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Volume 111 (1992)
- Issue 6 Pages 693-
- Issue 5 Pages 553-
- Issue 4 Pages 425-
- Issue 3 Pages 283-
- Issue 2 Pages 141-
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Volume 111, Issue 5
Displaying 1-26 of 26 articles from this issue
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Jun Hirabayashi, Motohide Satoh, Yuji Ohyama, Ken-ichi Kasai1992 Volume 111 Issue 5 Pages 553-555
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo carbohydrate-binding proteins (subunit molecular masses, 32 and 16 kDa, respectively) were isolated for the first time from a nematode, Caenorhabditis elegans. They were specifically extracted with lactose and adsorbed on asialofetuin-Sepharose in the absence of a metal ion. Although these two proteins were co-eluted from a gel filtration column at a position corresponding to an apparent molecular size of 30 kDa under non-denaturing conditions, they could be separated by reversed-phase chromatography. The 32 kDa protein, the main component, was further characterized. Together with its solubility, saccharide specificity and metal independence, some other structural properties, including its amino acid composition, UV spectrum, and partial amino acid sequence, strongly suggested that the 32 kDa protein is a member of a class of soluble β-galactoside-binding lectins which had previously been only found in vertebrates.View full abstractDownload PDF (312K) -
Kazuaki Hiki, Ryuichi Hattori, Chuichi Kawai, Yoshiki Yui1992 Volume 111 Issue 5 Pages 556-558
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSNitric oxide synthase [EC 1. 14. 23] from the particulate fraction of rat cerebella was purified and characterized. The homogenate of rat cerebella was centrifuged to obtain a pellet, which was washed and incubated with Triton X-100 containing buffer. The enzyme activity appeared in the 100, 000×g supernatant after incubation with the detergent. The solubilized enzyme was then purified by sequential affinity chromatography using adenosine 2', 5'-diphosphate agarose and calmodulin Sepharose 4B, which gave a product that migrated as a single protein band on SDS/PAGE with a molecular mass of about 150 kDa. The purified enzyme exhibited an absolute requirement for FAD, in addition to NADPH and Ca2+/calmodulin. Thus, there is an insoluble nitric oxide synthase in rat cerebellum that has similar characteristics to the soluble type.View full abstractDownload PDF (325K) -
Yutaka Yoshida, Ji-Qun Cai, Shoichi Imai1992 Volume 111 Issue 5 Pages 559-562
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA plasma membrane Ca2+-pump ATPase preparation purified from porcine aorta was incubated with cGMP-dependent protein kinase (G-kinase) under the conditions under which dose-dependent stimulation of the enzyme by G-kinase was observed. Several proteins were phosphorylated, but two isoforms of plasma membrane Ca2+-pump ATPase with molecular masses of 135- and 145-kDa were not phosphorylated. The protein that was phosphorylated by G-kinase and identified in our previous study as the 135-kDa isoform of Ca2+-pump ATPase, on the basis of its almost identical mobility on SDS-PAGE, was found to be another protein with a molecular mass of 138 kDa. Fractionation of the enzyme preparation after incubation with G-kinase by a newly developed calmodulin affinity chromatographic method resulted in the separation of all the G-kinase substrates from the two isoforms of plasma membrane Ca2+-pump ATPase. These results suggest that the direct phosphorylation of the Ca2+-pump ATPase does not occur in association with the stimulation of the plasma membrane Ca2+-pump ATPase by G-kinase.View full abstractDownload PDF (898K) -
Kazuyoshi Uehara, Shyuichiro Matsubara, Kenji Kadomatsu, Jun-ichiro Ts ...1992 Volume 111 Issue 5 Pages 563-567
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMidkine (MK) is a product of a retinoic acid-responsive gene and a new heparin-binding growth/differentiation factor. The coding sequence of human MK was located on 1.5 kb DNA segment. The structure of the cloned human MK gene was determined and compared with that of the mouse gene. The coding sequence was divided into 4 exons, and each exon and exon-intron boundary was highly homologous to those of the mouse. Furthermore, 170 bases in the upstream region of the putative transcription initiation sites and 3 blocks of 200-350 bases in regions further upstream were highly conserved. These sequences are likely to be involved in developmentally regulated expression of MK.View full abstractDownload PDF (1191K) -
