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Volume 98 (1985)
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Volume 98, Issue 4
Displaying 1-35 of 35 articles from this issue
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Sumihiro HASE, Norihisa KIKUCHI, Tokuji IKENAKA, Kozo INOUE1985 Volume 98 Issue 4 Pages 863-874
Published: October 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHuman C 3, the third component of human complement, contained mannose and N-acetylglucosamine as sugar components. The sugar chains were liberated from the polypeptide chains by hydrazinolysis, and the free amino groups were N-acety-lated. The reducing end residues of the sugar chains thus obtained were tagged with 2-aminopyridine, and the pyridylamino (PA-) derivatives of sugar chains were separated by high-performance liquid chromatography. The structures of purified PA-sugar chains were analyzed by a combination of stepwise exoglycosidase digestions, size determination by paper electrophoresis, methylation analysis, Smith degradation, and partial acetolysis. These results showed that C 3 contained two high-mannose type sugar chains ranging from Man5GlcNAc2 to Man9GlcNAc2. Analyses of the sugar chains of α- and β-chains of C 3 indicated that the α-chain contained mainly Man8GlcNAc2 and Man9GlcNAc2, while the β-chain contained mainly Man5GlcNAc2 and Man6GlcNAc2.View full abstractDownload PDF (1790K) -
Kazuyuki SUGAHARA, Shigeyuki FUKUI, Ikuo YAMASHINA1985 Volume 98 Issue 4 Pages 875-885
Published: October 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSulfation of glycosaminoglycans (GAGs) secreted by baby hamster kidney (BHK) cells and the polyoma virus-transformants (PY-BHK) was investigated. It has been reported that chondroitin sulfate (CS) of cell membranes from PY-BHK cells is undersulfated compared to that from BHK cells (Cancer Res. 43, 2712-2717, 1983). In the first series of experiments of the present study, cells were incubated with [3 H] glucosamine and [35 S] sulfate, and GAGs isolated from the culture medium were examined. GAG composition was comparable between the BHK and PY-BHK cultures. Disaccharide analysis of the chondroitinase ACII digests of the hyaluronate lyase-resistant materials showed a high proportion (68% for BHK and 47% for PY-BHK) of ΔDi-0 S, with ΔDi-4 S (32% for BHK and 53% for PY-BHK) as the major sulfated disaccharide on the basis of 3H-radioactivities. The β-D-xyloside treatment did not alter the degree of undersulfation of the CS of either culture. In the second series of experiments, disaccharide analysis of the chon-droitinase ABC digests of unlabeled GAGs demonstrated similar disaccharide composition for the two cell types. The BHK and PY-BHK preparations showed 28 and 17% (mol percent) of ΔDi-0 S, 58 and 72% of ΔDi-4 S, and 14 and 11% of ΔDi-6 S, respectively. These results indicate a considerable degree of undersulfation of secretory CS from both cells, and a slightly higher degree, if any, of under-sulfation of secretory CS from BHK cells if compared between the two cell types, which is in contrast to the results reported for membrane CS.View full abstractDownload PDF (936K) -
Takao TAKI, Hidemi ISHIKAWA, Kozo IMAI, Akira YACHI, Makoto MATSUMOT1985 Volume 98 Issue 4 Pages 887-895
Published: October 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSGlycolipids of human cell lines of colonic adenocarcinoma (Colo 205 and BM 314), gastric tumor (AZ 521 and KATO-III), and lung tumor (A 549) were studied by the immunohistochemical fluorescence technique, flow cytometric analysis and immunostaining on thin layer chromatoplates with antibodies against ganglio-triaosylceramide (Gg3Cer), gangliotetraosylceramide (Gg4Cer), fucogangliotetrao-sylceramide (Fuc-Gg4Cer), blood group B active lipid, globopentaosylceramide (Gb5Cer) and lactoneotetraosylceramide (nLc4Cer). Anti-nLc4Cer antibody was the only antibody which reacted with all the tumor cell lines used.
The glycolipid fractions of each cell line separated by Iatrobeads column chro-matography were immunostained with the six antibodies mentioned above on thin layer plates. The presence of nLc4Cer was detected in all cell lines. On the other hand, Gg4Cer was detected in gastric tumor cell lines, and Gg3Cer was detected in AZ 521. Based on these results, the tumor cell lines were analyzed by flow cytometry using anti-nLc4Cer antibody. About 70% of total cells in each cell line were separated as nLc4Cer-expressing cells. The present findings, together with the occurrence of nLc4Cer in ascitic fluids of cancer patients (Taki, T., Kojima, S., Seto, H., Yamada, H., & Matsumoto, M. (1984) J. Biochem. 96, 1257-1265), suggest that nLc4Cer may be a tumor-associated lipid.View full abstractDownload PDF (3966K) -
Naoyuki MURAKAMI, Tetsuya KONISHI1985 Volume 98 Issue 4 Pages 897-907
Published: October 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effects of N, N'-dicyclohexylcarbodiimide (DCCD) on light-induced H+-trans-port and transmembrane electric potential (Δψ) formation were studied in the membrane vesicles of Halobacterium halobium R 1 M 1. In accordance with our previous finding of the existence of two DCCD-binding components in vesicle membrane using 14C-DCCD (Konishi & Murakami FEBS Lett. 169, 283-286 (1984)), DCCD inhibited the H+-influx process biphasically; that is, the H+-influx process which is electrically silent was initially inhibited at concentrations below 30 nmol of DCCD/mg vesicle protein, while another H+-influx process which is coupled to Δψ formation was secondarily inhibited above this concentration of DCCD. The latter H+-influx process was highly dependent on the Na+ concentration. The extents of Na+-dependent recovery of Δψ formation and H+-influx were quantitatively correlated. From these results, it was concluded that the second DCCD-sensitive H+-influx process which is coupled to Δψ formation is due to the hypothetical Na+/H+-antiporter postulated by Lanyi and MacDonald (Biochemistry 15, 4608-4614 (1976)). It was also found that Li+ can be substituted for Na+ in this system, as is the case with Na+/H+-antiporters found in other organisms.View full abstractDownload PDF (774K) -
Yoichi OHBA, Yusuke MIZUNO, Toshihide TAKASAWA, Toshihide TAKASAWA, Hi ...1985 Volume 98 Issue 4 Pages 909-919
