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Volume 115 (1994)
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- Issue 3 Pages 369-
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Volume 115, Issue 3
Displaying 1-40 of 40 articles from this issue
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Makoto Kimura, Yoshiaki Kouzuma, Kei Abe, Nobuyuki Yamasaki1994 Volume 115 Issue 3 Pages 369-372
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA Bowman-Birk family proteinase inhibitor (EBI) was isolated from the seeds of Erythrina variegata. The protein was purified by ion-exchange column chromatography on DEAE-cellulose followed by gel filtration on Sephadex G-75. The stoichiometry with trypsin was estimated to be 1:1, while that with chymotrypsin was not obvious, as determined from the titration patterns of its inhibitory activities. The complete amino acid sequence of EBI was determined by sequencing tryptic and chymotryptic peptides. The EBI protein consists of 61 amino acid residues, which is the shortest among the Bowman-Birk family inhibitors sequenced to date, and has a Mr of 6, 689. Comparison of this sequence with those of other leguminous Bowman-Birk family inhibitors revealed that EBI could be classified as a group II inhibitor, showing the best homology (67%) to the Bowman-Birk proteinase inhibitor from soybeans.View full abstractDownload PDF (407K) -
Tadashi Kobayashi, Naoko Tsugawa, Toshio Okano, Sonoko Masuda, Atsuko ...1994 Volume 115 Issue 3 Pages 373-380
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe binding properties, with blood proteins, and tissue distribution of 22-oxa-1α, 25-dihydroxyvitamin D3 (22-oxacalcitriol; OCT), a noncalcemic analogue of lα, 25-dihydroxy-vitamin D3 [ 1, 25(OH)2D3], in rats were investigated. The binding affinity of OCT to plasma vitamin D binding protein (DBP) is extremely low and OCT mainly circulates in the blood as an intact form nonspecifically bound to lipoproteins, especially to chylomicrons and low density lipoprotein (LDL). OCT intravenously injected into normal rats rapidly disappear-ed from the blood, and rapidly appeared in the bile as glucuronides of intact OCT and lαa, 3β, 20(S)-trihydroxy-9, 10-secopregna-5, 7, 10(19)-triene (23, 24, 25, 26, 27-pentanorOCT; pentanorOCT) as an OCT metabolite. When OCT or 1, 25(OH)2D3 was injected into normal rats, significant amounts of OCT and 1, 25(OH)2D3 were quickly detected in the thyroid and parathyroid glands, thymus, adrenals, liver, plasma, small intestine, kidneys, and calvar-ia. The detected amounts of OCT in the parathyroid glands, thymus, adrenals, liver, small intestine, and kidneys were significantly higher than the respective values for 1, 25(OH)2D3 2 and/or 10 min after injection, while those of OCT in the plasma and calvaria were significantly lower than those of 1, 25(OH)2D3. The in vivo rapid turn-over, nonspecific transportation, and incorporation of detectable amounts into the tissues are typical characteristics of OCT which may account for its specific activities.View full abstractDownload PDF (828K) -
Hiromu Takematsu, Takehiro Kawano, Susumu Koyama, Yasunori Kozutsumi, ...1994 Volume 115 Issue 3 Pages 381-386
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have proposed that CMP-N-acetylneuraminic acid (CMP-NeuAc) hydroxylation is mediated by an electron transport system consisting of cytochrome b5 (b5), b5 reducing factor(s), and CMP-NeuAc hydroxylase, all of which have been detected in the cytosolic fraction of mouse liver [Kozutsumi, Y., Kawano, T., Yamakawa, T., & Suzuki, A. (1990) J. Biochem. 108, 704-706]. In order to elucidate the reaction mechanism underlying CMP-NeuAc hydroxylation, the interaction between b5 and the hydroxylase was studied using a b5-immobilized affinity column. The enzyme activity was retarded on the b5 column in the presence of the substrate, CMP-NeuAc, but not in the presence of the reaction product, CMP-N-glycolylneuraminic acid (CMP-NeuGc). These findings suggest that the binding of CMP-NeuAc to CMP-NeuAc hydroxylase changes the conformation of the enzyme so as to construct a recognition site for b5, followed by the formation of a ternary complex through this domain. Then the transport of electrons from NAD(P)H to the enzyme through b5 takes place, CMP-NeuAc is converted to CMP-NeuGc, and finally the ternary complex dissociates into its components to release CMP-NeuGc. It is known that a soluble form of b5 is abundant in erythrocytes and is synthesized from a mRNA different from that for the microsomal form of b5. In order to determine the origin of b5 detected in the cytosolic fraction of mouse liver, the molecular forms of b5 mRNA expressed in mouse liver were analyzed. The polymerase chain reaction, with mouse liver cDNA and specific primers for the respective forms of b5 mRNA, detected only the mRNA encoding the microsomal form in mouse liver. These results suggest the possibility that b5 involved in the ternary complex formation in the cytosolic fraction of mouse liver originates from the microsomal form, which was solubilized after its synthesis. Possible participation of the microsomal b5 on the endoplas-mic reticulum in the hydroxylation reaction was also suggested by the results of an in vitro reconstitution assay involving isolated microsomes.View full abstractDownload PDF (1791K) -
Dipali Sinha, Xiaoxin Yang, Frances Emig, Edward P. Kirby1994 Volume 115 Issue 3 Pages 387-391
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo protease inhibitors (Inh2 and Inh3) from bovine plasma have been isolated and characterized. The apparent molecular weights of the two proteins are 56 and 58 kDa, respectively. Although Inh2 and Inh3 both inhibit trypsin and human neutrophil elastase, only Inh3 is a good inhibitor of chymotrypsin and cathepsin G. Inh3 is much more sensitive to oxidation than Inh2.One marine monoclonal antibody recognizes Inh3 but not Inh2. Inh3 resembles human α1-antitrypsin both structurally and functionally. Inh2, on the other hand, has some structural homology to human αl-antichymotrypsin, but its specificity does not correspond to that of either human α1-antitrypsin or human α1-antichymotrypsin.View full abstractDownload PDF (1440K) -
Shigeyuki Terada, Satoshi Fujimura, Hideki Katayama, Michiaki Nagasawa ...1994 Volume 115 Issue 3 Pages 392-396
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo subtilisin inhibitors (CLSI-II and -III) were purified from seeds of Canavalia lineata by extraction with water, ammonium sulfate precipitation, and chromatographies on DEAE-Toyopearl and hydroxyapatite. The two inhibitors have the same molecular weight of about 22, 000, and quite similar amino acid compositions. They contain five half-cystine residues and tend to dimerize through an intermolecular disulfide bridge due to the presence of a single cysteine residue. CLSI-III only inhibited subtilisin-type serine proteases, while CLSI-II showed a wider inhibitory specificity. Though the two inhibitors have almost identical thermal labilities, CLSI-II is more stable as to extreme pH than CLSI-III. They are considered to be Kunitz type inhibitors on the basis of several properties.View full abstractDownload PDF (600K) -
Shigeyuki Terada, Hideki Katayama, Koichiro Noda, Satoshi Fujimura, Ei ...1994 Volume 115 Issue 3 Pages 397-404
