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Volume 102 (1987)
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- Issue 5 Pages 963-
- Issue 4 Pages 673-
- Issue 3 Pages 445-
- Issue 2 Pages 243-
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Volume 102, Issue 4
Displaying 1-35 of 35 articles from this issue
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Makoto MATSUOKA, Yuriko KANO-MURAKAMI, Yoshiyuki TANAKA, Yoshihiro OZE ...1987 Volume 102 Issue 4 Pages 673-676
Published: October 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have cloned a full length cDNA for the small subunit of ribulose-1, 5-bisphosphate carboxylase from C4 monocot maize, determined the complete nucleotide sequence of this cDNA and deduced its amino acid sequence. The cDNA insert included 513 by of the coding region, and 65 and 252 nucleotides of the 5' and 3' untranslated regions, respectively. The transit and mature peptides have, respectively, 47 and 123 amino acids. Comparison with the small subunit genes from other plants revealed that the maize small subunit is similar to the wheat one, there being 73 % homology between the transit peptides and 64 % between the mature proteins. This indicates that there is no noteworthy difference between the C3 and C4 small subunit structures. Extreme codon bias was observed for this gene, and similar codon preferences are observed for other proteins highly expressed in maize leaf, light harvesting chlorophyll binding protein and phosphoenolpyruvate carboxylase. The results indicate that preferential codon usage for highly expressed genes occurs in maize leafView full abstractDownload PDF (326K) -
Tohru NAKANO, Kohji HANASAKI, Hitoshi ARITA1987 Volume 102 Issue 4 Pages 677-680
Published: October 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe rapid Ca2+ increase shown by quin2-loaded platelets was found to be an artifact, probably due to light scattering elicited by collagen. Further findings as to fura2- loaded platelets offered additional support, demonstrating that the initial activation of phospholipase A2 (PLA2) does not require cytoplasmic Ca2+ mobilization. A possible role of the Na+/H+ antiporter as a trigger for collagen-induced activation of PLA2 in rat platelets was presented for the first time.View full abstractDownload PDF (290K) -
Toshio TAKAGI, P.F. RAO, Hidehiro KUBOTA1987 Volume 102 Issue 4 Pages 681-684
Published: October 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA modernized schlieren optics was applied to follow protein bands visually during polyacrylamide gel electrophoresis. A band containing as little as 0.3 μg of a protein could be detected. Besides the protein bands, usually overlooked phenom-ena such as boundary migration of the buffer components could be visualized. As an example, electrophoretic patterns thus obtained for SDS-polyacrylamide gel electrophoresis with a discontinuous buffer system are presented. The use of a split-type colored filter was found to be useful for coloring a particular location on the gel red or blue depending on the sign of the refractive index gradient. This means of detection also seems useful in that, firstly, it makes electrophoretic mobility measurement more quantitative and, secondly, it allows localization of a protein band and sampling in the intact state.View full abstractDownload PDF (751K) -
Tadao HASHIMOTO, Yukuo YOSHIDA, Kunio TAGAWA1987 Volume 102 Issue 4 Pages 685-692
Published: October 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA regulatory subunit of yeast mitochondrial ATP synthase, 9K protein, formed an equimolar complex with Fl-ATPase in the presence of ATP and Mg2+, indicating that the binding of the protein to the enzyme took place in a similar manner to that of ATPase inhibitor. The ATP-hydrolyzing activity of Fl-ATPase decreased 40% on binding of the 9K protein, and the remaining activity was resistant to external ATPase inhibitor. The apparent dissociation constant of the Fl-ATPase-9K complex was determined by gel permeation chromatography to be 3.7 × 10-6 M, which was in the same order of magnitude as that of enzyme-ATPase inhibitor complex (4.2 × 10-6 M). When added simultaneously the binding of the inhibitor and 9K protein to Fl-ATPase were competitive and the sum of their bindings did not exceed 1 mol per mol of enzyme. However, the binding of each protein ligand to Fl-ATPase took more than 1 min for completion, and when one of these two proteins was added 10 min after the other, it did not replace the other. These observations strongly suggest that membrane-bound Fl-ATPase always binds to either the 9K protein or ATPase inhibitor in intact mitochondria and that the complexes with the two ligands are active and inactive counterparts, respectivelyView full abstractDownload PDF (594K) -
Satoko ISEMURA, Eiichi SAITOH, Kazuo SANADA1987 Volume 102 Issue 4 Pages 693-704
Published: October 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA cysteine proteinase inhibitor (designated as cystatin SA) was isolated from human whole saliva by procedures including chromatography on DE 32 and DEAE-Se-pharose CL-6B. The amino acid sequence determined by conventional methods showed sequence homology of 90 and 87% as compared with the sequences of cystatin S and cystatin SN, respectively, both of which are salivary inhibitors char-acterized previously. The new inhibitor consisted of 117 residues and had a pI value of 4.3. Cystatin SA inhibited ficin and papain more strongly than cystatin S or cystatin SN did. It also exhibited inhibitory activity toward dipeptidyl peptidase I but the activity was much weaker than those toward ficin and papain.View full abstractDownload PDF (1220K) -
Haruhiro MURATSUBAKI1987 Volume 102 Issue 4 Pages 705-714