Published: October 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo acylphosphatases, named Ch1 and Ch2, have been purified from chicken skeletal muscle. The molecular weights were determined to be 11, 900 and 12, 000 for Ch1 and Ch2, respectively, by sedimentation equilibrium. In the amino acid compositions of Ch1 and Ch2, two residues of histidine were contained in Ch2, but none in Ch1, and one residue of cysteine was contained in Ch1, but none in Ch2. There were 11 lysines and 6 arginines in Ch1, whereas there were 6 lysines and 11 arginines in Ch2. In addition, the contents of methionine, serine, glutamic acid and glutamine, and alanine were considerably different between Ch1 and Ch2. There were also differences in the peptide maps and carboxyl-terminal amino acid sequences (-Ser-Thr-Arg-Tyr-COOH for Ch1, and -Phe-Thr-Ile-Arg-Lys-COOH for Ch2). In the double immunodiffusion, Ch2 did not form a precipitin line with the rabbit anti-Ch1 antiserum. These results indicate that Ch1 and Ch2 are different, genetically specified isozymes of acylphosphatase of chicken skeletal muscle. Ch2 is considered to be a new type of acylphosphatase from skeletal muscle.View full abstractDownload PDF (3029K) -
Toshiharu YAGI, Jun KIRINO, Sinpei YAMAMOTO, Mitsuhiro NOZAKI1985 Volume 98 Issue 4 Pages 921-926
Published: October 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA simple and rapid preparation method for apoaspartate aminotransferase from Escherichia coli B was developed. A crude extract of the bacterial cells was treated batchwise with DEAE-cellulose. The enzyme fraction obtained was then applied to a pyridoxamine-Sepharose column. Apoaspartate aminotransferase was eluted with 50mM potassium phosphate buffer (pH 7.0), and found to be electrophoretically homogeneous. The apoenzyme preparation thus obtained showed very low holoenzyme activity (only 0.4% of the activity seen in the fully saturated condition with pyridoxal 5'-phosphate) and was successfully used for assaying pyridoxal and pyridoxamine 5'-phosphate.View full abstractDownload PDF (1110K) -
Tsuneo IMANAKA, Masako YAMAGUCHI, Shoji OHKUMA, Tatsuya TAKANO1985 Volume 98 Issue 4 Pages 927-931
Published: October 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe hydrolysis of tri-, di-, and monooleoylglycerols by highly purified lysosomal acid lipase from rabbit liver (Imanaka, T., et al. (1984) J. Biochem. 96, 1089-1101) was examined. The degradative products were separated by thin layer chromatography on silicic acid impregnated with boric acid. Trioleoylglycerol was hydrolyzed with intermediate accumulation of 1, 2 (2, 3)-dioleoylglycerol and 2-monooleoylglycerol, but no detectable production of the 1, 3-isomer. 1, 2 (2, 3)-Dioleoylglycerol was also hydrolyzed with the intermediate accumulation of 2-monooleoylglycerol. The 1 (3)-isomer of monooleoylglycerol was hydrolyzed exclusively, with no hydrolysis of the 2-isomer. These results indicate that the enzyme shows preference for 1 (3)-ester bonds and that the main lipolytic reaction sequence catalyzed is triacylglycerol---1, 2 (2, 3)-diacylglycerol---2-monoacylglycerol.View full abstractDownload PDF (373K) -
Ruka TAKEUCHI, Tsuneo IMANAKA, Shoji OHKUMA, Tatsuya TAKANO1985 Volume 98 Issue 4 Pages 933-938
Published: October 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCholesterol oleate liquid crystals were prepared in vitro as a model of lipid droplets accumulated in the atheromatous aorta. The hydrolysis of cholesterol oleate crystals by lysosomal acid lipase was examined in the presence and absence of various phospholipids. Phosphatidylserine markedly stimulated the hydrolysis of cholesterol oleate liquid crystals (20 times the basal value); it increased the Vmax value about 15 times and decreased the Km value to 1/20 times the basal value. The polar head group and the acyl chains of phosphatidylserine were required for its stimulation of hydrolysis.View full abstractDownload PDF (929K) -
Shigeru CHAEN, Mitsuyo SHIMADA, Haruo SUGI1985 Volume 98 Issue 4 Pages 939-947
Published: October 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFluorescence spectra of ANM-labeled, glycerinated rabbit psoas muscle fibers were recorded in relaxed, contracted, and rigor states. SDS polyacrylamide gel electrophoresis of the ANM-labeled muscle fibers indicated that proteins labeled with ANM were myosin heavy chain, C protein, and actin. In a relaxed state in the presence of ATP, myosin heavy chain was mainly labeled. During the transition from rigor to the relaxed or contracted state, there was a blue shift (about 5 nm) of the ANM emission spectrum. Similar experiments with FAM (N-(3-fluoranthyl)-maleimide)-labeled muscle fibers showed that these fluorescence changes were not artifacts due to the movement of muscle fibers. The fibers labeled in the ATP relaxing solution showed a marked decrease in both isometric force and unloaded shortening velocity (V0), while in the fibers labeled in the rigor solution isometric tension was not markedly suppressed, though V0 decreased to the same extent as in the fibers labeled in the ATP relaxing solution. Fluorescence spectra of ANM-labeled HMM in different states were also measured. A fluorescence enhancement and a blue shift (about 5 nm) of the emission maximum were observed in HMM+ MgATP or HMM+MgATP+F-actin in comparison with HMM+F-actin. These results suggest that the fluorescence spectra of the ANM-labeled muscle fibers reflect their conformational changes between the rigor state (in the absence of MgATP) and the relaxed or contracted state (in the presence of MgATP).View full abstractDownload PDF (980K) -
Hajime FUJIO, Yutaka TAKAGAKI, Youn-Mun HA, Elvira Missako DOI, Amin S ...1985 Volume 98 Issue 4 Pages 949-962
Published: October 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo types of antibodies were differentiated in conventional guinea pig anti-hen egg-white lysozyme (HEL) antisera. The specificities of both antibodies were directed to the loop I region (mainly directed to Cys64-Cys80 loop) but the antibodies were distinct in respect of reactivities with native HEL. One type of antibody reacted with HEL and loop-peptides of HEL but not with the completely reduced and carboxymethylated form of loop-peptides (native conformation specific antibody: NC-Ab). On the other hand, the other type of antibody did not react with HEL but reacted with loop-peptides and also with the completely reduced and carboxymethylated form of loop-peptides (non-native conformation specific antibody: NNC-Ab). The percentage of NNC-Ab in loop I reactive antibody fraction from pooled guinea pig anti-HEL antisera obtained by two different immunization methods was about 25%. Since the affinities of the NNC-Ab to loop-related peptides were higher by one order of magnitude than those of the NC-Ab to the same peptides, care is necessary in evaluating antigenic determinants in native protein.