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe amino acid sequences of two subtilisin inhibitors (CLSI-II and -III) from Canavalia lineata seeds were determined by manual Edman degradation using the DABITC/PITC double coupling method after enzymatic digestions and CNBr degradation. CLSI-II and -III consist of 190 and 183 amino acids, respectively, and have identical amino acid sequences except for in the C-terminal regions: an elongated sequence, Gly-Thr-Ile-Arg-Ser-Asp-Gly, was found at the C-terminus of CLSI-II. A short-chain analog protein without an N-terminal Asn residue was also detected in each inhibitor preparation. The inhibitors showed significant homology to Kunitz type inhibitors, but differed from them with respect to the half-cystine content. Among five half-cystine residues present in CLSI-III, two disulfide bonds link Cys44 to Cys88 and Cys142 to Cys149, Cys106 being present as a free cysteine residue. Phenylglyoxal treatment abolished the inhibitory activity of CLSI-III, indicating the participation of an Arg residue in the interaction with the enzyme. The reactive-site peptide bond was deduced to be Arg68-Gly69.View full abstractDownload PDF (756K) -
Qizhi Hu, Erika Ishii, Yasuhito Nakagawa1994 Volume 115 Issue 3 Pages 405-408
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSChanges in the fatty acid composition of choline glycerophospholipid (CGP) and ethanolamine glycerophospholipid (EGP) in the liver, heart, kidney, testis, spleen, and brain of rats were examined at various stages of streptozotocin-induced diabetes. Diabetes of prolonged duration. (8 weeks) caused profound alterations in the fatty acid composition of phospholipids in these tissues, the most consistent of which were a decrease in the relative level of arachidonic acid and an increase in that of linoleic acid. Although a decrease in the relative proportion of arachidonic acid in CGP was a common feature seen in all tissues after long-term diabetes, the profile of the reduction during the progress of diabetes differed among the tissues. A rapid decrease was found in the liver and plasma at an early stage of diabetes (7 days), while the reductions level of arachidonic acid in CGP of the heart, kidney, and testis appeared at a later stage of diabetes (3 weeks). CGP from the brain retained the normal amount of arachidonic acid over the first 4 weeks of diabetes and no consistent changes in the fatty acid composition of phospholipids were found during this time. A slight change in the level of arachidonic acid composition in CGP from the brain was found after 8 weeks of diabetes. The present study indicates that the extent of reduction in the levels of arachidonic acid of CGP in the tissues examined was dependent on the duration of diabetes in every case.View full abstractDownload PDF (456K) -
Hwei-Ling Peng, Ming-June Hsieh, Chih-Ling Zao, Hwan-You Chang1994 Volume 115 Issue 3 Pages 409-414
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe deaD gene of Klebsiella pneumoniae was isolated and its nucleotide sequence determined. the K. pneumoniae gene is highly homologous with the Escherichia coli analog throughout most of the coding region. The deduced primary sequence of the K. pneumoniae deaD gene product is 659 amino acids in length, in contrast with the 571 amino acids of the E. coli deaD product published previously. Sequence comparison revealed several differences near the 3' end of the deaD genes which result in the frame-shift effect. The3' end sequence of the E. coli deaD gene was therefore analyzed to verify the discrepancy. Our result indicates that the E. coli deaD gene encodes a product of comparable size to the K. pneumoniae DeaD protein, and the carboxyl terminal, sequences of the two proteins are highly homologous. In vivo expression of the K. pneumoniae deaD gene in E. coli yielded a 65-kDa protein. Primer extension analysis of the mRNA from K pneumoniae identified a major transcription start site at an A residue 44 nt upstream of the first in-frame ATG codon.View full abstractDownload PDF (3214K) -
Keiko Sakai, Tadahiko Fujii, Toshihiko Hayashi1994 Volume 115 Issue 3 Pages 415-421
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPurified plasma fibronectin in Tris-buffered saline aggregated on incubation at 37&C in the presence of dithiothreitol without the presence of cells. On SDS polyacrylamide gel electrophoresis without reduction, dimeric fibronectin showed a 460 kDa band, while the protein band of aggregated fibronectin remained at the top of the running gel. The aggregate was a disulfide-bonded multimer, since both the dimeric and the multimeric fibronectins migrated as 230 kDa polypeptides after reduction. The multimer formation required SH reagent and proceeded faster with higher SH concentration, suggesting the occurrence of a disulfide exchange reaction during the aggregation. Since dimeric fibronectin with carboxymethylated sulfhydryl groups also formed multimers under the same condition, the free sulfhydryl groups of dimeric fibronectin may not be involved in the multimer formation, suggesting involvement of disulfide exchange from intramolecular bonds to intermolecular bonds. The multimerization was not influenced by Na+, Ca2+, or EDTA, while urea-treated fibronectin required a higher concentration of dithiothreitol for multimer formation. Fibronectin partially degraded by m-calpain did not form multimers. The multimeric fibronectin retained heparin-binding and cell attachment activities, but had lost gelatin-binding activity. Involvement of the terminal regions containing type I and type II repeats was suggested in the interaction of pFN leading to the multimerization.View full abstractDownload PDF (5337K) -
Goran Poznanovic, Vesna Grujic, Svetlana Ivanovic-Matic, Ljiljana Seva ...1994 Volume 115 Issue 3 Pages 422-428
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effect of the acute phase response on the affinity of binding between nuclear matrix proteins and the rat haptoglobin (Hp) gene region was examined. Nuclear matrices isolated from acute phase livers were enriched with the 5' Hp gene flanking region (-705/+159), but not with the spliced, protein-coding sequence. Reassociation experiments with isolated nuclear protein matrix spheres and end-labeled fragments I (-146/+156), II (-146/-541), and III (-541/-705) revealed that the matrix proteins displayed an increased binding potential during the acute phase response for all of the examined regions, this being most pronounced for fragment II. BAL 31 digestion of fragment II showed that the sequence element that was responsible for the increased association with nuclear matrix proteins during the acute phase response was a tract of 38 adenine bases. The DNA region established stable associations with nuclear lamin B (67 kDa, pI 5.7) in the controls, and with lamins A (69 kDa, pI 7.0), B, isoforms of lamin C (62 kDa, pI 6.55-6.95), and a 55-kDa (pI 5.9) polypeptide during the acute phase response. Sequence ABC (-165/-56), which overlaps fragments I and II and represents the Hp cis-acting element, did not bind to the non-histone nuclear matrix proteins.View full abstractDownload PDF (3229K) -
Kaoru Omichi, Sumihiro Hase1994 Volume 115 Issue 3 Pages 429-434
