巻号一覧
107 巻, 5 号
選択された号の論文の24件中1～24を表示しています
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Hiromi Kirino, Reiko Kuwahara, Nobuko Hamasaki, Tairo Oshima1990 年 107 巻 5 号 p. 661-665
発行日: 1990年
公開日: 2008/11/18
ジャーナル フリーThe effect of unusual polyamines, such as thermine, caldopentamine, caldohexamine, tris (3-aminopropyl) amine, or tetrakis (3-aminopropyl) ammonium, on the activities of various restriction endonucleases was investigated by using an Escherichia coli plasmid as a substrate, which contains a high GC content fragment from an extreme thermophile. Restriction enzymes used were SmaI, BanII, NoeI, RsaI, and TaqI. Most of the polyamines tested were inhibitory to the enzyme activities. The larger and more branched a polyamine was, the more the activities of nucleases were inhibited. The inhibition was positively correlated with the polyamine concentration. The sites protected by a polyamine were identical to those protected by other polyamines, and also identical to those which were less sensitive to the restriction enzyme in the absence of polyamines. No sequence specificity was seen among these sites.抄録全体を表示PDF形式でダウンロード (2627K) -
Yoshiro Kobayashi, Joost J. Oppenheim, Kouji Matsushima1990 年 107 巻 5 号 p. 666-670
発行日: 1990年
公開日: 2008/11/18
ジャーナル フリーThe effect of phosphorylation of pre interleukin 1α (IL 1α) on its association with various phospholipids was investigated. We prepared genetically engineered truncated human pre IL 1α (residues 64 to 271) and phosphorylated this pre IL 1α in vitro by using the catalytic subunit of cAMP-dependent protein kinase. Phosphorylated truncated pre IL 1α selectively binds to acidic phospholipids including phosphatidic acid, phosphatidylserine, and phosphatidylinositol, but not to other phospholipids (phosphatidylcholine and phosphatidylethanolamine). This binding required divalent cations: Ca2+ or Mn2+, but not Mg2+. In order to obtain half-maximal binding of pre IL 1α to phosphatidic acid or phosphatidylserine, Ca2+ between 5 and 100μM was required. Unphosphorylated pre IL 1α did not bind to phosphatidylserine, indicating that phosphorylation is required for this binding. Phosphorylated pre IL 1α did not bind to intact peripheral blood mononuclear cells irrespective of lipopolysaccharide stimulation, but did bind to membrane vesicles prepared from these cells in the presence of calcium. Furthermore, phosphorylated pre IL 1α bound only to inside-out ghosts, but not right-side-out ghosts, prepared from human red blood cells. Taken together, these data suggest that phosphorylated pre IL 1α binds to the inner surface of plasma membrane in a Ca2+- and phospholipid-dependent manner.抄録全体を表示PDF形式でダウンロード (559K) -
Kingo Takiguchi, Hiroshi Hayashi, Eiji Kurimoto, Sugie Higashi-Fujime1990 年 107 巻 5 号 p. 671-679
発行日: 1990年
公開日: 2008/11/18
ジャーナル フリーWe have compared actin-activated myosin ATPase activity, myosin binding to actin, and the velocity of myosin-induced actin sliding in order to understand the mechanism of myosin motility. In our in vitro assay, F-actin slides at a constant velocity, regardless of length. The F-actin could slide over myosin heads at KC1 concentrations below a critical value (60mM with myosin and HMM, 100mM with S-1), and the sliding velocities were quite similar below the critical KC1 concentration. However, at KC1 concentrations close to the critical value, the sliding F-actin is attached to only one or a few particular points on the surface, each of which perhaps consists of a single head of myosin. The KATPase values for actin-activated ATPase were_??_30μM for S-1 and _??_200μM with HMM below the critical KC1 concentration, and _??_5, 000μM above the critical KC1 concentration. This increase in KATpase is due to a drastic reduction in the binding affinity of myosin heads to F-actin, as determined by a proteolytic digestion method and direct observation by fluorescence microscopy. We also show that the Vmax of actin-activated myosin ATPase activity decreases steadily with increasing KC1 concentration, even though the velocity of F-actin sliding remains unchanged. This result provides evidence that the ATPase activity is not necessarily linked to motility. We discuss possible models that do not require a tight coupling between myosin ATPase and motility.抄録全体を表示PDF形式でダウンロード (3724K) -
Tamotsu Taketomi, Atsushi Hara, Yukie Kutsukake, Eiko Sugiyama1990 年 107 巻 5 号 p. 680-684
発行日: 1990年
公開日: 2008/11/18
