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Volume 81 (1977)
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- Issue 5 Pages 1181-
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- Issue 2 Pages 277-
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Volume 81, Issue 5
Displaying 1-49 of 49 articles from this issue
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Sumihiro HASE, Yoshio MATSUSHIMA1977 Volume 81 Issue 5 Pages 1181-1186
Published: May 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn acidic polysaccharide fraction composed of glucose and N-acetylmannosaminuronic acid with a small portion of peptidoglycan was isolated by enzymic digestion and subsequent ECTEOLA-cellulose chromatography from the cell walls of Micrococcus lysodeikticus. On mild acid treatment, the fraction became Morgan-Elson positive and formed the Morgan-Elson chromogen on heating with phosphate buffer (pH 7). The product of mild acid treatment released inorganic phosphate on treatment with phosphomonoesterase. After gel-chromatog-raphy on Sephadex G-25 and DE-32, the acidic polysaccharide fraction contained less glucosa-mine than muramic acid. By reduction of this fraction with borohydride, a part of the glucos-amine was converted into glucosaminitol. Based on these results, it is suggested that the acidic polysaccharide is linked to glucosamine by a (1-3) linkage, which is linked to the 6 posi-tion of a muramic acid residue by a phosphodiester inkage.View full abstractDownload PDF (405K) -
Kiyoshi ISHIKURA, Yasunori NITTA, Takehiko WATANABE1977 Volume 81 Issue 5 Pages 1187-1192
Published: May 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. The hydrolytic reaction of phenyl β-maltoside catalyzed by saccharifying α-amylase [EC 3. 2. 1. 1] of Bacillus subtilis was studied at 25°C and pH 5.4, by measuring the total reducing power and the amount of phenol liberated, and by thin layer chromatography.
2. The enzyme hydrolyzed phenyl β-maltoside at the glucosidic linkage between the two glu-cose residues to form D-glucose and phenyl β-D-glucoside. Besides these products, maltose, maltotriose, and phenyl β-maltotrioside were also observed as reaction products. The identifi-cation of phenyl β-maltotrioside is described in detail. The formation of these products was attributed to the transglycosylation reaction of the enzyme.
The time course of reaction as followed by reducing power measurement showed an induction period of several minutes.View full abstractDownload PDF (1503K) -
Kazuko ÔBA, Seiji MURAKAMI, Ikuzo URITANI1977 Volume 81 Issue 5 Pages 1193-1201
Published: May 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSLactate dehydrogenase [L-lactate: NAD oxidoreductase, EC 1. 1. 1. 27] was isolated from sweet potato root tissues. Two species of the enzyme (isozymes I and II) were separated by DE-52 cellulose column chromatography from healthy, cut, and black-rot diseased tissues. Isozymes I and II were purified from healthy and diseased tissues, respectively. Reduction of pyruvate by NADH with either isozyme I or II was inhibited by pyruvate at high concentrations, by NAD+ and by several mononucleotides. Isozyme I was inhibited by a lower concentration of adenine nucleotide than isozyme II, and Km for pyruvate was increased markedly at acidic pH in the case of isozyme I, but only slightly in the case of isozyme II. The molecular weights of both isozymes were determined to be 150, 000 and they were found to be charge isomers by polyacrylamide gel electrophoresis. The enzyme activity increased in response to infection by black-rot fungus but decreased in response to cutting.View full abstractDownload PDF (591K) -
Mariko SAITO, Satoshi TOYOSHIMA, Toshiaki OSAWA1977 Volume 81 Issue 5 Pages 1203-1208
Published: May 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThree radioactive glycopeptides were isolated from human peripheral lymphocytes stimulated with Wistaria floribunda mitogen in the presence of D-[14C]glucosamine hydrochloride by mild trypsin digestion followed by gel filtration and preparative high-voltage paper electrophoresis. The carbohydrate compositions of these glycopeptides suggest that one has a sugar chain of the type found in serum glycoproteins, consisting of sialic acid, galactose, N-acetylglucosamine, mannose, and fucose in a molar ratio of 2:2:4:2:1, and the other two have sugar chains like those of mucins, consisting of sialic acid, galactose, and N-acetylgalactosamine in a molar ratio of 1 or 2:1:1. The results of enzymic degradation with purified glycosidases indicate that these sugar chains are similar in structure to their counterparts in human erythrocyte membranes.View full abstractDownload PDF (450K) -
I. Partial Purification and PropertiesYoshio OKAWA, Tsutomu YAMAGUCHI1977 Volume 81 Issue 5 Pages 1209-1215
Published: May 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. A search for a long chain fatty acyl sterol-ester hydrolase in microorganisms led to the isolation from soil of five strains belonging to Fusarium sp. which produced strong activity in the culture medium.
2. The cholesterol esterase from Fusariurre oxysporum IGH-2 was purified about 270-fold by means of CaCl2 precipitation and Sephadex G-75 column chromatography.
3. The cholesterol esterase was activated by adekatol and Triton X-100. It was inhibited by lecithin and lysolecithin, and completely inactivated by heat treatment (60°C for 30min, at pH 7.0).
4. The optimum pH of the enzyme was found to be around 7.0.
5. Among various cholesterol esters tested, cholesterol linoleate was the most suitable sub-strate.
6. Cholesterol esters in serum were also hydrolyzed by this enzyme.View full abstractDownload PDF (483K) -
Nariko KAWAMURA, Tamotsu TAKETOMI1977 Volume 81 Issue 5 Pages 1217-1225
Published: May 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTotal gangliosides were isolated from grey and white matter of animal brains and human brains at various ages, including the pathological brain of a patient with late infantile amaurotic family idiocy (Bielschowsky-Jansky disease) by a new procedure. Total lipids extracted from brain tissues were directly applied to a column of Silica Gel G-Silica Guhr G (1:1, w/w) slurried with chloroform-methanol-28% ammonia (32:8:1, by vol) without Folch's partition. After the total lipids, with the exception of gangliosides, had been eluted with the same solvent, the total gangliosides were eluted with chloroform-methanol-water (60:35:8, by vol). The total gangliosides were subjected to mild alkaline methanolysis and dialyzed against deionized water, then the ganglioside fraction thus obtained was rechromatographed in the same way. The content of gangliosides in various brains was 400-500μg for grey matter and 150-200μg for white matter (amount of long chain base per gram of fresh tissue). GDla and GMI accounted for about 30-40% of the total gangliosides in grey matter, while in white matter GMI was the major ganglioside (>50%): it was particularly noteworthy that sialosylgalactosyl ceramide accounted for 10-20% of the total gangliosides in human white matter. The long chain bases of individual gangliosides in human brains contained both C18 and C20 sphingosine in different ratios, and the ratio of C20 to C18 sphingosine increased with age in the order GM3<GM2<GM1<GDla<GDlb_??_GTl. The ratios of individual gangliosides in the patient with Bielschowsky-Jansky disease were 2 to 3 times lower than those of age-matched controls.View full abstractDownload PDF (2592K) -
Shinji YOKOYAMA, Masaharu TAKAYAMA, Yasuo AKANUMA1977 Volume 81 Issue 5 Pages 1227-1230
Published: May 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA sonicated dispersion of [14C] lecithin was incubated with high density lipoproteins (HDL) coupled to Sepharose. After washing the gels thoroughly with a buffer, the gels were incubated with low density lipoproteins (LDL); [14C] lecithin was transferred from the sonicated dispersion via HDL-Sepharose to the LDL.
