A yeast vacuolar protease, carboxypeptidase Y (CPY), is known to be involved in the C-terminal processing of peptides and proteins; however, its real function remains unclear. The CPY biosynthetic pathway has been used as a model system for protein sorting in eukaryotes. CPY is synthesized as a prepro-form that travels through the ER and Golgi to its final destination in vacuoles. In the course of studies on the transport mechanism of CPY, various post-translational events have been identified, e. g. carbohydrate modification and cleavage of the pre-segments. In addition, sorting signals and various sorting vehicles, similar to those found in higher eukaryotic cells, have been found. The catalytic triad in the active site of CPY makes this enzyme a serine protease. A unique feature distinguishing CPY from other serine proteases is its wide pH optimum, in particular its high activity at acidic pH. Several structural properties which might contribute to this unique feature exist such as a conserved free cysteine residue in the S₁ substrate binding pocket, a recognition site for a C-terminal carboxyl group, and a disulfide zipper motif. The structural bases in CPY functions are discussed in this article.

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A calcium binding protein with a molecular mass of 40 kDa (CBP40), the gene product of plasmodial-specific LAV1-2 of Physarum polycephalum, was crystallized in the presence of EDTA. The crystals diffracted X-rays up to a resolution of 3.0 A. They belonged to the trigonal space group, P3₂21 (or P3121), with unit cell dimensions of a=b=64.4 A and c=207.2 Å. Ca²⁺-bound crystals were obtained by soaking in a CaCl₂ solution, which gave diffraction data of similar quality. The Ca²⁺-soaked crystals belonged to the same space group as those crystallized in the presence of EDTA with unit cell dimensions of a=b=64.4 Å and c=209.4 Å.

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Fe-S proteins acquire Fe-S clusters by an unknown post-translational mechanism. To study the in vivo synthesis of the Fe-S clusters, we constructed an experimental system to monitor the expressed ferredoxin (Fd) as a reporter of protein-bound Fe-S clusters assembled in Escherichia coli. Overexpression of five Fds in a T7 polymerase-based system led to the formation of soluble apoFds and mature holoFds, indicating that assembly of the Fe-S cluster into apoFd polypeptides is a rate-limiting step. We examined the coexpression of the E. coli ORF1-ORF2-iscS-iscU-iscA-hscB-hscA-fdx-ORF3 gene cluster, which has recently been suggested to be involved in the formation or repair of Fe-S protein [Zheng, L., Cash, V. L., Flint, D. H., and Dean, D. R. (1998) J. Biol. Chem. 273, 13264-13272], with reporter Fds using compatible plasmids. The production of all five reporter holoFds examined was dramatically increased by the coexpression of the gene cluster, and apparent specificity to the polypeptides or to the type of Fe-S clusters was not observed. The increase in holoFd production was observed under the coexpression conditions in all culture media examined, with either 2×YT medium or Terrific broth, and with or without supplemental cysteine or iron. These results indicate that the proteins encoded by the gene cluster are involved in the assembly of the Fe-S clusters in a wide variety of Fe-S proteins.

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A cysteine protease, phytolacain R from full-growth greenish fruits of pokeweed, Phytolacca americana L, was purified to electrophoretic homogeneity by a simple purification procedure employing CM-Sepharose ion-exchange chromatography. The enzyme was present in low content in the young fruits about 50 d after flowering but gradually accumulated in growing fruits. Its molecular mass was estimated to be ca. 23 kDa by SDS-PAGE, and its sugar content was zero. Its amino acid sequence was established by automated sequence analysis of the peptides obtained by cleavage with Achromobacter protease I, chymotrypsin, trypsin, and cyanogen bromide. The enzyme is composed of 218 amino acid residues, of which it shares 110 residues (50%) with papain, 104 (47%) with actinidain, and 87 (40%) with stem bromelain. The amino acid residues forming the substrate-binding the S₂ pocket of papain, Tyr61, Tyr67, Pro68, Trp69, Val133, and Phe207, were predicted to be replaced by Gly, Trp, Met, His, Ala, and Met in phytolacain R, respectively. As a consequence of these substitutions, the S₂ pocket is expected to be less hydrophobic in phytolacain R than in papain.

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Porcine cardiac myosin monomers in equilibrium with filaments under physiological conditions were observed to have two conformations, extended and folded forms, upon electron microscopy and gel filtration HPLC. The conformational state was independent of ATP and the phosphorylation of regulatory light chain. The folded monomers of cardiac myosin were mainly in an open conformation with only one bend in the tail, and may not trap the hydrolysis products of ATP, as assessed by single turnover experiments. These properties are similar to those of the folded monomers of rabbit skeletal myosin [Katoh, T., Konishi, K., and Yazawa, M. (1998) J. Biol. Chem. 273, 11436-11439]. The conformational states of skeletal and cardiac myosin monomers were not affected by pH between 7.0 and 8.5. Although significant disassembly of filaments and thus an increase in the monomer concentration were observed with an increase in pH. The results indicate that the pH-dependent change in filament assembly is due to a shift of equilibrium between the filaments and extended monomers toward filament disassembly. The Mg²⁺-ATPase activity of these myosin monomers decreased with a decrease in the salt concentration below _??_0.1M, suggestive of the formation of a closed conformation similar to the conformation of 10S smooth myosin. The results suggest that the conformational change from the extended to the folded form is a common property of various myosin IIs.