ジャーナル フリーThin layer chromatography of lysosulfatide showed anomalous Rf-values in contrast with such lysosphingolipids as glucopsychosine and galactopsychosine with neutral, acidic, and alkaline developing solvents. This was thought to be due to the presence of oppositely charged sulfate and amino groups in the lysosulfatide. In the negative mode of fast atom bombardment mass spectrometry, the lysosulfatide showed the pseudo molecular ion (M+H)- peak at m/z 540 and sulfate ion peak at m/z 97, whereas in the positive mode, it showed not only the pseudo molecular ion (M + H)+ peak at m/z 542, but also the major peaks of protonated psychosine at m/z 462 and fragment ions of dehydrated sphingosine at m/z 282 and 264. 13C-NMR signals of all carbons of lysosulfatide were determined by using distortionless enhancement by polarization transfer. The difference in chemical shifts of ring carbons of galactose residue between lysosulfatide and galactopsychosine was largest at C-3 (downfield shift), thereby indicating the location of the sulfate group to be at C-3 of galactose. This conclusion is supported by the 1H-NMR spectra of the lysosulfatide and galactopsychosine. Thus, the chemical structure of lysosulfatide was confirmed by fast atom bombardment mass spectrometry and 13C- and 1H-NMR spectroscopy. Furthermore, 13C-NMR signals of C-1 to C-5 of the sphingosine moiety showed significantly different chemical shifts between the lysosulfatide and galactopsychosine. These differences suggested that C-1 to C-5 of sphingosine might be influenced by intramolecular or inter-molecular interaction between the sulfate group of the galactose residue and the amino group of sphingosine. In particular, the order of the absolute value of difference in chemical shift (|Δδ|) being C-1>C-3>C-5 in the sphingosine between lysosulfatide and galactopsy-chosine suggested that the sulfate group might influence the carbons at C-1, -3, and -5, inducing a zigzag conformation of sphingosine in lysosulfatide. Also, the downfield shift of protons at C-1, -3, and -5 of sphingosine in lysosulfatide relative to sulfatide indicated that the removal of fatty acid from sulfatide might similarly induce a zigzag conformation of sphingosine in lysosulfatide. The interaction between the sulfate and amino groups was also suggested by the 1H-NMR spectra of the lysosulfatide and galactopsychosine, because the former showed no amino proton signal.抄録全体を表示PDF形式でダウンロード (1838K) -
Yasunori Kushi, Kiyoshi Ogura, Chiaki Rokukawa, Shizuo Banda1990 年 107 巻 5 号 p. 685-688
発行日: 1990年
公開日: 2008/11/18
ジャーナル フリーBlood group A-active glycosphingolipids from human erythrocyte membranes were identified by the combination of thin-layer chromatography and matrix-assisted secondary ion mass spectrometry (TLC/SIMS). Partially purified lipid extracts were chromato-graphed by TLC and then blood group A-active glycolipids were detected by TLC-immuno-staining assay using anti-A antibody. The parts of the plates which contained the same Rf area as anti-A positive spots were cut out and subjected to direct SIMS analysis. The TLC/ SIMS spectra were quite similar to those obtained by ordinary SIMS. Detailed information, such as molecular weight, molecular species, ceramide portion, and oligosaccharide sequence, was obtained. Also, peracetylated blood group A-active glycolipids were analyzed in a similar manner. After the position of A-active glycolipids on a TLC plate was confirmed by in situ deacetylation and TLC-immunostaining, acetylated A-active glyco-lipids were also analyzed by the TLC/SIMS. Enhanced sensitivity was obtained with peracetylated glycolipids. Consequently, small amounts of unpurified bioactive glyco-lipids can be readily analyzed by TLC/SIMS.抄録全体を表示PDF形式でダウンロード (2645K) -
Teruo Miyazawa, Toshihide Suzuki, Kenshiro Fujimoto, Takashi Kaneda1990 年 107 巻 5 号 p. 689-693
発行日: 1990年
公開日: 2008/11/18
ジャーナル フリーThe formation and accumulation of phospholipid hydroperoxides, especially of phos-phatidylcholine hydroperoxide (PCOOH), a primary peroxidation product of phos-phatidylcholine (PC), in livers of carbon tetrachloride-intoxicated rats was investigated. PCOOH in liver and blood plasma was measured by a chemiluminescence-high-perform-ance liquid chromatography procedure originally developed by Miyazawa et al. (Anal. Lett.20, 915, 1987; Free Radical Biol. Med. 7, 209, 1989). Male Sprague-Dawley rats (120g body wt., 5 weeks of age) were used in the experiments. The amount of PCOOH in the liver of control rats (CCl4-untreated) was 160±20pmol/100mg protein (mean±SD) and the PCOOH/PC molar ratio was 1.1±0.1 ×10-5. In CCl4 (0.1ml/100g body wt.)