All issues
Volume 80, Issue 1
Displaying 1-24 of 24 articles from this issue
- |<
- <
- 1
- >
- >|
-
Yoshinori ENDO, Akira KOBATA1976 Volume 80 Issue 1 Pages 1-8
Published: July 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe culture medium of Diplococcus pneumoniae contains enzymic activity that cleaves Galβ1→3Ga1NAc from desialized human erythrocyte membrane glycoprotein. The enzyme was purified 180-fold by ammonium sulfate fractionation, gel filtration through a Sephadex G-200 column, and DEAE A-25 Sephadex chromatography. The purified enzyme liberates Galβ1→3Ga1NAc from glycopeptides and glycoproteins with Ga1β1→3GalNAcαl→Ser and Thr moieties.
The optimum pH of this enzyme is 6.0. Using glycopeptides obtained by trypsin digestion of human erythrocyte membrane glycoprotein as a substrate, a Km of 0.20mM (on the basis of the amount of Galβl→3Ga1NAc residues) was obtained.
So far, the enzyme appears to have a strict specificity for Galβ1→3Ga1NAcα→Ser and Thr structures, because no oligosaccharides larger than trisaccharides were liberated from porcine submaxillary mucin.View full abstractDownload PDF (625K) -
Takao KIYOHARA, Tadao TERAO, Kohei SHIOIRI-NAKANO, Toshiaki OSAWA1976 Volume 80 Issue 1 Pages 9-17
Published: July 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThree β-N-acetylhexosaminidases [EC 3.2.1.52] and one β-galactosidase [EC 3.2.1, 23] were purified from the culture filtrate of Streptococcus 6646 group K by a combination of column chromatographies on p-aminophenyl β-D-thiogalactopyranoside-substituted Sepharose and N-(p-aminophenyl)oxamic acid-substituted Sepharose.
These β-N-acetylhexosaminidases showed optimal activities between pH 5.0 and 5.5 and could hydrolyze synthetic and glycopeptidic substrates. Glycolipids such as GM2, asialo-GM2, and globoside I were not susceptible to these β-hexosaminidases.
β-Galactosidase, which was purified more than 11, 000-fold, had a substrate spe-cificity rather similar to that of β-galactosidase from E. coli. This enzyme was inhibited by EDTA and activated by Mn2+, Ca2+, and Mg2+.
Problems pertinent to the application of affinity chromatography to the purification of glycosidases are also discussed.View full abstractDownload PDF (617K) -
Hiroshi NAGANO, Hiroyuki KIUCHI, Yasuko ABE, Ryoiti SHUKUYA1976 Volume 80 Issue 1 Pages 19-26
Published: July 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn the hepatic cytosol fraction of bullfrog, Rana catesbeiana, an alkaline RNase [EC 3.1.4.22] exists in two forms. One is the free form of RNase, which elutes from a carboxymethyl-cellulose column at a concentration of 0.2 M NaCl. The other is a masked or latent form (RNase-RNase inhibitor complex) which is not adsorbed on the carboxymethyl-cellulose column and which can be converted to the free form of RNase by the addition of p-chloromercuribenzoate. Electrophoretically pure RNase was obtained by the following procedure. The unadsorbed fraction of hepatic cytosol on a column of carboxymethyl-cellulose was treated with p-chloromercuribenzoate and then applied to a second carboxymethyl-cellulose column.
The molecular weight of RNase was determined to be approximately 12, 000 by gel filtration and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. From the results of gel filtration, the molecular weight of the RNase-RNase inhibitor complex was 130, 000.
The RNase hydrolyzed poly C, poly U, and poly I, but not poly A or poly G. When poly C was used as a substrate, 2', 3'-cyclic CMP as an intermediate and 3'-CMP as a final product were identified. The results of amino acid analysis indicated the presence of an unusual component. The general properties of the RNase and the RNase-RNase inhibitor complex are also reported.View full abstractDownload PDF (1278K) -
Fuyuhiko TAMANOI, Tsuneko UCHIDA, Fujio EGAMI, Tairo OSHIMA1976 Volume 80 Issue 1 Pages 27-32
Published: July 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. 3'-Guanylyl-ethanol, 3'-guanylyl-propanol, and 3'-guanylyl-a-glycerol were synthe-sized by ribonuclease N1 [EC 3.1.4.8] using guanosine 2', 3'-cyclic phosphate as a phosphate donor and various alcohols as phosphate acceptors. The yields of these phosphodiesters were 15%, 13.5%, 38.2%, respectively, with respect to phosphate donor under the optimum conditions. No phosphodiester was synthesized when 2-propanol was used as a phosphate acceptor. Thus, primary alcoholic hydroxyl groups may be regarded as the preferred phosphate acceptor.