The immunization of guinea pigs with Ploop I•II [sequence 57-107 (Cys 64-Cys 80, Cys 76-Cys 94)] evoked an antibody population having specificity similar to but not identical with that of the NNC-Ab type anti-loop I antibody in conventional anti-HEL antisera.View full abstractDownload PDF (1244K) -
Virinder K. MOUDGIL, Shaun P. HEALY, Virendra B. SINGH1985 Volume 98 Issue 4 Pages 963-973
Published: October 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAliquots of rat liver cytosol glucocorticoid-receptor complexes (GRc) were transformed by an incubation with 8-10mM ATP at 0°C and were compared with those transformed by an exposure to 23°C. The extent of receptor transformation was measured by chromatography of the samples over columns of DEAE-Sephacel. The ATP-transformed complexes, like those which were heat-transformed, exhibited lower affinity for the positively charged ion-exchange resin and were eluted with 0.12M KCI (peak-I): the nontransformed complexes appeared to possess higher affinity and required 0.21M KCI (peak II) for their elution. As expected, the receptor in the peak-I exhibited the DNA-cellulose binding capacity and sedimented as 4S in sucrose gradients. Peak II contained an 8-9S glucocorticoid receptor (GR) form that showed reduced affinity for DNA-cellulose. Presence of sodium tungstate (5mM) prevented both heat and ATP transformation of the GRc resulting in the elution of the complexes in the region of nontransformed receptors. When parallel experiments were performed, binding of the cytosol GRc to rat liver nuclei or DNA-cellulose was seen to increase 10-15 fold upon transformation by heat or ATP: tungstate treatment blocked this process completely. The transformed and nontransformed GRc were also differentially fractionated by (NH4)2SO4: tungstatetreated (nontransformed) receptor required higher salt concentration and was precipitated at 55% saturation. In addition, the GRc could be extracted from DNA-cellulose by an incubation of the affinity resin with sodium tungstate resulting in approximately 500-fold purification of the receptor with a 30% yield. These studies show that the nontransformed, and the heat-, salt-, and ATP-transformed GRc from the rat liver cytosol can be separated chromatographically, and that the use of tungstate facilitates the resolution of these different receptor forms. In addition, extraction of the receptor from DNA-cellulose by tungstate provides another new and efficient method of partial receptor purification.View full abstractDownload PDF (1659K) -
Tadashi YOSHIMOTO, Kazuo KAWAHARA, Futoshi MATSUBARA, Kunio KADO, Dais ...1985 Volume 98 Issue 4 Pages 975-979
Published: October 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe inhibitory effects of proline-containing peptides and their derivatives on prolyl endopeptidases from Flavobacterium meningosepticum and bovine brain were compared. Replacement of the carboxyl terminal proline in N-blocked peptides with prolinal resulted in remarkable decreases in K1 values for both prolyl endopeptidases. Further reduction of the prolinal to prolinol led to a decrease in their inhibitory effects. Z-Pro-, Z-Val-, and Suc-Pro-prolinals were similarly inhibitory for both the enzymes with K1 values of nM order. However, the inhibitory effects of Z-Pyr-prolinal and Boc-Pro-prolinal on these enzymes were significantly distinguished: they strongly inhibited the mammalian prolyl endopeptidase with K1 values of nM order, while the K1 values of these compounds for the microbial enzyme were only of μM order. These results suggest that there are some structural differences in the S2 and S3 subsites between the two enzymes, though their substrate specificities are apparently indistinguishable.View full abstractDownload PDF (398K) -
Hideyo HASUMI, Etsuko NAGATA, Satoshi NAKAMURA1985 Volume 98 Issue 4 Pages 981-990
Published: October 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe gross protein structure, the microenvironment of the iron-sulfur cluster, and the effect of neutral salts on the molecular structure of spinach ferredoxin were studied by CD and absorption spectroscopy in the alkaline pH range.
