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Hiroyuki Nakaura, Fumi Yanaga, Iwao Ohtsuki, Sachio Morimoto
1999 Volume 126 Issue 3 Pages
457-460
Published: 1999
Released on J-STAGE: November 18, 2008
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The functional effects of two missense mutations in human cardiac troponin T, PhellOlle and Glu244Asp, associated with familial hypertrophic cardiomyopathy were examined by exchanging the bacterially expressed and purified mutant troponin T into rabbit cardiac skinned muscle fibers. Both mutations significantly increased the maximum force without affecting the cooperativity. The Glu244Asp mutation also increased the Call sensitivity of the force generation, as in the case of other mutations associated with a poor prognosis. On the other hand, the PhellOlle mutation, associated with a favorable prognosis, had no effect on the Ca
2+ sensitivity. The results strongly support the hypothesis that increased Call sensitivity is responsible for the pathogenesis of hypertrophic cardiomyopathy with a poor prognosis caused by mutations in troponin T.
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Chao-Tsai Liao, Wen-Horng Wang, Hou-Shan Yang, Jia-Perng Chen, Ban-Yan ...
1999 Volume 126 Issue 3 Pages
461-469
Published: 1999
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The promoter -10 binding region of the
Bacillus subtilis δ
A factor forms an amphiphilic α-helix with three conserved isoleucines located at four-residue intervals. To investigate the structural and functional roles of the three isoleucine residues, we constructed a series of
sigA mutants with single and double Ile-to-Ala substitutions on the hydrophobic face of this α-helix and isolated intragenic revertants with either same-site or second-site suppressor that partially restores the structural stability and transcription activity of the mutant δ
A factors. Our data show that the three conserved isoleucine residues (Ile-194, Ile-198, and Ile-202) are involved in the hydrophobic core packing of δ
A; they affect differentially and additively the structure and function of δ
A, with the central isoleucine residue (Ile-198) playing the most important role. By analogy with the crystal structure of a δ
70 peptide, it is apparent that interdigital interactions exist between the three conserved isoleucine residues and certain hydrophobic amino acids in region 2.1 of δ
A. They include at least the van der Waals contacts between Ile-194 and both Leu-145 and Ile-149, between Ile-198 and both Ile-149 and Tyr-153, as well as between Ile-202 and Tyr-153. The same-site suppressors, Val-194 and Val-198, restore the structural stability and transcription activity of δ
A by repacking the hydrophobic core of δ
A. The second-site suppressor (S291F) appears to be allele-specific, but it is not as effective as the same-site suppressors in restoring δ
A structure and function.
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Yuji Honda, Mitsunori Kirihata, Tamo Fukamizo, Satoshi Kaneko, Ken Tok ...
1999 Volume 126 Issue 3 Pages
470-474
Published: 1999
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4-Methylumbelliferyl β-chitotrioside ((GlcN)
3-UMB) was prepared from 4-methylumbel-liferyl tri-
N-acetyl-β-chitotrioside [(GlcNAc)
3-UMB] using chitin deacetylase from
Colletotrichum lindemuthianum, and hydrolyzed by chitosanase from
Streptomyces sp. N174. The enzymatic deacetylation of (GlcNAc)
3-UMB was confirmed by
1H-NMR spectroscopy and mass spectrometry. When the (GlcN)
3-UMB obtained was incubated with chitosanase, the fluorescence intensity at 450 nm obtained by excitation at 360 nm was found to increase with proportion to the reaction time. The rate of increase in the fluorescence intensity was proportional to the enzyme concentration. This indicates that chitosanase hydrolyzes the glycosidic linkage between a GlcN residue and UMB moiety releasing the fluorescent UMB molecule. Since (GlcN)
3 itself cannot be hydrolyzed by the chitosanase, (GlcN)
3-UMB is considered to be a useful low molecular weight substrate for the assay of chitosanase. The
kcat and
Km values obtained for the substrate (GlcN)
3-UMB were determined to be 8.1×10
-5 s
-1 and 201 μM, respectively. From TLC analysis of the reaction products, the chitosanase was found to hydrolyze not only the linkages between a GlcN residue and UMB moiety, but also the linkages between GlcN residues. Nevertheless, the high sensitivity of the fluorescence detection of the UMB molecule would enable a more accurate determination of kinetic constants for chitosanases.
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Chiho Yoshinaga, Hideyuki Mukai, Masanao Toshimori, Masaaki Miyamoto, ...