2. 3'-Guanylyl-glucose and 3'-guanylyl-ribose were synthesized using glucose and ribose as phosphate acceptors. Under the optimum conditions, the yields of guanylyl-glucose amounted to 52.0%, while that of guanylyl-ribose was much lower. The guanylyl-glucose can be regarded as 3'-guanylyl-6-glucopyranose, based on the results of periodate oxidation.
3. Neither hydroxyamino acids (serine and threonine) nor N-acetylserinamide could be phosphorylated under the conditions used for the above phosphorylations.
4. 3'-Guanylyl-glycerol obtained as above was hydrolyzed by snake venom phospho-diesterase to produce glycerol 3-phosphate. The latter consisted of L-glycerol 3-phos-phate (ca. 17%) and the D-isomer (ca. 83%). Ribonuclease N1 thus catalyzes an asymmetric synthesis.View full abstractDownload PDF (1273K) -
The “Single Addition” of 2'(3)-O-Dihydrocinnamoyl-nucleoside 5'-Diphosphate to a Primer Oligonumleotide Catalyzed by a Thermophilic Polynucleotide PhosphorylaseYo KIKUCHI, Keiko HIRAI, Kinumi SOMENO, Kenji SAKAGUCHI1976 Volume 80 Issue 1 Pages 33-37
Published: July 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSeveral oligonucleotides of defined sequence were synthesized using 2'(3')-O-dihydro-cinnamoyl-nucleoside 5'-diphosphates (DHC-NDP) as substrates for polynucleotide phosphorylase [EC 2.7.7.8] from Thermus thermophilus. The enzyme catalyzed the transfer of one nucleotidyl residue from each of the 2'(3')-O-dihydrocinnamoyl esters of CDP, UDP, and GDP to the 3'-terminus of the primer triadenosine diphosphate, (Ap)2A. The products were shown to be (Ap)3C, (Ap)3U, and (Ap)3G by enzymatic analysis.View full abstractDownload PDF (374K) -
Masachika IRIE, Kazuko OHGI1976 Volume 80 Issue 1 Pages 39-43
Published: July 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn order to investigate the base specificity of the minor RNase [EC 3.1.4.23] from Aspergillus saitoi, the kinetic constant of the enzyme was measured with 16 dinu-cleoside phosphates (XpY's) as substrates at pH 5.5 and 25°, The maximum rates of transesterification of GpY's were in the range of 10, 000 to 2, 800 and were markedly larger than those of other XpY's, including XpG's. The average Km values of UpY, CpY, ApY, and GpY increased in the order A, C, U, and G. This order coincides with that of the rates of release of 4 common nucleotides from RNA by RNase Ms (the rates decreased in the order 3'-GMP, 3'-AMP, 3'-CMP, and 3'-UMP), except for the case of GpY. Therefore the rates of release of nucleotides seem to be dependent on the affinity constant of the X base in XpY, except in the case of GpY. The high rate of release of guanylic acid from RNA was explained by the findings that higher rates of hydrolysis of GpY's compensate for their lower affinity to the enzyme. These results suggested that the base specificity was rather dependent upon the X nucleotide in XpY.
