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Volume 107 (1990)
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- Issue 4 Pages 507-
- Issue 3 Pages 331-
- Issue 2 Pages 173-
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Volume 107, Issue 2
Displaying 1-26 of 26 articles from this issue
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Yasuyuki Suzuki, Koji Yoshida, Toshihide Ichimiya, Tetsuya Yamamoto, H ...1990 Volume 107 Issue 2 Pages 173-179
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSContrapsin and two isoforms, F (fast) and S (slow), of α-1-antiproteinase (also called α-l-proteinase inhibitor) were isolated in an apparently homogeneous state from plasma of inflamed guinea pigs. Contrapsin inactivated trypsin, but did not significantly affect chymotrypsin, pancreatic elastase, or pancreatic kallikrein. On the other hand, both isoforms of α-1-antiproteinase inhibited trypsin, chymotrypsin, and elastase, but not plasma or pancreatic kallikrein. The S isoform of α-1-antiproteinase was present in barely detectable amounts in healthy animals, but increased markedly when the acute-phase reaction was induced by subcutaneous injection of turpentine. On the other hand, the plasma levels of the F isoform, contrapsin, and α-macroglobulin showed moderate (1. 5 to 2. 3-fold.) elevation during the acute-phase reaction. In contrast to the previous findings that rats and rabbits contain two different α-macroglobulins, one of which is an acute-phase reactant while the other is not, inflamed guinea pigs contained only one species of α-macroglobulin. Murinoglobulin, the most prominent acute-phase negative protein in both mice and rats, showed no significant change in guinea pigs. These results indicate that guinea pig plasma contains four major trypsin inhibitors, i. e., contrapsin, α-1-antiproteinase, α-macroglobulin, and murinoglobulin, the properties of which are very similar to those of the respective mouse homologues, but that the acute-phase response of these inhibitors differs greatly from that of the homologous proteins in rats or mice.View full abstractDownload PDF (2605K) -
Yoshio Nakano, Masahiro Itoh, Eiichi Tanaka, Kinuko Kimura1990 Volume 107 Issue 2 Pages 180-183
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBacillus cereus glutamine synthetase was modified by reaction with a fluorescent SH reagent, N-[[(iodoacetyl)amino] ethyl] -5-naphthylamine-l-sulfonic aicd (IAEDANS.), or an ATP analog, 5'-p-fluorosulfonylbenzoyladenosine (FSBA). The locations of the specific binding sites of these reagents were identified. IAEDANS inactivated Mg2+-dependent activity and activated Mn2+-dependent activity. FSBA inactivated only Mn2+-dependent activity. Mg2+plus Mn2+-dependent activity was inactivated by IAEDANS or FSBA. Amino acid sequence analysis of the single AEDANS-labeled proteolytic fragment showed the cysteinyl residue at position 306 to be the site of modification. Cys 306 is one of three cysteines that are unique to Bacillus glutamine synthetase. The result suggested that the cysteine has a role in the active site of the enzyme. We also report that the amino acid residue modified by FSBA was the lysyl residue at position 43.View full abstractDownload PDF (483K) -
Shigeyuki Imamura, Shigeru Ueda, Michinao Mizugaki, Akihiko Kawaguchi1990 Volume 107 Issue 2 Pages 184-189
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe multienzyme complex for fatty acid oxidation was purified from Pseudomonas fragi, which was grown on oleic acid as the sole carbon source. This complex exhibited enoyl-CoA hydratase [EC 4.2.1.17], 3-hydroxyacyl-CoA dehydrogenase [EC 1.1.1.35], 3-oxoacyl-CoA thiolase [EC 2.3.1.16], cis-3, trans-2-enoyl-CoA isomerase [EC 5.3.3.3], and 3-hydroxy-acyl-CoA epimerase [EC 5.1.2.3] activities. The molecular weight of the native complex was estimated to be 240, 000. Two types of subunits, with molecular weights of 73, 000 and 42, 000, were identified. The complex was composed of two copies each of the 73, 000- and 42, 000-Da subunits. The β-oxidation system was reconstituted in vitro using the multienzyme complex, acyl-CoA synthetase and acyl-CoA oxidase. This reconstituted system completely oxidized saturated fatty acids with acyl chains of from 4 to 18 carbon atoms as well as unsaturated fatty acids having cis double bonds extending from odd-numbered carbon atoms. However, unsaturated fatty acids having cis double bonds extending from even-numbered carbon atoms were not completely oxidized to acetyl-CoA: about 5 mol of acetyl -CoA was produced from 1 mol of linoleic or α-linolenic acid, and about 2 mol of acetyl-CoA from 1 mol of γ-linolenic acid. These results suggested that the 3-hydroxyacyl-CoA epimerase in the complex was not operative. When the epimerase was by-passed by the addition of 2, 4-dienoyl-CoA reductase to the reconstituted system, unsaturated fatty acids with cis double bonds extending from even-numbered carbon atoms were also completely degraded to acetyl-CoA.View full abstractDownload PDF (711K) -
