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Volume 112 (1992)
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Volume 112, Issue 5
Displaying 1-24 of 24 articles from this issue
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Toshihide Hashimoto, Ken-ichirou Morohashi, Koichi Takayama, Shin-ichi ...1992 Volume 112 Issue 5 Pages 573-575
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe previously reported the presence of six different cis-acting elements (Ad1 to Ad6) in the promoter region of the bovine CYP11B gene. Although the Adl site (TGACGTGA) was similar to a palindromic CRE (TGACGTCA), two other upstream sequences, Ad3 and Ad4, were identified as the cAMP response sequences of the gene. We analyzed the functional relationship between the Adi site and the upstream elements. Mutation analyses of the Adl site indicated that the 5' half of the site (TGACG) was important for the transcription of the gene in vitro. In Y-1 cells, a plasmid with a mutated Adl showed no response to cAMP. The effect of the mutation at the Adl site on the cAMP response was almost the same as that of the deletion of Ad3 and Ad4, although the role of each element seemed to be different. These results indicated that both the Ad1 site and the upstream elements, Ad3 and Ad4, were necessary for the full response to cAMP of the CYP11B gene. When the Adi site in the promoter region was replaced with a palindromic CRE, elevated transcription activity was detected both in vitro and in vivo. Two kinds of CREBs (43 and 47 kDa) purified from a HeLa cell nuclear extract bound to the Adi site. The binding of the palindromic CRE to the nuclear factor (s) was stronger than that of Ad1.View full abstractDownload PDF (2845K) -
Mizuho Une, Noboru Izumi, Tsuneo Imanaka, Takahiko Hoshita1992 Volume 112 Issue 5 Pages 576-577
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWhether 3α, 7α, 12α-trihydroxy-5β-cholestanoic acid (THCA) was converted into cholic acid in human skin fibroblasts was examined. THCA was incubated with subcellular fractions of cultured skin fibroblasts in the presence of NAD+, ATP, CoA, and Mg2+. The reaction products were analyzed by thin-layer chromatography and high-performance liquid chromatography after p-bromophenacyl ester derivatization. The highest specific activity was found in the light mitochondrial fraction (2.71nmol/mg protein/h). The specific activity was about 9-fold higher than that in heavy mitochondrial fraction. The peroxisomel fraction prepared from the light mitochondrial fraction by sucrose gradient centrifugation was also able to catalyze the conversion of THCA into cholic acid. The specific activity in this fraction was a further 2.2-fold higher than that in the light mitochondrial fraction. These results suggest that cultured human skin fibroblasts are able to convert THCA into cholic acid, and that the activity exists in peroxisomes.View full abstractDownload PDF (256K) -
Hiroyuki Kanehara, Hiroshi Yokota, Masahiko Sato, Akira Yuasa1992 Volume 112 Issue 5 Pages 578-582
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA UDP-glucuronyltransferase isoform glucuronizes phenolic xenobiotics such as 4-nitrophenol, and an isoform glucuronizing 4-hydroxybiphenyl has also been found in rat liver. We purified a UDP-glucuronyltransferase isoform glucuronizing 4-hydroxybiphenyl from bovine liver microsomes by solubilization with 0.7% sodium cholate followed by three column chromatographic separations using DEAE-Toyopearl 650S, UDP-hexanolamine Sepharose 4B, and hydroxyapatite. The purified bovine liver 4-hydroxybiphenyl UDP-glucuronyltransferase (named Bovine 4HBGT) had glucuronidation activities toward 4-hydroxybiphenyl and 4-methylumbelliferone but had little activity toward 4-nitrophenol and 1-naphthol. The apparent molecular mass of Bovine 4HBGT was 54, 000 Da on SDS-PAGE, and this was decreased to 50, 000 Da by digestion with endo-β-N-acetylglucosaminidase H. These data suggest that Bovine 4HBGT consists of a 50, 000 Da polypeptide and a high mannose type oligosaccharide chain(s) of about 4, 000 Da. The NH2-terminal sequence of GT-3 was GKVLVWPVDFSXWINI. These properties of Bovine 4HBGT were very similar to those of rat UDP-glucuronyltransferase glucuronizing xenobiotics. However, the NH2-terminal sequence of Bovine 4HBGT had higher homology with that of rat liver 4-hydroxybiphenyl UDP-glucuronyltransferase than with that of rat liver 4-nitrophenol UDP-glucuronyltransferase.View full abstractDownload PDF (1647K) -