The LDL fraction thus prepared showed no contamination with lecithin dispersion or HDL. The lecithin: cholesterol acyltransferase (LCAT) reaction could be completely inhib-ited during preparation, and the net recovery of radioactivity in LDL was 16%. of that of the original lecithin dispersion.
The [14C] lecithin in the washed HDL-Sepharose was shown to be a substrate of the LCAT reaction in vitro.View full abstractDownload PDF (281K) -
Norio SUZUKI, Shizue KAWASHIMA, Sumiko FUKUSHIMA, Nobuo UETA, Hiroshi ...1977 Volume 81 Issue 5 Pages 1231-1236
Published: May 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe chemical and physical properties of bullfrog serum low density lipoprotein (LDL) were investigated. On a weight percentage basis, LDL contained cholesterol ester, 30.3%; choles-terol, 5.6%; triglyceride, 12.5%; phospholipids, 23.3%; and protein, 22.4%.
The fatty acid compositions of triglycerides and major phospholipids from the bullfrog serum LDL were quite similar to those of human serum LDL. However, the fatty acid composition of the chlesterol ester from the bullfrog serum LDL was quite different from that of the human serum LDL.
Th average particle weight, determined by gel filtration, was 2×106 daltons. This value is very close to that of human LDL. In the fluorescence emission spectrum of bullfrog serum LDL, the emission maximum was 324nm.
The amino acid composition of the apo-LDL resembled that of human apo-LDL.View full abstractDownload PDF (380K) -
Tsuneko UCHIDA, Chiyoko FUNAYAMA-MACHIDA1977 Volume 81 Issue 5 Pages 1237-1246
Published: May 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. Using reverse transesterification by three RNases with different base specificities, that is RNases U2, N1, and B. subtilis RNase, a method for the systematic synthesis for all 16 possible dimers and 50 trimers out of 64 in all was established. 2. The yield of dimer was dependent on the ratio of phosphate acceptor to donor (A/D) and the total substrate concentration, in addition to various other conditions. A/D and the accep for concentration were preferably more than 10 and 0.25M, respectively. Therefore, the yield of dimer was dependent on the solubility of the acceptor, when it was less than 0.25M. 3. Trinucleotide were also synthesized with RNases U2 or N1 as follows; I. X-Y>p+N or II. X>p+Y-N. The mode and yield of trimer synthesis were quite similar to those of the corresponding dimer synthesis with the same joining site sequence. 4. B. subtilis RNase was not useful for trimer synthesis, because the rate of degradation of dinucleotide used as the acceptor or donor was faster than that of reverse transesterification. 5. The yield of dimer synthesis was 50-70% using a pyrimidine derivative as an acceptor and 10-30%. using a purine derivative. The yields of trimer in type I reaction were 60-80% in cases with a pyrimidine derivative and 10-30% in cases with a purine derivative, while those in type II reaction were about 20% with pyrimidine as the Y base in acceptor Y-N, and about 10% with purine.View full abstractDownload PDF (751K) -
Masao ONO, Masaya KAWAKAMI1977 Volume 81 Issue 5 Pages 1247-1252
Published: May 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMouse myeloma cells, MOPC-31C, were incubated in the presence of 2-thiouridine and newly-synthesized RNA which appeared to contain 2-thioUMP as a constituent was separated from preexisting RNA by affinity chromatography using organomercurial agarose as a support. Both pH and salt concentration greatly affected the specific adsorption of the newly-synthesized RNA on the column. Under optimal conditions the rate of adsorption of the newly-synthe-sized RNA on the column was proportional to the logarithmic concentration of 2-thiouridine in the culture medium. Furthermore, at a given concentration of 2-thiouridine in the medium, a shorter incubation period caused a reduction of the rate of RNA adsorption on the column. The molecular size distributions of both total RNA and the adsorbed fraction, synthesized during 30min in the presence of 2-thiouridine, were similar to that of RNA synthesized in the absence of the drug.View full abstractDownload PDF (418K) -
Hirobumi TERAOKA, Mari SHIMOYACHI, Kinji TSUKADA1977 Volume 81 Issue 5 Pages 1253-1260
Published: May 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDNA ligases have been purified 1, 500-fold from cytoplasmic fraction and 114-fold from 0.15M NaCl extract of rat-liver nuclei. These enzymes catalyze the formation of a phosphodiester linkage between the 3'-hydroxyl group and the 5'-phosphoryl group of an interrupted strand in a DNA duplex. The enzyme from each fraction requires ATP and Mg2+ (or Mn2+) for activity, and is inhibited reversibly by p-chloromercuribenzoate. The cytoplasmic and nuclear enzymes are distinguished from each other by the following criteria: apparent molecular weight; sedi-mentation coefficient; charge properties; Km for Mg2+ (or Mn2+); Km for ATP; effect of ionic strength on the molecular and kinetic properties.View full abstractDownload PDF (674K) -
Hiroshi SHINKAI, Yutaka NAGAI1977 Volume 81 Issue 5 Pages 1261-1268
Published: May 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCollagenase released from embryonic and adult human skin explants has been studied with special reference to the latency of the enzyme.
1) Embryonic human skin explants showed a much higher capacity for collagenase produc-tion than did adult skin, on the basis of unit weight of tissue.
2) Culture medium from embryonic skin explants contained latent collagenase at almost twice the concentration of the active form. No appreciable amount of latent enzyme was observed in the adult skin system.
3) The molecular weights of active and latent collagenases were about 40, 000 and 50, 000, respectively.
4) The latent collagenase was found to be activated by simple passage through a Sephadex G-50 column after adding NaI to a final concentration of 3M. The degree of activation pro-duced by this treatment was as high as that by limited proteolysis with trypsin. It was con-cluded that no activating enzyme system was involved in the activation of latent collagenase during NaI treatment, and that the latent enzyme was composed of an enzyme-inhibitor complex.
5) The physiological significance of latent enzyme in the regulation of collagenase activity in vivo is discussed.View full abstractDownload PDF (537K) -
Hiroshi NAKATANI, Hideshi FUJIWAKE, Keitaro HIROMI1977 Volume 81 Issue 5 Pages 1269-1272
Published: May 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBenzeneboronic acid (BBA), a possible transition-state analog for serine proteases, was found to inhibit Asp. melleus semi-alkaline protease [EC 3. 4. 21. 15]. The pH dependence of inhibitor constants was studied by the pH-stat method using N-acetyl-L-tyrosine ethyl ester as a sub-strate at 25°C. From the pH dependence of the association constant (reciprocal inhibitor constant), a pK value of 6.6, which may be attributable to the catalytic histidine residue of the enzyme, was estimated. The BBA-enzyme interaction was studied kinetically by the tempera-ture-jump method. Apparent association and dissociation rate constants were determined at pH 6.5.View full abstractDownload PDF (215K) -
Evidence for a Conformational Change in a Tetrahedral Adduct of α-ChymotrypsinKazumitsu HANAI1977 Volume 81 Issue 5 Pages 1273-1283
Published: May 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe interaction of α-chymotrypsin [EC 3. 4. 21. 1] with a transition state analog inhibitor, benzeneboronic acid (BBA), was investigated by the temperature jump method using pH in-dicator. The relaxation signal due to the BBA-enzyme interaction was found to be independent of the BBA concentration in the concentration range examined, in contrast to the BBA-subtilisin and BBA-chymotrypsinogen systems, in which the relaxation time was dependent on the BBA concentration.
