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Volume 102, Issue 1
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Koichi KATO, Mamoru NAKANISHI, Yoji ARATA, Reiko TESHIMA, Tadao TERAO, ...1987 Volume 102 Issue 1 Pages 1-4
Published: July 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSUsing a digital imaging fluorescence microscope, we have detected a rapid transient increase in the free cytosolic calcium concentration in a single rat basophilic leukemia cell (RBL-2H3) after antigen stimulation. Calcium ions were transported very rapidly (within 1 s) after a lag time (about 10 s at 37°C) from the external environment into the cytoplasm. On the basis of the present experimental results we conclude that the gradual changes in the overall fluorescence intensity observed for a cell suspension are due to the distribution of different lag times shown by different cells as to the calcium influx through membrane calcium channels.View full abstractDownload PDF (905K) -
Kohji HANASAKI, Tohru NAKANO, Hitoshi ARITA1987 Volume 102 Issue 1 Pages 5-8
Published: July 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe formation of thromboxane A2 (TXA2) in collagen-stimulated rat platelets was successfully divided into two stages, an initial and a second one, by the specific TXA2 receptor antagonist, ONO3708. In the presence of this antagonist, only the initial TXA3 production was observed, without the subsequent platelet shape change and aggregation. Collagen causes the specific cleavage of arachidonic acid from phos-phatidylinositol (PI) in the initial stage, whereas in the absence of the antagonist, it caused decrease in the arachidonic acid levels in phosphatidylethanolamine (PE) and PI with concomitant formation of the respective lysoforms. These results demonstrate that phospholipase A (PLA) preferentially acts on PI to release ara- chidonic acid which leads to the initial TXA2 production, which might be a trigger for the second release of arachidonic acid from PE and PI.View full abstractDownload PDF (313K) -
Shoichi ISHIURA, Harumi ANRAKU, Isao KAMO, Hirotaka KOIZUMI, Kiichi AR ...1987 Volume 102 Issue 1 Pages 9-12
Published: July 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA Ca-dependent erythrolytic protein (perforin) was isolated from a cytotoxic T-cell line (CTLL2). Cellular extracts were fractionated on DEAE-cellulose and hydro-phobic Phenyl-Sepharose columns. Lytic activity was tightly bound to the hydrophobic column and was eluted with 50% ethyleneglycol. The erythrolytic activity was dependent on the concentration of Ca2+ ions, and heparin accelerated the lysis of erythrocytes by perforin 10-fold, with a half maximal concentration of 12ng/ml. The activity was strongly inhibited by micromolar concentrations of heavy metal ions, such as Zn2+ and Fe2+, and glycylarginine-methylcoumarinamide (Gly-Arg-MCA) in the presence of 100 ng/ml heparin.View full abstractDownload PDF (298K) -
Kiyoshi OKANO, Yosuke AOKI, Tohru SAKURAI, Masayuki KAJITANI, Shozo KA ...1987 Volume 102 Issue 1 Pages 13-16
Published: July 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMedullasin, an inflammatory serine protease in bone marrow cells, modifies thefunctions of natural killer cells, monocytes, and granulocytes. We have cloned a medullasin cDNA from a human acute promyelocytic cell (ML3) cDNA library using oligonucleotide probes synthesized from the information of N-terminal amino acid sequence of natural medullasin. The cDNA contained a long open reading frame encoding 237 amino acid residues beginning from the second amino acid of natural medullasin. The deduced amino acid sequence of medullasin shows a typical serine protease structure, with 41 % homology with pig elastase 1.View full abstractDownload PDF (922K) -
Kazuhiro KOHAMA, Tomoko KOHAMA, John KENDRICK-JONES1987 Volume 102 Issue 1 Pages 17-23
Published: July 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have established a quick method for preparing Physarum myosins whose actin-activated ATPase activities are inhibited by μM levels of Ca2+ (from plasmodial stage: Kohama, K. & Kendrick-Jones, J. (1986) J. Biochem. 99, 1433-1446; and from amoebal stage: Kohama, K., Takano-Ohmuro, H., Tanaka, T., Yamaguchi, Y., & Kohama, T. (1986) J. Biol. Chem. 261, 8022-8027). N-Ethylmaleimide alkylates sulfhydryl (SH) groups on the heavy chains in the heads of the plasmodial myosin. The actin-activated ATPase activity of the modified myosin was significantly decreased when assayed in low Ca2+ concentrations. Moreover, the activity remained low even when the Ca2+ concentrations was increased, i.e., the myosin was desensitized. For complete desensitization, about 4 mol SH per mol myosin (500, 000 Mr) must be modified. These residues are probably the “reactive thiols” which have been predicted from primary structure studies to be conserved among myosins of higher and lower eukaryotes. Ultraviolet absorption spectra of the modified and intact myosins showed a peak at 277 nm. The height of this peak in intact myosin was reduced when the Ca2+ concentration was increased. This Ca-induced reduction was hardly detectable in the modified myosin although Ca-binding activity to myosin did not appear to be affected by the modification. We interprete these results that Ca2+ may change the conformation of the myosin heavy chain by binding to myosin and speculate that impairment of this process upon modification could cause the desensitization to Ca2+ in the ATPase activity.View full abstractDownload PDF (1789K) -
