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Volume 128, Issue 1
Displaying 1-18 of 18 articles from this issue
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Kikuo Ogata, Rie Ohno, Kazuo Terao, Yaeta Endo2000 Volume 128 Issue 1 Pages 1-9
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTreatment of 30S-5SRNP with I M Cs2SO4 at 2°C overnight followed by sucrose densitygradient centrifugation yielded particles smaller than 30S-5SRNP, designated as CsSparticles. CsCl density-gradient centrifugation of CsS-particles showed the homogeneity of the particles containing about half the amount of proteins in 30S-5SRNP particles. The particles contained 18SrRNA, 5SRNP and about half the number of proteins in 30S-5SRNP. The ATPase activity of freshly prepared CsS-particles was about half the original 30S-5SRNP level although it was unstable even at 2°C. Poly (U) slightly enhanced the activity, and phe-tRNAphe, stimulated it concentration-dependently. EF-lα alone enhanced it, and in combination with poly (U) and phe-tRNAphe stimulated it markedly. EF-2 alone markedly increased it. The activity with the full components for elongation described above became very high, being comparable to that of the original 30S-5SRNP and twice that of 40S subunits. A two-dimensional electrophoretogram of the protein in CsS-particles revealed 9 small subunit protein species, in addition to L5, which included proteins interacting with mRNA and two elongation factors. Taken together with the results of our preceding study indicating the participation of ATPase of 80S ribosomes in peptide elongation, the present results indicate CsS-particles may be a part of the ATPase centre of 80S ribosomes.View full abstractDownload PDF (3439K) -
Makoto Kakinuma, Akimasa Hatanaka, Hideto Fukushima, Misako Nakaya, Ka ...2000 Volume 128 Issue 1 Pages 11-20
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSVarious recombinant light meromyosin (LMM) fragments were prepared from cDNAs encoding the 10°C and 30°C types of myosin heavy chain isoforms predominantly expressed in fast skeletal muscles of the 10°C-and 30°C-acclimated carp, respectively. These included three kinds of quarter fragments, 1/4-, 2/4-, and 4/4-quarter, composed of residues 1-130, 131-270, and 401-563 from the N-terminus, respectively, as well as three halves, N-, M-, and C-half fragments, containing residues 1-301, 131-400, and 302-563, respectively, and 69K fragments of residues 1-525. Unfortunately, in spite of extensive efforts, the 3/4-quarter fragment was not expressed for both 10°C and 30°C types in our expression system using Escherichia coli. All the LMM fragments except for the 10-and 30-2/4 quarters for the 10°C and 30°C types, respectively, exhibited a typical pattern of α-helix in CD spectrometry. When these were subjected to differential scanning calorimetry (DSC), 30°C-type LMM fragments were all found to be more thermostable than the 10°C-type counterparts. To identify amino acid substitutions responsible for different thermostabilities between the 10°C- and 30°C-type LMMs, six mutant proteins were prepared, mainly focusing on substitutions in the C-terminal half of LMM, and subjected to DSC and CD analyses. For three mutants in which two residues of the 10°C type were replaced by those of the 30°C type, 10-S355T/T361A, 10-M415L/L417V, and 10-S535A/ H536Q, the endothermic peaks in DSC increased by 1.4-2.0°C from that of the original 10°C type. The Tm values for two single-residue substitutions, 10-H449R and 10-T491I, shifted 0.8 and 1.3°C higher than that for the 10°C-type LMM, respectively, whereas the last mutant, 10-G61V, showed no change in thermostability. The finding that the difference in Tm values for major endothermic peaks from the 10-69K and 30-69K fragments was 4.6°C, which roughly corresponds to that between the original 10°C and 30°C types, suggested that the eight substitutions located in the C-terminal region of the 69K fragments (residues 302-525) are major candidates for the residues responsible for the difference in thermostability between the 10°C- and 30°C-type LMMs.View full abstractDownload PDF (3649K) -
Kunihiko Gekko, Tadashi Kamiyama, Eiji Ohmae, Katsuo Katayanagi2000 Volume 128 Issue 1 Pages 21-27
