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Volume 94 (1983)
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Volume 94, Issue 6
Displaying 1-45 of 45 articles from this issue
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Shoichi NAKASHIMA, Hiroshi KAMIKAWA, Zen-ichi OGITA1983 Volume 94 Issue 6 Pages 1723-1730
Published: December 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn the IgG antibody response to bacterial α-amylase (BαA) assayed by the enzymatic procedure, C3H/He (C3) mice were high and C57BL/6 (B6) mice were low responders. High responsiveness was inherited as a dominant characteristic in (B6XC3)F1, hybrid mice.
In these strains, the primary antibody response was analyzed for heterogeneity by isoelectric focusing (IEF). The IEF spectra were visualized with the use of the capacity of antibody to inhibit the amylase activity of antigen. Increases in the antigen dose and in the time interval between immunization and bleeding resulted in increases in antibody titers accompanied by strong staining of focused antibodies and by the expansion of the pH range where antibodies were focused.
High responsiveness in C3 and F1 hybrid mice was also associated with the increase in intensity of stain and the rapid expansion of pH range of focused antibodies.
Another strain difference was noted in the isoelectric point (pI) values of antibodies taken early in the primary response. B6 antisera contained those fractions of antibodies focusing over a more alkaline area than C3 antibodies. A similar strain difference in the pI values of antibodies occurred in the response to an irrelevant antigen, Taka-amylase A (TAA), suggesting that the hypervariable regions of antibody molecules play no major part in the strain difference observed. Antisera from F1 hybrid mice displayed bands covering the combined pH ranges of B6 and C3 spectrotypes.View full abstractDownload PDF (1824K) -
Yoichi IIDA, Fukashi SASAKI1983 Volume 94 Issue 6 Pages 1731-1738
Published: December 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMost genes of higher eukaryotes are discontinuous. The DNA which codes for a protein is interrupted by introns. In the nucleus, certain machinery is supposed to recognize the exon-intron and intron-exon junctions. Applying computer searching to eighteen genes from various biological species, we examined what kinds of patterns or nucleotide sequences are necessary and sufficient to recognize the splice junctions. We propose that four common patterns of AG/GTA, /GTAAGT, RG/GTGAG and AG/GTXXGT, where R=A or G and X=A, T, G, or C, are often used as signals for exon-intron junctions. This proposal is based on the facts that they are not found in the exons of the genes examined and on the assumption that the recognition machinery scans the mRNA precursor from the 5'-end to the 3'-end.View full abstractDownload PDF (638K) -
Hitoshi AOSHIMA1983 Volume 94 Issue 6 Pages 1739-1751
Published: December 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSome basic inhibition schemes for acetylcholine receptor (AChR)-mediated ion translocation in the presence of agonist, cholinergic ligand, and inhibitors are proposed on the basis of the minimum reaction scheme (Hess, G. P., Cash, D. J., & Aoshima, H. (1983) Annu. Rev. Biophys. Bioeng. 12, 443-473). Equations for the rate coefficients of ion flux before and after desensitization, JA and JD, and for desensitization, α, were derived from each scheme, assuming that binding of inhibitors to AChR does not affect the values of rate and equilibrium constants of cholinergic ligand to the receptor.
In the presence of inhibitors, AChR-mediated transmembrane Li+ influx caused by carbamylcholine (Carb) was measured by using Electrophorus electricus membrane vesicles, a simple filtration assay and flame emission spectroscopy. The dependence of the ratios of rate coefficients on the concentration of cholinergic ligand and inhibitors was examined in detail. The inhibition constants of d-tubo-curarine and caffeine were estimated to be about 31 nM and 0.84mM, respectively, on the basis of a simple competitive inhibition scheme, and that of procaine was estimated to be 0.11mM on the basis of a simple noncompetitive one.View full abstractDownload PDF (957K) -
Takashi TAKAGI, Kazuhiko KONISHI1983 Volume 94 Issue 6 Pages 1753-1760
Published: December 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe amino acid sequence of ascidian troponin C has been determined. It is composed of 155 amino acid residues and contains two tryptophan residues which have not been found in other troponin C sequences so far determined. The N-terminus is acetylated, and the molecular mass was calculated to be 17, 794.
Calcium binding sites I and III of ascidian troponin C appear to have lost the ability to bind calcium, and the total number of binding sites is reduced to two.View full abstractDownload PDF (567K) -
Masami HORIKOSHI, Hiro-omi TAMURA, Kazuhisa SEKIMIZU, Masuo OBINATA, S ...1983 Volume 94 Issue 6 Pages 1761-1767
Published: December 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe DNA binding subunits of RNA polymerase II from Ehrlich ascites tumor cells were investigated in the following three ways.
(1) RNA polymerase II was dissociated in urea and the binding of the dissociated subunits to DNA was investigated.
(2) The RNA polymerase II: DNA complex was dissociated progressively with various concentrations of urea, and the subunits firmly attached to DNA were investigated.
(3) RNA polymerase II was dissociated into subunits in a SDS-polyacrylamide gel containing urea and blotted onto a nitrocellulose filter. The filter was then incubated with 32P-nick-translated DNA to identify the DNA binding subunits.