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To determine the role of the polyol metabolizing pathway under hyperglycemic conditions, the effects of aldose reductase (AR) on the cellular functions of pancreatic β-cells were examined. Stable transfectants of rat AR cDNA were obtained with a pancreatic β-cell line, HIT, in which a negligible amount of AR was originally expressed. Overproduction of AR triggered DNA fragmentation, as judged with the TUNEL method and agarose gel electrophoresis. Morphological analysis by electron microscopy also clearly showed apoptosis of the AR-overexpressing HIT cells. Induction by interleukin-1 β of gene expression such as those of an inducible form of nitric oxide synthase (NOS-II) and Mn-superoxide dismutase (Mn-SOD), was much lower in the transfectants than in the control cells, while the expression of constitutively expressed genes such as those for Cu, Zn-superoxide dismutase and insulin was not changed. The susceptibility to interleukin-1 β stimulation of the expression of the NOS II and Mn-SOD genes was due to suppressed NF-κB activity, which is essential for the expression of these genes. In addition, the intracellular NADPH/NADP⁺ ratio was considerably lower in the AR-transfected cells than in control cells. Thus, the overexpression of AR in pancreatic β-cells induced apoptosis that may be caused by a redox imbalance.

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During a large-scale in vitro translation analysis of a human full-length cDNA bank, we found many clones producing in vitro translation products showing ladder bands on a fluorogram with the equidistance of about 9 kDa at the position larger than the molecular mass expected from the open reading frame. We have analyzed a clone showing a typical pattern of the ladder bands. This clone encoded a 188-amino acid polypeptide containing a putative transmembrane domain. A green fluorescent protein-tagged polypeptide expressed in COS7 cells was localized in the endoplasmic reticulum and the Golgi apparatus. The ladder bands were observed in a rabbit reticulocyte lysate system, but not in a wheat germ extract system. Addition of the glutathione S-transferase-fused ubiquitin into the lysate caused upward shifts of the ladder bands. Addition of microsomal membranes prevented the formation of the ladder bands. Time course experiments demonstrated that the in vitro translation products increased in the presence of microsomal membranes, but were gradually degraded in their absence. These results suggest that the ladder formation resulted from the ubiquitination of misfolded polypeptide that failed to translocate to its proper position, and that an exclusion mechanism of misfolded membrane protein works in the rabbit reticulocyte lysate system.

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Collagen type I extracted with acid or digested with pepsin forms fibrils under physiological conditions, but this ability is lost when the collagen is treated with alkaline solution or digested with matrix metalloproteinase 1 (MMP1). When acid-soluble collagen was incubated with alkali-treated collagen, the fibril formation of acid-soluble collagen was inhibited. At 37°C, at which alkali-treated collagen is denatured, the lag time was prolonged but the growth rate of fibrils was not affected. At 30°C, at which the triple helical conformation of alkali-treated collagen is retained, the lag time was prolonged and the growth rate reduced. Heat-denatured alkali-treated collagen and MMP1-digested fragments have no inhibitory effect on the fibril formation of acid-soluble collagen. This means that the triple helical conformation and the molecular length are important factors in the interaction of collagen molecules and that alkali-treated collagen acts as a competitive inhibitor for fibril formation of collagen. We found that alkali-treated collagen and MMP1-digested fragments form fibrils that lack the D periodic banding pattern and twisted morphology under acidic conditions at the appropriate ionic strength. We also calculated the relative strengths of hydrophobic and electrostatic interactions between collagen molecules. When the hydrophobic interaction between linear collagen molecules was considered, we found a pattern of periodic maximization of the interactive force including the D period. On the other hand, the electrostatic interaction did not show the periodic pattern, but the overall interaction score affected fibril formation.

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We have cloned the human UDP-N-acetylglucosamine (UDP-GlcNAc) transporter cDNA, which was recognized through a homology search in the expressed sequence tags database (dbEST) based on its similarity to the human UDP-galactose transporter. The chromosomal location of the UDP-GlcNAc transporter gene was assigned to chromosome 1p21 by fluorescence in situ hybridization (FISH). The transporter was expressed ubiquitously in every tissue so far examined. Expression of the transporter cDNA in CHO-Kl cells in its native and in a C-terminally HA-tagged form indicated that the human UDP-GleNAc transporter was localized in the Golgi apparatus. The membrane vesicles prepared from yeast cells expressing the cDNA product exhibited UDP-GleNAc-specific transporting activity. Comparison among UDP-galactose, CMP-sialic acid, and UDP-GIcNAc transporters from several organisms enabled us to identify residues highly conserved among the transporters and residues specific for each group of transporters.