Yoshio Yamakawa, Tamotsu Omori-Satoh1992 Volume 112 Issue 5 Pages 583-589
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe complete amino acid sequence of an antihemorrhagic factor, HSF, in the serum of the Japanese Habu snake, Trimeresurus flavoviridis, has been determined. The protein is composed of 323 amino acid residues and contains three asparagine-linked oligosaccharide chains at positions 123, 185, and 263. The molecule contains two copies of the cystatin domain in the N-terminal portion up to position 240, and these domains show a remarkable sequence homology (about 50%) to those of plasma glycoproteins such as α2-HS (human) and fetuin (bovine) and to a lesser extent to that of HRG (human). The amino acid sequence of the noncystatin region towards the C-terminus is unique, showing no significant homology with those of the corresponding regions of α2-HS and fetuin. In spite of the presence of cystatin domains, HSF does not inhibit cysteine proteinases such as papain and cathepsin B but does inhibit several metalloproteases in Habu venom. The results suggest that HSF is the first protein found to be functionally related to metalloproteinase inhibitors among the structurally homologous proteins with a double-headed cystatin domain, and is a member of a novel family (family 4) with divergent functions of the cystatin superfamily proteinase inhibitors. Although HSF possesses similar physicochemical properties to those of oprin, a snake venom metalloproteinase inhibitor with antihemorrhagic activity isolated from opossum serum [Catanese & Kress (1992) Biochemistry 31, 410-418], its primary structure is strikingly different from that of oprin.View full abstractDownload PDF (904K) -
Takuma Nakajima, Shiho Tsunoda, Susumu Nakada, Shigekazu Nagata, Kinic ...1992 Volume 112 Issue 5 Pages 590-597
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn expression vector, pF1900M, which expresses a cloned gene at a high level in quiescent mammalian cells was constructed using the rat fibronectin (FN) promoter. Human interferon γ (HuIFN-γ) cDNA inserted downstream of the FN promoter in pF1900M was introduced into rat 3Y1 cells and several IFN-producing cell lines were established. These cells secreted a low level of IFN when they were growing but secreted at a high level after they had reached confluence. The level was further increased when the confluent cells were maintained in low-serum medium and a cell line, I7, produced 4×105IU/ml of IFN, comparable to that produced by genetically engineered Escherichia coli in 2 days. The IFN-producing ability of I7 cells could be maintained by successive replacements of low-serum medium for at least 2 weeks. HuIFN-γ secreted into the medium had a molecular weight range of 22, 000 to 25, 000, similar to that of IFN-γ produced by human lymphocytes. The N-linked glycosylation of HuIFN-γ seemed to occur properly, since treatment of the IFN with N-glycanase resulted in a reduction of molecular weight to 17, 000, which corresponds to that calculated from the deduced amino acid sequence of HuIFN-γ.View full abstractDownload PDF (3172K) -
Akio Sugihara, Masahiko Ueshima, Yuji Shimada, Susumu Tsunasawa, Yoshi ...1992 Volume 112 Issue 5 Pages 598-603
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA thermostable lipase from Pseudomonas cepacia has been purified to homogeneity as judged by SDS-PAGE and isoelectric focusing. The purification included treatment of the culture supernatant with acrinol, hydrophobic interaction chromatography, and gel filtration. The enzyme was a monomeric protein with Mr of 36, 500 and pI of 5.1. The optimal pH at 50°C and optimal temperature at pH 6.5 were 5.5-6.5 and 55-60°C, respectively, when olive oil was used as the substrate. Simple triglycerides of short and middle chain fatty acids (C≤12) were the preferred substrates over those of long chain fatty acids. The enzyme cleaved all the ester bonds of triolein, with some preference for the 1, 3-ester bonds. The enzyme retained all its activity even after incubation at 75°C (pH 6.5) for 30 min. Further, the activity was not impaired during 21h storage at pH 6.5 in 40% water-miscible solvents including methanol, ethanol, acetone, acetonitrile, dimethylformamide, dimethylsulfoxide, and dioxane. The addition of dimethylsulfoxide or acetone to the assay mixture in the range of 0-35% stimulated the enzyme, whereas benzene or n-hexane had an inhibitory effect. These properties together with the N-terminal amino acid sequence confirmed that the enzyme differs from the known Pseudomonas sp. lipases.View full abstractDownload PDF (1598K) -