Published: October 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWhen baker's yeast grown aerobically on ethanol as a carbon source was anaerobi-cally cultured in a medium containing glucose, the activity of a cytoplasmic fumarate reductase irreversibly catalyzing the conversion of fumarate to succinate increased, reaching about 3 times the original activity after 12h, while the activity of succinate dehydrogenase was almost lost after 10h. These results indicate that the citrate cycle is partially modified to become a reductive pathway leading to succinate during the anaerobic cultivation. In non-proliferating cells grown anaerobically on glucose, the rates of accumulating succinate and pyruvate were decreased and increased, respectively, with increasing concentrations of L-aspartate or NH4Cl in the medium containing glucose as a substrate. These changes were accompanied with increase in the cellular content of aspartate, an inhibitor of pyruvate carboxylase that is involved in supplying the intermediates of the citrate cycle, and pyruvate, a sub-strate of the enzyme. The aminotransferase inhibitor, aminooxyacetate, prevented the changes in succinate accumulation and cellular aspartate following the addition of NH4Cl. The addition of L-glutamate caused a marked increase in the rate of succinate accumulation without changing the cellular content of aspartate. Neither L-glutamate nor L-aspartate had the ability to produce succinate. The rate of glucose consumption was not changed upon adding these nitrogen compounds. Similar findings were also observed in experiments using proliferating cells. This report presents evidence that in cells containing a large amount of the fumarate reductase, the production of succinate from glucose is regulated by the cellular level of aspartate through the pyruvate carboxylase reaction and that glutamate regulates the succinate production by a mechanism distinct from that involved in the regulation by L-aspartate.View full abstractDownload PDF (756K) -
Nobukazu KASHIMA, Hiroshi MATSUI, Seiko YAMASAKI, Nobuo KONDO, Junji H ...1987 Volume 102 Issue 4 Pages 715-724
Published: October 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMurine interleukin-2 (MIL-2) cDNA was inserted into an expression vector carry-ing an Escherichia coli tryptophan promoter and was expressed in E. coli. Recom-binant MIL-2 produced by E. coli supported the growth of murine CTLL-2 cells, but not that of human T-cell blasts. Recombinant MIL-2 strongly inhibited the binding of recombinant human IL-2 (HIL-2) to murine responder cells, but only very weakly inhibited the binding to human responder cells. Moreover, recom-binant MIL-2 induced secondary alloantigen specific cytotoxic T lymphocytes (2°CTL) from memory CTL and activated natural killer (NK) cells in murine systems in the same manner as recombinant HIL-2. The results suggest that the species hierarchy (that MIL-2 derived from native cell culture does not act on human T-cells) is due to the protein moiety, not the sugar moiety, and is to be ascribed to the difference in binding affinity of MIL-2 and HIL-2 to murine and human responder cells respectively, and that recombinant MIL-2 shares identical biological and im-munological activities with recombinant HIL-2. Thus, MIL-2 might be a convenient tool for extensive studies of the pharmacological and physiological activities of IL-2 in murine models.View full abstractDownload PDF (702K) -
Nobukazu KASHIMA, Hiroshi MATSUI, Seiko YAMASAKI, Nobuo KONDO, Junji H ...1987 Volume 102 Issue 4 Pages 725-732
Published: October 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMurine interleukin-2 (MIL-2) cDNA deleted of 11 repeats of a CAG sequence, and that encoded from 33Met to 149G1n were inserted into an expression vector carrying an Escherichia coli tryptophan promoter and were expressed in E. coli, respectively. Recombinant MIL-2 deleted of 11 glutamine repeats (MIL-2(-Gln)) supported the growth of murine CTLL-2 cells but recombinant MIL-2 initiated from 34Asp (34Asp-MIL-2) did not. The growth of human T-cell blasts was not supported by MIL-2(-Gln) or 34Asp-MIL-2. MIL-2(-Gln) had identical biological and immunological activities with intact MIL-2, but 34Asp-MIL-2 did not. These results suggest that the consecutive glutamine repeats do not play a role in the biological and immu-nological activities of MIL-2, but that the peptide sequence around them does, and the species hierarchy that MIL-2 does not act on human lymphocyte is not due to the presence of glutamine repeats in MIL-2.View full abstractDownload PDF (521K) -
Izumi KUMAGAI, Shuichi KOJIMA, Eisuke TAMAKI, Kin-ichiro MIURA1987 Volume 102 Issue 4 Pages 733-740
Published: October 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn expression plasmid for hen egg-white lysozyme in Saccharomyces cerevisiae was constructed by inserting almost full-length cDNA (about 600 base pairs) encoding hen egg-white pre-lysozyme into a yeast expression vector, pAM82. The hen lysozyme was expressed under the control of the repressible acid phosphatase pro-moter of pAM82 in S. cerevisiae. About half of the expressed lysozyme was secreted in the yeast growth medium as a precise mature protein which exhibited specific activity consistent with that of authentic hen egg-white lysozyme. The replacement of Trp 62 of hen egg-white lysozyme with a tyrosine residue was per-formed by site-directed mutagenesis using a 19-mer oligodeoxyribonucleotide. The mutant lysozyme with Tyr 62 was found to exhibit enhanced bacteriolytic activity.View full abstractDownload PDF (1295K) -
Norimasa TAKIZAWA, Hiraku SAITOH, Yasunori KIHIRA, Kaoru MIYAZAKI, Tom ...1987 Volume 102 Issue 4 Pages 741-753
Published: October 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDNA polymerase A (I or major) and its stimulative factor were purified from 15-20kg wet weight of baker's yeast by several procedures, which were varied in order to examine the possible occurrence of proteolysis. The extraction was carried out in the presence of 10 or 3mM phenylmethylsulfonyl fluoride (PMSF), followed by either batchwise adsorption-elution or column chromatography on DEAE-Sepharose (rapid or time-consuming, respectively). These early steps were followed by column chromatographies on DEAE-, CM-, and heparin-Sepharoses, phosphocellulose, and Sephacryl S-300. Preparations of the polymerase obtained by all the procedures described above showed a single protein band at Mr of about 145, 000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), unless they had been treated with 2-mercaptoethanol (ME). After ME treatment, however, they showed two protein bands at Mr of about 145, 000 and 75, 000 in SDS-PAGE, except for those obtained by the procedure involving 10mM PMSF and the batchwise adsorption-elution. All the preparations described above showed practically the same specific activity. This indicates that in intact cells, the polymerase consisted of a single peptide with Mr of about 145, 000, and that after cell disruption, it was artificially hydrolyzed