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In order to determine the functional role of the procathepsin L propeptide region for the preparation of active recombinant rat cathepsin L (CL), cDNAs encoding two short-length propeptides (C-terminal 2 and 27 residues) and the full-length (96 residues) one plus the entire CL were expressed as two soluble fusion proteins with a fragment of maltose-binding protein and an insoluble fusion protein with glutathione-S-transferase in Escherichia coli, respectively. After refolding of the insoluble fusion protein, each gene product was purified to homogeneity by amylose or glutathione-Sepharose-4B affinity column, and digestion with factor Xa and α-thrombin under alkaline conditions (pH_??_8.0) led to the elution of two pure short-length procathepsin Ls (PCLs) and a full-length one, respectively. The enzymatic activity, estimated by hydrolytic assaying of benzoxycarbonyl-Phe-Arg-7-(4- methyl)coumarylamide under acidic conditions (pH 5.5), indicated that the two shortlength PCLs exhibited in a great loss of the activity, as compared with the full-length PCL. The CD spectra of the short-length PCLs were different from that of the full-length one. The present results clearly show that the full-length propeptide is essential for construction of the active tertiary structure of CL at the stage of recombinant protein expression, although the expression of CL itself in E. coli does not require the propeptide. Based on the tertiary structure of PCL, the propeptide region necessary for the construction of the CL active structure has been discussed.

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Sphingosine 1-phosphate (Sph-1-P) is considered to play a dual role in cellular signaling, acting intercellularly as well as intracellularly. In this study, we examined the role of Sph-1-P as a signaling molecule in human platelets, using DL-threo-dihydrosphingosine (DHS) and N, N-dimethylsphingosine (DMS), inhibitors of Sph kinase and protein kinase C. Both DMS and DL-threo-DHS were confirmed to be competitive inhibitors of Sph kinase obtained from platelet cytoplasmic fractions. In intact platelets labeled with [³H]Sph, stimulation with 12-O-tetradecanoylphorbol 13-acetate or thrombin did not affect [³H]-Sph-1-P formation. While both DMS and DL-threo-DHS inhibited not only [³H]Sph-1-P formation but also protein kinase C-dependent platelet aggregation, staurosporine, a potent protein kinase inhibitor, only inhibited the protein kinase C-dependent reaction. Hence, it is unlikely that Sph kinase activation and the resultant Sph-1-P formation are mediated by protein kinase C in platelets. Furthermore, Ca²⁺ mobilization induced by platelet agonists that act on G protein-coupled receptor was not affected by DMS or DL-threo-DHS. Our results suggest that Sph-1-P does not mediate intracellular signaling, including Ca²⁺ mobilization, in platelets.

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To elucidate the roles of conserved Asp residues of Bacillus cereus sphingomyelinase (SMase) in the kinetic and binding properties of the enzyme toward various substrates and Mg²⁺, the kinetic data on mutant SMases (D126G and D156G) were compared with those of wild type (WT) enzyme. The stereoselectivity of the enzyme in the hydrolysis of monodispersed short-chain sphingomyelin (SM) analogs and the binding of Mg²⁺ to the enzyme were not affected by the replacement of Asp126 or Asp156. The pH-dependence curves of kinetic parameters (1/K_m and k_cat) for D156G-catalyzed hydrolysis of micellar SM mixed with Triton X-100 (1:10) and of micellar 2-hexadecanoylamino-4-nitrophenylphosphocholine (HNP) were similar in shape to those for WT enzyme-catalyzed hydrolysis. On the other hand, the curves for D126G lacked the transition observed for D156G and WT enzymes. Comparison of the values and the shape of pH-dependence curves of kinetic parameters indicated that Asp126 of WT SMase enhances the enzyme's catalytic activity toward both substrates and its binding of HNP but not SM. The deprotonation of Asp126 enhances the substrate binding and slightly suppresses the catalytic activity toward both substrates. Asp156 of WT SMase acts to decrease the binding of both substrates and the catalytic activity to HNP but not SM. From the present study and the predicted three-dimensional structure of B. cereus SMase, Asp126 was thought to be located close to the active site, and its ionization was shown to affect the catalytic activity and substrate binding.

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Cytochrome bd-type ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli contains two hemes b (b₅₅₈ and b₅₉₅) and one heme d as redox metal centers. To clarify the structure of the reaction center, we analyzed the fully oxidized enzyme by visible and EPR spectroscopies using fluoride ion as a monitoring probe. The visible spectral changes upon fluoride-binding were typical of ferric iron-chlorine species, indicating heme d as a primary binding site. The negative peak at 645 nm in the difference spectrum indicates that heme b₅₉₅ also provides the low-affinity fluoride-binding site. Fluoride-binding caused a complete disappearance from the EPR spectra of the low-spin signals ascribable to heme d and spectral changes in both rhombic and axial high-spin signals. After fluoride-binding, each component of the rhombic high-spin signal showed superhyperfine splitting arising from the interaction of the unpaired spin of the heme d iron with the nuclear magnetic moment of ¹⁹F. The axial high-spin species was converted to a new rhombic high-spin species assignable to heme b₅₉₅-fluoride. The g=2 component of this new species also gave ¹⁹F-superhyperfine splitting. These results indicate that both heme d and heme b₅₉₅ can coordinate with a fluoride ion with different affinities in the fully oxidized state.