1999 Volume 126 Issue 3 Pages
475-484
Published: 1999
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PKN is a fatty acid- and Rho GTPase-activated protein kinase whose catalytic domain in the carboxyl terminus is homologous to those of protein kinase C (PKC) family members. The amino terminal region of PKN is suggested to function as a regulatory domain, since tryptic cleavage or the binding of Rho GTPase to this region results in protein kinase activation of PKN. The structural basis for the regulation of PKN was investigated by analyzing the activity of a series of deletion/site-directed mutants expressed in insect cells. The amino-terminally truncated form of PKN (residue 455-942) showed low basal activity similar to that of the wild-type enzyme, and was arachidonic acid-dependent. However, further deletion (residue 511-942) resulted in a marked increase in the basal activity and a decrease in the arachidonic acid dependency. A (His)
6-tagged protein comprising residues 455-511 of PKN (designated His-Iα) inhibited the kinase activity of the catalytic fragment of PKN in a concentration-dependent manner in competition with substrate (
Ki=0.6±0.2 μM). His-Iα also inhibited the activity of the catalytic fragment of PRK2, an isoform of PKN, but had no inhibitory effect on protein kinase A or protein kinase Cδ. The IC
50 value obtained in the presence of 40 μM arachidonic acid was two orders of magnitude greater than that in the absence of the modifier. These results indicate that this protein fragment functions as a specific inhibitor of PKN and PRK2, and that arachidonic acid relieves the catalytic activity of wild-type PKN from autoinhibition by residues 455-511 of PKN. Autophosphorylation of wild-type PKN increased the protein kinase activity, however, substitution of Thr64, Ser374, or Thr531 in the regulatory region of PKN with alanine, abolished this effect. Substitution of Thr774 in the activation loop of the catalytic domain of PKN with alanine completely abolished the protein kinase activity. These results suggest that these phosphorylation sites are also important in the regulation of the PKN kinase activity. Potential differences in the mechanism of activation between the catalytic regions of PKN and PRK2 are also discussed.
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Takanori Iwata, Kentaro Sakai, Masayuki Hori, Shunya Uchida, Takae Tow ...
1999 Volume 126 Issue 3 Pages
485-493
Published: 1999
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It has been reported that the urinary excretion of
N-acetyl-β-D-glucosaminidase (NAG), a lysosomal enzyme, transiently increases in human after treatment with human parathyroid hormone (hPTH) (1-34). We report here that hPTH (1-34) caused transient changes in the size and density of rat renal lysosomes following urinary excretion of NAG and other lysosomal enzymes tested. Percoll density gradient centrifugation revealed that hPTH (1-34) slightly but significantly increased the fraction of high density lysosomes (around 1.12 g/ml) 5-10 min after the treatment with hPTH (1-34), with a concomitant decrease in the fraction of intermediate density lysosomes (1.07-1.08 g/ml). On electron micrographs, some lysosomes in proximal tubules but not in distal tubules showed a change in morphology from circular to oval, and became enlarged and electron-dense 5-10 min after the treatment with hPTH (1-34). These responses to bPTH (1-34) were also reversible and transient. NAG excreted in urine after treatment with hPTH (1-34) had the molecular mass of a mature form in lysosomes and/or endosomes and was not a prepro- and/or pro-form of the enzyme. Thus, the changes in the density and size of renal lysosomes appear to be associated with the exocytosis of lysosomal enzymes by hPTH (1-34).
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Akihiko Tsuji, Shigeru Yoshida, Shin-ichi Hasegawa, Miwa Bando, Ichiro ...
1999 Volume 126 Issue 3 Pages
494-502
Published: 1999
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PACE4 (SPC4) is a member of the mammalian subtilisin-like proprotein convertase (SPC) family, which participates in maturation of precursor proteins. PACE4 is expressed at high levels in the anterior pituitary, central nervous system, the developing olfactory bulb, heart, and liver. Recently, we determined the gene structure of human PACE4. [Tsuji
et al. (1997)
J. Biochem. 122, 438-452]. The 5'-flanking region of PACE4 gene contains 12 E-boxes (E1 to E12) within 1 kb upstream of the transcription initiation site. To examine the function of these E-box elements in the regulation of PACE4 expression, deletion and mutation constructs of the 5'-flanking region were ligated to the luciferase gene and analyzed for promoter activity in HepG2 and GH4C1 cells, which express PACE4 at high level. Some differences were observed in the activity of each promoter construct between HepG2 and GH4C1 cells, although the overall profiles of activity for the promoter fragment series were similar regardless of cell type. We showed that the basal promoter activity of the PACE4 gene is first determined by sequences lying between -315 and -1 by and further regulated by positive and negative elements in the upstream region. Site-directed mutagenesis of E-boxes in these regulatory elements showed that the E10 E-box act as positive regulator, whereas an E-box cluster (E4-E9) acts as a negative regulator in both cells. E2 E-box acts as a positive regulator only in HepG2 cells. Other E-boxes (E1, E3, and E12) had no effect on the promoter activity. These results indicate that E-box elements play a critical role in controlling PACE4 expression in HepG2 and GH4C1 cells and that PACE4 expression is regulated by a mechanism distinct from that of other SPC family proteases.
