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Volume 86 (1979)
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- Issue 4 Pages 833-
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Volume 86, Issue 4
Displaying 1-40 of 40 articles from this issue
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Masato NOGUCHI, Tadashi YOSHIDA, Goro KIKUCHI1979 Volume 86 Issue 4 Pages 833-848
Published: October 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBiliverdin reductase was purified from pig spleen soluble fraction to a purity of more than 90 % as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was a monomer protein with a molecular weight of about 34, 000. Its isoelectric point was at 6.1-6.2. The enzyme was strictly specific to biliverdin and no other oxidoreductase activities could be detected in the purified enzyme preparation. The purified enzyme could utilize both NADPH and NADH as electron donors for the reduction of biliverdin. However, there were considerable differences in the kinetic properties of the NADPH-dependent and the NADH-dependent biliverdin reductase activities: Km for NADPH was below 5 μm while that for NADH was 1.5-2mM; the pH optimum of the reaction with NADPH was 8.5 whereas that of the reaction with NADH was 6.9; Km for biliverdin in the NADPH system was 0.3 μm whereas that in the NADH system was 1-2 μm. In addition, both the NADPH-dependent and NADH-dependent activities were inhibited by excess biliverdin, but this inhibition was far more pronounced in the NADPH system than in the NADH system. IXα-biliverdin was the most effective substrate among the four biliverdin isomers, and the di-methylester of IXα-biliverdin could not serve as a substrate. Biliverdin reductase was also purified about 300-fold from rat liver soluble fraction. The hepatic enzyme was also a monomer protein with a molecular weight of 34, 000 and showed properties quite similar to those of the splenic enzyme as regards the biliverdin reductase reaction. The isoelectric point of the hepatic enzyme, however, was about 5.4. It was assumed that NADPH rather than NADH is the physiological electron donor in the intracellular reduction of IXα-biliverdin. The stimulatory effects of bovine and human serum albumins on the biliverdin reductase reactions were also examined.View full abstractDownload PDF (1926K) -
I. Conformational Dynamics of EnzymeHitomi FUKUZAWA, Kenji AKI1979 Volume 86 Issue 4 Pages 849-856
Published: October 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe dynamic structures of two major forms (LD (I) and LD (II)) of pig heart lipoamide dehydrogenase, resolved by TEAE-cellulose column chromatography, were studied by fluorescence depolarization. FAD and ANM were used as an intrinsic and an extrinsic fluorescent probe, respectively.
In the experiments with bound FAD of lipoamide dehydrogenase, no thermal dependence of the fluorescence depolarization of either enzyme was observed and the values of polarization were close to the theoretical maximum value of 0.5.
Both enzymes contained two reactive thiol groups which differed in their reactivities toward ANM.
When the enzymes were labeled with one mol of ANM per mol of enzyme, the rotational relaxation times of LD (I) and LD (II) were found to be 18 ns and 196 ns, respectively. These findings indicate that the segment of LD (I) labeled with ANM fluctuates in the order of nanoseconds, whereas this segment of LD (II) is fixed rigidly. On the other hand, when the enzymes were labeled with two mol of ANM per mol of enzyme, both enzymes showed the composite result of fluorescence depolarization due to the motilities of the segment of enzyme and the whole enzyme molecule. These findings indicate that both LD (I) and LD (II) have the non-equivalent motilities of segments containing one reactive thiol group between the two monomers. In other words, the segment containing the ANM binding site of the one monomer is flexible and this segment of the other monomer is fixed rigidly in both enzymes.View full abstractDownload PDF (542K) -
Kazuo UEDA, Tamitaro MIZUNOYA1979 Volume 86 Issue 4 Pages 857-862
Published: October 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA detailed in vitro study was made of the riboflavin sensitized photodestruction of mitomycin C. The dependences of the quantum yield in the system were examined on the introduction of various amounts of quenchers, such as halogen ion, paramagnetic ion, 1, 4-diazabi-cyclo[2, 2, 2]octane, 2, 6-di-tert-butyl-p-cresol, and p-hydroquinone.
The results are consistent with a mechanism involving oxygen molecule in the excited singlet state as the photochemically reactive species. The rate constant of the reaction between the excited singlet oxygen and mitomycin C was calculated to be 8.9 × 109 M-1•s-1View full abstractDownload PDF (364K) -
II. Regulation of Protein and Amino Acid MetabolismKeiji TANAKA, Kyoichi KISHI, Akira ICHIHARA1979 Volume 86 Issue 4 Pages 863-870
Published: October 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe nitrogen balance of adult rat hepatocytes in primary culture was calculated from changes in the concentrations of amino acids, urea and ammonia in the medium. When cells from 3-day cultures were incubated in Hanks' solution they released amino acids, whereas when they were incubated in medium containing amino acids in excess of 0.5 mm they consumed amino acids and formed urea. These catabolic and anabolic states of the cells were stimulated by glucagon and insulin, respectively.
Analysis showed that the relative amounts of individual amino acids released in salt solution were very similar to their relative amounts in liver proteins, except that the amounts of alanine, glutamate, aspartate, proline, and arginine released were relatively low. These amino acids were all used for gluconeogenesis, except arginine, which was converted to orni-thine. During culture in Williams' medium E, the concentrations of glutamine, alanine, glycine, arginine, and aspartate in the medium decreased greatly; those of histidine, proline, and glutamate did not change; that of ornithine increased; and those of other amino acids decreased moderately. Glucagon markedly stimulated the consumptions of alanine, gluta-mine, and glycine, whereas dexamethasone stimulated the uptakes of both essential and non-essential amino acids only in Williams' medium E. The uptakes of amino acids exceeded the amounts used for protein synthesis; the excess amounts taken up were metabolized by other pathways, such as glucose formation.