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We have isolated a 3.9-kbp-long cDNA and approximately 93 kbp long genomic DNA encoding a membrane guanylyl cyclase (designated OlGC 1) from the medaka fish Oryzias latipes, and determined their nucleotide sequences. The open reading frame for the OlGC 1 cDNA predicts a protein of 1, 055 amino acids. Phylogenetic analysis indicates OlGC 1 to be a medaka fish homolog of mammalian guanylyl cyclase B (GC-B), a member of the natriuretic peptide receptor family. Northern blot analysis demonstrated 3.9 kb OlGC 1 transcripts in the eye and brain, while reverse transcription-polymerase chain reaction (RT-PCR) analysis showed OlGC 1 transcripts in a number of adult peripheral organs as well as embryos during early stages of development. The OlGC 1 gene consists of 22 exons with an exon/intron organization similar to that of the human and rat GC-A genes, except that OlGC 1 has several very large introns. The OlGC 1 gene has no apparent TATA box or CAAT box in the region 1.2 kbp upstream of the putative transcription initiation point, but several consensus sequences for cis-regulatory elements, including an NF-IL 6-binding element, a shear stress responsive element, and multiple GM-CSF-binding elements, are present in that region.

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The production of phospholipid hydroperoxide and aldehydic phospholipid was examined in human red blood cell (RBC) membranes after peroxidation with 2, 2-azobis (2-amidinopropane) dihydrochloride (AAPH) or xanthine/xanthine oxidase (XO/XOD/Fe³⁺). Both radical-generation systems caused a profound decrease in the amount of polyunsaturated fatty acid (PUFA) in choline glycerophospholipid (CGP) and induced formation of peroxidized CGP in RBC membranes to different extents. No consistent generation of peroxidized lipids from CGP was evident after peroxidation with XO/XOD/Fe³⁺, which caused the apparent decomposition of phospholipids and the formation of large amounts of thiobarbituric acid-reactive substance (TBARS). On the other hand, CGP hydroperoxide was formed as a primary product of peroxidation with AAPH. Aldehydic CGP was also detected as a secondary product of hydroperoxide decomposition in AAPH-Peroxidized RBC membranes. Aldehydic CGP was preferentially generated from arachidonoyl CGP rather than from linoleoyl CGP in AAPH-peroxidized membranes. AAPH mainly oxidized CGP to hydroperoxide and aldehydic phospholipids. The sum of hydroperoxide and aldehyde of CGP corresponded to the loss of CGP due to peroxidation by AAPH. This result indicates that CGP was mainly converted into these two oxidized phospholipids in AAPH-perox-idized RBC membranes.

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Skinned fibers prepared from rabbit fast and slow skeletal and cardiac muscles showed acidotic depression of the Ca²⁺ sensitivity of force generation, in which the magnitude depends on muscle type in the order of cardiac>fast skeletal>slow skeletal. Using a method that displaces whole troponin-complex in myofibrils with excess troponin T, the roles of Tn subunits in the differential pH dependence of the Ca²⁺ sensitivity of striated muscle were investigated by exchanging endogenous troponin I and troponin C in rabbit skinned cardiac muscle fibres with all possible combinations of the corresponding isoforms expressed in rabbit fast and slow skeletal and cardiac muscles. In fibers exchanged with fast skeletal or cardiac troponin I, cardiac troponin C confers a higher sensitivity to acidic pH on the Ca²⁺ sensitive force generation than fast skeletal troponin C independently of the isoform of troponin I present. On the other hand, fibres exchanged with slow skeletal troponin I exhibit the highest resistance to acidic pH in combination with either isoform of troponin C. These results indicate that troponin C is a determinant of the differential pH sensitivity of fast skeletal and cardiac muscles, while troponin I is a determinant of the pH sensitivity of slow skeletal muscle.

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The mammalian ATP/ADP translocator isoform-2 (ANT2) gene is growth-activated. Regulation of the gene appears to involve Sp1, as an essential activator, and a suppressor through an Sp1 core element next to the transcription start. We show here that the proximal promoter also binds AP-2 strongly and specifically to two sites, one of which overlaps the Sp1 proximal suppressor site. AP-2 binds with an affinity of 10 to15 fold higher than that of Sp1. AP-2 alone does not alter the ANT2 promoter activity in transfected SL2 cells, but enhances the Sp1-dependent activation of the promoter several fold. Enhancement by AP-2 is observed only when Spl is limiting for transcription activation. These data suggest that the cellular AP-2/Sp1 ratio might influence ANT2 expression in developing or differentiating cells.

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Connective tissue growth factor (CTGF) is a novel cysteine-rich, secreted protein. Recently, we found that inhibition of the endogenous expression of CTGF by its antisense oligonucleotide and antisense RNA suppresses the proliferation and migration of vascular endothelial cells. In the present study, the following observations demonstrated the angiogenic function of CTGF in vitro and in vivo: (i) purified recombinant CTGF (rCTGF) promoted the adhesion, proliferation and migration of vascular endothelial cells in a dose-dependent manner under serum-free conditions, and these effects were inhibited by anti-CTGF antibodies; (ii) rCTGF markedly induced the tube formation of vascular endothelial cells, and this effect was stronger than that of basic fibroblast growth factor or vascular endothelial growth factor; (iii) application of rCTGF to the chicken chorioallantoic membrane resulted in a gross angiogenic response, and this effect was also inhibited by anti-CTGF antibodies. (iv) rCTGF injected with collagen gel into the backs of mice induced strong angiogenesis in vivo. These findings indicate that CTGF is a novel, potent angiogenesis factor which functions in multi-stages in this process.

