isoform is generated through alternative splicing of the primary transcript.View full abstractDownload PDF (2676K) -
Mikio Kataoka, Ken'ichi Mihara, Fumio Tokunaga1993 Volume 114 Issue 4 Pages 535-540
Published: October 01, 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe solution structure and calcium-dependent structural changes of recoverin, a 23 kDa calcium binding protein of vertebrate photoreceptors, have been studied by small-angle X-ray scattering and CD, as well as the effect of N-terminal myristoylation. The CD spectrum is not affected by N-terminal myristoylation, but strongly affected by Ca2+, indicating that N-terminal myristoylation alone does not cause a conformational change. The major conformational change in recoverin induced by Ca2+ is characterized as a decrease in the α-helical content of the protein and an increase in global size upon removal of Ca2+. In the presence of Ca2+, unmyristoylated recoverin is monomeric and globular in solution, while N-terminal myristoylation brings about aggregation. In the absence of Ca2+, unmyristoylated recoverin tends to aggregate, while myristoylated recoverin becomes monomeric and globular. These observations indicate that recoverin changes its surface properties depending on both calcium binding and N-terminal myristoylation. Melittin interacts non-specifically only with the myristoylated recoverin in the absence of Ca2+. This may be indicative of the properties of the interaction between recoverin and its normal physiological target enzyme.View full abstractDownload PDF (633K) -
Tomoko Kuroiwa, Masao Sakaguchi, Katsuyoshi Mihara, Tsuneo Omura1993 Volume 114 Issue 4 Pages 541-546
Published: October 01, 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIt is widely accepted that a proteinous channel participates in the co-translational trans-location of proteins across the endoplasmic reticulum (ER) membrane. Truncated mRNAs encoding N-terminal 70, 75, 88, and 110 amino acids of interleukin 2 were translated with wheat germ cell-free system in the presence of rough microsomal membrane (RM), and integral membrane proteins were probed with the translocating nascent peptides by using a cross-linking reagent (DSS). Two membrane proteins, 9 kDa (Cp9, cross-linking partner 9 k) and 39 kDa (Cp39, cross-linking partner 39 k), were cross-linked with the 75 amino acids nascent peptide. When NEM-treated RM was used for the translocation reaction, neither Cp9 nor Cp39 proteins were cross-linked. When the translation products were treated with puromycin before the cross-linking, both proteins were not cross-linked. The cross-linked products of Cp9 and Cp39 were not extracted by alkaline extraction of the membrane, not sensitive to endoglycosidase H, and did not bind to Con A-Sepharose. These results indicate that both of the cross-linking partners were nonglycosylated integral membrane proteins. Cp39 was cross-linked with the 70, 88, and 110 amino acid nascent peptides as well as the 75 amino acid peptide, whereas Cp9 reacted only with the nascent peptides consisting of 70 and 75 amino acid residues. Even after the digitonin treatment of the RM with the translocating intermediates, the cross-linked products with Cp9 and Cp39 were detected. Cp9 and Cp39 seem to be tightly associated with the ribosome-nascent peptide complex. These results indicate that Cp39 is identical with mammalian Sec61p, whereas Cp9 is a newly identified protein.View full abstractDownload PDF (2453K) -
Shigeki Sugiura, Kazuo Yamaguchi1993 Volume 114 Issue 4 Pages 547-552
Published: October 01, 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe Rep protein, a replication initiator of plasmid pSC101, also functions as an autorepressor for its own structure gene, rep, through binding to the operator which consists of imperfectly dyad-symmetric (pseudo-symmetric) sequences, IR-1 and IR-2. In order to define the DNA binding motif of Rep, we have analyzed its binding affinity for each half repeat of the IR sequences and found that the right half of IR-2 was the preferred sequence for Rep, although its affinity was much lower than those of the IR sequences. Next, dimeric sequences of each half repeat with different configurations, head to head (the same configuration as natural IRs), head to tail, and tail to tail, were constructed and their affinities for Rep were examined. Almost all of sequences with the head-to-head configuration had binding affinity for Rep but these, including even the symmetric sequence of the right half of IR-2, exhibited lower Rep-binding abilities than natural IR sequences. We presume a pseudo-symmetric sequence, 5' GGNNTAGNNATTNNNATNN (N) CTAGNCC 3', to be the Rep-binding motif from a sequence comparison. Some single-base substitution experiments confirmed the motif and suggested that each subunit of the Rep dimer recognizes each half repeat of IR asymmetrically.View full abstractDownload PDF (1401K) -