These results show that metabolism of proteins in these cells is affected by the amino acids and hormones present in the medium and mimics that in vivo. Thus this system should be useful for comprehensive studies on the control of liver proteins and amino acid metabolism.View full abstractDownload PDF (580K) -
Hideo SAWADA, Akira HARA, Fumiko KATO, Toshihiro NAKAYAMA1979 Volume 86 Issue 4 Pages 871-881
Published: October 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSNADPH-dependent enzymatic reduction of aromatic aldehydes and ketones observed in the cytosol of guinea pig liver was mediated by at least three distinct reductases (AR 1, AR 2, and AR 3), which were separated by DEAE-cellulose chromatography. By several procedures AR 2 and AR 3 were purified to homogeneity, but AR 1 could be purified only 30-fold because of the small amount. These enzymes were found to have similar molecular weights of 34, 000 to 36, 000 and similar Stokes radii of about 2.5 nm. AR 3 was identical to aldehyde reductase [EC 1.1.1.2] in substrate specificity for aromatic aldehydes and D-glucuronate and specific inhibition by barbiturates. AR 1 and AR 2 acted on aromatic ketones and cyclohexanone as well as aromatic aldehydes at optimal pHs of 5.4 and 6.0, respectively, and were immunochemically distinguished from AR 3. AR 1 was the most sensitive to sulfhydryl reagents, and AR 2 was more stable at 50°C than the other enzymes. Similar heterogeneity was observed in the kidney enzymes, but other tissues had little aldehyde reductase activity and contained only AR 3. In addition, lung contained a high molecular weight aromatic ketone reductase different from the above reductases.View full abstractDownload PDF (2077K) -
Hideo SAWADA, Akira HARA, Masakazu HAYASHIBARA, Toshihiro NAKAYAMA1979 Volume 86 Issue 4 Pages 883-892
Published: October 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo NADPH-dependent aromatic aldehyde-ketone reductases purified from guinea pig liver catalyzed oxidoreduction of 17β-hydroxysteroids and 17-ketosteroids. One enzyme efficiently oxidized 5β-androstanes and reduced 17-ketosteroids of A/B cis configuration, whereas the other enzyme efficiently oxidized 5'-androstanes and equally reduced both 5'-and 5'-androstanes of 17-ketosteroids. However, aromatic aldehydes and ketones, and 3-ketosteroids were irreversibly reduced by the two enzymes. The two enzymes utilized NADP+ or NADPH as cofactor, but little activity with NAD+ or NADH was found. Phosphate ions enhanced the NAD+-dependent dehydrogenase activity and NADH-dependent reductase activity of the two enzymes, whereas the activities with NADP+ and NADPH were not affected. The ratios of the two activities of ketone reduction and 17β-hydroxysteroid oxidation of the two enzymes were almost constant during the purification steps after the two enzymes had been separated by DEAE-cellulose chromatography. By kinetic studies and electrophoresis and isoelectric focusing experiments it was confirmed that both of the two enzymes were responsible for the reduction of aldehydes, ketones, and ketosteroids and for the oxidation of 17β-hydroxysteroids. These results indicate that 17β-hydroxysteroid dehydrogenases may play important roles in the metabolism of exogeneous aldehydes and ketones as well as steroids.View full abstractDownload PDF (1082K) -
Kimitsuna WATANABE, Yoshiyuki KUCHINO, Ziro YAMAIZUMI, Mayumi KATO, Ta ...1979 Volume 86 Issue 4 Pages 893-905
Published: October 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe nucleotide sequence of formylmethionine tRNA from an extreme thermophile, Thermus thermophilus HB8, was determined by a combination of classical methods using unlabeled samples to determine the sequences of the oligonucleotides of RNase T1 and RNase A digests and a rapid sequencing gel technique using 5'-32P labeled samples to determine overlapping sequences. Formylmethionine tRNA from T. thermophilus is composed of two species, tRNAMetf1 and tRNAMetf2. Their nucleotide sequences are almost identical, and are also almost identical with that of E. coli tRNAMetf, except for slight modifications and replacements. Both species have modifications at three points which do not exist in E. coli tRNAMetf: 2'-O-methylation at G19, N-1-methylation at A59 and 2-thiolation at T55. Moreover U51 in E. coli tRNAMetf is replaced by C51 in both species, so that a G-C pair is formed between this C51 and G65. tRNAMetf2 has a reversed G-C pair at positions 52 and 64 compared with those in tRNAMetf1 and E. coli tRNAMetf. Other regions are mostly the same as those in all prokaryotic initiator tRNAs so far reported. The thermostability of these thermophile initiator tRNAs is discussed in relation to their unique modifications.View full abstractDownload PDF (1799K) -
Motoharu HIRAI, Teruo NAKAJIMA1979 Volume 86 Issue 4 Pages 907-913
Published: October 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe biochemical mechanisms of the renal toxicity of 5-hydroxy-L-tryptophan to rats were studied using Wistar and Sprague Dawley rats, which had different LD50 values. When the amino acid was injected intraperitoneally, Wistar rats, which had a low LD50 value of 5-hydroxy-t-tryptophan, excreted larger amounts of serotonin and smaller amounts of 5-hydroxyindole acetic acid into the urine than Sprague Dawley rats, which had a high LD50 value. The activity of renal aromatic L-amino acid decarboxylase was higher in Wistar rats than in Sprague Dawley rats, while the activity of renal aromatic amino acid transaminase was in an opposite relationship. The activity of renal monoamine oxidase was almost the same in both strains and the activity of renal UDP glucuronyltransferase in Wistar rats was higher than that in Sprague Dawley rats. Since the renal damage caused in rats by 5-hydroxy-L-tryptophan was very similar to that caused by serotonin, the amine formed from the administered amino acid was thought to be an important factor for the renal necroses, and the difference in serotonin formation from the administered precursor amino acid may be one of the important factors leading to the difference in LD50 values in the two strains of rats.View full abstractDownload PDF (543K) -
Mitsuyoshi MORII, Shoji ODANT, Tokuji IKENAKA1979 Volume 86 Issue 4 Pages 915-921
Published: October 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe release of a peptide (molecular weight: about 3, 600) was observed during complex formation between human arantitrypsin (αl-AT) and bovine a-chymotrypsin, when monitored by gel-electrophoresis in the presence of sodium lauryl sulfate. Release of the peptide was proportional to the extent of complex formation. Peptides of the same molecular eight were also released during the complex formation of αl-AT with bovine trypsin or porcine elastase.