These procedures all showed that the largest subunit a of RNA polymerase II had strong affinity to DNA. It was found that a portion of subunits b and c could be recovered in DNA fraction when analyzed by procedures (1) and (2), but no significant DNA binding activity was detected when analyzed by procedure (3), suggesting that these subunits have either a much weaker affinity toward DNA compared to a or have affinity to a itself.View full abstractDownload PDF (2176K) -
Ken-ichiro HIGASHI, Yutaka KIRINO1983 Volume 94 Issue 6 Pages 1769-1779
Published: December 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe Ca2+-ATPase of sarcoplasmic reticulum from rabbit skeletal muscle was incorporated into vesicles made from dimyristoylphosphatidylcholine or dipalmitoyl-phosphatidylcholine. The Ca2+-ATPase activity of these reconstituted membranes became appreciable above 20°C and 30°C, respectively, in accord with the results of previous investigators. Measurement by the spin-labeling technique of the fluidity of the bulk lipid revealed the gel-to-liquid crystalline phase transition at 29°C and 39°C, respectively, while the fluidity of the boundary lipid in both samples was found to be low throughout the temperature range studied. The rotational mobility of the Ca2+-TPase protein in both samples, measured by saturation transfer electron spin resonance, was also very low throughout the temperature range studied and its temperature-dependence did not show any break or jump corresponding to the phase transition of the bulk lipid. On the other hand, the structural fluctuation of the Ca2+-ATPase protein in dimyristoylphosphatidylcholine-recombinant, measured in terms of hydrogen-deuterium exchange reaction kinetics, showed a jump at about 27°C, apparently in accordance with the phase transition of the bulk lipid. Results obtained in this study suggested that the Ca2+-ATPase protein molecules are in an aggregated state in these reconstituted membranes and that the Ca2+-ATPase activity is neither directly correlated to the fluidity of the boundary lipid nor to the rotational mobility of the Ca2+-ATPase, contrary to the suggestions of previous investigators (Hesketh et al. (1976) Biochemistry 15, 4145-4151; Hidalgo et al. (1978) J. Biol. Chem. 253, 6879-6887).View full abstractDownload PDF (874K) -
Seiichi KAWASHIMA, Kazutomo IMAHORI1983 Volume 94 Issue 6 Pages 1781-1787
Published: December 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDNA-histone complexes were reconstituted from DNA and acid-extracted core histones and the products were characterized by micrococcal nuclease digestion to examine whether proper nucleosome structure had been reconstituted. No nucleosome structure was produced starting from the mixture of acid-extracted histones and purified DNA in 2 M NaCl-5 M urea, while the reassociation of chromatin by the same procedures was successful. This was due to the inappropriate conformation of acid-extracted histones, which was preserved in 2 M NaCl even in the presence of 5 M urea. If acid-extracted histones were reannealed from the completely denatured state, such as in 5 M urea, 6 M guanidine hydrochloride or 0.6 M NaCl-5 M urea, reconstitution of nucleosome structure was always successful.View full abstractDownload PDF (1304K) -
Katsura IZUI, Yoshihiro MATSUDA, Isamu KAMESHITA, Hirohiko KATSUKI, Al ...1983 Volume 94 Issue 6 Pages 1789-1795
Published: December 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn an attempt to investigate the topography of the catalytic site of phosphoenol-pyruvate (PEP) carboxylase [EC 4.1.1.311] of Escherichia coli, the inhibitor constants (K1) for more than 20 compounds were determined with the reaction system containing dioxane, a non-physiological activator of the enzyme. The K1 values for the compounds lacking methylene-, carboxylate-, or phosphate groups were all more than 10-fold larger than the Km value for PEP, indicating the significant contribution of these groups to the binding of PEP with the enzyme. The K1 value for L-phospholactate (0.30mM) was almost equal to the Km value for PEP (0.25mM), whereas that for n-phospholactate (0.99mM) was about 3-fold larger than the Km value. It was presumed that PEP binds with the enzyme on its si-side. Among 6 PEP homologs, the K1 values for phosphoenol α-ketobutyrate (0.024mM) and phosphoenol α-ketovalerate (0.034mM) were about one-tenth the KM value, indicating the presence of a hydrophobic pocket around the binding site of the methylene group of PEP, where the carboxylation reaction is supposed to occur. DL-Phosphomalate, a presumptive carboxylated substrate, was a weak inhibitor with a K1 value of 2.20mM.View full abstractDownload PDF (567K) -
Kaoru OMICHI, Tokuji IKENAKA1983 Volume 94 Issue 6 Pages 1797-1802
Published: December 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTransglycosylation reactions of α-amylases from human pancreatic juice and saliva were examined by using O-6-deoxy-6-[(2-pyridyl) amino] -α-D-glucopyranosyl-(1→4)-O-α-D-glucopyranosyl-(1→ 4)-O-α-D-glucopyranosyl-(1→ 4)-O-α-D-glucopyranosyl-(1→4)-D-glucopyranose as a substrate and O-α-D-glucopyranosyl-(1→4)-O-α-D-glucopyranosyl-(1→4)-1-deoxy-l-[(2-pyridyl) amino] -D-glucitol as an acceptor. The transfer reaction was estimated by quantitation of O-α-D-glucopyranosyl-(1→4)-1-deoxy-l-[(2-pyridyl) amino] -D-glucitol produced by the enzymes from the transfer products, because the acceptor was not hydrolyzed. The amount of O-α-D-gluco-pyranosyl-(1→4)-1-deoxy-l- [(2-pyridyl) amino] -D-glucitol in the digest with pancreatic a-amylase was six times that in the digest with salivary α-amylase at the stage when the substrate was completely consumed, and the difference increased gradually on further incubation. The phenomenon can be applied to differentiate the two α-amylases in human serum.View full abstractDownload PDF (966K) -
Seiichi YOKOE, Minoru TANAKA, Hiroshige HIBASAMI, Jun NAGAI, Kunio NAK ...1983 Volume 94 Issue 6 Pages 1803-1808
Published: December 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTobacco mosaic virus RNA, forming 40 S or 80 S initiation complexes with wheat germ ribosomes, was covalently bound to 18S ribosomal RNA by the photoreaction with an RNA cross-linking agent, 4'-aminomethyl-4, 5', 8-trimethylpsoralen (AMT). Synthetic polyribonucleotide, poly (A, U), with the cap structure m7GpppGmC at the 5'-terminal was also cross-linked to 18 S ribosomal RNA in 40 S or 80 S complexes with ribosomes by the AMT photoreaction. Polyuridylic acid with the same 5'-cap structure, forming 40 S complexes but not 80 S complexes with ribosomes, was most efficiently cross-linked to 18 S ribosomal RNA by the psoralen photoreaction. These results suggest that the interactions between mRNA and 18 S rRNA are not necessarily of strict complementarity but occur during formation of the complexes in eukaryotes. The 40 S complexes would be then converted to 80 S complexes in the presence of the AUG initiation codon or AUG-like triplets containing A and U on the polyribonucleotide chains which interact with 18 S ribosomal RNA.View full abstractDownload PDF (429K) -
Kazumichi KURODA, Shun-ichi OHNISHI1983 Volume 94 Issue 6 Pages 1809-1813
Published: December 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPhosphatidylcholine (PC) transfer activity was found in human erythrocyte hemolysate. The transfer activity was assayed by the ESR peak height increase when spin-labeled PC vesicles were incubated with egg yolk PC vesicles. The transfer activity was isolated from hemoglobin by an ion exchange chromatography followed by gel filtration. The partial purification resulted in a 405-fold increase in the specific transfer activity compared with that of the hemolysate. The molecular weight of the PC transfer protein was estimated to be 23, 000 by gel filtration. The transfer activity was inactivated by heat-treatment at 75°C for 10 min. Phosphatidylserine vesicles strongly inhibited the activity. Half-maximal inhibition occurred on addition of 0.24 mol% of phosphatidylserine vesicles to the incubation mixture. Ca2+ restored the activity. The transfer protein was quite similar to the PC transfer protein obtained from bovine liver cytosol.View full abstractDownload PDF (384K) -
Koichiro TANAKA, Tomisaburo KAKUNO, Jinpei YAMASHITA, Takekazu HORIO1983 Volume 94 Issue 6 Pages 1815-1826