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The effects of vanadate were examined on Ca²⁺-activated force and phosphorylation of 20-kDa myosin light chain in membrane-permeabilized rabbit aortic smooth muscle strips. Addition of vanadate during maximum contraction reduced the force in a dose-dependent manner, and inhibited it almost completely at 1mM. Two-dimensional polyacrylamide gel electrophoretic analyses revealed that vanadate also reduced the phosphorylation of 20-kDa myosin light chain in a dose-dependent manner from _??_50% in the absence of vanadate to _??_20% in the presence of 1mM vanadate. The effects of 1mM vanadate on purified myosin light chain kinase and phosphatase were then examined using purified myosin as substrate, and it was found that vanadate neither inhibited myosin light chain kinase nor activated myosin light chain phosphatase. These results indicate that the reduction in the 20-kDa myosin light chain phosphorylation level by vanadate may be effected through its inhibition of the force generation in skinned smooth muscle strip, as evidenced by the finding that vanadate eliminated the enhancement of myosin light chain kinase activity brought about by the interaction between purified myosin and actin.

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The possible involvement of lecithin-cholesterol acyltransferase (LCAT) inthe metabolism of oxidized phosphatidylcholine (PC) in plasma was investigated. A variety of oxidized products are formed from PC following oxidation of low density lipoproteins (LDL). A significant increase in LDL oxidation levels in patients with familial LCAT deficiency (FLD) has been previously demonstrated by a sensitive sandwich ELISA for oxidized LDL using the monoclonal antibody DLH3 which recognizes oxidized products of PC. In the present study, we found that LCAT produces various metabolites from oxidized PC and that oxidized PC molecules in LDL particles serve as substrates. When the neutral lipid fraction was separated by TLC after the incubation of oxidized 1-palmitoy1-2-[1-¹⁴C]linoleoyl PC with human plasma, a number of radioactive bands were formed in addition to cholesteryl ester. These products were not formed from native 1-palmitoy1-2[1-¹⁴C]linoleoyl PC. Plasma from FLD patients also failed to form the additional products from oxidized PC. The addition of dithio-bis(nitrobenzoate) (DTNB), an LCAT inhibitor, or the inactivation of LCAT activity by treating the plasma at 56°C for 30min abolished the generation of these products from oxidized PC. The activity was recovered in the high density lipoprotein (HDL) fraction but not in the LDL fraction separated from normal plasma. When Ipalmitoy1-2-[1-¹⁴C](9-oxononanoyl) PC and 1-stearoy1-2-[1-₁₄C] (5-oxovaleroyl)PC, PC oxidation products that contain short chain aldehydes, were incubated with human plasma, radioactive products in the neutral lipid fraction were observed on TLC. LDL containing oxidized PC was measured by sandwich ELISA using an anti-apolipoprotein B antibody and DLH3. The reconstituted oxidized PC-LDL particles were found to have lost their ability to bind DLH3 upon incubation with HDL, while the reactivity of the reconstituted oxidized PC-LDL remained unchanged in the presence of DTNB. These results suggest that LCAT is capable of metabolizing a variety of oxidized products of PC and preventing the accumulation of oxidized PC in circulating LDL particles.

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We previously demonstrated that amino acid residues Gln62 (P3), Phe63 (P2), Leu64 (P1), and Phe67 (P3') in the primary binding loop of Erythrina variegata chymotrypsin inhibitor (ECI), a member of the Kunitz inhibitor family, are involved in its strong inhibitory activity toward chymotrypsin [Iwanaga et al. (1998) J. Biochem. 124, 663-669]. To determine whether or not these four amino acid residues predominantly contribute to the strong inhibitory activity of ECI, they were simultaneously replaced by Ala. The results showed that a quadruple mutant, Q62A/F63A/L64A/F67A, retained considerable inhibitory activity (K₁, 5.6×10^-7M), indicating that in addition to the side chains of these four amino acid residues, the backbone structure of the primary binding loop in ECI is essential for the inhibitory activity toward chymotrypsin. Two chimeric proteins, in which the primary binding loops of ECI and ETIa were exchanged: an isoinhibitor from E. variegata with lower chymotrypsin inhibitory activity, were constructed to determine whether the backbone structure of the primary binding loop of ECI was formed by the amino acid residues therein, or through an interaction between the primary binding loop and the residual structure designated as the “scaffold.” A chimeric protein, ECI/ETIa, composed of the primary binding loop of ECI and the scaffold of ETIa showed weaker inhibitory activity (K₁, 1.3×10^-6M) than ECI (K₁, 9.8×10^-8 M). In contrast, a chimera, ETIa/ECI, comprising the primary binding loop of ETIa and the scaffold of ECI inhibited chymotrypsin more strongly (K₁, 5.7×10^-7M) than ETIa (K₁, 1.3×10^-6M). These results indicate that the intramolecular interaction between the primary binding loop and the scaffold of ECI plays an important role in the strong inhibitory activity toward chymotrypsin. Furthermore, surface plasmon resonance analysis revealed that the side chains on the primary binding loop of ECI contribute to both an increase in the association rate constant (k_on) and a decrease in the dissociation rate constant (k_off) for the ECI-chymotrypsin interaction, whereas the backbone structure of the primary binding loop mainly contributes to a decrease in the dissociation rate constant.