The peptide released from the complex with bovine α-chymotrypsin was composed of 32 amino acid residues, which did not correspond to the composition of any 32 amino acid segment in the bovine α-chymotrypsin sequence. The N- and C-terminal sequences of the peptide were determined to be H-(Ser)-Ile-Pro-Pro-Glu- and -Gln-Lys-OH, respectively. Though there was some uncertainty as to the N-terminal sequence, it is quite different from that of the original α1-AT molecule, and showed a similarity to the sequences of the leaving group sides of the reactive sites in some legume proteinase inhibitors. The C-terminal 2 residues were identical with those of native α1-AT. These results suggest that the peptide was released from the C-terminal region of α1-AT upon interaction with α-chymotrypsin.
It is tempting to suggest that α1-AT inhibits a serine proteinase by the acyl enzyme mechanism at a residue adjacent to the amino group of the N-terminus of this peptide and that this peptide is liberated as a leaving group in the enzymic process.View full abstractDownload PDF (963K) -
Lyang-ja LEE, Akira KIMURA, Tatsurokuro TOCHIKUR1979 Volume 86 Issue 4 Pages 923-928
Published: October 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn enzyme having both UDP-glucose (UDP-Glc) and UDP-galactose (UDP-Gal) pyro-phosphorylase activities was purified to homogeneity from Bifidobacterium bifidum. The molecular weight of the enzyme was about 200, 000 and it appeared to be composed of four identical subunits. The purified enzyme showed almost the same reactivity towards UDP-Glc and UDP-Gal, and showed about 10% of this activity towards UDP-xylose at 8mM. The enzyme required magnesium ions for maximum activity. The apparent equilibrium constants were about 2.5 for UDP-Glc pyrophosphorolysis and 1.1 for UDP-Gal pyrophos-phorolysis. The nzyme activities were inhibited by various nucleotides (product or substrate analogs). Some sugar phosphates, such as fructose 6-P, erythrose 4-P, and 3-phos-phoglycerate, stimulated the activities.
These properties are discussed in relation to the significance of the enzyme in galactose metabolism of B. bifidum.View full abstractDownload PDF (407K) -
Akemichi UENO, Michiaki KOHNO, Akira ISHIHAMA, Kumao TOYOSHIMA1979 Volume 86 Issue 4 Pages 929-942
Published: October 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA procedure was established whereby most of the major viral proteins were isolated to
apparent homogeneity in biologically and immunologically active forms from a single batch of avian sarcoma virus QV2. For the initial step of purification, gently disrupted virions were fractionated by CsC1 centrifugation into envelope proteins, RNA-dependent DNA polymerase, and viral core proteins. Further purification of envelope glycoproteins and DNA polymerase was performed by affinity chromatography on agarose columns cross-linked with plant lectins and poly(C), respectively. On the other hand, core proteins were fractionated by a combination of gel filtration and ion-exchange column chromatography into components p27, p19, and p15. The core protein p15 thus isolated retained proteolytic activity even after storage for 6 months.