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo address the effects of single amino acid substitutions on the flexibility of Escherichia coli dihydrofolate reductase (DHFR), the partial specific volume (_??_°) and adiabatic compressibility (_??_°) were determined for a series of mutants with amino acid replacements at Gly 67 (7 mutants), Gly 121 (6 mutants), and Ala 145 (5 mutants) located in three flexible loops, by means of precise sound velocity and density measurements at 15°C. These mutations induced large changes in _??_° (0.710-0.733 cm3-g-1) and _??_° (-1.8×10-6-5.5×10-6 bar-1) from the corresponding values for the wild-type enzyme (_??_°=0.723 cm3.g-1, _??_s°=1.7×10-6 bar-1), probably due to modifications of internal cavities. The _??_s° value increased with increasing v°, but showed a decreasing tendency with the volume of the amino acid introduced. There was no significant correlation between _??_s° and the overall stability of the mutants determined from urea denaturation experiments. However, a mutant with a large _??_s° value showed high enzyme activity mainly due to an enhanced catalytic reaction rate (kcat) and in part due to increased affinity for the substrate (Km), despite the fact that the mutation sites are far from the catalytic site. These results demonstrate that the flexibility of the DHFR molecule is dramatically influenced by a single amino acid substitution in one of these loops and that the flexible loops of this protein play important roles in determining the enzyme function.View full abstractDownload PDF (1173K) -
Tadashi Nakai, Hisashi Mizutani, Ikuko Miyahara, Ken Hirotsu, Sou Take ...2000 Volume 128 Issue 1 Pages 29-38
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS4-Amino-4-deoxychorismate lyase (ADCL) is a member of the fold-type IV of PLP dependent enzymes that converts 4-amino-4-deoxychorismate (ADC) to p-aminobenzoate and pyruvate. The crystal structure of ADCL from Escherichia coli has been solved using MIR phases in combination with density modification. The structure has been refined to an R-factor of 20.6% at 2.2 Å resolution. The enzyme is a homo dimer with a crystallographic twofold axis, and the polypeptide chain is folded into small and large domains with an interdomain loop. The coenzyme, pyridoxal 5'-phosphate, resides at the domain interface, its re-face facing toward the protein. Although the main chain folding of the active site is homologous to those of D-amino acid and L-branched-chain amino acid aminotransferases, no residues in the active site are conserved among them except for Arg59, Lys159, and Glu193, which directly interact with the coenzyme and play critical roles in the catalytic functions. ADC was modeled into the active site of the unliganded enzyme on the basis of the X-ray structures of the unliganded and liganded forms in the D-amino acid and L-branched-chain amino acid aminotransferases. According to this model, the carboxylates of ADC are recognized by Asn256, Arg107, and Lys97, and the cyclohexadiene moiety makes van der Waals contact with the side chain of Leu258. ADC forms a Schiff base with PLP to release the catalytic residue Lys159, which forms a hydrogen bond with Thr38. The neutral amino group of Lys159 eliminates the α-proton of ADC to give a quinonoid intermediate to release a pyruvate in accord with the proton transfer from Thr38 to the olefin moiety of ADC.View full abstractDownload PDF (2129K) -
Yoshitaka Tanaka, Rie Tanaka, Takahiro Kawabata, Youichiro Noguchi, Ma ...2000 Volume 128 Issue 1 Pages 39-48
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCathepsin B, a lysosomal cysteine protease, is synthesized as a glycoprotein with two N-linked oligosaccharide chains, one of which is in the propeptide region while the other is in the mature region. When cultured rat hepatocytes were labeled with [32P]phosphate, 32P-labeled cathepsin B was immunoprecipitated only in the proform from cell lysates and medium. Either Endo H or alkaline phosphatase treatment of 32P-labeled procathepsin B demonstrated the acquisition of a mannose 6-phosphate (Man 6-P) residue on high mannose type oligosaccharides. To identify the site of phosphorylation, immunoisolated 35S- or 32P-labeled procathepsin B was incubated with purified lysosomal cathepsin D, since cathepsin D cleaves 48 amino acid residues from the N-terminus of procathepsin B, in which one N-linked oligosaccharide chain was also included [Kawabata, T. et al. (1993) J. Biochem. 