-dosed rats, the liver PCOOH was 289±65 pmol/100mg protein (PCOOH/PC =2.4±0.4×10-5), 764±271 pmol/100mg protein (PCOOH/PC =5.2±1.7×10-5), and 856±165pmo1/100mg protein (PCOOH/PC=6.0±0.8×10-5) at 6h, 24h, and 1 week after the dose, respectively. Under such conditions, the liver phosphatidylethanolamine hydroperoxide (PEOOH) level was not altered and the concentration was less than 100pmol/ 100mg protein even after the dose. The increments of liver PCOOH were suppressed 56% by the oral supplementation of DL-a-tocopherol (5mg/100g body wt./day) for a week before CC14 administration. A relatively larger amount of PEOOH was found after stimulation of PC hydroperoxidation in the liver of rats with a large amount of CC14 (0.25ml/100g body wt.) rather than with the small amount of CC14 (0.1ml/100g body wt.). On the other hand, the amount of plasma PCOOH was less than 10nM in the control rats and also in the CO, (0.1ml/100g body wt.)-treated rats at 6 and 24h after the dose, but 1 week after the dose, plasma PCOOH showed a large increase to 135±9nM. The results indicate that PC is the most susceptible lipid class to lipid hydroperoxidation in vivo, and dietary α-tocopherol inhibits the PC hydroperoxidation.抄録全体を表示PDF形式でダウンロード (594K) -
Tomiko Asano, Masato Nagahama, Kanefusa Kato1990 年 107 巻 5 号 p. 694-698
発行日: 1990年
公開日: 2008/11/18
ジャーナル フリーThe localization of GTP-binding protein (G-protein) subunits, G0α, G12α and β, in subcel-lular fractions of rat cerebral cortex was determined by means of immunoassays specific for the respective subunits. High concentrations of all three subunits were observed in both crude mitochondrial and microsomal fractions. Muscarinic cholinergic receptors were also densely localized in these fractions. Then the crude mitochondrial and microsomal frac-tions were subfractionated by sucrose density gradient centrifugation. Each fraction obtained was evaluated morphologically by electron microscopy and biochemically by determination of membrane markers. The crude mitochondrial fraction was subfractionat-ed into myelin, synaptic plasma membrane, and mitochondrial fractions. All the G-protein subunits examined and muscarinic receptors were exclusively localized in the synaptic plasma membrane fraction. Among the submicrosomal fractions, the heavy smooth-surfaced microsomal fraction showed the highest concentrations of all G-protein subunits and receptors, while the rough-surfaced microsomal fraction contained low amounts of them. The heavy smooth-surfaced microsomal fraction also contained high specific activity of (Na+-K+)-ATPase, a marker of the plasma membrane. These results indicated that the Goα, G12α and β subunits are mainly localized in the plasma membrane in the brain.抄録全体を表示PDF形式でダウンロード (1299K) -
Qiulin Li, Naoki Yamamoto, Akira Inoue, Seiji Morisawa1990 年 107 巻 5 号 p. 699-702
発行日: 1990年
公開日: 2008/11/18
ジャーナル フリーWe report that long-chain fatty acyl-CoAs are potent inhibitors of the thyroid hormone (T3) receptor isolated from rat liver nuclei. Both saturated and unsaturated fatty acyl-CoAs were similarly potent. Fifty per cent inhibition of T3 binding by the receptor was observed at an oleoyl-CoA concentration as low as 1.3μM, and the affinity of oleoyl-CoA for the receptor (K1) was estimated to be 0.45μM. Fatty acyl-CoAs also promoted dissociation of the hormone bound to the receptor. The action of fatty acyl-CoAs was competitive for the hormone binding site, resulting in a reduction in the receptor's affinity for T3. These observations suggest that fatty acyl-CoAs modulate the binding of the thyroid hormone to its nuclear receptor, in vitro. Whether or not such events occur in vivo remains to be determined.抄録全体を表示PDF形式でダウンロード (378K) -
Yuji Shimada, Akio Sugihara, Taro Iizumi, Yoshio Tominaga1990 年 107 巻 5 号 p. 703-707
発行日: 1990年
公開日: 2008/11/18
ジャーナル フリーGeotrichum candidum produces two extracellular lipases, I and II. A lipase II cDNA clone was isolated from a cDNA library by colony hybridization using the 32P-labeled fragment of lipase I cDNA isolated previously. The nucleotide sequence of lipase II cDNA determined by the dideoxy chain terminating method includes the N- and C-terminal amino acid sequences of lipase II, and the overall amino acid composition deduced from the cDNA coincides with that deduced on amino acid analysis of this protein. The cloned lipase II cDNA codes a protein of 544 amino acids and a part of the signal sequence of 13 amino acids. The peptide chain lengths of lipases I and II are the same, their overall identity being 84%. Furthermore, four Cys residues are completely conserved, which may participate in the formation of disulfide bridges. A homology search indicated that the G. candidum lipases and Candida cylindracea lipase are homologous enzymes and that they are members of the cholinesterase family.抄録全体を表示PDF形式でダウンロード (600K) -
Shuichi Furuta, Hiroaki Hayashi1990 年 107 巻 5 号 p. 708-713
発行日: 1990年
公開日: 2008/11/18