The present study also demonstrated that QV2 p19 is structurally altered from the corresponding protein of avian myeloblastosis virus (AMV), a reference avian leukosis-sarcoma virus having a well-characterized polypeptide composition.View full abstractDownload PDF (4172K) -
Susumu IWASA, Hayao UENO, Tsutomu MIYA, Mitsuhiro WAKIMASU, Koichi KON ...1979 Volume 86 Issue 4 Pages 943-949
Published: October 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn enzyme immunoassay of pancreatic glucagon was established by using E. coli β-D-galactosidase [EC 3.2.1.23] as a marker. In order to increase the sensitivity of the immuno-assay, different peptides obtained from glucagon fragments were used to produce the enzyme conjugate and the immunogen. Antiserum N6E raised against C-terminal fragment peptide (15-29) could be diluted to more than 1:100, 000 in the assay and was highly specific for pancreatic glucagon. The antiserum reacted well with the C-terminal fragment peptide (21-29) as well as another fragment peptide (15-29) and pancreatic glucagon. The enzyme immunoassay using antiserum N6E and fragment peptide (21-29)-enzyme conjugate could detect as little as 1 to 2 pg of glucagon. The mean recovery of glucagon added to serum specimens was 104% and the coefficients of variation were 3.7-14.5% (within assay) and 9.0-18.5% (between assay).View full abstractDownload PDF (458K) -
Ryuichi MASAKI, Keishiro WADA, Hiroshi MATSUBARA1979 Volume 86 Issue 4 Pages 951-962
Published: October 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo ferredoxin-NADP+ reductases (FNRs I and II) [EC 1.6.7.1] were purified from a blue-green alga, Spirulina platensis, by (NH4)2SO, fractionation, gel filtration on Sephadex G-100 and DEAE-Sephadex A-50 chromatography. FNRs I and II were both FAD-containing enzymes with molecular weights of 33, 000, and could photochemically reduce NADP+ to the same extent in the presence of S. platensis ferredoxin, using FNR-depleted membrane fragments of S. platensis. They had similar physical and enzymatic properties, except for chemical properties such as the amino (N)-terminal sequences and the patterns of their peptide maps. The significance of the presence of two FNRs in S. platensis as well as of the multiple forms found in other organisms is discussed.View full abstractDownload PDF (1843K) -
Masuyuki KATSUMATA, Mary K. BAKER, Allen S. GOLDMAN1979 Volume 86 Issue 4 Pages 963-970
Published: October 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA method of regression analysis for Scatchard plots of the cooperative type (1-15) was derived from Deming's method (16) which is applicable to implicit functions, f(X, Y), in which both variables, X and Y, are subject to error. Deming's method involves the L coefficient, (∂f/∂X)2/Wx+(∂f/∂Y)2Wy, whose value varies with the unit of concentration. When the observations fit the theoretical curve well, the variation with the concentration unit is negligible, whereas the variation is appreciable when the observations fit badly. In order to standardize the effect of the unit, we introduced a hypothetical unit of concentration, which makes the mean values of the observations, _??_ and _??_, identical. Assuming two binding sites in one receptor protein, we applied the method to dexamethasone binding to the hepatic cytosol receptor protein of mice, strains A/J, C57 B10 and B10A. The Kd values of the first binding site of A/J and B10A mice were calculated to be significantly smaller than that of B10 mice. However, the Kd value of the second site of A/J mice was significantly larger than the corresponding values of B10 and B10A mice, and the value of B10A was larger than that of B10 mice. The differences in the Kd values may explain the genetic differences in the susceptibility to glucocorticoid-induced cleft palate among the three strains, since the affinity of the first binding is higher in the A/J and B10A strains, which have higher susceptibility than B10.View full abstractDownload PDF (540K) -
Fumie YAMASAKI, Hidenori HAYASHI1979 Volume 86 Issue 4 Pages 971-977
Published: October 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe sites of action of cyclic adenosine 3', 5'-monophosphate (cAMP) in phosphodiesterase [EC 3.1.4.17] induction in Dictyostelium discoideum were studied. When cAMP was added to the cell suspension from the start of the incubation, the effect of the cyclic nucleotide on the cellular enzyme-induction did not appear for 30min, then occurred abruptly. From experiments on the addition of cAMP to the cell incubation mixture at various times, preparations for the synthesis of the enzyme appear to occur during the period from 20min to 30min after the start of the incubation. After the addition of cycloheximide at 30min, the enzyme was degraded very rapidly. The half-life of phosphodiesterase was roughly 21min in the presence of cAMP, and 12min in its absence. The degradation rates became approximately the same on removing cAMP. When daunomycin and actinomycin D were added to cells previously stimulated with cAMP, phosphodiesterase was still synthesized at a higher rate than in cells not pretreated with cAMP.
These results suggest that cAMP acts at two sites at least, i.e., on enzyme synthesis at the transcription level, and in suppressing the degradation of phosphodiesterase.View full abstractDownload PDF (474K) -
Takeshi MIZUNO, Makoto KAGEYAMA1979 Volume 86 Issue 4 Pages 979-989
Published: October 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe outer membrane of Pseudomonas aeruginosa PAO contains six major proteins (proteins D, E, F, G, H, and I). Two of them (protein F and protein H) were found to be retained by the peptidoglycan layer when cell envelopes were extracted with 2% sodium dodecyl sulfate (SDS) solution at 35°C. At higher temperatures (>55°C), no proteins were retained by peptidoglycan.
By making use of this property, purification of protein F and protein H was achieved. Three other major outer membrane proteins, D, E, and I were also isolated and characterized. Their amino acids compositions were determined.
Circular dichroism spectra of these isolated proteins were measured in SDS solution. Protein F was rich in β-structure, while protein I was rich in α-helix. When isolated protein F was heated (100°C-15min) in SDS solution, the circular dichroism spectrum changed significantly. In parallel with the conformational change, the electrophoretic mobility of protein F on urea-SDS polyacrylamide gel also changed. These results indicate that protein F is a so-called heat-modifiable protein.View full abstractDownload PDF (2566K) -
Takeshi MIZUNO1979 Volume 86 Issue 4 Pages 991-1000
Published: October 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSProtein H, one of the major outer membrane proteins of Pseudomonas aeruginosa, shows an interesting interaction with the peptidoglycan layer of the cell. It is retained by peptidoglycan after extraction of the cell envelope with SDS solution at 35°C. A protein of the same molecular weight (21, 000) as protein H was found in the peptidoglycan-associated fraction of Escherichia coli K-12 prepared under the same conditions. This protein, designated here as protein 21K, was purified from the cell envelope of E. coli, and the properties of two proteins (protein H and protein 21K) were compared. By gas chromatographic analysis of purified protein 21K and protein H, it was found that both contained covalently linked fatty acid. In isotopic labeling experiments, [14C]palmitic acid and [2-3H]glycerol were incorporated into both proteins H and 21K. These two proteins showed similar amino acid compositions, but no apparent correlation was found between protein 21K or protein H and Braun's lipoprotein. These results suggest that protein 21K of E. coli K-12 and protein H of P. aeruginosa PAO are a novel type of lipoprotein. All nine gram-negative bacteria tested, including enteric and non-enteric bacteria, contained a similar protein of apparent molecular weight 21, 000 as a peptidoglycan-protein complex.View full abstractDownload PDF (1799K) -
Akihiko NAKANO, Mitsuo TASUMI, Kitao FUJIWARA, Keiichiro FUWA, Tatsuo ...1979 Volume 86 Issue 4 Pages 1001-1011
Published: October 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe spin-lattice and spin-spin relaxation rates were measured of the Gly Ca and Tyr aryl protons of glycyl-L-tyrosine (Gly-Tyr) bound to manganese(II)-substituted carboxypeptidase A (MnCPA) in aqueous solution. The temperature and frequency dependences of the relaxation rates were analyzed using the Solomon-Bloembergen-Morgan equations. The binding modes of MnCPA with Gly-Tyr in solution are different from that of ZnCPA in crystals.
