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Volume 100 (1986)
- Issue 6 Pages 1403-
- Issue 5 Pages 1109-
- Issue 4 Pages 825-
- Issue 3 Pages 521-
- Issue 2 Pages 259-
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Volume 100, Issue 5
Displaying 1-33 of 33 articles from this issue
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Tomohiro MEGA, Yasuki HAMAZUME, Yeong-Man NONG, Tokuji IKENAKA1986 Volume 100 Issue 5 Pages 1109-1116
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo determine the phosphate binding sites in hen egg white riboflavin binding protein (RBP), a highly phosphorylated peptide, which consisted of 23 amino acid residues including eight phosphoserines, was isolated from the tryptic digest of reduced and carboxymethylated RBP. The conditions of the β-elimination-addition reaction to convert phosphoserine residues in the peptide to cysteic acids, S-methylcysteines, alanines, and β-methylaminoalanines (DL-α-amino-β-methylamino propionic acid) were examined. These converted peptides were purified by HPLC and subjected to Edman degradation. The results of Edman degradation indicated that the S-methylcysteine derivative of the peptide gave the most satisfactory result for deter-mining the phosphate binding sites in the peptide. The phosphorylation sites of the peptide determined by the method mentioned above are as follows: 182 His-Leu-Leu-Ser 185 -Glu-Ser(P)-Ser(P)-Glu-Glu 190 -Ser(P)-Ser(P)-Ser( P)-Met-Ser(P) 195- Ser(P)-Ser(P)-Glu-Glu-. These studies indicated that the conversion of phosphoserines in phos-phoproteins to S-methylcysteines followed by Edman analysis was a useful method for the elucidation of the phosphorylation sites in phosphopeptides.View full abstractDownload PDF (603K) -
Makoto ODA, Kiyoshi SATOUCHI, Kojiro YASUNAGA, Kunihiko SAITO1986 Volume 100 Issue 5 Pages 1117-1123
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA mixture of rabbit polymorphonuclear leukocytes (PMNs) and platelets at con-centrations of 5×106 PMN and 3.5×108 platelets/ml Tyrode's solution was stim-ulated with the chemotactic peptide, formyl-methionyl-leucyl-phenylalanine (FMLP). A micromolar concentration of FMLP elicited an immediate weak aggregation, followed by a strong aggregation with a time lag of about 1min. Microscopic examination showed that the immediate aggregation was due to PMNs and the delayed one was more complex and involved platelets. The delayed aggregation was dependent upon the concentrations of both the PMNs and FMLP. The delayed aggregation was completely blocked by pretreatment of the PMN-platelet mixture with 8μM CV-3988, a specific receptor antagonist of 1-alky1-2-acetyl-sn-glycero-3- phosphocholine (AGEPC), or by the application of platelets desensitized to AGEPC. The time course of AGEPC production by PMNs was well matched to that of the biphasic aggregation response. Furthermore, nordihydroguaiaretic acid inhibited both the AGEPC production by PMNs and the delayed aggregation in a similar dose-dependent manner. These result demonstrate that AGEPC, newly-generated by PMNs under FMLP-stimulation, is of primary importance in platelet aggregation in a PMN-platelet mixed system.View full abstractDownload PDF (1009K) -
Saad TAYYAB, Mohammad Abul QASIM1986 Volume 100 Issue 5 Pages 1125-1136
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSUsing succinic anhydride, six succinylated derivatives of bovine serum albumin having percent modification in the range of 23-87% were prepared and their physi-cochemical and immunological properties were studied. Measurements of Stokes radius, frictional ratio, UV spectra, solvent perturbation, solubility, and immu-nological cross-reactivity against anti-bovine serum albumin antiserum revealed that the protein undergoes gradual changes in its native conformation with increase in the degree of succinylation. These changes were less marked below 50% modi-fication but became pronounced above 50% modification. However, even the maximally modified preparation (87%) contained a significant amount of folded structure. Interestingly, though the measurements of various molecular properties revealed significant changes in 23-49% modified preparations, the solubility param-eters for these preparations which were obtained at high ionic strength were indis-tinguishable from those of the native protein. The various results taken together suggest that at lower degrees of chemical modification, the conformational changes were produced mainly because of an increase in electrostatic free energy, whereas at higher degrees of modification, steric hindrance in addition to the electrostatic factor seems to make a substantial contribution to the conformational changes in the modified proteins.View full abstractDownload PDF (2168K) -
Akiko BABA, Taka-aki NAKAMURA, Masao KAWAKITA1986 Volume 100 Issue 5 Pages 1137-1147
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSarcoplasmic reticulum membrane vesicles from rabbit skeletal muscle were treated with iodoacetamide (IAA) at pH 7.0 and 30°C. At 1.0mM IAA, 1 mol of IAA per mol of ATPase peptide was bound in 1h. Under these conditions, IAA was attached specifically to the B-tryptic fragment portion of the peptide. The binding of IAA did not affect the Ca2+-transporting activity of ATPase. Three fluorescent derivatives of iodoacetamide, 5-(2-acetamidoethyl)aminonaphthalene-l-sulfonate (IAEDANS), 5-iodoacetamido fluorescein (IAF), and 5-iodoacetamido eosin (IAE), were also tested for reactivity toward sarcoplasmic reticulum ATPase at 30°C and pH 7.0. In 1 h at 50μM concentration, each of these fluorescent labels modified ATPase to a labeling density of 1 mol per mol of ATPase. Neither IAEDANS nor IAF at this labeling density affected Ca2+-transporting activity, but IAE reduced it to 20% of the untreated control. The target site of IAEDANS at this labeling density was located exclusively on the B-fragment portion, as was the case with IAA, but IAF label was found on both Al and B fragments after limited tryptic digestion. IAEDANS was used as a B-fragment portion-directed conformational probe of Ca2+-transport ATPase, and an increase in fluorescence intensity accom-panying ECa1-P formation was detected. The fluorescence enhancement was abolished when ECa1•ADPβS was formed by adding ADPβS to preformed ECa1-P. This suggests that the conformation of ATPase in the neighborhood of the IAEDANS binding site may be altered in response to the dissociation of ADP from the phos-phorylated intermediate.View full abstractDownload PDF (2203K) -