Fumiyasu Fukumori, Naomi Takeuchi, Toshihiko Hagiwara, Hirokazu Ohbaya ...1990 Volume 107 Issue 2 Pages 190-196
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAleuria aurantia lectin (AAL) is a protein composed of two identical subunits having no carbohydrate chain and shows sugar-binding specificity for L-fucose. Full-length cDNA encoding for the lectin has been isolated from a λgt11 library, screened with an antiserum directed against AAL. The cDNA clone contained 1, 370 nucleotides and an open reading frame of 939 nucleotides encoding 313 amino acids. The amino-terminal sequence (residues 1-30) of the lectin isolated from the mushroom coincided with the deduced amino acid sequence starting from proline at the 2nd residue, indicating that the mature AAL consists of 312 amino acids. Its molecular weight is calculated to be 33, 398. The deduced amino acid sequence shows that AAL includes six internal homologous regions, and has considerable homology with a hemagglutinin from a Gram-negative bacterium, Myxococcus xanthus, which forms a fruiting body. No significant homology was observed with higher plant or animal lectins. The recombinant AAL produced by Escherichia coli JM109 carrying the AAL expression plasmid pKA-1 [Fukumori, F. et al. (1989) FEBS Lett. 250, 153-156] was purified from the cell lysate by affinity chromatography using a fucose-starch column, and hundreds of milligrams of the lectin was obtained. The recombinant lectin showed the same biochemical characteristics and sugar binding specificity as did the natural AAL.View full abstractDownload PDF (1921K) -
Yotaro Konishi, Yukie Okawa, Setsuko Hosokawa, Ken Fujimori, Hidetsugu ...1990 Volume 107 Issue 2 Pages 197-201
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA single intraperitoneal injection of acarbose (400 mg/kg, ) into rats caused lysosomal accumulation of glycogen in the liver, mimicking the cytological characteristics of human glycogen storage disease type II (Pompe's disease). The animal model is therefore useful for studying the pathogenesis of the disease. In the present study, we applied this model to examine the lysosomal hydrolytic pathway of glycogen in vivo. To quantify the lysosomal glycogen, the lysosome-rich fraction was rapidly prepared from liver homogenate by agglutination in the presence of Ca2+. Then the fraction was treated with α-amylase in isotonic medium to remove cytosolic glycogen, followed by transfer to hypotonic conditions in the presence of Triton X-100 to destroy total glycogen. The amount of lysosomal glycogen was calculated from the difference between the glycogen levels measured before and after the treatment under hypotonic conditions, and then it was corrected based on measurements of the intactness (%) of lysosomes and the recovery (%) of the lysosomal marker enzyme (βNAGase). We observed no measurable lysosomal glycogen in normal liver by this method, and this was confirmed by electron microscopy. After administration of acarbose, the lysosomal glycogen level increased to 2. 5mg/g liver within 2days, and then decreased gradually at a rate of 0. 4mg/day/g. The accumulation of glycogen in the lysosomes at an initial velocity of 1. 5mg/day/g liver may be considered as the amount of glycogen that would normally be degraded by acid α-glucosidase. Therefore, assuming that the liver breaks down about 40mg glycogen/day/g, we estimated that about 3% of the glycogen would be hydrolyzed by the lysosomal pathway.View full abstractDownload PDF (1973K) -
Kuniharu Ohnishi, Atsuko Nakazima, Keiko Matsubara, Kazuyoshi Kiritani1990 Volume 107 Issue 2 Pages 202-208
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe liv gene cluster responsible for encoding the high-affinity branched-chain amino acid transport proteins in Salmonella typhimurium was mapped in the 7.6-kilobase HindIII-SacI segment of plasmid pMN12 by utilizing the gene dosage effect. By subcloning and biochemical analysis, the livB and livC structural genes encoding the leucine-, isoleucine-, valine-, threonine-binding protein (LIVT-BP.) and the leucine-specific binding protein (L-BP.), respectively, were localized within the 3, 617-base HindIII-BstEII segment. Upon determining the nucleotide sequence of the 3, 617 bases, we found that the coding sequence of the livB gene (1, 095 base pairs) starts at the position 355 and specifies the precursor LIVT-BP of 365 amino acid residues, and the livC gene (1, 107 base pairs) starts at the position 2, 452 and encodes the precursor L-BP of 369 amino acid residues. The two genes, separated by a 1-kilobase intergenic region, each possess potential promoters and rhoindependent transcriptional terminators. The mature LIVT-BP and L-BP are produced by removing the putative 21 and 23 signal peptides from the respective precursors. In comparison with the analogous two binding proteins from Escherichia coli K-12, strong homologies are observed.View full abstractDownload PDF (1895K) -
Nobuhisa Iwata, Norihisa Inazu, Tetsuo Satoh1990 Volume 107 Issue 2 Pages 209-212
