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Volume 109 (1991)
- Issue 6 Pages 799-
- Issue 5 Pages 667-
- Issue 4 Pages 499-
- Issue 3 Pages 371-
- Issue 2 Pages 199-
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Volume 109, Issue 3
Displaying 1-22 of 22 articles from this issue
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Harumi Takada, Tohru Yoshimura, Toshihisa Ohshima, Nobuyoshi Esaki, Ke ...1991 Volume 109 Issue 3 Pages 371-376
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe gene encoding the thermostable phenylalanine dehydrogenase [EC 1. 4. 1.-] of a thermophile, Thermoactinomyces intermedius, was cloed and its complete DNA sequence was determined. The phenylalanine dehydrogenase gene (pdh) consists of 1, 098 nucleotides and encodes 366 amino acid residues corresponding to the subunit (Mr 41, 000) of the hexameric enzyme. The amino acid sequence deduced from the nucleotide sequence of the pdh gene of T. intermedius was 56.0 and 42.1% homologous to those of the phenylalanine dehy-drogenases of Bacillus sphaericus and Sporosarcina ureae, respectively. It shows 47.5% homology to that of the thermostable leucine dehydrogenase from B. stearothermophilus. The pdh gene was highly expressed in E. coli JM109, the amount of phenylalanine dehydrogenase produced amounting up to about 8.3% of that of the total soluble protein. We purified the enzyme to homogeneity from transformant cells in a day, with a 58% recovery.View full abstractDownload PDF (666K) -
Shuichi Kojima, Yoshitaka Nishiyama, Izumi Kumagai, Kin-ichiro Miura1991 Volume 109 Issue 3 Pages 377-382
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIt has been shown that the P1 site (the center of the reactive site) of protease inhibitors corresponds to the specificity of the cognate protease, and consequently specificity of Streptomyces subtilisin inhibitor (SSI) can be altered by substitution of a single amino acid at the PI site. In this paper, to investigate whether similar correlation between inhibitory activity of mutated SSI and substrate preference of protease is observed for subtilisin BPN', which has broad substrate specificity, a complete set of mutants of SSI at the reaction site PI (position 73) was constructed by cassette and site-directed mutagenesis and their inhibitory activities toward subtilisin BPN' were measured. Mutated SSIs which have a polar (Ser, Thr, Gln, Asn), basic (Lys, Arg), or aromatic amino acid (Tyr, Phe, Trp, His), or Ala or Leu, at the PI site showed almost the same strong inhibitory activity toward subtilisin as the wild type (Met) SSI. However, the inhibitory activity of SSI variants with an acidic (Glu, Asp), or aΒ-branched aliphatic amino acid (Val, Ile), or Gly or Pro, at P1 was decreased. The values of the inhibitor constant (K1) of mutated SSIs toward subtilisin BPN' were consistent with the substrate preference of subtilisin BPN'. A linear correlation was observed between log(1/K1) of mutated SSIs and log(1/Km, ) of synthetic substrates. These results demonstrate that the inhibitory activities of P1 site mutants of SSI are linearly related to the substrate preference of subtilisin BPN', and indicate that the binding mode of the inhibitors with the protease may be similar to that of substrates, as in the case of trypsin and chymotrypsin. On the other hand, the Cys73 mutant showed only temporary inhibition after mixing of the SSI and subtilisin BPN', which may be due to irregular disulfide bridge formation in the SSI molecule.View full abstractDownload PDF (2113K) -
Takako Nishiya1991 Volume 109 Issue 3 Pages 383-388