in a limited fashion into two peptides with Mr of about 75, 000, which were still active and were linked to each other through a disulfide bond. Preparations of the factor obtained by all the procedures described above showed a single protein band at Mr of about 20, 000 in SDS-PAGE before and after ME treatment. The relative activities of the purified polymerase were (100%), 123, 21, 37, 196, and 38 with native and denatured salmon sperm DNA, native and denatured calf thymus DNA, poly(dA-dT), and poly(dA)•oligo(dT)10, respectively. With the addition of the purified factor, they were 173, 272, 173, 217, 173, and 247%, respectively, i.e., significantly stimulated. The purified factor also stimulated the activity of calf thymus DNA polymerase α by 150% with denatured salmon sperm DNA; Km was about 5 × 10-10M, practically the same as that of yeast DNA poly-merase A. However, it hardly influenced the activities of Escherichia coli enzyme I or Micrococcus luteus enzyme.View full abstractDownload PDF (1563K) -
Tetsuya UEDA, Takashi IDEGUCHI, Naruki KAINO, Tomisaburo KAKUNO, Jinpe ...1987 Volume 102 Issue 4 Pages 755-765
Published: October 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe molecular organization of photochemical reaction (PR) complex in chromatophores from Rhodospirillum rubrum was studied by a combination of proteolytic analysis with proteinase K followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunochemical analysis with rabbit polyclonal antibodies against its five subunits (H, M, L, a, and β). The preparations used for comparison were reaction center complex (RC) (composed of H, M, and L), PR complex, and chromatophores (closed membranous vesicles of polar lipid bilayer having PR complex buried in the membrane). 1. RC was bound with anti-H, anti-M, and anti-L antibodies, whereas PR complex and chromatophores were bound with anti-H and anti-β antibodies, but not with the other antibodies. 2. With PR complex, H (Mr 31, 000 (31K)) was rapidly degraded into two peptides with Mr of 16K and 14.5K (abbreviated as 16K and 14.5K, respectively), M (27K) into 25.5K, and β (11K) into 10K. Significantly later, the 25.5K of M was degraded into 24K, L (23K) into 19K, and α (12K) into 11K. With chromatophores, H and β were degraded in a manner similar to that with PR complex, whereas M, L, and α were not degraded at all. With RC, H, M, and L were rapidly degraded. 3. With RC, the activity for photooxidation of P870 (photochemical activity) was hardly affected till H, M, and L had been degraded into <10K, 24K, and 19K, respectively. With PR complex, the absorbance spectrum due to the bacteriochlorophylls of lightharvesting complex-1 composed of α and β (LH1-Bch1) changed in parallel to the degradation of α or 10K (a part of β). 4. Together with the previous results (Ueda et al. (1985) J. Biochem. 98, 1487-1498), the present findings suggest that:
1) RC is directly surrounded by 12 α and further by 12 β; 2) H and β are mostly and partially exposed, respectively, on the outer surface of the membranous vesicle; 3) a small part of M is exposed on the inner surface of the membranous vesicle.View full abstractDownload PDF (3244K) -
Yuko NAGAMINE, Michito SUMIKAWA, Kaoru OMICHI, Tokuji IKENAKA1987 Volume 102 Issue 4 Pages 767-775
Published: October 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFive modified maltooligosaccharides, phenyl O-6-amino-6-deoxy-α-D-glucopyranosyl-(1→4)-O-α-D-glucopyranosyl- (1→4)-O-α-D-glucopyranosyl-(1→4)-α-D-glucopy-ranoside(AG4P), phenyl O-(α-D-glucopyranosyluronic acid)-(1→4)-O-α-D-gluco-pyranosyl-(1→4)-O-α-D-glucopyranosyl-(1→4)-α-D-glucopyranoside (CG4P), phenyl O-6-amino-6-deoxy-α-D-glucopyranosyl-(1→4)-O-α-D-glucopyranosyl-(1→4)-O-α-D-glucopyranosyl-(1→4)-O-α-D-glucopyranosyl-(1→4)-α-D-glucopyranoside (AG5P), phenyl O-(α-D-glucopyranosyluronic acid)-(1→4)-O-α-D-glucopyranosyl-(1→4)-O-α-D-glucopyranosyl-(1→4)-O-α-D-glucopyranosyl-(1→4)-α-D-glucopyranoside (CG5P), and phenyl O-6-deoxy-6-[(2-pyridyl)amino]-α-D-glucopyranosyl-(1→4)-O-α-D-glucopyranosyl-(1→4)-O-α-D-glucopyranosyl-(1→4)-α-D-glucopyranoside (FG4P), were prepared to examine the active site of Taka-amylase A (TAA) [EC 3.2.1.1, Aspergillus oryzae]. Phenyl α-maltotetraoside (G4P) was predominantly hydrolyzed by TAA to maltose and phenyl α-maltoside (G2P). While G2P, phenyl α-glucoside (GP), and phenol were liberated from AG4P in the ratio of 7:63:30. G4P, phenyl a-maltotrioside (G3P), G2P, and GP were liberated from G 5P in the ratio of 1:20:73:6, but AG5P was almost completely hydrolyzed to modified maltotriose and G2P. On the hydrolysis of CG4P and CG5P, no remarkable change was observed except for a decrease in the relative reaction rates compared with G4P and G5P, respectively. When FG4P and G4P were hydrolyzed in the pH range of 4.5-6.0, the molar ratio of the hydrolysis products of G4P remained almost constant. However, that in the case of FG4P changed with pH, i.e., GP was predominantly formed at lower pHs, while the formation of G2P increased at higher pHs. These results suggest the presence of acidic amino acids at subsites S3 and S4 in the active site of TAA that interact with amino or pyridylamino groups of the substrates.View full abstractDownload PDF (686K) -
Yoshihiro FUKUMORI, Kazuya WATANABE, Tateo YAMANAKA1987 Volume 102 Issue 4 Pages 777-784
Published: October 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCytochrome c oxidase (cytochrome a03-type) [EC 1. 9. 3. 1] was purified from Erythrobacter longus to homogeneity as judged by polyacrylamide gel electrophoresis, and some of its properties were studied. The spectral properties of the oxidase closely resembled those of mitochondrial and other bacterial cytochromes aa3. The enzyme showed absorption peaks at 430 and 598nm in the oxidized form, and at 444 and 603 nm in the reduced form. The CO compound of the reduced enzyme showed peaks at 432 and 600nm. The enzyme oxidized eukaryotic ferrocytochromes c more rapidly than E. longus ferrocytochrome c. The reactions catalyzed by the enzyme were 50% inhibited by 0.7μM KCN. The enzyme contained 1g atom of copper and 1g atom of magnesium per mol of heme a. The enzyme molecule seemed to be composed of two identical subunits, each with a molecular weight of 43, 000.View full abstractDownload PDF (1771K) -
Nobuhiro HOSHINO, Ryo NAKAJIMA, Isao YAMAZAKI1987 Volume 102 Issue 4 Pages 785-791