Sunna HAUSCHILDT1986 Volume 100 Issue 5 Pages 1149-1154
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFeeding a 17.5% amino acid diet to rats results in inactivation of the hepatic branched-chain 2-oxoacid dehydrogenase complex. Reactivation occurs when preincubating mitochondria in the presence of 0.3mM ATP, ADP, and AMP. The effect of AMP is assumed to be due to de novo formation of ADP. NaF (25mM) blocks reactivation suggesting the involvement of a protein phosphatase in the activation process. At high nucleotide concentrations (3mM) the enzyme is inactive. In the presence of Mg2+ ions nucleotide induced activation is further increased. Mg2+ ions themselves influence the equilibrium state of the enzyme complex. Low concentrations (1mM) favor inactivation while high concentrations (10mM) stimulate activation of the enzyme suggesting that Mg2+ ions may act by regulating the associated kinase and phosphatase.View full abstractDownload PDF (462K) -
Hitoshi SOHMA, Fumi MORITA1986 Volume 100 Issue 5 Pages 1155-1163
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA protein kinase activity phosphorylating regulatory light chain-a (RLC-a) of scallop smooth muscle myosin was found to be present in scallop smooth muscle homogenate. The kinase was purified to homogeneity and named RLC-a myosin kinase (aMK). aMK was extracted from the muscle homogenate with a low salt solution and was purified by successive DE-32 ion exchange chromatography, gel filtration on Ultrogel AcA 44, and affinity chromatography on Sepharose 4B-6- arninohexy1-1-pyrophosphate. The molecular weight of aMK was estimated to be 40-kDa from the mobility on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and 35-IcDa from the elution volume on Sephadex G-150 gel filtration. The phosphorylation site of RLC-a by aMK was determined to be Ser residue(s). Only RLC-a was phosphorylated; the other regulatory light chain, RLC-b, was not. The phosphorylatable Ser of RLC-a is, therefore, considered to be Ser-11, which is located in the N-terminal region having a different amino acid sequence from that of RLC-b. RLC-a was phosphorylated by aMK 3 times faster in the free state than in the bound state to myosin. aMK does not require cal-modulin and is rather inhibited by CaCl2.View full abstractDownload PDF (2337K) -
Kuniaki MUKAI, Hiroshi MATSUBARA1986 Volume 100 Issue 5 Pages 1165-1173
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe autoxidizability of beef heart cytochrome c1 was investigated in terms of the integrity of the binding of the hinge protein to the heme subunit. Cytochrome c1 was isolated as a subcomplex consisting of the heme subunit and the hinge protein. Treatment of the cytochrome c1 subcomplex with p-chloromercuribenzoate (pCMB) under mild conditions lessened the binding strength between the two subunits. They were dissociated on polyacrylamide gel electrophoresis (PAGE) under non-denaturing conditions, but were not separated by gel filtration chromatography. The pCMB-treated subcomplex had a slight autoxidizability. This was repressed to the level of the native subcomplex, when the mercurial compound bound to the subcomplex was removed by the addition of 2-mercaptoethanol. Concomitantly, the less stable binding between the subunits was apparently reversed to the native state. After pCMB treatment of the subcomplex, the heme subunit recovered from PAGE showed marked autoxidizability, even if it was treated with 2-mercapto-ethanol. Addition of cholate repressed the autoxidizability of the heme subunit after the removal of the mercurial compound. These results confirmed that the stable binding of the hinge protein to the heme subunit was essential for the non-autoxidizability of cytochrome c1 subcomplex. In addition, it was suggested that cysteinyl residues in the subcomplex must be involved to a great extent in the stable binding between the two subunits.View full abstractDownload PDF (1806K) -
Keiichi NOMURA, Bunzo MIKAMI, Yuhei MORITA1986 Volume 100 Issue 5 Pages 1175-1183
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe interaction of soybean β-amylase with glucose was investigated by inhibition kinetics studies and spectroscopic measurements. The inhibition type, inhibitor constant (K1) and dissociation constant (Kd) of β-amylase-glucose complex were dependent on pH. At pH 8.0, glucose behaved as a competitive inhibitor (K1=34mM). Binding of glucose produced a characteristic difference spectrum and a change of circular dichroism (CD) at pH 8.1. By using difference absorbance at 292nm and difference ellipticity at 290 nm, Kd values for β-amylase-glucose complex were determined to be 45 and 46mM, respectively. In contrast to pH 8.0, glucose behaved as a mixed-type inhibitor (K1=320mM) at pH 5.4. The Kd values obtained from the difference spectrum were increased by lowering the pH from 8. The pH dependence of the K1 and Kd values suggested that one ionizable group of pK=8.0, which is shifted to 6.9 by the binding of glucose, controls the binding affinity of glucose. The binding of glucose competed with the binding of cyclohexaamylose and maltose at pH 8.0. The modification of S1-1 groups of the enzyme affected the binding of glucose but did not affect the binding of maltose or cyclohexaamylose at pH 8.0. It was concluded from these results that the binding site of glucose is different from that of maltose and cyclohexaamylose. Presumably, glucose may bind to the subsite 1 of soybean β-amylase.View full abstractDownload PDF (667K) -
Shigeru IKUMA, Munetoshi KIYOTA, Chiaki SETOYAMA, Kazunori SHIMADA1986 Volume 100 Issue 5 Pages 1185-1192