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSInteractions of melittin and/or phospholipase A2, (PLA2) with circular dichroism (CD)-active phospholipid, bis(4'-n-octanoxyazobenzene-4-carboxyl)-L-α -phosphatidylcholine (CDPC), were studied. In the presence of melittin at a lipid-to-melittin molar ratio (Ri) of 5, multilamellar dispersion, composed of CDPC and dipalmitoylphosphatidylcholine with molar ratio of 1, underwent morphological change to form small melittin-lipid particles. When PLA2 was added to the melittin-lipid particles at 37°C, the CD band at 222 nm exhibited a remarkable enhancement depending on Ri, indicating the formation of melittin-PLA2-lipid complex. After a 30 min incubation of melittin-PLA2-lipid complex at 45°C in the presence of Ca2+, the CD band at 222 um was still enhanced and a new positive band at 356 nm was observed. On the other hand, in the absence of Ca2+, the CD enhancement characteristic of melittin-PLA2-lipid complex disappeared after the incubation at 45°C. These results suggest that the melittin-PLA2-lipid complex did not undergo any drastic morphological change upon PLA2-catalyzed hydrolysis of lipid, and that Ca2+ is indispens-able in order that the melittin-PLA2-lipid complex remains intact and PLA2 exerts efficient hydrolytic activity in the melittin-PLA2-lipid complex.View full abstractDownload PDF (578K) -
Yoshihiko Ohyama, Osamu Masumoto, Emiko Usui, Kyuichiro Okuda1991 Volume 109 Issue 3 Pages 389-393
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo solve the problem of whether a common enzyme catalyzes both 5β-cholestane-3α, 7α, 12α-triol 27-hydroxylation and 25-hydroxylation of 1α-hydroxyvitamin D3 (a synthetic compound used therapeutically for vitamin D-deficient diseases) in rat liver mitochondria, enzymological and kinetic studies were performed. A cytochrome P-450 was purified from female rat liver mitochondria based on these catalytic activities and it was found that the two enzyme activities accompanied each other at all purification steps. The 5β-cholestane-3α, 7α, 12α-triol 27-hydroxylation activity of the final preparation had a turnover number of 36 min-1, and the value of the corresponding 1α-hydroxyvitamin D3 25-hydroxylation activity was 1.4min-1. When the enzyme was partially denatured by heating at different temperatures, both enzyme activities declined in a parallel fashion. Treatment of the enzyme with N-bromosuccinimide decreased both enzyme activities in a similar manner. 5β-Cholestane-3α, 7α, 12α-triol competitively inhibited 25-hydroxylation of lα-hydroxy-vitamin D3, and vice versa. From these results it was concluded that 5β-cholestane-3α, 7α, 12α-triol 27-hydroxylation and lα-hydroxyvitamin D3 25-hydroxylation are catalyzed by a common enzyme in rat liver mitochondria.View full abstractDownload PDF (571K) -
Yoshihisa Tomioka, Kazuyuki Aihara, Akihiko Hirose, Takanori Hishinuma ...1991 Volume 109 Issue 3 Pages 394-398
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe subcellular distribution of Δ3, Δ2-enoyl-CoA isomerase [EC 5.3.3.8] and the inducing effect of clofibrate, a peroxisomal proliferator, on the enzyme activity were examined in rat liver. From the results of spectrophotometric investigation of the fractions, which were prepared by sucrose discontinuous gradient centrifugation from the light mitochondrial fraction, the isomerase activity was found in the fractions enriched in mitochondria and those enriched in peroxisomes of the control and the clofibrate treated rat livers. The anti-isomerase antibody reacted with both the mitochondrial isomerase and the perox-isomal isomerase, revealing a single band with an apparent molecular weight of 30, 000. However, the isomerase was induced by clofibrate administration mainly in the mito-chondrial fraction. These results suggest that Δ3, Δ2-enoyl-CoA isomerase is located in the mitochondria and the peroxisomes of the normal rat liver, and that the isomerase in the mitochondria is induced by clofibrate administration.View full abstractDownload PDF (955K) -
Masashi Kimura, Masafumi Hirono, Tohru Takemasa, Yoshio Watanabe1991 Volume 109 Issue 3 Pages 399-403
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSChange in actin mRNA level was investigated in the course of synchronous division in Tetrahymena induced by intermittent heat treatment. The level of actin mRNA decreased from just after the end of the heat treatment (EHT) to 45 min after EHT, and then promptly increased before synchronous division at 75 min after EHT. In contrast, levels of the total RNA and mRNAs of Tetrahymena calmodulin and calcium-binding protein of 25 kDa (TCBP-25) increased gradually from EHT to synchronous division. Drastic change in mRNA level before synchronous division seems to be unique to actin mRNA. From the effects of actinomycin D (50 μg/ml) on both synchronous division and actin mRNA level, the increase in actin mRNA level starting from 45 min after EHT is speculated to be prerequisite for the oncoming synchronous division. The results of a nuclear run-off experiment supported the above speculation.View full abstractDownload PDF (540K) -