Yuichi OGASAWARA, Tohru KOMIYA, Takashi OBINATA1987 Volume 102 Issue 1 Pages 25-30
Published: July 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTroponin T (TNT) expressed in the developing chicken cardiac muscle was examined by immunoblotting combined with two-dimensional electrophoresis (2-D PAGE) and peptide mapping. When the whole lysate of the neonatal heart was examined by 2-D PAGE, two TNT variants were detected on the gel by monoclonal antibody to TNT. Expression of the two variants was developmentally regulated: one isoform (type I) was expressed from embryonic through neonatal stages, and the other (type II) from the late embryonic stage through adulthood during cardiac muscle development. The type-I isoform, but not type-II isoform, was also ex- pressed transiently in chicken skeletal muscle at embryonic stages. As judged from the peptide maps, the two isoforms differed in the N-terminal region but not in the C-terminal region.View full abstractDownload PDF (2583K) -
Etsuo YOKOTA, Issei MABUCHI, Hidemi SATO1987 Volume 102 Issue 1 Pages 31-41
Published: July 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWhen 21S dynein ATPase [EC 3.6.1.3] from sea urchin sperm flagellar axonemes was mixed with the salt-extracted axonemes, the ATPase activity was much higher than the sum of ATPase activities in the two fractions, as reported previously (Gibbons, I. R. & Fronk, E. (1979) J. Biol. Chem. 254, 187-196). This high ATPase level was for the first time demonstrated to be due to the activation of the 21S dynein ATPase activity by the axonemes. The mode of the activation was studied to get an insight into the mechanism of dynein-microtubule interaction. The salt-extracted axonemes caused a 7- to 8-fold activation of the 21S dynein ATPase activity at an axoneme : dynein weight ratio of about 14 : 1. The activation was maximal at a low ionic strength (no KCl) at pH 7.9-8.3. Under these conditions, 21S dynein rebound to the salt-extracted axonemes. The maximal binding ratio of 21S dynein to the axonemes was the same as that observed in the maximal activation of 21S dynein ATPase. The sliding between the outer doublet microtubules in the trypsintreated 21S dynein-rebound axonemes took place upon the addition of 0.05-0.1 mM ATP in the absence of KC1. During the sliding, the rate of ATP hydrolysis was at the same level as that of the 21S dynein activated by the salt-extracted axonemes. However, it decreased to the level of 21S dynein alone after the sliding. These results suggested that an interaction of the axoneme-rebound 21S dynein with B-subfibers of the adjacent outer doublet microtubules in the axoneme causes the activation of the ATPase activity.View full abstractDownload PDF (2784K) -
Yukichi HARA, Osamu URAYAMA, Kiyoshi KAWAKAMI, Hiroshi NOJIMA, Hideaki ...1987 Volume 102 Issue 1 Pages 43-58
Published: July 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA rat brain cDNA library was screened by using as a probe a fragment of cDNA encoding the alpha-subunit of human Na+, K+-ATPase. Two different cDNA clones were obtained and analyzed. One of them was concluded to be a cDNA encoding the alpha-subunit of the weakly ouabain-sensitive rat kidney-type Na+, K+-ATPase. The deduced amino acid sequence consists of 1, 018 amino acids. The alpha-subunit of the rat kidney-type Na+, K+-ATPase shows 97% homology in amino acid se-quence with the alpha-subunit of human, sheep, or pig enzyme and 87% with that of Torpedo. Based on a comparison of the amino acid sequence at the extracellular domain of the alpha-subunit between weakly ouabain-sensitive rat kidney-type enzyme and the ouabain-sensitive human, sheep, pig, or Torpedo enzyme, it was proposed that only two significant amino acid replacements are unique to the rat kidney-type alpha-subunit. Another cDNA clone obtained showed 72% homology in nucleotide sequence with the former cDNA coding the alpha-subunit of the rat kidney-type Na+, K+-ATPase and the deduced amino acid sequence exhibited 85% homology with that of the alpha-subunit of rat kidney-type Na+, K+-ATPase.View full abstractDownload PDF (1076K) -
Shinobu WATARAI, Shizuo HANDA, Takushi TADAKUMA, Tatsuji YASUDA1987 Volume 102 Issue 1 Pages 59-67