Inhibited enzymes with a tetrahedral adduct at Ser 195 were found to exhibit relaxation signals due to a unimolecular isomerization similar to that of the BBA-enzyme system even in the absence of BBA, while those with a trigonal adduct did not except for the carbamylated enzyme. The relaxation signal in DIP-α-chymotrypsin disappeared upon the photooxidation of His 57. The DIP-zymogen exhibited no relaxation signal under similar conditions, under which relaxation could be detected for DIP-α-chymotrypsin, but a relaxation signal appeared on activation by trypsin.
These observations indicate that the tetrahedral adduct at Ser 195 of α-chymotrypsin has at least two different conformations. His 57 and perhaps the oxyanion hole are involved in the interaction in the tetrahedral adduct of the enzyme. Possible participation of the confor-mational change in the α-chymotrypsin-catalyzed reaction is discussed.View full abstractDownload PDF (852K) -
Genichiro OSHIMA, Kinzo NAGASAWA1977 Volume 81 Issue 5 Pages 1285-1291
Published: May 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe activity of carboxypeptidase A [EC 3. 4. 12. 2] was inhibited by 3-phenylpropionate deriva-tives (p-aminocinnamate, 3-p-aminophenylpropionate and 3 p-acetylaminophenylpropionate), and to investigate its use as a ligand for affinity chromatography 3-p-aminophenylpropionate was directly and indirectly coupled to Sepharose 4B. Carboxypeptidase A was adsorbed only on 3-p-aminophenylpropionate bound to the gel through p-phenylenediamine as aspacer.
Carboxypeptidase A from pancreas was purified by a combination of this affinity adsorbent and ion exchange chromatography. The purified carboxypeptidase A had a homogeneity similar to that of a commercial product, as judged by disc gel electrophoresis.
The carboxypeptidase activity of Pronase was slightly retarded on the gel column, but could not be separated from its caseinolytic activity. Angiotensin I-converting enzyme [peptidyl dipeptide hydrolase, EC 3. 4. 15. 1] obtained from hog kidney cortex was not bound to the gel.View full abstractDownload PDF (1178K) -
Yukio IKEHARA, Kimimitsu ODA, Keitaro KATO1977 Volume 81 Issue 5 Pages 1293-1297
Published: May 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMinor contaminants occasionally found in conventionally prepared rat serum albumin were easily and completely removed by concanavalin A-Sepharose chromatography. The unad-sorbed fraction from a concanavalin A-Sepharose column contained albumin which was homo-geneous on polyacrylamide gel electrophoresis. The recovery of albumin from rat serum was approximately 30%.
Approximately 2% of the added protein obtained as an albumin peak in DEAE-cellulose chromatography was adsorbed on and eluted with α-methyl-D-glucoside from the concanavalin A-Sepharose column, and resolved into three components by gel electrophoresis. There was one major glycoprotein, possibly α1-antitrypsin, and two minor proteins one of which was albumin.View full abstractDownload PDF (1791K) -
Atsushi IKAI1977 Volume 81 Issue 5 Pages 1299-1305
Published: May 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCattle and squid opsins were found to be associated with phospholipids after extensive dialysis of the salt-free digitonin extract of rhodopsin against 30% aqueous 2-chloroethanol (v/v) at pH 2.5. The approximate sizes of opsin-lipid complexes were estimated by sedimentation studies to be around 110, 000 and 150, 000 daltons, respectively, for the cattle and the squid opsin. Phospholipids did not dissociate from opsin even in 80% 2-chloroethanol. The complexes were purified by passage through a Sephadex G-200 gel column equilibrated with 30% 2-chloroethanol. The optical properties of the complex suggested the presence of β-conformation and a small amount of α-helix in solubilized cattle opsin.View full abstractDownload PDF (514K) -
Hiroaki KAWAGUCHI, Haruhiko NODA1977 Volume 81 Issue 5 Pages 1307-1317
Published: May 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThermal denaturation of cytochromes c of horse, cow, and Candida krusei in aqueous guanidine hydrochloride in the neutral pH region was studied by means of absorption and optical rotation measurements. The values of standard free energy change upon denaturation were estimated over the temperature range from 3 to 51°C. Large differences in the heat capacity of the native and denatured states amounting to several kcal/mol. deg were obtained for all three kinds of cytochromes c. These lead to a change in the sign of both the enthalpy and entropy change of denaturation, with maximum stability of the native state at l2°C for horse and bovine cytochromes c and at 9°C for Candida krusei.View full abstractDownload PDF (777K) -
Masao KAJIYOSHI, F. Koichi ANAN1977 Volume 81 Issue 5 Pages 1319-1325
Published: May 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCD and MCD spectra of bovine liver catalase and its derivatives were studied over the wave-length range from 250 to 500nm. Native catalase showed a large negative CD peak in the Soret region which was comparable with that of other hemoproteins, except for methemoglobin and metmyoglobin. The Soret CD peak of the peroxide derivatives of catalase was similar to that of native catalase, while the Soret MCD peak of native catalase was smaller in magnitude than that of other hemoproteins. The MCD peak of the cyanide complex of catalase showed a magnitude about half that of methemoglobin and metmyoglobin.
The difference in the magnitude of the MCD peak among hemoproteins is thought to be caused by an endogeneous ligand other than iron-histidine interaction at the fifth coordination position. The magnitude of the MCD peak of low-spin ferric derivatives in the Soret region was considerably larger than that of high-spin ferric derivatives. Conversion of native catalase to the peroxide compound I caused only a small change in the MCD spectrum in the Soret region, while reduction of compound I to compound II was accompanied by a major change in the Soret MCD spectrum.
In the near ultraviolet region, CD and MCD spectra of native catalase and its derivatives did not exhibit any evidence of a heme-related peak, the magnitude of which was not altered by ligand substitution. It is suggested that a heme iron-amino acid residue (s) interaction other than heme-histidine interaction is present.View full abstractDownload PDF (506K) -
Masao KAJIYOSHI, F. Koichi ANAN1977 Volume 81 Issue 5 Pages 1327-1333
Published: May 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCD and MCD spectra of human methemoglobin and its derivatives were studied with special reference to the relation of CD and MCD spectra to the magnetic moments. The intensities of MCD peaks of methemoglobin derivatives at 270nm were inversely proportional to their magnetic susceptibility, and there was a linear relationship between magnetic ellipticity and magnetic susceptibility. The MCD peak intensity of methemoglobin peroxide compound was close to that of methemoglobin cyanide complex, suggesting that the methemoglobin peroxide compound is in a low-spin state.View full abstractDownload PDF (462K) -
Ken-ichi ARAI, Takao ARAI, Masao KAWAKITA, Yoshito KAZIRO1977 Volume 81 Issue 5 Pages 1335-1346
Published: May 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe conformation and conformational stability of the polypeptide chain elongation factors Tu (EF-Tu) and Ts (EF-Ts) have been investigated by means of hydrogen-tritium exchange, optical rotatory dispersion and fluorescence spectroscopy, and the following results were obtained.