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAs retinoic acid (RA) and dibutyryl cAMP (cAMP) treatment induces differentiation of mouse teratocarcinoma F9 cells into parietal endoderm cells in vitro, we initiated studies on the molecular mechanisms underlying early mammalian cell differentia-tion in this system. We constructed cDNA libraries on the poly(A)+RNAs ex-tracted from the undifferentiated F9 cells, and screened for cDNA sequences ex-pressed abundantly in F9 cells, but not in terminally differentiated mouse parietal endoderm PYS-2 cells. Six different cDNA clones were isolated and characterized. The levels of RNAs hybridizable to these clones were at most 5 to 24% in the PYS-2 cells when compared with those in the undifferentiated F9 cells. The six clones were classified into two groups on the basis of their responses to the RA and cAMP treatment. In F9 cells, the levels of RNAs hybridizable to the first group, which contained four clones, were decreased within 72 h after the addition of RA and cAMP, while those of the second group, which contained the remaining two clones, did not decrease significantly. One of the first group clones, named pF9-1, corre-sponded to the mouse “early transposon-like elements” and another, named pF9-4, hybridized to multi-size RNAs extracted from the undifferentiated F9 cells. The mouse genomic DNA sequences hybridizable to pF9-4 were repeated approximately 5, 000 times, and comprise a new gene family, the expression of which is develop-mentally regulated in mouse F9 cells.View full abstractDownload PDF (1780K) -
Yuji SUGITA, Yasuko NAKANO, Motowo TOMITA1986 Volume 100 Issue 5 Pages 1193-1200
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMild trypsinization of human erythrocyte stroma solubilized CR1 (complement receptor type 1, C3b/C4b receptor) without significant loss of decay-accelerating activity to C5 convertases on hemolytic intermediate cells (EAC 1-3b, P). The solubilized CR1 was purified using DEAE-Sephacel, C3-Sepharose, and anti-CR1- Sepharose column chromatographies. The purified material showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under non-reducing conditions, and its molecular weight was determined to be 175K, about 20K smaller than native CR1. Because the purified sample was separated into the several seg-ments by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions, the molecule is considered to be nicked and those segments are associated by disulfide bonds. These results mean that a large portion of the CR1 molecule is present outside of the plasma membrane of erythrocytes, and the intramembranous and cytoplasmic domains are not necessary for decay-accelerating activity.View full abstractDownload PDF (1059K) -
Kiyomi YOSHIZUMI, Kiyosuke KAMIYAMA, Tiee-Cherng SHIEH, Shuji TANAKA, ...1986 Volume 100 Issue 5 Pages 1201-1206
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMethyl Nα-acetyl-2-(alkylthio)-L-tryptophanoates bearing different alkylthio groups were synthesized and employed as substrates for α-chymotrypsin and Carlsberg subtilisin in an attempt to investigate the properties of the hydrophobic pocket or cleft (S1 subsite) of the enzymes which accommodates the side-chain of the P1 amino acid residue of the substrates. The derivatives with ethylthio, 2-hydroxyethylthio, 2, 3-dihydroxypropylthio, 2-aminoethylthio, carboxymethylthio, 2-carboxyethylthio, 1, 2-dicarboxyethylthio, and 2-amino-2-carboxyethylthio (cysteinyl-S) groups were hydrolyzed by α-chymotrypsin but with kcat/Km values 4.6 to 15 times smaller than that of methyl Nα-acetyl-L-tryptophanoate, due mainly to larger Km values. The glutathionyl derivative was only weakly bound to the enzyme. Analyses of the kinetic parameters suggested that the S1 pocket of α-chymotrypsin is rather more spacious than has been supposed and is able to interact flexibly with substrates so as to orient the scissile bond to the catalytic residues. On the other hand, none of the derivatives were hydrolyzed by Carlsberg subtilisin but they all inhibited the enzyme with K1 values which are generally smaller than the Km values for Nα-acetyl-L-aromatic (modified aromatic) amino acid methyl esters. The S1 cleft of Carlsberg subtilisin interacts rather strongly with the derivatives but lacks the flexibility neces-sary for catalysis.View full abstractDownload PDF (455K) -
Mitsu SUZUKI, Hirotaka YAMAMOTO, Michinao MIZUGAKI1986 Volume 100 Issue 5 Pages 1207-1213
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSLipase [triacylglycerol lipase, EC 3.1.1.3] has been purified to homogeneity from Rhizopus japonicus NR 400 by chromoatography on hydroxylapatite, octyl-Sepharose and Sephacryl S-200. It showed a molecular weight of about 30, 000 by SDS-PAGE and a specific activity of 68, 900 units/mg protein. The enzyme catalyzed the hy-drolysis of tricapryn and tricaprylin rapidly in comparison with other triglycerides. This lipase had an optimum pH of around 5, and albumin enhanced its activity between pH 3 and 8. The composition of fatty acids liberated from linseed oil by the lipase was similar to that in the case of pancreatic lipase. The lipase activity was not affected by the addition of 1mM metal ions or bile salts. Stimulation of the lipase activity was observed upon addition of albumin to the reaction mixture. Immunotitration experiments were also performed with antibodies raised against the purified lipase.View full abstractDownload PDF (1731K) -
Shuji MITA, Shuichiro MAEDA, Kazunori SHIMADA, Shukuro ARAKI1986 Volume 100 Issue 5 Pages 1215-1222
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe distribution of prealbumin mRNA in various tissues of a control subject and an individual with familial amyloidotic polyneuropathy (FAP) was investigated using a cloned human prealbumin cDNA as a probe. Prealbumin mRNAs were detected in the total RNAs from liver and choroid plexus of brain, but not in those from brain, heart, kidney, and thyroid gland. The level of prealbumin mRNA in the choroid plexus was about 100-fold higher than that in the liver. We detected no differences in the sizes of prealbumin mRNAs in the liver and choroid plexus of the control subject, or in those of the individual with FAP, and found that the prealbumin mRNAs in these tissues had been transcribed from a common tran-scription initiation site. The levels of prealbumin mRNAs in the livers of four control subjects and those of six individuals with FAP were much the same. Because all the individuals with FAP so far examined were heterozygous for the prealbumin gene, carrying one normal and one mutant gene, the levels of the normal and mutant prealbumin mRNAs in the liver and the brain tissue containing choroid plexus were separately estimated and were found to be approximately equal.View full abstractDownload PDF (3479K) -