Published: July 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSLiposomes were applied to the immunization with GgOs4, Cer and screening for production of monoclonal antibody to GgOse4Cer. Four-week-old and 22-week-old Balb/c mice were immunized wit GgOse4Cer and Salmonella minnesota R595 lipopolysaccharides incorporated liposomes which were composed of dipalmitoyl-phosphatidylcholine and cholesterol. Since antibody response to GgOse4Cer was higher in 22-week-old than 4-week-old Balb/c mice after immunization, 22-week-old Balb/c mice were used for the immunization prior to generation of the monoclonal antibodies to GgOse4Cer. The screening of monoclonal antibodies was performed by complement-dependent liposome immune lysis assay using GgOse4Cer-containing liposomes. Six kinds of monoclonal antibodies, AG-1, -2, -3, -4, -5, and -6, of the IgM class were established. The specificities of the monoclonal antibodies ob-tained were defined by complement-dependent liposome immune lysis assay using various glycosphingolipids incorporated in liposomes and by thin-layer chromatography (TLC) with immunostaining. All of the monoclonal antibodies reacted only with GgOse4Cer in the liposome immune lysis assay. In addition, the monoclonal antibodies reacted only with GgOse4Cer in the TLC immunostaining. However, none of the monoclonal antibodies obtained was capable of removing natural killer activity from C3H/He mice spleen cell suspensions in vitro. Liposomes may be useful in the procedures of immunization and screening for generation of antiserum and monoclonal antibody to GSLs.View full abstractDownload PDF (678K) -
Kensuke BABA, Masayuki OKADA, Takehiro KAWANO, Hiroto KOMANO, Shunji N ...1987 Volume 102 Issue 1 Pages 69-74
Published: July 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA glycine-rich antibacterial protein with a molecular mass of 7, 000, termed sarcotoxin III, was purified to homogeneity from the hemolymph of third instar larvae of Sarcophaga peregrina. When the hemolymph was fractionated, this protein was recovered in the same fraction as sarcotoxin I, a group of potent antibacterial proteins that have been purified. But, it was clearly different from sarcotoxin I in amino acid composition and molecular mass. Sarcotoxin III was shown to be induced in the hemolymph in response to injury of the larval body wall.View full abstractDownload PDF (1028K) -
Keiko IWAMI, Yoshiko DOHI, Tadashige MORIYAMA, Kozo HAMAGUCHI1987 Volume 102 Issue 1 Pages 75-82
Published: July 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCa2+ or Cd2+ binding and the conformational change induced by the metal binding in two frog bone Gla-proteins (BGP, termed BGP-1 and BGP-2) were studied by equilibrium dialysis and CD measurement. By CD measurement in the far-ultraviolet region, the a-helix content of both apoBGPs was found to be 8%. Binding of both Ca2+ and Cd2+ was accompanied with a change in the CD spectrum, and the a-helix content increased to 15 and 25% for BGP-1 and BGP-2, respectively. CD measurement in the near-ultraviolet region indicated that the environment of aromatic amino acid residues in the protein molecule was changed by metal binding. Equilibrium dialysis experiments indicated that each of these two protein binds specifically 2mol of Ca2+, and nonspecifically an additional 3-4mol of Ca2+ in 0.02M Tris-HCl/0.15M NaCl (pH 7.4), at 4°C. According to the two separate binding sites model, BGP-1 has 1 high-affinity Ca2+ binding site (Kd1=0.17mM) and 1 low-affinity site (Kd2=0.29mM), and BGP-2 contains 1 high-affinity site (Kd1=0.14mM) and 1 low-affinity site (Kd2=0.67mM). In addition, 2 Cd2+ bound to a high-affinity binding site on BGP-1 with Kd1, of 10.4μM, and 1 Cd2+ bound to a low-affinity binding site with Kd2, of 41.5μM. On the other hand, BGP-2 had three classes of binding sites and 1 CD2+ bound to each binding site with Kd1=3.6μM, Kd2=16.3μM, Kd3=51.7μM, respectively. Parallel analysis of Ca2+ binding and CD titration at 222nm with Ca2+ revealed that conformational change was maximum at 4.9mM Ca2+, that is, sufficient to bind more than 5mol of Ca2+ to BGPs. In contrast, cooperativity of Cd2+-induced conformational change was observed when the first Cd2+ bound to BGP, and maximum CD change occurred at 100μM Cd2+, that is, sufficient to bind 2mol of Cd2+ to independent binding sites.View full abstractDownload PDF (580K) -
Atsushi HARA, Tamotsu TAKETOMI1987 Volume 102 Issue 1 Pages 83-92