1. Free EF-Tu not liganded with guanine nucleotide rapidly exchanges its bound tritium atoms with hydrogen atoms in the medium, only 12 tritium atoms being retained in the protein after back-exchange for 10 hat 10°C. The rate of exchange was found to be markedly reduced by guanine nucleotides. Thus, in the presence of 20μM GDP or 50μM GTP, as many as 40 tritium atoms were retained under the same conditions. Even at 22°C, where the exchange in free EF-Tu becomes exceedingly rapid, about 25 tritium atoms remained associated with the protein after incubation for about 6 h. The kinetic distribution of very slowly exchanging hydrogen atoms, estimated by interrupted-flow gel filtration, was almost identical with both EF-Tu•GDP and EF-Tu•GTP.
2. From the optical rotatory dispersion spectra, the α-helical contents of EF-Tu•GDP, EF-Ts, and EF-Tu•EF-Ts were estimated to be 24, 52, and 33%, respectively. The spectra of EF-Tu•GDP and EF-Tu•GTP were almost identical over the range of wavelength from 220 to 350nm. The reduced mean residue rotation at 233nm of free EF-Tu decreased in the presence of guanine nucleotide ligands.
3. The intrinsic fluorescence of tryptophan is markedly quenched in EF-Tu•GDP and EF-Tu EF-Ts in their native conformation. The fluorescence emission at 340nm characteristic of tryptophan increased about 30 percent as a result of conversion of EF-Tu GDP to EF-Tu•GTP or free EF-Tu without any accompanying change in fluorescence from tyrosine. Upon denaturation of the protein, fluorescence of tryptophan further increased with a shift of the emission maximum to 335nm.
These results indicate that the binding of GDP or GTP causes a gross conformational change in EF-Tu, stabilizing its secondary and tertiary structure, increasing its α-helical con-tent, and reducing the motility of the protein core. The shielding of tryptophan fluorescence is more marked in EF-Tu•GDP than in EF-Tu•GTP, indicating that the conformations of these two forms of EF-Tu are different.View full abstractDownload PDF (890K) -
IX. Role of Methionine in Polypeptide Chain InitiationTerufumi SAKAMOTO, Tokuhiko HIGASHI1977 Volume 81 Issue 5 Pages 1347-1355
Published: May 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSInitiation with methionine of the synthesis of rat liver catalase [EC 1. 11. 1. 6] has been investi-gated. Analysis of the N-terminal residue of nascent catalase peptides labeled in vivo with injected radioactive amino acids, including [3H] methionine, indicated a remarkably high con-tent of methionine. By fractionating [3H] methionine-labeled nascent catalase according to chain length, it was found that peptides of shorter chain length contained more N-terminal methionine relative to total methionine incorporated. In addition, only a small amount of [3H] methionine was detected as the N-terminal amino acid when newly completed catalase was examined by Edman degradation. These results indicate that the synthesis of liver catalase is initiated with methionine, and suggest the presence of a mechanism for its subsequent removal from the N-terminal position.
Catalase was also synthesized in a cell-free system directed by the catalase mRNA, using [3H] Met-tRNAf or [3H] Met-tRNAm. The results obtained in such in vitro experiments were in good agreement with those from in vivo studies, and further showed that the N-terminal methionine was provided by a specific initiator tRNA, i.e. tRNAfMet.View full abstractDownload PDF (629K) -
Koji YODA, Kazuo NAGAI, Gakuzo TAMURA1977 Volume 81 Issue 5 Pages 1357-1365
Published: May 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSLarge amounts of extra proteins, X (in the envelope fraction) and X' (in the cytoplasmic fraction), were detected by SDS-polyacrylamide gel electrophoresis when DNA of Escherichia coli was damaged. These two proteins had the same apparent molecular weight (approxi-mately 40, 000) and were produced under identical conditions, including requirement for the recA+ and lexA+ genotype. Sucrose density gradient centrifugation revealed that protein X' consisted of relatively large and heterogeneous aggregates in the cytoplasmic fraction; the distribution of protein X in the envelope was not determined. As proteins X and X' were shown to be equivalent, it is suggested that they are identical. Co-precipitation of the aggregates of protein X' with the envelope led to the appearance of protein X in the envelope fraction.View full abstractDownload PDF (1214K) -
Kinuko KIMURA, Tetsuhisa GOTO1977 Volume 81 Issue 5 Pages 1367-1373
Published: May 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDyhydrodipicolinate reductases were purified 100-fold from crude extracts of B. cereus and B. megaterium and their properties were compared with those of the reductase from B. subtilis. The molecular weights of the reductases of B. cereus and B. megaterium were found to be 155, 000 and 150, 000, respectively. These reductases were shown to be free of flavin, unlike the B. subtilis enzyme, which contains flavin. Both NADPH and NADH acted as coenzymes forthese two reductases, NADPH being three or four times more effective than NADH. The Km values for NADPH and dihydrodipicolinate were 8μM and 62μM, respectively, with B. cereus reductase, and 13μM and 59μM with B. megaterium reductase. The pH optima of the enzymes from B. cereus and B. megaterium were pH 7.4 and 7.2, respectively. The reductases were inhibited by dipicolinate noncompetitively with respect to dihydrodipicolinate and the Ki values were 85μM and 140μM, respectively. Lysine and diaminopimelate were not inhibitory. The properties of the reductases from B. cereus and B. megaterium were similar, but they differed considerably from those of the B. subtilis enzyme. However, all three Bacillus reductases were markedly inhibited by dipicolinate, unlike the enzyme from E. coli.View full abstractDownload PDF (459K) -
Yoshihisa NAKANO, Hiroko TOKUNAGA, Shozaburo KITAOKA1977 Volume 81 Issue 5 Pages 1375-1381
Published: May 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBacillus cereus strain K-22 produced two distinct ω-amino acid transaminases, one catalyzing the transamination between β-alanine and pyruvic acid and the other that between γ-amino-butyric acid and α-ketoglutaric acid. The two enzymes were partially purified and separated from each other by various chromatographies. β-Alanine: pyruvic acid transaminase and γ-aminobutyric acid: α-ketoglutaric acid transaminase were induced by the addition of β-alanine and γ-aminobutyric acid, respectively, to the growth medium.
β-Alanine transaminase showed an optimum pH of 10.0 and optimum temperature of 35°C, and its Km values for β-alanine and pyruvic acid were both 1.1mM. γ-Aminobutyric acid, ε-aminocaproic acid, 2-aminoethylphosphonic acid, and propylamine showed about 30-40% of the activity of β-alanine as amino donors, and oxalacetic acid was as good an amino acceptor as pyruvic acid. The optimum pH and temperature of γ-aminobutyric acid trans-aminase were 9.0 and 50°C, respectively, and its Km value for α-aminobutyric acid was 2.8mM, while that for α-ketoglutaric acid was 2.3mM. γ-Aminobutyric acid and δ-aminovaleric acid were good amino donors but other ω-amino acids were virtually inactive with γ-aminobutyric acid transaminase; α-ketoglutaric acid, and to a lesser extent glyoxylic acid, were active amino acceptors. Sulfhydryl reagents specifically activated γ-aminobutyric acid transaminase.View full abstractDownload PDF (570K) -
II. Kinetic Behavior of Various Types of Chelating Agents towards Bovine Carbonic AnhydraseYoshinori KIDANI, Junzo HIROSE1977 Volume 81 Issue 5 Pages 1383-1391
Published: May 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn order to investigate the kinetics and mechanism of the removal of zinc ions from bovine carbonic anhydrase [EC 4. 2. 1. 1] (BCA), several chelating agents with various stability con-stants were used to remove zinc from BCA. The second-order rate constants (kapp) of zinc removal from BCA were found to be in the following order; 2, 6-pyridinedicarboxylic acid>>2-pyridinecarboxylic acid>2, 4-pyridinedicarboxylic acid>2, 3-pyridinedicarboxylic acid_??_1, 10-phenanthroline_??_5-methyl-1, 10-phenanthroline>>2, 2'-bipyridine. With similar chelating agents the greater the stability constant, the faster was the rate of removal of zinc ions from BCA. With EDTA, trans-1, 2-cyclohexanediaminetetraacetic acid, and nitrilotriacetic acid, the rate of zinc ion removal from the native enzyme was governed by the rate of spontaneous dissociation of the zinc enzyme.