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe tissue distribution of carbonyl reductase in ovary and liver of various animal species was investigated by measuring the reduction of 13, 14-dihydro-15-keto-prostaglandin F2α, a specific substrate for rat ovarian carbonyl reductases, and by means of Western blotting analysis using anti-rat ovarian carbonyl reductase antibody. The highest ovarian carbonyl reductase activity towards 13, 14-dihydro-15-keto-PGF2α was found in rat among ten animal species tested, followed by hamster and monkey. The immunoreactive protein was detected in hamster and monkey ovaries. Although carbonyl reductase activity towards 13, 14-dihydro-15-keto-PGF2α was not detectable in non-pregnant rabbit ovary, pregnant rabbit ovary showed not only moderate activity but also immunoreactivity with anti-rat ovarian carbonyl reductase antibody. On the other hand, carbonyl reductase activity towards 13, 14-dihydro-15-keto-PGF2α was detected in hepatic tissue of all the species tested, except for rat and left-eye flounder. Immunoreactive proteins were present in hepatic tissue of various species that exhibited measurable carbonyl reductase activity towards 13, 14-dihydro-15-keto-PGF2α.View full abstractDownload PDF (2935K) -
Akio Matsukage, Yoshio Nishimoto1990 Volume 107 Issue 2 Pages 213-216
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHighly purified preparations of chick embryo DNA polymerase γ contained 3'→5'exonuclease activity which might be responsible for the exonucleolytic proofreading during DNA synthesis [Kunkel, T. A. & Soni, A. (1988) J. Biol. Chem. 262, 4450-4459]. A rabbit antibody produced against highly purified chick DNA polymerase γ precipitated 3'→5' exonuclease activity to the same extent as DNA polymerase γ activity. Furthermore, the antibody neutralized the two enzyme activities to an equal extent. However, the exonuclease activity was more resistant than DNA polymerase γ activity to thermal treatment at 50°C, although both activities were partially protected with polynucleotides. The results obtained suggest that these two enzymes are associated as a single enzyme complex or that the two activities reside in a single molecule, and the active site of DNA polymerase γ and 3'→5' exonuclease are, although not identical, closely correlated.View full abstractDownload PDF (2695K) -
Takako Nishiya, Sharmin Ahmed1990 Volume 107 Issue 2 Pages 217-221
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe circular dichroim (CD) active phospholipid bis(4'-n-octanoxyazobenzene-4-carboxyl)-L-α-phosphatidylcholine (CDPC) was used to study the effects of carboxymethyl-chitin (CM-chitin) on the membrane dynamics. For this purpose, CD and electronic spectra were observed for a mixture of CM-chitin and liposomes composed of CDPC and egg lecithin (EPC) (50% CDPC-EPC-liposomes), and for a mixture of CM-chitin, 50% CDPC-EPC-liposomes, and EPC-liposomes. CM-chitin at concentrations higher than ca.10-5M induced the increase of absorbance at 600 nm synchronized with the dilution of CDPC in 50% CDPC-EPC-liposomes by EPC matrix, indicating that CM-chitin induces the fusion among a few liposomes and/or the lipid transfer through the transient liposome fusion. Tempera-ture dependent CD spectra of a mixture of the 50% CDCP-EPC-liposomes and CM-chitin suggested that even high concentration of CM-chitin has no significant perturbation of the lipid organization in the membranes. The effects of CM-chitin on the leakage of [3H] sucrose from the EPC-liposomes were also studied. By the presence of 5×10-6M CM-chitin, the half-life for leakage of [3H]sucrose in plasma was increased by 4 times. This effect of CM-chitin was reduced by chitinase, while no effect was observed with lysozyme, suggesting that CM-chitin molecules are adsorbed on the liposome surface and stabilize the liposomes against the plasma proteins.View full abstractDownload PDF (601K) -
Norio Muto, Tomoe Nakamura, Itaru Yamamoto1990 Volume 107 Issue 2 Pages 222-227
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have previously found that some mammalian tissue homogenates can catalyze a unique transglucosylation from maltose to L-ascorbic acid (AA), resulting in a chemically stable AA derivative, L-ascorbic acid α-glucoside (AAG.). In the present study, the enzyme responsible for this transglucosylation was isolated from rat intestinal membrane. The formation of AAG was determined by HPLC with an ODS column. The specific activity of AAG-forming enzyme was increased in parallel with that of α-glucosidase (maltose hydrolase) during the purification, and two neutral α-glucosidases, termed α-glucosidases I and II, were purified to apparent homogeneity. Their enzymological properties showed that they corresponded to maltase [EC 3. 2. 1. 20] and sucrase-isomaltase complex [EC 3. 2. 1. 48/10], respectively. Both enzymes could form AAG by splitting only maltose among the disaccharides examined, although α-glucosidase I possessed a considerably higher activity than the other enzyme. Both AAG formation and maltose hydrolysis were dependent on incubation temperature with the maximal activity at 60°C, but there was an apparent difference between their pH optima. AAG thus formed could also be hydrolyzed by the purified enzymes. From these results, it is concluded that membrane-bound neutral α-glucosidases from rat intestine have site-specific transglucosylase activity to form nonreducing AAG which is distinct from L-ascorbic acid-6-O-α-D-glucoside.View full abstractDownload PDF (1600K) -