Published: July 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSGlycosphingolipids in serum and lipoproteins from Watanabe hereditable hyperlipidemic rabbit (WHHL rabbit), which is an animal model for human familial hypercholesterolemia (FH), were analyzed for the first time in this study. Chylomicrons and very low density, low density, and high density lipoproteins contained sulfatide as a major glycosphingolipid (12nmol/μmol total phospholipids (PL) in chylomicrons, 19nmol/μmnol PL in VLDL, 18nmol/μmol PL in LDL, and 14nmol/μmol PL in HDL) with other minor glycosphingolipids such as glucosylceramide, galactosylceramide, GM3 ganglioside, lactosylceramide, and globotriaosylceramide. The concentration of sulfatide as a major glycosphingolipid in WHHL rabbit serum (121nmol/ml) was much higher than that in normal rabbit serum (3nmol/ml). Fatty acids of the sulfatides comprised mainly nonhydroxy fatty acids (C22, 23, and 24) and significant amounts of hydroxy fatty acids (about 10%), whereas long chain bases of the sulfatides comprised mostly (4E)-sphingenine with a significant amount of 4D-hydroxysphinganine (about 10%). Furthermore, sulfatides in the liver and small intestine from normal and WHHL rabbits (where serum lipoproteins are produced) were determined to amount to 260nmol/g liver in WHHL rabbit, 104nmol/g liver in control rabbit, 99.6nmol/g small intestine in WHHL rabbit, and 31.2nmol/g small intestine in control rabbit. Ceramide portions of the sulfatides in the liver were mainly composed of (4E)-sphingenine and nonhydroxy fatty acids, while those in the small intestine were mainly composed of 4D-hydroxysphinganine and hydroxy fatty acids. These results indicated that the sulfatides of serum lipo-proteins were mostly derived from the liver (90% of the total), and that the remaining sulfatides (10% of the total) might be derived from the small intestine. These two sulfatides, which have different ceramide portions, could be useful markers for metabolic and biosynthetic studies of various lipoproteins in WHHL rabbit, and thus would be helpful to further elucidate the relationship between hypercholesterol-emia and atherosclerosis in the rabbit.View full abstractDownload PDF (2395K) -
Toshiyuki MIYATA, Sigeharu TERUKINA, Michio MATSUDA, Ayatsugu KASAMATS ...1987 Volume 102 Issue 1 Pages 93-101
Published: July 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSStructural analyses of fibrinogens from patients with congenital dysfibrinogenemia, designated as fibrinogens Kawaguchi and Osaka, have been performed to identify thedifference responsible for the lack of fibrinopeptide A release. For the structural studies, a new strategy was employed. Amino acid sequence analysis of one of the lysyl endopeptidase-peptides isolated from the abnormal fibrinogens indicated that in both fibrinogens, arginine-16 of the Aa chain had been replaced by cysteine. To characterize the chemical nature of the sulfhydryl group of cysteine-16, a tryptic peptide containing cysteine-16 of the Aa chain was prepared from intact fibrinogen Kawaguchi. The amino acid composition and the molecular weight determination of this aberrant peptide revealed that it was a dimeric NH2-terminal peptide corresponding to residues 1-19 derived from the abnormal Aa chain. These results indicate that the half-cystine at position 16 in the abnormal Aa chain forms an intramolecular disulfide bridge with the same residue in the other abnormal Aa chain and that fibrinogen Kawaguchi is a homo dimer composed of two identical abnormal halves.View full abstractDownload PDF (667K) -
Masao KAWAKITA, Tetsuro YAMASHITA1987 Volume 102 Issue 1 Pages 103-109
Published: July 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe reactive sulfhydryl group (SHD) (Kawakita et al. (1980) J. Biochem. 87, 609-617) which is essential for the decomposition of the E-P intermediate of Ca2+-transporting ATPase of the rabbit skeletal muscle sarcoplasmic reticulum has been identified. One sample of sarcoplasmic reticulum membranes was reacted for 3 min with 0.4 mM N-[3H]ethylmaleimide at pH 7.0 at 30°C to a labeling density of 1 mol/mol ATPase without loss of the Ca2+-transporting activity. Another sample of the membranes was treated similarly with non-radioactive N-ethylmaleimide and then labeled with 0.4 mM N-ethy1[14C]maleimide for 17 min. An extensive loss of the Ca2+-transporting activityoccurred during the period of this radio-labeling, thus substantiating the 14C-labeling of SHD. The labeled membranes were digested by thermolysin, and the labeled peptides were fractionated by gel filtration and reversed-phase HPLC. Two major radioactive peptides were present in both 3H- and 14C-labeled thermolytic digests, and each of the major components of 14C-labeled pep-tides had a counterpart in the major components of 3H-labeled peptides which behaved identically on HPLC. The major 14C-labeled peptides were purified and found to be identical with the two SHN peptides, TL-I and TL-II (Saito-Nakatsuka et al. (1987) J. Biochem. 101, 365-376), and 0.5 mol/mol ATPase each of Cys344 and Cys364 was assigned as SHD. It seems that the Ca2+-transport system retains its activity while either of the two Cys residues is unoccupied, but loses it when both of them are modified with N-ethylmaleimide.View full abstractDownload PDF (566K) -
Yoshimasa SAITO, Yoshinori ISHII, Satoshi KOYAMA, Kiyoshi TSUJI, Hisas ...1987 Volume 102 Issue 1 Pages 111-122