113, 389-394]. Treatment of intracellular 35S-labeled procathepsin B with a molecular mass of 39-kDa with cathepsin D resulted in the production of the 31-kDa intermediate form, but the 32P-label incorporated into procathepsin B disappeared after treatment with cathepsin D. These results indicate that the phosphorylation of procathepsin B is restricted to an oligosaccharide chain present in the propeptide region. Interestingly, cathepsin B sorting to lysosomes was not inhibited by NH4Cl treatment and about 90% of the intracellular procathepsin B initially phosphorylated was secreted into the medium without being dephosphorylated intracellularly, and did not bind significantly to cation-independent-Man 6-P receptor, suggesting the failure of Man 6-P-dependent transport of procathepsin B to lysosomes. Additionally, about 50% of the newly synthesized 35S-labeled cathepsin B was retained in the cells in mature forms consisting of a 29-kDa single chain form and a 24-kDa two chain form, while part of the procathepsin B was associated with membranes in a Man 6-P-independent manner. Taken together, these results show that in rat hepatocytes, cathepsin B is targeted to lysosomes by an alternative mechanism (s) other than the Man 6-P-dependent pathway.View full abstractDownload PDF (3663K) -
Kyung-Jin Kim, Pao-Yu Chi, Kwang-Woo Hwang, Benjamin C. Stark, Dale A. ...2000 Volume 128 Issue 1 Pages 49-55
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe bacterium, Vitreoscilla, produces a ΔμNa+ across its membrane during respiration. A key enzyme for this function is the cytochrome bo terminal oxidase which, when incorporated into synthetic proteoliposomes, pumps Na+ across the membrane upon the addition of a substrate. A Vitreoscilla cytochrome bo knock out (cyo-) mutant was isolated by transposon mutagenesis using pUT-mini-Tn5Cm. The membranes of this mutant lacked the characteristic 416 nm peak and 432 nm trough in CO difference spectra, which are clearly visible in spectra of the Vitreoscilla wild-type, but peaks at 627, 560, and 530 nm in reduced minus oxidized difference spectra indicate that cytochrome bd is still present. The specific NADH oxidase and ubiquinol-1 oxidase activities of the cyo- mutant membranes were less than those of Vitreoscilla wild-type and Escherichia coli membranes, and the stimulation of these activities of the mutant and E. coli membranes by 75 mM NaCl was approximately 50% less than that of Vitreoscilla wild-type membranes. The ubiquinol-1 oxidase activity of the cyo- mutant membranes was inhibited by 10 mM KCN to a lesser degree than that of the Vitreoscilla wild-type and E. coli membranes (50, 80, and 85%, respectively). This result is also consistent with the cyo mutant membrane fragments containing only the cytochrome bd terminal oxidase, which is known to be less sensitive to KCN. Although the maximum respiration and growth of the cyo- mutant were less than those of the wild-type, this mutant is still capable of growing with cytochrome bd alone.View full abstractDownload PDF (2268K) -
Maki Kondo, Takashi Yamaoka, Soichi Honda, Yoshihiro Miwa, Rumi Katash ...2000 Volume 128 Issue 1 Pages 57-64
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe recently showed that an increased supply of purine nucleotides increased the growth rate of cultured fibroblasts. To understand the mechanism of the growth rate regulation, CHO Kl (a wild type of Chinese hamster ovary fibroblast cell line) and CHO ade -A (a cell line deficient in amidophosphoribosyltransferase, a rate-limiting enzyme of the de novo pathway) were cultured under various conditions. Moreover, a defective de novo pathway in CHO ade -A cells was exogenously restored by 5-amino-4-imidazole-carboxamide riboside, a precursor of the de novo pathway. The following parameters were determined: the growth rate of CHO fibroblasts, the metabolic rate of the de novo pathway, the enzyme activities of amidophosphoribosyltransferase and hypoxanthine phosphoribosyltransferase, the content of intracellular nucleotides, and the duration of each cell-cycle phase. We concluded the following: (i) Purine de novo synthesis, rather than purine salvage synthesis or pyrimidine synthesis, limits the growth rate. (ii) Purine nucleotides are synthesized preferentially by the salvage pathway as long as hypoxanthine is available for energy conservation. (iii) The GTP content depends on the intracellular ATP content. (iv) Biosynthesis of purine nucleotides increases the growth rate mainly through ATP production and promotion of the G1/S transition.View full abstractDownload PDF (740K) -