Published: December 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSReaction center of chromatophores of Rhodospirillum rubrum consists of three kinds of protein, H-, M-, and L-subunit, and is bound with many other kinds of protein to form a larger protein complex (PRU; photoreaction unit), which contains all the bacteriochlorophyll. In the present study, purified PRU was dissociated in a stepwise manner in the presence of various mixtures of lithium dodecyl sulfate, sodium cholate and/or sodium deoxycholate, and separated into five, smaller protein complexes (PL1, PL2, PL3, PL4, and PL4') by high-speed molecular-sieve chromatography. The protein complexes were analyzed for molecular mass (Mm), protein composition, and molecular weights of the constituent proteins by the chromatography described above and by lithium or sodium dodecyl sulfate-polyacryl-amide gel electrophoresis. The results suggest that PRU consisted of 1 molecule each of 40 K, 39 K, 31 K (H-subunit), 25 K (M-subunit), and 22 K (L-subunit), about 12 molecules each of 12K (light-harvesting bacteriochlorophyll-protein) and 11 K, and about 6 molecules each of IOK and 9 K (the protein nomenclature refers to the apparent molecular weights); the measured and calculated Mm values were 650 K and 547 K, respectively. The compositions of the other protein complexes were as follows. PL1=PRU-10K-9K (measured & calculated Mm, 520K & 409K); PL2=PLI-39K (340K & 267K); PL3=PL2-40K (160K & 147K); PL4=PL3-31K-25K (90K & 82K); PL4'=31K+25K+22K (inactivated reaction center) (90K & 78K). The molar ratios of 12 K and 11 K to 25 K were lower in the dissociated protein complexes than in PRU, and they differed from one complex to another. The locations of the constituent proteins in PRU are discussed.View full abstractDownload PDF (912K) -
Seiko SHIGETA, Hiroshi KUBOTA, Hisashi TAMURA, Satoru OKA1983 Volume 94 Issue 6 Pages 1827-1832
Published: December 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn α-mannosidase was isolated from the extract of acetone powder of CHCl3 treated internal organs of the sea-squirt, Styela plicata. The enzyme was purified 4, 110-fold in 11% yield. The preparation was fairly homogeneous on disc and SDS-polyacrylamide gel electrophoreses and Sephadex G-200 chromatography. The enzyme had an estimated molecular weight of 275, 000 by gel chromatography and 70, 000 by SDS-polyacrylamide gel electrophoresis, and was therefore considered to be a tetramer. The optimum pH for the enzyme activity was 3.4 but the stable pH range was from 4 to 6. The isoelectric point was 5.0. The enzyme was activated by Zn2+ but inhibited by Cu2+, Fe2+, Hg2+, and EDTA. The isolated enzyme released mannose not only from stem bromelin glycopeptide and ovalbumin glycopeptide but also from yeast mannan.View full abstractDownload PDF (846K) -
Masakazu HIRASAWA-SOGA, Goro TAMURA, Shigeo HORIE1983 Volume 94 Issue 6 Pages 1833-1840
Published: December 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSInteractions of ferredoxin-linked nitrite reductase (NiR) from spinach with its substrate were studied by spectrophotometry and electron spin resonance (ESR) spectroscopy. Siroheme was extractable from NiR with 2.5% (w/v) trichloroacetic acid (TCA) and with acetone containing 0.01 N HCl. The addition of nitrite or sulfite to these extracts resulted in shifts of the absorption spectra of siroheme. The HCl-acetone extract showed ESR signals of symmetrical high spin heme, which disappeared on addition of nitrite. Spectral titration indicated a high affinity of extracted siroheme to nitrite and sulfite. The addition of nitrite or sulfite to protoheme dissolved in 0.01 N HCl-acetone did not cause a shift of the absorption spectrum.
The extractability of siroheme with 0.01 N HCl-acetone was suppressed by the addition of nitrite to the NiR preparation. Moreover, a substrate-induced difference spectrum with peaks at about 295 and 287 nm was observed on addition of nitrite to NiR.
These observations indicated an intrinsic strong affinity of siroheme to nitrite and sulfite, formation of rhombicity of siroheme by binding to the protein moiety, and also a probable conformational change of NiR on binding to the substrate.
In agreement with previous reports, ESR signals of the heme-NO complex were observed with NiR in the presence of nitrite, methyl viologen (MV), and dithionite. In the present study, the same signals of similar intensity were also observed on omission of MV, under which conditions no catalytic reduction of nitrite occurred. Furthermore, the signal of the heme-NO complex was not observed when MV was replaced by spinach ferredoxin.
Based on the present results, the roles played by the siroheme prosthetic group and the protein moiety of NiR in determining its substrate specificity are discussed. The possible reason for the dependence of heme-NO signals on the kind of electron donors used is also discussed.View full abstractDownload PDF (676K) -
Yasunori KUSHI, Shizuo HANDA, Hideki KAMBARA, Kenichi SHIZUKUISHI1983 Volume 94 Issue 6 Pages 1841-1850
Published: December 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAcidic glycosphingolipids were analyzed by field desorption (FD-MS) and secondary ion mass spectrometry (SI-MS) using the primary ion Xe+ with a glycerol matrix.
In the analysis of underivatized gangliosides by FD-MS, the fragment corresponding to the asialo residue resulting from the cationized cluster ion (M+Na)+ was the base peak, and ions due to cleavage at the glycosidic linkages were detected, as in the neutral glycosphingolipids. In the case of sulfatide, the ceramide fragment showed the highest intensity in the spectrum. In SI-MS spectra of acidic glycosphingolipids, (M+Na)+, (M+2Na-H)+, and (M+K)+ were continuously detected as relatively high intensity ions during analysis of gangliosides and sulfatide. Other ions were mostly similar to those obtained by FD-MS.
In FD-MS spectra of permethylated gangliosides, the cationized molecular ion (M+Na)+ was the base peak, and fragment ions due to asialo gangliosides were prominent. Other peaks were hard to detect. In SI-MS, molecular ions (M+H)+ and (M+H-32)+ and other ions due to cleavage of the glycosidic linkages were clearly detected. In this case, the sensitivity was greatly improved. Ions due to the non reducing end sugars were clearly detected, because of the relatively low intensity of ion peaks due to the glycerol matrix.
It is concluded that the combination with FD-MS and SI-MS is particularly useful for the determination of molecular weight, sugar sequence and ceramide structure with sample amounting to only a few Fig order.View full abstractDownload PDF (645K) -
Yasuko KAWAMURA, Satoshi NAKAMURA1983 Volume 94 Issue 6 Pages 1851-1856
Published: December 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe kinetics of assembly of oxygenated hemoglobin from isolated α and β chains was investigated under various buffer conditions by use of a circular dichroism (CD) stopped-flow apparatus.
The difference CD spectra of hemoglobin against its constituent chains were independent of the buffer conditions, while the time courses of the Soret CD after mixing equimolar amounts of the α and β chains changed with the buffer conditions. The time courses were analyzed on the basis of a scheme which included a monomertetramer equilibrium of the β chain (β4_??_4β), dissociation of the β4 (β4→4β), and a second-order combination of a and β monomers (α+ β→αβ).
The analysis showed that buffer conditions affected the dissociation of the β4 rather than the monomer combination: The rate of the dissociation of the β4 accelerated with decreasing phosphate concentration, while the rate of the monomer combination was less sensitive to the phosphate concentration. This result indicates that the stability of the β4 depends on the phosphate concentration. It was furthermore suggested that the inorganic phosphate was bound to the β4 with an association constant of 133 M-1 and a Hill coefficient of 1.2.View full abstractDownload PDF (415K) -
Koichiro OMORI, Shigeru TAKEMURA, Kyoko OMORI, Tomohiro MEGA, Yutaka T ...1983 Volume 94 Issue 6 Pages 1857-1866
Published: December 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS(Na+, K+) ATPase from dog kidney was solubilized and denatured by SDS treatment, then applied to a Con A- and WGA-Sepharose column. While the α subunit of the ATPase had no affinity for either of the lectin-Sepharoses, the β subunit specifically bound to WGA-Sepharose and was eluted with N-acetylglucosamine. This property was utilized for the isolation of the a and β subunits by using lectin-Sepharoses and SDS-polyacrylamide gel electrophoresis. The amino acid composition of the α subunit thus isolated was in reasonable agreement with the data reported by Kyte (Kyte, J. (1972) J. Biol. Chiem. 247, 7642-7649). The amino acid and carbohydrate compositions of the β subunit were, however, different from his data. The β subunit contained little histidine (0.1 mol/100 mol amino acid) and a very large amount of carbohydrates (33%). The antibody raised against α or β subunit reacted specifically with the corresponding subunit and with proteasefragmented α subunit and neuraminidase-treated β subunit, respecively, but no cross-reactivity was observed between the two subunits. These results indicate that our α and β subunits were highly purified.View full abstractDownload PDF (3220K) -
Shugo WATABE, Kanehisa HASHIMOTO, Shizuo WATANABE1983 Volume 94 Issue 6 Pages 1867-1875
Published: December 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMyosins were isolated from the ordinary (white) and dark (red) muscles of yellowtail, Seriola quinqueradiata, and the pH-dependency of ATPase activity, along with some physicochemical properties, was examined.