Keiko Masuda-Momma, Tatsuya Hatanaka, Kuniyo Inouye, Kenji Kanaori, At ...1993 Volume 114 Issue 4 Pages 553-559
Published: October 01, 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSKinetic analysis was performed on the interaction between subtilisin BPN' and recom-binant species of a proteinaceous proteinase inhibitor, Streptomyces subtilisin inhibitor (SSI), of which the P1 site amino acid residue, Met 73, was replaced by site-directed mutagenesis. The inhibitor constant, K1•was determined from the residual enzyme activity by using a peptide substrate. The rate constant of binding, kon, and the rate constant of dissociation, koff, were determined from a progress curve of the substrate hydrolysis in the presence of the inhibitor by using newly derived equations. A recombinant SSI in which Met 73 was replaced by Ile showed an affinity (1/Ki) toward subtilisin BPN' of only about 7% of that of the wild-type SSI, and the kinetic analysis revealed that the increase of koff was responsible for this difference. The affinity of other SSI mutants in which Met 73 was replaced by Glu or Asp decreased significantly as pH became increasingly alkaline. The decrease in the affinity of these recombinants was due to the decrease of kon rather than the increase of koff. Stopped-flow studies revealed that the binding reaction was reconcilable with a two-step mechanism, and the kinetic parameters for each step were obtained for the binding of the enzyme and recombinant SSIs.View full abstractDownload PDF (731K) -
Osamu Yamaguchi, Makoto Kimura, Masato Araki, Nobuyuki Yamasaki, Yoshi ...1993 Volume 114 Issue 4 Pages 560-566
Published: October 01, 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe previously isolated galactose-specific isolectins, EVLI, EVLII, and EVLIII, from the Erythrina variegata seeds [ J. Chromatogr. 597, 207-211 (1992) ]. The amino acid sequences of the two subunits, A- and B-subunits, of which the isolectins are composed, were determined. A comparison of the amino acid sequences of tryptic peptides from the two subunits revealed seven amino acid substitutions. Among them, Asn46, to which the oligosaccharide chain is linked in the A-subunit, is replaced by Asp46 in the B-subunit, causing the B-subunit to lack one glycosylation site. The N-linked oligosaccharides of these subunits were also analyzed. The N-linked oligosaccharides were first liberated by hydrazinolysis. After N-acetylation, the reducing ends of the oligosaccharides were coupled with 2-aminopyridine, and then the pyridylamino (PA-) derivatives were purified by gel filtration and BPLC on an ODS-silica column. One major sugar chain, accounting for more than 98% of the total, was purified from both species. The structure of this major sugar chain was established to be Manα6 (Manα3)(Xylβ2) Manβ4GlcNAcβ4 (Fucα3) G1cNAc. This finding that there is no structural difference of the sugar chains linked to the two subunits of E. variegata lectins, together with the results of amino acid sequence comparisons, indicates that the difference in molecular mass of these two subunits results almost wholly from the difference in the number of oligosaccharides linked to them.View full abstractDownload PDF (605K) -
Toshihide Nishimura, Norimatsu Takeshita, Hiroyasu Satow, Kaoru Kohmot ...1993 Volume 114 Issue 4 Pages 567-569
Published: October 01, 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe previously reported the partial sequence of a cloned genomic DNA, mC26, which codes for a protein highly expressed in the lactating mouse mammary gland [mC26: Satow et al. (1986) J. Biochem. 99, 1639-1643; partial sequence: Kawamura et al. (1987) J. Biochem. 101, 103-110]. In this study, we sequenced the Eco RI-Hin dIII fragment (5, 394 bp) of this gene and found that this gene contains a sequence completely (100%) homologous to the cDNA sequence currently reported to code for GlyCAM-1, a putative ligand for L-selectin. We show by means of an RNA protection assay that the mRNA of this gene is expressed in the mammary glands of lactating mice as well as in the lymph nodes. Semi-quantitative analysis of expression of the mC26 gene in the mammary glands revealed that the amount of mRNA was not detectable in the early stage of pregnancy, increased in the late stage, and remained quite abundant during lactation. The potential role of this gene highly expressed in the mammary gland in a stage-specific and tissue-specific manner is discussed.View full abstractDownload PDF (1340K) -
Takashi Kumazaki, Kenta Kajiwara, Shuichi Kojima, Kin-ichiro Miura, Sh ...1993 Volume 114 Issue 4 Pages 570-575