ジャーナル フリーCatalase is a characteristic enzyme of peroxisomes. To study the molecular mechanisms of the biogenesis of peroxisomes and catalase in a less complex system than rat liver cells, we expressed recombinant rat catalase in Escherichia coli, which has no peroxisomes. The concentration of recombinant catalase produced in E. coli transformed with the expression vector carrying the complete coding region of rat catalase cDNA was about 0.1% of the total soluble protein. The recombinant catalase was purified by DEAE-cellulose column chromatography followed by acidic ethanol precipitations. The properties of rat liver catalase and those of the recombinant were similar with respect to molecular mass, catalytic properties, profiles of absorption spectra, and iron contents. The NH2-terminal amino acid sequence of the purified recombinant catalase, as determined by Edman degradation, was in complete agreement with the amino acid sequence predicted from the nucleotide sequence of rat catalase cDNA, except that the first initiator methionine was not detected. The COOH-terminal amino acid sequence was determined by carboxypeptidase A digestion and the sequence, -Ala-Asn-Leu-OH, matched the predicted COOH-terminal amino acid sequence of rat catalase. Recombinant rat catalase gave almost the same multiple protein bands on native polyacrylamide gel isoelectric focusing as observed with authentic rat liver catalase.抄録全体を表示PDF形式でダウンロード (2505K) -
Katsuyuki Mukai, Yoshihiro Fukumori, Tateo Yamanaka1990 年 107 巻 5 号 p. 714-717
発行日: 1990年
公開日: 2008/11/18
ジャーナル フリーThe electron transport system (with cytochrome aa3) coupled to the oxidation of methanol in Methylobacterium extorquens AM1 (former Pseudomonas AM1) was reconstituted with highly purified constituents of the system. A mixture of 2.7μM methanol dehydrogenase, 3.2μM cytochrome cH, and 71nM cytochrome c oxidase (=cytochrome aa3) consumed oxygen at a lower rate in the presence of methanol, while its activity was enhanced 3-fold by the addition of 1.4μM cytochrome cL (74 mol of O2 consumed/mol of heme a of cytochrome c oxidase per min). Further addition of amicyanin to the above mixture did not affect the activity. Although ammonium ion greatly activated the activity of methanol dehydrogenase, the ion had little effect on the oxygen consumption activity of the above mixture. On the basis of the results obtained in the present study, an electron transport system is proposed for the oxidation of methanol in M. extorquens AM1.抄録全体を表示PDF形式でダウンロード (436K) -
Kiyoshi Nagata, Frank J. Gonzalez, Yasushi Yamazoe, Ryuichi Kato1990 年 107 巻 5 号 p. 718-725
発行日: 1990年
公開日: 2008/11/18
ジャーナル フリーFour microsomal cytochrome P-450s (P-450), all of which are active testosterone 6β-hydroxylases, were purified to electrophoretic homogeneity from livers of phenobarbitaltreated (P-4506β-1and P-4506β-3) or dexamethasone-treated adult male rats (P-4506β-2 and P-4506β-4). Purified P-4506β-1, P-4506β-2, P-4506β-3, and P-4506β-4, had apparent molecularweights of 52, 000, 51, 000, 52, 000, and 52, 500 as assessed by sodium dodecyl sulfate - polyacrylamide gel electrophoresis. Absolute spectra revealed that all four P-450 forms had characteristic low-spin spectral patterns in their fully oxidized states. P-4506β-1 and P-4506β-3 displayed spectra of the reduced carbonyl complex with λmax at 447nm. P-4506β-2 and P-4506β-4 showed λmax at 446 and 448nm, respectively. Antibodies raised against each P-450 recognized all forms, although differences were observed with respect to the extents of cross-reactivities on Western blots. Form-specific peptide fragments were also detected among the four P-450 proteins after partial protease-digestion. P-4506β-1 was identical to P-4506β-3 in the first 26 residues of the NH2-terminal amino acid sequence, but differed by 13 residues from P-4506β-2 The amino-terminal sequence of P-4506β-2 was unique and was not identical with those of any rat P-450 previously reported. This P-450 form was detected in the livers of untreated male rats and was induced by treatment with dexamethasone, but not with phenobarbital. All four purified preparations catalyzed testosterone 6β-hydroxylation in a reconstituted system containing cytochrome b5, extracted microsomal lipids and sodium cholate. Rates of hydroxylation were 35.3, 23.7, 13.8, and 9.6nmol/min/ nmol P-450, respectively, with P-4506β-1, P-4506β-2, P-4506β-3, and P-4506β-4, In addition, all antibodies against each of the four P-4506β forms inhibited more than 90% of the formation of 6β-hydroxytestosterone in hepatic microsomes of untreated rats. These results indicate that at least four distinct yet structurally related forms of P-450, including a new form, P-4506β-2 exist in rat livers, and all of them catalyze testosterone 6β-hydroxylation.抄録全体を表示PDF形式でダウンロード (4140K) -
Yasuzo Nishina, Kyosuke Sato, Kiyoshi Shiga1990 年 107 巻 5 号 p. 726-731
発行日: 1990年
公開日: 2008/11/18