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Kunio Fujiwara, Yukinobu Masuyama, Shiroki Yagisawa, Toshio Tanabe, Ma ...
1999 Volume 126 Issue 3 Pages
503-509
Published: 1999
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Among five monoclonal antibodies (AHA-1 to 5 mAbs) prepared against glutaraldehyde (GA)-conjugated histamine (HA) in our previous study, only mAb AHA-2 was found to detect HA specifically in rat brain neurons by an immunocytochemistry method (ICC) using GA as a tissue fixative. All the other mAbs, except for AMA-5, reacted with HA in the enterochromaffin-like cells (ECL cells) of rat stomach [Fujiwara
et al. (1997) Histochem.
Cell Biol. 107, 39-45]. Enzyme-linked immunosorbent assay (ELISA) binding and inhibition tests demonstrated that AHA-2 is specific for HA, with almost no detectable crossreaction with any other established or putative amino acid neurotransmitters, LH-RH, TRH, or peptides with N-terminal histidines. ELISA assays also suggested that the AHA-2 mAb recognizes a HA epitope structure different from the one recognized by the AHA-1 mAb. The immunostaining patterns with AHA-2 mAb, as seen in the five subgroups of the tuberomammillary nuclei in the rat posterior hypothalamus, were very similar to those described by Inagaki
et al. [(1988)
Brain Res. 439, 402-405; (1990)
Exp. Brain Res. 80, 374-380] and Panula
et al. [(1984) Proc. Natl. Acad. Sci. USA 81, 2572-2576; (1988)
J. Histochem. Cytochem. 36, 259-269] using polyclonal anti-HA serum. However, it was also noted that moderate numbers of immunoreactive nerve fibers projected into the median eminence. The present HA ICC method using AHA-2 mAb allows highly sensitive HA detection in brain, and thus might permit detailed studies of HA localization hitherto impossible using previously available anti-HA polyclonal antibodies produced against carbodiimide-conjugated HA.
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Motonari Tsubaki, Tatsushi Mogi, Hiroshi Hori
1999 Volume 126 Issue 3 Pages
510-519
Published: 1999
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Cytochrome
bd-type ubiquinol oxidase contains two hemes
b (
b558 and
b595) and one heme
d as the redox metal centers. To clarify the structure of the reaction center, we analyzed
Escherichia coli cytochrome
bd by visible absorption, EPR and FTIR spectroscopies using azide and cyanide as monitoring probes for the exogenous ligand binding site. Azide-binding caused the appearance of a new EPR low-spin signal characteristic of ferric iron-chlorin-azide species and a new visible absorption band at 647 nm. However, the bound azide (
14N
3) anti-symmetric stretching infrared band (2, 010.5cm
-1) showed anomalies upon
15N-substitutions, indicating interactions with surrounding protein residues or heme
b595 in close proximity. The spectral changes upon cyanide-binding in the visible region were typical of those observed for ferric iron-chlorin species with diol substituents in macrocycles. However, we found no indication of a low-spin EPR signal corresponding to the ferric iron-chlorin-cyanide complexes. Instead, derivative-shaped signals at
g=3.19 and
g=7.15, which could arise from the heme
d(Fe
3+)-CN-heme
b595 (Fe
3+) moiety, were observed. Further, after the addition of cyanide, a part of ferric heme d showed the rhombic high-spin signal that coexisted with the
gz=2.85 signal ascribed to the minor heme
b595-CN species. This indicates strong steric hindrance of cyanide-binding to ferric heme d with the bound cyanide at ferric heme
b595.
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Tomoyasu Nishizawa, Munehiko Asayama, Kiyonaga Fujii, Ken-ichi Harada, ...