The K1 values of various nucleotides and nucleosides towards RNase Ms were measured. These compounds inhibited the RNase competitively. Although the inhib-itory effect depends on the bases, sugars and location of phosphate, when the location of phosphate on the sugar was the same, the K1 values of ribonucleotides decreased in the order U, G, C, and A and those of deoxyribonucleotides decreased in the order T, G, C, and A. The dependence of the inhibitory effect of ribonucleosides on the bases was similar to that of ribonucleotides, but that of deoxyribonucleosides was in the order dT, dA, dG, and dC.View full abstractDownload PDF (387K) -
Aritake MIZUNO1976 Volume 80 Issue 1 Pages 45-52
Published: July 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe incorporation of serine and ethanolamine into phospholipids in rabbit retinal subcellular fractions and in excised retinas was studied in vitro, and some enzymic properties of the incorporation of phospholipid bases by base exchange were examined in the microsomal fraction. The retina was found to have a higher rate of base exchange for the incorporation of phospholipid bases than other tissues. The retinal microsomal fraction possessed the highest specific activity of base exchange, while the rod outer segment had very little activity. These results suggest that the phos-pholipids in the rod outer segment may be transferred from the inner segment of the photorecepter cell. The apparent Km values for serine and ethanolamine in the microsomal fraction decreased with decreasing Ca2+ concentration. Although no further increase of incorporation of serine and ethanolamine occurred after 40 min in the microsomal fraction, continuous incorporation of both bases into phospholipids was seen for 3 hr in excised retina. Illumination did not significantly affect the incorporation of serine and ethanolamine in excised retina or in the rod outer segment fraction. Base exchange reaction thus may not play a direct role in the visual process.View full abstractDownload PDF (578K) -
Mieko OSHIMA1976 Volume 80 Issue 1 Pages 53-59
Published: July 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSGlucosyl sphingosine was isolated from the spleen of a patient with adult-type Gaucher's disease. The yield of purified glucosyl sphingosine was 9.3 nmoles/g wet tissue.
The gas chromatography-chemical ionization mass spectrum of acetylated glucosyl sphingosine showed peaks due to the presence of ions formed by successive loss of acetic acid (mass 60) from the molecular ion (QM+, 714).
Fragments from acetylated sugar, m/e 331, and from the sphingosine residue, m/e 306, were also detected.
The GC-CI mass spectrum of the trimethylsilyl derivative of glucosyl sphingosine showed peaks due to the molecular ion (QM+, 822), ions from the sugar moiety (mle 361, 271), and ions from the sphingosine base (m/e 264, 280).
Fragmentation analysis of the purified sample by GC-EI and GC-CI mass spectro-metry confirmed the structure glucopyranosyl(1→1)-l, 3-dihydroxy-2-amino-4-octadecene.View full abstractDownload PDF (1060K) -
IV. Inactivation of the Acid Protease from Mucor pusillus by Acid Protease-specific InhibitorsKenji TAKAHASHI, Wen-Jong CHANG, Kei ARIMA1976 Volume 80 Issue 1 Pages 61-67
Published: July 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMucor pusillus acid protease was rapidly inactivated with 1:1 stoichiometry by reac-tion with diazoacetyl-DL-norleucine methyl ester (DAN) in the presence of cupric ions. Cupric ions were essential for this inactivation. The rate of inactivation was maximal at around pH 6 when the enzyme was mixed with DAN and cupric ions without prior mixing of the reagents, and at pH 5.3 when DAN and cupric ions were mixed and incubated before addition to the enzyme solution. In both cases, the rate of inactivation decreased as the pH was either increased or decreased. The amino acid composition of an acid hydrolysate of the DAN-modified enzyme was indistinguishable from that of the native enzyme except for the incorporation of about one norleucine residue per molecule of protein.
The enzyme was also inactivated by reaction with 1, 2-epoxy-3-(p-nitrophenoxy)-propane (EPNP). At the stage of about 90% inactivation, 1.50 residues of EPNP were incorporated per molecule of protein and the rate of inactivation followed pseudo-first order kinetics. The optimal pH for the inactivation was pH 3.0 and the rate of inactivation decreased as the pH was either increased or decreased.
Furthermore, the enzyme was strongly inhibited by pepstatin, and the reactions of DAN and of EPNP were also inhibited significantly by prior treatment of the enzyme with pepstatin.