ジャーナル フリーTrigonelline, i. e., N-methylnicotinate, which has a zwitterionic structure similar to a substrate D-amino acid, is a useful active site probe for D-amino acid oxidase (DAO). The affinity of trigonelline for DAO in the deprotonated state at the enzyme bound FAD 3-imino group is higher than in the neutral state, contrary to in the case of benzoate, which is a competitive inhibitor and is in a monoanionic form. The time course of the absorbance change was monitored for the binding of DAO with trigonelline by means of a stopped-flow technique. The reaction, on monitoring at 507 nm, was found to be biphasic at pH 8.3, with fast and slow phases. The dissociation of the 3-imino proton of the enzyme bound FAD was observed in the same time course as the slow phase. These results suggest that the positive charge of trigonelline exists near the 3-imino group of the enzyme bound FAD and interacts repulsively with the proton of the 3-imino group. The absorption spectra of the DAO-trigonelline complex at various pHs also support this hypothesis. In the catalysis of DAO, a similar mechanism may be involved, that is, the positive charge of a D-amino acid may interact repulsively with the 3-imino proton of the enzyme bound FAD, and this interaction may be important for the catalysis.抄録全体を表示PDF形式でダウンロード (707K) -
Takuya Fukuoka, Shigeki Moriya, Hiroshi Yoshikawa, Naotake Ogasawara1990 年 107 巻 5 号 p. 732-739
発行日: 1990年
公開日: 2008/11/18
ジャーナル フリーBacillus subtilis DnaA protein was overproduced by a recombinant plasmid containing B. subtilis dnaA gene in a mutant Escherichia coli strain which is deficient in its own DnaA and RNaseH. The protein was purified to near homogeneity as judged by SDS-PAGE analysis. The purified protein binds preferentially to DNA fragments which are derived from flanking regions of the B. subtilis dnaA gene and contain various numbers of the repeat of 9 nucleotides, TTATCCACA, and closely related sequences. The purified protein binds ATP with high affinity (Kd=0.02μM) and ADP with less affinity, but does not bind cAMP. ATP stimulates the binding of the DnaA protein to the repeated sequences. DNaseI footprinting experiments demonstrated that the DnaA bound first to the consensus 9-mer and then to sequences differing by one base from the consensus. Sequences differing by two bases from the consensus were bound by the DnaA only when they were located contiguous to the strong DnaA-boxes. The three DnaA-box clusters, incA, incB, and incC, derived from the replication origin region of the B. subtilis chromosome showed different levels of growth inhibition when they were introduced into B. subtilis. We demonstrated by assaying competition for DnaA-binding among the DnaA-box clusters that there is a good correlation between the degree of growth inhibition by DnaA-box clusters in vivo and their strength of binding to the DnaA protein in vitro.抄録全体を表示PDF形式でダウンロード (2608K) -
Yutaka Sanai, Kinichiro Oda, Yoshitaka Nagai1990 年 107 巻 5 号 p. 740-742
発行日: 1990年
公開日: 2008/11/18
ジャーナル フリーThe effect of the expression of the human adenovirus type 12 E1A gene on ganglioside composition of rat 3Y1 cells was investigated using cell lines established by introduction of recombinant vector DNA containing the E1A 13S- or 12S-mRNA cDNA placed downstream of the hormone-inducible promoter of mouse mammary tumor virus. Ganglioside GD3 was newly detected after switching on of either 13S- or 12S-mRNA cDNA by addition of dexamethasone. A kinetic study indicated that GD3 was expressed 8 h after additon of dexamethasone. Gangloside GM2 was also accumulated by induction of 13S-mRNA of adenovirus E1A gene. The results indicated that 13S- or 12S-mRNA product alone has the ability to induce GD3 ganglioside in rat 3Y1 cells.抄録全体を表示PDF形式でダウンロード (1684K) -
Motomi Enomoto, Haiou Pan, Fujio Suzuki, Masaharu Takigawa1990 年 107 巻 5 号 p. 743-748
発行日: 1990年
公開日: 2008/11/18
ジャーナル フリーWe have demonstrated that high concentrations of retinoic acid (RA) inhibit expression of the differentiated phenotypes of rabbit costal chondrocytes in culture [M. Takigawa et al. (1980) Proc. Natl. Acad. Sci. U. S. 77, 1481-1485]. In this study we examined the effects of low concentrations of RA on rabbit costal chondrocytes cultured in medium containing vitamin A-deficient serum. In vitamin A-deficient medium, chondrocytes isolated from growth cartilage (GC) proliferated only very slowly, and RA strongly stimulated their proliferation. This stimulatory effect was observable at a concentration of 10-10M RA and maximal at a concentration of 10-8M. RA at 10-8M did not change GC cells from a typical polygonal shape to fibroblast-like cells or inhibit their synthesis of type II collagen. Moreover, RA-treated cells did not synthesize type I collagen. RA inhibited