Kazuya Yamanishi, Masayuki Takahashi, Tsutomu Nishida, Yasukazu Ohmoto ...1991 Volume 109 Issue 3 Pages 404-409
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA human truncated macrophage colony-stimulating factor (M-CSF) encoding the amino acid residues from 3 to 153 of the native M-CSF was expressed by using a two-cistron expression system in Escherichia coli. The truncated M-CSF found in inclusion bodies was renatured and had CSF activity. Purification, which included a QAE-ZeTa preparative cartridge concentration step followed sequentially by HPLC on TSK-gel Phenyl-5PW and TSK-gel DEAE-5PW columns, gave an overall yield of 63.8%. The purified truncated M-CSF had a specific activity of 4×107 units/mg of protein. Peptide mapping of a lysylendo-peptidase digest by reversed-phase HPLC confirmed the amino acid sequence predicted from the cDNA sequence. SDS-PAGE of the purified truncated M-CSF gave a single band at 17 kDa under reducing conditions and at 32 kDa under non-reducing conditions. Activated Thiol-Sepharose 6B column chromatography and other experiments failed to detect any free cysteine residue in spite of the existence of 7 cysteine residues in the truncated M-CSF subunit. These results indicate that it is a dimeric structure linked by one or more intermolecular disulfide bonds.View full abstractDownload PDF (1393K) -
Kaoru Omichi, Sumihiro Hase, Tokuji Ikenaka1991 Volume 109 Issue 3 Pages 410-415
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe active site of human salivary α-amylase is composed of tandem subsites (S3, S2, Sl, S1', S2', etc.) geometrically complementary to several glucose residues, and the glycosidic linkage of the substrate is split between S1 and S1'. As a matter of convenience, the subsites to which the non-reducing-end part (glycone) and the reducing-end part (aglycone) of the substrate being hydrolyzed are bound are named the glycone-binding site (S3, S2, S1) and the aglycone-binding site (S1', S2'), respectively. The features of the aglycone-binding site of human salivary α-amylase were examined by means of transglycosylation reaction using phenyl α-maltoside (GGΦ: G-G-Φ) and its derivatives (GAGΦ: G-AG-Φ, GCGΦ: G-CG-Φ, AGGΦ: AG-G-Φ, and CCGΦ: CG-G-Φ) in which one of the glucose residues (G) has been converted to 6-amino-6-deoxy-glucose (AG) or glucuronic acid (CG) residue as the acceptor. A fluorogenic derivative of maltotetraose, p-nitrophenyl O-6-deoxy-6- [(2-pyri-dyl)amino]-α-D-glucopyranosyl-(1→4)-O-α-D-glucopyranosyl-(1→4)-O-α-D-glucopyranosyl-(1→4)-α-D-glucopyranoside (FG4P, FG-G-G-G-P), was used as the substrate. HSA catalyzed both hydrolysis of FG4P to FG3 (FG-G-G) and p-nitrophenylα-glucoside (G-P) and transfer of the FG3 residue of FG4P to the acceptors. Transfer to GAGΦ occurred more effectively than to GGΦ. Transfers to GCGΦ and CGGΦ were less than to GGΦ and very little transfer to AGGΦ occurred. When transglycosylation reaction occurs, the acceptors should approach the aglycone-binding site (S1', S2') and their reducing-end and non-reducing-end glucose residues interact with S2' and S1', respectively. The results obtained suggested that AG and CG residues were hardly bound to S1' owing to the protonated amino group and carboxylate groups and that a negative charge of an acidic amino acid residue of the enzyme was near to the C-6 hydroxy group of the glucose residue bound to S2'.View full abstractDownload PDF (673K) -
Tamo Fukamizo, Sachio Goto1991 Volume 109 Issue 3 Pages 416-420