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Ipriflavone (7-isopropoxy-3-phenyl-4H-1-benzopyran-4-one) is a synthetic flavonoid that has been shown to stimulate the activity of osteoblasts. We show here that ipriflavone also promotes the deposition of calcium and the formation of mineralized nodules by newborn rat calvarial osteoblast-like (ROB) cells as well as the activity of alkaline phosphatase. We reported previously that endothelin-1 inhibits the differentiation of ROB cells [Y. Hiruma et al. (1998) J. Cardiovasc. Pharmacol. 31, S521-S523]. Therefore, we examined the effects of ipriflavone on the expression of endothelin receptors in ROB cells by polymerase chain reaction-Southern blot analysis and in binding assays with ¹²⁵I-labeled endothelin-1. Ipriflavone reduced levels of endothelin ET_A receptors (to 48% of the control level) in ROB cells around day 7 in our standard cultures, while it had no apparent effect on the expression of the mRNA for the endothelin ET_A receptor. By contrast, treatment with 10^-7M endothelin-1 on days 6 through 9 alone suppressed mineralization by ROB cells. Ipriflavone also reduced the ability of endothelin-1 to inhibit mineralization by ROB cells. These results suggest that the acceleration of osteoblastic differentiation by ipriflavone might be due, at least in part, to a time-specific down-regulation of endothelin receptors.

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An antagonist specific for the thrombin receptor is expected to be a remedy for thrombosis. Structure-activity studies of thrombin receptor-tethered ligand SFLLRNP have revealed the importance of the Phe-2-phenyl group in receptor recognition and the replacement of the Phe-2 by para-fluorophenylalanine [(p-F)Phe] was found to enhance its activity [Nose, T. et al. (1993) Biochem. Biophys. Res. Commun. 193, 694-699]. In order to obtain a small sized antagonist, a series of (p-F)Phe derivatives was designed and synthesized novel structural elements essential for receptor interactions being introduced at both the N and C-termini. β-Mercaptopropionyl (βMp) or its derivative activated by S-3-nitro-2-pyridinesulphenyl (Npys) was introduced at the N-terminus, and phenylmethyl amines were coupled to the Cterminus. All compounds were inactive when assayed for human platelet aggregation, indicating that they are not agonists. β-Mercaptopropionyl derivatives were also inactive as antagonists. However, Npys-containing analogs were found to inhibit the agonist activity of SFLLRNP. In particular, SNpys-βMp-(p-F)Phe-NH-R [R =-CH(C₆H₅)₂ and -CH₂-CH-(C₆H₅)₂] potently suppressed platelet aggregation. The results suggested that (p-F)Phe can be used as a structural core to construct an effective antagonist conformation.

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LIM-homeodomain (LHX) transcription factors play critical roles in cell fate determination during development, in particular, in CNS. The transcriptional activity of several LHX proteins is postulated to be regulated by interaction with an LIM-domain binding protein, Ldb1. We have now identified a novel LHX molecule, termed Lhx6.1, that is closely related to a recently reported Lhx6 molecule. The Lhx6.1 transcript is found in several restricted regions in the developing CNS, mostly within the embryonic forebrain. We further show that Lhx6.1 interacts with Ldb1 through tandem LIM-domains, implying transcriptional regulation of Lhx6.1 by Ldb1.

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Secretion of triglycerides by the liver in ruminants as components of very low density lipoproteins particles is low as compared with that in primates or rodents. The rate-limiting steps for the hepatic export of very low density lipoproteins have been studied in liver slices to determine the origin of the low lipotropic capacity of calf liver compared to that of rat liver. The rates of production of apolipoprotein B (apo B) and albumin as well as the rate of secretion of VLDL-apolipoproteins were measured during 12-h incubation of liver slices in organo-culture using [³⁵S] methionine-cysteine labeling. Hepatic apo B production was similar in the two animal species but the VLDL-apolipoprotein secretion rate for calf liver slices amounted to only 20% of that observed for rat liver slices. Although calf and rat liver slices synthesized similar amounts of total protein, the hepatic production of albumin, measured in cells and media, was much higher in calf than rat liver slices (around 2.7-fold), whereas the rate secretion of albumin was similar in the two species. Our results showed that the slow rate of secretion of VLDL by calf liver cells was not consecutive to a low rate of synthesis of apo 13 but rather to a defect in VLDL assembly and/or secretion.