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSOligosaccharides are often converted to fluorogenic pyridylamino-oligosaccharides (PA-oligosaccharides) to be analyzed sensitively. A method for determining the glycosidic linkage position to the PA-reducing-end residue was developed with PA-disaccharides as model compounds. Periodate oxidation of PA-disaccharides was carried out at 0&C for 15min or at 4&C for 40 h, and the reaction mixtures were reduced with borohydride. The fluorogenic products obtained at 4&C for 40 h were purified by reversed phase HPLC, and the fractions collected were hydrolyzed with acid. The hydrolysates were analyzed by reversed phase HPLC. PA-glyceraldehyde was formed from 2-substituted PA-disaccharides with PA-hexose, PA-threose (or PA-erythrose) from 3-substituted ones, and PA-glycolaldehyde from 4- or 6-substituted ones. HPLC analysis of the products obtained at 0&C for 15min revealed a difference between 4- and 6-substituted ones. PA-glyceraldehyde was formed from 6-substituted ones, but not from 4-substituted ones. The linkage position, therefore, can be determined by analyzing fluorogenic product (s). As for PA-disaccharides with PA-N-acetylglucosamine, the linkage position can be simply determined by analysis of 40-h oxidation-reduction mixtures. 2-Acetamido-2-deoxy derivatives of PA-threose, PA-xylose, and PA-glyceraldehyde were formed from 3-, 4-, and 6-substituted ones, respectively. The linkage position analysis was successfully applied to determination of the structures of two Fuc-Man-PAs produced through the transglycosylation action of bovine kidney α-L-fucosidase.View full abstractDownload PDF (514K) -
Tsunehiro Aki, Akihiro Fujikawa, Takeshi Wada, Toshihiko Jyo, Seiko Sh ...1994 Volume 115 Issue 3 Pages 435-440
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn immunoreactive clone, which shares no homology with the major allergens, Der f I and Der f II, was isolated by screening of a Dermatophagoides farinae cDNA library with rabbit antiserum raised against an extract of the house dust mite and sera from patients with mite allergy. The deduced amino acid sequence of the cDNA of the clone is significantly homologous, up to 65.5%, to the carboxyl-terminal region of the heat shock cognate protein (hsc) 71 in the heat shock protein (hsp) 70 family. A pool of the recombinant protein-specific IgE purified from mite-allergic sera recognized a 67 kDa protein on a blot of ATP-binding components partially purified from the mite body extract, while the antibody did not bind to the major allergens. We conclude that the recombinant protein, one of several important allergens, may be a protein in the heat shock protein 70 family from the house dust mite, D. farinae.View full abstractDownload PDF (2029K) -
Kiyomitsu Nara, Shiho Ito, Teizo Ito, Yohko Suzuki, Magdy A. Ghoneim, ...1994 Volume 115 Issue 3 Pages 441-448
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSElafin was shown to be a new type of proteinase inhibitor which has an anchoring sequence. Human elafin, a potent inhibitor specific for elastase and proteinase 3, has a unique repeating sequence in its prosegment that is rich in Gln and Lys residues. The prosegment, termed “cementoin, ” exhibits high homology with the repetitive element of seminal vesicle clotting protein, which is known as a good substrate for prostate transglutaminase. The cross-linking of cementoin by tissue transglutaminase showed that the cementoin moiety is indeed a preferable substrate for transglutaminase. In addition, transglutaminasemediated cross-linking between cementoin and laminin was observed in vitro, suggesting that cementoin has the ability to covalently attach to other extracellular matrix proteins. To determine whether or not this type of covalent gluing of elafin through the cementoin moiety occurs in vivo, we determined the molecular size of cementoin-elafin in the trachea mucous epithelium by Western blotting; the rationale of this approach is that (i) the trachea is the richest source of cementoin-elafin, as shown below, and (ii) if cementoin-elafin is covalently associated with other proteins, it should migrate as a higher Mγ species on SDS-polyacrylamide gel electrophoresis; cementoin-elafin immunoreactivity was indeed detected at a position corresponding to 50 kDa, a value much higher than that of its monomeric form. RNase protection analysis and immunohistochemical staining revealed that cementoin-elafin is densely distributed in the skin and trachea, and moderately in the stomach, duodenum and small intestine. These sites of localization are consistent with the locations where elastic fibers are abundant. These findings suggest that the cementoin moiety serves as molecular glue for anchoring elafin to extracellular matrix proteins in order to protect elastic tissues. Elafin may therefore represent a new class of proteinase inhibitors.View full abstractDownload PDF (3145K) -
Sumio Ohtsuki, Ko-ichi Homma, Shoichiro Kurata, Hiroto Komano, Shunji ...1994 Volume 115 Issue 3 Pages 449-453
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA prolyl endopeptidase that hydrolyses Suc-Gly-Pro-MCA (Suc, succinyl; MCA, methylcoumaryl-7-amide) was purified to near homogeneity from NIH-Sape-4 cells derived from the flesh fly (Sarcophaga peregrina). The molecular mass of the purified enzyme was 84 kDa, and its activity was inhibited almost completely by 1mM diisopropyl fluorophosphate (DFP). Immunoblotting and DFP-labeling experiments revealed that the leg imaginal discs of Sarcophaga contained this enzyme as a major serine proteinase. This prolyl endopeptidase is suggested to be involved in the differentiation of imaginal discs, because 2mM DFP and 0.1mM N-benzyloxycarbonyl-thioprolyl-thioprolynal-dimethylacetal (ZTTA), a specific inhibitor for prolyl endopeptidase, inhibited differentiation of the imaginal discs from the eversion to the elongation stage.View full abstractDownload PDF (1578K) -
Yousuke Nakamura, Daitatsu Kai, Shunji Kaya, Yoshikazu Adachi, Kazuya ...1994 Volume 115 Issue 3 Pages 454-462
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPhospholipase A2 [EC 3. 1. 1. 4] treatment of pig kidney Na+, K+-ATPase [EC 3. 6. 1. 3] labeled with fluorescene probes at the α-chain reduced the extent of the fluorescence intensity change of an N-[p-(2-benzimidazolyl) phenyl] maleimide (BIPM) probe at Cys-964 to below one-third of the control level accompanying the accumulation of phosphoenzymes. However, it only induced a slight decrease in that of a fluorescence isothiocyanate (FITC) probe at Lys-501 with a large decrease in the rate of change. The addition of phosphatidylserine (PS) or phosphatidylinositol (PI) to the phospholipase-treated BIPM-FITC-labeled enzyme increased the rate of the FITC fluorescence change. Phospholipase treatment of the BIPM-enzyme greatly reduced the Na+, K+-ATPase activity. The addition of PS or PI to the treated enzyme induced reactivation. These data and others suggest that Cys-964 and Glu-953 (Rb+ protectable dicyclohexyl carbodiimide binding site) are located in the vicinity of the surface area of the enzyme where hydrocarbon chains of phospholipids are present, and conserved H-bonding amino acids, Thr-955 and Ser-962, are located rather near the center of a domain forming a cation binding route or cage with other hydrophobic transmembrane segments. These data may indicate that the interaction between the BIPM probe and the hydrocarbon chains of phospholipids changes in such a way as to sense the change in the binding state of various ligands accompanying the sequential appearance of reaction intermediates of the enzyme.View full abstractDownload PDF (1309K) -
Kyoko Shinagawa, Masanao Ohya, Tsutomu Higashijima, Kaori Wakamatsu1994 Volume 115 Issue 3 Pages 463-468