Kunio AOKI, Takashi IDEGUCHI, Tomisaburo KAKUNO, Jinpei YAMASHITA, Tak ...1986 Volume 100 Issue 5 Pages 1223-1230
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIt is known that the halophilic green alga Dunaliella tertiolecta grows under hyper-tonic conditions (with NaCI), which induce the intracellular accumulation of high concentrations of glycerol in order to counterbalance the osmotic change. The effects of NaC1 and glycerol on the photosynthetic oxygen-evolving activity of thylakoid membranes prepared from D. tertiolecta were investigated in relation to the dissociation of the membranes. It was found that proteins with Mr of 24, 000, 17, 000, and 13, 000 were dissociated from thylakoid membranes of D. tertiolecta by washing with 1M NaCI, whereas the photosynthetic oxygen-evolving activity was stimulated 2-fold by 0.1-1.5M NaCI. The antibodies against spinach 24K and 17K proteins did not cross-react with Dunaliella 24K and 17K proteins, respectively. The salt-tolerant feature of the oxygen-evolving activity with Dunaliella thylakoid membranes may be related to the difference of the properties of these two proteins between D. tertiolecta and spinach. When the membranes were washed with 1M Tris, proteins with Mi. of 50, 000 and 31, 000 were also dissociated in addition to the 24K and 17K proteins described above. The antibody against spinach 33K protein cross-reacted with 31K protein of D. tertiolecta, showing that Dunaliella 31K protein corresponds to spinach 33K protein. When the membranes were treated with a mixture of 1% cholate and 2% deoxycholate, the oxygen-evolving activity was completely depressed, but the depressed activity was significantly restored by organic solvents. Glycerol and dimethylsulfoxide were the most effective for the restora-tion. The restoration of the oxygen-evolving activity was also observable with membranes treated with the other detergents such as digitonin, MEGA-8, Triton
X-100, Nonidet P-40, and octyl glucoside. High concentrations of glycerol and dimethylsulfoxide stimulated the NaCl-stimulated activity a further twofold with untreated thylakoid membranes. Possibly, the components associated with the oxygen-evolving activity were able to act more effectively in the presence of high concentrations of NaC1 and glycerol. It was found, from the results of polyacryl-amide gel disk electrophoresis, that the membrane proteins were appreciably dis-sociated by Triton X-100 or octyl glucoside. Accordingly these detergents may be available for preparing PS II reaction center complex.View full abstractDownload PDF (1823K) -
Kazuya TANIGUCHI, Kuniaki SUZUKI, Toshio SASAKI, Hirokata SHIMOKOBE, S ...1986 Volume 100 Issue 5 Pages 1231-1239
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAddition of up to 300μM ATP in the presence of 2M NaCl with MgCl2 to pig kidney Na+, K+-ATPase treated with N-[p-(2-benzimidazolyl)phenyl]maleimide seemed to be insufficient to saturate the rate of the fluorescence decrease. How-ever, both the extent of the decrease and the amount of phosphoenzyme at a steady state were saturated below 20μM ATP. Addition of Mg2+ with Na+ to the enzyme preincubated with 20 to 600μM ATP gave nearly the same rate constant, which was below 50% of that obtained by adding 300μM ATP to the Na+-form enzyme in the presence of Mg2+. High concentrations of ATP affected neither the rate of light-scattering change (Taniguchi, K. et al. (1986) J. Biol. Chem. 261, 3272-3281) after ADP-sensitive phosphoenzyme formation (E1P) nor that of the breakdown of E1P. A stoichiometric amount of [32P]P1 was liberated from [32P]E1P.
The data suggested that ATP did not bind to E1P in such a way as to increase the extent of phosphorylation further or to accelerate dephosphorylation. The data also suggested that the reason for the large difference in the apparent affinity of ATP as evaluated from the rate and the extent of fluorescence change is the large dissociation constant for ATP of a Michaelis complex.View full abstractDownload PDF (759K) -
Yoshiaki KOMIYA, Neil A. COOPER, Antony D. KIDMAN1986 Volume 100 Issue 5 Pages 1241-1246
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFollowing a single intraperitoneal injection of β, β'-iminodipropionitrile (EDPN) simultaneously with or 5 weeks after injection of [35S]methionine into the ventral horn area of rat spinal cord, the changes of slowly migrating axonal proteins were analyzed electrophoretically up to 10 weeks after labeling, and the following results were obtained. 1. After a single injection of IDPN, only the transport of neuro-filament proteins is inhibited, leaving that of tubulin and actin almost unaffected, though a small portion of the former was retarded through the interaction with neurofilaments. The inhibitory effect of IDPN on neurofilament transport is not a complete blockage, but a slowing of the rate of transport to about a half of the control with a possible short halting period just after IDPN treatment. 2. The dose-response data indicate a threshold between 0.5 and 1.0 g/kg of body weight, increasing the dose above which does not further affect the neurofilament transport. 3. The transport of neurofilaments is uniformly impaired by IDPN along the whole axon.View full abstractDownload PDF (1720K) -
Takeyuki KOHNO, Eiji ISHIKAWA1986 Volume 100 Issue 5 Pages 1247-1251
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA highly sensitive enzyme immunoassay of anti-insulin antibodies in guinea pig serum is described. Guinea pig anti-insulin serum was diluted to various extents with nonspecific guinea pig serum and incubated with insulin. After incubation, free insulin was separated from insulin-anti-insulin antibody complex by treatment with dextran-charcoal. Anti-insulin antibodies in the complex were dissociated from insulin by incubation with 0.23M HC1 and inactivated. The amount of dissociated insulin was measured by sandwich enzyme immunoassay using anti-insulin IgG-coated polystyrene balls and affinity-purified anti-insulin Fab'-horse-radish peroxidase conjugate. The detection limit of anti-insulin antibodies in guinea pig serum was 6.7pg/assay or 150 ng/liter of serum. The present enzyme immunoassay was 10, 000-fold more sensitive than the previously described enzyme immunoassay, in which insulin-coated polystyrene balls were incubated with diluted guinea pig anti-insulin serum and subsequently with rabbit (anti-guinea pig IgG) Fab'-horseradish peroxidase conjugate.View full abstractDownload PDF (398K) -