glycosamino-glycan (GAG) synthesis by the cells dose-dependently, but did not change the distribution profile of proteoglycan monomers as determined by glycerol gradient centrifugation. The inhibitory action of RA on GAG synthesis was reversible: after removal of RA from the culture, the rate of GAG synthesis increased within 2 days. In contrast, resting cartilage (RC) cells proliferated well in vitamin A-deficient medium without addition of RA, and RA (10-8M) stimulated their proliferation only slightly. Furthermore, the inhibitory effect of RA on GAG synthesis in RC cells was much weaker than that in GC cells. These observations suggest a physiological role of RA in cartilage in stimulating the proliferation of GC cells without causing drastic change in their differentiated phenotypes.抄録全体を表示PDF形式でダウンロード (2109K) -
Yukihiko Adachi, Jayanta Roy-Chowdhury, Namita Roy-Chowdhury, Rolf Kin ...1990 年 107 巻 5 号 p. 749-754
発行日: 1990年
公開日: 2008/11/18
ジャーナル フリーIn order to characterize the mechanism for bilirubin transport in the liver, the uptake of bilirubin diglucuronide (BDG) into purified sinusoidal plasma membrane vesicles was investigated. BDG uptake was saturable, and was inhibited by sulfobromophthalein and unconjugated bilirubin, but was not affected by sodium taurocholate. BDG uptake was sodium-independent and was stimulated by intravesicular bilirubin or BDG (transstimulation). BDG transport showed strong potential sensitivity; vesicle inside-negative membrane potential created by different anion gradients inhibited BDG uptake whereas vesicle inside-positive membrane potential generated by potassium gradients and valinomycin markedly stimulated BDG transport. These data suggest that BDG, sulfo-bromophthalein, and probably unconjugated bilirubin share a common transporter in liver cells which is sodium independent, membrane-potential-dependent and capable of exchange. The direction of transport in vivo may be governed by the intracellular concentration of BDG and of other yet unidentified organic anions sharing this transporter.抄録全体を表示PDF形式でダウンロード (742K) -
Shoichi Imai, Yutaka Yoshida, Hsiao-Tung Sun1990 年 107 巻 5 号 p. 755-761
発行日: 1990年
公開日: 2008/11/18
ジャーナル フリーTo elucidate the regulation mechanisms for sarcolemmal Ca2+-pumping ATPase of vascular smooth muscle, the preparation of the membrane fraction of porcine aorta with which the enzyme activity could be analyzed was attempted. A Ca2+-activated, Mg2+-dependent ATPase ((Ca2++Mg2+)-ATPase) activity with high affinity for Ca2+(Km =79±18nM) was found in a sarcolemma-enriched fraction obtained from digitonin-treated microsomes that possessed the essential properties of plasma membrane (PM) Ca2+-pumping ATPases, as determined for the erythrocyte and cardiac muscle enzymes. The activity was stimulatd by calmodulin and inhibited by low concentrations of vanadate. Saponin had a stimulatory effect on it. The existence of the PM enzyme in the membrane fraction was substantiated by the Ca2+-dependent, hydroxylamine sensitive phosphorylation of a 130K protein, which could be selectively enhanced by LaCl3. The enzyme activity was potentiated by either cGMP or a purified G-kinase. Purified protein kinase C potentiated the enzyme activity. However, none of these agents stimulated the activity of the enzyme purified from microsomes by calmodulin affinity chromatography. The results suggest that the sarcolem-mal Ca2+-pumping ATPase of vascular smooth muscle is regulated by these protein kinases not through phosphorylation of the enzyme itself but through phosphorylation of mem-brane components(s) other than the enzyme. Phosphatidylinositol phosphate was found to stimulate the enzyme, suggesting its role in mediation of the stimulatory effects of the protein kinases.抄録全体を表示PDF形式でダウンロード (1720K) -
Atsuo Kimura, Shun-ichi Takewaki, Hirokazu Matsui, Masaki Kubota, Seiy ...1990 年 107 巻 5 号 p. 762-768
発行日: 1990年
公開日: 2008/11/18
ジャーナル フリーThe substrate specificity of honeybee α-glucosidase I, a monomeric enzyme, was kinetically investigated. Unusual kinetic features were observed in the cleavage reactions of sucrose, maltose, p-nitrophenyl α-glucoside, phenyl α-glucoside, turanose, and maltodextrin (DP=13). At relatively high substrate concentrations, the velocities of liberation of fructose from sucrose, glucose from maltose, p-nitrophenol from p-nitrophenyl α-glucoside, and phenol from phenyl α-glucoside were accelerated, and so the Lineweaver-Burk plots were convex, indicating negative kinetic cooperativity: the Hill coefficients were calculated to be 0.50, 0.64, 0.50, and 0.67 for sucrose, maltose, p-nitrophenyl p-glucoside, and phenyl α-glucoside, respectively. For the degradation of turanose and maltodextrin, the enzyme showed a sigmoidal