Hwei-Ling Peng, Tzu-Fun Fu, Sou-Feng Liu, Hwan-You Chang1992 Volume 112 Issue 5 Pages 604-608
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe entire galactose (gal) operon of Klebsiella pneumoniae was isolated and functionally analyzed in Escherichia coli. The genes encoding galactokinase (galK), galactose-l-phosphate uridyltransferase (galT), and UDP-galactose-4-epimerase (galE) were mapped by complementation analysis. The gene order E-T-K was found to be identical to that of Salmonella spp. and E. coli. Analysis of the nucleotide sequence in the control region revealed significant homology with that of E. coli. Two major sites for transcriptional initiation, both mapped to a cytosyl residue, were identified by primer extension. When the operon is expressed in E. coli, the K. pneumoniae gal gene products make up about 30% of the total cellular proteins. The presence of a powerful promoter responsible for high level synthesis of the gal proteins was also demonstrated using β-galactosidase as reporter.View full abstractDownload PDF (2995K) -
Yasushi Masuzawa, Teruo Miyauchi, Masahiro Hamanoue, Shinji Ando, Junk ...1992 Volume 112 Issue 5 Pages 609-615
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe peanut agglutinin (PNA)-binding site is protein-bound Galβ1→3GalNAc, and is a tumor-associated carbohydrate marker expressed in many human carcinomas. PNA-binding glycoproteins isolated from KATO-III human gastric carcinoma cells were deglycosylated by trifluoromethanesulfonic acid, and rabbit antibodies against the core proteins were used to screen a λgtll expression library constructed from these cells. Two different core proteins were identified by this approach. One was polymorphic epithelial mucin (PEM), initially found in breast carcinomas. PEM mRNA was expressed in normal tissues of the stomach, colon, and lung, but not in the small intestine, thyroid, and spleen. High levels of PEM mRNA were detected in some nude mouse-transplanted carcinomas, i.e. colorectal, pancreatic, stomach, and lung carcinomas. The other core protein was a novel one called MGC-24, which has a molecular mass of 24 kDa, is rich in hydroxyl amino acids and cysteine, and lacks repeating motifs. The mature MGC-24 glycoprotein behaved as a high-molecular-mass one upon SDS-polyacrylamide gel electrophoresis even after neuraminidase treatment. Treatment with endo-α-N-acetylgalactosaminidase in the absence of neuraminidase significally changed the staining pattern by anti-MGC-24, confirming that MGC-24 carried PNA-binding sites. MGC-24mRNA was intensely expressed in normal tissues of the colon, small intestine and thyroid, and in some nude mouse-transplanted colorectal and pancreatic adenocarcinomas.View full abstractDownload PDF (4951K) -
Norifumi Tanida, Naohito Ohno, Yoshiyuki Adachi, Motohiro Matsuura, Ma ...1992 Volume 112 Issue 5 Pages 616-623
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRecent studies by our group suggested that lysozyme has a high affinity for bacterial lipopolysaccharide (LPS) of both the smooth and rough forms, and inhibits various immunomodulatory activities of LPS. GLA60 is a synthetic monosaccharide analogue of bacterial lipid A well known as having most of the activities of lipid A with very low toxicity. In this study, we characterized the interaction of lysozyme with GLA60 in comparison to that with Escherichia coli 0111 LPS (smooth form) by means of an immunopharmacological approach. Using dansylated lysozyme (DNS-LZM) as a probe, LZM was found to bind to GLA60. The mitogenic and polyclonal B-cell activating activities were significantly reduced by complex formation. However, there was no inhibitory