Hideo Nishitani, Hideki Kobayashi, Motoaki Ohtsubo, Takeharu Nishimoto1990 Volume 107 Issue 2 Pages 228-235
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe isolated a cDNA clone from Xenopus cDNA libraries for the homolog of the human RCC1 gene, which is considered to be a regulator for the onset of chromosome condensation. The cloned Xenopus RCC1 cDNA encoded a protein of 424 amino acids which had the same seven homologous repeated domains of about 60 amino acids as found in human RCC1 cDNA. The overall identity of amino acid sequence between the human and Xenopus RCC1 protein was 76%. Specifically, in the repeated domain the amino acid sequence was highly conserved between both species. The identity of amino acids in this region was 82%. In the N-terminal region, albeit the overall identity was low (35%), some positively charged amino acids were conserved. The transcript for Xenopus RCC1 gene with the length of 2.2-kb was detected in both Xenopus oocytes and cultured somatic cells, A6. The antibody prepared against Xenopus RCC1 protein produced in E. coli recognized 45 and 46 kDa proteins not only in Xenopus oocytes and A6 cells, but also in human and hamster cells. Xenopus RCC1 cDNA complemented the tsBN2 mutation, depending on the amount of its product in tranformants. Thus, the RCC1 protein was suggested to regulate the onset of chromosome condensation in the eukaryote from amphibian to mammalian cells.View full abstractDownload PDF (4345K) -
Yuji Ito, Hidenori Yamada, Shinzo Nakamura, Taiji Imoto1990 Volume 107 Issue 2 Pages 236-241
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe lysozyme (rabbit kidney lysozyme) from the homogenate of rabbit kidney (Japanese white) was purified by repeated cation-exchange chromatography on Bio-Rex 70. The amino acid sequence was determined by automated gas-phase Edman degradation of the peptides obtained from the digestion of reduced and S-carboxymethylated rabbit lysozyme with Achromobacter protease I (lysyl endopeptidase). The sequence thus determined was KIYERCELARTLKKLGLDGYKGVSLANWMCLAKWESSYNTRATNYNPGDKSTDYGIFQINSRYWCNDGKTPRAVNACHIPCSDLLKDDITQAVACAKRVVSDPQGIRAWVAWRNHCQNQDLTPYIRGCGV, indicating 25 amino acid substitutions from human lysozyme. The lytic activity of rabbit lysozyme against Micrococcus lysodeikticus at pH 7, ionic strength of 0. 1, and 30°C was found to be 190 and 60% of those of hen and human lysozymes, respectively. The lytic activity-pH profile of rabbit lysozyme was slightly different from those of hen and human lysozymes. While hen and human lysozymes had wide optimum activities at around pH 5.5-8.5, the optimum activity of rabbit lysozyme was at around pH 5.5-7.0. The high proline content (five residues per molecule compared with two prolines per molecule in hen or human lysozyme) is one of the interesting features of rabbit lysozyme. The transition temperatures for the unfolding of rabbit, human, and hen lysozymes in 3M guanidine hydrochloride at pH 5.5 were 51.2, 45.5, and 45.4°C, respectively, indicating that rabbit lysozyme is stabler than the other two lysozymes. The high proline content may be responsible for the increased stability of rabbit lysozyme.View full abstractDownload PDF (690K) -
Mikio Kato, Kazuya Nishikawa, Masahiro Uritani, Masazumi Miyazaki, Sho ...1990 Volume 107 Issue 2 Pages 242-247
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBy utilizing an enzymatically reconstructed tRNA variant containing an altered anticodon sequence, we have examined the different biochemical behavior of translation between the Watson-Crick type and the wobble type base pair interactions at the first anticodon position. We have found that the Watson-Crick type base pair has an advantage in translation in contrast to the wobble type base pair by comparing the efficiency of transpeptidation of native tRNAPhe (anticodon; GmAA.) with its variant tRNA (anticodon; AAA) in the poly(U)-programmed ribosome system. Thomas et al. [Proc. Natl. Acad. Sci. U. S. (1988) 85, 4242-4246] showed that the wobble codon at the ribosomal A-site accepted its cognate tRNA less efficiently than the Watson-Crick base pairing codon. We report here that the wobble interaction at the ribosomal P-site also affected the rate of translation. This variable translational rate may be a mechanism of gene regulation through preferential codon usage.View full abstractDownload PDF (696K) -
Yohei Natori, Yasunobu Kano, Fumio Imamoto1990 Volume 107 Issue 2 Pages 248-255