Hiroo YONEZAWA, Koji OKAMOTO, Kazuhiko TOMOKIYO, Nobuo IZUMIYA1986 Volume 100 Issue 5 Pages 1253-1259
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo elucidate the mode of antibacterial action by gramicidin S (GS), a detailed experi-ment on GS distribution on bacteria cells was carried out. 14C-Labeled gramicidin S ([14C]GS) was incubated with cells of Gram-positiveBacillus subtilis and Gramnegative Escherichia coli, and the amount of [14C]GS adsorbed on the cells was measured. Adsorption on B. subtilis cells was observed from 1 ileml of [14C]GS. As the concentration of [14C]GS increased, the amount adsorbed on B. subtilis increased discontinuously, producing a curve which had three plateaus. On the other hand, [14C]GS was not easily adsorbed on E. coli cells at lower concentrations, but the amount adsorbed increased above 6ilg/ml, and the cells were temporarily saturated with GS at 10μg/ml, which is the minimum inhibitory concentration for E. coli. The amount of [14C]GS adsorbed on the protoplast membrane of B. subtilis was the same as that of natural cells. However, the amount of [14C]GS adsorbed on the cell wall dropped to about 20% of that of natural bacteria. These facts indicate that GS is adsorbed on the cell membrane of bacteria particularly. The uptake of amino acid or glucose in B. subtilis was inhibited by GS. Therefore, it is concluded that GS damages the phospholipid bilayer of the cell membrane by ad-sorption, and prevents the functioning of the cell membrane. The amount of [14C]GS adsorbed on the spheroplast membrane of E. colt increased remarkably as compared with natural cells, even at a lower concentration of GS. The poor GS adsorption on E. coli cells may be due to the permeability barrier of the E. coli cell wall.View full abstractDownload PDF (595K) -
Shoichi OHKUBO, Hidekazu IWASAKI, Hiroshi HORI, Syozo OSAWA1986 Volume 100 Issue 5 Pages 1261-1267
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe nucleotide sequences of 5S rRNA from seven denitrifying bacteria have been determined. Based on these sequences and those reported in the literature (in-cluding two denitrifiers), a phylogenic tree of 104 eubacterial 5S rRNA sequences has been constructed to establish the position of the denitrifying bacteria. These bacteria belong to either one of the three major subgroups of gram-negative bacteria. The grouping based on 5S rRNA sequences is almost compatible with the type of the nitrite reductases, with the one apparent exception of Paracoccus denitrificans ATCC 13543. Moreover, the separation time of most of the denitrifying bacteria from other non-denitrifying bacteria belonging to the same subgroup is recent. These results suggest that the denitrifying systems in these bacteria would have developed polyphyletically, and not so anciently, during eubacterial evolution.View full abstractDownload PDF (530K) -
Mitsue TAKEDA, Eiko UENO, Yuzo KATO, Kenji YAMAMOTO1986 Volume 100 Issue 5 Pages 1269-1277
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn erythrocyte membrane-associated cathepsin D-like acid proteinase, termed “EMAP, ” was purified to homogeneity from freshly collected rat blood in a yield of 60-65%. The molecular weight of the enzyme was determined to be 80, 000-82, 000 by Sephadex G-100 gel filtration. The enzyme was inhibited strongly by pepstatin and partially by HgC12, Pb(NO3)2, and iodoacetic acid. The preferred substrate for the enzyme was hemoglobin. The enzyme also hydrolyzed serum albumin and casein, but to lesser extents, with an optimum pH of 3.5-4.0. However, it could not hydrolyze leucyl-2-naphthylamide, benzyloxycarbonyl-Phe-Arg-4- methy1-7-coumarylamide or other synthetic substrates at pH values ranging from 3.5 to 9.5. The enzyme was very similar to human EMAP in a number of enzymatic properties, whereas it differed from rat cathepsin D in several respects, such as pH stability, molecular weight, isoelectric point, and chromatographic properties. Immunologically, the enzyme cross-reacted with the rabbit antibody prepared against human EMAP. The patterns of immunoelectrophoresis, immunoblotting, and immunoprecipitation of the enzyme were remarkably similar, if not identical, to those of human EMAP. In contrast, rat EMAP showed no reaction with the rabbit antibody raised to rat spleen cathepsin D. These results indicate that EMAP is a unique cathepsin D-like acid proteinase different from ordinary cathepsin D.View full abstractDownload PDF (2429K) -
Makoto DAIMON, Ken-ichi TSUTSUMI, Kiichi ISHIKAWA1986 Volume 100 Issue 5 Pages 1279-1286
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDNA methylation was studied as a potential factor for the regulation of tissue-specific and developmentally specific expression of the rat aldolase B gene. We examined cytosine methylation in the HpaII and HhaI recognition sequences in the aldolase B gene in aldolase expressing and nonexpressing tissues and cells. Out of the 15 methyl-sensitive restriction sites examined, the sites in the 3'-half and 3'-flanking regions were found to be heavily methylated in all the tissues or cells, regardless of the level of aldolase B gene expression. However, the methylation pattern in the region immediately upstream and in the 5'-half of the gene exhibited tissue-specificity: the site located about 0.13 kb upstream of the cap site (just next to the CCAAT box), and the sites in the first intron (intron 1) were heavily methyl-ated in nonexpressing cells and tissues (ascites hepatoma AH130 and brain), whereas those in an expressing tissue (liver) were considerably less methylated. These results suggest that cytosine methylation at the specific sites in the 5'-flanking and 5'-half regions of the gene is associated with repression of the gene activity. However, the gene is still substantially methylated in the fetal liver on day 16 of gestation, when it is in a committed state for rapid activation in the period immediately after-wards (Numazaki et al. (1984) Eur. J. Biochem. 152, 165-170). This suggests that demethylation of the methylated cytosine residues in the specific gene region is not necessarily required before activation of the gene during development, but it may occur along with or after the activation.View full abstractDownload PDF (1841K) -