1. The ordinary muscle myosin contained three kinds of light chain (A1, DTNB and A2 light chains), the molecular weights of which were 28, 000, 20, 000, and 16, 000, respectively. The dark muscle myosin possessed only two kinds of light chain (D1 and D2), the molecular weights of which were 26, 000 and 20, 000 respectively. These ordinary and dark muscle myosins resemble the fast and slow muscle myosins of the higher vertebrate, respectively, in light chain pattern.
2. The pH optima of the ordinary muscle myosin Ca2+-ATPase activity appeared at 6-6.5 and 9-10, irrespective of whether the enzyme reaction was started by the addition of ATP to the preincubated reaction mixture containing myosin (method I), or vice versa (method II).
In the case of the dark muscle myosin, a small peak appeared at around pH 8.5 on the alkaline side when the activity was assayed by method I, whereas a prominent peak appeared at around 9.5 when it was assayed by method II, suggesting instability of this myosin under alkaline conditions. In connection with this, the reaction mixture at pH 9.5 showed a very small and slow increase in turbidity, suggesting a change in the physical state of myosin.
3. The ordinary muscle myosin exhibited approximately three times higher actinactivated Mg2+-ATPase activity than the dark muscle myosin. Superprecipitation activity was also higher in the former than the latter actomyosin. However, both actomyosins showed similar pH-superprecipitation activity profiles.View full abstractDownload PDF (1062K) -
Hazime SAITO, Yoshikazu SUGIMOTO, Ryoko TABETA, Sintaro SUZUKI, Giichi ...1983 Volume 94 Issue 6 Pages 1877-1887
Published: December 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have analyzed the manner of incorporation of bile acid into lipid bilayers and resultant perturbation of the bilayer structure with lower bile acid/lipid ratios relevant to the physiological conditions (_??_1mM) by 2H and 31P NMR methods, as an aid to understanding the possible role as an endogenous tumor promoter in colon cancer besides the primary physiological function of solubilizing lipids. On the basis of the 2H quadrupole splittings of [6, 6, 7, 7, 8-2H5] deoxycholate and [11, 11, 12, 12-2H4] chenodeoxycholate in the presence of lamellar multibilayers of egg yolk lecithin, these bile acids were found to be incorporated in such a manner that the B-D rings lie parallel with the normal of the bilayers when the ratio of the bile acid to lipid is low (<0.11). When the ratio is increased, these bile acid molecules are not dispersed entirely in the bilayer but aggregate to form micelles with lipids. Further, we studied the resultant perturbation of the multibilayers of egg yolk lecithin analyzed by using the 2H quadrupole splitting of [18, 18, 18-2H3]stearic acid as a probe and by 31P chemical shift anisotropy. We found that the bilayer structure is retained even at the bile acid-to-lipid ratio of 0.25, although a small amount of an isotropic phase appeared such as small vesicles and micelles. The molecular ordering of fatty acyl chains was rather enhanced by the presence of 1mM deoxycholate in erythrocyte ghosts as seen from the 2H quadrupole splitting of [16, 16, 16-2H3]-palmitic acid, although deoxycholate caused hemolysis in this condition. The former observation can be explained by the way the lipid-protein interaction is modified by deoxycholate located in the interface between the lipids and proteins.View full abstractDownload PDF (736K) -
Tadashi YOSHIMOTO, Futoshi MATSUBARA, Eriko KAWANO, Daisuke TSURU1983 Volume 94 Issue 6 Pages 1889-1896
Published: December 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSProlidase [iminodipeptidase, EC 3. 4. 13. 9] was highly purified from the cytosol fraction of bovine small intestine by a series of column chromatographies on DEAE-Toyopearl, Sephadex G-150, PCMB-T-Sepharose and hydroxyapatite. The puri-fied enzyme appeared homogeneous as judged by disc gel electrophoresis. The enzyme was most active at pH 7.2 with Gly-Pro as substrate. It was stable between pH 5.5 and 8.5 for 30min at 30°C and retained half of the activity after 15 min at 40°C. It was completely inactivated by p-chloromercuribenzoate (PCMB) but not inhibited by diisopropylphosphorofluoridate (DFP), phenylmethane sulfonylfluoride (PMSF) and metal chelators. Its amino acid composition was determined. Its molecular weight was estimated to be 116, 000 by gel filtration on Sephadex G-150 and 56, 000 by sodium dodecyl sulfate (SDS) gel electrophoresis, suggesting that it is a dimer. It hydrolyzed dipeptides represented as X-Pro (X =amino acid).View full abstractDownload PDF (964K) -
Etsuo YOKOTA, Koscak MARUYAMA1983 Volume 94 Issue 6 Pages 1897-1900
Published: December 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBy using isolated actin bundles of brush border microvilli of chicken intestinal epithelial cells, it was clearly visualized that muscle β-actinin caps the pointed end of an actin filament, whereas cytochalasin D masks the barbed end. The growth rate at the barbed end in the presence of β-actinin was markedly slower than in its absence.View full abstractDownload PDF (923K) -
Teruhiro TAKABE, Hiroshi ISHIKAWA, Satsuki NIWA, Shigeru ITOH1983 Volume 94 Issue 6 Pages 1901-1911
Published: December 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTreatment of isolated spinach thylakoid fragments with Triton X-100 followed by repeated sucrose density gradient centrifugations and Sephacryl S-300 and DEAE-Sephacel chromatographies yielded a highly purified P700-chlorophyll α protein complex which consists of five polypeptides. The protein complex is virtually free of chlorophyll b (Chl α/Chl b>10) with approximately 30 chlorophylls per P700, and contains iron-sulfur centers A, B, and X.