In the pH range of 7-11, the existence of reversible isomerization which consisted of at least two proton dissociation processes was indicated by the statical CD and absorption spectra. The CD changes in the visible and far-UV regions were dramatic upon elevation of the pH from neutral to alkaline, indicating a significant alteration of the microenvironment of the cluster and a decrease in the ordered secondary structures. The absorption change in the visible region due to pH elevation was small but clearly observed with a high signal-to-noise ratio. The numbers of protons involved in the respective processes and the apparent pK values obtained from the pH-dependence of the CD changes were in good agreement with those obtained from the pH-dependence of the absorption changes in the visible region. In addition, the rate constants obtained from the time courses of the CD and absorption changes agreed with one another. By the addition of 1M NaCl, the CD and absorption spectra at alkaline pH were reversed almost to those at neutral pH without significant pH change. On the other hand, above pH 11, ferredoxin was found to be irreversibly denatured. Based on analyses of the statical CD and absorption spectra and of the time courses of the CD changes, the probable mechanism of the isomerization was considered to be as follows:
_??_
where H stands for a proton, N-form for native ferredoxin at neutral pH, N*-form for alkaline ferredoxin below pH 11 which still has the iron-sulfur cluster but with disordered secondary structures of the polypeptide chain, and D-form for completely denatured ferredoxin above pH 11.
These results lead to the conclusions that (1) the interaction between the protein moiety and the iron-sulfur cluster is essential for maintaining the native ferredoxin structure, and (2) neutral salts protect the polypeptide chain from unfolding through electrostatic interaction with the ionized side chains, resulting in the stabilization of ferredoxin.View full abstractDownload PDF (837K) -
Itaru YAMAMOTO, Eiji MATSUURA1985 Volume 98 Issue 4 Pages 991-998
Published: October 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHybridoma cell lines secreting antibodies for vitamin D3 metabolites have been generated by fusing splenocytes from BALB/c mice immunized with 3β-glutaryl-25-hydroxyvitamin D3 conjugated to bovine serum albumin (3β-glu-25-OH-D3-BSA) and Sp2/O-Ag14 myeloma cells. Purification of monoclonal antibodies from culture media or ascites fluids was accomplished by procedures including affinity chromatography on Protein A-Sepharose 4 B. Each monoclonal antibody was analyzed as to its affinity and specificity by equilibrium dialysis and an enzyme immunoassay (EIA) based on a double antibody system. It was demonstrated that clone 1C2-60 produced an antibody highly specific to lα, 25-dihydroxyvitamin D3 (calcitriol), and the clone 2B3-66 antibody was reactive to 25-hydroxyvitamin D3 and similar structural compounds. These two monoclonal antibodies produced by 1C2-60 and 2B3-66 were determined to belong to the IgG2a class, and their affinity constants (Ka) with 3β-glu-25-OH-D3 were demonstrated to be 3.6×109 M-1 and 2.9×109 M-1, respectively, at 4°C. The characteristics of these monoclonal antibodies were compared with those of conventional antibodies raised in mice and rabbits. Finally, by using monoclonal antibody 1C2-60, a sensitive EIA has been developed that can detect 10 pg of calcitriol.View full abstractDownload PDF (619K) -
Hiroaki SAWAI, Kaichiro ISHIBASHI, Masao ITOH, Shoichi WATANABE1985 Volume 98 Issue 4 Pages 999-1005
Published: October 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA novel 125I-labeled derivative of 2', 5'-triadenylate 5'-triphosphate, pppA2'p5'A2'p5'A, with high specific radioactivity was synthesized by coupling of periodateoxidized pA2'p5'A2'p5'A with β-alanyltyrosine methyl ester followed by 5'-triphosphorylation and iodination with 125I. Antisera toward 2', 5'-oligoadenylate 5'-triphosphate were produced in rabbits by immunization with the conjugate of pppA2'p5'A2'p5'A2'p5'A with bovine serum albumin, and an antiserum with high specificity and high sensitivity for 2', 5'-oligoadenylates was selected and tested extensively. Radioimmuno assaying of 2', 5'-oligoadenylates was carried out by a competitive double antibody method in which the amount of the antibody bound to the 125I-labeled probe was measured after precipitation with goat anti-rabbit IgG. The concentration of pppA2'p5'A2'p5'A required for 50% inhibition of the binding between the antiserum and the probe was 0.6 nM. The cross reactivity of the antiserum with the 3', 5'-triadenylate was more than 10, 000 times weaker compared to in the case of 2', 5'-oligoadenylates. Very low or no cross reaction was observed with ATP, AMP, and adenosine. The radioimmuno assay using the 125I-labeled compound and the antiserum allows the direct analysis of 2', 5'-oligoadenylates in the range of 4 fmol to 1 pmol (0.04-10 nM in a 100 μl sample). This assay was applied to the measurement of the activity of 2', 5'-oligoadenylate synthetase in cells stimulated by interferon. The properties of the 125I-labeled derivative of pppA2'p5'A2'p5'A are described.View full abstractDownload PDF (566K) -
Tetsuyuki KOBAYASHI, Ichiro KUDO, Hiroshi HOMMA, Ken KARASAWA, Keizo I ...1985 Volume 98 Issue 4 Pages 1007-1016
Published: October 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe genes coding for the phospholipid degradation enzymes in E. coli, detergentresistant (DR-) phospholipase A (pldA) and lysophospholipase L2 (pldB), were cloned together on the plasmid pKO1 (Homma, H., Kobayashi, T., Ito, Y., Kudo, I., Inoue, K., Ikeda, H., Sekiguchi, M., & Nojima, S. (1983) J. Biochem. 94, 2079-2081). To study their gene organization, a transducing λ phage, λpldApldB, carrying both the pldA and pldB genes was constructed in vitro from plasmid pKO1. Viable deletion mutants of λpldApldB were isolated by EDTA killing, and their deleted DNA regions were determined by electron microscopic analysis of appropriate heteroduplexes. The activities of DR-phospholipase A and lysophospholipase L2 were also measured in lysates of cells infected with the deletion phages. The DNA region essential for the expression of each lipolytic activity was determined. In addition, proteins coded by the bacterial DNA on the plasmids containing the pldA-pldB region to various extents were detected by the maxicell system. The results showed that the product of the pldB gene is a protein with molecular weight of 40, 000. It was also shown that the pldB gene is located at a region about 3 kilobase from the pldA gene.View full abstractDownload PDF (1338K) -