effect on GLA60 induced production of IL-1 and TNF of macrophages. Interestingly, the activities of macrophages induced by the complex were found to be significantly higher than those induced by GLA60 itself. In contrast, the activities of 0111 LPS were significantly inhibited by LZM. Since the GLA60-LZM complex produced a turbid suspension but the 0111 LPS-LZM complex remained soluble, we consider that the activities of GLA60 alone were mediated by the common functional LPS receptor for dispersed form in both macrophages and B-lymphocytes, but activation of macrophages by the complex was mediated either by another LPS receptor not present in B-lymphocytes or through the phagocytic function of macrophages.View full abstractDownload PDF (946K) -
Tsuneaki Koike, Naoki Harada, Takeshi Yoshida, Minoru Morikawa1992 Volume 112 Issue 5 Pages 624-630
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo calcium binding proteins, MRP-8 and MRP-14, are specifically synthesized in human myeloid cells. This paper shows that Me2SO, all-trans-retinoic acid (RA) and 1α, 25-dihydroxyvitamin D3, (1α, 25(OH)2D3), but not 12-O-tetradecanoyl phorbol-13-acetate (PMA) are potent inducers of MRP-8/14 protein complex in human leukemic cells. Transforming growth factor-β1 (TGF-βl) is shown to enhance the inductive effect of RA and 1α, 25(OH)2D3. We have examined the possibility that MRP expression is regulated through the protein kinase pathway. Both cytosolic and membrane-bound protein kinase C (PKC) activities increased during differentiation by RA and 1α, 25(OH)2D3. PMA-treatment led to a decrease of cytosolic PKC activity and an increase of membrane-bound PKC activity in the presence of these differentiation inducers, while PMA alone resulted in low cytosolic and high membrane-bound PKC activities. PKC inhibitor H7 inhibited MRP synthesis in HL-60 cells treated with RA and 1α, 25(OH)2D3. These results suggest that cytosolic PKC activity may be involved in a stimulatory pathway of MRP synthesis and that protein phosphorylation reactions may play important roles in MRP expression during myelocytic differentiation.View full abstractDownload PDF (2987K) -
Osamu Sato, Yoshiaki Nonomura, Sumiko Kimura, Koscak Maruyama1992 Volume 112 Issue 5 Pages 631-636
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe molecular shape of dystrophin has been reported to be a 175 nm flexible rod [Pons, F. et al. (1990) Proc. Natl. Acad. Sci. USA 87, 7851-7855] or a 120nm dumbbell [Murayama, T. et al. (1990) Proc. Jpn. Acad. 66B, 96-99]. The present work revealed that 100nm flexible rods with or without spheres were predominant in highly purified dystrophin preparations. When the sample was subjected to gel filtration, dystrophin oligomers were isolated just after the void volume and the fraction largely consisted of dumbbell-shaped molecules. From various rotary-shadowed images, it was suggested that dystrophin is a rod with spheres at both ends, approximately 110nm long and 2nm wide. It appeared that this monomer binds to another monomer in a staggered way, forming a dimer, and the dimers associate with each other side-by-side, forming a dumbbell-shaped tetramer, 130nm long and 5nm wide. The tetramers form an end-to-end aggregate. It seemed that the dumbbell structure was not affected by alkaline (pH 11) treatment to dissociate dystrophin associated glycoproteins, but was deteriorated by detergent, NP-40, Triton X-100, or CHAPS, used for solubilization of membrane-bound dystrophin.View full abstractDownload PDF (6388K) -
Masaki Edamatsu, Masafumi Hirono, Yoshio Watanabe1992 Volume 112 Issue 5 Pages 637-642
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSLocalization of Tetrahymena profilin was examined by an immunofluorescence method. In interphase Tetrahymena cells, immunofluorescence for profilin was diffusely distributed in the cytoplasm, while in dividing cells, additional intense fluorescence was observed in the division furrow. From the result of immunofluorescence localization using cytoskeletal cell models, a significant fraction of profilin appeared to become insoluble in association with a cytoskeletal structure just beneath the division furrow during cytokinesis, although remaining profilin existed as a soluble form in the cytoplasm. Double immunofluorescence staining with anti-profilin and anti-actin antibodies revealed that the localization of profilin in the division furrow coincided with that of contractile ring microfilaments in terms of both position and timing. This is the first report describing the coexistence of profilin with actin filaments in the division furrow, implying the possible involvement of profilin in assembly and disassembly of contractile ring microfilaments in the process of cytokinesis.View full abstractDownload PDF (4594K) -