At pH values higher than 6, divalent cations, but not monovalent or trivalent cations, efficiently accelerated the electron transfer from reduced spinach plastocyanin to the photooxidized P700 in the P700-chlorophyll α protein complex. At pH values lower than 6, the reaction rate drastically increased with decreasing pH with a maximum at about pH 4.3 without cations. Divalent salts as well as monovalent or trivalent salts decreased the P700 reduction rate at low pH, indicating the involvement of electrostatic interaction in those pH regions. The rate of electron transfer from plastocyanin to the photooxidized P700 in the reaction center protein, which consists of only the largest peptide subunit and no iron-sulfur centers, was reduced only 50% at pH 7.0 in the presence of MgCl2 as compared to the case of P700-chlorophyll α protein complex. Essentially similar effects of pH and metal ions on this electron transfer reaction were observed as in the case of P700-chlorophyll α protein complex. These results strongly suggest that plastocyanin donates electrons directly to the largest peptide of P700-chlorophyll α protein complex and the observed effects of pH and cations are mainly due to the interaction between the largest peptide of P700-chlorophyll α protein complex and plastocyanin. The four small subunits in the protein complex seemed to have only a minor role in the reaction with plastocyanin.View full abstractDownload PDF (1526K) -
Katsufumi SAKYO, Jur, -ichi KOBAYASHI, Akira ITO, Yo MORI1983 Volume 94 Issue 6 Pages 1913-1923
Published: December 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo metal dependent proteases were investigated in rabbit uterus using a synthetic substrate, 2, 4-dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg (Dnp-peptide). One was extracted by homogenization in 50mM Tris-HCl/0.25% Triton X-10D/100mM CaCl2, pH 7.4, from rabbit uterus, and the other from the insoluble fraction by heating at 60°C for 4min in 50mM Tris-HCl/100mM CaCl2, pH 7.4. Both enzymes were partially purified by gel filtration, ion-exchange chromatography and chromatofocusing, and further characterized. The soluble enzyme was a metal dependent peptidase, and hydrolysed 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg as well as Dnp-peptide. Its molecular weight was about 7.0×104, and the cleavage sites for Dnp-peptide were Gln-Gly and possibly Gly-Ile in the ratio of 3:1. On the other hand, the enzyme extracted from the insoluble fraction was present as a latent form, and was found to be activated by 4-aminophenylmercuric acetate but not by trypsin. The activated enzyme hydrolysed gelatin, in addition to Dnp-peptide, indicating that the enzyme is a gelatinase. The molecular weight was about 7.4×104 for the active form, and the cleavage site for Dnp-peptide was only the Gly-Ile bond. The rabbit uterine metal dependent peptidase obtained here had negligible activity on gelatin, but once it had been cleaved by the above gelatinase, the presence of metal dependent peptidase accelerated the action of gelatinase. Thus, the actions of both enzymes on gelatin were found to be synergistic.View full abstractDownload PDF (1487K) -
Mikiharu YOSHIDA, Osamu MINOWA, Koichi YAGI1983 Volume 94 Issue 6 Pages 1925-1933
Published: December 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe Ca2+ binding to plant (wheat germ) calmodulin was measured in 0.1 M NaCl by a flow-dialysis method. The four macroscopic binding constants best fitted to the data were 0.20, 0.25, 0.025, and 0.0024 μM-1. The cysteine residue of this calmodulin is located at the 27 th position from the NH2-terminal (Yazawa, M. et al. (1982) Abstr. 33 th Conf. Protein Structure pp. 9-12, Osaka). According to the quantitative analysis of the reaction of 5, 5'-dithiobis (2-nitrobenzoic acid) (DTNB) with Cys 27, the calmodulin which binds 3 Ca2+ showed the minimum reactivity with DTNB. This suggests that the site for the third Ca2+ binding is located close to Cys 27. Cys 27 was spin-labeled with N-(2, 2, 6, 6-tetramethyl-4-piperidine-1-oxyl)maleimide, and its ESR spectrum was measured in the presence of Mn2+ and/ or Ca2+. The rotational relaxation time of the label (1.2 ns) was increased by about one-tenth with 1 to 2 mol of bound Ca2+, but was unchanged with Mn2+. On the other hand, Mn2+ induced a remarkable quenching of the spectrum. From the decrease in the peak heights of the ESR spectrum, the distance between the label and the first bound Mn2+ was estimated to be 0.8 nm. It is concluded that the first Mn2+ binds to a domain near the NH, -terminal. The difference UV absorption spectrum induced by Mn2+ was similar to that induced by Ca2+. However, the amount of Mn2+ needed to saturate the difference spectrum was 1 mol more than the amount of Ca2+. These results indicate that the high affinity site for Mn2+ is in the NH2-terminal half region, while the sites for Ca2+ are in the COOH-terminal half region.View full abstractDownload PDF (623K) -
Shoji OHKUMA, Yoshinori MORIYAMA, Tatsuya TAKANO1983 Volume 94 Issue 6 Pages 1935-1943
Published: December 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSStudies were carried out on the electrogenicity of the lysosomal proton pump using dipropylthiadicarbocyanine iodide (diS-C3-(5)) as a membrane potential probe. Pure lysosome preparations (tritosomes) quenched the fluorescence of diS-C3-(5). The quenching correlated well with the potassium ion diffusion potential (inside negative) generated by K+ with or without valinomycin. The quenching caused by lysosomes was reversed by lipophilic cations, tetraphenylarsonium (TPA) or triphenylmethylphosphonium (TPMP). Mg-ATP also reversed the quenching, which was inhibited by a protonophore, 3, 5-di-tert-butyl-4-hydroxybenzylidene-malononitrile (SF-6847). The properties of the ATP-induced recovery of the quenching were exactly the same as those of ATP-induced acidification, as measured with fluorescein-isothiocyanate-dextran (FD) (Ohkuma, S., et al. (1982) Proc. Natl. Acad. Sci. U. S. 79, 2758-2762) and acridine orange (Moriyama, Y., et al. (1982) J. Biochem. 92, 1333-1336), except replacement of the anion by an impermeable one enhanced ATP-induced recovery of quenching, but reduced ATP-induced acidification. Amines which dissipate ΔpH across the lysosomal membrane also enhanced the Mg-ATP-induced fluorescence recovery. These results suggest that isolated lysosomes exhibit an inside negative membrane potential, especially in low K+ medium, mostly due to the K+-diffusion potential, and that the Mg-ATP-driven proton pump causes membrane depolarization (in the direction of inside positive). These possibilities were supported by results on the uptake of the radioactive membrane-permeant ions [3H] TPMP and [14C] SCN. The present results provide evidence for the electrogenic nature of the lysosomal proton pump.View full abstractDownload PDF (670K) -
Hidenari TAKAHARA, Yoshihiro OIKAWA, Kiyoshi SUGAWARA1983 Volume 94 Issue 6 Pages 1945-1953
Published: December 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe preceding paper described the identification and some properties of peptidylarginine deiminase, which catalyzes the deimination of arginyl residues in protein, from rabbit skeletal muscle, kidney, brain, and lung. In the present work we purified peptidylarginine deiminase from rabbit skeletal muscle with a 16% yield by 7 steps. The purification involved ion-exchange chromatography on DEAE-Sephacel, gel filtration on Bio-Gel A-0.5 m, and affinity chromatography on soybean trypsin inhibitor-Sepharose 4B and aminohexyl-Sepharose 4B. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate. The molecular weight of the enzyme was estimated to be about 83, 000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis and 130, 000-140, 000 by gel filtration on Sephadex G-200. The isoelectric point was 5.3 and the amino acid composition was also determined. The enzyme preferably catalyzed the formation of citrulline derivatives from arginine derivatives in which both the amino and carboxyl groups were substituted and showed the highest activity towards Bz-L-Arg-O-Et among the arginine derivatives tested. The Km value for Bz-L-Arg-O-Et was found to be 0.50×10-3 M. The enzyme also showed marked activities towards native protein substrates, such as protamine sulfate, soybean trypsin inhibitor, histone and bovine serum albumin.View full abstractDownload PDF (1305K) -
Masato UMEDA, Shoshichi NOJIMA, Keizo INOUE1983 Volume 94 Issue 6 Pages 1955-1966