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe lysozyme-catalyzed reaction of chitooligosaccharide was carried out in a continuous flow system in which the solution of substrate, chitooligosaccharide [(G1cNAc)n], flowed into the lysozyme solution in an ultrafiltration apparatus and the products were filtered off. The filtrate was continuously collected in test tubes with the aid of a fraction collector. The product distribution in each fraction was analyzed by high performance gel filtration. Using (GlcNAc)5 as the substrate, the concentrations of products, (GlcNAc)1-4, increased gradually and came to the steady state when the volume of the outflow amounted to sixfold of the inside volume. Before reaching the steady state, the product distribution was quite different from that observed in the closed reaction system, in which the reaction species are not exchangeable through the boundary of the system. The outflows of (GlcNAc)3-5 were delayed in comparison with those of GlcNAc and (GlcNAc)2. The delay period increased with the decrease in substrate concentration, and was shortened by using the [Asp 101 or Trp 62]-modified lysozyme instead of the native lysozyme. These results suggest that the delay in the (GlcNAc)3-5 outflows is caused by the nonproductive binding of the oligosac-charide to the lysozyme molecule. The profile of the flow reaction yields information not only on the catalytic efficiency but also on the substrate binding efficiency of the lysozyme.View full abstractDownload PDF (592K) -
Yoshinori Kumazawa, Hyouta Himeno, Kin-ichiro Miura, Kimitsuna Watanab ...1991 Volume 109 Issue 3 Pages 421-427
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe present study shows unilateral aminoacylation specificity between bovine mitochon-dria and eubacteria (Escherichia coli and Thermus thermophilus) in five amino acid-specific aminoacylation systems. Mitochondrial synthetases were capable of charging eubacterial tRNA as well as mitochoildrial tRNA, whereas eubacterial synthetases did not efficiently charge mitochondrial tRNA. Mitochondrial phenylalanyl-, threonyl-, arginyl-, and lysyl-tRNA synthetases were shown to charge and discriminate cognate E. coli tRNA species from noncognate ones strictly, as did the corresponding E. coli synthetases. By contrast, mitochondrial seryl-tRNA synthetase not only charged cognate E. coli serine tRNA species but also extensively misacylated noncognate E. coli tRNA species. These results suggest a certain conservation of tRNA recognition mechanisms between the mitochondrial and E. coli amionoacyl-tRNA synthetases in that anticodon sequences are most likely to be recognized by the former four synthetases, but not sufficiently by the seryl-tRNA synthetase. The unilaterality in aminoacylation may imply that tRNA recogni-tion mechanisms of the mitochondrial synthetases have evolved to be, to some extent, simpler than their eubacterial counterparts in response to simplifications in the species-number and the structural elements of animal mitochondrial tRNAs.View full abstractDownload PDF (837K) -
Yoshiharu Ohoka, Shin-ichi Imai, Tohru Kozasa, Tomohiko Maehama, Katsu ...1991 Volume 109 Issue 3 Pages 428-435
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA GTP-binding protein with an Mr of 24, 000 was purified from a cholate extract of bovine brain membranes in addition to the previously reported αβγ-trimeric GTP-binding proteins (G proteins). Partial amino acid sequence analysis of the purified 24-kDa protein revealed that it was not identical to any of the low Mr GTP-binding proteins already reported, but similar to the rac-gene products serving as the substrate of an ADP-ribosyltransferase (C3) purified from the culture medium of Clostridium botulinum type C. However, the 24-kDa protein was not ADP-ribosylated by the botulinum C3 enzyme. The 24-kDa protein was purified as a nucleotide-free form and characterized by the following unique properties distinct from those of αβγ-trimeric G proteins. (1) Mg2+ was essentially required for nucleotide binding to the 24-kDa protein; there was a progressive increase in its binding affinity for nucleotides as the concentration of the divalent cation was increased. (2) Nucleotides previously bound to the 24-kDa protein were rapidly dissociated from the protein in Mg2+-free medium, in accord with the fact that the protein was indeed purified as a nucleotide-free form with Mg2+-free solutions. (3) The 24-kDa protein apparently exhibited much lower GTPase activity than do αβγ-trimeric G proteins because the product GDP was released from the 24-kDa protein in exchange for the substrate GTP only at a very low rate. Based on these findings, a possible role of the 24-kDa protein in cellular signalling is discussed in comparison with well characterized αβγ-trimeric G proteins.View full abstractDownload PDF (1363K) -