Tetsuyuki KOBAYASHI, Ichiro KUDO, Ken KARASAWA, Hiroshi MIZUSHIMA, Kei ...1985 Volume 98 Issue 4 Pages 1017-1025
Published: October 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe nucleotide sequence of the pldB gene of Escherichia coli K-12, which codes for lysophospholipase L2 located in the inner membrane, was determined. The deduced amino acid sequence of lysophospholipase L2 contains 340 amino acid residues, resulting in a protein with a molecular weight of 38, 934. It is characterized by a high content of arginine residues (36 out of 340 residues). The amino acid sequence near the NH2-terminus of the protein is composed of a large number of polar or charged amino acid residues, suggesting that this region cannot be a signal peptide. The hydropathy profile of the deduced amino acid sequence of lysophospholipase L2 was studied. Most of the region was rather hydrophilic, and there was no stretch of hydrophobic amino acid region, such as might be predicted to traverse the lipid bilayer. These results are consistent with the experimental observation that lysophospholipase L2 is extracted by salt solution from the membrane fraction, and it may be classified as a peripheral membrane protein. Computer analysis showed that there is no homology in amino acid sequences between lysophospholipase L2 and other extracellular phospholipases, as well as detergent-resistant phospholipase A, which is another membrane-bound phospholipase in E. coli and whose DNA sequence was determined (Homma, H., Kobayashi, T., Chiba, N., Karasawa, K., Mizushima, H., Kudo, I., Inoue, K., Ideka, H., Sekiguchi, M., & Nojima, S. (1984) J. Biochem. 96, 1655-1664). This is the first report of the primary structure of a lysophospholipase.View full abstractDownload PDF (692K) -
Shuichi KAMINOGAWA, Atsushi ENOMOTO, Jun-ichi KURISAKI, Kunio YAMAUCHI1985 Volume 98 Issue 4 Pages 1027-1032
Published: October 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo monoclonal antibodies (mAb 23E5 and 32A8) to hen's egg ovomucoid (OM), which causes hen's egg allergy and has trypsin inhibitory activity, were prepared and purified. Their affinity to the three separate domains of the ovomucoid, which are homologous in primary structure and are designated as DI, DII, and DIII, was studied by a competitive radioimmunoassay. MAb 23E5 bound to OM more efficiently than to DI, DTI, or DIII-2 (with carbohydrate), but reacted with DIII-1 (free from carbohydrate) more efficiently than with OM. Except for the binding to OM, mAb 32A8 bound to DIII-2 most efficiently and to DIII-1 least efficiently, suggesting that this antibody recognized the carbohydrate moiety of DIII. MAb 32A8 inhibited the trypsin inhibitory activity of OM, whereas mAb 23E5 had no effect on it. These monoclonal antibodies should be useful for analyzing the antigenic determinants and trypsin inhibitory activity of ovomucoid.View full abstractDownload PDF (444K) -
Shigeru HOSHIKO, Nack Kie SUNG, Teruhiko BEPPU1985 Volume 98 Issue 4 Pages 1033-1040
Published: October 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA sequential reaction was suggested for the conversion of L-alloisocitrate to α-oxoglutarate by an enzyme complex of L-alloisocitrate dehydrogenase and oxalosuccinate decarboxylase from Pseudomonas strain No. 2, during which oxalosuccinate was not released from the enzyme-substrate complex. The stereochemistry of oxalosuccinate formed by L-alloisocitrate dehydrogenase and decarboxylated by oxalosuccinate decarboxylase was opposite to that of the substrate for D-isocitrate dehydrogenase. Incubation of L-alloisocitrate with the dehydrogenase and decarboxylase in deuterium oxide provided [3-2H]-α-oxoglutarate, the configuration of which turned out to be the same as that produced by D-isocitrate dehydrogenase from D-isocitrate. The data suggested that enol form of α-oxoglutarate was involved as an intermediate in decarboxylation of oxalosuccinate by oxalosuccinate decarboxylase. L-Alloisocitrate dehydrogenase was shown to react with pro-S proton of NADH.View full abstractDownload PDF (584K) -
Masaru KURIYAMA, Ryo-ichi HIWATARI, Toshio ARIGA, Yoshiyuki SAKANO, Ju ...1985 Volume 98 Issue 4 Pages 1041-1047
Published: October 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSNeutral oligosaccharides were isolated from urine of an adult patient with glycogen storage disease type II, a deficiency of lysosomal acid α-glucosidase, by chromatography on columns of activated charcoal, Dowex 50×2 and Dowex 1×2. Total neutral oligosaccharides in the urine of the patient were increased about 5-fold as compared with those in normal controls. The most accumulated oligosaccharide was separated by Bio-Gel P-2 column chromatography, and finally purified by paper chromatography. Based on various studies, including carbohydrate analysis, chemical ionization mass spectrometry, fast atom bombardment mass spectrometry, degradation by glucoamylase and isopullulanase, and methylation analysis, the structure of this oligosaccharide was deduced to be Glcα1→ 6Glcα1→4Glcα1→4Glc. This oligosaccharide appears to be accumulated in urine of the patient with acid α-glucosidase deficiency as an end product of the hydrolysis of glycogen.View full abstractDownload PDF (2105K) -
Masaru KURIYAMA, Toshio ARIGA, Susumu ANDO, Minoru SUZUKI, Takamichi Y ...1985 Volume 98 Issue 4 Pages 1049-1054
Published: October 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe describe the structures of two positional isomers of sialylheptasaccharide isolated from the urine of a patient with sialidosis with partial deficiency of β-galactosidase. Based on structural studies including compositional sugar analysis, exoglycosidase digestion, chemical ionization mass spectrometry, proton nuclear magnetic resonance spectrometry, and methylation analysis, their structures were deduced to be as follows:
1. AcNeuα2→6Galβ1→4GlcNacβ1→2Manα1→3(Manα1→6)Manβ1→4GlcNac
2. AcNeuα2→6Galβ1→4GlcNacβ1→2Manα1→ 6(Manα1→3)Manβ1→4GlcNac
Sialyloligosaccharide 1 has previously been found in the urine and liver of patients with mucolipidosis I and II and sialidosis, but sialyloligosaccharide 2 has not been found yet in human urine. These two sialyloligosaccharides could not be completely separated by any chromatographic procedures tested. The analytical techniques, including methylation study and NMR spectroscopy, could not clearly detect the differences between them. However, α-mannosidase treatment gave important information for the structural analyses of these sialyloligosaccharides.View full abstractDownload PDF (1294K) -
Yoshihiro FUKUMORI, Taketomo FUJIWARA, Yumiko OKADA-TAKAHASHI, Yasuo M ...1985 Volume 98 Issue 4 Pages 1055-1061
Published: October 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA peroxidase was purified from Halobacterium halobium L-33 to an electrophoretically homogeneous state and some of its properties were studied. The enzyme showed an absorption peak at 406 nm in the oxidized form and peaks at 440, 558, and 591 nm in the reduced form. The difference spectrum, reduced+CO minus reduced, of the enzyme showed peaks at 425, 538, and 577 nm and troughs at 444, 562, and 596 nm. These spectral properties were apparently similar to those of “cytochrome a1” except for the occurrence of the peak at 558 nm in the reduced form. The molecular weight of the enzyme was 110, 000 and the enzyme possessed one unit of protoheme in the molecule.
The activity to oxidize guaiacol in the presence of H2O2 of the peroxidase was about one-twentieth of that of horseradish peroxidase. The enzyme also showed a catalase activity one-fourth as active as that of liver catalase. The reactions catalyzed by the enzyme were strongly inhibited by KCN.View full abstractDownload PDF (458K) -
Hiroshi NARITA, Hwa Jin PARK, Ken-ichi TANAKA, Takao MATSUURA, Makoto ...1985 Volume 98 Issue 4 Pages 1063-1068
Published: October 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWhen human platelets (5×108/ml) were stimulated by a threshold concentration of collagen (2 μg/ml), a lag period of about 60 s was seen before the initiation of release reaction and aggregation. Breakdown of [32P] phosphatidylinositol 4, 5-bisphosphate was seen within 10 s after the addition of collagen. The concentration of intracellular free Ca2+ (monitored by Quin II) rose from 80 nM to 145 nM within 10 s after stimulation by collagen. However, a lag period of about 50 s remained. The rise was not blocked by indomethacin. It was supposed that the initial Ca2+ mobilization by myo-inositol 1, 4, 5-trisphosphate was too small to cause aggregation. Thromboxane A2 was gradually accumulated during the lag period and then abruptly increased in parallel with aggregation. These events were completely inhibited by 10 μM indomethacin. Thus, aggregation appeared to be dependent on the generation of thromboxane A2. Addition of 25 nM A23187 at 10 s after stimulation by collagen shortened the lag period before initiation of the abrupt thromboxane A2 generation, secretion and aggregation, whereas 25 nM A23187 could not cause these reactions in the absence of collagen. Accordingly, the lag period is assumed to be required for accumulation of free Ca2+ to the threshold for aggregation of platelets. It is considered that thromboxane A2 plays a central role in Ca2+ mobilization during stimulation of human platelets by collagen.View full abstractDownload PDF (1243K) -
Fumiaki SHONO, Kazushige YOKOTA, Shozo YAMAMOTO1985 Volume 98 Issue 4 Pages 1069-1077
Published: October 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA solid-phase enzyme immunoassay for thromboxane B2 was developed using a conjugate of thromboxane B2 and β-galactosidase. Anti-thromboxane B2 IgG was bound to a polystyrene tube, and the enzyme-labeled and unlabeled thromboxane B2 were allowed to react in a competitive manner with the immobilized antibody. Then, the specifically bound β-galactosidase was assayed fluorimetrically, and the enzyme activity was correlated with the amount of unlabeled thromboxane B2. By using a calibration curve, thromboxane B2 was determined in the range of 20 fmol-14 pmol. 2, 3-Dinor- and 2, 3, 4, 5-tetranor-thromboxane B2 cross-reacted with thromboxane B2 to the extents of 18.6% and 0.4%, respectively. Most prostaglandins and their metabolites tested showed cross-reactivities of less than 1%. In application of the method to human blood and urine, an octadecylsilyl silica column was utilized for extraction and concentration of thromboxane B2. The crude extract contained a substance (s) which disturbed the enzyme immunoassay and gave an apparently high value of thromboxane B2, and the interfering substance was separated from thromboxane B2 by reverse-phase HPLC. Various amounts of authentic thromboxane B2 added to the purified material from human plasma could be determined by the enzyme immunoassay with a recovery of about 80%, and the results correlated well with the values obtained by radioimmunoassay (γ=0.979). When the extract from human urine was analyzed by reverse-phase HPLC, the 2, 3-dinor metabolite rather than thromboxane B2 was the predominant compound detected by the enzyme immunoassay.View full abstractDownload PDF (769K) -
Yoshio YAO, Keishiro WADA, Yuichiro TAKAHASHI, Sakae KATOH, Hiroshi MA ...1985 Volume 98 Issue 4 Pages 1079-1082
Published: October 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe sulfhydryl groups of three ferredoxin-NADP+ oxidoreductases [EC 1. 18. 1. 2] (FNR) two from blue-green algae, Spirulina sp. and Synechococcus sp., respectively, and one from spinach, were analyzed by carboxymethylation and titration with sulfhydryl reagents under different conditions. Five sulfhydryl groups in Spirulina and spinach enzymes and four in Synechococcus enzyme were found to be in reduced forms. There was no disulfide bond in any FNR tested. The results in the present experiment contradict those reported by Karplus et al. [Biochemistry 23, 6576-6583 (1984)].View full abstractDownload PDF (262K) -
Akira MURAYAMA, Fumio FUKAI1985 Volume 98 Issue 4 Pages 1083-1090