Published: October 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe phenol oxidation catalyzed by horseradish peroxidase (HRPZ) is slowed down by the presence of excess H2O2. This inhibition is due to accumulation of Compound III, which is a catalytically sluggish form of HRP. When HRP is polymerized through covalent bonds, Compound III becomes unstable and the peroxidase activity is less sensitive to excess H2O2. Under suitable experimental conditions, the phenol oxidation is increased by about 20-fold upon polymerization of the enzyme. This fact represents the principle of a homogeneous enzyme immunoassay reported by Hoshino et al. (J. Biochem. 97, 113-118 (1985)). The ratio of the peroxidase activities of monomeric and polymerized HRPs is 1:4 when phenol is replaced by resorcinol, and the difference is no larger when guaiacol and catechol are used as electron donors.View full abstractDownload PDF (423K) -
Tadashi SAKAI, Katsuko WATANABE, Hiroshi KAWATSU1987 Volume 102 Issue 4 Pages 793-796
Published: October 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn unknown bilirubin conjugate was detected by HPLC analysis in the bile of yellowtail, Seriola quinqueradiata. The retention time of this unknown conjugate was identical with that of authentic ditaurobilirubin, a bilirubin conjugated with 2 mol of taurine. The retention times of the ethyl anthranilate diazo derivatives of both bilirubin conjugates were the same. The azo derivatives of this unknown conjugate were purified by Amberlite XAD-2 treatment and preparative TLC and hydrolyzed with 6 N HC1 at 105°C for 12h. The hydrolyzates obtained were analyzed with an amino acid analyzer. The ninhydrin-positive substance in the hydrolyzates was purified by column chromatography and analyzed by TLC and mass spectrometry. The results of these analyses indicated that this ninhydrin-positive substance was taurine. Therefore, this unknown bilirubin conjugate was identified as ditaurobilirubin.View full abstractDownload PDF (282K) -
Yasuo MUKOHATA, Manabu YOSHIDA1987 Volume 102 Issue 4 Pages 797-802
Published: October 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCell envelope vesicles of Halobacterium halobium synthesize ATP by utilizing base-acid transition (an outside acidic pH jump) under optimal conditions (1 M NaCl, 80 mM MgC12, pH 6.8) even in the presence of azide (a specific inhibitor of F0F1-ATPase) (Mukohata & Yoshida (1987) J. Biochem. 101, 311-318). An azideinsensitive ATPase was isolated from the inner face of the vesicle membrane, and shown to hydrolyze ATP under very specific conditions (1.5 M Na2SO4, 10 mM MnCl2, pH 5.8) (Nanba & Mukohata (1987) J. Biochem. 102, 591-598). This ATPase activity could also be detected when the vesicle components were solubilized by detergent. The relationship between ATP synthesis and the membrane-bound ATPase was investigated by modification of the vesicles with 7-chloro-4-nitrobenzo-2-oxa-1, 3-diazole (NBD-Cl) or N-ethylmaleimide (NEM). The inhibition pattern of ATP synthesis in the modified vesicles and that of ATP hydrolysis of the solubilized modified vesicles were compared under the individual optimum conditions. The inhibition patterns were almost identical, suggesting that the ATP synthesis and hydrolysis are catalyzed by a single enzyme complex. The ATP synthase includes the above ATPase (300-320 kDa), which is composed of two pairs of 86 and 64 kDa subunits. This is a novel H+-translocating ATP synthase functioning in the extremely halophilic archaebacterium. This “archae-ATP-synthase” differs from F0F1ATPase/synthase, which had been thought to be ubiquitous among all respiring organisms on our biosphere.View full abstractDownload PDF (466K) -
Ryo TAGUCHI, Hiroh IKEZAWA1987 Volume 102 Issue 4 Pages 803-811
Published: October 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe properties of acetylcholinesterase solubilized from bovine erythrocyte membrane by phosphatidylinositol (PI)-specific phospholipase C of Bacillus thuringiensis or with a detergent, Lubrol-PX, were studied. The activity of Lubrol-PX-solu-bilized acetylcholinesterase was broadly distributed in the fractions having Ve/Vo=1.0-2.0 in gel filtration on a Sepharose 6B column. The intermediary fractions (Ve/Vo=1.3-1.7) were collected as “the middle active Sepharose 6B eluate” and characterized on the basis of enzymology and protein chemistry. When this eluate was treated with PI-specific phospholipase C, the major activity peak was obtained in the later fractions with Ve/Vo=1.75-2.0 on the same column chromatography. Lubrol-solubilized and phospholipase C-treated acetylcholinesterase preparations were different in the thermostability, the elution profiles of chromatography on Mono Q, butyl-Toyopearl and phenyl-Sepharose columns, and the affinity to phos-pholipid micelles. On treatment with PI-specific phospholipase C, Lubrol-solubilized acetylcholinesterase became more thermostable. The phospholipase C-treated enzyme was eluted at lower NaCl concentration from the Mono Q column than the Lubrol-solubilized enzyme. The most important difference was observed in the hydrophobicity of these two enzyme preparations. The Lubrol-solubilized enzyme shows high affinity to phospholipid micelles and hydrophobic adsorbents such as butyl-Toyopearl and phenyl-Sepharose. However, this hydrophobicity was lost when acetylcholinesterase was solubilized from bovine erythrocyte membrane by PI-specific phospholipase C. The presence of myo-inositol was confirmed in the purified preparation of acetylcholinesterase by gas chromatography (GC)-mass spectrometry (MS). Furthermore, the monomeric form of acetylcholinesterase was directly released from erythrocyte ghosts by phospholipase C, after reduction with 2-mercaptoethanol and alkylation with monoiodoacetic acid. These results indicate that the diacylglycerol moiety of the PI molecule in Lubrol-solubilized and membrane-bound acetylcholinesterase plays a role as a hydrophobic anchor binding this enzyme protein to other membrane components, and that each subunit of acetylcholinesterase is covalently anchored to the erythrocyte membrane through a PI molecule.View full abstractDownload PDF (744K) -
Haruhiko AOYAGI, Sannamu LEE, Tstsuhiko KANMERA, Hisakazu MIHARA, Tets ...1987 Volume 102 Issue 4 Pages 813-820