Hitoshi Takamura, Makoto Kito1991 Volume 109 Issue 3 Pages 436-439
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA highly sensitive method was developed for quantitative analysis of phospholipid molecular species. Diradylglycerols prepared from phospholipids with phospholipase C were converted to the anthroyl-diradylglycerol derivatives, which could be separated into molecular species and sensitively quantified by reverse-phase HPLC using a fluorescence detector. All the molecular species of the derivatives had the same peak area per mole, and the peak areas were proportional to the amounts of the derivatives. Quantification could be carried out at the femtomole level.View full abstractDownload PDF (423K) -
Hiroshi Nakatani, Kazuhiko Ishikawa1991 Volume 109 Issue 3 Pages 440-443
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA numerical computing method to estimate pK and heat of ionization directly from initial velocity data was developed for systematic analysis of rate parameters. Values of rate and thermodynamic parameters were determined together with standard deviations, without using Arrhenius and van't Hoff plots. Distributions of pK and heat of ionization of small-molecular derivatives of aliphatic carboxylates, imidazoles, thiols, and aliphatic amines as possible models for catalytic residues of enzymes were analyzed from a ther-modynamic point of view. Each group was classified in a parallelogram without overlapping, except for a part of the imidazole and thiol groups. It was shown that values of pK and heat of ionization of carboxylates and histidine residues involved in the catalytic activity of enzymes determined from pH-profile experiments can be classified into similar, though somewhat extended, areas to those of the corresponding small-molecular derivatives. Identification of catalytic residues using values of pK and heat of ionization is proved to be a reliable method when the procedure is properly used.View full abstractDownload PDF (445K) -
Koei Hamana, Masaru Niitsu, Keijiro Samejima, Shigeru Matsuzaki1991 Volume 109 Issue 3 Pages 444-449
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTriamines such as norspermidine, spermidine, and homospermidine and tetraamines such as norspermine, spermine, thermospermine, and aminopropylhomospermidine were found to be distributed ubiquitously in the eight extremely thermophilic (growing at 70°C) Thermus species tested. Three linear pentaamine (caldopentamine, homocaldopentamine, and thermopentamine), two linear hexaamines (caldohexamine and homocaldohexamine), two tertiary branched tetraamines (N4-aminopropylnorspermidine and N4-aminopropyl-spermidine), and quaternary branched pentaamines such as N4-bis(aminopropyl)norsper-midine andN4-bis(aminopropyl)spermidine were detected in T. thermophilus HB8, T. filiformis Wai33 Al, T. flavus AT-62, and T. caldophilus GK24. The linear hexaamines and branched polyamines were absent in T. aquaticus YT-1, T. sp. X-1, T. sp. T2, and T. sp. T351, in which linear pentaamines were minor components. Moderately thermophilic Thermus ruber and Thermus sp. K-2 contained putrescine, spermidine, norspermidine, homospermidine, spermine, norspermine, thermospermine, and aminopropylhomosper-midine. No pentaamines, hexaamines, or branched polyamines were found in these two moderately thermophilic Thermus species. On the other hand, moderately thermophilic, acidophilic Acidothermus cellulolyticus was devoid of all of the polyamines.View full abstractDownload PDF (593K) -
Yoshimi Bando, Kenji Aki1991 Volume 109 Issue 3 Pages 450-454