These results suggest that the enzyme may have two essential carboxyl groups at the active site, one reactive with DAN in the presence of cupric ions and the other with EPNP, and that pepstatin binds part of the active site to inhibit the reactions with DAN and EPNP as well as the enzyme activity.View full abstractDownload PDF (533K) -
Masanobu JANADO, Kazuko SHIMADA, Naomi HORIE, Toshiro NISHIDA1976 Volume 80 Issue 1 Pages 69-71
Published: July 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have previously suggested the involvement of both hydrogen bonding and hydro-phobic bonding in the adsorption of sulfanilamide on Bio-Gel beads. In the present study, we closely examined the concentration dependence of the binding curve and our proposed binding model has been corroborated. For comparison, binding param-eters and thermodynamic data pertaining to the sulfanilamide-Sephadex system have been also evaluated.View full abstractDownload PDF (223K) -
Shigekazu NAGATA, Kentaro IWASAKI, Yoshito KAZIRO1976 Volume 80 Issue 1 Pages 73-77
Published: July 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSince it was shown that the low molecular weight form of the eukaryotic polypeptide chain elongation factor 1 (EF-lα) is highly unstable but could be stabilized by the addition of 25% (v/v) glycerol to all the buffer solutions (Nagata, S., Iwasaki, K., & Kaziro, Y. (1976) Arch. Biochem. Biophys. 172, 168-177), its distribution in several tissues has been investigated under conditions where it is stabilized. The results obtained indicate that EF-la is present as a predominant species in all the tissues examined, such as pig liver, rat liver, rabbit reticulocytes, and Artemia sauna cysts. Furthermore, it is suggested that the high molecular weight form of the elongation factor 1 contains a new elongation factor, EF-1β, in addition to EF-lα, while the low molecular weight form contains little EF-1β.View full abstractDownload PDF (347K) -
I. Splitting Enzyme 1Toshiyuki TEZUKA, Kenzo TONOMURA1976 Volume 80 Issue 1 Pages 79-87
Published: July 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn enzyme (S-1) which catalyzes the splitting of carbon-mercury linkages of organo-mercury compounds was purified about 24-fold from the cell-free extract of mercury-resistant Pseudomonas K-62 strain by treatment with streptomycin, precipitation with ammonium sulfate, and successive chromatography on Sephadex G-150, DEAE-Sephadex, and DEAE-cellulose. A purified preparation of the enzyme showed a single band on polyacrylamide gel electrophoresis, and was colorless. The molecular weight of the enzyme was estimated to be 19, 000, and Km was 5.3×10-5 M for p-chloromercuribenzoic acid (PCMB). The temperature and pH optimum for the reaction were 50° and 7.0, respectively. The enzyme was capable of catalyzing the decomposition of methylmercuric chloride (MMC), ethylmercuric chloride (EMC), phenylmercuric acetate (PMA), and PCMB in the presence of a sulfhydryl compound to form a mercuric ion plus methane, ethane, benzene, or benzoic acid, respectively. The mercuric ion thus formed was reduced to metallic mercury by metallic mercury-releasing enzyme (MMR-enzyme).View full abstractDownload PDF (1215K) -
Renata DABROWSKA, Zoya PODLUBNAYA, Ewa NOWAK, Witold DRABIKOWSKI1976 Volume 80 Issue 1 Pages 89-99
Published: July 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. The TN-T and TN-I components of troponin both interact with tropomyosin and cause its precipitation in 0.1M KCl at neutral pH. The precipitate contains both end-to-end and side-by-side aggregates of tropomyosin molecules.
2, The TN-T and TN-I components change the band pattern of tropomyosin paracrystals formed in MgCl2 solutions, although in different ways. TN-T causes the formation of hexagonal net structures, double-stranded net or paracrystals which result from the collapse of the double-stranded net. TN-l at pH 7.9 causes the formation of paracrystals with a 400 A periodic band pattern and a 200 A repeat. The same band pattern can also be seen in tropomyosin paracrystals formed at pH values below 6.0.
3. The TN-C component does not precipitate tropomyosin in 0.1M KCl. The aggregates of tropomyosin obtained with either TN-T or TN-I can be solubilized by the addition of TN-C. No interaction of TN-C was observed with tropomyosin paracrystals formed in the presence of MgCl2.View full abstractDownload PDF (4281K) -
IV. “Abnormal” Reactivity of S1 Thiol and Conformational Changes around S2 ThiolKenji TAKAMORI, Kimiyo A. KATO, Takamitsu SEKINE1976 Volume 80 Issue 1 Pages 101-110
Published: July 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe flexibility of the tertiary structure around the active site of myosin ATPase [EC 3. 6. 1. 3] was studied using the reactivity of two specific thiol groups, S1 and S2, as a structural probe. The following four maleimide derivatives were used as thiol-directed reagents : N-ethylmaleimide (NEM), N-(4-methoxy-2-benzimidazolyl methyl) maleimide (MBM), N-(p-(2-benzimidazolyl)phenyl)maleimide (BIPM) and N-(4-dimethyl-amino-3, 5-dinitrophenyl)maleimide (DDPM).