Jun-ichi Kawano, Tsutomu Oinuma, Tatsuo Nakayama, Tatsuo Suganuma1992 Volume 111 Issue 5 Pages 568-572
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSβ1→4 Galactosyltransferase was purified from rat liver microsomes. Catalytic properties of the enzyme resembled those of previously purified soluble and membrane-bound β1→4 galactosyltransferases. The enzyme purified in the present study showed a major band around a molecular weight of 53, 000 on SDS-PAGE. The NH2-terminal sequence of the enzyme was determined up to the 20th residue. The sequence was identical to the amino acid sequence from Ala-13 to Lys-32 deduced from mouse β1→4 galactosyltransferase cDNA. These results suggest that most of the mature enzyme in rat liver microsomes is produced by removal of the N2-terminal 12 amino acids from a precursor polypeptide.View full abstractDownload PDF (867K) -
Takahisa Masaki, Misako Matsumoto, Isao Nakanishi, Reiko Yasuda, Tsuka ...1992 Volume 111 Issue 5 Pages 573-578
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSProteolytic inactivation of C4b is a crucial step for regulation of the classical complement pathway. A plasma protease factor I and membrane cofactors, C3b/C4b receptor (CR1) and membrane cofactor protein (MCP), participate in the regulation of cell-bound C4b although the physiological potency of these cofactors remains unknown. We have examined the optimal conditions of the factor I-mediated C4b regulatory system using purified cofactors. CR1 being a cofactor at a cofactor/C4b ratio <0.1 (w/w), fluid phase C4b, and methylaminetreated C4 (C4ma) were degraded by factor I into C4bi: minimal C4d was generated in the fluid phase. Liposome-bound C4b (LAC4b), on the other hand, was degraded into C4c and C4d. CR1 showed two optimal pHs (6.0 and 7.5) for fluid phase C4b, but one (6.0) for LAC4b, and in both cases low conductivity conditions enhanced the C4bi generation. CR1 cofactor activity was barely influenced by the NP-40 concentration. On the other hand, MCP degraded C4b and C4ma, as a factor I-cofactor, more efficiently into C4c and C4d. Though MCP cofactor activity, like that of CR1, was enhanced under low conductivity conditions, it has only one optimal pH, 6.0, in both fluid and solid phases. Furthermore, as in the case of C3b cleavage, a sufficient NP-40 concentration to solubilize membrane was needed for MCP to express full cofactor activity for C4b, in contrast to CRl. MCP was less potent for C4b inactivation than for C3b inactivation, while CR1 acted as a slightly more effective cofactor for C4b cleavage than for C3b cleavage. C4 isotypic variants (C4A and C4B) did not modulate MCP/CR1 cofactor activity except that CR1 and factor I more efficiently cleaved C4ma of C4B than that of C4A in the fluid phase. Covalent C4b dimer, an element of a C5 convertase of the C3-independent classical pathway, was degraded similarly by factor I and either of CR1 or MCP. The results imply that although MCP and CR1 serve as C4b regulatory factor I-cofactors, their C4b inhibitory profiles are different from each other, and in fluid and solid phases their activities are not sufficient to block the classical pathway.View full abstractDownload PDF (2217K) -
Joseph Vamecq, Nathalie Mestdagh, Jean-Pierre Henichart, Jacques Poupa ...1992 Volume 111 Issue 5 Pages 579-583
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe enzymic synthesis, transfer, and utilization of glycolyl-CoA (i.e. 2-hydroxyacetyl-CoA) have been studied in rat and mouse livers. On the one hand, these tissues contain the enzyme activities allowing the synthesis of glycolyl-CoA from fatty acids (palmitate ω-hydroxylase, ω-hydroxypalmitoyl-CoA synthetase, and mitochondrial β-oxidation of ω-hydroxypalmitoyl-CoA) and 3-hydroxypyruvic acid (oxidation by intact mitochondria). On the other hand, three types of glycolyltransferase activities can be demonstrated in rodent livers, depending on either carnitine, glucosamine, or glucosamine-6-phosphate. The subcellular distributions of these glycolyltransferase activities are similar to those of the corresponding acetyltransferase counterparts. Concerning carnitine glycolyltransferase, the activity is widely distributed in the subcellular fractions, pointing out its occurrence in most cell compartments. By contrast, the glucosamine and glucosamine-6-phosphate glycolyltransferase activities were located preferentially in the microsomal fraction. The condensation between glycolyl-CoA and glucosamine (or glucosamine-6-phosphate) raises the interesting question of the nature and the role of the resulting glycolylglucosamine molecule, especially in an alternative N-glycolylneuraminic acid synthesis pathway.View full abstractDownload PDF (550K) -