Published: October 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMolybdate was shown to have complex effects in modulating the molecular organization of the constituents of the estrogen receptor (ER) system of porcine uterus. We showed previously the presence of one basic ER molecule (vero-ER) (sedimentation coefficient, 4.5 S; Stokes radius, 44 A) and ER-binding factors (ERBFs) [“8 S” ER-forming factor (“8 S” ER-FF), (component A) •(component B)6; “6 S” ER-FF, (component B)6; “5 S” ER-FF, component A] in the porcine uterus [Fukai, F. & Murayama, A. (1981) J. Biochem. 95, 1697-1704]. Molybdate regulates the specific interaction of vero-ER with ERBFs in a complex way. The apparent Kd value (6.7×10-10M) of vero-ER with “8 S” ER-FF in the presence of molybdate (30mm) was decreased remarkably as compared with that (2.7×10-9M) in the absence of molybdate. In contrast, the apparent Kd value (3.7×10-9M) of vero-ER with “5 S” ER-FF observed in the presence of molybdate (30mM) was increased over ten-fold as compared with that in the absence of molybdate. Meanwhile, the affinity (Kd, 5×10-9M) of vero-ER for “6 S” ER-FF was scarcely influenced by molybdate. These results reveal the mechanism by which molybdate selectively stabilizes “8 S” ER. Molybdate further affected the molecular constitution of ERBFs. The dissociation of “8 S” ER-FF into component A and component B, which takes place under hypertonic (0.4M KCl) conditions at higher temperature (25°C), was suppressed almost completely by molybdate (30mM). The previously reported inhibiting effect of molybdate on the formation of “5 S” ER can be attributed to both the inhibition of the formation of “5 S” ER-FF and to the decrease of the affinity of vero-ER for “5 S” ER-FF. Molybdate did not influence the affinity of ER for estradiol.View full abstractDownload PDF (594K) -
Fumio KISHI, Atsushi NAKAZAWA1985 Volume 98 Issue 4 Pages 1091-1095
Published: October 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDuring chick embryogenesis, the mRNA for myokinase, as determined by in vitro translation, appears on the 16 th day. The mRNA levels do not change drastically in succeeding stages and increase immediately after hatching. In contrast, myokinase activity and its protein gradually increase from the 16 th day. Thus, myokinase mRNA that is translatable in vitro seems to accumulate transiently in the late stage of embryogenesis. In sucrose density gradient centrifugation, the mRNA sedimented as one peak with a coefficient of 10 S.View full abstractDownload PDF (1988K) -
Toshiro YANO, Ikuo FUNAKOSHI, Ikuo YAMASHINA1985 Volume 98 Issue 4 Pages 1097-1107
Published: October 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSNucleotide pyrophosphatase was purified from human placenta to near homogeneity with a specific activity of about 500-fold over the Triton extract of the homogenate. Purification was achieved most effectively by successive chromatographic steps with AMP-agarose and ADP-agarose columns, based on the affinity of the enzyme towards 5'-adenylate and adenosine 3', 5'-diphosphate, and a lectin-Sepharose column, based on the glycoprotein nature of the enzyme. The purified enzyme was found to be essentially homogeneous on SDS-polyacrylamide gel electrophoresis with a mobility corresponding to 130 K. The purified enzyme was found to hydrolyze a wide variety of nucleotides, i.e. 3'-phosphoadenosine 5'-phosphosulfate (PAPS), adenosine 5'-phosphosulfate (APS), NADH, ATP, nucleotide sugars, oligonucleotides, and p-nitrophenyl-thymidine 5'-phosphate (PNTP). From the oligonucleotides, the enzyme produced 5'-phosphates. Mg2+ was required for full activity. Glycine and sulfhydryl compounds such as 2-mercaptoethanol and 2, 3-dimercapto-l-propanol were inhibitory. Most of these properties are common to nucleotide pyrophosphatases [EC 3. 6. 1. 9] and type I (5'-phosphate forming) phosphodiesterases [EC 3. 1. 4. 1] from various sources. The relevance of this enzyme to a unique genetic disease, Lowe's syndrome, is discussed.View full abstractDownload PDF (2426K) -
Akiko KUMAGAI, Shunsuke TAKEMOTO, Koji OKAMOTO, Ikuo TAKEUCHI1985 Volume 98 Issue 4 Pages 1109-1115
Published: October 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBy means of two-dimensional gel electrophoresis, we analyzed proteins present in a slug-shaped tissue mass of D. discoideum and examined the changes in their amounts after disaggregation of the slugs. Of approximately one hundred polypeptides, six were found to decrease in amount after disaggregation. The decreases of four polypeptides were inhibited by the presence of 1mM cAMP or 250 μg/ml cycloheximide. The decreases of the two other proteins were not suppressed by cAMP or cycloheximide. The patterns of proteins present in vegetative and aggregative cells were also examined. None of the six proteins which showed a decrease after slug disaggregation was found in vegetative or preaggregative cells. These results indicate that both synthesis and degradation of these proteins are controlled by cell-cell contact.View full abstractDownload PDF (2576K) -
Ken KARASAWA, Ichiro KUDO, Tetsuyuki KOBAYASHI, Takao SA-EKI, Keizo IN ...1985 Volume 98 Issue 4 Pages 1117-1125
Published: October 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSLysophospholipase L2, which is bound to the inner membrane of Escherichia coli K-12, was produced in a large amount in cells bearing its cloned structural gene. Starting from these cells, the lysophospholipase L2 was purified approximately 700-fold to near homogeneity by solubilization with KCl, ammonium sulfate fractionation, chromatofocusing in the presence of a zwitterionic detergent, CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), and heparin-Sepharose affinity column chromatography. The final preparation showed a single protein band with a molecular weight of 38, 500 daltons in SDS-polyacrylamide gel electrophoresis. The amino acid sequence of the NH2-terminal portion of the purified enzyme was determined. It was in complete agreement with that deduced from the nucleotide sequence of the structural gene, pldB [Kobayashi, T., Kudo, I., Karasawa, K., Mizushima, H., Inoue, K., & Nojima, S. (1985) J. Biochem. 98, 1017-1025.]