1. Mn(II)-coordinated water of MnCPA is not excluded by the binding of Gly-Tyr substrate molecules.
2. The Gly carbonyl group does not coordinate tightly to the metal ion of MnCPA. The Gly Ca protons of Gly-Tyr in the productive binding site are appreciably mobile.
3. A non-productive loose binding of another Gly-Tyr molecule is suggested by simulation of the temperature and frequency dependences of the proton relaxation rates.View full abstractDownload PDF (828K) -
Sadaaki KOMURA, Kiyoshi KURAHASHI1979 Volume 86 Issue 4 Pages 1013-1021
Published: October 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn L-2, 4-diaminobutyric acid activating enzyme was found in crude extracts of Aerobacillus polyaerogenes, which produces polymyxin E1, and E2. The enzyme was partially purified by sonication of the cells, followed by ultracentrifugation, ammonium sulfate fractionation, and DEAE-cellulose column chromatography. In addition to L-2, 4-diaminobutyric acid, the enzyme activated L-leucine and L-threonine, which are constituent amino acids of polymyxin E. All three amino acids were bound to the enzyme as thioesters. These results suggest that polymyxin is synthesized by a multienzyme thiotemplate mechanism, in the same way as gramicidin S, tyrocidines, bacitracins, and gramicidin A.View full abstractDownload PDF (654K) -
Evidence for a Dimer Structure of the ATPaseFuminori KUDOH, Shinji NAKAMURA, Motonori YAMAGUCHI, Yuji TONOMURA1979 Volume 86 Issue 4 Pages 1023-1028
Published: October 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSNa+, K+-dependent ATPase [EC 3.6.1.3] was purified from porcine kidney by the method of Lane et al. [(1973) J. Biol. Chem. 248, 7197-7200] with slight modifications [Yamaguchi, M. & Tonomura, Y., (1979) J. Biochem. 86, 509-523].
The amounts of a phosphorylated intermediate (EP) and ouabain bound to the enzyme during the ATPase reaction were measured in 2.1mM MgCl2 and various concentrations of NaCl and KC1 at pH 7.5 and 20°C. In the presence of NaCl and the absence of KCI, the molar ratio of the amounts of EP and bound ouabain was 1 : 2. In the presence of both NaCl and KCl, it was 1 : 1. In both cases, the amount of bound ouabain was equal to that of EP in the absence of ouabain.
These findings suggest that the functional unit of the transport ATPase is a dimer.View full abstractDownload PDF (396K) -
Motonari TSUBAKI, Kiyoshi NAGAI1979 Volume 86 Issue 4 Pages 1029-1035
Published: October 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn order to clarify the role of salt-bridges in hemoglobin, the oxygen equilibrium curves and electron paramagnetic resonance (EPR) spectra of cobalt-iron hybrid hemoglobins were determined.
The EPR spectra of deoxy α(Co)2β(Fe)2 could be interpreted as a mixture of two distinct paramagnetic species: one showed a maximum of the first derivative spectrum at g=2.39 and the other at g=2.33. The oxygen equilibrium curves of the hybrid indicated that the former is assignable to the T structure and the latter to the R structure.
The cooperativity of oxygen binding of α(Co)2β(Fe)2 was abolished by removal of Arg-141α. The EPR spectrum of stripped deoxy des-Arg α(Co)2β(Fe)2 exhibited a maximum at g=2.33, which is characteristic of the R structure, regardless of the pH. Addition of inositol hexaphosphate (IHP) to des-Arg α(Co)2β(Fe)2 restored the cooperativity of oxygen binding, which implies that the deoxygenated form of des-Arg α(Co)2β(Fe)2 is converted to the T structure upon addition of IHP. However, the EPR signal at g=2.39 was not restored upon conversion to the T structure by addition of IHP.