Hiroshi Takagi, Shino Arafuka, Masayori Inouye, Makari Yamasaki1992 Volume 111 Issue 5 Pages 584-588
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSubtilisin from a wide variety of Bacillus species has been extensively investigated as a promising target for protein engineering. In this study, we analyzed the substrate specificity of B. subtilis subtilisin E based on the structi'lre of a new alkaline elastase produced by the alkalophilic Bacillus strain Ya-B, which has very high elastolytic activity. Despite the high homology of the primary sequences of both enzymes (54% identical), alkaline elastase was found to lack four consecutive amino acids which, in subtilisin, have been shown by X-ray analysis to lie close to the P1 binding cleft. To examine the influence of such a deletion in subtilisin on its substrate specificity, we constructed several mutants missing four amino acids by site-directed mutagenesis. When assayed with synthetic peptides, elastin and casein as substrates, a mutant lacking Ser161-Thr162-Ser163-Thr164 showed considerably lower specific activity toward the substrates for subtilisin, and its substrate specificity approached that of alkaline elastase. The results indicate that the deletion in subtilisin E influences the catalytic efficiency as well as the P1 specificity, and that this region is, in part, responsible for the difference in specificity between the two enzymes.View full abstractDownload PDF (668K) -
Hideo Akiyama, Shozo Kanai, Hitoshi Ozawa1992 Volume 111 Issue 5 Pages 589-593
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn improved strategy for fluorescence-labeled dideoxy chain termination sequencing involving restriction enzyme-digested DNA fragments as primers, which are prepared from the DNA to be sequenced, is described. By using modified nucleoside triphosphates for strand protection in chain termination reactions, newly synthesized chains were detached from a primer at the regenerated recognition site by means of suitable restriction enzyme digestion. The digests could be analyzed with commercial automated DNA sequencers. Thus, by using restriction DNA fragments (double-stranded) as primers, sequence information was obtained from both “minus” and “plus” single-stranded DNA templates without subcloning. Nor is the synthesis of oligonucleotide primers needed. This method, named “Multi-Priming Sequencing, ” was proven to be time-saving, economical, and effective compared to conventional methods.View full abstractDownload PDF (1434K) -
Hiroyuki Matsushita, Eiji Itagaki1992 Volume 111 Issue 5 Pages 594-599
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe variation with pH of kinetic parameters was examined for 3-ketosteroid-Δ1-dehydrogenase from Nocardia corallina. The Vmax/Km profile for 4-androstenedione indicates that activity is lost upon protonation of a cationic acid-type group with a pK value of 7.7. The enzyme was inactivated by diethylpyrocarbonate at pH 7.4 and the inactivation was substantially prevented by androstadienedione. Analyses of reactivation with neutral hydroxylamine, pH variation, and spectral changes of the inactivated enzyme revealed that the inactivation arises from modification of a histidine residue. Studies with [14C] diethylpyrocarbonate provided support for the idea that the 1-2 essential histidine residues are essential for the catalytic activity of the enzyme. Dye-sensitized photooxida-tion led to 50% inactivation of the enzyme with the decomposition of two histidine residues. This inactivation was also prevented by androstadienedione. Dancyl chloride caused a loss of the enzyme activity. Modifiers of glutamic acid, aspartic acid, cysteine, and lysine did not affect the enzyme activity. Butanedione and phenylglyoxal in the presence of borate rapidly inactivated the enzyme, indicating that arginine residues also have a crucial function in the active site. The data described support the previously proposed mechanism of β-oxidation of 3-ketosteroid.View full abstractDownload PDF (690K) -
Eiichi Hoshino, Takahisa Kanda, Yasushi Sasaki, Kazutosi Nisizawa1992 Volume 111 Issue 5 Pages 600-605