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe previously showed that peptides corresponding to the N-terminal parts of the third intracellular loops of turkey and hamster β-adrenergic receptors (tuβI3N and haβI3N, respectively) can activate the Gs protein (one of the GTP-binding regulatory proteins which couples to the β-adrenergic receptor) reconstituted in phospholipid vesicles, and also that such activation can be greatly enhanced by a modification which increases the hydrophobicity of the peptides. These observed phenomena suggest that the interaction with phospholipid membranes is important for the activity of these peptides; hence, in the present study we employed circular dichroism to analyze the interaction of the synthetic peptides corresponding to the intracellular loops of G protein-coupled receptors with phosphatidylserine/phosphatidylcholine mixed vesicles. The tuβI3N and haβI3N peptides were subsequently found to take on an α-helical conformation upon binding with the vesicles, whereas those corresponding to the intracellular loops of m1 and m2 muscarinic acetylcholine receptors in contrast did not interact with the vesicles. The positions of several side chains of the membrane-bound loop peptides were also determined. Our results show for the first time the interaction occurring between the intracellular loops of β-adrenergic receptors and a phospholipid membrane.View full abstractDownload PDF (713K) -
Jun Uematsu, Yuji Nishizawa, Arnoud Sonnenberg, Katsushi Owaribe1994 Volume 115 Issue 3 Pages 469-476
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHemidesmosomes (HDs) are specialized cell-substrate junctions with distinct cytoplasmic plaques where intermediate filaments (IFs) are anchored. In our previous work, we described two types of HDs in terms of their molecular constituents, i.e. type I HD and type II HD [Hieda, Y., Nishizawa, Y., Uematsu, J., & Owaribe, K. (1992) J. Cell Biol. 116, 1497-1506]. In the present study we further characterized type II HDs in cultured cells, comparing their composition and function with those of conventional type I HDs. Although the two bovine mammary gland epithelial cell lines, BMGE+H and BMGE-H, were derived from the same tissue, their cell-substrate adhesion properties are markedly different. Immunological examination showed that BMGE-H cells express HD1 and the integrin α6β4 complex but not bullous pemphigoid antigens, while BMGE+H cells express all these components, i.e. the former have type II HDs and the latter have type I HDs. GoH 3, a monoclonal antibody to the integrin α6 subunit, inhibited BMGE-H cell adhesion to laminin as a substrate, as also observed for BMGE+H cells. These and electron microscopic results indicate that BMGE-H cells form type II HD-like structures containing HD1 and α6β4 which are associated with IFs. This structure mediates adhesion of the cell to laminin. This is the first demonstration of an adhesion function for type II HDs in cultured cells.View full abstractDownload PDF (5710K) -
Kyoji Horie, Seiji Nishiguchi, Shuichiro Maeda, Kazunori Shimada1994 Volume 115 Issue 3 Pages 477-485
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe relationship between DNA structure of replacement vectors and gene targeting efficiency was studied using positive-negative selection. The vectors contained pBR 322 DNA, a bacterial neomycin-resistance gene (neo) for positive selection, a herpes simplex virus (HSV) thymidine kinase gene (tk) for negative selection, and a mouse genomic fragment, including exons 1 to 3 of the transthyretin (ttr) gene. The neo gene that confers G 418 resistance was inserted into the second ttr exon, and the HSV-tk gene that confers gancyclovir (GANG) sensitivity was added to the 3' end of the ttr fragment. The vectors were linearized by digesting with restriction enzyme (s) and transfected into mouse embryonal carcinoma F 9 cells. In this system, the enrichment by GANC selection as well as the frequency of gene targeting was increased by placing the pBR 322 DNA at the 3' end of the HSV-tk gene. Adding one more HSV-tk gene at the 5' end of the ttr fragment did not increase the enrichment by GANC selection. This enrichment factor was also increased by reducing the size of the ttr fragment present between the two selection markers. However, it decreased the frequency of gene targeting and, overall, it did not increase the efficiency of isolating targeted clones. When structures of the vector DNA fragments present in 20 G 418-resistant and GANC-resistant non-targeted clones were examined by Southern blot analysis, the inefficiency of GANC selection proved to be mostly caused by exonucleolytic degradation of HSV-tk genes progressing from ends of the vectors.View full abstractDownload PDF (3911K) -
Atsushi Inanobe, Katsunobu Takahashi, Toshiaki Katada1994 Volume 115 Issue 3 Pages 486-492
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSGTP-binding proteins (G proteins), predominantly located at the inner surface of the plasma membranes of mammalian cells, dissociate into their constituent α and βγ subunits upon stimulation of G protein-coupled receptors by agonists. In the present studies, cytoplasmic proteins which might have an affinity for the dissociated βγ subunits were investigated by means of βγ subunit-immobilized affinity-column (βγ-immobilized column) chromatography. When soluble fractions obtained from various materials including rat liver, bovine brain, and HL-60 cells were applied to a βγ-immobilized column, some proteins were specifically eluted from the column with high-salt and detergent-containing solutions. One of the βγ subunit-binding proteins, of which the molecular weight was approximately 93, 000 on SDS-PAGE, appeared to be commonly present in all tissues tested. The 93-kDa βγ-binding protein was identified as 90-kDa heat shock protein, hsp 90, based on the findings of its partial amino acid sequences and its immunoreactivity to a monoclonal anti-hsp 90 antibody. The brain hsp 90 inhibited βγ-supported pertussis toxincatalyzed ADP-ribosylation of a subunits. The hsp 90 was also capable of binding to βγ subunits which had been reconstituted into phospholipid vesicles. The binding of hsp 90 to βγ subunits was inhibited by the addition of GDP-bound α subunits, but not by GTPγS-bound ones. These results suggested that hsp 90 could associate functionally with free βγ subunits dissociated from trimeric G proteins in vitro.View full abstractDownload PDF (4406K) -
Shiho Yamaguchi, Ayako Inazu, Yoshihiro Deyashiki, Toshihiro Nakayama, ...1994 Volume 115 Issue 3 Pages 493-496
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe stereochemical course in the enzymatic oxidation of trans-dihydrodiols of benzene and naphthalene by dimeric dihydrodiol dehydrogenase of monkey kidney was compared with that by monomeric dihydrodiol dehydrogenase of rat liver. The monkey kidney and rat liver enzymes each oxidized about half of the racemic dihydrodiol of benzene added to the reaction mixture, but almost all the substrate was disappeared in the reaction mixture containing both enzymes. The CD spectra of the unreacted dihydrodiols of benzene and naphthalene in reaction mixtures containing the rat liver enzyme showed the negative sign of Cotton effect, while those in reaction mixtures containing the monkey kidney enzyme gave the positive sign of Cotton effect. Thus, the monkey kidney dimeric enzyme selectively oxidized (-)-[1R, 2R]-dihydrodiols of aromatic hydrocarbons, in contrast to the stereospecificity of the rat liver enzyme for the (+)-[1S, 2S]-isomers. The (+)-[1S, 2S]- and (-)-[1R, 2R]-dihydrodiols of benzene were separately prepared from the racemic form by using the two enzymes, and were used as substrates to determine the stereospecificity of dihydrodiol dehydrogenases from other mammalian tissues. The dimeric enzymes from pig liver and rabbit lens also exhibited specificity for the (-)-isomer, which was opposite to that of the monomeric enzymes from human and mouse liver, although aldehyde reductase and aldose reductase oxidized both (+)- and (-)-isomers.View full abstractDownload PDF (510K) -