curve in v versus s plots and thus catalyzed the reaction with positive kinetic cooperativity. The Lineweaver-Burk plots were concave and the Hill coefficients were 1.2 and 1.5 for turanose and maltodextrin, respectively. These unique properties cannot be interpreted by the reaction mechanism that Huber and Thompson proposed: (1973) Biochemistry 12, 4011-4020. The rate parameters for the hydrolysis of sucrose, maltose, p-nitrophenyl α-glucoside and phenyl α-glucoside were estimated by extrapolat-ing the linear part of the Lineweaver-Burk plots at low substrate concentrations. The ratios of the Vmax values for maltose, sucrose, kojibiose, p-nitrophenyl α-glucoside, phenyl α-glucoside, phenyl α-maltoside, and malto-triose, -tetraose, -pentaose, -hexaose, -heptaose and -octaose, were estimated to be 100: 95 : 81 : 126: 85 : 86 : 113: 117: 111 : 104: 95 : 95, and the Km values for these substrates were 0.85, 4.2, 25, 0.31, 0.60, 0.41, 0.62, 2.0, 8.0, 15, 30, and 33mM, respectively. Based on the rate parameters for maltooligosaccha-rides, the subsite affinities (Ais) in the active site of the enzyme were evaluated. Subsites 1, 2, and 3 having positive A1 values (A1, A2, and A3 : 1.34, 5.37, and 0.269 kcal/mol, respective-ly) were considered to be effective for the binding of substrate to the active site.抄録全体を表示PDF形式でダウンロード (703K) -
Narito Morii, Yasuhiro Ohashi, Yasuo Nemoto, Motohatsu Fujiwara, Yasuc ...1990 年 107 巻 5 号 p. 769-775
発行日: 1990年
公開日: 2008/11/18
ジャーナル フリーBotulinum C1 neurotoxin and C3 exoenzyme were purified to apparent homogeneity from the culture filtrate of Clostridium botulinum type C strain 003-9. Both preparations catalyzed ADP-ribosylation of the same substrate, the Mr 22, 000 rho gene product (Gb). When the light and heavy chains of C1 toxin were separated, ADP-ribosyltransferase activity in the toxin was quantitatively recovered in the light chain fraction. Anti-C1 toxin antiserum precipitated the ADP-ribosyltransferase activity and the neurotoxicity of C1 toxin in parallel, whereas it had no effect on C3 exoenzyme. On the other hand, anti-C3 exoenzyme antiserum precipitated the ADP-ribosyltransferase activities of both C3 exoen-zyme and C1 toxin. This antibody, however, did not precipitate the neurotoxicity of C1 toxin. The ADP-ribosyltransferase in C1 toxin was quantitatively adsorbed onto the anti-C3 antibody column and separated from the majority of C1 toxin protein. The enzyme was then eluted with acidic urea and Western blotting analysis of this eluate revealed the appearance of a protein band positively stained with anti-C3 antibody at a position similar to that of C3 exoenzyme. Quantitative determination by enzyme-linked immunosorbent assay showed that the C3-like immunoreactivity is present in the C1 toxin molecules at the molecular ratio of 1 to 1, 000. These results suggest that the ADP-ribosyltransferase activity in C1 toxin is expressed by a C3-like molecule which is present in a small amount in the toxin preparation and appears to bind to the toxin component(s). The above results also indicate that the ADP-ribosyltransferase in C1 toxin is not related to its neurotoxin action.抄録全体を表示PDF形式でダウンロード (2919K) -
Masaaki Kuboki, Hidemi Ishii, Mutsuyoshi Kazama1990 年 107 巻 5 号 p. 776-780
発行日: 1990年
公開日: 2008/11/18
ジャーナル フリーThe calpain-binding components on the plasma membrane were characterized using calpain I. 125I-labeled calpain was bound to inside-out membrane vesicles from human erythrocyte in a Ca2+-dependent manner, but not to right-side-out membrane vesicles. The maximum binding was observed at more than 5μM Ca2+. The binding amount of calpain to the inside-out membrane vesicles was decreased when the vesicles were pretreated with 100μg/ml of trypsin, chymotrypsin, elastase, or pronase P for 30 min at 37°C, although the binding to the vesicles pretreated with 200μg/m1 of phospholipase A2 or C was not affected. Calpain-binding proteins in the membrane were analyzed by using a modified immunoblot-ting for calpain. Immunostained bands of 240, 220, 89, 72, 52, and 36 kDa were detected as the calpain-binding proteins in the native membrane. All of these bands had disappeared in trypsin-treated membrane. The disappearance of bands was dose-dependent with respect to trypsin and paralleled the reduction of binding amount of calpain to the trypsinized membrane. In calpain-treated membrane, the 240 and 36 kDa bands were retained in the blotting, though the other bands disappeared dose-dependently with respect to calpain. These results suggested that the significant component in the inner surface of plasma membrane for binding of calpain was proteinaceous and the calpain-binding proteins could be classified into two species, i. e. substrates of calpain (220, 89, 72, and 52 kDa protein) and non-substrates (240 and 36 kDa protein).抄録全体を表示PDF形式でダウンロード (1554K) -