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFive trp genes, trpD, trpC, trpF, trpB, and trpA, of Lactobacillus casei were cloned by transformation of tryptophan auxotrophic mutants of the respective trp genes in Escheri-chia coli. These trp genes appear to constitute an operon and are located in the above order in a segment of DNA of 6, 468 base pairs. The entire nucleotide sequence of this DNA segment was determined. Five contiguous open reading frames in this segment can encode proteins consisting of 341, 260, 199, 406, and 266 amino acids, respectively, in the same direction. The amino acid sequences of these proteins exhibit 25.5-50.2% homology with the amino acid sequences of the corresponding trp enzymes of E. coli. Two trp genes, trpC and trpF, from L. casei can complement mutant alleles of the corresponding genes of E. coli. However, neither the trpA gene nor the trpB gene of L. casei can complement mutations in the E. coli trpA gene and the trpB gene, respectively, suggesting that the protein products of the L. casei and E. coli trpA and trpB genes, respectively, cannot form heterodimers of trypto-phan synthetase with activity. Other features of the coding and flanking regions of the trp genes are also described.View full abstractDownload PDF (715K) -
A. K. M. Abdus Sattar, Naoko Yamamoto, Tadashi Yoshimoto, Daisuke Tsur ...1990 Volume 107 Issue 2 Pages 256-261
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSProlyl endopeptidase [EC 3.4.21.26] was purified to homogeneity from the culture filtrate of Agaricus bisporus by a procedure that comprised ammonium sulfate fractionation, anion-exchange chromatographies on DEAE-Toyopearl and DEAE-Sephadex, hydrox-ylapatite chromatography, and high-performance liquid chromatography (HPLC) on a TSKgel G 2000 SW column. The overall activity recovery was 8. 6%. The enzyme was most active at or around pH 7. 5 and was stable in the range of pH 5-9 when checked with Z-Gly-Pro-β-naphthylamide as a substrate. The isoelectric point of the enzyme was about 4. 8. The enzyme was a monomeric protein of molecular weight 78, 000±2, 000 as judged by gel permeation chromatography on Sephadex G-150 and electrophoresis on sodium dodecyl sulfate (SDS.) polyacrylamide gel. The enzyme hydrolyzed Pro-X bonds and at least five subsites (S3, S2, S1, S1', and S2'.) were found to be involved in enzyme-substrate binding. Among them, S2, S1, and S1' subsites of the enzyme showed high stereospecificity. The enzyme was strongly inhibited by diisopropylfluorophosphate (DFP.), Z-Gly-Pro-CH2C1, Z-Pro-prolinal, Z-Pro-pyrrolidine, Z-Thiopro-pyrrolidine, Z-Pro-thiazolidine, Z-Thiopro-thiazolidine, and p-chloromercuribenzoate (PCMB), while it was not inhibited by phenyl-methylsulfonyl fluoride (PMSF), E-64, iodoacetamide, or metal chelators. Although the A. bisporus enzyme showed no immunological cross reaction with anti-bovine prolyl endo-peptidase antiserum, the other characteristics were quite similar to those of mammalian and plant enzymes.View full abstractDownload PDF (2445K) -
Haruyuki Atomi, Mitsuyoshi Ueda, Masaki Hikida, Tadashi Hishida, Yutak ...1990 Volume 107 Issue 2 Pages 262-266
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA genomic DNA clone encoding isocitrate lyase, a key enzyme of the glyoxylate cycle and a peroxisomal enzyme of the n-alkane-assimilating yeast Candida tropicalis has been isolated with a cDNA probe from the yeast λEMBL library. Nucleotide sequence analysis of the genomic DNA clone disclosed that the region coding isocitrate lyase had a length of 1, 650 base pairs, corresponding to 550 amino acids (61, 602 Da.). RNA blot analysis demon-strated that only one kind of mRNA (2 kb.) supposed to be transcribed from this gene was present in the cells. A comparison of the amino acid sequences was made with the isocitrate lyase of castor bean, one of the glyoxysomal enzymes, and the enzyme of E. coli. The isocitrate lyases of C. tropicalis and castor bean had high homology, and the presence of some amino acid stretches conserved in all three enzymes suggests that these might be involved in the catalysis of the common reaction. There was an insertion common to the isocitrate lyases of C. tropicalis and castor bean, which is of interest concerning their evolution. In the C-terminal region, a characteristic sequence similar to that previously proposed as the import signal to peroxisomes was present.View full abstractDownload PDF (577K) -
Mauno Vihinen, Pauli Ollikka, Jukka Niskanen, Peter Meyer, Ilari Suomi ...1990 Volume 107 Issue 2 Pages 267-272