Weijia OU, Akio ITO, Ken-ichirou MOROHASHI, Yoshiaki FUJII-KURIYAMA, T ...1986 Volume 100 Issue 5 Pages 1287-1296
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn the presence of a membrane-permeable metal chelator, bovine adrenal cortex mitochondria imported P-450(SCC) precursor without processing of the amino-terminal extension peptide. The imported precursor was bound to the matrix side surface of the inner membrane. When the inhibition due to the metal chelator was removed, the imported precursor was processed to the mature form. Unprocessed precursor was also detected in mitochondria when the import reaction was carried out at relatively low temperature. These results suggest that the translocation of P-450(SCC) precursor across mitochondrial membranes is independent of its pro-cessing to the mature form. Both membrane-bound and solubilized P-450(SCC) could be cleaved by trypsin into two fragments with molecular weights of 29 kDa and 26 kDa, respectively, suggesting a two-domain structure of the molecule. The in vitro-imported and processed P-450(SCC) was also cleaved by trypsin in the same way. This finding indicated that the in vitro-imported and processed P-450(SCC) has the same conformation as the native form.View full abstractDownload PDF (4406K) -
Eva STEFANSKI, Waldemar PRUZANSKI, Berit STERNBY, Peter VADAS1986 Volume 100 Issue 5 Pages 1297-1303
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA soluble phospholipase A2 (PLA2) was purified 4, 500-fold from human rheumatoid synovial fluid. Preparative sodium dodecyl sulfate polyacrylamide gel electropho-resis yielded two bands of PLA2 activity of molecular weights 15, 000 and 17, 000 and p1 4.2-5.0. Purified PLA2 had absolute 2-acyl specificity, and hydrolyzed phosphatidylcholine with optimal activity at pH 7.5-8.0 and phosphatidylethanol-amine with optimal activity at pH 7.0. Human synovial fluid PLA2 did not cross-react with anti-human pancreatic PLA2, as tested by radioimmunoassay.View full abstractDownload PDF (1117K) -
Yasuo OGAWA, Nagomi KUREBAYASHI, Hikaru HARAFUJI1986 Volume 100 Issue 5 Pages 1305-1318
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn order to obtain a better understanding of the mechanism of the function of frag-mented sarcoplasmic reticulum (FSR), we examined the binding of β, γ-methylene [3H]adenosine triphosphate (AMPOPCP), an unhydrolyzable ATP analogue, and 45Ca to FSR from bullfrog skeletal muscle. In medium containing 100mM KCl and 20mM Tris-maleate (pH 6.80) on ice, FSR has a single class of [3H]AMPOPCP-binding sites which amount to 4.4-8.6 nmol/mg protein (usually about 7nmol/mg protein). The affinity was in the range of 6.2-12.3 × 103 M-1 in the absence of Ca2+. Ca2+ increased the affinity for AMPOPCP without changing the total number of binding sites, whereas Mel- decreased it. The change of the affinity is due to the direct effect of Ca2+ and Mg2+ on FSR. The possibility that Mg-AMPOPCP, Ca-AMPOPCP, and free AMPOPCP might have different affinities to FSR is excluded. The extent of Ca2+-I-induced enhancement in AMPOPCP binding is de-pendent not only on Ca2+ concentration but also on the concentration of AMPOPCP. The binding sites for AMPOPCP are likely to be the ATP-binding sites on Ca2+ I-ATPase protein on the basis of several lines of evidence, including competition between ATP, ADP, or AMP. FSR also binds 7-13nmol Ca/mg protein (usually about 8nmol/mg protein) with the affinity of 4-14 × 104 M-1 in the absence of the nucleotide in a similar medium containing 4mM MgCl2. The ratio of Ca-binding sites to AMPOPCP-binding sites is mostly 1, but occasionally 2, corresponding to the ratio of Ca accumulated to ATP hydrolyzed by frog FSR. In the presence of a sufficient amount of the nucleotide, the affinity for Ca2+ was also increased. These findings are well explained by the random sequence binding model of Cat I and
AMPOPCP, which bind to FSR with positive cooperative interaction between them. However, high concentrations of the nucleotide result in a negative cooperative interaction in the nucleotide binding in the presence of Ca2+, whereas no coopera-tivity is observed in the absence of Ca2+. Stimulation of Ca binding by AMPOPCP is also correspondingly affected. Comparative studies show that rabbit skeletal muscle FSR, in contrast to the frog one, shows negative cooperativity in its inter-actions with Ca2+ and AMPOPCP under some conditions and that the ratio of Ca-binding sites to AMPOPCP-binding sites is 2, corresponding to the well-known stoichiometry with ATP.View full abstractDownload PDF (1022K) -
Yasuo OGAWA, Hikaru HARAFUJI1986 Volume 100 Issue 5 Pages 1319-1328
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn order to obtain a better understanding of Ca2+-activated ATP hydrolysis by sarcoplasmic reticulum, the transient kinetics of phosphorylated intermediate (EP) formation was examined with different sequences of addition of Ca2+ and ATP to GEDTA-added (Ca2+-free) fragmented sarcoplasmic reticulum (FSR) from bullfrog skeletal muscle. With a short delay line (10-20ms), the addition sequence of ATP followed by Ca2+ gives rise to a faster EP formation without any lag time. In the reverse sequence of ligand addition, a lag time of 2.5-3ms was consistently observed irrespective of ATP concentration, and the rate of EP formation was lower. As the preincubation time with Ca2+ became longer, the rate constant for EP formation and the maximum level of EP attainable increased even in the presence of fixed concen-trations of Ca2+ and ATP. The rate constant per the unit concentration of EP, however, remained constant, indicating second-order kinetics between Ca2+-activated FSR and ATP. With a preincubation time of 83.6ms, no lag time was observed. In the addition sequence of ATP and 83.6ms later Ca2+, a burst in EP formation was observed, which was followed by a usual exponential time-course of EP forma-tion. Similar determinations in the presence of various concentrations of ATP led us to the following estimates of the rates of the reactions leading to EP formation at 15°C.