The rate constants for the removal of zinc ions from BCA were governed by the affinity of the chelating agents for the metal ion and the conformation of the chelating agents. Based on these findings, reaction pathways for various chelating agents are proposed.View full abstractDownload PDF (603K) -
II. Affinity Labeling of Phosphoglycerate Kinase with a Reactive AMP AnalogKoichi SUZUKI, Chikahiko EGUCHI, Kazutomo IMAHORI1977 Volume 81 Issue 5 Pages 1393-1399
Published: May 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAffinity labeling of yeast and B. stearothernnophilus phosphoglycerate kinases with a reactive AMP analog, N6-(p-bromoacetaminobenzyl)-AMP was examined. Complete loss of enzyme activity was observed when I mol of the reagent had reacted per mol of either enzyme. Re-sults on the effect of pH and substrate addition on the inactivation, titration of SH groups before and after modification, and kinetic studies with AMP analogs suggest that the modification occurs at one amino group at or near the subtrate binding site. General affinity labeling of kinases is discussed based on the results obtained.View full abstractDownload PDF (491K) -
Chikahiko EGUCHI, Koichi SUZUKI, Kazutomo IMAHORI1977 Volume 81 Issue 5 Pages 1401-1411
Published: May 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn order to obtain a better understanding of the allosteric site of rabbit muscle phosphorylase b, nine AMP analogs having a bulky hydrophobic benzene ring were synthesized and tested for activity as activators or inhibitors. N6-Benzyl-AMP derivatives activated phosphorylase b to the same extent as AMP but bound to the enzyme more tightly than AMP. N6-p-nitro-benzyl-AMP had the highest affinity (Ka=7.7×10-7M) for the AMP site. In an atempt to irreversibly modify the allosteric site of phosphorylase b, N6-p-bromoacetaminobenzyl-AMP was synthesized. Phosphorylase b was maximally activated upon incorporation of 1.0 mol of N6-p-bromoacetaminobenzyl-AMP per enzyme subunit, and its activity was approximately 90% of that of native phosphorylase b measured in the presence of AMP. The modified enzyme showed characteristics (e.g., kinetic parameters, stability, solubility, inhibition by glucose-6-phosphate, and state of aggregation) quite similar to those observed for the native enzyme in the presence of AMP. These results indicate that the AMP site of phosphorylase was specifically labeled by N6-p-bromoacetaminobenzyl-AMP. The nature of the allosteric site of phosphorylase b is discussed based on the results obtained.View full abstractDownload PDF (905K) -
Yukio NISIMOTO, Fumio TAKEUCHI, Yukio SHIBATA1977 Volume 81 Issue 5 Pages 1413-1425
Published: May 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSL-Kynurenine 3-hydroxylase [EC 1. 14. 1. 2] has been solubilized with Triton X-100 and purified to homogeneity from rat liver mitochondria. The purified enzyme exists in aqueous media as an oligomeric aggregate and in 0.2% Triton X-100 as a dimer with a molecular weight of about 345, 000. Its monomeric molecular weight is approximately 160, 000, and 4 mol of FAD are associated with 1 mol of the monomeric unit of the enzyme. The addition of FAD to the apoenzyme resulted in remarkable increase in both L-kynurenine 3-hydroxylase activity and FAD fluorescence. The enzyme molecule migrated as a single band in polyacrylamide gel electrophoresis and in chromatography on a Sephadex G-200 column, and was resolved into two bands only after complete denaturation by high concentrations of SDS or urea. SDS-polyacrylamide gel electrophoresis in the presence of SDS at a concentration of more than 1 and Sephadex G-100 gel chromatography in the presence of 5M urea caused cleavage of the monooxygenase molecule into a protein having a molecular weight of about 137, 500 and a smaller fragment. Upon dissociation into these fragments, the enzyme activity was completely lost. The visible absorption spectrum of the oxidized holo-L-kynurenine 3-hydroxylase showed an absorption maximum at 408nm with a prominent shoulder at around 445 run. In contrast to the oxidized form, the dithionite-reduced form had peaks at about 420 and 554nm. In spite of its electrophoretic homogeneity, appreciable amounts of cytochrome b5-like heme-protein were detected in our highly purified preparation of L-kynurenine 3-hydroxylase. The purified L-kynurenine 3-hydroxylase catalyzed the reductions by NADH or NADPH of DCPI and ferricyanide.View full abstractDownload PDF (1543K) -
Kosuke ICHIHARA, Emi KUSUNOSE, Masamichi KUSUNOSE, Tatsuo MORI1977 Volume 81 Issue 5 Pages 1427-1433
Published: May 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMycobacterium lepraemurium, strain Hawaii, grown on 1% Ogawa egg yolk medium contain-ing hemin, was extremely rich in superoxide dismutase [EC 1. 15. 1. 1]. This enzyme accounted for at least 7% of total proteins in the crude extracts, as determined by immunological procedures. The enzyme was purified about 18.5-fold from the crude extracts by streptomycin treatment, ammonium sulfate fractionation, and Sephadex G-100 gel filtration. The homo-geneity of the purified enzyme was established by polyacrylamide gel electrophoresis, analytical ultracentrifugation, and immunodiffusion. The molecular weight of the enzyme was estimated to be approximately 45, 000 by sedimentation equilibrium analysis, whereas that of the subunit was 22, 000 as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis. The enzyme was found to contain 1.29g atom of manganese per mol by atomic absorption spectroscopy. In addition, a small but significant amount of iron was found. The amino acid composition was similar to that of the superoxide dismutase from Mycobacterium smegmatis. Superoxide dismutase is the first enzyme which has been isolated and characterized from M. lepraemurium.View full abstractDownload PDF (2138K) -
Tsutomu UNEMOTO, Noriko KANBAYASHI, Maki HAYASHI, Makoto HAYASHI1977 Volume 81 Issue 5 Pages 1435-1445
Published: May 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSGlucose 6-phosphate dehydrogenase was partially purified from a marine Vibrio alginolyticus. The enzyme specifically required NADP+ as a coenzyme and showed no significant activity toward glucose. Its molecular weight was approximately 100, 000 and it consisted of two sub-units. The optimum activity was obtained at pH 9.0 and at an ionic strength of 0.1. Polyvalent anions such as sulfate, phosphate, and bicarbonate also stimulated the activity as a result of the increase in ionic strength. High concentrations of salts, and particularly chaotropic anions, inhibited the activity and the order of inhibitory effect followed the chaotropic sequence: SCN->NO3->Br-Cl-. The inhibition by chaotropic anions was not due to dissociation of the active form of the enzyme to its inactive subunits.