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe adsorption mode of two highly purified cellulases, exo- and endo-type cellulases, from Irpex lacteus (Polyporus tulipiferae) was investigated by using pure cellulosic materials with different crystallinity as substrates. Adsorption of the two enzymes on the substrates was found to fit the Langmuir-type adsorption isotherm. Maximum amount of adsorbed enzyme obtained from the Langmuir plots showed an inverse correlation to the crystallinity of the substrate with both enzymes, and this value of endo-type cellulase was less dependent on the degree of crystallinity of substrates than that of exo-type cellulase, whose isotherms reached saturation in the range of low enzyme concentrations. The two enzymes showed relatively high affinities for all the substrates and their affinities increased with increasing crystallinity, but this tendency was less marked with endo-type cellulase than with exo-type one. In addition, large negative values of free energy change were observed on the adsorption of both enzymes, and the values became more negative with increasing crystallinity. Consequently, both cellulases showed high adsorption on crystalline cellulose and the adsorption process became smoother with increasing crystallinity. The adsorption of the two types of cellulases was endothermic with an increase in entropy, especially for amorphous cellulose, suggesting the occurrence of water release from the substrates during enzyme adsorption. In addition, the changes in thermodynamic parameters (ΔH, ΔS, and ΔG) in adsorption of exo-type cellulase were larger than in that of endo-type enzyme.View full abstractDownload PDF (572K) -
Yoshihide Ikeuchi, Kazunori Iwamura, Tomoaki Machi, Takaaki Kakimoto, ...1992 Volume 111 Issue 5 Pages 606-613
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effects of the neutral salt concentration, pH, and coexistence of myosin on the denaturation of F-actin without ATP at low temperature were studied using the DNase I inhibition assay. The percent denaturation of F-actin gradually increased with a decrease in pH from 8.0 to 5.2, on incubation for 2 weeks in the presence of 50mM KCl at 0°C. This change was much faster in 0.5M KCl and more than 75% of the F-actin became denatured on incubation for 1 week at pH 5.2. The buffer composition was found to exert a strong influence on the denaturation of F-actin. That is, there was a tendency for the denaturation of F-actin at pH6.0 to be faster in MES[2-(N-morpholino)ethanesulfonic acid]-NaOH buffer than in sodium phosphate buffer, the critical concentrations of actin in 0.5M KCl being 0.31mg/ml for MES-NaOH buffer and 0.15mg/ml for sodium phosphate buffer. A sigmoidal relationship was found between the percent denaturation of F-actin and the KCl concentration added, the greatest change occurring at KCl concentrations between 0.25 and 0.75M. The time courses of the denaturation of F-actin showed that the percent denaturation rose at first and that in time the rate of the increase decreased. In the case of pH 8.0 and 0.5M KCl, it took about 1 week for the denaturation rate to begin to drop. The pH of 6.0 further promoted the instability of F-actin exposed to high KCl concentrations. When a constant concentration of F-actin (2mg/ml) was mixed with various concentrations of heavy meromyosin (HMM), a small amount of HMM markedly accelerated the denaturation of F-actin. The maximum denaturation was observed at an HMM/actin weight ratio of 1:100, at 0.5M KCl and pH 8.0. A decrease in the KCl concentration to 50mM made it shift toward a higher HMM/actin weight ratio. A large amount of HMM effectively protected F-actin against the denaturation by overcoming the destabilizing effect of a high salt concentration or low pH. A scheme for interpretation of the destabilizing effect of a small amount of HMM was proposed.View full abstractDownload PDF (846K) -
Takao Taki, Masanori Kuroyanagi, Hisashi Yoshioka, Shizuo Handa1992 Volume 111 Issue 5 Pages 614-619
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSpectra of 125MHz 13C-nuclear magnetic resonance (NMR) of glycosphingolipids, GicCer, GalCer, sulfatide, LacCer, and nLc4Cer have been studied, and the following results were obtained. (i) Signals of ring carbons of each sugar component are distributed in a wide field (50-110 ppm) and clearly separated. (ii) Chemical shifts of anomeric carbon (C1) and methylene carbon (C6) of sugars are far from those of other methine carbons of sugars and characteristic of sugar components, which makes it possible to identify each sugar component and its molar raito. (iii) The downfield shifts (about 6-9 ppm) of α-carbon signals involved in the glycosidic linkages and upfield shifts (about 1.5-2ppm) of the neighboring β-carbons, which are known as glycosylation shifts, could be observed. (iv) Characteristic shifts of aglycon signals caused by the presence of an OH group at the α-position of fatty acid were assigned. These observations are useful for the characterization of glycosphingolipid structures.View full abstractDownload PDF (457K) -