Published: October 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSix peptide fragments, SEP6-11, SEP6-15, SEP1-11, SEP1-15, SEP1-20, and SEP25-39, corresponding to the residues 6-11, 6-15, 1-11, 1-15, 1-20, and 25-39, respectively, of the extension peptide of cytochrome P-450(SCC) precursor were chemically synthesized by the solution method. CD spectra of the peptides indicated that SEP1-15 and SEP1-20, which inhibit the import of the precursor, held a random conformation in a Tris-HCI buffer and a partially ordered conformation (like a-helix or type II' β-turn) in a buffer containing acidic liposomes. Slightly changed spectra were observed for SEP1-15 and SEP1-20 in the buffer containing neutral liposomes and in MeOH, respectively. Other peptides which show weak or no inhibitory effect had almost random conformations in these solvents. The hydrophobic moments of SEP1-15 and SEP1-20 when they take a-helical conformation are very small, suggesting that amphiphilic helical property is not always necessary for the import of the precursor, although partially ordered conformation seems to be required. The ability of SEP1-15 and SEP1-20 to induce leakage of carboxyfluorescein from the inside of dipalmitoyl-DL-a-phosphatidylcholine (DPPC) or DPPC-dipalmitoyl-DL-a-phosphatidylglycerol (3:1) vesicles was much greater than that of other peptides. The leakage induction ability of the peptides qualitatively parallels the degree of their inhibition of the import of the precursor. Perturbation of the membranes caused by the action of the peptides in their partially ordered form may be important for the import of the precursor into mitochondria.View full abstractDownload PDF (565K) -
Shigeki FURUYA, Masayoshi OKADA, Akio ITO, Haruhiko AOYAGI, Tatsuhiko ...1987 Volume 102 Issue 4 Pages 821-832
Published: October 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSVarious portions of the extension peptides of P-450(SCC) precursor were chemically synthesized. The effects of these peptides on the import of enzyme precursors into mitochondria were examined. Peptides SEPIAS and SEP1-20, corresponding to the amino terminal portion of the extension peptides, strongly inhibited the import of P-450(SCC) precursor into mitochondria. These peptides were effective at concentrations above 30μM, and complete inhibition was observed at 100μM. SEP1-11, which is shorter than SEP1-15 and SEP1-20, showed very weak inhibition. SEP25-39, which corresponds to the carboxy terminal portion of the extension pep-tide, did not affect the import of the precursor. The import of P-450(11β) and adrenodoxin precursors were also inhibited by SEP1-15. Another peptide, AEP1-14, which corresponds to the amino terminal portion of the extension peptide of adrenodoxin precursor, was also synthesized. The peptide inhibited the import of both adrenodoxin and P-450(SCC) precursors into mitochondria. The import of malate dehydrogenase was also inhibited by SEP1-15 and AEP1-14. The rate of the internalization of the precursor into mitochondria was decreased by the peptides. The amount of the precursor bound to the surface of mitochondria and the processing of adrenodoxin precursor were not affected. The respiratory activities of isolated mitochondria were not influenced by SEP1-15 even at 100μM. We conclude that the inhibitory activities of the synthetic partial extension peptides on the import of enzyme precursors into mitochondria require the presence of about fifteen amino acid residues in the amino terminal portion of the extension peptides, and the inhibition of the import by the peptides was dependent on the blockage of the internalization of the precursors into mitochondria.View full abstractDownload PDF (3600K) -
Takeo KUMAMOTO, Ken-ichirou MOROHASHI, Akio ITO, Tsuneo OMURA1987 Volume 102 Issue 4 Pages 833-838
Published: October 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe precursor of cytochrome P-450(SCC) (preP-450(SCC)), an inner membrane protein of adrenal cortex mitochondria, has an extension peptide consisting of 39 amino acids which is thought to play an essential role in the import of the precursor into mitochondria. The amino terminal portion of the extension peptide contains three positively charged amino acid residues, Arg(4), Arg(9), and Lys(14). To investigate their role in the import of preP-450(SCC) into mitochondria, they were replaced by other amino acids, Ser or Thr, by site-directed mutagenesis. The import of mutated preP-450(SCC)s with single amino acid substitution was much less efficient than with the original precursor. The mutated preP-450(SCC)s with two or three substitutions were not imported. These results suggest that the positively charged amino acid residues in the amino terminal portion of the extension peptide are essential for the import of preP-450(SCC) into mitochondria.View full abstractDownload PDF (1155K) -
Yuko KANO, Yoshiyuki SAKANO, Daisaburo FUJIMOTO1987 Volume 102 Issue 4 Pages 839-842
Published: October 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCross-linking occurred rapidly in collagen when it was treated with ascorbate-copper ion systems. Sodium dodecyl sulfate-polyacrylamide gel electrophoretic patterns of CNBr peptides suggest that in some respects the modifications by the ascorbate-copper ion systems mimicked those that occurred in collagen with agingView full abstractDownload PDF (1630K) -
Susumu IMAOKA, Tetsuya KAMATAKI, Yoshihiko FUNAE1987 Volume 102 Issue 4 Pages 843-851
Published: October 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSix rat hepatic cytochromes P-450, named P-450IF-1-6, were purified from hepatic microsomes of immature female rats by high-performance liquid chromatography with anion-exchange, cation-exchange, and hydroxylapatite columns. The purified forms, except for P-450IF-4, gave a single protein-staining band on SDS-polyacryl-amide gel electrophoresis, with a minimum molecular weight of 50, 000 for P-450IF-1, 49, 000 for P-450IF-2, 47, 000 for P-4501F-3, 53, 500 for P-4501F-5, and 54, 000 for P-450IF-6. The CO-reduced spectral maximum of these forms was at 450 nm for P-450IF-1, 448 nm for P-4501F-2, 451 nm for P-4501F-3, 449 nm for P-4501F-4, 449 nm for P-4501F-5, and 450 nm for P-450IF-6. All of these cytochromes had the low-spin state of heme in the oxidized form. P-450IF-4 had high metabolic activity for both benzphetamine and 7-ethoxycoumarin. P-4501F-5 had moderate activity toward 7-ethoxycoumarin. P-4501F-3 catalyzed the hydroxylation of testosterone at the 7a-position effectively, but the other forms did not hydroxylate testosterone. Analysis of the NH2-terminal sequence showed that P-450IF-1, 2, 3, 5, and 6 differed structurally from each other. The sequences of P-450IF-1 and IF-2 were somewhat homologous, but the NH2-terminal sequences of the other forms were all different. Based on these results, we concluded that P-450IF-1 corresponded to one of the phenobarbital-inducible forms in male rat liver. P-4501F-2 was a female-specific form and its concentration was low. P-4501F-5 and IF-6 were new forms not purified previously.View full abstractDownload PDF (678K) -