Kouichi Mizuno, Koji Kimura, Yuji Arai, Tsutomu Kawasaki, Hiroaki Shim ...1992 Volume 112 Issue 5 Pages 643-651
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFour forms of branching enzyme, termed RBEl, RBE2 (a mixture of RBE2A and RBE2B), RBE3, and RBE4, were apparently separated by DEAE-cellulose column chromatography of soluble extract from immature rice seeds, and each of these four forms was further purified by gel-filtration. RBE1, RBE2A, and RBE2B were the predominant forms of the enzyme. The molecular size, amino-terminal amino acid sequence, and immunoreactivity with anti-maize branching enzyme-I (BE-I) antibody were identical among these three forms, except that the molecular mass of RBE2A was almost 3 kDa higher than those of RBE1 and RBE2B. These results indicate that RBE1, RBE2A, and RBE2B are the same (termed rice BE-I). The cDNA clones coding for rice BE-I have been identified from a rice seed library in λgtll, using the maize BE-I cDNA as a probe. The nucleotide sequence indicates that rice BE-I is initially synthesized as an 820-residue precursor protein, including a putative 64- or 66-residue transit peptide at the amino terminus. The rice mature BE-I contains 756 (or 754) amino acids with a calculated molecular mass of 86, 734 (or 86, 502) Da, and shares a high degree of sequence identity (86%) with the maize protein. The consensus sequences of the four regions that form the catalytic sites of amylolytic enzymes are conserved in the central region of the rice BE-I sequence. Thus, rice BE-I as well as the maize protein belongs to a family of amylolytic enzymes.View full abstractDownload PDF (3311K) -
Akiko Takasuga, Kenzoh Banba, Katsuhiko Yoshino, Hiroshi Izutsu, Masah ...1992 Volume 112 Issue 5 Pages 652-657
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA pentapeptide which potently inhibits primary IgE antibody formation, Asp-Ser-Asp-Gly-Lys (DSDGK), has been efficiently produced with the aid of the dihydrofolate reductase (DHFR) handle [M. Iwakura, et al. (1992) J. Biochem. 111, 37-45]. The genes coding fused proteins comprising DHFR and multimeric forms of DSDGK, namely, DHFR-(DSDGK)3, DHFR-(DSDGK)14, and DHFR-(DSDGK)28, were constructed and expressed in Escherichia coli. The C-terminal peptides attached to DHFR did not affect the expression or the function of the DHFR handle, even when the length of the C-terminal peptide was as long as 160 amino acid residues. The fused proteins were easily purified by methotrexate affinity chromatography, one of the major advantages of the DHFR handle. The fused proteins were digested with trypsin and the monomeric peptide, DSDGK, was purified by HPLC. The yields of the peptide were estimated to be 11, 43, and 99mg per 1 gram of the total cell proteins from E. coli cells producing DHFR-(DSDGK)3, DHFR-(DSDGK)14, and DHFR-(DSDGK)28, respectively.View full abstractDownload PDF (1446K) -
Kazuo Yamasaki, Taibo Yamamoto1992 Volume 112 Issue 5 Pages 658-664
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn the preceding paper, we suggested that 1mol Ca2+-ATPase of sarcoplasmic reticulum (SR) contains 0.5mol of high-affinity vanadate binding sites as well as 0.5mol of lowaffinity vanadate binding sites [Yamasaki, K. & Yamamoto, T. (1991) J. Biochem. 