Toru Hisabori, Hiromi Kobayashi, Chitose Kaibara, Masasuke Yoshida1994 Volume 115 Issue 3 Pages 497-501
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSF1-ATPase isolated from plasma membrane of a thermophilic Bacillus strain PS3 (TF1) has very little or no endogenously bound adenine nucleotides. However, it can bind one ADP per mol of the enzyme on one of three β subunits to form a stable TF1•ADP complex when incubated with a high concentration of ADP [Yoshida, M. & Allison, W. S. (1986) J. Biol. Chem. 261, 5714-5721 ]. The same TF1•ADP complex was recovered after filling all ADP binding sites with [3H] ADP and repeated gel filtration. Direct binding assay revealed that the TF1•ADP complex had lost the highest affinity site for TNP-ADP. When a substoichiometric amount of TNP-ATP was added, the complex hydrolyzed TNP-ATP slowly (single site hydrolysis), like native TF1•However, this hydrolysis was not promoted by chase-addition of excess ATP. The optimal pH of the ATPase activity of TF1 or the TF1•ADP complex measured with a short reaction period, 6.5, was lower than the reported value, 9.0, under the steady-state condition. Although the bound ADP was released from the complex only when the enzyme underwent multiple catalytic turnover, the rate of this release was much slower than the turnover. These results suggest that when one ADP binds to a site on one of the β subunits and stays there for a long time, the enzyme will change form and the bound ADP will become a special species which is not able to be directly involved in the enzyme catalysis. This binding site for ADP appears to be the first site responsible for the single-site catalysis reaction observed for native TF1.View full abstractDownload PDF (654K) -
Kimiko Fukuda, Masao Ichinose, Hidetoshi Saiga, Koichiro Shiokawa, Sad ...1994 Volume 115 Issue 3 Pages 502-506
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSEmbryonic chick pepsinogen (ECPg) is one of the pepsinogen isozymogens and its expression is restricted to epithelial cells of the embryonic chick proventriculus (glandular stomach). To examine whether DNA methylation is involved in the regulation of organspecific and developmental stage-specific expression of ECPg gene, we analyzed the extent of methylation of ECPg gene in normal embryonic and hatched chick organs using methylation-sensitive restriction enzymes. In the proventriculus some CCGG sites underwent demethylation in the gene region after the onset of transcription of the ECPg gene. By contrast, these sites were kept methylated throughout the development in the other organs which do not express ECPg gene. GCGC sites in the gene region became methylated in organs which do not express the ECPg gene, after the initiation of transcription of the ECPg gene in the proventriculus. In the proventriculus, GCGC sites, which were methylated in other organs, were kept unmethylated throughout the development. The methylation state of CpG sites showed no change in the proventriculus of a chick 2 weeks after hatching when the expression of the ECPg gene had completely ceased. The data presented here demonstrate that the DNA methylation is involved in the regulation of organ-specific expression, but stage-specific expression might be brought about by some other mechanisms.View full abstractDownload PDF (2887K) -
Kuniyo Inouye, Atsushi Osaki, Ben'ichiro Tonomura1994 Volume 115 Issue 3 Pages 507-515
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCu, Zn-superoxide dismutase (SOD) of bovine erythrocytes is a dimeric enzyme of identical subunits associated through unusually strong noncovalent interactions. It is known not to be dissociated into subunits even in 8M urea for 72 h at 25&C [Malinowski, D. P. & Fridovich, I. (1979) Biochemistry 18, 5055-5060]. Effects of urea, temperature, and SOD concentration on the inactivation and dissociation into subunits were examined. The activation energy of the inactivation of SOD (at 0.05mg/ml) was 64 kcal/mol at pH 7.8, and was decreased to 40 kcal/mol in the presence of urea (2.0-7.3M). The apparent first-order rate constant (kapp) of the inactivation by urea was dependent on the SOD concentration [SOD] during the urea treatment, and SOD showed a higher resistance to the inactivation with increase in the concentration. Dissociation of SOD was monitored by gel filtration HPLC. When SOD solutions of various concentrations were incubated in 6M urea at 45&C, two monomer species (M 1 and M 2) were observed in addition to dimer (D). The dimer maintained full activity, while the monomers did not show the activity. The peak areas of these species were changed depending on the SOD concentration during urea treatment; at over 15mg/ml, almost all SOD was eluted as D, and with a decrease in the SOD concentration, the peak area of D decreased and concomitantly the monomers appeared. M 2 could be the sole form in infinitely diluted SOD solution, and D was considered to be converted to M 2 through M 1. The SOD concentration giving 50% D ([SOD]1/2) was 1.0mg/ml. At 25&C, pH 7.8, with or without the treatment in 6M urea, concentration-dependent dissociation was observed with [SOD]1/2 of 0.3-0.4mg/ml, without any loss of activity. In this case, appearance of M 1 was almost negligible and the dissociation seems to proceed directly from D to M 2. It is suggested that SOD exists mostly in monomer under the assay conditions, and when incubated below 45&C, SOD monomer expresses activity even after treatment in 6M urea.View full abstractDownload PDF (961K) -
Hiroya Obama, Shuichiro Matsubara, Jean-Louis Guénet, Takashi M ...1994 Volume 115 Issue 3 Pages 516-522
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMidkine (MK) is a novel heparin-binding growth/differentiation factor coded by a retinoic acid-responsive gene. MK cDNA probe reacts with two bands, a 4 kb one and a 3 kb one, upon Southern blot analysis of Hindill fragments of mouse genomic DNA: the midkine gene (Mdk) is on the 4 kb fragment. Sequence analysis of the 3 kb fragment revealed that it has an Mdk-related sequence (Mdk-rs) highly homologous to MK cDNA, three mouse Aluequivalent repeats and seven A+T-rich segments. The Mdk-rs carried an inserted microsatellite DNA, is flanked by imperfect direct repeats observed in many retroposons, and lacks introns. Interspecific hybrid analysis revealed that Mdk-rs is located on chromosome 11, while Mdk is known to be on chromosome 2. The evolutional velocity of Mdk-rs was calculated to be 11 times higher than that of mouse Mdk. These features suggest that Mdk-rs is a processed pseudogene generated in the mouse genome. The 3 kb fragment with Mdk-rs, which is rich in inserted DNA sequences probably due to the presence of A+T-rich segments, may be a hot spot for amplification and evolution of genomic DNA. Mdk-rs was estimated to have been generated about 19.1 million years ago. A chicken protein retinoic acid-induced, heparin-binding protein (RIHB), is highly homologous to MK, and its divergence from human MK was estimated to have occurred about 250 million years ago, suggesting that RIHB is the chicken homolog of MK. Thus, so far there are only two established protein members, MK and heparin-binding, growth-associated molecule (HB-GAM)/pleiotrophin (PTN) in the MK family.View full abstractDownload PDF (784K) -