1999 Volume 126 Issue 3 Pages
520-529
Published: 1999
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Peptide-synthetase-encoding DNA fragments were isolated by a PCR-based approach from the chromosome of
Microcystis aeruginosa K-139, which produces cyclic heptapeptides, 7-desmethylmicrocystin-LR and 3, 7-didesmethylmicrocystin-LR. Three open reading frames (
mcyA, mcyB, mcyC) encoding m
-icrocy
-stin synthetases were identified in the gene cluster. Sequence analysis indicated that McyA (315 kDa) consists of two modules with an
N-methylation domain attached to the first and an epimerization domain attached to the second; Mcyll (242 kDa) has two modules, and McyC (147 kDa) contains one module with a putative C-terminal thioesterase domain. Conserved amino acid sequence motifs for ATP binding, ATP hydrolysis, adenylate formation, and 4'-phosphopantetheine attachment were identified by sequence comparison with authentic peptide synthetase. Insertion mutations in
mcyA, generated by homologous recombination, abolished the production of both microcystins in
M. aeruginosa K-139. Primer extension analysis demonstrated lightdependent mcy expression. Southern hybridization and partial DNA sequencing analyses of six microcystin-producing and two non-producing
Microcystis strains suggested that the microcystin-producing strains contain the mcy gene and the non-producing strains can be divided into two groups, those possessing no mcy genes and those with mcy genes.
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Haruko Ueda, Takeshi Saitoh, Kyoko Kojima, Haruko Ogawa
1999 Volume 126 Issue 3 Pages
530-537
Published: 1999
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An
N-acetylglucosamine (GlcNAc)/
N-acetylneuraminic acid-specific lectin from the fruiting body of
Psathyrella velutina (PVL) is a useful probe for the detection and fractionation of specific carbohydrates. In this study, PVL was found to exhibit multispecificity to acidic polysaccharides and sulfatides. Purified PVL and a counterpart lectin to PVL in the mycelium interact with heparin neoproteoglycans, as detected by both membrane analysis and solid phase assay. The pH-dependencies of the binding to heparin and GIcNAc
5-6 differ. The heparin binding of PVL is inhibited best by pectin, polygalacturonic acid, and highly sulfated polysaccharides, but not by GlcNAc, colominic acid, or other glycosaminoglycans. Sandwich affinity chromatography indicated that PVL can simultaneously interact with heparin- and GIeNAc-containing macromolecules. Extensive biotinylation was found to suppress the binding activity to heparin while the GIeNAc binding activity is retained. On the other hand, biotinyl PVL binds to sulfatide and the binding is not inhibited by GlcNAc,
N-acetylneuraminic acid, or heparin. These results indicate that PVL is a multi-ligand adhesive lectin that can interact with various glycocon-jugates. This multispecificity needs to be recognized when using PVL as a sugar-specific probe to avoid misleading information about the nature of glycoforms.
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Margarita Saiz, Young-Tae Ro, Dyann F. Wirth, Jean L. Pattersont
1999 Volume 126 Issue 3 Pages
538-544
Published: 1999
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Leishmaniavirus (LRV) is a double-stranded RNA (dsRNA) virus that persistently infects some strains of the protozoan parasite, Leishmania. LRV generates a short transcript, corresponding to the 5' end of the positive-sense RNA (320 nt),
via a cleavage event mediated by the viral capsid protein on the full-length positive sense RNA transcript. To address the possibility that the RNA cleavage represents a regulatory mechanism for maintaining persistent infection, the interactions between Leishmania cytoplasmic proteins and
in vitro synthesized viral transcripts were studied. In gel mobility shift experiments, three specific RNA/protein complexes were formed between cellular proteins and the cleaved viral transcript, and three major proteins were labeled by UV cross-linking. No protein binding activity was observed for either the short (320 nt) or full-length RNA transcripts. However, the two cleavage reaction products were able to form stable RNA/RNA complexes. We present a model in which the virus is targeting its own transcript for cleavage to promote binding of host factors to cryptic domains inaccessible in the full-length transcript.
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Sabato D'Auria, Roberto Nucci, Mose' Rossi, Enrico Bertoli, Fabio Tanf ...
1999 Volume 126 Issue 3 Pages
545-552
Published: 1999
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β-Glycosidase from the extreme thermophilic archaeon
Sulfolobus solfataricus is a tetrameric protein with a molecular mass of 240 kDa, stable in the presence of detergents, and with a maximal activity at temperatures above 95°C. Understanding the structureactivity relationships of the enzyme under different conditions is of fundamental importance for both theoretical and applicative purposes. In this paper we report the effect of methanol, ethanol, 1-propanol, and 1-butanol on the activity of S. solfataricus 6-glyco-sidase expressed in
Escherichia coli. The alcohols stimulated the enzyme activity, with 1-butanol producing its maximum effect at a lower concentration than the other alcohols. The structure of the enzyme was studied in the presence of 1-butanol by circular dichroism, and Fourier-transform infrared and fluorescence spectroscopies. Circular dichroism and steady-state fluorescence measurements revealed that at low temperatures the presence of the alcohol produced no significant changes in the tertiary structure of the enzyme. However, time-resolved fluorescence data showed that the alcohol modifies the protein microenvironment, leading to a more flexible enzyme structure, which is probably responsible for the enhanced enzymatic activity.