It is therefore concluded that the EPR spectrum of the deoxy α(Co) subunit depends both on the quaternary structure and on the localized strain at the heme.View full abstractDownload PDF (502K) -
Yozo GOTOH, Shigeru SAITO, Atsushige SATO1979 Volume 86 Issue 4 Pages 1037-1040
Published: October 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFibroblast-like cells were isolated from dental pulp of rabbit incisor, and were shown to synthesize and secrete procollagen. Analysis of the secreted procollagen and their CNBr peptides indicated that the prominent form was type I procollagen. Our results also suggested the presence of type III procollagen as a minor component synthesized by pulp cells in vitro.View full abstractDownload PDF (263K) -
Takahide WATANABE, Takao NAKAMURA1979 Volume 86 Issue 4 Pages 1041-1047
Published: October 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSLinoleate hydroperoxide, purified by silica gel chromatography and at concentrations of 70-100 nmol/mg mitochondrial protein, activated state 4 respiration and Mg-ATPase activity of mitochondria to levels of 80% and 25%, respectively, of those induced by 300μM DNP, and completely inhibited oxidative phosphorylation. These effects are the same as those caused by linoleate, but the hydroperoxide caused more rapid degeneration of the activated respiration of mitochondria than linoleate. Further addition of the hydroperoxide induced oligomycin-insensitive Mg-ATPase to a level 3 times that obtained with DNP, accompanied by clearing of the mitochondrial suspension and release of malate dehydrogenase from the matrix. The extent of the effects caused by the methyl ester of linoleate hydroperoxide was much less than those by the free acid.View full abstractDownload PDF (527K) -
Yasunori KOZUTSUMI, Nobuyuki ITOH, Kazuhisa FUJIMOTO, Toshisuke KAWASA ...1979 Volume 86 Issue 4 Pages 1049-1054
Published: October 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMucopolysaccharides were isolated from rat liver mitochondria which had been labeled with 35S-sulfate. They were prepared from trichloroacetic acid (TCA)-insoluble and -soluble fractions of lipid-free mitochondria. These fractions were digested with pronase exhaustively, and the mucopolysaccharides were recovered in the void volume fractions of gel filtration of the pronase digests on Sephadex G-50, monitored by radioactivity determination.
Identification of these mucopolysaccharides was based on electrophoresis on cellulose acetate film using three different media, enzymatic and chemical degradations specific to each type of mucopolysaccharide, using chondroitinases, heparitinase, and nitrous acid.
From the TCA-insoluble fraction, chondroitin sulfate A and dermatan sulfate were obtained in a ratio of about 1:2, based on 35S-radioactivities, whereas the TCA-soluble fraction yielded chondroitin sulfates A/C, dermatan sulfate, and heparan sulfate in a ratio of about 1:3:12. The total amount of mitochondrial mucopolysaccharides was about 3mg/g protein, distributed between the TCA-insoluble and -soluble fractions in a ratio of about 1:3.View full abstractDownload PDF (484K) -
Hiroshi NORMA, Haruhiko NODA1979 Volume 86 Issue 4 Pages 1055-1065
Published: October 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe kinetics of denaturation by guanidine hydrochloride (GuHCl) of a thermostable phosphoglycerate kinase (PGK) extracted from Thermus thermophilus and of yeast PGK at neutral pH were studied by circular dichroism. Denaturation by GuHCl proceeded as a first-order reaction. The activation free energy of the denaturation reactions (ΔGf≠) in the absence of GuHCl was estimated to be 32.7kcal/mol for T. thermophilus PGK and 27.9kcal/mol for yeast PGK (at 25°C). Measurements of the rate constants at various temperatures indicated that ΔGf≠ has maximum values at 29°C for T. thermophilus PGK and at 20°C for yeast PGK, and that the temperature dependences of ΔGf≠, ΔHf≠, and ΔSf≠ for T. thermophilus PGK are smaller than those of yeast PGK. Values of ΔSf≠ for thermal denaturation for both PGK's are approximately 200 e.u.View full abstractDownload PDF (562K) -
Hiroyuki KOMIYA, Shosuke TAKEMURA1979 Volume 86 Issue 4 Pages 1067-1080
Published: October 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSStarting from low molecular weight RNA obtained from rainbow trout (Sahno gairdnerii) liver, 5S ribosomal RNA (rRNA) was highly purified by successive chromatography on columns of DEAE-Sephadex A50 and Sephadex G100. Products of complete and partial digestions of this RNA with pancreatic ribonuclease (RNase A) [EC 3.1.4.22] and RNase T1, [EC 3.1.4.8] were isolated and sequenced by conventional and high-performance liquid chro-matography (HPLC) procedures. The nucleotide sequence of this RNA thus established was compared with those of five other vertebrate 5S rRNAs, and the rates of base substitution per site per year were found to be nearly constant in these RNAs. The analyses of the partial digests of the trout 5S rRNA revealed several sites susceptible to RNase attack, which could be accounted for by the secondary structure model for eukaryotic 5S rRNAs proposed by Nishikawa and Takemura (1).View full abstractDownload PDF (890K) -
Hiroyuki KOMIYA, Kazuya NISHIKAWA, Kazuko OGAWA, Shosuke TAKEMURA1979 Volume 86 Issue 4 Pages 1081-1088
Published: October 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSConditions were established for the separation and quantitative determination of ribonucleosides, mono- and oligo-ribonucleotides by high-performance liquid chromatography (HPLC) on columns of AS-Pellionex SAX and AL-Pellionex WAX. By combining a high-speed UV spectrum monitor with an HPLC apparatus, products of RNase digestions of oligonucleotides and 5S ribosomal RNA (rRNA) were identified by measuring their UV spectra under continuous solvent flow, and also from their retention times on the columns (positions of elution). It took only 10 to 30 min for one chromatography run and required less than 0.01, A260 unit of sample per nucleotide material in each peak.View full abstractDownload PDF (553K) -
Yohtalou TASHIMA, Hideo MIZUNUMA, Masahiro HASEGAWA1979 Volume 86 Issue 4 Pages 1089-1099
Published: October 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA simple procedure has been developed for the purification of mouse liver and kidney fructose-1, 6-bisphosphatase. In addition to the conventional method, including substrate elution from phosphocellulose, Blue Sepharose column chromatography made the purification procedure highly reproducible. The enzyme from rabbit liver was also purified by this method with a small modification. The isolated preparation was electrophoretically homogeneous. The mouse liver enzyme was identical with the kidney enzyme, and different from the rabbit liver enzyme electrophoretically. The structural properties and the amino acid composition were similar to those of this enzyme from other mammalian livers; the molecular weight was 143, 000, subunit size was 37, 500, S20, w was 7.0, and partial specific volume was 0.74. Cysteine and methionine residues amounted to 5-6 mol per subunit. Tryptophan was not detected. The Km value for fructose-1, 6-bisphosphate was 1.3 μm. The K1 value for AMP was 19 μM. EDTA strongly activated the activity of the mouse liver enzyme at neutral pH. A partial proteolytic digestion of the mouse liver enzyme decreased the activity at neutral pH, and increased it at alkaline pH.View full abstractDownload PDF (1895K) -
Tateo YAMANAKA, Minoru SHINRA, Koko TAKAHASHI, Mineo SHIBASAKA1979 Volume 86 Issue 4 Pages 1101-1108
Published: October 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHydroxylamine oxidoreductase [EC 1.7.3.4] of Nitrosomonas europaea was purified to an electrophoretically homogeneous state and some of its properties were studied.