Published: December 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe HVJ-mediated interaction of liposomes containing glycophorin with human erythrocytes was studied. A ternary complex of erythrocyte-HVJ-liposome was formed on incubation at 4°C. Spin-labeled phosphatidylcholine and spin-labeled galactosylceramide, both of which were incorporated into liposomes, were effectively transferred into erythrocytes upon shifting-up of the incubation temperature to 37°C. This finding indicates that fusion between glycophorin liposomes and erythrocytes occurs in the presence of HVJ. Fusion could be also inferred from the transfer of radio-labeled lipids embedded in the liposomal membranes to erythrocyte membranes as well as uptake of solutes entrapped in the aqueous space of the liposomes by erythrocytes. The total amount of liposomes fused with erythrocytes depended on the glycophorin content of liposomes and the amount of HVJ added. No transfer was observed when trypsinized HVJ was used in place of HVJ, suggesting that the fusion is F protein dependent.View full abstractDownload PDF (1008K) -
Kazuyuki NAGAMATSU, Yuji MIYAZAWA1983 Volume 94 Issue 6 Pages 1967-1971
Published: December 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe A76 or A73 nucleotide at the 3'end of tRNAPhe was modified with the fluorescent reagent of proflavine (PF). The distance between the fluorophore of the 3'end and the Y base was measured by singlet-singlet energy transfer under the conditions of 10mM and 0.01mM Mg2+. The distance obtained at 10mM Mg2+ is very close to that obtained by the X-ray diffraction method, while the distance at 0.01mM Mg2+ is significantly smaller. The difference in the distance is explained as a result of destabilization of the tertiary structure with reduction of the Mg2+ concentration. The calculated distance between A73 and A76 shows the stacked conformation of the CCA strand. Fluorescent quenching experiments showed that the degree of stabilization of 3'end A76 by stacking is lower than that of A73. The removal of the CCA segment causes a difference not only in the thermal melting curves but also in the fluorescent wavelength of the Y base at 0.01mM Mg2+. The results suggest that the 3'end CCA strand has a helical structure and contributes to the stabilization of the whole structure of tRNAPhe.View full abstractDownload PDF (385K) -
Shin NAKAMURA, Osamu TAKENAKA, Kenji TAKAHASHI1983 Volume 94 Issue 6 Pages 1973-1978
Published: December 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAmino acid sequences of fibrinopeptides A and B from savanna baboons, Papio anubis and Papio hamadryas, and highland baboon, Theropithecus gelada, were established. The sequences of the fibrinopeptides A from the three baboons were identical: Ala-A_??_p-Thr-Gly-Glu-Gly-A_??_p-Phe-Leu-Ala-Glu-G_??_y-Gly-Gly-Val-A_??_g. The fibrinopeptides B were composed of 9 residues and demonstrated the sequence: Asn-Gln-Glu-Gly-L_??_u-Phe-_??_-Gly-A_??_g, where_??_=Arg in P. anubis, His in P. hamadryas, and Gly in Th. gelada. Position-3 of the B peptides was the only replacement site observed among the 25 amino acid residues in both fibrinopeptides from the baboons. Based on these sequences, a molecular phylogeny for the three species of baboons was deduced. The evolutionary rates of the peptides B of the baboons and macaques were also estimated. It was observed that the fibrinopeptides changed at an uneven rate during the evolution of old-world monkeys, i.e., baboons and macaques.View full abstractDownload PDF (419K) -
On the Structures of Flavoprotein D-Amino Acid Oxidase Purple Intermediates. A Resonance Raman StudyYasuzo NISHINA, Kiyoshi SHIGA, Retsu MIURA, Hiromasa TOJO, Miho OHTA, ...1983 Volume 94 Issue 6 Pages 1979-1990
Published: December 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSResonance Raman (RR) spectra were obtained in H2O or D2O solution for the purple intermediates of n-amino acid oxidase (DAO) with isotopically labeled substrates, i.e., [1-13C]-, [2-13C]-, [3-13C]-, [15N]-, and [3, 3, 3-D3]alanine; [carboxyl-13C]- and [15N]proline. RR spectra were also measured for the intermediates of DAO reconstituted with isotopically labeled FAD's, i.e., [4a-13C]-, [4, 10a-13C2]-, [2-13C]-, [5-15N]-, and [1, 3-15N2] FAD in D2O. The isotopic shift of the 1692 cm-1 band upon [15N]- or [2-13C]-substitution of alanine indicates that the band is due to the C=N stretching mode of an imino acid derived from n-alanine, i.e., α-imino-propionate. The 1658 cm-1 band with D-proline was also assigned to the C=N stretching mode of an imino acid derived from D-proline, i.e., Δ1-pyrrolidine-2-carboxylate, since the band shifts to 1633 cm-1 upon [15N]-substitution and its stretching frequency is generally found in this frequency region. Since the band shifts to low frequency in D2O, the imino acid should have a protonated imino group such as the C=H form. The intense band at 1363 cm-1 with D-alanine was assigned to a mixing of the CO2- symmetric stretching and CH3, symmetric deformation modes in a-iminopropionate, based on the isotope effects. The 1359 cm-1 band with D-proline has probably contributions of CO2- symmetric stretching and CH2 wagging, considering the isotope effects with [carboxyl-13C]proline. The 1359 cm-1 band with D-proline was split into 1371 cm-1 and 1334 cm-1 bands in D2O. As this splitting of the 1359cm-1 band with D-proline in D2O can not be interpreted only by the replacement of the C=-H proton by deuterium, the carboxylate of the imino acid probably interacts with the enzyme through some proton (s) exchangeable by deuterium (s) in D2O. The bands around 1605 cm-1 which shift upon [4a-13C]- and [4, 10 a-13C2]-labeling of FAD are derived from a fully reduced flavin, because the isotopic shifts of the band are very different from those of the bands of oxidized or semiquinoid flavin observed near 1605 cm-1. These features of RR spectra suggest that the plane containing C(l0a)=C(4a)-C(4)=O of reduced flavin and the plane containing H-N=C-COO- of an imino acid face each other in close contact for the generation of the charge transfer interaction between them, and that the CO2- of an imino acid interacts with the enzyme through some proton (s).View full abstractDownload PDF (858K) -
Eiichi SAITOH, Satoko ISEMURA, Kazuo SANADA1983 Volume 94 Issue 6 Pages 1991-1999
Published: December 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThree basic proline-rich peptides were newly isolated from human parotid saliva, and designated as P-G, P-H, and P-I. The amino acid sequence of P-H was determined to be Ser-Pro-Pro-Gly-Lys-Pro-Gln-Gly-Pro-Pro-Gln-Gln-Glu-Gly-Asn-Asn-Pro-Gln-Gly-Pro-Pro-Pro-Pro-Ala-Gly-Gly-Asn-Pro-GIn-GIn-Pro-GIn-AIa-Pro-Pro-Ala-Gly-Gln-Pro-Gln-Gly-Pro-Pro-Arg-Pro-Pro-GIn-Gly-G ly-Arg-Pro-Ser-Arg-Pro-Pro-Gln by conventional methods. The amino terminal ten residues of P-H were the same as those of proline-rich peptides P-D, P-E, and P-F reported previously. Comparison of the amino acid sequences between P-H and P-D revealed that there are two deletion parts and several amino acid substitutions in the sequence of P-H. Homology between P-H and P-D was as high as 70%.View full abstractDownload PDF (1272K) -
Shigeru UTSUMI, Tomohiko MORI1983 Volume 94 Issue 6 Pages 2001-2008
Published: December 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe constituent subunits of 11S globulin of broad bean, legumin, were separated into basic subunits (BS, a mixture of BSI, BSII, and BSIII) and acidic subunits (ASI, ASII, and ASIII). The 11S components were formed in reconstitution reactions from combinations of BS and one or two each of the acidic subunits. The reconstituted 11S components were similar to the native legumin; they all consisted of acidic (A) and basic (B) subunits linked by disulfide bridges in the ratio of 1:1 and had the 6 (AB) structure. In the reconstitution of 11S components, ASI preferentially selected BSI from among the three kinds of BS, and ASH and ASIII exhibited selectivities for BSII and BSIII, respectively. The same selectivities were observed in the reconstitution reaction containing all subunits and in the renaturation reaction from the reduced-denatured state. The selectivity of each acidic subunit for basic subunits coincides with the combination of acidic and basic subunits in the native legumin. The 11S component was reconstituted from any combination of the intermediary subunits examined. This may be one of the reasons for the occurrence of heterogeneity of legumin molecular species.View full abstractDownload PDF (3264K) -
Keiichiro HASHIMOTO, Keizo INOUE, Shoshichi NOJIMA, Takushi TADAKUMA, ...1983 Volume 94 Issue 6 Pages 2009-2014
Published: December 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe binding of liposomes sensitized with 2, 4-dinitrophenyl-6-N-aminocaproylphos-phatidylethanolamine (DNP-Cap-PE) to MOPC-315 cells, which secrete and bear on their surfaces anti-DNP immunoglobulins, was studied.