Published: October 01, 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have unexpectedly found that Streptomyces subtilisin inhibitor (SSI) and some other similar serine protease inhibitors produced by Streptomycetes strongly inhibit Streptomyces griseus metallo-endopeptidase II (SGMP II) [Kajiwara, K. et al. (1991) J. Biochem. 110, 350-354]. In order to elucidate the mode of their unusual interaction with SGMP II in more detail, we prepared twelve kinds of SSI analogues, in which one or two amino acid residues in the peptide segment from Thr64 to Va174 of wild-type SSI had been replaced or deleted by site-directed mutagenesis, and determined the dissociation constants of their complexes with SGMP II. Six analogues among them showed dissociation constants one order of magnitude lower than that of the wild type. Three had higher values. The results suggest that at least some residues in this segment are interacting with SGMP II in the complex. We also prepared an SSI mutant in which the disulfide bridge between Cys71 and Cys101 had been eliminated by replacing the two Cys residues with Ser residues. This mutated SSI inhibited SGMP II as strongly as the wild-type SSI did. While peptide bonds in the wild-type molecule did not suffer from the hydrolytic action of SGMP II except those at the amino-terminal fragile portion, the Pro72-Met73 bond of the mutant was specifically cleaved by the enzyme. This peptide bond, therefore, seems to play the role of the reactive site in the interaction of SSI with SGMP II.View full abstractDownload PDF (634K) -
Takeo Yamaguchi, Masaki Matsumoto, Eiji Kimoto1993 Volume 114 Issue 4 Pages 576-581
Published: October 01, 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe investigated the hemolytic properties under high pressure (200 MPa) of human eryth-rocytes in which sialic acids and glycopeptides had been removed from membrane surface by using neuraminidase and proteolytic enzymes such as trypsin and chymotrypsin, respectively. The degree of hemolysis increased in proportion to the amounts of sialic acids or glycopeptides released from intact erythrocytes. Studies of the time course of hemolysis showed that upon enzymatic digestion erythrocyte membranes became more fragile against high pressure. Such fragility decreased in the presence of chlorpromazine and trifluo-perazine but was unaffected by chlorpromazine methiodide or indomethacin. Furthermore, the effect of cross-linking of membrane proteins by diamide on the fragility was examined. The degree of hemolysis at 200 MPa increased upon removal of sialic acids from red cells in which spectrin is mainly cross-linked, but did not upon enzymatic digestion of red cells in which glycophorins, in addition to cross-linking of themselves, are included in the large-molecular-weight aggregates formed by cross-linking of the membrane skeleton with transmembrane proteins. In the latter case, however, upon reduction of the cross-linking by dithiothreitol the effect of enzymatic digestion appeared again. On the other hand, such an enzymatic digestion effect on osmotic hemolysis was not observed either in intact erythrocytes or in diamide-treated red cells. These results suggest that the interaction of the cytoplasmic domains of glycophorins with cytoskeletal proteins may be weakened by enzymatic digestion of the exofacial domains of glycophorins.View full abstractDownload PDF (2411K) -
Kimihide Hayakawa, Narihiro Minami, Shoichiro Ono, Yuichi Ogasawara, T ...1993 Volume 114 Issue 4 Pages 582-587
Published: October 01, 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA monoclonal antibody (McAb) specific for actin depolymerizing factor (ADF) was prepared. With this and previously prepared anti-cofilin McAb (MAB-22) and other antibodies, the expression of cofilin and ADF in the muscles of dystrophic (NH-413) chicken and dystrophic (C57BL/6Jdy/dy) mice was compared with that in normal control animals by immunoblotting and immunocytochemical methods. Since cofilin expression is down-regulated during normal postnatal development of skeletal muscles [Abe et al. (1989) J. Biochem. 106, 696-702], cofilin was detected in the breast (pectoralis) muscle of normal adult chicken and the leg (femoris and tibialis anterior) muscles of normal mice only at a low level. ADF was not detectable in adult skeletal muscles. However, a significant increase of cofilin amount, but not of ADF amount, was observed in these muscles of the dystrophic animals, when the symptom of muscular dystrophy became evident. In order to localize cofilin in individual muscle fibers, serial cryosections of the dystrophic chicken muscle were examined with anti-cofilin antibody (MAB-22). The antibody stained cells of different size in the dystrophic muscle, indicating that cofilin expression was induced in the regenerating muscle cells as well as in the pre-existing myofibers. We suggest that cofilin is involved in disassembly or reorganization of actin in the dystrophic muscle.View full abstractDownload PDF (3607K) -