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe relationship between structure, activity, and stability of the thermostable Bacillus stearothermophilus α-amylase was studied by site-directed mutagenesis of the three most conserved residues. Mutation of His-238 to Asp involved in Ca2+ and substrate binding reduced the specific activity and thermal stability, but did not affect the pH and tempera-ture optima. Replacement of Asp-331 by Glu in the active site caused almost total inactiva-tion. Interestingly, in prolonged incubation this mutant enzyme showed an altered end-product profile by liberating only maltose and maltotriose. Conservative mutation of the conserved Arg-232 by Lys, for which no function has yet been proposed, resulted in lowered specific activity: around 12% of the parental enzyme. This mutant enzyme had a wider pH range but about the same temperature optimum and thermal stability as the wild-type enzyme. Results obtained with different mutants were interpreted by computer aided molecular modeling.View full abstractDownload PDF (2866K) -
Toshimasa Yasuhara, Hideyoshi Yokosawa, Shin-ichi Ishii1990 Volume 107 Issue 2 Pages 273-279
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn aminopeptidase showing broad substrate specificity was purified to electrophoretic homogeneity from spermatozoa of the sea urchin, Strongylocentrotus intermedius. It is a single chain protein (Mr=110, 000) with an isoelectric point of 5. 2 and shows the highest activity in a pH range between 7.0 and 7.5. Ni2+, Cu2+, Zn2+, and Hg2+, as well as 1, 10-phenanthroline and p-chloromercuribenzoate, inhibit the enzyme irrespective of the substrates used, but Ca2+, Mn2+, Mg2+, and Co2+ modified the activity differently depending on the nature of the substrate. The effect of Ca2+ was most marked; it stimulated the activity toward some 4-methylcoumaryl-7-amide (MCA.) substrates (for example leucine MCA), whereas it depressed the activity toward some other substrates such as arginine-MCA and lysine-MCA in a competitive manner. The rate of enzymatic hydrolyis determined for a mixture of leucine-MCA and arginine-MCA, in respect to the release of their common product (7-amino-4-methylcoumarin.), was in good agreement with the value calculated on the assumption that these two substrates compete with each other for a single active site of the enzyme. Furthermore, the enzyme showed an identical K 1 value for each of the competitive inhibitors examined, irrespective of the type of substrate. Ca2+ also influenced the activities toward various peptide substrates in a dual way similar to that observed on the MCA substrates. These results indicate that the sea urchin sperm aminopeptidase has an active site that alters its substrate preference depending on the Ca2+ concentration of the reaction medium.View full abstractDownload PDF (832K) -
Yasushi Kikuta, Emi Kusunose, Tsuyoshi Okumoto, Ichiro Kubota, Masamic ...1990 Volume 107 Issue 2 Pages 280-286
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo forms of cytochrome P-450 (P-450), designated as P-450LpGAω1 and P-450LpGAω2, have been purified to specific contents of 17.9 and 11.1 nmol P-450/mg protein, respectively, from liver microsomes of rabbits treated with di(2-ethylhexyl.)phthalate (DEHP), a peroxisomal proliferator. The purified P-450LpGAω1 and P-450LpGAω2 were found to have apparent molecular weights of 52, 500 and 53, 000, respectively. They showed absorption maxima at 451 and 450 nm in the carbon monoxide-difference spectra for their reduced forms, respectively. The two P-450s both efficiently catalyzed the ω-hydroxylation of prostaglan-dins A1 (PGA1) and A2 (PGA2), as well as the ω- and (ω-1)-hydroxylation of fatty acids such as laurate, myristate, and palmitate. In a reconstituted system, various metal ions such as Na+ and Mg2+ stimulated these reactions. The P-450s exhibited no detectable activity toward several xenobiotics tested. The two P-450s showed different peptide map patterns following limited proteolysis with Staphylococcus aureus V8 protease or papain. The NH2-terminal amino acid sequences (ALNPTRLPGSLSGLLQVAGL and ALSLTRLPGSFS-GFLQAxGLLGLLL) of P-450LpGAω1, and P-450LpGAω2 were identical at 18/20 and 19/24 positions with that of the lung prostaglandin ω-hydroxylase from pregnant rabbits, respectively. An antibody against P-450LpGAω2 recognized a 52, 000-53, 000 molecular weight protein(s) in rabbit liver microsomes. The intensity of the immunoblot was significantly increased in liver microsomes from rabbits treated with DEHP, but not with phenobarbital or 3-methylcholanthrene. The laurate and PGA1 ω-hydroxylase activities of liver mi-crosomes from both untreated and DEHP-treated rabbits were markedly inhibited by pretreatment with the antibody against P-450LpGAω1 or P-450LpGAω2. The results suggest that the two P-450s are responsible for a major portion of the fatty acid and PGA ω-hydroxylase activities in rabbit liver microsomes. To our knowledge, this is the first time that P-450s specific for ω-hydroxylase activities have been isolated from liver microsomes of DEHP-treated rabbits.View full abstractDownload PDF (2756K) -
Michio Yazawa, Fumiko Matsuzawa, Koichi Yagi1990 Volume 107 Issue 2 Pages 287-291