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Yuko Takaoka, Satoru Niwa, Hiroichi Nagai
1999 Volume 126 Issue 3 Pages
553-558
Published: 1999
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This study was conducted to investigate the mechanism of interleukin-1β (IL-1β)-induced IL-6 production in human osteoblasts (MG-63 cells). Stimulation with IL-1β resulted in the production of IL-6 and prostaglandin E
2 (PGE
2). IL-6 production gradually increased and peaked 96 h after stimulation. IL-6 mRNA was detected between 4 and 72 h after IL-1β stimulation. The patterns of PGEZ production and the expression of cyclooxygenase-2 (COX-2) mRNA were biphasic after stimulation. Actinomycin D, cycloheximide, indomethacin, and NS-398 (COX-2 inhibitor) suppressed the production of IL-6 and PGE
2. Anti -PGE
2, antibody markedly reduced the production of IL-6. In addition, stimulation with 17-pbenyl-PGE
2, a PGE receptor-1 (EP-1 receptor) agonist, led to the expression of IL-6 mRNA after pretreatment with IL-1β. These findings indicate that IL-1β-induced IL-6 production in MG-63 cells involves the following sequence of steps: IL-1β-induced COX-2 activation, PGE
2, production, and EP-1 receptor signaling prior to IL-6 production.
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Tadashi Yoshimoto, Tsutomu Kabashima, Kouichirou Uchikawa, Takahiko In ...
1999 Volume 126 Issue 3 Pages
559-565
Published: 1999
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Prolyl aminopeptidase from
Serratia marcescens specifically catalyzes the removal of N-terminal proline residues from peptides. We have solved its three-dimensional structure at 2.3 Å resolution by the multiple isomorphous replacement method. The enzyme consists of two contiguous domains. The larger domain shows the general topology of the α/β hydrolase fold, with a central eight-stranded β-sheet and six helices. The smaller domain consists of six helices. The catalytic triad (Ser113, His296, and Asp268) is located near the large cavity at the interface between the two domains. Cys271, which is sensitive to SH reagents, is located near the catalytic residues, in spite of the fact that the enzyme is a serine peptidase. The specific residues which make up the hydrophobic pocket line the smaller domain, and the specificity of the exo-type enzyme originates from this smaller domain, which blocks the N-terminal of P1 proline.
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Keishi Narita, Shin-ichi Ohnuma, Tokuzo Nishino
1999 Volume 126 Issue 3 Pages
566-571
Published: 1999
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Geranyl diphosphate synthase catalyzes the condensation of isopentenyl diphosphate with dimethylallyl diphosphate to give a C
10 compound, geranyl diphosphate, which is a precursor of all monoterpenoids. However, the gene has not been isolated from any organisms. To examine the possibility that geranyl diphosphate synthase has evolved from a common ancestor of the prenyltransferase family and to predict the active site structure, we tried to convert
Bacillus stearothermophilus farnesyl diphosphate synthase to geranyl diphosphate synthase, according to our previous findings. Several mutated farnesyl diphosphate synthases that have single amino acid substitutions before the first aspartaterich motif were constructed. A mutated enzyme that has the replacement of serine by phenylalanine at the fourth position before the motif exclusively produced geranyl diphosphate when dimethylallyl diphosphate was used as the primer, and hardly accepted geranyl diphosphate as a primer, indicating that this mutation causes the conversion to geranyl diphosphate synthase. This result supports the idea that the product specificities of all members of the
E-prenyltransferase family are mainly defined by a few structural features: the amino acids at the fourth position and the fifth position before the first aspartate-rich motif, and the insertion of two amino acids in the motif. This suggests that natural geranyl diphosphate synthases might have an active site structure similar to that of the mutated enzyme.