Hiroto Kawashima, Takeomi Murata, Kazuo Yamamoto, Akihiro Tateishi, Ta ...1992 Volume 111 Issue 5 Pages 620-622
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA simple method for the release of oligosaccharides from glycoproteins separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) has been developed. Asialo-α1-acid glycoprotein, which was tritiated at the nonreducing terminal D-galactopyranosyl residue by reduction with sodium borotritide after incubation with D-galactose oxidase, was used as a model compound. After electrophoretic separation of the glycoprotein, oligosaccharides were released by the use of a gas-phase hydrazinolysis apparatus. In the first method, the gel was stained with Coomassie Blue and the glycoprotein together with the gel was directly subjected to gas-phase hydrazinolysis after removal of water in a P2O5 desiccator. The recovery of released oligosaccharides was 25.9±2.4%, based on the amount of the glycoprotein loaded on the gel within the range of 3.5-28.5μg. In the second method, the glycoprotein was electroblotted onto an Immobilon transfer membrane and was visualized by staining with Coomassie Blue. A small piece of the membrane with the corresponding band was cut out, dried in a desiccator and subjected to gas-phase hydrazinolysis. In this case, the recovery of released oligosaccharides was 15.2±1.0%. These procedures, particularly the first one, should be widely applicable for the isolation of oligosaccharides from glycoproteins separated by SDS-PAGE.View full abstractDownload PDF (335K) -
Hideo Katoh, Shinji Satomura, Shuji Matsuura1992 Volume 111 Issue 5 Pages 623-626
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA convenient method for the analysis of the binding properties of lectin with fluorogenic sugar chains is described. A lectin (concanavalin A or Datura stramonium agglutinin) was mixed with pyridylaminated sugar chains in buffer and the free chains obtained were isolated by membrane ultrafiltration. The amount of free sugar chains in the filtrate was measured by high-pressure liquid chromatography. The binding constants with the sugar chains, reaction kinetics, and other properties of these lectins were easily investigated. The method is simple and could be used to study the characteristics of any lectin in native form.View full abstractDownload PDF (385K) -
Shitsuw Shikata, Kohei Shimada, Hiroshi Kataoka, Sueharu Horinouchi, T ...1992 Volume 111 Issue 5 Pages 627-632
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe Serratia marcescens serine protease gene encoding a 1, 045-amino-acid precursor protein of 112 kDa directs excretion of the mature protease of ca. 58 kDa through the outer membrane of Escherichia coli. A typical signal peptide of 27 amino acids and a large COOH-terminal domain of the precursor are both functionally essential for the excretion of the mature protease into the medium. Sequence analysis of the fragment peptides of the mature protease as well as site-directed mutagenesis indicated that the COOH-terminus of the mature enzyme was Asp645. By using the polyclonal antibody against the 112-kDa precursor protein, not only the intact preursor but also two proteins, C-1(40 kDa) and C-2 (38 kDa), corresponding to the processed COOH-terminal domains were detected in the insoluble fraction of E. coli cells. Further fractionation by sucrose density gradient centrifugation showed that C-1 and C-2 were localized in the outer membrane. The NH2-terminal residues of C-1 and C-2 were determined to be Ala702 and Phe717, respectively. All these data suggest that the precursor is cleaved at three positions, between Asp645-Ser646, Glu701-Ala702, and Gly716-Phe717, probably by the self-processing activity in the normal excretion pathway through the outer membrane.View full abstractDownload PDF (1481K) -
Brigitte Frorath, Charles C. Abney, Mirco Scanarini, Heike Berthold, N ...1992 Volume 111 Issue 5 Pages 633-637