The kinetic mechanism of this enzyme followed a sequential ordered mechanism with NADP+ combining with the free enzyme first and NADPH being the last product to be re-leased. From kinetic analyses, it was found that the activation of the enzyme on increasing the ionic strength is due to a decrease in the Km for G-6-P, whereas the inhibitory effect of high concentrations of salts or chaotropic anions is due to an increase in the dissociation constant of NADP+-enzyme complex (Kia). The effect of NO3- in increasing the Kia, value was found to be due to a decrease in the rate constant for the formation of NADP+-enzyme complex, without significantly affecting the rate constant for its dissociation.View full abstractDownload PDF (829K) -
Hiroshi YOSHIDA, Hiroshi SAGAMI, Shigeo OIKAWA1977 Volume 81 Issue 5 Pages 1447-1454
Published: May 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMidgut glands of abalone Haliotis discus contained two acid phosphatases [orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3. 1. 3. 2] separable by phosphocellulose column chromatography. They were designated as acid phosphatases I and II in order of elution and were purified 99- and 290- fold, respectively. Purified acid phosphatase II was nearly homogeneous as judged by polyacrylamide gel electrophoresis. The substrate specificity of acid phosphatase I was narrow, whereas that of acid phosphatase II was broad. Good substrates for acid phosphatase I included p-nitrophenyl phosphate, phosphoenolpyruvate, inorganic pyrophosphate, and nucleoside di- and triphosphates. The acid phosphatases did not require any metal ion for maximum activity and were inhibited by Zn2+, Cu2+, and Hg2+. Fluoride and arsenate were potent inhibitors of both enzymes. The pH optima of acid phos-phatases I and II were 5.9 and 5.5, respectively. The molecular weights of acid phosphatases I and II were estimated to be 28, 000 and 100, 000, respectively, by gel filtration on Sephadex G-100. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggested that acid phosphatase II consists of two identical subunits.View full abstractDownload PDF (564K) -
Yasuzo NISHINA, Kihachiro HORIIKE, Kiyoshi SHIGA, Yoshihiro MIYAKE, To ...1977 Volume 81 Issue 5 Pages 1455-1463
Published: May 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe quenching of tryptophanyl fluorescence of native and denatured D-amino acid oxidase from hog kidney was measured. About 60% of the tryptophanyl fluorescence of the native apoenzyme was quenched by iodide at pH 8.3, and 25°C. All of the tryptophanyl fluorescence. of the apoenzyme in 6M guanidine hydrochloride was quenched. The tryptophanyl fluores-cence quenching of the holoenzyme by 1-methyl nicotinamide chloride was low in comparison with that of the apoenzyme. These results of the quenching experiments are discussed based on the intermolecular collision quenching mechanism.
By measuring the fluorescence intensities of the tryptophanyl residues and FAD of the holoenzyme solution, and the fluorescence polarization of the holoenzyme solution contain-ing halide anions such as iodide, bromide, chloride, or fluoride, we found that FAD dissociates from the holoenzyme in the presence of iodide, bromide, or chloride, and the ability to dissociate FAD from the holoenzyme decreases in order iodide, bromide, and chloride. However, fluoride seems to enhance the association reaction of FAD with the apoenzyme. These results were consistent with the visible absorption spectra and derivative spectra of free FAD and the holoenzyme in the presence and absence of halide anions.View full abstractDownload PDF (666K) -
Circular Dichroism Spectra of D-Amino Acid Oxidase ComplexesKiyoshi SHIGA, Kihachiro HORIIKE, Yasuzo NISHINA, Akio ISOMOTO, Toshio ...1977 Volume 81 Issue 5 Pages 1465-1472
Published: May 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSVisible circular dichroism spectra (CD spectra) of D-amino acid oxidase complexes with ring-substituted benzoate derivatives (quasi-substrates) were measured and compared with CD spectra of the enzyme complex with benzoate (“benzoate complex”) to study the effects of sub-stituted groups on the interactions between D-amino acid oxidase and quasi-substrates.
CD spectra of the enzyme complexes with o-, m-, and p-fluorobenzoate and o-, m-, and p-chlorobenzoate (“o-, m-, and p-fluorobenzoate complexes” and “o-, m-, and p-chlorobenzoate complexes”) showed patterns similar to that of the benzoate complex. However, the CD spectra of the chlorobenzoate complexes, particularly o- and m-chlorobenzoate complexes, differed more from that of the benzoate complex than did those of the fluorobenzoate complexes. This might be due to a stronger steric effect at the active site of the enzyme caused by the larger chlorine atom. The CD spectra of the m- and p-methylbenzoate complexes and the o-, m-, and p-hydroxybenzoate complexes were different from that of the benzoate complex. They showed negative ellipticity around 500nm and had crossover points which did not exist for the benzoate complex, at 482nm for p- and 475nm for m-methylbenzoate complexes and 470nm for o-, 455nm for m- and 492nm for p-hydroxybenzoate complexes. Furthermore, these CD spectra were similar to those of the “dehydrogenase type” of flavoenzymes, although D-amino acid oxidase belongs to the “oxidase type”.
The patterns of CD spectra of m- and p-nitrobenzoate complexes differed from those of all the other complexes mentioned above.
We concluded that the gross modifications of CD spectra by substituted groups were determined by the group species with further minor modifications due to its o-, m- or, p-posi-sion. These findings suggest that each substituted group interacts with the enzyme at the active site in a specific mode. Furthermore, the drastic differences were largely in the spectral regions corresponding to the lowest transition of absorption spectra; this was consistent with the hypothesis that this band is affected by the “through-space” interaction.View full abstractDownload PDF (543K) -
Discrimination of Allosteric SitesMinoru MORIKAWA, Katsura IZUI, Hirohiko KATSUKI1977 Volume 81 Issue 5 Pages 1473-1485
Published: May 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRevertants having mutationally altered phosphoenolpyruvate (PEP) carboxylases were isolated from PEP carboxylase-lacking E. coli K12 strains. For comparison of the sensitivities of the altered enzymes to allosteric effectors with those of the wild enzyme, the effects of the effectors on the enzyme activities were measured at 8mm PEP by “radioisotopic assay.” Some of the altered enzymes were apparently desensitized to L-aspartate, acetyl-CoA, fructose 1, 6-bisphos-phate and/or fatty acid, in various combinations. In this connection, it appeared possible that an alteration of the enzyme affinity for PEP might have given rise to the apparent desensitization. We therefore adopted the “equivalent method, ” in which the enzyme activities were measured at concentrations of PEP giving a definite saturation (25% saturation). Kinetic studies were carried out on the partially purified ER1-70 enzyme, which was adopted as an example of the altered enzymes, in comparison with the wild enzyme by the “equivalent method” and by the “standard method, ” in which the enzyme activities were measured optically at 1mm PEP. Measurements of the ER1-70 enzyme activities by the “equivalent method” showed that only the sensitivity to laurate or dioxane was lost, and those to the other effectors were not lost, contrary to the results obtained by the “standard method.” This accorded with the results of “radioisotopic assay, ” in which the concentration of PEP used was in a range of appropriate saturation. The concentrations of PEP for half-maximal saturation of the altered enzymes were about the same as or less than that of the wild enzyme (5-10mm), as far as tested.