Masayuki Seki, Junn Yanagisawa, Takeo Kohda, Tadao Sonoyama, Michio Ui ...1994 Volume 115 Issue 3 Pages 523-531
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDNA-dependent ATPase activities in crude extracts prepared from HeLa cells were separated into five peaks designated Q 1 to Q 5 by FPLC Mono Q column chromatography. In our previous study, we observed that crude extracts prepared from xeroderma pigmentosum complementation group C (XP-C) cells contained no DNA-dependent ATPase activity at the peak position of Qi and exhibited a broader peak with higher activity than normal Q 2 at the peak position of Q2 [Yanagisawa, J., Seki, M., Ui, M., & Enomoto, T. (1992) J. Biol. Chem. 267, 3585-3588]. We have purified two DNA-dependent ATPases Q1 and Q2 from HeLa cells and characterized their properties in order to obtain a means to discriminate ATPase Q1 from Q2 in XP-C cells. The apparent molecular masses of Q1 and Q2 on SDS-polyacrylamide gel electrophoresis were 73 and 100 kDa, respectively. The two enzymes required a divalent cation for activity. DNA-dependent ATPase Q 1 hydrolyzed ATP and dATP and Q2 hydrolyzed ATP preferentially among the nucleotides tested. Both enzymes preferred single-stranded DNA as a cofactor. The DNA-dependent ATPase activity of Q2 was inhibited by 90% in the presence of 200mM NaCl, whereas that of Q 1 was not affected by NaCl at concentrations up to 200mM. Both enzymes had DNA helicase activity, that of Q1 being more resistant to NaCl than that of Q2. The DNA helicase activity of Q2 was about 150-fold higher than that of Q1, when compared with units of ATPase activity. The direction of unwinding for Q 1 was from 3' to 5' and that for Q2 was 5' to 3' with respect to the DNA to which the enzymes bound. Examinations based on the differences in the properties of the two enzymes have indicated that DNA-dependent ATPase Q1 is altered in XP-C cells, resulting in the overlapping elution of Q 1 and Q 2.View full abstractDownload PDF (3209K) -
Takashi Kumazaki, Shin-ichi Ishii, Hideyoshi Yokosawa1994 Volume 115 Issue 3 Pages 532-535
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSInactivation of Streptomyces griseus metallo-endopeptidase II (SGMPII) by ClCH2CO-DL-(N-OH) Leu-OCH3 and by CICH, CO-DL-(N-OH) Leu-Ala-Gly-NH2 was studied kinetically. These reagents cause irreversible inhibition of the enzyme in a pseudo-first order reaction, and the inhibition reaction exhibits saturation kinetics. The second-order rate constants for inactivation of SGMPII by ClCH2CO-DL-(N-OH) Leu-OCH, and by CICH2CO-DL-(N-OH) Leu-Ala-GIy-NH2 were measured to be 0.12 and 8.9M-1•S-1, respectively. The order of affinities of metallo-endopeptidases towards these irreversible inhibitors is thermolysin>SGMPII>Pseudomonas aeruginosa elastase. A competitive inhibitor of SGMPII, L-Val-L-Trp, protects the enzyme against inactivation by CICHZCO-DL-(N-OH)-Leu-Ala-Gly-NH2 in a competitive manner. Furthermore, the pH profile of the inactivation closely resembles that for the hydrolysis of synthetic peptide substrates by the enzyme. These findings suggest that these reagents bind reversibly and react irreversibly at the active site of the enzyme.View full abstractDownload PDF (473K) -
Ichiro Tokimitsu, Shingo Tajima1994 Volume 115 Issue 3 Pages 536-539
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSElastin synthesis in cultured smooth muscle cells was inhibited by one fourth in the presence of 0.1M K+ in the medium. The degree of inhibition paralleled the decrease in the steady-state levels of elastin mRNA. The inhibition of elastin synthesis was blocked by addition of 1 μM nifedipine, a Ca2+ antagonist. Comparable inhibition of elastin synthesis was observed by addition of A 23187, a Ca2+ ionophore. In contrast, collagen synthesis and thymidine uptake were stimulated threefold and twofold respectively in the presence of 0.1M K+ with a concomitant increase in collagen mRNA. The stimulation of collagen synthesis was also blocked by nefedipine. These results indicate that K+ modulates elastin and collagen synthesis and their gene expression reciprocally, and these effects are mediated by Ca2+ influx. Thus K+ exerts profound effects on the composition of extracellular matrices in aorta.View full abstractDownload PDF (2538K) -
Toshiyasu Endo, Yukiko Ishibashi, Satoshi Shiokawa, Yasuyuki Fukumaki, ...1994 Volume 115 Issue 3 Pages 540-544
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSVarious chemicals which are known to have positive effects on differentiation of some erythroid cell lines were tested on a human chronic myelogenous leukemia cell line, KU-812 F. Succinic acid, 5-azacytidine, daunomycin, and hemin showed a positive effect. Among them, hemin and 5-azacytidine were the most effective inducers for erythroid differentiation of KU-812 F cells. Dimethylsulfoxide, cytosine arabinofuranoside, and sodium n-butyrate showed no effect. In addition, subclone KU-812 F/33 derived from the KU-812 F cell line showed differential expression of the β- and γ-globin genes in the presence of either 2 μM 5-azacytidine or 40 μM hemin. Hemoglobin synthesis in differentiated KU-812 F/33 cells was analyzed by isoelectric focusing gel electrophoresis, and S 1 mapping analysis of β- and γ-globin mRNA was performed. After treatment with 5-azacytidine, the β-globin gene expression was predominantly enhanced (18.75-fold higher level of β-globin mRNA). After treatment with hemin, the most notable increase was in the γ-globin gene expression (1.83-fold higher level of γ-globin mRNA), while no increment of β-globin was observed.View full abstractDownload PDF (2601K) -
Kazumasa Miyamoto, Shigenori Kanaya, Kosuke Morikawa, Hachiro Inokuchi1994 Volume 115 Issue 3 Pages 545-551
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo establish a system for overproduction of the ferrochelatase [EC 4. 99. 1. 1] from Escherichia coli, a plasmid designated pFC 3 was constructed. The 35-kDa protein was accumulated in E. coli DH5α cells that harbored pFC 3 to a level equal to approximately 9% of the total protein (roughly 50mg/liter) upon thermal induction. This 35-kDa protein was identified as the ferrochelatase of E. coli by Western blotting and amino-terminal amino acid sequence analysis. The protein with ferrochelatase activity was purified from the cells by three simple steps with a yield of 17%. The optimum pH of the purified enzyme was around 8.0. The molecular weight of the enzyme was estimated to be 35-kDa from column chromatography on Sephacryl S-300, a value consistent with that estimated from SDS-polyacrylamide gel electrophoresis, suggesting that the enzyme is a monomer. The isoelectric point of the enzyme was approximately 4.7. Determination of the far-ultraviolet circular dichroism spectrum allowed us to calculate the α-helix and β-sheet contents of the enzyme as 10±0.2 and 39±0.2%, respectively. High-level production of the ferrochelatase from E. coli will greatly facilitate detailed structural analysis of this protein.View full abstractDownload PDF (1689K) -
Zong Xuan Jin, Tetsuo Nakajima, Masaaki Morisawa, Hiroshi Hayashi1994 Volume 115 Issue 3 Pages 552-556