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAutoantibodies directed against the thyroid peroxidase (TPO), the thyroid microsomal antigen, are widely used to diagnose human autoimmune thyroid disease. A cloned 3.088 kb cDNA coding for the entire mature human TPO was isolated from a cDNA library derived from a pathological thyroid gland of a Graves' disease patient and used further to generate a so-called TPO epitope cDNA library in order to map linear autoantigenic epitopes involving a recombinant molecular biology approach. The TPO epitope cDNA library consisting of randomly fragmented cDNA sequences inserted in the expression vector pGEX-2T was expressed in Escherichia coli and screened with characterized anti-TPO autoantisera from Hashimoto's disease patients. All the sera were positively tested with a purified thyroid microsomal antigen fraction (TMA/TPO). Only about 1% of examined autoantisera were able to recognize bacterial expressed recombinant TPO representing sequential antigenic determinants. A corresponding autoantigenic epitope with 61 amino acids in length was located at the C-terminus of human TPO.View full abstractDownload PDF (1773K) -
Masahiro Iwakura, Teruo Tanaka1992 Volume 111 Issue 5 Pages 638-642
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe Bacillus subtilis dihydrofolate reductase (DHFR) gene was expressed in Escherichia coli. The gene product was purified to homogeneity by Butyl-Toyopearl, Toyopearl HW55, and DEAE-Toyopearl column chromatographies, and its molecular properties were compared to those of E. coli DHFR. The specific enzyme activity of the B. subtilis DHFR was 240units/mg under the standard assay conditions, being about four times higher than that of the E. coli DHFR. Km for coenzyme NADPH was 20.7μM, a value about three times larger than that of E. coli, whereas Km (1.5μM) for the substrate, dihydrofolate, was similar to that of E. coli DHFR. This seems to reflect the low homology of the amino acid sequence in residues 61-88 of the two DHFRs where one of the NADPH binding sites is located [Bystrof, C. & Kraut, J. (1991) Biochemistry 30, 2227-2239]. Similar to the E. coli DHFR [Iwakura, M. et al. (1992) J. Biochem. 111, 37-45], the extension of amino acid sequences at the C-terminal end of the B. subtilis DHFR could be attained without loss of the enzyme function or decrease of the protein yield. Thus, the DHFR is useful as a carrier protein for expressing small polypeptides, such as leucine enkephalin, bradykinin, and somatostatin.View full abstractDownload PDF (1472K) -
Jörg Peters, Saori Takahashi, Masazumi Tada, Yoshihiro Miyake1992 Volume 111 Issue 5 Pages 643-648
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSmGK-6-derived true tissue kallikrein was shown to be synthesized in mouse pituitary AtT-20 cells. This cell line, which is capable of processing other prohormones, only partially processed the proform of kallikrein to its active form, secreting it predominantly as the proform. The secretion of the active form was stimulated in response to a secretagogue, 8-bromo-cyclic AMP. These results imply that not only cellular elements capable of directing the processing of the proform to the active form and the intracellular transport of the kallikrein, but also a pathway that regulates the release of the active form may be present in the AtT-20 cells, thus the availability of this cell line for investigation of biosynthetic and secretory processes for tissue kallikrein in vivo being suggested.View full abstractDownload PDF (999K) -
Javier Naval, Miguel Calvo, Jorge Laborda, Pierre Dubouch, Monique Fra ...1992 Volume 111 Issue 5 Pages 649-654
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSα-Fetoprotein and albumin, two members of a multigene family, reversibly bind fatty acids with high affinity. The origin of α-fetoprotein (AFP) and albumin present in fetal tissues other than the liver and yolk sac is a subject of controversy. In this work, we have searched for the presence of the albumin and AFP mRNA molecules in different fetal organs of the baboon (Papio cinocephalus), using a highly sensitive gel-blot hybridization assay with human albumin and AFP eDNA probes. Large amounts of albumin and AFP mRNA molecules were found in the fetal liver; significant quantities were also present in the gastrointestinal tract and in the kidney. No detectable levels were found in the other tissues examined (brain, skin, spleen, pancreas, muscle, heart, thymus, placenta, and amnion). After injection of radiolabeled AFP into pregnant baboons, all fetal tissues took up the protein. White adipose tissue, kidney, intestine, lung, liver, and cerebral cortex showed a great uptake of exogenous AFP. [14C]Docosahexaenoic acid (22:6, n-3), injected at the same time, was actively transferred from the maternal compartment across the placenta and incorporated into cellular lipids by all fetal tissues and particularly by liver (around 70% of total incorporation). The levels of [14C] docosahexaenoic acid per gram of tissue increased in the order: maternal blood<placenta<fetal liver, indicating a selective accumulation of this fatty acid by the fetus. These results indicate that intracellular AFP in non-hepatic tissues of the developing baboon is, for the most part, of plasma origin.View full abstractDownload PDF (2448K) -