Published: July 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe high-level synthesis of a-human atrial natriuretic polypeptide hormone in Escherichia coli has been achieved based on the idea that the yield of a small, basic and unstable polypeptide, such as the natriuretic polypeptide, would be improved by fusion with an appropriate protective polypeptide to construct a neutral fused polypeptide. We prepared an expression vector, pCLaHtrp3t, coding a neutral polypeptide containing 130 amino acid residues in which the polypeptide hormone was fused to a newly designed protective polypeptide through lysine as an enzymatically cleavable residue. The fused polypeptide was synthesized at the high level of 32% of total cellular proteins and at 4.7 × 106 molecules per single cell. It was recovered as cellular insoluble fraction and purified to homogeneity. For the isolation of the peptide hormone from the resultant fused polypeptide, Achromobacter protease I, a lysine-specific endopeptidase was used, because it has sufficient activity even in 8 Murea. The recombinant natriuretic polypeptide was indistinguishable from native a-human atrial natriuretic polypeptide as regards amino acid sequence as well as biological activity.View full abstractDownload PDF (1426K) -
Kazuya ONOMICHI, Yuzuru ETO, Hiroshiro SHIBAI1987 Volume 102 Issue 1 Pages 123-131
Published: July 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSExpression of human interleukin 2 (IL-2) at high levels has been achieved in Chinese hamster ovary (CHO) cells by amplification of transfected sequences. Plasmids containing the human IL-2 cDNA or genomic DNA and mouse dihydrofolate reductase (DHFR) cDNA were transfected into DHFR-negative CHO cells. Trans-formants expressing DHFR were selected in media lacking nucleosides, and cells which amplified both DHFR and IL-2 genes were obtained by exposure to increasing methotrexate (MTX) concentrations. These cell lines constitutively expressed elevated levels of IL-2 at a concentration of 2mg/liter. These cell lines continued to produce IL-2 stably through at least 1 month, even in the absence of MTX.View full abstractDownload PDF (2333K) -
Tetsuo MAITA, Hirofumi ONISHI, Eiko YAJIMA, Genji MATSUDA1987 Volume 102 Issue 1 Pages 133-145
Published: July 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSChicken gizzard myosin was modified with N-iodoacetyl-N'-(5-sulfo-l-naphthyl)-ethylenediamine (IAEDANS) in the presence of ATP and in 0.15 M KCl, where the myosin assumed 10S conformation. From the tryptic digest of the modified myosin, a fluorescent fragment (24 kilodaltons) was isolated by gel filtration on a Sephadex G-100 column followed by chromatography on a CM 52 column. The amino acid sequence of the fragment was analyzed by conventional methods, and was: (S, Z) K-P-L-S-D-D-E-K-F-L-F-V-D-K-N-F-V-N-N-P-L-A-Q-A-D-W-S-A-K-K-L-V-W-V-P-S-E-K-H-G-F-E-A-A-S-I-K-E-E-K-G-D-E-V-T-V-E-L-Q-E-N-G-K-K-V-T-L-S-K-D-D-I-Q-K-M-N-P-P-K-F-S-K-V-E-D-M-A-E-L-T-C-L-N-E-A-S-V-L-H-N-L-R-E-R-Y-F-S-G-L-I-Y-T-Y-S-G-L-F-C-V-V-I-N-P-Y-K-Q-L-P-I-Y-S-E-K-I-I-D-M-Y-K-G-K-K-R-H-E-M-P-P-H-I-Y-A-I-A-D-T-A-Y-R-S-M-L-Q-D-R-E-D-Q-S-I-L-C-T-G-E-S-G-A-G-K-T-E-N-T-K-K-V-I-Q-Y-L-A-V-V-A-S-S-H-K-G-K. The amino-terminus was blocked, and the fragment was assigned as an amino-terminal part of the heavy chain of gizzard myosin. Position 127 was occupied by ε-N-trimethyllysine. Trp-130 of rabbit skeletal myosin heavy chain, which was reported to cross-link to an azide derivative of ATP by Okamoto and Yount (Proc. Natl. Acad. Sci. U. S. 82, 1575-1579 (1985)), was replaced by glutamine in gizzard myosin. Cys-93 of the fragment is the amino acid residue whose reaction with IAEDANS alters the ATPase activity of gizzard myosin (Onishi, H. (1985) J. Biochem. 98, 81-86).View full abstractDownload PDF (872K) -
Hyeun Wook CHANG, Ichiro KUDO, Motowo TOMITA, Keizo INOUE1987 Volume 102 Issue 1 Pages 147-154
Published: July 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPeritoneal exudate produced in rat injected with caseinate contained extracellular phospholipase A2. The activity required Ca2+ ion and had a pH optimum of 9 (Chang, H. W., Kudo, I., Hara, S., Karasawa, K., & Inoue, K. (1986) J. Biochem. 100, 1099-1101). This phospholipase A2 was purified about 14, 000-fold to near homogeneity by the sequential use of column chromatography on Sephadex G-75, Toyopearl HW-65, and TSK ODS-120T reverse-phase HPLC. The final preparation showed a single protein band on SDS-polyacrylamide gel electrophoresis, and its molecular weight was estimated to be approximately 13, 500. The enzyme hydrolyzed phosphatidylethanolamine (PE) and phosphatidylserine (PS) more effectively than phosphatidylcholine (PC). When 1-acyl-2-[1-14C] linoleoyl-sn-glycero-3-phospholipids were used as a substrate, the apparent Km values were 0.027mM with PE, 0.032mM with PS, and 0.1mM with PC, and the Vmax values were 105 μmol/min/mg with PE, 71 μmol/min/mg with PC. The enzyme activity was inhibited by p-bromophenacyl bromide, dithiothreitol, and mepacrine. The amino acid sequence of the NH2-terminal portion and the amino acid composition of the purified enzyme were deter-mined. They were different from those of rat pancreatic phospholipase A2, but very similar to those of phospholipase A2 secreted from rat platelets.View full abstractDownload PDF (1265K) -