110, 915-921]. In the present study, we examined the effects of vanadate binding to the high- and low-affinity sites upon phosphorylation of the enzyme by inorganic phosphate (P1). When vanadate was added to the reaction medium in which the Ca2+-ATPase had been phosphorylated by P1 in the absence of Ca2+, the steady-state level of phosphoenzyme (E2P) decreased due to inhibition of its formation. The decrease of E2P after addition of vanadate exhibited biphasic kinetics consisting of an initial fast decay process followed by a slower first-order decay process. The size of the fast E2P decay, which was estimated by extrapolating the slow phase decay to time 0, varied depending on the vanadate concentration with a dissociation constant of 17μM, and reached maximum at 50μM vanadate. The maximum value of the fast E2P decay was almost equal to the initial E2P level. The initial fast decay of EZP was competitively prevented by P1 with a dissociation constant of 7.4mM, which was very close to Km for the E2P formation under similar conditions. These observations suggested that vanadate inhibits E2P formation by competition with P1 at a phosphorylation site on the Ca2+-ATPase. The slow first-order decay of E2P corresponded well to the vanadate binding to the high-affinity site of the Ca2+-ATPase. When SR was incubated with 5μM vanadate prior to the phosphorylation, the amount of E2P decreased to below 5% of the initial level in 10min. Since vanadate binding to the low-affinity site was negligibly small at this concentration, the inhibition of E2P formation was considered to be caused by vanadate binding to the high-affinity site. P1 also interrupted the slow decay of E2P which was induced by vanadate binding to the high-affinity site. The apparent dissociation constant of P1 binding under this condition was estimated to be about 0.4mM which is much lower than the Km value for the E2P formation. Based on these results, we discuss the involvement of dimeric interaction of the Ca2+-ATPase in the E2P formation.View full abstractDownload PDF (793K) -
Sakae Tsuda, Saburo Aimoto, Kunio Hikichi1992 Volume 112 Issue 5 Pages 665-670
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe Ca2+- and Mg2+-dependence of the interaction between rabbit skeletal muscle troponin C (TnC) and a 21 residue peptide corresponding to 96-116 of troponin I (denoted as CN4) was examined by means of 1H-NMR spectroscopy. The spectral changes of TnC with 4 mol of Ca2+ (Ca4TnC) and TnC with 4 mol of Mg2+ (Mg4TnC) were observed as a function of CN4 concentration. As CN4 was added to Ca4TnC, resonances of the following residues changed in chemical shift: Tyr10, Phe23, Phe72, Ala106, G1y108, Tyr109, IlellO, His125, G1y144, Ile146, Phe102 or Phel5l, and Phe148 located in the N- and C-domains of Ca4TnC. Such CN4-induced change was also observed for resonances of Phe19, 26, and 75 in the N-domain of Ca4TnC by means of NOESY and HOHAHA experiments. The presence of CN4 increased the native-to-unfolded transition temperature of the N-domain of Ca4TnC. On the basis of these results, we conclude that CN4 binds to both the C- and N-domains of Ca4TnC ([CN4]:[ThC]=1:1) and stabilizes the structure of the N-domain. The CN4-binding constant was estimated to be 1.1×105M-1. As CN4 was added to Mg4TnC, chemical shift change was observed for resonances of Phe99, Tyr109, and Ile110 in the C-domain, while no change was observed for resonances arising from the N-domain. The presence of CN4 did not change the thermal stability of the N- and C-domains of Mg4TnC. The CN4-binding constant of Mg4TnC was obtained as 0.9×104M-1, which is one-tenth of that of Ca4TnC. These results suggest that CN4 binds only to the C-domain of Mg4TnC and does not stabilize the structure of the N-domain.View full abstractDownload PDF (711K) -
Eun-Ok Lee, Jin-Gu Kim, Jong-Duk Kim1992 Volume 112 Issue 5 Pages 671-676
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe vesicle-to-micelle transition of immunoliposomes formed by dioleoylphosphatidylethanolamine (DOPE) and palmitoyl-immunoglobulin G (p-IgG) was investigated in the presence of bile salts and conjugated bile salts. Turbidity and the release of calcein from liposomes were measured as a function of the amount of bile salts added and compared with the solubilizing profiles of the salts according to the number and configurational state of hydroxy groups in the cholate. The solubilizing phenomena by bile salts conjugated with glycine or taurine were investigated in comparison with non-conjugated bile salts. The solubilizing effect of bile salts on the bilayer of immunoliposomes increased remarkably with the number of hydroxy groups, but was not influenced by the configurational state of the hydroxy