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Hajime J. Yuasa, Jos A. Cox, Takashi Takagi
1999 Volume 126 Issue 3 Pages
572-577
Published: 1999
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Calcium vector protein (CaVP) is an EF-hand Ca
2+-binding protein, which is unique to the protochordate, amphioxus. CaVP is supposed to act as a Ca
2+ signal transductor, but its exact function remains unknown. Not only its function but also its exact evolutionary relationship to other Ca
2+ -binding proteins is unclear. To investigate the evolution of CaVP, we have determined the complete sequences of Ca VP cDNAs from two amphioxus species,
Branchiostoma lanceolatum and
B. floridae, whose open reading frame cDNA and amino acid sequences show 96.5 and 98.2% identity, respectively. We have also elucidated the structure of the gene of
B. floridae CaVP, which is made up of seven exons and six introns. The positions of four of the six introns (introns 1, 2, 3, and 5) are identical with those of calmodulin, troponin C, and the Spec protein of the sea urchin. These latter proteins belong to the so-called troponin C superfamily (TnC superfamily) and thus CaVP likely also belongs to this family. Intron 6 is positioned in the 3' noncoding region and is unique to
CaVP, so it may represent a landmark of the CaVP lineage only. The position of intron 4 is not conserved in the genes of the TnC superfamily or CaVP, and seems to result from either intron sliding or the addition of an intron (randomly inserted into or close to domain III) to the genes of the TnC superfamily during their evolution.
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Kyoko Matsumoto, Mizue Morioka, Hajime Ishikawa
1999 Volume 126 Issue 3 Pages
578-583
Published: 1999
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A GroEL homolog produced by
Buchnera, an intracellular symbiotic bacterium of aphids, is not only a molecular chaperone but also a novel phosphocarrier protein, suggesting that this protein plays a role in a signal transducing system specific to bacteria living in an intracellular environment. This prompted us to look into phosphocarrier proteins of
Buchnera that may be shared in common with other bacteria. As a result, no evidence was obtained for the presence of sensor kinases of the two-component system in
Buchnera, which are found in many bacteria. It is possible that the lack of sensor kinases is compensated for by the mulitifunctional GroEL homolog in this symbiotic bacteria. In contrast, we successfully identified three phosphotransferase system genes, ptsH, ptsl, and
crr in
Buchnera, and provide evidence for their active expression. While the deduced amino acid sequences of these gene products, histidine-containing phosphocarrier protein, Enzyme I, and Enzyme III were similar to their counterparts in
Escherichia coli, the predicted isoelectric points of the
Buchnera proteins were strikingly higher. It was also suggested that
Buchnera Enzyme I, when produced in
E. coli, is able to accept the phosphoryl group from phosphoenolpyruvate, but not from ATP.
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Kaori Kunichika, Yoshio Hashimoto, Taiji Imotol
1999 Volume 126 Issue 3 Pages
584-590
Published: 1999
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To collect folding information, we screened and analyzed the recombinant hen lysozyme mutants which were not secreted from yeast. As model mutants, Leu8Arg, Ala10Gly, and Met12Arg were prepared by site-directed mutagenesis and analyzed as to whether they were secreted from yeast or not. Consequently, Ala10Gly was found to be secreted from yeast, but Leu8Arg and Met12Arg were not. Next, these mutants were expressed in
Escherichia coli and refolded in vitro. As a result, Ala10Gly folded as the wild-type did. Leu8Arg efficiently refolded in renaturation buffer containing glycerol. Met12Arg did not refold even in the presence of glycerol. These results show that the Ala10Gly mutation does not affect folding or stability, that Leu8Arg is too unstable to be secreted from yeast, and that Met12Arg may be very unstable or the mutation affects the folding pathway. We screened the mutants that were not secreted by yeast from a randomly mutated lysozyme library, and obtained Asp18His/Leu25Arg and Ala42Val/Ser50Ile/Leu56Gln. These two mutants were expressed in
E. coli and then refolded in the presence of urea or glycerol. These mutants were refolded only in the presence of glycerol. Each single mutant of Asp18His/Leu25Arg and Ala42Val/Ser50Ile/Leu56Gln was independently prepared and folded
in vitro. The results showed that Leu25Arg and Leu56Gln were the dominant mutations, respectively, which cause destabilization. These results show that the mutant lysozymes which were not secreted from yeast may be unstable or have a defect in the folding pathway. Thus, we established a screening system for selecting mutants which are unable to form a stable structure from random mutants.
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Akihiko Tsuji, Emi Hashimoto, Takayuki Ikoma, Takazumi Taniguchi, Kenj ...