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSen-affil=Phosphorylation of a tyrosine residue within a 15-kDa protein has been found to play a role in the initiation of flagellar movement of quiescent spermatozoa of the rainbow trout, Salmo gairdneri [Morisawa & Hayashi (1986) Biomed. Res. 6, 181-184; Hayashi et al. (1987) J. Biol. Chem. 262, 16692-16698]. In order to find a more accessible source of the 15-kDa protein for biochemical studies, phosphorylation of proteins was studied in other organs from other species. Cell-free extracts from spermatozoa and testes of Salmonidae were prepared and all catalyzed the cAMP-dependent, Mgll-requiring, but Ca2+-independent phosphorylation of the 15-kDa proteins. Protein from the testes of a white salmon, Oncorhynchus keta, was isolated by conventional purification methods. The 15-kDa protein in the cell-free extract was found to be complexed with several other proteins such that the 15-kDa protein and its phosphorylating activity were copurified. Isolation of a sufficient amount of the complex for the preparation of antibodies against the various constituents is now possible.View full abstractDownload PDF (4192K) -
Mikio Tomida, Yuri Yamamoto-Yamaguchi, Motoo Hozumi1994 Volume 115 Issue 3 Pages 557-562
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThree cDNAs for mouse differentiation-stimulating factor (D-factor)/leukemia inhibitory factor (LIF) receptor were isolated from a cDNA library prepared from the liver of a pregnant mouse. A probe for screening was prepared by the RT-PCR method using human cDNA sequences as primers. The mouse D-factor receptor cDNA encoded 1, 092 amino acids, which had a marked homology with the human counterpart and consisted of signal sequence, extracellular, transmembrane, and cytoplasmic domains. The WSXWS motif found in members of the cytokine receptor family was also present in the extracellular domain of the mouse D-factor receptor. A second form of cDNA that had a 501 by insertion was isolated. The insertion introduced a stop codon so that the mRNA encoded the soluble receptor lacking transmembrane and intracellular domains. Because the insertion contained polyadenylation signals, two different sizes of mRNA encoding the soluble receptor were produced, depending on whether or not it utilized these signals. Transcripts utilizing these signals were 2.6-3 kb in size, and were very abundantly expressed in the liver. Transcripts that did not use these signals were longer than 5 kb and of similar size to the mRNA for the cellular receptor.View full abstractDownload PDF (1538K) -
Masatora Iwashina, Takeshi Mizuno, Shigehisa Hirose, Teizo Ito, Hiromi ...1994 Volume 115 Issue 3 Pages 563-567
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo define the ligand-binding site of natriuretic peptide receptor (NPR), amino acid substitutions were carried out by site-directed mutagenesis at selected residues of bovine NPR-C. The mutant receptors were expressed in COS-1 cells and the effect of the mutations on the binding of ligand was analyzed by binding assay, affinity labeling, and immunoblotting. The replacement of His145-Trp146 (HW) by Leu145-Leu146 in the extracellular domain markedly reduced binding affinity, whereas mutations at charged amino acid residues in the vicinity of HW, such as Glu133 and Asp147, had little effect. Mutation of cysteine residues forming the Cys104-Cys132 and Cys209-Cys257 loops to Ser caused a reduction in the affinity of the receptor to bind ANP. These results suggest that HW residues and the two disulfidelinked loops in the extracellular domain contribute significantly to the ligand binding to NPR.View full abstractDownload PDF (1816K) -
Keiji Miyazawa, Shin-ichi Kawaguchi, Akihiro Okamoto, Ryuichi Kato, To ...1994 Volume 115 Issue 3 Pages 568-577
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSEscherichia coli aspartate aminotransferase (AspAT) and E. coli aromatic amino acid aminotransferase (AroAT) have almost identical and high activities toward acidic amino acid substrates. AroAT also has high activity toward aromatic amino acid substrates. The two proteins have 44% amino acid sequence homology. In order to study the mechanism responsible for the different substrate specificities of these aminotransferases, chimeric enzymes of AspAT and AroAT were constructed using homologous recombination in E. coli cells. Five chimeric enzymes were obtained, even though the nucleotide sequence homology between the two parent enzymes was as low as about 50%. The yields of the legitimate chimeric genes were related to the lengths of the homologous region between the two parent genes. Homologous recombination occurred in the region where more than eight nucleotides out of ten were identical. The substrate specificity of the chimeric enzymes suggest that not only the amino acid residues in the active site but also those distant from the active site contribute to the substrate specificity of the parental aminotransferases.View full abstractDownload PDF (1073K) -
Yoshinori Takei, Katsunobu Takahashi, Yasunori Kanaho, Toshiaki Katada1994 Volume 115 Issue 3 Pages 578-583
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSNuclear protein transport was inhibited in permeabilized HeLa cells which had been prepared after culture with pertussis toxin, suggesting that the pertussis toxin-sensitive protein (s) might be involved in the nuclear protein transport. To investigate the mechanism whereby pertussis toxin inhibited the nuclear protein transport, an accumulation of proteins containing a nuclear localization signal sequence (NLS) into isolated rat liver nuclei was investigated. The NLS-containing protein accumulation required ATP and cytosolic proteins, and was temperature- and wheat germ agglutinin-sensitive as had been observed in permeabilized cells. Non-hydrolyzable GTP analogues, such as guanosine 5'-(γ-thio) triphosphate and guanosine 5'-(β, γ-methylene) triphosphate, but not ATP analogues, inhibited the NLS-containing protein accumulation in the isolated nuclei. The NLS-containing protein accumulation was also inhibited by prior treatment of the nuclei with pertussis toxin plus NAD, and the effect of pertussis toxin was blocked when guanosine 5'-(γ-thio) triphosphate was simultaneously added during the pretreatment with pertussis toxin. The inhibition induced by pertussin toxin and the blockage by guanosine 5'-(γ-thio) triphosphate were well correlated to ADP-ribosylation of 40-kDa protein in nuclear fraction. These results suggested that the nuclear pertussis toxin-sensitive GTP-binding protein is involved in a pathway of nuclear protein transport.View full abstractDownload PDF (3081K) -
Kyoko Hiramatsu, Sachiko Kamei, Masayuki Sugimoto, Kenji Kinoshita, Ke ...1994 Volume 115 Issue 3 Pages 584-589
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe developed an improved system for the simultaneous measurement of free and acetylated polyamines, which comprised a HPLC pump, a separation column, an enzyme reactor, and an electrochemical detector, connected in series. Polyamines were separated with an isocratic elution system, and the separated polyamines were introduced into the enzyme reactor, in which they were deacetylated and oxidized to generate hydrogen peroxide. The amount of hydrogen peroxide generated was then determined with the electrochemical detector. Analysis of a mixture of nine standard polyamines including both free forms and acetylated derivatives with this method revealed that the analytical variables were satisfactory. For the analysis of polyamines in urine, pretreatment of samples with a weakly acidic ion exchange resin was necessary to reduce the interfering substances present in the urine. On successive determinations of polyamines in a urine sample, the coefficients of variation obtained were below 5.4%, except that for spermine (27.6%), and the analytical recovery rates were above 90%, except that for acetylputrescine (78.5%). The correlation coefficient between the total polyamine content in urine estimated by our method and that obtained by means of a commercially available enzymatic assay system was calculated to be 0.98, and the regression equation was expressed as y=0.81x+0.89.View full abstractDownload PDF (664K) -