The above results strongly suggest that PEP carboxylase of E. coli has independent allosteric sites for each of the four effectors. The effectiveness of the “equivalent method” for comparison of the alterations of sensitivities to allosteric effectors between the altered and wild enzymes is also discussed.View full abstractDownload PDF (1050K) -
Jun-ichi HAYASHI, Tadashi ISHIMODA, Tamio HIRABAYASHI1977 Volume 81 Issue 5 Pages 1487-1495
Published: May 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. Tropomyosins from chicken cardiac, skeletal, and gizzard muscles were each resolved into two subunits by polyacrylamide gel electrophoresis in a system containing sodium dodecylsulfate (SDS), urea and sodium borate, and were designated Cl C2, S1 S2, and G1 G2, respectively, in descending order of mobility on electrophoresis. S1, S2, G1, and G2 were prepared as pure samples by electrophoresis.
2. The apparent molecular weights of C (C1+C2), S1, S2, G1, and G2 were calculated to be 36, 000, 36, 000, 37, 500, 36, 000, and 40, 000, respectively, based on SDS gel electrophoretic mobility according to the method of Weber and Osborn. C and S1 showed nearly the same mobility in all electrophoretic systems tried. S1 and Gl, which comigrated in an SDS-sodium borate system, showed different mobilities upon addition of 5M urea to the system.
3. Immunological evidence presented indicates that each subunit has a specific antigenic site (s) in addition to an identical one (s) in common with the others.
4. As each tropomyosin subunit formed two precipitin lines with the homologous antiserum, as many as ten kinds of subunits may exist in chicken muscles.View full abstractDownload PDF (1979K) -
Norio KATOH, Shuichiro KUBO1977 Volume 81 Issue 5 Pages 1497-1503
Published: May 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMyosin from rabbit stomach was highly purified by ammonium sulfate fractionation in the presence of ATP and MgCl2, ultracentrifugation and Sepharose 4B chromatography.
The myosin composed of one heavy and two light chains as determined by SDS-gel electro-phoresis. The molecular weights of the light chains were the same as those of gizzard myosin, about 20, 000 and 17, 000, respectively.
The pH-activity curve and the KCl concentration dependency of Ca-ATPase of the stomach myosin were similar to those of other smooth muscle myosins.
The stomach myosin was more resistant to pepsin digestion than skeletal myosin. Other proteolytic enzymes, trypsin, chymotrypsin, papain, and nagarse, digested the myosin in the same way as skeletal myosin.View full abstractDownload PDF (2006K) -
Naotaka HAMASAKI, Akio TOMODA, Hiroaki HARASAKI, Shigeki MINAKAMI1977 Volume 81 Issue 5 Pages 1505-1509
Published: May 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAccumulation of exogenous phosphoenolpyruvate against the concentration gradient was observed when human red cells were incubated in an acidified isotonic sucrose medium. Fluoride increased the apparent accumulation by inhibition of the intracellular metabolic interconversion of the phosphate compound. The accumulation appeared to be specific for phospho-enolpyruvate and the accumulation rate for 3-phosphoglycerate, which has a molecular size and pKa similar to those of phosphoenolpyruvate, was less than one-tenth of the rate for phosphoenolpyruvate. Red cells incubated in the acidified sucrose medium tended to adhere to each other when examined with a scanning electron microscope.View full abstractDownload PDF (815K) -
Atsuko NAKAGAWA, Makoto NAKAO1977 Volume 81 Issue 5 Pages 1511-1515
Published: May 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPhlorizin at 2×10-4M inhibited Na+ and Rb+-activated ATPase activities in human red cell membranes by 43%. It inhibited the 86Rb uptake activity of erythrocytes by only 15%. 86Rb uptake into resealed ghosts was inhibited strongly when phlorizin and ATP were preloaded in the ghosts before resealing. Na, K-ATPase activity in the resealed ghosts was also inhibited in the presence of phlorizin inside but not outside the ghosts. These findings suggested that the phlorizin site is located inside the cell.View full abstractDownload PDF (316K) -
XVIII. Properties of an Isoleucine Carrier in the Cytoplasmic Membrane Vesicles of Escherichia coliIchiro YAMATO, Yasuhiro ANRAKU1977 Volume 81 Issue 5 Pages 1517-1523
Published: May 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe properties of the carrier for isoleucine in Escherichia coli were studied using cytoplasmic membrane vesicles (IM vesicles) prepared by the method of Yamato, Anraku, and Hirosawa (J. Biochem. 77, 705 (1975)). The IM vesicles exhibited respiration-dependent isoleucine transport activity which was more than 30-fold higher than that of “Kaback vesicles” prepared by our hand from the same strains of E. coli K12. The isoleucine carrier activity of IM vesicles was inhibited by norleucine but not by threonine. The carrier was driven by proton motive force.
Mutants were isolated which had lost the carrier activity for isoleucine, as judged by assay with IM vesicles. Using these mutants, the effects of binding proteins specific for branched chain amino acids on the translocation of substrate in IM vesicles were studied. Leucine-isoleucine-valine-threonine-binding protein (LIVT-binding protein) stimulated the initial rate of isoleucine uptake by IM vesicles only when the vesicles possessed carrier activity and it did not affect the Kt value for entry of substrate. This evidence suggests the partial reconstitution of the osmotic shock-sensitive transport reaction in which the binding protein seems to affect the carrier activity with turnover ability.View full abstractDownload PDF (555K) -
Shigeyuki ICHIHARA, Shoji MIZUSHIMA1977 Volume 81 Issue 5 Pages 1525-1530
Published: May 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSOuter membrane proteins of various strains of Escherichia coli were compared using three different systems of sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The outer membranes of E. coli K-12, E. coli B, and E. coli J-5 had distinctive protein compositions.
As regards proteins which interact with peptidoglycan, E. coli K-12 contained O-8 and O-9, while E. coli B possessed one protein which migrated to the position of O-9. Although E. coli J-5 possessed two such proteins, O-8' and O-9', their positions on polyacrylamide gel were different from those of O-8 and O-9. Protein O-7, which migrates slightly more slowly than O-8, was found specifically in E. coli K-12.
Proteins O-10 and O-11 were found in all strains tested, although the relative amounts were different depending on the strain.
Strains of E. coli K-12 and E. coli J-5 gave three major bands, O-2a, O-2b, and O-3, in the region of high molecular weight. These proteins were repressed by iron in the cultivation media. Strains of E. coli B, on the other hand, gave only O-2b and O-3. E. coli J-5 gave two other major bands in this region, but the amounts were not controlled by iron in the cultivation media.View full abstractDownload PDF (2184K) -
Akio ITO, Toyoko KUWAHARA, Yoichiro MITSUNARI, Tsuneo OMURA1977 Volume 81 Issue 5 Pages 1531-1541
Published: May 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. The subcellular distribution of sulfite oxidase was investigated in homogenates of rat liver. The activity, as measured by sulfite-dependent cytochrome c reduction, exhibited a structure-linked latency. The distribution pattern of the activity unmasked by hypotonic treatment was quite similar to that of a mitochondrial marker enzyme, succinate-cytochrome c reductase. The activity after the hypotonic treatment was further activated by low concentrations of detergents such as sodium deoxycholate, and this detergent-unmasked activity was mainly localized in microsomes. 80% of the fully unmasked activity was localized in mitochondria and 10% each in microsomes and soluble fraction.