The molecular weight of the enzyme as determined by gel filtration on Sephadex G-150 and by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate is 175, 000-180, 000, while the minimum molecular weight per heme determined from the dry weight and heme content is 17, 500. The enzyme is a C-type cytochrome; its reduced form shows absorption peaks at 418 (γ peak), 521 (β peak), 553 (α peak), and 460 nm (due to an unidentified chromophore). Although the α peak at 553 nm has a shoulder at 559 nm, the enzyme does not possess protoheme or a cytochrome b subunit. It seems likely that the enzyme molecule possesses heme c molecules in different states.
The enzyme reacts rapidly with various eukaryotic cytochromes c, but does not react with “bacterial-type” cytochromes c. Although the enzyme does not react with cytochrome c-552 (N. europaea), another C-type cytochrome of the organism, cytochrome c-554 (N. euro-paea) acts as an electron acceptor for the enzyme.View full abstractDownload PDF (592K) -
Purification and Some PropertiesKaoru RIKITAKE, Imao OKA, Makoto ANDO, Tadashi YOSHIMOTO, Daisuke TSUR ...1979 Volume 86 Issue 4 Pages 1109-1117
Published: October 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCreatinine amidohydrolase [EC 3.5.2.-; creatininase] was purified in an overall yield of 11% from cell-free extract of Pseudomonas putida var. naraensis, strain C-83, by column chro-matographies on sarcosine-HM-Sepharose and DEAE-cellulose, and gel filtration on Sephadex G-200. The purified enzyme was homogeneous as judged by disc gel electrophoresis. The enzyme catalyzed the reversible conversion of creatinine to creatine with an optimal pH of 7-9. Km values for creatinine and creatine were 26mM and 0.13M, respectively. The enzyme was also active toward glycocyamidine, though the reaction rate was quite low, but it was completely inert toward hydantoin and its derivatives. The molecular weight of the enzyme was estimated to be 175, 000 by ultracentrifugal analysis and the subunit molecular weight estimated by SDS-polyacrylamide gel electrophoresis was 23, 000, suggesting that the enzyme is composed of eight subunit monomers. The isoelectric point was 4.7 as judged by iso-electric focusing experiments. The enzyme was markedly inactivated by heavy metal ions, N-bromosuccinimide, ethoxyformic anhydride, and dye-sensitized photooxidation, and also partially by metal chelators. The enzyme was found to contain about one gram atom of zinc per subunit monomer. The metal-free, inactive enzyme was prepared and could be reactivated by the addition of Mn2+, Co2+, Mg2+, Fe2+, Ni2+, and Zn2+ in that order of de-creasing effectiveness. These results indicate that metal is intimately involved in the creatini-nase activity of P. putida.View full abstractDownload PDF (1169K) -
Kiyoshi KONDO, Shoji TAJIMA, Ryo SATO, Kozo NARITA1979 Volume 86 Issue 4 Pages 1119-1128
Published: October 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe primary structure of the membrane-binding segment of rabbit cytochrome b5 has been determined. This segment, prepared by trypsin digestion of the intact protein, consists of 43 amino acid residues and corresponds to the COOH-terminal end (residues 91-133) of the parent molecule. Deduction of the primary structure was based on automated sequence analysis of the whole segment as well as manual and dansyl-Edman degradations of peptide fragments produced by CNBr cleavage and partial acid hydrolysis. The sequence obtained is: Leu -Ser -Lys -Pro -Met -GIu -Thr -Leu -Ile -Thr-Thr-Val-Asn-Ser-Asn-Ser-Ser-Trp-Trp-Thr -Asn-Trp-Val-Ile-Pro-Ala-Ile-Ser-Ala-Leu-Ile-Val-Ala-Leu-Met-Tyr-Arg-Leu-Tyr-Met-Ala-Asp-Asp. This sequence is 63 to 81% homologous with respect to those determined for the membrane-binding segments of equine, porcine and bovine cytochrome b5. The interaction of this segment with phospholipid bilayer membranes is discussed, and a prediction of its secondary structure is also presented.View full abstractDownload PDF (723K) -
Inactivation, and Protection with Detergents and PhospholipidsYumiko TAMORI, Masahiro NISHIJIMA, Shoshichi NOJIMA1979 Volume 86 Issue 4 Pages 1129-1138
Published: October 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA crude preparation of membrane-bound phospholipase A (detergent-resistant) in Escherichia coli K-12 cells was found to be quite stable or even apparently activated on incubation at 100°C, but became strikingly thermolabile when it was highly purified and Triton X-100 was removed from the purified enzyme preparation. The rate of inactivation showed a biphasic temperature dependence: inactivation was rapid at 37°C and also above 70°C. Inactivation above 70°C changed the mobility of the enzyme on sodium dodecyl sulfate/polyacrylamide gel electrophoresis, but inactivation at 37°C did not affect the electrophoretic mobility. Triton X-100 effectively protected the enzyme against inactivation at 37°C. The concentration required for the protection of the enzyme was more than its critical micelle concentration. Phospholipids, such as phosphatidylethanolamine, phosphatidylglycerol, cardiolipin, phosphatidylcholine, lysophosphatidylethanolamine, and lysophosphatidylcholine, also protected the enzyme against inactivation at 37°C. These results suggest that the binding of hydrophobic compounds stabilizes the enzyme.View full abstractDownload PDF (1190K) -
Possible Involvement of Some the Most Reactive SH GroupsTakashi SHIMIZU1979 Volume 86 Issue 4 Pages 1139-1145
Published: October 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn apparent ‘triphasic’ alteration of 30S dynein ATPase activity was produced by treatment with various amounts of NEM when the modification and subsequent ATPase assay were carried out at pH 7.4 and pH 10-10.2, respectively.