Min-Qian Tang, Seiichi Ando, Shoji Yamada, Seiichi Hayashi1992 Volume 111 Issue 5 Pages 655-661
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSEel liver glutamate dehydrogenase (GDH) [EC 1. 4. 1. 3] was eightfold activated by trypsin and the molecular weight of the subunit of the native GDH decreased from 54, 000 to 50, 000. The C-terminal amino acid of both subunits was Thr. One peptide was released after proteolysis of the native GDH by trypsin and purified by anhydrotrypsin agarose and reversed-phase HPLC. The isolated peptide consisted of 39 amino acids and its amino acid sequence was as follows: H2NS-E-A-V-E-K-E-D-D-P-N-F-F-K-M-V-E-G-F-F-D-K-G-A-A-I-V-E-N-K-L-V-E-E-D-L-K-T-R-COOH. The peptide contained the N-terminal of the native GDH and its molecular weight was calculated to be 4, 413. We concluded that the trypsin-catalyzed activation was caused by release of this peptide from the native GDH. p-Chloromercuribenzoic acid inhibited the activity of the trypsin-treated GDH, but stimulated that of the native GDH. The response of trypsin-treated GDH to ADP and GTP was decreased compared with that of the native GDH.View full abstractDownload PDF (2535K) -
Thomas Sickmann, Bianka Weske, Roger D. Dennis, Carsten Mohr, Herbert ...1992 Volume 111 Issue 5 Pages 662-669
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAs a first approach to testing the working hypothesis that glycosphingolipids are functionally involved in the ontogeny of insects, their chemical distribution in larval organs was determined and any stadium-correlated differences documented. Selected organs, i.e., the fatbody, striated muscle, intestinal tract, salivary glands, imaginal discs, and central nervous system, were dissected from seven-day-old larvae of the blowfly, Calliphora vicina, and their glycolipids isolated. Two-dimensional, high-performance thin-layer chromatography was used to separate the neutral and acidic glycolipids of each organ. Significantly different total glycolipid component-patterns were obtained for the individual organs, whereby, except for a number of additional uncharacterized components in the intestinal tract, the neutral glycolipids of all organs were found to be qualitatively similar. However, major quantitative differences between the selected organs were found in their total glycolipid-carbohydrate contents, as well as the respective quantitative neutral glycosphingolipid-component distributions. The acidic glycolipids showed pronounced qualitative as well as quantitative organ-dependent variations. Whereas the highest proportion of uncharged glycolipids was characteristic of the fatbody, a high proportion of zwitterionic glycolipid-components was observed to be typical of the central nervous system and imaginal discs, i.e., of organs persisting during larval life and throughout metamorphosis. Imaginal disc glycolipids were distinguished by their high content of acidic glycolipids, a putative reflection of the functional role of these glycoconjugates in regulated cell reorganization during metamorphosis.View full abstractDownload PDF (4315K) -
Kei-ichi Enjyoji, Hisao Kato1992 Volume 111 Issue 5 Pages 670-675
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESST-Kininogen is one of the acute phase proteins, and is a precursor of T-kinin and a cysteine protease inhibitor. Two homologous T-kininogens (TI- and TII-kininogens) were isolated from microsomal fraction of inflamed rat liver, by chromatographies on columns of DEAE-Sepharose CL-6B and DEAE-5PW and by affinity chromatography on a column of anti T-kininogen monoclonal antibody. The amino terminal amino acid sequences of the two microsomal pyridylethylated T-kininogens after pyroglutamyl aminopeptidase treatment were identical with those of TI- and TII-kininogens from inflamed rat plasma. Microsomal T-kininogens moved faster on SDS-PAGE after treatment with endoglycosidase H. The amounts of microsomal TI- and TII-kininogens in inflamed and non-inflamed rat liver were quantitated by immunoblotting of homogenates of liver microsomes using anti T-kininogen rabbit antiserum. The amounts of microsomal T-kininogens were increased in inflamed rat liver, but the ratio of the amounts of TI-kininogen to TII-kininogen was not different in the inflamed and non-inflamed rat liver. On the other hand, TII-kininogen was not significantly detected in non-inflamed rat plasma. These results indicate that the secretion of one of the T-kininogens, TII-kininogen, into plasma may be prevented by some unknown mechanism.View full abstractDownload PDF (3393K) -