The binding was affected by cholesterol content, phospholipid composition and hapten density of liposomes: The binding of distearoylphosphatidylcholine liposomes sensitized with 5 mol% hapten to the cells increased with increasing cholesterol content in liposomes. The amount of liposomes composed of phospholipid with a higher transition temperature (such as distearoylphosphatidylcholine), which bound to MOPC-315 cells, was much higher than that of liposomes composed of phospholipid with a lower transition temperature (such as egg yolk phosphatidyl-choline). The amount of distearoylphosphatidylcholine liposomes containing equimolar cholesterol, which bound to the cells at 0°C, increased with increasing amount of the hapten in liposomes up to 2.5 mol%. The binding became maximum at 2.5 mol% and decreased with higher hapten density in liposomes. The immunogenicity of hapten-sensitized liposomes is known to be affected by the liposomal composition such as cholesterol content, phospholipid composition and hapten density. This model study suggests that the binding of liposomes to cells is important for expressing the immunogenicity of hapten-sensitized liposomes.View full abstractDownload PDF (509K) -
Masatoshi KATO1983 Volume 94 Issue 6 Pages 2015-2022
Published: December 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHeparin, a naturally occurring mucopolysaccharide, was found to be a potent inhibitor of L-arginyl-tRNA:protein arginyltransferase [EC 2. 3. 2. 8]. It was demonstrated that heparin was not an acceptor of arginine, but an inhibitor competing with arginyl-tRNA. The K1, value of heparin was 1.5 μm, while the Km value of arginyl-tRNA was 0.5 μM. The inhibition was specific for heparin among the mucopolysaccharides examined. The N-sulfate groups were indispensable for heparin to inhibit the arginyltransferase activity. Arginyltransferases from various origins were commonly inhibited by heparin. Polyamines such as spermine and spermidine were also found to inhibit the transferase activity, competing with arginyl-tRNA. An extremely high specific activity of 2.3 μmol/min/mg was achieved by the application of these inhibitors to the purification of the transferase by affinity chromatography.View full abstractDownload PDF (575K) -
Yasufumi MINAMI, Hikoichi SAKAI1983 Volume 94 Issue 6 Pages 2023-2033
Published: December 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe previous paper (Minami, Y., et al. (1982) J. Biochem. 92, 889-898) demonstrated that the neurofilament preparation obtained from porcine brain promotes tubulin polymerization, thereby leading to network formation in vitro. Since this preparation fractionated by gel filtration and centrifugation was contaminated mainly by tubulin, the neurofilaments thus prepared were solubilized in a 6M urea solution, further purified by hydroxyapatite column chromatography, and reconstituted into neurofilaments. This highly purified neurofilament was found to retain the ability to stimulate microtubule assembly and to cause gelation. Next we separated the purified neurofilament into the individual triplet subunits, referred to as 200 K, 150 K, and 70 K proteins, by DEAE-cellulose (DE-52) column chromatography in the presence of 6M urea. By measuring viscosity and turbidity changes, it has been found that the activity to stimulate polymerization of tubulin is due to the 200 K polypeptide, while the 150 K protein was less active to promote tubulin polymerization and the 70 K protein was totally inactive.View full abstractDownload PDF (2749K) -
Hideaki OTSUKA, Akemi SUZUKI, Tamio YAMAKAWA1983 Volume 94 Issue 6 Pages 2035-2041
Published: December 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn a previous paper (Otsuka, H. & Yamakawa, T. (1981) J. Biochem. 90, 247-254), we reported the separation of acidic glycolipids by droplet counter-current chromatography using 500 columns with a commercially available DCC apparatus and described the precise conditions of the separation. In this paper, separation of neutral glycolipids from rabbit and human erythrocytes by DCC is described. For efficient separation of neutral glycolipids, addition of a definite amount of benzene was required. The solvent system of chloroform: benzene: methanol: water=50:50:70:20 gave clear separation of these glycolipids and further modification of this solvent system to chloroform:benzene:methanol:water=50:25:65:30 gave satisfactory results for the separation of Globoside I from human erythrocytes due to the difference between the amide linked fatty acids. Also under the same conditions, the clear separation of two peaks with blood group A activity was demonstrated by a hemagglutination inhibition test.View full abstractDownload PDF (1054K) -
Yasuhiro HASHIMOTO, Akemi SUZUKI, Tamio YAMAKAWA, Nobumoto MIYASHITA, ...1983 Volume 94 Issue 6 Pages 2043-2048
Published: December 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSGM2 containing NeuGc was a major ganglioside in the liver of most inbred strains of mouse; A/J (H-2a), C57BL/10 (H-2b ), BALB/c (H-2d), C3H/He (H-2k), etc., but GM1 (NeuGc) and GD1a (NeuGc) in addition to GM2 (NeuGc) were detected in the liver of several strains such as SWM/Ms, SJL/J (H-2S), SL/QDJ (H-2s), SWR/J (H-2s), PL/J (H-2u), and RFM/Ms (H-2f) and a wild-derived strain as well, M. Cas-Qzn (H-2wcl, tentative designation). GM1 (NeuGc) and GDla (NeuGc) were also expressed in the liver of several strains of H-2 congenic mouse; B10. Cas-Qzn (H-2wcl), B10. S (H-2s), B10. G (H-2q), A. SW/Sn (H-2s), and C3H. JK (H-2J), although the respective inbred-partner strains had only GM2 (NeuGc) as a major component. This difference of the ganglioside composition between H-2 congenic and inbred-partner strains suggests that a gene for expression of GM1(NeuGc) and GDla (NeuGc) is closely linked to the H-2 complex. The locus was mapped at the left outside the H-2 complex on chromosome 17 by analysis of H-2 recombinant mice.View full abstractDownload PDF (1106K) -
Osamu OTSURU, Hideaki OTSUKA, Takeshi KASAMA, Yousuke SEYAMA, Tatsuo S ...1983 Volume 94 Issue 6 Pages 2049-2054
Published: December 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA major lipid class in the Harderian glands of the Mongolian gerbil was investigated. The IR and 1H-NMR spectra suggested that it was a wax-like compound. Fatty acids present were capric, lauric, myristic, palmitic, stearic, arachidic, and behenic acids in ratios of 3.0%, 16.3%, 16.5%, 29.5%, 5.1%, 16.4%, and 9.8%, respectively. Odd-numbered, branched chain and unsaturated fatty acids were not present in large amounts. The structure of the alcohol moiety was elucidated to be 2, 3-alkanediol with carbon chain lengths from C12, to C22 by GC-MS of the TMS and isopropylidine derivatives. Lemieux-von Rudloff oxidation of these alcohols confirmed the 2, 3-diol structure, giving fatty acids two carbon units shorter.View full abstractDownload PDF (372K) -