The ATP-activation route is much faster and more relevant to the physiologically important Ca-uptake process.View full abstractDownload PDF (726K) -
Shinpei YAMADA, Junichi FUJII, Hajime KATAYAMA1986 Volume 100 Issue 5 Pages 1329-1342
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn order to characterize the phosphoenzymes (EPs) formed from MgATP and CaATP as substrates, the effects of Mg2+ and Ca2+ outside SR vesicles on the hydrol-ysis rates of EPs were examined by using purified and unpurified Ca-ATPases of sarcoplasmic reticulum (SR) at low [γ-32P]ATP (4-10μM), 0.1M KCl, pH 7.0, and 0°C. When the phosphorylation reaction was stopped by adding an excess of EDTA over Ca and Mg, two components of EP, EPfast (rate constant, kfast=15-20 min-1), and EPsiow (kslow =0.3-0.4 min-1), were recognized in the time course of EP decomposition. These two rates did not depend on the Ca2+ or Mg2+ con-centration in the medium during the phosphorylation reaction, although the pro-portions of EPfast and EPslow essentially depended on the concentrations of MgATP and CaATP in the phosphorylation reaction medium. The proportion of EPfast increased with increasing [MgATP]/[CaATP] in the medium, whereas that of EPslow decreased. The rate of EPslow hydrolysis in the presence of excess EDTA was basically the same as that of EP formed from CaATP. These results suggest that EPfast and EPslow are derived from MgATP and CaATP, respectively, and EPtast is a reaction intermediate with Mg bound at the substrate site ( MgEP), while the main EPslow is a reaction intermediate with Ca bound at the substrate site (CaEP) which is readily converted to metal-free EP by EDTA addition (Shigekawa et al., (1983) J. Biol. Chem. 258, 8698-8707). Mg2+ added outside SR vesicles stimulated the conversion of CaEP to MgEP and inhibited the hydrolysis of MgEP in the relatively high concentration range (K(Mg) =7.9mM). Ca2+ added outside SR vesicles stimulated the conversion of MgEP to CaEP and inhibited the conversion of CaEP to MgEP by Mg2+ addition. The Ca2+ outside SR vesicles did not essen-tially affect the hydrolysis of MgEP. These results suggest that the interconversion between MgEP and CaEP takes place during the reaction by exchange of the diva-lent cation on the substrate site. The following scheme is proposed.View full abstractDownload PDF (1101K) -
Takao AKAMA, Kenneth M. YAMADA, Nobuko SENO, Isamu MATSUMOTO, Ichiro K ...1986 Volume 100 Issue 5 Pages 1343-1351
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe cell-adhesive glycoprotein vitronectin in human plasma was characterized with a monospecific anti-vitronectin antibody. Vitronectin, a mixture of monomeric 75 and 65kDa polypeptides, was found to have different ratios of amounts of 75 and 65kDa polypeptides in immunoblots of sera from various healthy human donors. Two states of vitronectin were previously reported; the open state binds to heparin, but the cryptic state does not (Hayashi et al. (1985) J. Biochein. 98, 1135-1138). The anti-vitronectin antibody was suggested to react more strongly with the open state of vitronectin than with the cryptic state. To quantitate all vitronectin regardless of its state, an enzyme-linked immunosorbent assay of vitro-nectin was developed based on prior boiling of vitronectin-containing samples in 2% (w/v) sodium dodecyl sulfate and 40mM dithiothreitol to destroy conformational differences. About 12-20% of the vitronectin molecules in plasma were found to bind to heparin-Sepharose under physiological conditions. Vitronectin in plasma bound 30-fold more efficiently to heparin immobilized by amino groups than by carboxyl groups. Its affinity for heparin was higher than for chondroitin sulfate A or C, or dermatan sulfate. Vitronectin was also found to contain covalently-linked small polypeptides of 15 and 13kDa. These light chains seemed to be disulfide-bonded to the 65 kDa polypeptide, and might be endogenously derived from nicks in the carboxy-terminal portion of the 75kDa polypeptide in plasma.View full abstractDownload PDF (1933K) -
Kaoru OMICHI, Tokuji IKENAKA1986 Volume 100 Issue 5 Pages 1353-1358
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSp-Nitrophenyl 0-6-deoxy-6-[(2-pyridyl)amino]-α-D-glucopyranosyl-(1→4)-0-α-D-glucopyranosyl-(1→4)-O-α-D-glucopyranosyl-(1→4)-O-α-D - glucopyranosyl - (1→4)- α-o-glucopyranoside (FG5P) is hydrolyzed by human pancreatic α-amylase (HPA) or salivary α-amylase (HSA) to 0-6-deoxy-6-[(2-pyridyl)amino]-α-D-glucopyranosyl-(1→4)-O-α-D-glucopyranosyl-(1→4)-D-glucose (FG3) and p-nitrophenyl α-malto-side or to 0-6-deoxy-6-[(2-pyridyl)amino]-α-D-glucopyranosyl-(1→4)-O-α-D-gluco-pyranosyl-(1→4)-O-α-D-glucopyranosyl-(1→4)-D-glucose (FG4) and p-nitrophenyl α-glucoside. The use of α-D-glucosidase (maltase) [EC 3.2.1.20] of Saccharomyces carlsbergensis and oligo-1, 6-glucosidase (isomaltase) [EC 3.2.1.10] of bakers' yeast as coupled enzymes differentiates between the two reactions, because α-D-glucosidase liberates p-nitrophenol from both p-nitrophenyl α-glucoside and p-nitrophenyl α-maltoside, but oligo-1, 6-glucosidase liberates it only from p-nitrophenyl α-glucoside. HPA produces more FG4 and p-nitrophenyl α-glucoside than HSA. Taking advan-tage of the differences in the action of the two amylases and in the substrate specificity of the coupled enzymes, we have developed a new colorimetric differential rate assay of α-amylases in human serum.View full abstractDownload PDF (428K) -
Tatsumi MATSUMOTO, Yoshikazu EMI, Shun-ichirou KAWABATA, Tsuneo OMURA1986 Volume 100 Issue 5 Pages 1359-1371
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThree forms of cytochrome P-450, tentatively designated P-450(M-l), P-450(M-2), and P-450(M-3), and one form of cytochrome P-450, P-450(F-1), were purified from the liver microsomes of untreated male and female rats, respectively. Each purified form of the cytochrome showed a single protein band on SDS-polyacrylamide gel electrophoresis, and gave a minimum molecular weight of 51, 000 for P-450(M-1), 48, 000 for P-450(M-2), 49, 000 for P-450(M-3), and 50, 000 for P-450(F-1). The carbon monoxide-difference spectra of reduced P-450(M-1), P-450(M-2), P-450(M-3), and P-450(F-1) showed an absorption maximum at 451, 451, 448, and 449 nm, re-spectively. Judging from the absolute absorption spectra, the four forms of cyto-chrome P-450 were of low-spin type in the oxidized forms. The antibodies against P-450(M-2) did not crossreact with the other forms in the Ouchterlony double diffu-sion test, whereas the immunodiffusion test showed immunocrossreactivity between P-450(M-1) and P-450(F-1), P-450(M-1) and P-450(M-3), and P-450(M-3) and P-450(F-1). The NH2-terminal amino acid sequences of the four forms confirmed that they were different molecular species, although significant homology was noticed among P-450(M-1), P-450(M-3), and P-450(F-1). The quantitation of P-450(M-1) and P-450(F-l) in liver microsomes by quantitative immunoprecipitation confirmed that these two forms of cytochrome P-450 were developmentally induced in male and female rats, respectively. P-450(M-2) was also developmentally induced in male rats. In a reconstituted system containing NADPH and NADPH-cytochrome P-450 reductase, P-450(M-1) oxidized benzphetamine at a high rate, whereas the other forms had low activity toward benzphetamine. None of the four forms