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Cytochrome bo is the heme-copper terminal ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli, and functions as a redox-coupled proton pump. As an extension to our mutagenesis and Fourier-transform infrared studies on ion pumps, we examined the effects of subunit I mutations on redox-linked protein structural changes in cytochrome bo. Upon photo-reduction in the presence of riboflavin, Y288F and H333A showed profound effects in their peptide backbone vibrations (amide-I and amide-II), probably due to the loss of Cu_B or replacement of high-spin heme o with heme B. In the frequency region of protonated carboxylic C=O stretching vibrations, negative 1, 743cm^-1 and positive 1, 720cm^-1 bands were observed in the wild-type; the former shifted to 1, 741cm^-1 in E286D but not in other mutants including D135N. This suggests that Glu286 in the D-channel is protonated in the air-oxidized state and undergoes hydrogen bonding changes upon reduction of the redox metal centers. Two pairs of band shifts at 2, 566(+)/2, 574(-) and 2, 546(+)/2, 556(-)cm^-1 in all mutants indicate that two cysteine residues not in the vicinity of the metal centers undergo redox-linked hydrogen bonding changes. Cyanide had no effect on the protein structural changes because of the rigid local protein structure around the binuclear center or the presence of a ligand(s) at the binuclear center, and was released from the binuclear center upon reduction. This study establishes that cytochrome bo undergoes unique redox-linked protein structural changes. Localization and timeresolved analysis of the structural changes during dioxygen reduction will facilitate understanding of the molecular mechanism of redox-coupled proton pumping at the atomic level.

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Troponin is the regulatory protein of striated muscle. Without Ca²⁺, the contraction of striated muscle is inhibited. Binding of Ca²⁺ to troponin activates contraction. The location of troponin on the thin filaments and its relation to the regulatory mechanism has been unknown, though the Ca²⁺-induced dislocation of tropomyosin has been studied. By binding troponin(C+I) to actin in an almost stoichiometric ratio and reconstituting actintropomyosin-troponin(C+I) filaments, we reconstructed the three-dimensional structure of actin-tropomyosin-troponin(C+I) with or without Ca²⁺ from electron cryomicrographs to about 2.5 or 3 nm resolution, respectively. Without Ca²⁺, the three-dimensional map reveals the extra-density region due to troponin(C+I), which extends perpendicularly to the helix axis and covers the N-terminal and C-terminal regions of actin. In the presence of Ca²⁺, the C-terminal region of actin became more exposed, and troponin(C+I) became V-shaped with one arm extending towards the pointed end of the actin filament. This structure can be considered to show the location of troponin(C+I) in at least one of the states of skeletal muscle thin filaments. These Ca²⁺-induced changes of troponin(C+I) provide a clue to the regulatory mechanism of contraction.

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Soybean agglutinin (SBA), is a noncovalently bound tetramer comprised of four identical subunits having a single N-glycan chain, Man₉GlcNAc₂, that is known to be essential for regeneration of the functional tetrameric structure from unfolded subunits. In this study, SBA was found to have strong affinity for concanavalin A, indicating that the N-glycans are extensively solvent-exposed. The susceptibilities of the N-glycans to a-mannosidase and endo-β-N-acetylglucosaminidase revealed that their distal areas have nonreducing ends embedded among the subunits, whereas their proximal regions are solvent-exposed. Endo-β-N-acetylglucosaminidase-digested SBA was unable to retain its conformation and gradually unfolded. Periodate-oxidized SBA, whose N-glycans closely correspond to the invariant pentasaccharide core, tended to dissociate into the subunits, but permitted to stay as folded monomers. This SBA species was capable of refolding from unfolded subunits but unable to form the functional tetramer. It seems probable that the proximal regions of the N-glycans function in the formation and stabilization of the subunit conformation, whereas the branches outside the invariant cores stabilize the tetrameric structure.

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The superoxide dismutase (SOD) gene of Aeropyrum pernix, a strictly aerobic hyperther-mophilic archaeon, was cloned and expressed in Escherichia coli, and its gene product was characterized. The molecular mass of the protein, based on the deduced amino acid sequence, was 24.6kDa. The sequence showed overall similarity to the sequences of known Mn- and Fe-SODs. The metal binding residues conserved in Mn- and Fe-SODs were also found in A. pernix SOD. When the SOD gene was expressed in E. coli cells, the product formed a homodimer, and contained both Mn and Fe. Metal reconstitution experiments showed that A. pernix SOD is cambialistic, i.e. active with either Fe or Mn. The specific activities were 906 U/mg with Mn and 175 U/mg with Fe. No loss of activity of Mn-recon-stituted SOD was observed at 105°C even after 5h incubation. Sodium azide, an inhibitor of SODs, did not inhibit the Mn-reconstituted SOD from A. pernix even at concentrations up to 400mM. This SOD from an aerobic hyperthermophilic archaeon, Aeropyrum pernix, was extremely thermostable and active with either Mn or Fe. With Mn as a metal cofactor, it was more thermostable, and less sensitive to sodium azide and sodium fluoride than with Fe.