Satoru SUZUKI, Shunji IZUTA, Chikao NAKAYAMA, Mineo SANEYOSHI1987 Volume 102 Issue 4 Pages 853-857
Published: October 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSVarious 5-substituted 1-β-D-arabinofuranosyluracil 5'-triphosphates (H, methyl, ethyl, n-propyl, n-butyl, (E)-bromovinyl, styryl, and β-phenylethyl derivatives) were prepared and their inhibitory effects on two different herpes virus-induced DNA polymerases (OMV and HCMV) were studied. These dTTP analogues inhibited the incorporation of [3H]dTMP into DNA in vitro. Among them, analogues having a vinyl group at the 5-position were strongly active against DNA polymerases induced on herpes virus infection. Kinetic analysis showed that the inhibition by the analogues was essentially competitive with respect to the substrate, dTTP. The K1 values (μM) for AraUTP (2.4), AraTTP (1.0), BVAUTP (0.8), and StUAUTP (0.8) were smaller than the Km value (μM) for dTTP (3.4), but those for AraEtUTP, AraPrUTP, and AraBuUTP (5-14) were larger than the Km for dTTP in the case of HCMV-induced DNA polymerase. In contrast to these results, OMV-induced DNA polymerase seemed to be more resistant to these inhibitors than HCMV-induced DNA polymerase. However, the mode of the structure of substituent groups at the 5-position of base moieties is almost the same for the two DNA poly-merases, except for in the case of AraUTP itself.View full abstractDownload PDF (348K) -
Munehito IDEISHI, Masaharu IKEDA, Kikuo ARAKAWA1987 Volume 102 Issue 4 Pages 859-868
Published: October 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDepartment of Internal Medicine, Fukuoka University School of Medicine
Submandibular gland kallikrein [EC 3.4.21.8] of male Sprague-Dawley rats was purified by chromatography on soybean trypsin inhibitor (SBTI)-CH-Sepharose 4B, DEAE-Sephadex A-50, aprotinin-CH-Sepharose 4B and Sephadex G-100 columns and preparative isoelectrofocusing. The molecular weight of the kallikrein was estimated to be 30, 000 by Sephadex G-100 gel filtration and 29, 000 by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Isoelectric points ranged from pH 3.55 to 4.30. The kinin formed at pH 8 by this kallikrein from bovine low molecular weight (LMW) kininogen showed the same behavior as lysyl-bradykinin on HPLC in a solution of ammonium biphosphate containing acetonitrile. At physiological pH, this kallikrein also generated angiotensin II, a potent vasopressor, from human plasma protein. Rat submandibular gland kallikrein differs from tonin in the isoelectric point, the optimal pH for angiotensin II formation and the type of kinin formed. The tissue kallikrein might play a role in the regulation of local blood flow in view of its ability to form both vasoconstrictive and vasodilatory peptides.View full abstractDownload PDF (1499K) -
Yoshiaki KOMIYA, Neil A. COOPER, Antony D. KIDMAN1987 Volume 102 Issue 4 Pages 869-873
Published: October 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA single intraperitoneal injection of β, /β'-iminodipropionitrile (IDPN) at a dose of 1.5g/kg was given to 4-week-old rats. Immediately following, or at 1, 2, 3, 5, 10, and 15 weeks after IDPN injection, [35S]methionine was introduced into the anterior horn area of the lumbar cord. Labeled axonal proteins in the sciatic nerve were analyzed electrophoretically and fluorographically at 5, 10, and 15 weeks post-labeling. Labeled neurofilament proteins halt for a short period just after IDPN injection, then continue migrating distally, though at a slower rate, and finally the transport of affected neurofilament proteins completely recovers by 6 weeks post-labeling.View full abstractDownload PDF (876K) -
Masafumi YOHDA, Masasuke YOSHIDA1987 Volume 102 Issue 4 Pages 875-883
Published: October 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSingle-site catalysis by F1-ATPase from a thermophilic bacterium PS3 (TF1) was examined by incubating the enzyme with a submolar amount of radioactive ATP. The profile of single-site catalysis by TF1 at 23°C was different from that of beef heart mitochondrial F1-ATPase (MF1). ATP hydrolysis on the enzyme and release of the products was rapid, and subsequent addition of non-radioactive ATP (cold chase) did not promote the hydrolysis of radioactive ATP, indicating that the rate-limiting step was not the step of product release but the step of ATP binding to the enzyme. Thus, the characteristic features of so-called uni-site catalysis were not observed. At 60°C, whether in the presence or absence of phosphate ion, a small amount of bound [α, γ-32P]ATP and cold chase promotion were observed. However, since bound 32P1, was not detected by centrifugal gel filtration, it is not yet certain whether TF1 has typical uni-site characteristics. Based on the hydrolytic turnover rate for single-site catalysis and analysis of the kinetics of steady-state catalysis, it is proposed that single-site catalysis is dominant even in steady-state catalysis at ATP concentrations of less than about 20μM.View full abstractDownload PDF (657K) -
Norimitsu L. SATO1987 Volume 102 Issue 4 Pages 885-892