showed high activity toward benzo(a)pyrene. P-450(M-1) catalyzed the hydroxyla-tion of testosterone at the 16a and 2a positions, whereas P-450(M-2) catalyzed the 15a hydroxylation of the same substrate.View full abstractDownload PDF (2838K) -
Masanobu KAWAKAMI, Toshio MURASE, Shun ISHIBASHI, Natsuko MORI, Fumima ...1986 Volume 100 Issue 5 Pages 1373-1378
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effects of insulin and dexamethasone on the secretion of lipoprotein lipase (LPL) by mouse peritoneal macrophages were examined in vitro. Macrophages from either normal or thioglycollate-primed mice continuously secreted LPL into the culture medium. The time courses and the amounts of enzyme secretion and the responses to the hormones were essentially the same in resident or thioglycollate-primed macrophages. Insulin did not enhance the secretion of this enzyme by macrophages, even though a marked effect of this hormone on the enzyme in adipose tissue has been well established. Dexamethasone, which has been reported to stimulate the secretion of LPL in the heart, suppressed the secretion of LPL by macrophages. The present study is the first report to deal with the effect of hor-mones on the secretion of LPL by macrophages, and clearly demonstrates that insulin does not play an important role in the regulation of LPL activity in macro-phages. Dexamethasone also showed a different effect on macrophage LPL com-pared to that on the enzyme in other tissues. This difference in the regulation of LPL may be relevant to the possibly different role of this enzyme in macrophages as compared to other tissues such as adipose tissue, muscle, or heart.View full abstractDownload PDF (445K) -
Akiyoshi TANAKA, Masahide ITO, Keitaro HIROMI1986 Volume 100 Issue 5 Pages 1379-1385
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe binding of glucono-1, 5-lactone (gluconolactone) with almond β-glucosidase was studied at pH 5.0 and 25°C, in the absence and presence of glucose, by monitoring the enzyme fluorescence as a probe. From the results of fluorometric titration, the dissociation constant Kd and the maximum fluorescence intensity increase (percent) of the enzyme-gluconolactone complex relative to the enzyme alone, JFmax, were determined to be 12.7μM and 14.7%, respectively. From the study of the temperature dependence of Kd, ΔG°, ΔH° and ΔS° for the binding were evaluated to be- 6.7 kcal mol-1, -3.5 kcal mol-1, and 10.8 e.u. (cal mol-1 deg-1), respectively, at 25°C. The analysis of the fluorometric titration data in the presence of glucose revealed that these ligands bind competitively to the enzyme, probably at the same site. The results of a stopped-flow kinetic study are consistent with the following two-step mechanism:
(fast) (slow)
which indicates that gluconolactone (L) and the enzyme (E) transiently form a loosely bound complex, ELtr (k_i/k+i=4.5mM), in the first rapid bimolecular association step, and ELtr is converted into a more tightly bound complex EL (k+2=94s-1, k-2=0.36s-1) in the subsequent slow unimolecular process. The fluorescence intensity increase occurs solely in the latter step.View full abstractDownload PDF (492K) -
Tsutomu TSUJI, Toshiaki OSAWA1986 Volume 100 Issue 5 Pages 1387-1398
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHuman platelet membrane glycoproteins IUD (GPIIb) and Ina (GPIIIa), which have been proposed to be subunits of a receptor for fibrinogen, were purified from Triton X-100-solubilized platelet membranes by affinity chromatography on a concanavalin A (Con A)-Sepharose column followed by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Compositional analyses of the purified glycoproteins showed that GPIIb and GPIIIa contain 15% and 18% carbohydrate by weight, respectively, which consists of galactose, mannose, glucosamine, fucose, and sialic acid. This suggested that these glycoproteins contained N-linked car-bohydrate chains. The carbohydrate chains were released from each glycoprotein by hydrazinolysis and then fractionated by ion-exchange chromatography on a Mono Q column. From each glycoprotein, mono-, di-, and trisialylated and neutral oligosaccharide fractions were obtained. The structures of these oligosaccharides were investigated by means of compositional and methylation analyses and digestion by exoglycosidase, and their reactivities to immobilized lectins were also examined. The neutral oligosaccharides, which comprised about 14% of the total oligosac-charides released from GPIIb and about 52% of that from GPIlla, were found to be of the high mannose-type, in that they contained 5 or 6 mannose residues. On the other hand, a major part of the acidic oligosaccharides was found to consist of typical bi- and triantennary complex-type sugar chains, and much smaller amounts of tetraantennary complex-type sugar chains, and complex-type sugar chains with a fucosyl residue at a N-acetylglucosamine residue in the peripheral portion or a bisecting N-acetylglucosamine at a β-mannosyl residue in the core portion were also detected. In conclusion, we found that GPIIb contained mainly complex-type sugar chains, whereas high mannose-type sugar chains were the predominant car-bohydrate units in GPIIIa, and that the detected differences in the carbohydrate moieties of GPIIb and GPIIIa were quantitative but not qualitative.View full abstractDownload PDF (1671K) -
Yoshihiko SUMI, Yuichi NAKAMURA, Nobuo AOKI, Masaharu SAKAI, Masami MU ...1986 Volume 100 Issue 5 Pages 1399-1402
Published: 1986
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA 1.7 kilobase cDNA clone isolated from a human liver cDNA library contained 822 nucleotides encoding the carboxyl-terminal 274 amino acid sequence of α2- plasmin inhibitor, a stop codon and a 3' noncoding region of 0.9 kilobases. The amino acid sequence deduced from the cDNA shows 29-31% homology with those of other plasma protease inhibitors. The inhibitor's putative reactive-site peptide bond was Met-Ser, and the Met residue was located at the 91st position from the carboxyl-terminal end. The plasminogen binding site was located in the carboxyl-terminal region.View full abstractDownload PDF (315K)
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