1. All the maleimide derivatives used activated the Cat2+-ATPase activity and inhibited the EDTA-ATPase activity, like NEM, indicating that they modified S1. The rate of modification of S1 by NEM and BIPM increased with increasing pH, while that by DDPM decreased. BIPM simultaneously modified S1 and S2.
2. Sl showed much higher reactivity toward the maleimides, except for BIPM, than did N-acetylcysteine (N-Ac-Cys) a low-molecular-weight model compound. The extremely small pKa value of S1, 6.28, accounted for this high reactivity. In addi-tion, the ATP-induced increase in its reactivity indicated that Sl was in a buried state. Kinetic analysis showed that the tertiary structure around Sl at alkaline pH differed from that at acidic pH.
3. The apparent rate constant of S2-modification with NEM was approximately one seven-hundredth and one four-hundredth of those of Sl and N-Ac-Cys, respectively. Fluorimetric studies using BIPM revealed that S2 in the buried state was exposed upon adding ATP; this was compensated by the burying of some other thiol group(s) (Sp). Non-linearity of the Arrhenius plots of the reaction rate of S2 suggested that the S2 region of myosin had different conformations at high and low temperatures, the transition temperature being 10-15°. This non-linearity completely disappeared in the presence of Mg2+-ATP. On the other hand, Arrhenius plots for the thiols reactive to BIPM did not show non-linearity in the presence or absence of ATP.View full abstractDownload PDF (791K) -
Yoh OKAMOTO, Koichi YAGI1976 Volume 80 Issue 1 Pages 111-120
Published: July 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSOne of the low molecular weight components of myosin, g2, was isolated by alkali treatment of myosin and was chemically modified with a spin label reagent, 4-maleimido-2, 2, 6, 6-tetramethylpiperidinooxyl. The label on g2 showed a rather weakly immobilized ESR spectrum and it was clearly affected by Ca2+; the half-maximal change was at around pCa 4. The spin-labeled g2 was incorporated into myosin by exchange with the intrinsic g2 of myosin in 0.6M KSCN or 4 M LiCl. The label on g2 became strongly immobilized on association with myosin. Under the conditions used, ESR spectral change due to Ca2+ occurred at two different concentration ranges, which were as low as pCa 8 and at around pCa 4.
Phosphorylated g2 was isolated from myosin after the protein kinase [EC 2.7.1.37]-catalyzed phosphorylation of myosin and it was also modified with the maleimide label. Dephosphorylation of the phosphorylated g2 was performed using E. coli alkaline phosphatase [EC 3.1.3.1]. The effects of Ca2+ on the ESR spectra of phosphorylated and dephosphorylated g2 were investigated in the state associated with myosin. A change in the ESR spectrum from strongly immobilized to weakly immobilized states was observed with both g2 chains on the addition of Ca2+. How-ever, the effective concentration ranges of Ca2+ were quite different; around pCa 4 for the phosphorylated g2 and around pCa 8 for the dephosphorylated g2. The results indicate that g2 undergoes a conformational change at physiological levels of Ca2+ sufficient to saturate troponin, but it does not do so after phosphorylation.View full abstractDownload PDF (1852K) -
Katsuhisa TAWADA, Akihiro YOSHIDA, Kyoko MORITA1976 Volume 80 Issue 1 Pages 121-127
Published: July 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWith the final aim of replacing myosin in a single muscle fiber, a technique for removing myosin almost completely from single fibers was developed and an attempt to “re-form” thick filaments in the myosin-free ghosts of single fibers was made.
Complete removal of myosin from single glycerol-treated rabbit psoas fibers with Hasselbach-Schneider solution was difficult. However, when skinned glycerol-treated fibers were used and 1% (v/v) Triton X-100 was added to the Hasselbach-Schneider solution, almost complete removal of myosin was possible. The myosin-free ghosts of skinned single fibers were very fragile but retained the overall structure. In the ghost fibers, Z-membranes and thin filaments remained.
The ghost fibers, after irrigation with myosin, underwent contraction upon addition of Mg-ATP. In the myosin-irrigated fibers, thick filaments were re-formed in lengths from one Z-membrane to the other Z-membrane of a sarcomere, running parallel to the thin filaments. The packing of these two filaments was not good. The isometric tension developed by the irrigated fibers upon addition of Mg-ATP was about 10% of the tension developed by untreated fibers. The weak tension developed by irrigated fibers is probably due to the irregular packing of the thick and thin filaments in the fibers.