Teruo Miyazawa, Toshihide Suzuki, Kenshiro Fujimoto, Mikio Kinoshita1993 Volume 114 Issue 4 Pages 588-591
Published: October 01, 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effects of a seleno-organic compound, Ebselen [2-phenyl-1, 2-benzoisoselenazol-3 (2H)-one], on phospholipid hydroperoxide levels in in vitro and in vivo systems were examined by using high-performance liquid chromatography with chemiluminescence detection (CL-HPLC) as a tool for measuring the hydroperoxide. In in vitro systems, the phos-phatidylcholine hydroperoxide (PCOOH) content in phosphatidylcholine (PC) liposomes was significantly reduced by co-incubation with exogenously added Ebselen; 85, 97, and 99.6% of PCOOH was eliminated with 1, 10, and 100μM Ebselen, respectively. In human plasma, 79, 87, and 86% of PCOOH was eliminated after the incubation with 1, 10, and 100μM Ebselen, respectively. In an in vivo study involving single oral administration of Ebselen (10, 30, and 100mg/kg rat) to rats, plasma PCOOH (48.8 nM for Ebselen-untreated rat) was significantly decreased to 26.3, 5.6, and 3.3 nM at 2h after the administration and 6.7, 7.4, and 4.8 nM at 4h after the administration, respectively. These in vivo effects on plasma PCOOH elimination depended on both the dose and the time after the administration. Maximum decrease of plasma PCOOH appeared around 2-4h after oral administration. The liver and red blood cell PCOOH levels were not affected by oral Ebselen under the present conditions. These findings indicate that the Ebselen may be a potent agent for lowering plasma PCOOH levels in vivo.View full abstractDownload PDF (389K) -
Takumi Hatayama, Yasuyuki Asai, Tohru Wakatsuki, Teruko Kitamura, Hiro ...1993 Volume 114 Issue 4 Pages 592-597
Published: October 01, 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSUpon exposure to the heavy metals copper and zinc, a large amount of 70, 000-Da heat shock protein (hsp70) was synthesized in normal, non-thermotolerant (NT) HeLa cells, whereas only a little increase of hsp70 synthesis was observed in thermotolerant (TT) cells. To determine the inhibition mechanism of hsp70 induction in the TT cells, we first analyzed the hsp70 mRNA of these cells. Hsp70 mRNA in the NT cells increased immediately after exposure to these metals. In TT cells, however, the increase of hsp70 mRNA was delayed, even though it eventually increased to a similar or slightly lower level compared with NT cells. Further analysis of the activation of heat shock transcription factor (HSF) showed that a significant activation of HSF was observed immediately after exposure to these metals in the NT cells, whereas the activation of HSF was initially repressed in the TT cells. Thus, the decreased induction of hsp70 synthesis observed in the TT cells seemed to be due to the reduced translation of hsp70 mRNA and also in part to the reduced activation of HSF. Furthermore, by the gel mobility shift assay using anti-hsp70 antibody, the association of copper- and zinc-activated HSF with hsp70 was observed in both NT and TT cells. The amount of HSF-hsp70 complex was prominent in TT cells, in which the hsp70 content was 5 to 10 times higher than that in the NT cells. These findings strongly suggest that the activity of HSF is negatively regulated by hsp70 in the TT cells.View full abstractDownload PDF (3495K) -
Kenji Akasaki, Masataka Fukuzawa, Hiroko Kinoshita, Koji Furuno, Hiros ...1993 Volume 114 Issue 4 Pages 598-604
Published: October 01, 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSOur previous studies provided evidence that a 107-kDa major lysosomal membrane glycoprotein termed lamp-1 shuttles between lysosomes and the plasma membrane along the endocytic pathway in rat hepatic cells [Furuno et al. (1989) J. Biochem. 106, 708-716; Furuno et al. (1989) J. Biochem. 106, 717-722]. In the present study, we investigated the movement of a 96-kDa major lysosomal membrane glycoprotein, referred to as lamp-2, and lysosomal acid phosphatase (LAP) in the endocytic membrane transport system of cultured rat hepatocytes. Fab' fragments of anti-lamp-2 and anti-LAP antibodies conjugated with horseradish peroxidase (HRP) were used as probes to analyze quantitatively the transport of these two membrane proteins from the cell surface to lysosomes. After the addition of HRP-anti-lamp-2 and anti-LAP Fab' fragments to the culture medium, the delivery of the antibody conjugates to lysosomes was examined by cell fractionation on a Percoll density gradient. The amount of these HRP tracers in the lysosomal fraction became larger as the period of cell incubation was increased. Km values for uptake of HRP-anti-lamp-2, and