Akira Yamada, Keiichi Takahashi1992 Volume 111 Issue 5 Pages 676-680
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSUnder in vitro movement assay conditions, actin filaments move about 10 times faster toward, than away from, the center of large bipolar thick filaments of molluscan smooth muscle. Using thick filaments isolated from the anterior byssus retractor muscle of Mytilus edulis, the two speed modes of movement were studied in detail. Some thick filaments crossed over each other on the surface of the assay chamber, allowing actin filaments that moved into the crossover region to transfer to other thick filaments. When an actin filament that had been moving in the low speed mode crossed over to another thick filament and the speed changed to fast, the entire actin filament started to move in the high speed mode at the moment of transfer of its leading end, leaving the trailing part still in contact with the original thick filament. This indicates that myosin cross-bridges interacting in the slow mode do not impose a significant load on the cross-bridges interacting in the fast mode. Assuming the theoretical model of Tawada and Sekimoto [Biophys. J. 59, 343-356 (1991)], we suggest that the magnitude of force developed, as well as the speed of unloaded movement, differs greatly, depending on the orientation of the myosin cross-bridges.View full abstractDownload PDF (1452K) -
Kei-ichi Enjyoji, Mitsuru Emi, Tsunehiro Mukai, Hisao Kato1992 Volume 111 Issue 5 Pages 681-687
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTissue factor pathway inhibitor (TFPI) is a factor Xa-dependent inhibitor for the factor VIIa-tissue factor complex. We isolated cDNA for rat TFPI by screening a λgt10 rat liver cDNA library. We determined the 1, 228 bp nucleotide sequence, comprising a 88 bp 5' non-coding region, a 906 bp open reading frame, and a 234 bp 3' non-coding region, which encodes a protein of 302 amino acid residues. On Northern blot analysis of rat TFPI mRNA, rat TFPI mRNA was detected as two forms with different molecular sizes, 4.0 and 1.4 kb, which were expressed abundantly in heart, lung, kidney, and aortic endothelial cells. The homology of the amino acid sequence of rat TFPI with those of human and rabbit TFPI was found to be 60.7 and 57.4%, respectively. The lengths of the three tandem Kunitz-type inhibitor domains were strictly conserved not only among TFPI from the three species, but also among other proteins containing Kunitz-type inhibitor domains. The homology of the Kunitz-type domains in TFPI among the three species was 57, 86, and 69% in the 1st, 2nd, and 3rd domains, respectively. There was no significant difference in hydropathy profiles of TFPI from man, rabbit, and rat.View full abstractDownload PDF (3279K) -
Tomoyuki Terada, Takuya Oshida, Masuhiro Nishimura, Hideki Maeda, Taiz ...1992 Volume 111 Issue 5 Pages 688-692
Published: 1992
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThioltransferase, an enzyme which catalyzes the thiol/disulfide exchange reaction in the presence of GSH, was purified to homogeneity on 15% SDS-PAGE from human (36, 000-fold purification) and bovine (23, 000-fold) erythrocyte hemolysates. These enzymes had similar properties in their monomeric structures (Mr=11, 000) and broad specificities for substrates ranging from low-molecular disulfides (S-sulfocysteine, cystamine, and cystine) to protein disulfides (trypsin and insulin). They were highly sensitive to SH-reagents (monoiodoacetic acid and mercuric chloride), but were protected from inactivation by the presence of disulfides (GSSG, cystamine, and cystine). Phosphofructokinase and pyruvate kinase that had been inactivated by disulfides were reactivated effectively by the addition of thioltransferase with GSH. In addition, disulfides in membrane proteins of human erythrocytes that have been oxidatively damaged by diamide treatment were reduced to the SH-free form more effectively by incubation with thioltransferase.View full abstractDownload PDF (1544K)
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