2. Sulfite oxidases present in mitochondria, microsomes, and soluble fraction showed the same behavior on DEAE-Sephadex column chromatography and gel filtration with Sephadex G-200, and gave identical Km values for sulfite and cytochrome c.
By using a rabbit antiserum against purified mitochondrial sulfite oxidase, immunochemical comparison was also carried out. Ouchterlony double diffusion tests in agar gel, quantitative immunoprecipitation tests, and inhibition studies of enzyme activity indicated that the antigenic properties of these three sulfite oxidase preparations were identical.
3. The sulfite-cytochrome c reductase activity in microsomes was in a latent state, and its activity was greatly stimulated by low concentrations of detergents or by sonication; the activation was accompanied by solubilization of the enzyme.
Antibody and trypsin failed to attack the enzyme in intact microsomes, whereas the solu-bilized enzyme was susceptible to inhibition by the antibody and to digestion by trypsin.
These findings suggest an internal localization of the sulfite oxidase in microsomal vesicles.View full abstractDownload PDF (1509K) -
III. Physicochemical Properties of Glycoprotein IISyoko KIDO, Masanobu JANADO, Hiroshi NUNOURA1977 Volume 81 Issue 5 Pages 1543-1548
Published: May 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA glycoprotein fraction (GP-II) has been isolated from the vitelline membrane of hen's egg and its physicochemical properties clarified. GP-II is composed of polypeptide (92%), neutral sugar (4%), hexosamine (3.3%), and sialic acid (0.6%). The constituent neutral sugars of this glycoprotein are fucose, mannose, and galactose, in a molar ratio of 2:6:5. An interesting feature of the amino acid composition of GP-II is the high proportion of proline. GP-II exists in an aggregated form and is hydrophobic in nature. Upon velocity sedimentation in 0.5 SDS solution, it showed a hypersharp boundary with an apparent sedimentation coefficient of 5.6 S. Reduction of GP-II, however, gave a single component of 3.6 S which seems to be a subunit of GP-II.View full abstractDownload PDF (940K) -
Naoko KONO, Iwao SHIMA, Goroku OHTA1977 Volume 81 Issue 5 Pages 1549-1555
Published: May 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAntibodies against non-histone chromosomal proteins from 89Sr-induced osteogenic sarcoma (mouse) were prepared by immunization of rabbits. The. immunoreactivity of this antigen was then compared with those of non-histone chromosomal proteins from Ehrlich ascites tumor, normal mouse liver, and calf thymus by the method of quantitative microcomplement fixation. The non-histone chromosomal proteins of 98Sr-induced osteogenic sarcoma, fractionated by hydroxylapatite chromatography, exhibited significant affinity for the antibodies. Similar proteins from Ehrlich ascites tumor, normal mouse liver, or calf thymus were virtually inactive, indicating the tissue-specificity of 89Sr-induced osteogenic sarcoma proteins.View full abstractDownload PDF (476K) -
Kanefusa KATO, Yoshitaka HAMAGUCHI, Sachiyo OKAWA, Eiji ISHIKAWA, Keik ...1977 Volume 81 Issue 5 Pages 1557-1566
Published: May 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA highly sensitive sandwich enzymoimmunoassay of macromolecular antigens using the Fab' of rabbit antibody coupled to β-D-galactosidase together with rabbit antibody IgG loaded on silicone pieces is described. The Fab' fragments of rabbit antibody IgG are conjugated with β-D-galactosidase from Escherichia coli using N, N'-o-phenylenedimaleimide. Small silicone pieces are loaded with rabbit antibody IgG by simple adsorption. A wide range of concentrations of rabbit antibody IgG fraction (2 to 2000μg/ml) is effective for loading. The adsorption is sufficiently stable for immunoassay and the amount of rabbit antibody IgG loaded can be controlled. The smallest amounts of ornithine δ-aminotransferase from rat liver, human IgG, and DNP-human IgG that could be determined were 0.03, 0.3, and 0.03 fmol, respectively. The sensitivity of the assay for these antigens is affected mainly by the non-specific binding of complexes to the solid phase and by the ability of antigen molecules, when adsorbed on the solid phase, to bind with the complexes specifically. The assay with rabbit antibody IgG loaded on silicone pieces is simpler and more sensitive than that with rabbit antibody IgG coupled to Sepharose 4B and is as sensitive as that with rabbit antibody IgG coupled to glass rods. Suitable silicone pieces can be more readily prepared in the laboratory than glass rods. The F(ab')2, not Fab, fragments may also be used to load silicone pieces in a similar manner for immunoassay.View full abstractDownload PDF (794K) -
Shin NAKAMURA, Takashi MORITA, Sadaaki IWANAGA, Makoto NIWA, Kenji TAK ...1977 Volume 81 Issue 5 Pages 1567-1569
Published: May 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn endotoxin-activated hemocyte lysate from horseshoe crab (Tachypleus and Limulus) was found to hydrolyze specifically BZ-Ile-Glu-Gly-Arg-p-nitroanilide, which was recently intro-duced as the substrate for assay of the blood coagulation factor, Factor Xa. Further, this amidase activity increased by increasing the concentration of bacterial endotoxin (Salmonella minnesota R595) added to the lysate. Thus, the measurement of the amidase activity in the hemocyte lysate can be very useful to detect and determine the endotoxin.View full abstractDownload PDF (206K) -
Kunio OHYAMA, Ichiro KANEKO, Toshio YAMAKAWA, Tadao WATANABE1977 Volume 81 Issue 5 Pages 1571-1574
Published: May 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo enzymatically active forms of valyl-tRNA synthetase [EC 6. 1. 1. 9] were found in the cells of Bacillus subtilis. The aminoacylation activities of the two forms were altered during the sporulation of B. subtilis. The tRNA's acylated by these enzymes were analyzed by methylated albumin-Kieselguhr column chromatography.View full abstractDownload PDF (227K) -
Noriko NAKAYA, Yoshioi SAWASAKI, Hirobumi TERAOKA, Hiroshi NAKAJIMA, K ...1977 Volume 81 Issue 5 Pages 1575-1577
Published: May 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDNA ligase was isolated from rat brain and characterized. In developing rat brain the DNA ligase activities of the cerebellum increased after birth, at least 5 fold more than those of non-cerebellar parts. The DNA ligase activity in the cerebellum reached a maximum about 6 days after birth and then decreased towards maturation. The DNA ligase from the cerebellum can be fractionated into three molecular forms. Aging of the extracts leads to the conversion of the high molecular weight form into smaller weight forms.View full abstractDownload PDF (250K) -
Merrill OVERTURF, Robert DRUILHET, Walter M. KIRKENDALL1977 Volume 81 Issue 5 Pages 1579-1581
Published: May 25, 1977
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effects of several low molecular streptomyces protease inhibitors on human kidney renin activity have been observed. This study shows that, in addition to pepstatin A, leupeptin, antipain, and SP-I also inhibit human renin activity in vitro.View full abstractDownload PDF (182K)
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