Published: October 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWhen the cytosol of Ehrlich ascites tumor cells was fractionated by chromatofocusing in the pH range of 9 to 6, two active peaks (I and II) of tRNA nucleotidyltransferase were obtained. Fraction I was a multiple complex with a high molecular weight (M. W.>300K) and fraction II comprised components derived from fraction I. Fraction II was separated into tRNA nucleotidyltransferase (M. W., ca. 46, 000) and nucleosidediphosphate kinase (M. W., ca. 74, 000) by subsequent Sephacryl S-200 chromatography. The two enzymes appeared to be associated loosely with each other. Using the above fraction II or a mixture of the purified tRNA nucleotidyltransferase and nucleosidediphosphate kinase, it was possible to effectively synthesize the 3'-terminal -pCpCpA of tRNA in a reaction mixture containing [3H] CDP plus XTP or [3H] ADP plus XTP as substrate. Among the XTPs investigated, dTTP was most effective. In addition, it was found that [3H] AMP+XTP also serves as a substrate. [14C] CMP plus XTP, however, was not utilized. From the antagonism of cold CDP against [3H] CTP, and that of cold ADP and AMP against [3H] ATP with the purified tRNA nucleotidyltransferase, the affinity of CDP to the enzyme was estimated to be 1/100 of that of CTP, while the affinities of ADP and AMP to the enzyme were 3 and 30 times higher, respectively, than that of ATP, suggesting that the subsite which binds ATP also binds ADP or AMP. The tRNA nucleotidyltransferase, which had bound ADP or AMP, could not completely synthesize the 3'-terminus of tRNA. Thus, the possible involvement of nucleosidedi-phosphate kinase, which catalyzes the γ-P transfer from other excess XTPs to ADP or AMP on the tRNA nucleotidyltransferase, is suggested to participate in the regulatory mechanism as to the synthesis of the tRNA 3'-terminus according to the level of the nucleotide pool in mammalian cells.View full abstractDownload PDF (658K) -
Keisuke HIRAI, Yoshihiro MATSUDA, Hachiro NAKAGAWA1987 Volume 102 Issue 4 Pages 893-902
Published: October 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSGMP synthetase was found in the cytosolic fraction of Yoshida sarcoma ascite cells. However, prolonged centrifugation resulted in precipitation of the enzyme On sucrose density gradient centrifugation of a crude extract of Yoshida sarcom ascites cells, a part of this enzyme showed high sedimentability at low ionic strengtl On the basis of these observations, GMP synthetase was purified from Yoshid sarcoma ascites cells by means of procedures including centrifugal fractionation The purified enzyme was shown to be homogeneous on SDS-polyacrylamide electrophoresis and isoelectric focusing in polyacrylamide gel. The molecule weight of the GMP synthetase was estimated to be 78, 000 by SDS-polyacrylamid gel electrophoresis and gel filtration on Sephadex G-150. Its isoelectric point was estimated to be 5.6. The Km values of this enzyme for XMP, ATP, and glutamir were calculated to be 4. 6, 120, and 300 μM, respectively. Although ammonia coul substitute for glutamine as a donor of the amino group, the Km value was as hig as 120mM, indicating that it cannot be considered to be a physiological substrata This enzyme showed high activity only in the presence of Mg2+, and very low activit in the presence of other divalent cations. Inhibition by nucleoside monophosphah was not significant. The enzyme required reduced sulfhydryl compounds for its activityView full abstractDownload PDF (1190K) -
Junji TAKAYA, Koichiro OMORI, Shigeru TAKETANI, Yohnosuke KOBAYASHI, Y ...1987 Volume 102 Issue 4 Pages 903-911
Published: October 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe (H+, K+) ATPase-enriched microsomal fraction prepared from hog gastric mucosa by sucrose density gradient centrifugation was effectively solubilized with Emulgen, with apparent preservation of the enzyme activity, and then the ATPase was highly purified by polyethylene glycol fractionation, and Blue Sepharose CL-6B and amino-hexyl Sepharose chromatographies. The purified enzyme showed a single band, with an apparent molecular mass of _??_94 kDa, on SDS-PAGE, and exhibited both K+-ATPase and K+-stimulated-p-nitrophenyl phosphatase (pNPPase) activities. The optimum pH for the ATPase activity was 7.0. Amino acid analysis of the purified enzyme showed that it contains a large amount of hydrophobic amino acid (42%) and a small amount of glucosamine and galactosamine. The rabbit antibody monospecific for the ATPase, in the Ouchterlony double immunodiffusion and Western blotting tests, markedly inhibited both the K+-F-ATPase and K+-pNPPase activities.View full abstractDownload PDF (2313K) -
Toshio SHIMADA, Hisao KATO, Sadaaki IWANAGA1987 Volume 102 Issue 4 Pages 913-921
Published: October 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effect of zinc ions on the surface-mediated activation of factor XII and prekallikrein was studied, using the contact system reconstituted with the purified proteins from bovine and human plasmas. The sulfatide-mediated activation of factor XII and prekallikrein in the presence of high-molecular-weight (HMW) kininogen was remarkably accelerated by 10-5M zinc ions. This accelerating effect was observed only in the presence of HMW kininogen. The kinetic analysis of the accelerating effect of zinc ions demonstrated that zinc ions reduce the Km values and increase the Vmax values on the activation of factor XII by kallikrein and on the activation of prekallikrein by factor XIIa. The value of Vmax/Km increased 26.4-fold in the former reaction and 2.8-fold in the latter reaction, indicating that zinc ions accelerate mainly the activation of factor XII by kallikrein. In the presence of 5×10-4M zinc ions, typical difference spectra due to a red shift of tryptophan and/or tyrosine residues were observed for HMW kininogen and its derivatives but not low-molecular-weight (LMW) kininogen. Since the concentration of zinc ions required to induce the difference spectra is comparable with that to enhance the activation of factor XII and prekallikrein, it appears that there is some correlation between the conformational change of HMW kininogen and the enhancement of the activation.View full abstractDownload PDF (759K) -
Keiko NOHARA-UCHIDA, Masako OHASHI1987 Volume 102 Issue 4 Pages 923-932
Published: October 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFour major gangliosides isolated from frog liver were characterized by compositional analysis involving GLC and GC-MS, methylation analysis, chromium trioxide oxidation, and enzymatic hydrolysis. The results revealed that the most major ganglioside in the tissue was GM4 containing N -acetylneuraminic acid and the others were GM4 containing N-glycolylneuraminic acid, GD1a, and a fucosyl ganglioside which was tentatively assigned to be a-galactosyl a-fucosyl GM1. This is the first report describing the presence of GM4 containing N-glycolylneuraminic acid. The fatty acids in both GM4 were mainly a-hydroxylated, and those in the fucosyl ganglioside were exclusively nonhydroxy fatty acids. The GDla contained both nonhydroxy and a-hydroxy fatty acids in a ratio of about 3:2. The predomi-nant species were 22:0, 23:0, 24:0, and 24:1 in both species of the fatty acids. The long-chain bases of these four gangliosides consisted of C18-sphingosine and C18-phytosphingosine together with significant amounts of C16 to C19 dihydroxy and trihydroxy bases with iso and anteiso structures.View full abstractDownload PDF (2043K) -