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe calcium-dependent difference absorption spectrum of scallop calmodulin was mea-sured in the presence of mastoparan. The difference spectrum at 286 nm (ΔA286) showed biphasic response to Ca2+ concentration. The first change represents the conformational change around Tyr-138 and the second change may respond to an interaction between N-and C-domain of calmodulin which became apparent in the associated state with mastopar-an. Calmodulin-mastoparan complex was eluted from a gel filtration column after free calmodulin in the presence of Ca2+, which indicates a more compact structure of calmodulin-mastoparan complex than of free calmodulin. The biphasic response of ΔA286 was also observed with free calmodulin when the ionic strength was as low as 0.02 M NaCl. In the absence of NaCl, the Ca2+ dependence of ΔA288 was monophasic, assuming identical affinity of Ca2+ to both domains. Increase in the sensitivity of calmodulin to trypsin was observed with decrease in ionic strength. These results suggest an ionic-strength-depen-dent decrease in ordered structure of the connecting region. Calmodulin may change shape depending upon the ionic strength by bending at the connecting region. We assumed from the observations that calmodulin in solution may fluctuate between the two extreme shapes of the bent and the dumbbell structure. Target proteins may select and fix the specific bent structure for their activation.View full abstractDownload PDF (1422K) -
Yoshiharu Minamitake, Shiho Kodama, Toyoko Katayama, Hideki Adachi, Sh ...1990 Volume 107 Issue 2 Pages 292-297
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe complete peptide map of purified recombinant human interleukin 5 (rhIL-5) was determined to verify its primary structure, glycosylation sites, and disulfide bonding structure. Each peptide fragment generated by Achromobacter protease I (API) digestion was purified and characterized by amino acid analysis and amino acid sequence analysis. After digestion with API, we could identify all the peptides which were expected from human IL-5 cDNA sequence. The analyses of sulfhydryl content in rhIL-5 molecule and disulfide-containing peptide obtained from API digestion indicated that active form of rhIL-5 existed as an antiparallel dimer linked by two pairs of Cys-44 and Cys-86. In addition, we concluded that Thr-3 and Asn-28 were glycosylated. The results indicate that primary structure of rhIL-5 is highly homogeneous and observed heterogeneity is due to the difference in the content of carbohydrate.View full abstractDownload PDF (603K) -
Sabu Kasai, Hideko Nakano, Kazuya Maeda, Kunio Matsui1990 Volume 107 Issue 2 Pages 298-303
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFlavokinase (ATP:riboflavin 5'-phosphotransferase) [EC 2.7.1.26] was purified to appar-ent homogeneity from rat intestinal mucosa by fractionation with ammonium sulfate, gel filtration, and flavin affinity chromatography. The addition of ATP to the enzyme solution was necessary for its binding to the affinity gel. The apparent molecular weight of the enzyme was estimated to be 13, 500 by gel filtration on Sephadex G-100 and by SDS-PAGE. The properties of the enzyme, including its flavin specificity, were studied. Three types of riboflavin analogues were used for the flavin specificity study; namely, ones modified at the ribityl group, and at positions 3 and 8 of the isoalloxazine ring. Of the analogues modified at the ribityl group or position 3 of the isoalloxazine ring, only 2' -deoxyriboflavin was phosphorylated and then only weakly. On the other hand, most analogues modified at position 8 of the isoalloxazine ring were good substrates for the kinase, an appropriate increase in the substituent volume at position 8 of the isoalloxazine ring resulting in an increase in the Vmax value. In a previous paper on the mechanism of intestinal absorption of riboflavin, we proposed that one of the specific processes for the absorption of riboflavin is phosphorylation by flavokinase [Kasai, S. et al. (1988) J. Nutr. Sci. Vitaminol. 34, 265-280]. The present results support this conclusion because analogues that were absorbed at low concentrations through a process specific for riboflavin in our previous study were phosphorylated effectively by the enzyme, whereas those that were absorbed solely through simple diffusion at all concentrations were not phosphorylated or only phosphorylated weakly. The properties of the flavokinases from intestinal mucosa and liver were com-pared.View full abstractDownload PDF (717K) -
Bong Jin Lee, Sang Jong Lee, Fumiaki Hayashi, Hiroji Aiba, Yoshimasa K ...1990 Volume 107 Issue 2 Pages 304-309