Eiko Akaboshi, Paul Howard-Flanders1990 年 107 巻 5 号 p. 781-786
発行日: 1990年
公開日: 2008/11/18
ジャーナル フリーRecA protein mediates homologous pairing and strand exchange reactions between a circular duplex with a single strand gap and a linear duplex. We have used the DNase I footprinting method to analyze processes involving four strands during these reactions. We asked how the length of DNA protected by RecA protein changes as these reactions proceed. We compared two kinds of gapped DNAs. We found that RecA protein polymerizes rapidly in the forward direction (5' to 3' with respect to the single strand). We found, however, that polymerization in the reverse direction was more prominent with a duplex carrying a longer gap than one carrying a shorter gap. DNase I footprints showing protection by RecA protein were obtained only at limited nuclease concentrations, which in turn depended on the position of the end label and the stage of the strand exchange reaction. As judged by the concentrations of DNaseI good for footprinting, the extent of protection by RecA protein was greatest for (+) single-stranded DNA at its first binding site, next highest for heteroduplex containing this (+) strand, and least for the gapped homoduplex DNA. These differences in DNase I sensitivity can be explained in terms of differences in the accessibil-ity of various strands on the basis of a three-dimensional model for the strand exchange reaction.抄録全体を表示PDF形式でダウンロード (2890K) -
Taeko Miyagi, Junji Sagawa, Kimio Konno, Shizuo Handa, Shigeru Tsuiki1990 年 107 巻 5 号 p. 787-793
発行日: 1990年
公開日: 2008/11/18
ジャーナル フリーGanglioside-hydrolyzing sialidase activity was solubilized from rat brain particulate fraction by using Triton X-100 plus sodium deoxycholate. When chromatographed on AH-Sepharose 4B, the solubilized activity was resolved into two peaks, which were designated sialidases I and II in order of elution. The two sialidases were purified by using sequential chromatographies on Octyl-Sepharose CL-4B, Phenyl-Sepharose CL-4B, and Sephadex G-200. Sialidase II was purified further by Mono Q-FPLC. Overall purification was 450- and 2, 150-fold, for sialidases I and II, respectively. Purified sialidases I and II were maximally active at near pH 5.0 and exhibited Mr=70, 000 by gel filtration. Sialidase II hydrolyzed gangliosides but scarcely other substrates including 4-methylumbelliferyl-NeuAc (4MU-NeuAc). Sialidase II hydrolyzed oligosaccharides, glycoproteins, and 4MU-NeuAc although gangliosides appeared to be preferential substrates. Sialidase II cleaved GM2 much faster than sialidase I. An antibody raised in rabbits against sialidase II reacted with only sialidase I and an antibody against sialidase II reacted with only sialidase II. A subcellular distribution study suggested sialidase I in the synaptosomal membrane and sialidase II in the synaptosomal and lysosomal membranes, and this was verified by using the above antibodies.抄録全体を表示PDF形式でダウンロード (801K) -
Taeko Miyagi, Junji Sagawa, Kimio Konno, Shigeru Tsuiki1990 年 107 巻 5 号 p. 794-798
発行日: 1990年
公開日: 2008/11/18
ジャーナル フリーCytosolic sialidase was purified from rat skeletal muscle, and the purified enzyme migrated as a single band of Mr 43, 000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A polyclonal antibody raised against the enzyme inhibited and immunoprecipitated rat liver cytosolic sialidase as well as the muscle enzyme but failed to cross-react with the intralysosomal sialidase of rat liver and membrane sialidases I (synaptosomal) and II (lysosomal) of rat brain. The antibody against brain membrane sialidase I (anti-I) and that against sialidase II (anti-II), which could be useful to discriminate the two enzymes, did not cross-react with the intralysosomal and cytosolic sialidases of liver. Although more than 90% of liver plasma membrane sialidase was immunoprecipitated with anti-I, only 60% of liver lysosomal membrane sialidase was immunoprecipitated with anti-II, the remainder being immunoprecipitated with anti-I. In confirmation of these data, liver lysosomal membrane exhibited two peaks of ganglioside sialidase corresponding to the membrane sialidases I and II on Aminohexyl-Sepharose chromatography while only one peak of ganglioside sialidase corresponding to sialidase I was observed for liver plasma mem-brane. These results indicate that the four types of rat sialidase are proteins distinct from one another and that the three kinds of antisera described above are useful for discriminating these sialidases qualitatively and probably quantitatively.抄録全体を表示PDF形式でダウンロード (540K)
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