1999 Volume 126 Issue 3 Pages
591-603
Published: 1999
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PACE4 (SPC4), a member of the subtilisin-like proprotein convertase (SPC) family of proteases that cleave at paired basic amino acids, exhibits a dynamic expression pattern during embryogenesis and colocalizes with bone morphogenetic proteins (BMPs). Recently Cui
et al. reported that the ectopic expression of α1-antitrypsin variant Portland (α1-PDX), an engineered serpin that contains the minimal SPC consensus motif in its reactive loop, blocks the proteolytic activation of BMP4, leading to abnormal embryogenic development [Cui, Y.
et al. (1998)
EMBO J. 17, 4735-4743]. TGF β-related factors such as BMPs are synthesized as inactive precursors and activated by limited proteolysis at multibasic amino acids. Therefore, an α1-PDX-inhibitable protease is thought to participate in BMP activation. However, conflicting properties, including sensitivity to α1-PDX, have been reported for PACE4. In this study, we examined whether α1-PDX is responsible for the inhibition of PACE4 by measuring the protease/inhibitor complex directly. Here we show that α1-PDX has the ability to form an SDS-stable acyl-intermediate (180 kDa) with PACE4
in vivo and
in vitro. Further, we characterized the PACE4 secreted into the culture medium from Cos-1 cells by a specific immunological assay. An α1-PDX-insensitive and decanoyl-RVKR-chloromethylketone-sensitive 60-kDa protease (s) is greatly activated in conditioned medium by PACE4 overexpression, suggesting that the activation of an unknown protease (s) other than PACE4 is the cause of the variation in the properties of PACE4. PACE4 is a Ca
2+ -dependent protease with an optimal Ca
2+ requirement of 2mM, and shows its highest activity at weakly basic pH. PACE4 activity is completely inhibited by EDTA and EGTA, but not by leupeptin. These results show that PACE4 activity can be inhibited by α1-PDX as well as furin (SPC1) and suggest that the inhibition of PACE4-mediated activation of factors such as BMPs by α1-PDX causes abnormal embryogenic development.
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Tetsuto Nakagawa, Motohiro Tani, Katsuhiro Kita, Makoto Ito
1999 Volume 126 Issue 3 Pages
604-611
Published: 1999
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Sphingolipid ceramide
N-deacylase is an enzyme capable of hydrolyzing the
N-acyl linkages of ceramides of various sphingolipids. Recently, it was found that the enzyme catalyzes the reverse hydrolysis reaction in which free fatty acids are condensed to lyso-sphingolipids to produce sphingolipids. This paper describes a simple method for the synthesis of fluorescence-labeled sphingolipids utilizing the condensation reaction of the enzyme.
N-TFAc-aminododecanoic acids were efficiently condensed by the enzyme to the lyso-forms of GM1 and sphingomyelin in glycine buffer (pH 10). The reaction products,
N-TFAc-amino-GM1 and sphingomyelin, were obtained with overall yields of 60%. The purified products were identified to be ω-amino-GM1 and ω-amino-sphingomyelin, respec-tively, by TLC and FAB-MS or ESI-LC/MS analysis after removal of the
N-TFAc by mild alkaline treatment. NBD-labeled GM1 and sphingomyelin were prepared from ω-amino-GM1 and ω-amino-sphingomyelin by coupling with 4-fluoro-NBD. These fluorescence-labeled substrates, C12-NBD-GMI and C12-NBD-sphingomyelin, were hydrolyzed by endoglycoceramidase and sphingomyelinase, respectively, to produce NBD-dodecanoyl-sphingosines, but were resistant to hydrolysis by sphingolipid ceramide
N-deacylase. C12-NBD-sphingomyelin was found to be a better substrate than the commercially available C6-NBD-sphingomyelin for the assay of sphingomyelinase from various sources. We also describe a new method to detect GM1-binding proteins using fluorescence-labeled GM1.
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Takayuki Kawakami, Toshiaki Suzuki, Sung Hee Baek, Chin Ha Chung, Hiro ...
1999 Volume 126 Issue 3 Pages
612-623
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL
FREE ACCESS
The deubiquitinylating enzymes (DUBs), that release free ubiquitin (Ub) from its precursors or ubiquitinylated proteins, are known to comprise of a large protein family in eukaryotes, but those in mammalian tissues remain largely unknown. Here we report the existence of unexpectedly large species of DUBs in both soluble and membrane-bound fractions of bovine brain, based on their ability to cleave
125I-labeled Ub-fused aNH-MHIS-PPEPESEEEEEHYC (designated as Ub-PESTc). Two cytosolic enzymes, tentatively called sDUB-1 and sDUB-2, with molecular masses of about 30 kDa were purified to near homogeneity by Ub-Sepharose affinity chromatography. sDUB-1 and sDUB-2 corresponded to UCH-L3 and UCH-L1/PGP 9.5, respectively. Intriguingly, the particulate fraction of the brain homogenate was found to also contain strong activities against
125I-Ub-PESTc, which can be solubilized by treatment with 5% n-heptyl-β-D-thioglucoside and 1% Nonidet P-40, but not by washing with 1M NaCl. From the solubilized material, two new 30-kDa, membranous DUBs (called mDUB-1 and mDUB-2) were purified to apparent homogeneity by Ub-Sepharose chromatography. Two other Ub-aldehyde sensitive DUBS, designated as mDUB-3 and mDUB-4, were also partially purified by conventional chromatographic operations. These mDUBs differed from each other in substrate specificity and exhibited different characteristics from the sDUBs, revealing that they are a new type of membranebound DUB. These results indicate the presence of divergent DUBs in mammalian brain, which may contribute to regulation of numerous pivotal cellular functions mediated by the covalent modification of Ub.