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSeven well-resolved signals could be observed in the field lower than 9.5 ppm in the 1H-NMR spectrum of the H2O solution of cyclic AMP receptor protein (CRP). The signals of the tryptophan and histidine residues were identified on the basis of the CPMG spin echo spectra, the intra-residue NOE, 15N labeling, deuterium labeling, and the results of pH titration. The assignments of peaks to specific tryptophan and histidine residues are discussed in relation to the amino acid sequence and X-ray crystallographic data, and were confirmed by experiments involving partial subtilisin digestion. The four signals E (11.25 ppm), F (11.15 ppm), G (10.75 ppm), and H (10.65 ppm) were tentatively assigned to the resonances of the histidine residue at position 159, the arginine residue at position 82, and the tryptophan residues at positions 85 and 13, respectively. On the addition of cAMP and cGMP, signals F and G shifted up- and downfield respectively and conformational changes in the structure of CRP could be detected. The conformational transition mostly occurs when one cAMP molecule binds to one of the dimer subunits, but is completed only when both cAMP binding sites are saturated.View full abstractDownload PDF (597K) -
Tsukasa Seya, Michiyo Okada, Hiromi Nishino, John P. Atkinson1990 Volume 107 Issue 2 Pages 310-315
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSC3b/C4b receptor (CR1) and membrane cofactor protein (MCP) are integral membrane glycoproteins with factor I-dependent cofactor activity. They bind to C3b, allowing factor I to cleave C3b at two sites (first and second cleavage), which results in the generation of C3bi, a hemolytically inactive form which is a ligand for complement receptor type three (CR3). C3bi is further degraded by factor I and CR1 (third cleavage) to C3dg (a ligand for complement receptor type two, CR2) and C3c. Using two different substrates, fluid-phase C3b and cell-bound C3b, the cleavage of C3b by MCP and factor I was compared to that by CR1 and factor I under various conditions. The optimal pH for the first and second cleavage of either substrate was 6.0 for MCP and 7.5 for CR1. The third cleavage was mediated only by CR1 and factor I, the optimal pH being 8.0. Low ionic conditions enhanced the C3b binding and cofactor activity of both CR1 and MCP. The efficiency of binding C3b to CR1 or MCP was maximal at pH 6.2. The isoelectric point (pI) of MCP was acidic (_??_4.0), while that of CR1 was 6.8. Therefore, compared to CR1, MCP possesses distinct functional profiles relative to C3b-binding and factor I-cofactor activity.View full abstractDownload PDF (1737K) -
Teruo Miyauchi, Takuro Kanekura, Akihiro Yamaoka, Masayuki Ozawa, Sanz ...1990 Volume 107 Issue 2 Pages 316-323
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSLotus tetragonolobus agglutinin (LTA) binds preferentially to early embryonic cells in the mouse. The affinity-purified antibody raised against LTA receptors from embryonal carcinoma cells were used to screen a λgt11 expression library of F9 embryonal carcinoma cells, resulting in detection of a cDNA clone specifying a new glycoprotein termed “basigin.” The glycoprotein has been suggested to be a transmembrane one, and was found to be a new member of the immunoglobulin (Ig) superfamily. The molecular weight of basigin was largely in the range between 43, 000 and 66, 000, while that of the peptide portion with a putative signal sequence was inferred to be about 30, 000. Significant levels of basigin mRNA were detected not only in embryonal carcinoma cells, but also in mouse embryos at 9-15 days of gestation and in various organs of the adult mouse. The Ig-like domain of basigin is unique, since it has strong homology to both the β-chain of major his-tocompatibility class II antigen and the Ig V domain. The number of amino acids between the two conserved cysteine residues is intermediate between those of the Ig V and C domains. Therefore, basigin is an interesting protein in connection with the molecular evolution of the superfamily.View full abstractDownload PDF (4674K) -
Yasunobu Tsuji, Kenji Yamamoto, Tatsurokuro Tochikura, Tatsurokuro Toc ...1990 Volume 107 Issue 2 Pages 324-330
Published: 1990
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBacillus circulans isolated from soil was found to produce two types of α-L-fucosidase differing in substrate specificity. One was able to liberate L-fucose from porcine gastric mucin (PGM.), but not from artificial substrates, including p-nitrophenyl and methyl α-L-fucosides, while the other acted not on PGM but on p-nitrophenyl α-L-fucoside. The production of the former enzyme was enhanced about 150 times as much by PGM added to the medium as by glucose. The α-L-fucosidase acting on PGM was purified from the culture fluid obtained with PGM medium by ammonium sulfate fractionation and subsequent column chromatography. The purified enzyme was found to be homogeneous by PAGE and its molecular weight was estimated to be approximately 285, 000. The optimum pH was found to be 5.5 to 6.5 and the stable pH range was 4.5 to 9.0. The enzyme decomposed various blood group O(H) active substances such as PGM, human milk and human saliva, and moreover acted on A-, B-, and O-erythrocytes. The enzyme was shown to cleave α-(1→2)-, (1→3)-, and (1→4)-L-fucosidic linkages in various glycoproteins and oligosaccharides, but failed to hydrolyze α-(1→6)-L-fucosic linkages in 6-O-α-L-fucopyranosyl-N-acetylgluco-samine and intact bovine thyroglobulin.View full abstractDownload PDF (2003K)
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