Hiroaki Segawa, Tetsuro Yamashita, Masao Kawakita, Hideharu Taira2000 Volume 128 Issue 1 Pages 65-72
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe roles of N-linked glycosylation in the intracellular transport and fusion activity of the Sendai virus fusion (F) protein were studied. Each of three potential glycosylation motifs (designated g1, g2, and g3) in the F protein was mutated separately or in combination with the other sites. When the mutant F proteins were transiently expressed in COS cells, they showed significant changes in electrophoretic mobility, indicating that all three motifs in the F protein are glycosylated. Glycosylation-defective mutants which lacked the g2-oligosaccharide chain showed decreased immunoreactivity with a monoclonal antibody specific for the native conformation and were inefficiently transported to the cell surface. Such mutants, with the exception of a double mutant lacking gl and g2-oligosaccharide chains, were also not able to induce syncytia formation when cells expressing them plus the hemagglutinin-neuraminidase protein were treated with trypsin. Mutations at the other glycosylation sites did not significantly affect the immunoreactivity with the monoclonal antibody or the efficiency of intracellular transport of the F protein. These results indicate that the N-linked oligosaccharide chain attached at g2 is important for efficient intracellular transport and for the fusion activity of the F protein.View full abstractDownload PDF (3262K) -
Hisashi Mizutani, Ikuko Miyahara, Ken Hirotsu, Yasuzo Nishina, Kiyoshi ...2000 Volume 128 Issue 1 Pages 73-81
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe three-dimensional structure of the purple intermediate of porcine kidney D-amino acid oxidase (DAO) was solved by cryo-X-ray crystallography; the purple intermediate is known to comprise a complex between the dehydrogenated product, an imino acid, and the reduced form of DAO. The crystalline purple intermediate was obtained by anaerobically soaking crystals of oxidized DAO in a buffer containing excess D-proline as the substrate. The dehydrogenated product, Δ1-pyrrolidine-2-carboxylate (DPC), is found sandwiched between the phenol ring of Tyr 224 and the planar reduced flavin ring. The cationic protonated imino nitrogen is within hydrogen-bonding distance of the backbone carbonyl oxygen of Gly 313. The carboxyl group of DPC is recognized by the Arg 283 guanidino and Tyr 228 hydroxyl groups through ion-pairing and hydrogen-bonding, respectively. The +HN=C double bond of DPC overlaps the N (5)-C (4a) bond of reduced flavin. The electrostatic effect of the cationic nitrogen of DPC is suggested to shift the resonance hybridization of anionic reduced flavin toward a canonical form with a negative charge at C (4a), thereby augmenting the electron density at C (4a), from which electrons are transferred to molecular oxygen during reoxidation of reduced flavin. The reactivity of reduced flavin in the purple intermediate, therefore, is enhanced through the alignment of DPC with respect to reduced flavin.View full abstractDownload PDF (3281K) -
Jiro Takano, Masanori Watanabe, Kiyotaka Hitomi, Masatoshi Maki2000 Volume 128 Issue 1 Pages 83-92