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In a previous paper we showed that the B-pentamer of cholera toxin (CT-B) binds with reduced binding strength to different C (1) derivatives of N-acetylneuraminic acid (NeuAc) of the natural receptor ganglioside, GM 1. We have now extended these results to encompass two large amide derivatives, butylamide and cyclohexylmethylamide, using an assay in which the glycosphingolipids are adsorbed on hydrophobic PVDF membranes. The latter derivative showed an affinity approximately equal to that earlier found for benzylamide (_??_0.01 relative to native GM 1) whereas the former revealed a approximately tenfold further reduction in affinity. Another derivative with a charged C (1)-amide group, amino-propylamide, was not bound by the toxin. Toxin binding to C (7) derivatives was reduced by about 50% compared with the native ganglioside. Molecular modeling of C (1) and C (7) derivatives in complex with CT-B gave a structural rationale for the observed differences in the relative affinities of the various derivatives. Loss of or altered hydrogen bond interactions involving the water molecules bridging the sialic acid to the protein was found to be the major cause for the observed drop in CT-B affinity in the smaller derivatives, while in the bulkier derivatives, hydrophobic interactions with the protein were found to partly compensate for these losses.

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We developed an assay method using a novel quenched fluorescent substrate (QFS) flanking the β-cleavage site of amyloid precursor protein (APP), and purified a candidate β-secre-tase from bovine brain. N-terminal amino acid analysis showed the candidate to be thimet oligopeptidase (TOP). The cDNA for human TOP was cloned from a human brain cDNA library and expressed in COS cells. The enzyme was further purified on a Ni²⁺-agarose column. TOP cleaved the Swedish Alzheimer's substrate (SEVNLDAEFR) as well as the normal substrate (SEVKMDAEFR). We then coexpressed TOP with APP 695 in COS cells, collected transfected cells and conditioned media, and analyzed them by immunoblotting. The antibody against the specific secreted APP cleaved by β-secretase (sAPPβ) detected the secretion of sAPPβ only from APP/hTOP-overexpressing cells, and not from cells overex-pressing of antisense hTOP cDNA. Finally, we analyzed the immunolocalization of overex-pressed hTOP in COS cells. Most hTOP was localized in the nuclei, but a small amount was localized in the Golgi or other organelles around the nuclei. These results suggest that TOP has a β-secretase-like activity responsible for the processing of APP.

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In Burkholderia glumae (formerly named Pseudomonas glumae), isolated as the causal agent of grain rot and seedling rot of rice, oxalate was produced from oxaloacetate in the presence of short-chain acyl-CoA such as acetyl-CoA and propionyl-CoA. Upon purifica-tion, the enzyme responsible was separated into two fractions (tentatively named fractions II and III), both of which were required for the acyl-CoA-dependent production of oxalate. In conjugation with the oxalate production from oxaloacetate catalyzed by fractions II and III, acetyl-CoA used as the acyl-CoA substrate was consumed and equivalent amounts of CoASH and acetoacetate were formed. The isotope incorporation pattern indicated that the two carbon atoms of oxalate are both derived from oxaloacetate, and among the four carbon atoms of acetoacetate two are from oxaloacetate and two from acetyl-CoA. When the reaction was carried out with fraction II alone, a decrease in acetyl-CoA and an equivalent level of net utilization of oxaloacetate were observed without appreciable formation of CoASH, acetoacetate or oxalate. It appears that in the oxalate production from oxaloacetate and acetyl-CoA, fraction II catalyzes condensation of the two substrates to form an intermediate which is split into oxalate and acetoacetate by fraction III being accompanied by the release of CoASH.

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A ligand containing an SNpys group, i.e. 3-nitro-2-pyridinesulfenyl linked to a mercapto (or thiol) group, can bind covalently to a free mercapto group to form a disulfide bond via the thiol-disulfide exchange reaction. This SNpys chemistry has been successfully applied to the discriminative affinity labeling of μ and δ opioid receptors with SNpys-containing enkephalins [Yasunaga, T. et al. (1996) J. Biochem. 120, 459-465]. In order to explore the mercapto groups conserved at or near the ligand binding sites of three opioid receptor subtypes, we synthesized two Cys (Npys)-containing analogs of dynorphin A, namely, [D-Ala², Cys (Npys)⁸] dynorphin A-(1-9) amide (1) and [D-Ala², Cys (Npys)¹²] dynorphin A-(1-13) amide (2). When rat (μ and δ) or guinea pig (κ) brain membranes were incubated with these Cys (Npys)-containing dynorphin A analogs and then assayed for inhibition of the binding of DAGO (μ), deltorphin II (δ), and U-69593 (δ), the number of receptors decreased sharply, depending upon the concentrations of these Cys (Npys)-containing dynorphin A analogs. It was found that dynorphin A analogs 1 and 2 effectively label μ receptors (EC₅₀)=27-33 nM), but also label δ receptors fairly well (160-180 nM). However, for δ receptors they showed drastically different potencies as to affinity labeling; i.e., EC₅₀=210 nM for analog 1, but 10, 000 nM for analog 2. Analog 2 labeled κ receptors about 50 times more weakly than analog 1. These results suggested that dynorphin A analog 1 labels the Cys residues conserved in μ, δ, and κ receptors, whereas analog 2 only labels the Cys residues conserved in μ and δ receptors.

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