Yasunori Kondoh, Michi Kawase, Mayumi Hirata, Shinji Ohmori1994 Volume 115 Issue 3 Pages 590-595
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCarbon sources for D-lactate formation were investigated in vitro using 6, 000×g supernatant of rat liver homogenate and by rat liver perfusion in situ. As carbon sources, L-threonine, glucose, glycerol, acetone, and acetoacetate were tested. Glycerol was the best substrate for D-lactate formation via methylglyoxal in rat liver. Glucose was the second most preferred substrate, while L-threonine, acetone, and acetoacetate were poor substrates for D-lactate formation. Glycerol was several times more effective than normal as a substrate of D-lactate in the supernatants of liver homogenates of diabetic and starved rats, while it was less effective as a substrate of L-lactate. The glycerol kinase [EC 2. 7. 1. 30] activities in livers increased in the diabetic and starved states. These and other results can explain why the plasma concentration of D-lactate increases several-fold after running and why the D-lactate contents in plasma, liver, and skeletal muscle are markedly increases in diabetic and starved rats.View full abstractDownload PDF (625K) -
Kenji Ohwaki, Ryuzo Sakakibara, Masatsune Ishiguro1994 Volume 115 Issue 3 Pages 596-601
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn order to study the function of LH/CG receptor, we prepared four synthetic peptides (RP1, 2, 3, and 4 for residues 242-255, 49-66, 493-504, and 573-584) corresponding to extracellular domains of rat ovary LH/CG receptor and raised antibodies against RP 1 and RP 2 (antireceptor peptide IgGs). These peptides and IgGs were assayed for inhibition of 125I-labeled hCG binding to Leydig cell membrane receptors. However, neither the peptides nor the IgGs inhibited hCG binding to the receptor at doses up to 10-3 and 10-5M, respectively. On the other hand, although anti-receptor peptide IgGs did not show inhibition of hCG binding to the receptor, it was found that one of the IgGs (anti-RP1IgG) was bound to the cell membrane and stimulated testosterone production in rat Leydig cells. F (ab')2 fragment lacking the N-linked sugar chain in the IgG molecule, whose sugar chains are similar to those of hCG, was prepared from anti-RP 1 IgG. Anti-RP 1 F (ab')2 was still bound to the cell membrane but no longer stimulated testosterone production. These results suggested that when LH/CG receptor binds to a glycoprotein, even if it is different from the native ligand hCG, it may interact with sugar chains of the glycoprotein to cause signal transduction. Thus, a lectin-like domain in or near the receptor may play a key role in the receptor function.View full abstractDownload PDF (726K) -
Han Geuk Seo, Haruyuki Tatsumi, Junichi Fujii, Atsushi Nishikawa, Keii ...1994 Volume 115 Issue 3 Pages 602-607
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSNitric oxide synthase (NOS) has been purified over 6, 500-fold with a 3.4% yield from rat colorectum with 2', 5'-ADP-Sepharose, DEAE cellulose, and gel filtration. The purified enzyme gave a single band corresponding to an apparent molecular mass of 160 Dka on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When assayed in the requisite presence of L-arginine, CaCl2, NADPH, calmodulin, tetrahydro-L-biopterin, and FAD, the purified enzyme exhibited a specific activity of 328 nmol/min/mg L-citrulline formed and an apparent Km for L-arginine of 2.9 μM. Amino acid sequencing of 12 peptides revealed identical sequences to that of the neuronal type enzyme except for two altered amino acid residues. When partial reverse transcription-polymerase chain reaction of RNA from rat colorectum and cerebellum was performed using primers designed according to the amino acid sequences determined, these amino acid changes were found in both cDNA fragments, indicating the identity of the colorectal enzyme to the cerebellar one. A polyclonal antibody raised against NOS purified from rat cerebellum cross-reacted with the NOS from colorectum but not that from IFN-γ stimulated macrophage-derived cells, RAW 264.7. Immunohistochemical analysis of the colorectum using this specific antibody indicated that Auerbach's plexus is strongly immunoreactive, supporting the hypothesis that NO is an inhibitory transmitter for non-adrenergic and non-cholinergic nerves in the colorectum.View full abstractDownload PDF (3477K) -
Torn Kurome, Masahiko Katayama, Keiko Murakami, Kimikazu Hashino, Kyok ...1994 Volume 115 Issue 3 Pages 608-614
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo construct a mouse/human chimeric antibody, we cloned the genomic DNAs for Ig from a murine hybridoma that produces PL 7-6 monoclonal antibody specific to human P-selectin and expressed them in SP 2/0 myelomas using a series of pSV 2 vectors. Transfected cells that produce the mouse/human chimeric anti-human P-selectin antibody were geneticinselected and screened by an immunoassay using immobilized antigen. The chimeric antibody, cPL-2R1, expressed by the resultant clone has the murine Ig variable region and the human Ig constant region. The native antibody PL 7-6 and the chimeric antibody cPL-2R1 react equally with purified P-selectin and thrombin-stimulated platelets. Competitive inhibition tests demonstrated that the native antibody PL 7-6 and the chimeric antibody cPL-2 R 1 had identical affinity for purified human P-selectin. Thus, although human IgG1 constant region was substituted for the murine counterpart in this chimeric antibody, its specificity and binding affinity for P-selectin was not altered. This chimeric antibody may prove useful when employed in combination with imaging reagents or therapeutic drugs for targeting activated platelets or endothelium in patients with thrombosis or intravascular inflammation.View full abstractDownload PDF (3970K) -
Yukio Fujino, Tomoko Fujii, Tateo Daimon1994 Volume 115 Issue 3 Pages 615-621
Published: 1994
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe presence of a glycoprotein laminin in bovine adrenal chromal in granules was examined by SDS-PAGE followed by immunoblotting. The two chromaffin granule membrane fractions were obtained by linear sucrose gradient centrifugation followed by freezing and thawing and gel-filtration of the chromaffin granule-rich fraction, respectively. The purity of the granules in these fractions was examined by electron microscopy. These fractions contained laminin B chain-like immunoreactivity as a major immunoreactive component against anti-laminin. Laminin A chain-like immunoreactive protein was undetectable. The soluble fraction of the chromaffin granules contained no immunoreactive peptide. The presence of laminin-like immunoreactivity in the chromaffin granules was confirmed by immunocytochemical study. Laminin B chain-like immunoreactivity was also identified in the rat adrenal chromaffin granule fraction. Laminin A chain was hardly detected, as in the case of bovine adrenals. Structure of laminin in chromaffin granules in bovine and rat adrenals may be different from that of mouse Englebrethe-Holm-Swarm sarcoma laminin. The functional significance of laminin B chain-like protein in the granules is unknown at present.View full abstractDownload PDF (5254K)
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