The ghost fibers also contracted, though only slightly, upon addition of Mg-ATP after irrigation with heavy meromyosin.View full abstractDownload PDF (1625K) -
Tatsuhisa YAMASHITA, Etsuko SAWANOBORI-ISOBE, Matsuhiro MORI1976 Volume 80 Issue 1 Pages 129-134
Published: July 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. Serum alkaline phosphatase [EC 3. 1. 3. 1] was strongly inactivated by histidine during incubation at pH 8.0 and 45°; however, tryptic digestion of the serum strongly protected the enzyme against inactivation by histidine. In the absence of histidine, however, neither heat inactivation of the phosphatase nor the effect of trypsin [EC 3.4.21.4] was observed. Factors affecting the alkaline phosphatase inactivation were studied further.
2. The effect of trypsin on the histidine-induced heat inactivation differed consider-ably according to the tissue source of the enzyme, which suggests a possible method for distinguishing alkaline phosphatase isoenzymes.View full abstractDownload PDF (395K) -
Hideo SHIMADA, Yutaka ORII1976 Volume 80 Issue 1 Pages 135-140
Published: July 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDithionite reduced the heme c moiety of Pseudomonas nitrite reductase almost instantaneously, whereas the spectral change of heme d proceeded in two steps, requiring at least 15 min for completion. The final spectrum coincided well with that obtained by anaerobic reduction with ascorbate, during which a quasi oxidation-reduction equilibrium was established between the two heme groups. The difference in apparent redox potential was calculated to be 24 mV, heme d being more negative.
When the enzyme was supplemented with a reductant and molecular oxygen, an oxygenated intermediate appeared at the heme d moiety.View full abstractDownload PDF (418K) -
Morphology, Subunits, and Chemical CompositionYasuo KAGAWA, Nobuhito SONE, Masasuke YOSHIDA, Hajime HIRATA, Harumasa ...1976 Volume 80 Issue 1 Pages 141-151
Published: July 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. A stable membrane-bound ATPase [EC 3.6.1.3] (TF0•F1) capable of proton translocation in reconstituted vesicles was purified from the thermophilic bacterium PS3 cultured in medium containing L-[U-14C]amino acids.
2. TF0•1 was composed of a catalytic moiety (TF1) and a hydrophobic moiety (TF0). TF1 contained 3 polypeptide chains with molecular weights of 56, 000, 3 of 53, 000, 1 of 32, 000, 1 of 15, 500, and 1 of 11, 000. TF0 contained 1 chain of 19, 000, 2 of 13, 500, and 5 of 5, 400 daltons. TF1 was dissociated into subunits much less readily than F1.
3. TF1 consisted of 95A particles arrayed in hexagonal microcrystals. TF0•F1 consisted of a sphere (TF1) and a stalk plus base (TF0) which was buried in the membrane of the proton translocating vesicles.
4. Vesicles capable of energy transformation were formed when TF, came in contact with the surface of liposomes containing TF0. On addition of phospholipids, the helix content of TF0 increased 3-fold. The role of F0 in forming channels for protons is discussed.
5. The amino acid compositions of TF0, TF, , and TF0•F1 were compared. TF0 was not hydrophobic, despite its interaction with phospholipids. The phospholipid composition and other properties of the proton translocating vesicles were examined. Vesicles reconstituted from a mixture of phosphatidylethanolamine, phosphatidylgly-cerol, and cardiolipin in the same ratio as in the membranes had the highest activity.View full abstractDownload PDF (2262K) -
Ryoko KURIYAMA1976 Volume 80 Issue 1 Pages 153-165
Published: July 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAbout 10-20% of the total protein in the outer fiber fraction was solubilized by sonication in a solution containing 5mm MES, 0.5mm MgSO4, 1.0mm EGTA, 1.0mm GTP, and 0 or 50mm KCl at pH 6.7. The sonicated extract was shown by analytical centrifugation to consist largely of a 6 S component (tubulin dimer), having a molecular weight of 103, 000, as determined by gel filtration, and possessing a colchicine-binding activity of 0.8 mole per tubulin dimer.
The tubulin fraction failed to polymerize into microtubules by itself. Addition of a small amount of the ciliary outer fiber fragments or reconstituted short brain microtubules, however, induced polymerization, as demonstrated by viscosity of flow birefringence changes as well as light or electron microscopic observations. The growth of heterogeneous microtubules upon mixing outer fiber tubulin with DEAE-dextran-decorated brain microtubules was observed by electron microscopy.