The purified enzyme hydrolyzes 2-acyl glycerophosphoethanolamine (GPE) and 2-acyl glycerophosphocholine (GPC) more effectively than 1-acyl GPE and 1-acyl GPC, but does not attack diacylphospholipids. The enzyme also catalyzes the transfer of an acyl group from lysophospholipid to phosphatidylglycerol for formation of acyl phosphatidylglycerol. The acyl group was more effectively transferred from 2-acyl lysophospholipid than from the 1-acyl derivative. This enzyme was heat-labile and was inactivated at 55°C within 5min. The present paper shows clearly that lysophospholipase L2 is a different enzyme protein from lysophospholipase L1 which was formerly purified from the supernatant of the wild strain of E. coli K-12 homogenates [Doi, O. & Nojima, S. (1975) J. Biol. Chem. 250, 5208-5214].View full abstractDownload PDF (1338K) -
Ken EBISAWA, Yoshiaki NONOMURA1985 Volume 98 Issue 4 Pages 1127-1130
Published: October 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA Ca2+-dependent actin-severing 84 K Mr protein prepared from bovine aorta caused four-fold activation of smooth muscle actin-activated myosin ATPase at a 1/102 molar ratio to actin in the presence of tropomyosin and light chain kinase-calmodulin in a Ca2+-dependent manner, while it inhibited it markedly at a 1/5 molar ratio. Purified actin-tropomyosin filaments under the experimental ATPase conditions were distributed in a range of more than 10 μm in length and the addition of the 84 K Mr protein changed the filament length to around 1 μm at a 1/102 molar ratio to actin or less than 50 nm at a 1/5 molar ratio in the presence of Ca2+. However, the apparent length of actin filaments alone does not appear to be responsible for the activation of ATPase activity, since in the absence of tropomyosin, the ATPase activation was much less in spite of actin filament length changes. These results indicate the possibility that the 84K Mr protein plays an important role with tropomyosin in at least in vitro smooth muscle actin-myosin interaction.View full abstractDownload PDF (1141K) -
Toshihiro NAKAYAMA, Akira HARA, Kin-ya KARIYA, Hideo SAWADA1985 Volume 98 Issue 4 Pages 1131-1133
Published: October 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAldehyde reductases from several mammalian and avian tissues transferred the pro-4R hydrogen of NADPH to the substrate, whereas the stereospecificity of carbonyl reductases was not uniform being correlated with the ability to catalyze the oxidoreduction of hydroxysteroids.View full abstractDownload PDF (181K) -
Masao HAYASHI, Takao AKAMA, Ichiro KONO, Heihachiro KASHIWAGI1985 Volume 98 Issue 4 Pages 1135-1138
Published: October 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSVitronectin (serum spreading factor), a cell-adhesive glycoprotein present in mammalian serum, has previously been the subject of conflicting reports concerning its binding to heparin. Vitronectin purified from human plasma does not bind to heparin under physiological conditions, but it does so after treatment with denaturing agents including 8M urea or 6M guanidine-HCl, or heating at 100°C for 5min. These treatments seem to expose a heparin-binding site in vitronectin; this finding thus resolves the conflicts concerning this function.View full abstractDownload PDF (1164K) -
Yoshio KATSUYA, Kensaku HAMADA, Yasuo HATA, Nobuo TANAKA, Mamoru SATO, ...1985 Volume 98 Issue 4 Pages 1139-1142
Published: October 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPreliminary X-ray studies on Serratia protease have been carried out using crystallographic and small angle scattering techniques. The enzyme has been crystallized in three different crystalline forms by microdialysis and vapor diffusion methods using 50mM phosphate buffer, pH 6.0, at 24°C. They have orthorhombic space groups: C2221 for one form and P212121 for the other two forms. A small angle X-ray scattering study showed that the radius of gyration and the maximal dimension of the molecule in aqueous solution are 26.6 Å and 94.5 Å, respectively. The molecular weight of the enzyme was determined to be 45, 000-48, 000 by various physical methods.View full abstractDownload PDF (1130K) -
Yoh OKAMOTO, Takamitsu SEKINE1985 Volume 98 Issue 4 Pages 1143-1145
Published: October 01, 1985
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA rapid procedure for isolating subfragment one (SF1) from myosin was found. SF1 can be isolated specifically from proteolytic digests of myosin in the presence of a millimolar concentration of magnesium chloride. Under such ionic conditions all of the rod portion and undigested myosin is selectively precipitated. A nucleotide trapping experiment indicated how important quick preparation of SF1 is for maintaining the active site structure. This method can also be utilized in the preparation of heavy meromyosin.View full abstractDownload PDF (1097K)
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