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe oxidase reaction of lipoamide dehydrogenase with NADH generates superoxide radicals and hydrogen peroxide under aerobic conditions. ESR spin trapping using 5, 5-dimethyl-1-pyrroline-N-oxide (DMPO) was applied to characterize the oxygen radical species generated by lipoamide dehydrogenase and the mechanism of their generation. During the oxidase reaction of lipoamide dehydrogenase, DMPO-OOH and DMPO-OH signals were observed. The DMPO-OOH signal disappeared on addition of superoxide dismutase. These results demonstrate that the DMPO-OOH adduct was produced from the superoxide radical generated by lipoamide dehydrogenase. In the presence of dimethyl sulfoxide, a DMPO-CH3 signal appeared at the expense of the DMPO-OH signal, indicating that the DMPO-OH adduct was produced directly from the hydroxyl radical rather than by decomposition of the DMPO-OOH adduct. The DMPO-OH signal decreased on addition of superoxide dismutase, catalase, or diethylenetriaminepentaacetic acid, indicating that the hydroxyl radical was generated via the metal-catalyzed Haber-Weiss reaction from the superoxide radical and hydrogen peroxide. Addition of ferritin to the NADH-lipoamide dehydrogenase system resulted in a decrease of the DMPO-OOH signal, indicating that the superoxide radical interacted with ferritin iron.View full abstractDownload PDF (638K) -
Yasutomo Nomura, Mamoru Tamura1991 Volume 109 Issue 3 Pages 455-461
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThrough the use of a picosecond laser pulse of near-infrared light at 1, 064 nm, the temporal profile of the transmitted light through the anesthetized rat head has been investigated. The light intensity at a certain time after the input pulse was exponentially attenuated by the hemoglobin concentration with hematocrit values from 1.5 to 50%, although the transmitted pulse broadened markedly due to scattering by the cerebral tissue. The optical pathlength, which is required for quantitation of the absolute absorbance change, was directly determined, by the time of flight measurement of the light pulses, as the product of the velocity of light in tissue and time. The mean concentration of hemoglobin in the brain could be determined quantitatively by the use of this pathlength. The oxygen saturation of venous blood determined by our time of flight measurement was very close to that in the internal jugular vein determined directly with a gas analyzer. Thus, the picosecond laser technique is useful for quantifying the blood oxygenation in tissues.View full abstractDownload PDF (796K) -
Yoshikazu Uchida, Tadatomo Ogawa, Masao Iwamori, Yoshitaka Nagai1991 Volume 109 Issue 3 Pages 462-465
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSLipokeratinogenoside [N-(O-linoleoy1)-ω-hydroxy fatty acyl sphingosyl β-glucose] is one of the epidermosides which were found to be glycosphingolipids characteristic of the epidermis of mammalian skin. On the addition of lipokeratinogenoside to cultured rat keratinocytes (FRSK), the amount of keratin in the cells increased, 48 and 144 h after cultivation, to 1.4 to 1.8 times higher than that without the addition of lipokeratinogeno-side, and the number of cornified envelopes also significantly increased on cultivation of the cells with lipokeratinogenoside. Immunohistochemical staining with anti-keratin anti-body revealed that the cells cultivated with lipokeratinogenoside were densely covered with keratin in distinct contrast to the control cells. The same enhanced syntheses of keratin and cornified envelopes were observed on cultivation in the presence of TPA, which has been shown to elevate the intracellular Ca2+ -content and to translocate cytoplasmic protein kinase C to the plasma membrane in the initial stage of transmembrane signalling. Similarly, lipokeratinogenoside showed the ability to increase the intracellular Ca2+-content to the same extent as TPA did and to translocate protein kinase C to the membrane fraction. However, the above activities of lipokeratinogenoside decreased with removal of the linoleic acid moiety from lipokeratinogenoside with mild alkali, but linoleic acid alone did not show any activities, indicating that the lipokeratinogenoside molecule itself is required for expression of the activities.View full abstractDownload PDF (831K) -