Mikihiko NAITO, Ichiro KUDO, Yasuhito NAKAGAWA, Keizo WAKU, Hisao NOJI ...1987 Volume 102 Issue 1 Pages 155-162
Published: July 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCells of the human promyelocytic leukemia cell line HL-60 as well as HL-60 granulo-cytes induced in vitro by retinoic acid were examined for lipid composition. One of our original aims was to clarify how human granulocyte (differentiated HL-60 cells) synthesized enough precursors of lipid mediators, such as prostaglandins and/or platelet activating factor. Comparison studies yielded the following results. 1) After granulocyte differentiation, total phospholipid of HL-60 cells decreased to about 70 % of that of untreated cells, while the content of triglyceride increased to about 200% of the original level. 2) The subclass composition of ethanolamine-containing glycerophospholipid was greatly altered during differentiation; 1-alkeny1-2-acyl glycerophosphoethanolamine (GPE) increased to 166% of that in the unreated cells, while 1, 2-diacyl GPE decreased to 46% of the original value. The resultant profile became very similar to that of human peripheral polymorphonuclear leukocytes. 3) During differentiation, the amount of arachidonic acid stored in both phospholipid and triglyceride of retinoic acid-treated HL-60 cells significantly increased. Its distribution was also modified; arachidonic acid in 1, 2-diacyl GPE decreased to 63 %, while those of 1-alkeny1-2-acyl GPE, choline-containing glycero-phospholipids, and phosphatidylinositol increased to 169, 154, and 153 %, respectively. These results suggested that the regulatory mechanism of lipid turnover in HL-60 cells was modified during retinoic acid-induced granulocyte differentiation. The alterations were not enough to explain fully the capability of differentiated HL-60 cells to produce lipid mediators upon stimulation, but they were probably one of the factors that regulate these reactions.View full abstractDownload PDF (571K) -
Akira ENDO, Shigeo MURAKAWA, Hayato SHIMIZU, Yasushi SHIRAISHF1987 Volume 102 Issue 1 Pages 163-170
Published: July 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA collagenase active against native, insoluble collagen was isolated from the culture filtrate of a streptomycete which has been designated Streptomyces sp. C-51. Collagenase was produced by growing this strain in media containing gelatin. Purification by ammonium sulfate fractionation and chromatographies on DEAE-Toyopearl and DEAE-Cellulofine columns produced active enzyme which was free of contaminating proteins, including nonspecific proteinases, but which contained two active subspecies (I and II). Both subspecies were purified by preparative slab gel electrophoresis. The apparent molecular weights were 100, 000 for the homogeneous fraction I and 90, 000-110, 000 for the microheterogeneous fraction II. These two subspecies were most active at pH 8-9, very similar in amino acid composition and immunologically identical. Some other properties of the Streptomyces C-51 collagenase were compared with those of Clostridium histolyticum collagenase. Substrate specificity, insensitivity to N-ethylmaleimide and diisopropyl fluorophosphate, and sensitivity to certain metal ion complexing agents were similar for the collagenases from both microorganisms.View full abstractDownload PDF (2007K) -
Yoshihide NAKAO, Yasunori KOZUTSUMI, Ikuo FUNAKOSHI, Toshisuke KAWASAK ...1987 Volume 102 Issue 1 Pages 171-179
Published: July 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe carbohydrate portions of β-galactosidase from Aspergillus oryzae were found to be composed of two types of sugar chains. They were released equally well with endo β-N-acetylglucosaminidase H, but were distinct in their chain length. The long sugar chains (fraction I), corresponding to 4% of the total carbohydrate chains, were composed of galactomannan-type oligosaccharides, which consisted of man-nose, galactose, glucose, and glucosamine in the molar ratios of 30.0, 16.4, 1.4, and 2.1 per mol of aspartic acid, respectively. The short sugar chains (fraction II), corresponding to 96% of the total carbohydrate chains, consisted of mannose, galactose, glucose, and glucosamine in the molar ratios of 9.4, 0.6, 0.3, and 1.7 per mol of aspartic acid, respectively. Both types of sugar chains were fractionated into neutral and acidic subfractions. The neutral subfraction of fraction I (I-N), corresponding to 1% of the total carbohydrate chains, was very heterogeneous in length and was resistant to digestion with a-mannosidase and β-galactosidase. The neutral subfraction of fraction II (II-N), corresponding to 91 % of the total car-bohydrate, was composed of a mixture of oligosaccharides with oligomanneoside chains (Mann GlcNAcol). The major components were similar to high mannose-type oligosaccharides of mammalian origin in their composition and size (n=5-9). However, digestion of II-N with α1, 2-mannosidase produced considerable amounts of Man6G1cNAcol, an unusual product in the case of high mannose-type oligosaccharides of mammalian origin, in addition to the common one, Man5G1cNAcol. The following structure is proposed for Man6G1cNAcol:
The acidic subfraction of fraction I, corresponding to 1.3 % of the total carbohydrate chains, contained phosphate in mono- and diester linkages, whereas the acidic subfraction of fraction II, corresponding to 3 % of the total carbohydrate chains, contained phosphate only in monoester linkages.View full abstractDownload PDF (684K) -
Hiroshi UEMURA, Takamitsu SHIBA, Masayuki MACHIDA, Ikuo MATSUI, Yoshif ...1987 Volume 102 Issue 1 Pages 181-189
Published: July 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSENO1-'1acZ fusions with various lengths of the ENO1 5'-flanking region were constructed on various types of yeast plasmid vectors. The fully expressed level of βGal directed by ENO1-'1acZ fusions differed depending on the type of vector, but on any type of vector, βGal activity was not greatly influenced by the carbon source in the medium. The 86-bp DNA region of ENO1 at position -487 to -402 upstream of the initiation codon, in which we had previously delimited the positive regulatory region of ENO1 (Uemura, H., Shiba, T., Paterson, M., Jigami, Y., & Tanaka, H. (1986) Gene 45, 67-75), exerted its function without requiring precise location with respect to the TATA box. The action of the positive regulatory region was not affected by its orientation. In addition, the substitution of the UASs of PHO5, encoding repressible acid phosphatase, with the regulatory region of ENO1 changed the expression of PHO5-'lacZ gene to constitutive, irrespective of the concen-tration of inorganic phosphate in the medium. Furthermore, the GCR1 gene cloned in a multicopy plasmid increased the expression of the ENO1-'lacZ fused genes.View full abstractDownload PDF (1447K) -
Toshio SATOH, Felicia N. KURIHARA1987 Volume 102 Issue 1 Pages 191-197
Published: July 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDimethylsulfoxide (DMSO) reductase was purified to electrophoretic homogeneity from the periplasmic fraction of a photodenitrifier, Rhodopseudomonas sphaeroides f. s. denhrificans. The enzyme had a molecular weight of 82, 000 and had no subunit. It contained 1 mol of molybdenum per mol of enzyme, but iron and acid-labile sulfur were not present. The UV-visible spectrum showed only one absorption maximum at 280 nm. Denaturation of the enzyme released a molybdopterin cofactor, the fluorescence spectra of which were almost the same as those of a form B derivative of molybdopterin found in formate dehydrogenase. The Km value for DMSO was 15μM, which was much lower than that for trimethylamin-N-oxide (TMAO), whereas Vmax with TMAO was larger than that with DMSO.View full abstractDownload PDF (1091K) -
Toshiaki MIKI, Yutaka ORII, Yasuo MUKOHATA1987 Volume 102 Issue 1 Pages 199-209
Published: July 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBoth beef heart cytochrome oxidase and bacteriorhodopsin of Halobacterium halobium were reconstituted into liposomes by the sonication-cholate dialysis method. The proteoliposomes showed the respiratory control ratio of 4.2, and steady-state illumination of the vesicles lead to the 2.7-fold stimulation of the oxidase activity in the absence of uncouplers. The light-stimulated state 4 respiration increased with light intensity, but light had no effect on the oxidase activity that had been relieved by addition of uncouplers. Proteoliposomes with the photosensitive oxidase activity were also obtained when cytochrome oxidase vesicles were fused with bacterio-rhodopsin vesicles in the presence of calcium chloride, and the extent of photo-activation was maximally 1.4-fold. The light-induced respiratory release was observed even in the presence of valinomycin or nigericin, indicating that the oxidase activity was sensitive to both the membrane potential and the pH gradient. We propose as a mechanism of the respiratory control that the process of proton trans-port to thereaction center for water formation is the rate limiting step for the cyto-chrome oxidase activity.View full abstractDownload PDF (854K) -
Goro MIYAKODA, Akira YOSHIDA, Haruhiko TAKISAWA, Takao NAKAMURA1987 Volume 102 Issue 1 Pages 211-224