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCalpastatin, a specific inhibitor of calpain, consists of a unique N-terminal domain (domain L) and four repetitive protease-inhibitor domains (domains 1-4). The isolated cDNAs from various mammalian species have conspicuous differences in the regions encoding the N-terminal sequences and can be classified into four types. Mouse and bovine calpastatins (Type I and Type II, respectively), which also differ from each other in the uttermost N-terminal regions, possess longer domain L sequences than those of rabbit, pig, and human inhibitors (Type Ill). A sequence of a shorter isoform, registered in a DNA data bank, starts from a part of domain 2 with a different N-terminal sequence (Type IV). To clarify the source of this molecular diversity, we investigated the entire exon-intron organization of the mouse calpastatin gene. The previously obtained mouse calpastatin cDNA is encoded by as many as 31 exons including the first exon, designated lxa. Three additional exons specifying the N-terminal sequences of the other types (designated exons lxb, Iu, and 14t, respectively) were identified in the mouse genomic DNA sequence. While the mRNAs for Types I and III were expressed at high levels in liver, the Type II mRNA was abundant in heart and skeletal muscle and expressed at lower levels in liver, brain and testis. The Type IV mRNA was specifically expressed in testis among the tissues examined. These results suggest that the calpastatin isoforms possessing different N-terminal sequences are generated by alternative transcription initiation from their own promoters and skipping of the mutually exclusive exons.View full abstractDownload PDF (4983K) -
Chika Yamashita, Makiko Kudo, Hiroko Ishida, Mitsuhide Noshiro, Yuri A ...2000 Volume 128 Issue 1 Pages 93-99
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe role of serum insulin in regulating the expression level of hepatic sterol 14-demethylase P450 (CYP51) was examined. Administration of streptozotocin, which destroys pancreatic β-cells, caused reduction of CYP51 mRNA level in rats in parallel with the loss of serum insulin. Streptozotocin treatment also reduced the CYP51 activity. The decreased mRNA level and activity of the streptozotocin-treated rats were restored to the normal level within 24 h by repeated administration of insulin. CYP51 level of normal rats was insensitive to the circadian variation of serum insulin and insulin administration, and no significant difference was observed between the hepatic CYP51 activities of Sprague-Dawley and Wistar lean rats, although the serum insulin concentration of the latter was higher than the former. These facts indicate that the expression of hepatic CYP51 is maintained by serum insulin, and its lowest physiological level is sufficient for supporting the expression of CYP51. The responses of CYP51 expression to streptozotocin and insulin treatments were closely similar to those of the sterol regulatory element binding protein (SREBP)-1c expression [Shimomura et al. (1999) Proc. Nat. Acad. Sci. USA 96, 13656-13661]. Based on this fact, the possible contribution of SREBP-1c to the insulin-dependent expression of hepatic CYP51 gene was also discussed.View full abstractDownload PDF (3714K) -
Tsuyoshi Yamaguchi, Nobuyuki Takei, Kazuaki Araki, Kyoko Ishii, Takash ...2000 Volume 128 Issue 1 Pages 101-106
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA eDNA clone for a novel homologue to γ-glutamyl transpeptidase (γ-GTP), termed GTPH, was isolated from a rat brain expression cDNA library using antisera against total brain synaptosomal fractions. The cloned GTPH consists of 641 amino acid residues (78 kDa) and exhibits structural similarity with a conventional type of γ-GTP that is predominantly expressed in the liver: They share significant amino acid homology (33% identity, 73% similarity) spanning over the entire sequence. RNA analyses revealed that GTPH mRNA expression is found only in the nervous system, including all brain regions, eyes and peripheral ganglia, and increases during development. Endogenous GTPH protein is a membrane-bound glycoenzyme and migrates as 90-100 kDa in polyacrylamide gels. Taken together, GTPH is a novel form of a γ-GTP-like molecule expressed exclusively in the nervous system.View full abstractDownload PDF (1800K) -
Koichi Nishigaki, Mohammed Naimuddin, Keiichi Hamano2000 Volume 128 Issue 1 Pages 107-112