LAP Fab' fragments were 0.74 and 0.62μM, respectively, which were comparable to that of HRP-anti-lamp-1 Fab' (0.57μM). The endocytic process of the two HRP-antibodies continued for an extended period in the cells exposed to the protein synthesis inhibitor, cycloheximide. Furthermore, we measured the transit times of HRP-anti-lamp-1, anti-lamp-2, and anti-LAP Fab' fragments from the cell surface to lysosomes. Anti-lamp-1 and lamp-2 antibodies move to lysosomes at almost the same rates (t1/2=approximately 2.2h), while anti-LAP antibody is delivered to lysosomes with a shorter half time (t1/2=0.78h). Taken together, these results suggest that the three major lysosomal membrane proteins shuttle continuously between the cell surface and lysosomes in rat hepatocytes, but LAP cycles more rapidly than the lamp molecules in the endocytic vacuolar system.View full abstractDownload PDF (1591K) -
Kazuhiro Sogawa, Yasuo Kikuchi, Hiroaki Imataka, Yoshiaki Fujii-Kuriya ...1993 Volume 114 Issue 4 Pages 605-609
Published: October 01, 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have expressed truncated forms of BTEB and Spl in Escherichia coli and investigated the DNA-binding properties of the two proteins. The two proteins as well as their chimeric proteins protected the same DNA region in the BTE sequence (a GC box in the P-4501A1 gene) as examined by ortho-phenanthroline-Cu footprinting. The region overlapped nearly perfectly with the GC box consensus sequence. Methylation interference footprinting revealed that all the guanines within the region and two other guanines in the close vicinity interacted with the proteins. Competitive gel mobility shift assay using various synthetic oligonucleotides of the GC box sequences as the competitors demonstrated that BTEB and Sp1 have similar sequence specificities for DNA binding. We have purified the bacterially expressed BTEB and measured the dissociation constant of the BTEB-BTE complex using gel mobility shift assay. The dissociation constant was (3.0±1.0)×10-10M and was comparable to that of Sp1 binding to a GC box. Taken together, these findings indicate that the binding modes of BTEB and Sp1 to the GC box are similar to each other.View full abstractDownload PDF (4046K) -
Kei-ichi Uemura, Atsushi Hara, Tamotsu Taketomi1993 Volume 114 Issue 4 Pages 610-614
Published: October 01, 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMouse neuroblastoma NS-20Y cells were induced to grow neurites by removal of serum from the medium. The percentage of cells with neurites reached about 50-60% after 24h, whereas in medium containing 10% serum only a few cells (1-3%) were bearing neurites. Sphingosine inhibited the neuritogenesis in serum-free medium in a dose-dependent manner; a maximal effect was observed at 1-2μM. Sphingosine also caused retraction of neurites which had been induced to extend in serum-free medium. N, N-Dimethylsphingo-sine was 10 times more potent in preventing neurite outgrowth, and N-acetylsphingosine was 10 times less effective compared to sphingosine. However, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) did not inhibit the extension of neurites. Neurite outgrowth in serum-free medium and its inhibition by sphingosine were still observed in the cells made protein kinase C-deficient by prolonged treatment with phorbol ester. These results suggest that the effect of sphingosine is not mediated by inhibition of protein kinase C.View full abstractDownload PDF (1406K) -
Kyoto Nakashima, Akemi Sugiura, Takeshi Mizuno1993 Volume 114 Issue 4 Pages 615-621
Published: October 01, 1993
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSExpression of the Escherichia coli kdpABC operon, which is responsible for a high-affinity potassium-uptake system, is regulated in response to a change in the medium osmolarity. We present the first in vivo evidence that the putative osmosensor (kinase), KdpD, plays a crucial role in the osmotic regulation of kdpABC expression. We then isolated the intact form of KdpD from the cytoplasmic membrane with a mild non-ionic detergent, dodecyl maloside, as a soluble form. The solubilized KdpD itself did not exhibit the ability to phosphorylate the cognate response-regulator, KdpE. Upon reconstitution into liposomes, however, KdpD exhibited its kinase activity. Thus, using this partially purified form of KdpD, we succeeded in reconstituting KdpD-bearing liposomes that are fully functional in terms of their ability to phosphorylate the cognate positive regulator, KdpE. By adopting this in vitro method, some in vivo conditions were mimicked in order to address the molecular mechanism underlying signal transduction mediated by KdpD. Some catalytic properties of KdpD in liposomes are also presented.View full abstractDownload PDF (2186K)
- |<
- <
- 1
- >
- >|