Yasuo Shirakihara, Masafumi Yohda, Yasuo Kagawa, Ken Yokoyama, Masasuk ...1991 Volume 109 Issue 3 Pages 466-471
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFor a crystallization study, purification methods for F1 -ATPase from a thermophilic bacterium, PS3, and its major subunits, β and α, have been improved. The improvement depended on the introduction of dye-ligand chromatography columns to the previously adopted array of chromatography columns: a Blue-B (a blue dye bound to agarose) column was introduced for the F1 preparation, a Green-A column (a green dye attached to agarose) for the β subunit, and a Blue-A (another blue dye, Cibacron Blue 3GA, bound to agarose) column for the α subunit. The improved preparations of all the proteins had purities of nearly 99%. Using the highly purified preparations of the proteins, crystallization conditions were searched for in a systematic way. Large plate crystals (0.2 X 0.5 X 0.5 mm) of F1 were grown from a polyethylene glycol solution. However, neither of the subunits was crystallized, in spite of extensive search for crystallization conditions.View full abstractDownload PDF (2607K) -
Tarou Ogurusu, Shigeo Wakabayashi, Munekazu Shigekawa1991 Volume 109 Issue 3 Pages 472-476
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe used Mn2+ as an analogue for Mg2+ to examine the minimum requirement of divalent cations for the rapid turnover of the sarcoplasmic reticulum ATPase. We measured the binding of Ca2+ and Mn2+ to the purified Ca2+ -ATPase during steady-state hydrolysis of MnATP at 2°C and pH 7. In the presence of 20 μM Ca2+, Mn2+ was as effective as Mg2+ in stimulating ATPase activity and the maximal activation of ATP hydrolysis was observed at 0.1mM MnC12. Under these conditions, 2mol of Ca2+ were bound per mol of the ADP-sensitive phosphoenzyme, whereas no Ca2+ was bound to the ADP-insensitive phosphoenzyme. On the other hand, the stoichiometry for ATP-dependent binding of Mn2+ to these intermediates was about 1. We found that Mn2+ remained bound to the ADP-insensitive phosphoenzyme even in the presence of added chelator. In the absence of ATP, we detected a low level of Mn2+ binding, which reached 0.4 mol per mol of the phosphoryla-tion site at 0.1 mM free Mn2+. We present evidence that this extra Mn2+ binding did not affect the rate of decomposition of the ADP-sensitive phosphoenzyme, which was the rate-limiting step for ATP hydrolysis under the conditions used. All these results support our previous conclusion [Shigekawa, M., Wakabayashi, S., & Nakamura, II. (1983) J. Biol. Chem. 258, 14157-14161] that the minimum requirement of divalent cations for the rapid turnover of the Ca2+-ATPase is 3 mol per mol of the enzyme active site, of which two are Ca2+ ions bound at the transport sites, and that the metal moiety of the metal-ATP complex bound at the ATP site determines the catalytic rate of the reaction stepsView full abstractDownload PDF (621K) -
Yasuyuki Kitagawa, Nobuo Tanaka, Yasuo Hata, Masami Kusunoki, Gen-pei ...1991 Volume 109 Issue 3 Pages 477-485
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe three-dimensional structure of Cu, Zn-superoxide dismutase from spinach leaves has been determined by X-ray crystal structure analysis. The atomic coordinates were refined at 2.0 Å resolution using the Hendrickson and Konnert program for stereochemically restrained refinement against structure factors, which allowed the use of non-crystallographic symmetry. The crystallographic residual error for the refined model was 24.9%, with a root mean square deviation of 0.03Å from the ideal bond length and an average atomic temperature factor of 9.6Å2. A dimeric molecule of the enzyme is comprised of two identical subunits related by a non-crystallographic 2-fold axis. Each subunit of 154 amino acid residues is composed primarily of eight anti-parallel β-strands that form a flattened cylinder, plus three external loops. The