The Mg-ATPase activity was markedly inhibited by modification of the most reactive SH groups with 10 μM NEM, although the same treatment had no significant effect on the activity when assayed at neutral pH. Increasing the NEM concentration to 0.3 mm largely restored the enzyme activity, but a further increase in NEM concentration inhibited the enzyme activity again.
This unusual response of 30S dynein ATPase at pH 10-10.2 was accounted for by the results of Arrhenius plots of the enzyme activity at pH 10.1; the enzyme protein modified with not more than 10 μM NEM was not stable under the assay conditions (pH 10-10.2 at 25°C), whereas modification with 0.3 mm NEM stabilized 30S dynein against the assay con ditions.
The possible significance of the 10 μM NEM-induced inhibition of the 30S dynein alkaline ATPase activity is discussed in connection with the participation of SH groups of 30S dynein in the enzyme activity.View full abstractDownload PDF (475K) -
S. Tsuyoshi OHNISHI1979 Volume 86 Issue 4 Pages 1147-1150
Published: October 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA new method is introduced which allows the study of calcium-induced calcium release from fragmented sarcoplasmic reticulum. Results obtained with this method are in agreement with those obtained by previous investigators using skinned muscle fiber. It was also found that anesthtic drugs and alcohol increased the calcium- and caffeine-induced calcium release from the sarcoplasmic reticulum.View full abstractDownload PDF (332K) -
Hisahide HIURA, Tomisaburo KAKUNO, Jinpei YAMASHITA, Robert G. BARTSCH ...1979 Volume 86 Issue 4 Pages 1151-1153
Published: October 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWith Rhodospirillum rubrum, hydrogenase was found to exist partly as an extracellular enzyme in the culture medium. After 4-day cultivation, the total activity and the specific activity of the enzyme in the medium were about 10 times and 230 times as high as those in the crude extract obtained from disrupted cells. The time course for the production of hydrogenase during cultivation was studied.View full abstractDownload PDF (179K) -
Gen MATSUMOTO, Takaaki KOBAYASHI, Hikoichi SAKAI1979 Volume 86 Issue 4 Pages 1155-1158
Published: October 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMembrane excitability of the squid giant axon was destroyed by perfusing with a medium containing 0.2mM Ca ions that depolymerize microtubules. Restoration of the membrane excitability was achieved by perfusing the axon with a medium that contained purified tubulintyrosine ligase, tubulin, ATP, Mg2+, K+, cyclic AMP, and axonal 300K protein.View full abstractDownload PDF (432K) -
Masayoshi IWAKI, Hiroyuki KAGAMIYAMA, Mitsuhiro NOZAKI1979 Volume 86 Issue 4 Pages 1159-1162
Published: October 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe complete amino acid sequence of the β-subunit of protocatechuate 3, 4-dioxygenase is presented. The β-subunit contained 237 amino acid residues, 4 of which were methionines. Accordingly, cyanogen bromide cleavage of the S-carboxymethylated β-subunit produced five peptides. The sequences of these peptides were determined by analyses of the peptides obtained by tryptic, staphylococcal protease and thermolysin digestions. The alignment of the cyanogen bromide peptides was deduced by the use of overlapping peptides containing methionine which were obtained by tryptic digestion of the S-carboxymethylated β-subunit. The calculated molecular weight was 26, 588, which is close to the value estimated by acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate.View full abstractDownload PDF (240K) -
Hidemi ISHII, Tetsuya SUGA, Hidenori HAYASHI, Shinkichi NIINOBE1979 Volume 86 Issue 4 Pages 1163-1166
Published: October 01, 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effect of Triton WR-1339 on the rates of synthesis and degradation of hepatic catalase was examined. Triton WR-1339 was injected intraperitoneally into rats at a dose of 200mg per 100g body weight. Catalase activity decreased to about 35 % of that of the control at 42-48h after the injection and recovered to the normal level at 96 h. Other peroxisomal enzymes, D-amino acid oxidase and urate oxidase, showed similar patterns of the activities to those of catalase. During the first 48 h after the injection of Triton WR-1339, the rate of catalase synthesis (ks) fell to below a detectable value, while that of the degradation (kd) did not show any significant change. On the other hand, during the period 48-96h after the injection, the rate of the synthesis (ks) returned to the normal level though that of the degra-dation (kd) decreased to about 50% of the control.View full abstractDownload PDF (311K) -
1979 Volume 86 Issue 4 Pages 1167b
Published: 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDownload PDF (18K) -
1979 Volume 86 Issue 4 Pages 1167a
Published: 1979
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDownload PDF (18K)
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