group. The half-maximal concentration of bile salts, defined as the concentration giving the half-maximum turbidity of liposome solutions, decreased with hydrophobicity in the phosphatidylcholine (PC) bilayer. The increase in the hydrophobicity of bile salts induces the ability to permeabilize and solubilize phospholipid vesicles. In the case of PC or PE liposome bilayers with inserted protein, bile salts conjugated with taurine or glycine had lower hydrophobicity than non-conjugated bile salts and showed a lower half-maximal concentration. The conjugated bile salts are believed to interact with lipids and solubilize the bilayers, while the head groups of bile salts interact with the inserted protein and extract it from the lipid bilayer.View full abstractDownload PDF (723K) -
Tatsuo Kai, Yasushi Sugimoto, Takahiro Kusakabe, Rong Zhang, Katsumi K ...1992 Volume 112 Issue 5 Pages 677-688
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSGenomic clones encoding the Drosophila aldolase gene were isolated and the organization of the gene was determined. The protein-coding region spanning nearly 3.5 kb consists of five coding exons (exon 2, 3, 4α, 4β, and 4γ). The insect exon 2 corresponds to exons 2 to 7 of vertebrate aldolase genes and thus appears to have been formed by the fusion of these 6 exons into a single exon during evolution. The Drosophila aldolase gene is predicted to generate mRNAs for three isozymes (α-, β -, and γ-types) from the primary transcripts by alternative usage of the final three exons. The reverse transcriptase-PCR assay revealed the occurrence of mRNAs for the three isozymic forms at different developmental stages, and tissue-specific expression was also found to occur in adult flies. In addition to the usual type mRNA species for the α-, β -, and γ-isozymes, two novel forms of mRNAs, αβ- and βγ-type mRNAs, were detected tissue-specifically in adult flies, although their functions are unpredictable. The αβ-mRNA is an α-type mRNA in which exon 4β remains unspliced, while the N βγ-mRNA is a β-type mRNA with the exon 4γ remaining unspliced. Recombinant enzymes expressed in Escherichia coli were all active and exhibited different enzymatic properties.View full abstractDownload PDF (3553K) -
Tsuyoshi Hiraoka, Chihiro Katagiri1992 Volume 112 Issue 5 Pages 689-693
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe mechanism of the conversion of low-density lipophorin (LDLp) to high-density lipophorin (HDLp) in long-distance flight insects was investigated using a lipoprotein lipase from a bacterium, Alcaligenes sp. Diacylglycerol of LDLp was steadily hydrolyzed in vitro by the lipase, resulting in a 90% loss of diacylglycerol from LDLp during incubation. The “lipase-treated LDLp” thus obtained still contained associated apolipophorin-III (apoLp-III). These data suggest that the dissociation of apoLp-III is independent of the depletion of diacylglycerol from LDLp, and that the decrease in particle diameter caused by the depletion of diacylglycerol does not force the dissociation of apoLp-III from the lipophorin particle. Some physico-chemical properties of the lipase-treated LDLp were measured.View full abstractDownload PDF (2802K) -
Myeong-Jun Choi, Hyun-Sook Han, Hyoungman Kim1992 Volume 112 Issue 5 Pages 694-699
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWith the ultimate aim of targeting cancer drugs to malignant tissues, liposomes containing polymeric phosphatidylethanolamine and a fatty acid were prepared. For this purpose diacetylenic phosphatidylethanolamine (DAPE), a phosphatidylethanolamine containing diacetylene, was synthesized. Liposomes containing DAPE, fatty acid, and either phosphatidylethanolamine (PE) or phosphatidylethanolamine-β-oleoyl-γ-palmitoyl (POPE) were then prepared. Polymerization of DAPE was effected by UV illumination. The polymeric liposomes so obtained were stable at physiological pH but became leaky below pH 6.5. Of various compositions studied, the greatest pH-sensitivity was found with liposomes composed of 35 mol% DAPE, 35 mol% POPE, and 30 mol% saturated fatty acid.The presence of blood plasma albumin decreased vesicle stability while apolipoprotein A-I (apo A-I) had the opposite effect and plasma as a whole had a slightly stabilizing effect.View full abstractDownload PDF (761K) -