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSpecies identification is the basis of Biology and has been carried out based on phenotype. Although some genes, such as that for 16S rRNA, have been used for species confirmation, identification of species based only on genotype has never been done before, although recent whole genome sequencing studies have demonstrated it to be possible in principle. However, it is evidently unrealistic for routine experiments of species identification. This paper clarifies that a very limited amount of information derived from a genome sequence is sufficient for identifying the species. It also proves that Genome Profiling [Nishigaki, K., Amano, N., and Takasawa, T. (1991) Chem. Lett. 1097-1100], TGGE analysis of random PCR products, can not only fulfill such requirements, but also serve as a universal method to analyze species. Thus, this compact technology can be used in many fields of biology, especially in microbe-related disciplines such as microbial ecology and epidemiology where exact knowledge about all members of a population is essential but previously difficult to obtain. This is the first demonstration that genotype-based identification of species is possible using a simple and uniform protocol for all organisms.View full abstractDownload PDF (1667K) -
Jinhu Yin, Guozhong Jing2000 Volume 128 Issue 1 Pages 113-119
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA series of N-terminal fragments of staphylococcal nuclease with different chain lengths has been taken as an in vitro nascent peptide folding model. Previous studies have shown that nascent peptide folding of the nuclease may begin early in the synthetic process with the content of ordered secondary structure increases with increasing peptide chain length, and that conformational adjustments are observed at certain stages during nascent peptide folding. Here, we focus attention on the conformational changes in the later stage of nascent peptide folding of the nuclease when the N-terminal fragment elongates nearly to the C-terminus of the nuclease in order to determine the role of the C-terminal region of the nuclease in the formation of the integrated conformation of the nuclease. We compared the conformational features of SNase R and its larger N-terminal fragments SNR 135, SNR 139, SNR 140, and SNR 141 using circular dichroism spectra, ANS-binding fluorescence and intrinsic fluorescence spectra. The results show that Trp 140 is important for the enrichment of ordered secondary structure and for producing a greater ability to fold into a native-like conformation, but Ser 141 is essential for the formation of the integrated conformation of the nuclease with a tightly packed tertiary structure. Note that the addition of only one residue to the C-terminus of elongating peptide chain can cause a dramatic change in conformation. The data also show the occurrence of continuous adjustments in conformation during peptide elongation, even after a rigid tertiary structure has formed, suggesting that the last eight residues (residues 142-149), which are disordered at the C-terminus of the nuclease, also possess a structural role, forming the native tertiary structure to provide a framework for the active site, even though they are remote from the active site in both sequence and spatial structure.View full abstractDownload PDF (679K) -
Tomohiro Yamamoto, Ayumu Nozaki, Syunichi Shintani, Syuhei Ishikura, Y ...2000 Volume 128 Issue 1 Pages 121-128
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe NADP (H)-linked oxidoreductase activity of a major isozyme of human liver 3α-hydroxysteroid dehydrogenase was activated 5-, 4-, and 2-fold by D-thyroxin (T4), L-T4 and DL-3, 3', 5'-triiodothyronine (reverse T3), respectively. Kinetic analysis of the activation indicated that D-T4, L-T4, and reverse T3 are non-essential activators, showing binding constants of 1.5, 1.1, and 3.6 μM, respectively. Comparison of the effects of the T4 analogs on the activities of the mutant enzymes suggests that the binding site is composed of at least Lys-270, Arg-276, and the C-terminal loop of the enzyme. L-T3, DL-thyronine, and D-tyrosine had no effect on the enzyme, but 3, 5, 3', 5'-tetra-and 3, 5, 3'-tri-iodothyropropionic acids were potent competitive inhibitors with K1 values of 42 and 60 nM, respectively, with respect to the substrate. The inhibition constant was lowered upon the activation of the enzyme by D-T4, and the inhibition by the deamino derivatives of T4 and T3 disappeared upon modification of the C-terminal loop of the enzyme, but not upon replacement of Lys-270 or Arg-276 with Met. These results indicate that, depending on their structures, the T4 analogs bind differently to two distinct sites at the active center of the enzyme to produce stimulatory and inhibitory effects.View full abstractDownload PDF (2330K) -