Microtubules were reconstituted from outer fiber tubulin without addition of any nuclei fraction when a concentrated tubulin fraction was warmed at 35°. A few doublet-like microtubules or pairs of parallel singlet microtubules that were closely aligned longitudinally could be observed among many singlet microtubules.
Unlike outer fiber microtubules, the reconstituted polymers were depolymerized by exposure to Ca2+ ions, high or low ionic strength, colchicine, low temperature or SH reagents. No microtubules were assembled under these conditions.View full abstractDownload PDF (2652K) -
Masanori FUKUSHIMA, Hiroto OKAYAMA, Yohei TAKAHASHI, Osamu HAYAISHI1976 Volume 80 Issue 1 Pages 167-176
Published: July 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe localization of NAD+ glycohydrolase [EC 3.2.2.5] (NADase) in purified rat liver nuclei has been examined. Subnuclear fractionation revealed that at least 70% of the NADase in nuclei was associated with the nuclear envelope fraction. The nuclear envelope fraction was practically free of microsomal contamination as judged by electron microscopic morphometry and assays of microsomal marker enzymes. Therefore, NADase was found to be an integral component of the nuclear envelope. The enzymological properties of the nuclear envelope NADase were compared with those of the microsomal enzyme. The nuclear envelope NADase was identical to the microsomal enzyme in its Km for NAD+ (60 μm), pH optimum (pH 6.5), ratio of transglycosidase activity to NADase activity (about 0.5), thermal stability and sensitivity to various inhibitors. Thus, NADase is a common enzymic component of both the nuclear envelope and the endoplasmic reticulum.View full abstractDownload PDF (1423K) -
Tsukasa SUGANO, Masakazu SHIMADA, Hiroshi TATSUMI1976 Volume 80 Issue 1 Pages 177-186
Published: July 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBased on criteria such as the ADP/O ratio and respiratory control by ADP, the energy-coupling efficiency of brown adipose tissue mitochondria isolated from rats kept under normal environmental conditions for a long time decreased remarkably.The presence of bovine serum albumin, GTP, or ATP plus carnitine in the reaction medium markedly increased the efficiency of oxidative phosphorylation of brown adipose tissue mitochondria. Pre-treatment of brown adipose tissue mitochondria with 2% bovine serum albumin, GTP, or ATP plus carnitine caused a decrease in the amount of free fatty acids bound to the mitochondria from 13.1 to 7.0, 9.0, or 8.2μg per mg protein, respectively. Removal of the free fatty acids by means of these pre-treatments resulted in restoration of efficient oxidative phosphorylation; there was a correlation between the amount of free fatty acids removed and the degree of recovery in the respiratory control ratio. The elimination of only a frac-tion of the free fatty acids, as little as 4μg per mg protein, was sufficient to ensure respiratory control by ADP.
It appears that the free fatty acids which lie mainly outside the inner mito-chondrial membrane are responsible for the decrease in the efficiency of oxidative phosphorylation in brown adipose tissue mitochondria isolated from rats kept under normal environmental conditions.View full abstractDownload PDF (738K) -
Naomi AZUMA1976 Volume 80 Issue 1 Pages 187-189
Published: July 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSuperprecipitation was observed with abalone myosin and purified rabbit actin in the presence of calcium ions, but was not observed in the absence of calcium. The Mg-ATPase [EC 3.6.1.3] activity of abalone myosin and rabbit actin in the absence of calcium ions (EGTA present) showed about 60% inhibition as compared with values in the presence of calcium ions. The calcium sensitivity may be attributable to abalone myosin, as in the case of scallop myosin.View full abstractDownload PDF (677K) -
Kayoko TATEISHI, Hiroshi YAMAMOTO, Toshio OGIWARA, Chozo HAYASHI, Masa ...1976 Volume 80 Issue 1 Pages 191-194
Published: July 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA highly sensitive, reproducible method was established for enzyme-coupled im-munoassay of testosterone, involving coupling of testosterone to glucoamylase [EC 3.2.1.3] with a water-soluble coupling reagent carbodiimide. No enzyme activity was lost during this coupling procedure. The sensitivity of the method was comparable to that of competitive protein binding assay.View full abstractDownload PDF (275K)
- |<
- <
- 1
- >
- >|