Keiki SATO, Takuya IKEDA, Fumiko KAWAI, Yasuaki OSADA1987 Volume 102 Issue 4 Pages 933-938
Published: October 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA rat liver-specific antigen (RLSA) solubilized with the nonionic detergent non-anonyl-N-methylglucamide was purified through affinity column chromatography with a monoclonal antibody and by high-performance liquid chromatography with a hydroxylapatite column. The purified RLSA showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and its molecular weight was determined to be 105, 000 in the presence of 2-mercaptoethanol. The antigen was reactive to the Schiff reagent and contained glucosamine, but not galactosamine, indicating that the RLSA is a glycoprotein containing an asparagine-binding type of sugar chain.View full abstractDownload PDF (1015K) -
Fumio OKADA, Kazuhiko TAKAHASHI, Shigeharu NAGASAWA, Jiro KOYAMA1987 Volume 102 Issue 4 Pages 939-946
Published: October 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have investigated the effect of plasma kallikrein digestion upon hydrolytic activities of human C1s. Incubation of C1s (85 kDa) with plasma kallikrein led to progessive cleavages on the heavy chain to yield C1s-K1 (70 kDa) then C1s-K2 (53 kDa). Although these cleavages caused little change in the C2 hydrolytic and esterase activities of C1s, a marked loss in the C4 hydrolytic activity was observed. C1s-K1 and C1s-K2 were purified by DE-52 chromatography and it was found that the proteolysis of C1s into C1s-K1 was accompanied with a decrease in the C4 hydrolytic activity. Although the turnover numbers for the hydrolysis of C4 by C1s-K1 and C1s-K2 were almost the same as that of intact C1s, the K1s for C4 of C1s-K1 and C1s-K2 were found to be increased to 10 times that of intact C1s. This result suggests that the apparent decrease in the C4 hydrolytic activity upon plasma kallikrein digestion of C1s is not due to disruption in the active site but is due to decrease in the affinity between C4 and the C1s derivatives. In support of this assumption, C1s-K1 was found to be devoid of the ability to bind C4b-Sepharose. C1s is capable of forming a dimer through the C1s-binding domain in the N-terminal side of the heavy chain. Although Cis-K1 is still capable of forming a dimer, C1s-K2 fails to form a dimer, suggesting that the N-terminal C1s-binding site is released during cleavage of C1s-K1 into C1s-K2. These results suggest that the C4-binding site is located in the heavy chain and is independent of the C1s- and C2-binding sites.View full abstractDownload PDF (1796K) -
Issei MABUCHI, Robert E. KANE1987 Volume 102 Issue 4 Pages 947-956
Published: October 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe actin-based gel formed at 35°C in the cytoplasmic extract from eggs of a sea urchin, Tripneustes gratilla, contains several high-molecular-weight proteins. Among them, the 250K-molecular-weight protein was isolated and characterized. This protein migrated slightly more slowly than filamin from chicken gizzard upon poly-acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. It reacted only very weakly with antibodies against chicken gizzard filamin or against a high-molecular-weight actin-binding protein from Physarum plasmodia. It did not react with antibodies against chicken erythrocyte alpha-spectrin nor against the 220K protein from the same egg. A chemical crosslinking experiment revealed the pres-ence of dimers in the purified 250K protein preparation. A rotary shadowed specimen of such a preparation showed wavy single-stranded molecules 120-170nm long, having five to six globular domains, which may represent dimers. The appearance was different from that of spectrin or actin-binding protein from macrophage or chicken gizzard filamin. This protein increased the viscosity of F-actin solution. It bound to F-actin preferably at low KCl concentrations such as 20mM. The binding ability was not influenced by pH between 6.0 and 7.5, although it was somewhat reduced above pH 8.0. The binding was insensitive to low Ca ion concentrations. Electron microscopy using the negative staining technique supported the idea that this protein crosslinks actin filaments. In addition, a second protein from egg gels, with a reported molecular weight of about 220K (Kane, R. E., J. Cell Biol. 66, 305-315 (1975)), comigrated with human erythrocyte alpha-spectrin on an SDS-gel and reacted with antibodies against chicken erythrocyte alpha-spectrin. This suggests that this protein is a sea urchin egg spectrin. The role of these proteins in the cytoskeleton formation in the sea urchin egg is discussed.View full abstractDownload PDF (4262K) -
Masayuki MURATA, Yuki SUGAHARA, Sho TAKAHASHI, Shun-ichi OHNISHI1987 Volume 102 Issue 4 Pages 957-962
Published: October 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA twenty amino acid hydrophobic peptide with the same sequence as that of the HA2 N-terminal segment of influenza virus hemagglutinin was synthesized and studied as to its fusion activity. The peptide caused rapid and efficient fusion of egg yolk phosphatidylcholine sonicated vesicles at acidic pH but not at neutral pH. The threshold pH was ca. 6.2 and the maximum fusion occurred at pH 4.8, the half-maximal pH for fusion being 5.6. The pH dependence was similar to that of the parent virus. The fusion efficiency was dependent on the ratio of lipid to peptide, increasing with decreasing ratio. The fusion can be rapidly switched on and off by adjusting the pH, to the acidic side and neutral, respectively. The peptide with an acetylated or succinylated N-terminus also showed low pH-induced fusion activity but the pH range was shifted by ca. 1 unit to the acidic side. The results indicate that the HA2 hydrophobic segment in the virus fusion protein is directly involved in the fusion reaction and protonation of the acidic residues in the segment is required for the activity.View full abstractDownload PDF (1057K)
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