Published: July 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSpontaneously beating heart myocytes were prepared from adult rat ventricular tissues to study the correlation between β-adrenergic receptor-stimulated changes in contractile performance and protein phosphorylation in vitro. The plasma membrane of isolated myocardial cells was permeabilized by saponin in the presence of EGTA and Mg-ATP. The permeabilized myocytes, which formed a homogeneous cell population, retained the rod-cell morphology of heart cells in situ and showed spontaneous cyclic contractions. Their contractile activity in response to extracellularly added cAMP mimicked the effects caused by β-adrenergic stimulation of the whole heart: both the frequency and longitudinal velocity of free contraction and relaxation of the cells increased. Similar increases were observed when β-agonist, isoproterenol, and GTP were added to suspending medium. In addition, isoproterenol maximally enhanced the adenylate cyclase activity of the cells in the presence of GTP. Both of these effects of isoproterenol were completely blocked by the β-antagonist propranolol. cAMP-mediated phosphorylation of proteins in the permeabilized myocytes was investigated under conditions in which the beating frequency increased. cAMP elevated the phosphorylation level of five proteins; three of them with apparent molecular masses of 24, 15, and 12 kDa were mem-brane proteins and the other two with apparent molecular masses of 150 and 28 kDa were myofibrillar proteins. The 24-kDa phosphoprotein dissociated into 12-kDa molecules when boiled in sodium dodecyl sulfate, suggesting that these proteins are oligomeric and monomeric forms of phospholamban. The phospho-rylation of these five proteins was stimulated by isoproterenol. The effect of iso-proterenol was enhanced by GTP but completely blocked by propranolol. The time course of their phosphorylation correlated well with that of the increase in the beating frequency of the cells; both were measured after the administration of isoproterenol and GTP. When propranolol was added after the start of thestimulation by isoproterenol, only phospholamban and the 15-kDa protein were rapidly dephosphorylated in close correlation with the decrease of the beating frequency. These results demonstrate for the first time that the permeabilized myocytes retain the functional β-adrenergic receptor and cellular responses to β-adrenergic stimula-tion. They also suggest that cAMP-mediated phosphorylation of proteins, possibly phospholamban and/or the 15-kDa protein, is involved in the increased contractile activity of permeabilized heart cells.View full abstractDownload PDF (3188K) -
Joseph VAMECQ, Jean-Pierre DRAYE1987 Volume 102 Issue 1 Pages 225-234
Published: July 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSLong-chain monocarboxylic, ω-hydroxymonocarboxylic and dicarboxylic acids were activated approximatively at the same rate by rat liver homogenates into their CoA esters (2-3U/g liver). These acyl-CoA were substrates for rat liver peroxisomal β-oxidation. The distribution of the peroxisomal oxidation of these substrates was also studied in various tissues. Rat liver mitochondria were capable of oxidizing long-chain monocarboxyl- and ω-hydroxymonocarboxylyl-CoAs but not dicar-boxylyl-CoAs. When the mitochondrial preparations were incubated in coupling conditions, the addition ofeither free decanoic acid or free l0-hydroxydecanoic acid resulted in an increase of the oxygen uptake conversely to the addition of decanedioic acid. The comparative study of the chain-length substrate specificity of peroxisomal fatty acyl-CoA oxidase and mitochondrial fatty acyl-CoA dehydrogenase activities revealed that, actually, both types of organelles, peroxisomes and mitochondria, contain “oxido-reductases” active on long-chain onocarboxylyl-CoAs, ω-hydroxy-monocarboxylyl-CoAs and dicarboxylyl-CoAs.View full abstractDownload PDF (764K) -
Jean-Pierre DRAYE, Joseph VAMECQ1987 Volume 102 Issue 1 Pages 235-242
Published: July 01, 1987
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe interactions of 1-5mM valproic acid with the hepatic fatty acid oxidation are here described. Valproic acid was not substrate for hepatic peroxisomal fatty acid oxidation. Its activation outside the mitochondrial matrix compartment was poor when compared to that of octanoic acid, a fatty acid containing the same number of carbones. Valproic acid did not inhibit the fatty acyl-CoA oxidase nor the cyanide-insensitive acyl-CoA oxidation. Valproic acid inhibited the mitochondrial oxida-tions of both long-chain monocarboxylyl-CoAs and ω-hydroxymonocarboxylyl-CoAs. Valproic acid prevented the oxidation by coupled mitochondria of decanoic and 10-hydroxydecanoic acids. Both butyric and 4-hydroxybutyric acids were oxidized by coupled mitochondria. These activities were abolished by preincubating the enzyme source with valproic acid. Administration to rats of 0.5 % (w/w)- or 1% (w/w)-valproate containing diets were efficient in producing increased liver peroxisomal population and β-oxidation. Preliminary investigations on the effects of valproic acid on mitochondrial fatty acid oxidation as a function of the animal used for the experiments pointed out an association of the protection of the mitochondrial process against the toxicity of the drug with enhanced carnitine acyltransferase and acyl-CoA hydrolase activities.View full abstractDownload PDF (624K)
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