main-chain hydrogen bonds primarily link the β-strands. The overall structure of this enzyme is quite similar to that of the bovine dismutase except for some parts. The single disulfide bridge (Cys57-Cys146) and the salt bridge (Arg79-Asp101) maystabilize the loop regions of the structure. The Cu2+ and Zn2+ ions in the active site lie 6.1Å apart at the bottom of the long channel. The Cu2+ ligands (ND1 of His-46, and NE2 of His-48, -63, and -120) show an uneven tetrahedral distortion from a square plane. The Zn2+ ligands (ND1 of His-63, -71, and -80 and OD1 of Asp-83) show an almost tetrahedral geometry. The imidazole ring of His-63 forms a bridge between the Cu2+ and Zn2+ ions. The side chains and main chains of the metal-liganding residues are stabilized in their orientation by a complex network of hydrogen bonds.View full abstractDownload PDF (1145K) -
Akiko Fujiwara, Eigoro Tazawa, Ikuo Yasumasu1991 Volume 109 Issue 3 Pages 486-491
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn sperm of the echiuroid, Urechis unicinctus, respiration in the presence of CO was reversibly augmented by light irradiation in an examined range of wavelengths between 350 and 650 nm. The respiratory rate of sperm in the presence of CO was enhanced by light irradiation in proportion to the light fluence rate. A sharp and large peak was obtained at the wavelength of 430 nm in the action spectrum of photo-activated respiration of sperm in the presence of CO. Broad and small peaks were also found at around 530 and 570 nm. This action spectrum is similar in its profile to the absorption spectrum of reduced cytochrome b. Absorption ofphoton energy by reduced b-type cytochrome probably activates the redox reaction of this cytochrome to enhance the respiratory rate. Photo-activated respiration in the presence of CO was inhibited by antimycin A and cyanide. In this respiratory system, an electron equivalent is probably transferred through the mitochondrial respiratory chain between cytochrome b and cytochrome c and finally to molecular oxygen in the reaction catalyzed by the CO-insensitive terminal oxidase.View full abstractDownload PDF (819K) -
Mariko Shimamura, Tadahiko Hazato, Takao Iwaguchi1991 Volume 109 Issue 3 Pages 492-497
Published: 1991
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA membrane-bound enkephalin-degrading aminopeptidase was purified from the longitudinal muscle layer of the guinea pig small intestine by four steps of column chromatography using L-tyrosine β-naphthylamide. The molecular weight of the enzyme was estimated to be 105, 000 by gel filtration. The maximum activity was observed between pH 6.5 and 7.0. The Km, value for leucine-enkephalin was 137 μM. The aminopeptidase activity toward aminoacyl β-naphthylamide substrates was restricted to basic, neutral, and aromatic aminoacyl derivatives. No action was detected on acidic amino acid and proline derivatives. The enzyme was potently inhibited by the aminopeptidase inhibitors actinonin, amastatin, and bestatin, and bioactive peptides such as angiotensin III, substance P, and Met-Lys-bradykinin. The enzyme activity was also inhibited by the antibody against the purified serum enkephalin-degrading aminopeptidase of guinea pig at concentrations similar to those at which activity was observed toward serum enkephalin-degrading aminopeptidase and renal aminopeptidase M. The enzyme rapidly hydrolyzed Leu-enkephalin and Met-enkephalin with the sequential removal of the N-terminal amino acid residues. The enzyme also hydrolyzed two enkephalin derivatives, angiotensin III and neurokinin A. However, neurotensin, substance P, and bradykinin were not cleaved. These properties indicated that the membrane-bound enkephalin-degrading aminopeptidase in the longitudinal muscle layer of the small intestine is similar to the serum enkephalin-degrading aminopeptidase and resembles aminopeptidase M. It is therefore suggested to play an important role in the metabolism of some bioactive peptides including enkephalin in peripheral nervous systems in vivo.View full abstractDownload PDF (765K)
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