Yoshiyuki Endo, Hiroshi Nagai, Yoshihiko Watanabe, Kiyoshige Ochi, Tos ...1992 Volume 112 Issue 5 Pages 700-706
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAggregate formation of recombinant human erythropoietin (r-EPO) on heat-treatment was followed by gel permeation chromatography combined with a low-angle laser light scattering technique under various conditions with respect to pH and salt concentration in order to provide basic knowledge about the change strictly required to be monitored for medicinal proteins. When heated at 60°C at neutral pH, an aggregate with a limited size consisting of about 20 r-EPO molecules was formed. On heating at 50°C at acidic pH, aggregation was unlimited. The aggregation proceeded non-covalently in acidic pH, but in alkaline pH covalent bond formation was also involved. Increase in salt concentration enhanced the aggregation. Deglycosylation of the N-linked oligosaccharides made r-EPO remarkably susceptible to aggregation on heat-treatment, indicating that the carbohydrate chains are essential to the stability of r-EPO.View full abstractDownload PDF (818K) -
Chen-Sheng Liu, Po-Yao Kuo, Jin-Mei Chen, Shun-Wen Chen, Chen-Hsien Ch ...1992 Volume 112 Issue 5 Pages 707-713
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFrom the acidic components of Bungarus fasciatus venom, a very small amount (0.16%) of a novel phospholipase A2 was obtained. Both neurotoxicity and enzyme activity were found to be lacking. Amino acid sequence study showed that it has a normal backbone of group I snake venom phospholipase A2 with 118 amino acid residues. The lack of enzyme activity was attributed to its mutation of the indispensable Asp residue to an Ala residue, i.e., the usual His-Asp47 turned out to be His-Ala47. This is the eighth isoform of phospholipase A2 found from the venom of Bungarus fasciatus. Examination of structural homology with three other isoforms revealed 66% similarity at most.View full abstractDownload PDF (548K) -
Akiko Shiratsuchi, Showbu Sato1992 Volume 112 Issue 5 Pages 714-718
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAnthranilate synthase I (ASI) of Bacillus caldotenax, a thermophilic bacterium, was purified from a plasmid-bearing Escherichia coli and characterized. The molecular weight determination under native and denaturing conditions revealed that it was a monomeric enzyme of Mr=54, 000. The N-terminal amino acid sequence is the same as expected from DNA sequence of trpE except that the N-terminal methionine is lacking. All four cysteines in the molecule could be titrated with 5, 5'-dithiobis (2-nitrobenzoic acid) in more than 8M urea. The purified enzyme retained its full enzymatic activity after being heated at 60°C. Six mutated genes for the ASI with histidine in place of each conserved arginine, Arg321, Arg353, Arg358, Arg416, Arg429, and Arg452, were prepared by site-directed mutagenesis. All the mutated genes except one, the gene encoding an ASI mutant with histidine in place of Arg452 (R452H ASI) complemented an E. coli (trpE). The mutated ASIs were purified and compared with the wild type ASI. No distinctive differences in enzymatic properties were found between the wild type and the enzymatically active mutated ASIs. R452H ASI was enzymatically inactive, though its conformation seemed to be unchanged after the substitution based on CD spectra and the SH titration curve.View full abstractDownload PDF (1841K) -
Kristina Nilsson Ekdahl1992 Volume 112 Issue 5 Pages 719-723
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRat liver fructose-1, 6-bisphosphatase was phosphorylated by cAMP-dependent protein kinase to 2.6 mol phosphate/mol subunit but not by Ca2+/phopholipid-dependent and Ca2+/ calmodulin-dependent protein kinases. It was demonstrated that phosphorylation of Ser-341 and Ser-356, and to a much lower extent, Ser-338, was dependent on the presence of intact arginine residues. This observation implicates that the intact three-dimensional structure of the substrate is necessary for phosphorylation of Ser-356 since the closest arginine is located at a six amino acid residue distance.View full abstractDownload PDF (1639K)
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