Shushi Nagamori, Ken-ichi Nishiyama, Hajime Tokuda2000 Volume 128 Issue 1 Pages 129-137
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSecG, a membrane component of the protein translocation apparatus of Escherichia coli, undergoes membrane topology inversion, which is coupled to the membrane insertion and deinsertion cycle of SecA. Eighteen SecG derivatives possessing a single cysteine residue at various positions were constructed and expressed in a secG null mutant. All the SecG-Cys derivatives retained the SecG function, and stimulated protein translocation both in vivo and in vitro. Inverted membrane vesicles containing a SecG-Cys derivative were labeled with a membrane-permeable or -impermeable sulfhydryl reagent before or after solubilization with a detergent. The accessibility of these reagents to the cysteine residue of each derivative determined the topological arrangement of SecG in the membrane. Derivatives having the cysteine residue in the periplasmic region each existed as a homodimer crosslinked through disulfide bonds, indicating that two SecG molecules closely co-exist in a single translocation machinery. The crossfinking did not abolish the SecG function and the crosslinked SecG dimer underwent topology inversion upon protein translocation.View full abstractDownload PDF (4786K) -
Shin-ichi Wato, Kaeko Kamei, Tsutomu Arakawa, John S. Philo, Jie Wen, ...2000 Volume 128 Issue 1 Pages 139-144
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWhite kidney bean (Phaseolus vulgaris) contains two kinds of α-amylase inhibitors, one heat-stable (αAl-s) and one heat-labile (αAl-u). αAI-s has recently been revealed to be a tetrameric complex, α2β2, with two active sites [Kasahara et al. (1996) J. Biochem. 120, 177-183]. The present study was undertaken to reveal the molecular features of αAI-u, which is composed of three kinds of subunits, α, β, and γ. The γ-subunit, in contrast to the α- and β-subunits that are indistinguishable from the α- and β-subunits of αAl-s, was found to correspond to a subunit of an α-amylase inhibitor-like protein, which has been identified as an inactive, evolutionary intermediate between arcelin and the α-amylase inhibitor in a P. vulgaris defense protein family. The polypeptide molecular weight of αAI-u determined by the light-scattering technique, together with the polypeptide molecular weights of the subunits, suggests that αAl-u is a trimeric complex, αβγ. The inhibition of αAI-u by increasing amounts of porcine pancreatic α-amylase (PPA) indicates that an inactive 1:1 complex is formed between αAI-u and PPA. Molecular weight estimation of the complex by the light-scattering technique confirmed that it is a complex of αAl-u with one PPA molecule. Thus it seems probable that αAl-u is an evolutionary intermediate of the P. vulgaris α-amylase inhibitor.View full abstractDownload PDF (2075K) -
Keishi Sakaguchi, Nozomu Okino, Noriyuki Sueyoshi, Hiroyuki Izu, Makot ...2000 Volume 128 Issue 1 Pages 145-152
Published: 2000
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSEndoglycoceramidase (EGCase) is an enzyme capable of cleaving the glycosidic linkage between oligosaccharides and ceramides of various glycosphingolipids. We previously reported that the Asn-Glu-Pro (NEP) sequence is part of the active site of EGCase of Rhodococcus sp. strain M-777. This paper describes the molecular cloning of a new EGCase gene utilizing the NEP sequence from the genomic library of Rhodococcus sp. strain C9, which was clearly distinguishable from M-777 by 16S rDNA analysis. C9 EGCase possessed an open reading frame of 1, 446 by encoding 482 amino acids, and showed 78% and 76% identity to M-777 EGCase II at the nucleotide and amino acid levels, respectively. Interestingly, C9 EGCase showed the different specificity to the M-777 enzyme: it hydrolyzed b-series gangliotetraosylceramides more slowly than the M-777 enzyme, whereas both enzymes hydrolyzed a-series gangliosides and neutral glycosphingolipids to the same extent.View full abstractDownload PDF (1392K)
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