Takashi SASAKI, Nagahisa YOSHIMURA, Takanobu KIKUCHI, Michiyo HATANAKA ...1983 Volume 94 Issue 6 Pages 2055-2061
Published: December 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe structural relationship between calpain I (low Ca2+-requiring) and calpain II (high Ca2+-requiring) molecules and their respective larger (80 K) and smaller (30 K) subunit proteins of several non-muscular tissues and cells was studied by testing immunological cross-reactivities. In addition to qualitative analyses by a conventional double immunodiffusion method, quantitative data were obtained, for the first time, by enzyme-linked immunosorbent assays using affinity-purified anticalpain I and anti-calpain II immunoglobulins. The enzyme sources included rat kidney, porcine kidney and erythrocytes, and human erythrocytes. It was concluded that the 30 K subunits are immunologically almost indistinguishable between calpains, either I or II, not only from the same but also from different sources, while the 80 K subunits of different origins are immunologically related to variable extents but always with discrimination between calpain I and calpain II. Similarity of the 30 K subunit proteins and dissimilarity of the 80 K counterparts were further substantiated by their chromatographic and electrophoretic behaviors.View full abstractDownload PDF (1194K) -
Morio SETAKA, Noriko SATOH, Takao KWAN1983 Volume 94 Issue 6 Pages 2063-2066
Published: December 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA spin label technique was applied to examine the alignment of platelet membranes in the platelet-fibrin clot. The ESR spectrum of the clot in the control experiment was typical of isotropically oriented membranes, while the ESR spectrum of the clot which was kept isometric was quite different from that of the control experiment.
These spectra were simulated with a computer and explained to indicate that the shape of platelets in the clot was transformed to a cylindrical form under the isometric tension so that axes of all membrane cylinders were directed parallel to the generated force.View full abstractDownload PDF (279K) -
Kiyoshi SATOUCHI, Makoto ODA, Kojiro YASUNAGA, Kunihiko SAITO1983 Volume 94 Issue 6 Pages 2067-2070
Published: December 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe selected ion monitoring (SIM) technique was applied to determination of platelet-activating factor (PAF) or acetyl glyceryl ether phosphorylcholine (AGEPC). Two types of PAF, 1-hexadecyl- and 1-octadecyl-2-acetyl-sn-glyceryl-3-phospho-rylcholine (C16=0 AGEPC and C18=0 AGEPC), were found in human neutrophils on the challenge with ionophore A23187. The contents of C16=0 AGEPC in 1×107 neutrophil cells of four volunteers, respectively, were 47, 18, 59, and 73 ng and those of C18=0 AGEPC were 22, 4, 19, and 31 ng.View full abstractDownload PDF (247K) -
Tetsuji HIRAO, Kaoru HARA, Kenji TAKAHASHI1983 Volume 94 Issue 6 Pages 2071-2074
Published: December 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe substrate specificity of calcium-activated neutral protease (CANP) from monkey cardiac muscle was examined with various neuropeptides as substrates. The enzyme required mm order calcium ions for activation and had an enkephalinase activity, hydrolyzing Leu-enkephalin at the 1Tyr-2Gly and 3Gly-4Phe bonds. Furthermore, it showed the tendency to cleave especially the bonds around the paired basic amino acid residues in α- and β-neoendorphins and dynorphin (1-13), while it could not hydrolyze substance P.View full abstractDownload PDF (282K) -
Keiichi YAMAMOTO, Takamitsu SEKINE1983 Volume 94 Issue 6 Pages 2075-2078
Published: December 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo determine the spatial relationship between alkali light chain and actin in the actosubfragment-1 complex, we studied the cross-linking of actin and subfragment-1 with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. We found that (a) alkali light chain 1 was cross-linked to actin at two sites in the extrapeptide region, and (b) cross-linking of these two sites, especially the one which was very close to the NH2 terminal of the alkali light chain, to actin was inhibited drastically when the KCl concentration was increased from 0 to 100mM. Since the inhibition of cross-linking with carbodiimide reagent means separation of amino and carboxyl groups in alkali light chain and actin, we suggest that this decrease in electrostatic attraction is the reason why subfragment-1 with alkali light chain I has higher affinity to actin than subfragment-1 with alkali light chain 2 at low ionic strength but has almost the same affinity at moderate ionic strength.View full abstractDownload PDF (1771K) -
Hiroshi HOMMA, Tetsuyuki KOBAYASHI, Yuri ITO, Ichiro KUDO, Keizo INOUE ...1983 Volume 94 Issue 6 Pages 2079-2081
Published: December 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSE. coli bearing hybrid plasmid pKOI (Oeda et al. (1981) Mol. Gen. Genet. 184, 191-199) expressed a large amount of lysophospholipase L2 activity. When a mutant which was defective in lysophospholipase L2 activity was transformed with plasmid pKOI, it overproduced lysophospholipase L2 activity. The gene responsible for the lysophospholipase L2 activity was designated as pld B. On the same hybrid plasmid another gene (pld A) coding for detergent-resistant phospholipase A (DR-phospholipase A) was also identified. These facts together with the results of a Pl transduction experiment revealed that the pld B gene must be between the pld A and met E genes on the E. coli chromosome.View full abstractDownload PDF (216K) -
Sumiko KIMURA, Koscak MARUYAMA1983 Volume 94 Issue 6 Pages 2083-2085
Published: December 01, 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSConnectin, an elastic protein of striated muscle, was isolated in a native state from chicken breast muscle. Myofibrils were well washed with 5mM NaHCO3 and extracted with 0.1M sodium phosphate buffer, pH 5.6. After washing with water, myofibrils were extracted with 0.1M phosphate buffer, pH 6.6. The filtrate contained native connectin. The yield of connectin was as high as 200mg, starting from 50g of muscle. Since connectin had been isolated only in a denatured state before, the new preparation method enables investigation of possible interactions of connectin with other myofibrillar structural proteins.View full abstractDownload PDF (709K)
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