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Shingo Morii, Shizue Sawamoto, Yuhji Yamauchi, Masahiko Miyamoto, Masa ...
1999 Volume 126 Issue 3 Pages
624-631
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL
FREE ACCESS
Steroid monooxygenase of
Rhodococcus rhodochrous is a Baeyer-Villigerase catalyzing the insertion of an oxygen atom between the C
17- and C
20-carbons of progesterone to produce testosterone acetate. The 5.1-kbp-long
BamHI DNA fragment containing the steroid monooxygenase gene,
smo, was cloned from the chromosomal DNA and sequenced. The
smo gene is 1, 650 nucleotides long, starts with a TTG colon, and ends with a TGA codon. The deduced amino acid sequence indicates that the enzyme protein consist of 549 amino acid residues with a molecular mass of 60, 133. Thus, the molecular mass of the holoenzyme is 60, 919. The amino acid sequence is highly homologous (41.2% identity) to that of cyclohexanone monooxygenase of
Acinetobacter sp. In the upstream of the
smo gene, the genes of heat shock proteins,
dnaK, grpE, and
dnaJ, located on the complementary strand, and the DNA-inserts of pSMO and pD1, which contains the
ksdD gene, were joined at the BamHI site of the
dnaJ gene. The
smo gene was modified at the initiation codon to ATG and ligated with an expression vector to construct a plasmid, pSMO-EX, and introduced into Escherichia
coli cells. The transformed cells hyperexpressed the steroid monooxygenase as an active and soluble protein at more than 40 times the level in
R. rhodochrous cells. Purification of the recombinant monooxygenase from the
E. coli cells by simplified procedures yielded about 2.3 mg of enzyme protein/g wet cells. The purified recombinant steroid monooxygenase exhibited indistinguishable molecular and catalytic properties from those of the
R. rhodochrous enzyme.
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Yasumasa Nishito, Hirofumi Usui, Osamu Tanabe, Masahiro Shimizu, Masao ...
1999 Volume 126 Issue 3 Pages
632-638
Published: 1999
Released on J-STAGE: November 18, 2008
JOURNAL
FREE ACCESS
Human erythrocyte Mn
2+-dependent (C'A') and -independent (CA) protein-serine/threonine phosphatase (PP) 2A are composed of 34-kDa catalytic C' and C subunits, in which the metal dependency resides, and 63-kDa regulatory A' and A subunits, respectively. Each catalytic and regulatory subunit gave the same V8- and papain-peptide maps, respectively. Stoichiometric zinc and substoichiometric iron were detected in CA but not in C'A' [Nishito
et al. (1999)
FEBS Lett. 447, 29-33]. The Mn
2+-dependent protein-tyrosine phosphatase (PTP) activity of C'A' was about 70-fold higher than that of CA. Pre-incubation of CA with 25mM NaF changed CA to a Mn
2+-dependent form with higher PTP activity. The same NaF treatment had no effect on C'A'. Pre-incubation of C'A' with ZnCl
2, zinc-metallothionein, or FeCl
2 activated the Mn
2+-independent PP activity, but pre-incubation with FeCl
3 did not. Ascorbate in the pre-incubation and assay mixture significantly stimulated the effect of FeCl
2. Pre-incubation of C'A' with 5 μM ZnCl
2 and 15 μM FeCl
2 in the presence of 1mM ascorbate synergistically stimulated the Mu
2+-independent PP activity, with concomitant suppression of the Mn
2+-dependent PP and PTP activities. The PP and PTP activities of CA were unaffected by the same zinc and/or iron treatment. Micromolar concentrations of vanadate strongly inhibited the Mn
2+-dependent PP activity of C'A' but only slightly inhibited the PP activity of CA. Using the distinct effect of vanadate as an indicator, the interconversion between CA and C'A' with the above mentioned treatments was proved. These results support the notion that Mn
2+-independent CA is a Zn
2+- and Fe
2+-metalloenzyme, whose apoenzyme is Mn
Z+-dependent C'A'.
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