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Volume 93 (1983)
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Volume 93, Issue 5
Displaying 1-29 of 29 articles from this issue
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Masahiro TOMITA, Ryo TAGUCHI, Hiroh IKEZAWA1983 Volume 93 Issue 5 Pages 1221-1230
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSphingomyelinase of Bacillus cereus was specifically adsorbed onto sphingomyelin liposome in the presence of Ca2+ or with the coexistence of Ca2+ and Mg2+, but not onto the liposome of phosphatidylcholine. In the presence of Ca2+, the enzyme adsorption onto bovine erythrocytes and liposome increased with an increase in the amount of sphingomyelin. These results support that the major binding site for sphingomyelinase in the erythrocytes is sphingomyelin. The temperature-dependence of enzyme adsorption was not influenced by a change in ATP content of bovine erythrocytes. After treatment of red cells with neuraminidase or pronase, enzyme adsorption at 0°C lower than that at 37°C was observed. In unsealed or right-side-out ghosts, the difference between the enzyme adsorption at 37°C and that at 0°C became less pronounced than in the erythrocytes. Furthermore, the extent of enzyme adsorption onto sphingomyelin liposome at 0°C was almost equal to that at 37°C. The enzyme adsorption onto the erythrocyte membrane and liposome was always enhanced in the presence of Ca2+; in the presence of Mg2+ alone, adsorption was observed only for erythrocyte ghosts and the adsorbed enzyme was released from the membrane after extensive degradation of sphingomyelin by sphingomyelinase. With the coexistence of Ca2+ and Mg2+, the enzymatic hydrolysis of sphingomyelin proceeded rapidly for the attack against liposome and all the membranes tested, whereas in the presence of Mg2+ alone, hydrolysis was observed only for the action of liposome and ghosts. No appreciable hydrolysis of sphingomyelin was observed in the presence of Ca2+ nor in the absence of divalent metal ions.View full abstractDownload PDF (929K) -
Kunio TSURUGI, Mitsuru OYANAGI, Kikuo OGATA1983 Volume 93 Issue 5 Pages 1231-1237
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo examine whether serine proteases of rat liver chromatin are also involved in the degradation of newly synthesized and unbound ribosomal proteins and histones, like the nuclear thiol protease which we reported previously (Tsurugi, K. & Ogata, K. (1979) Eur. J. Biochem. 101, 205-213), in vivo experiments were carried out with serine protease inhibitor, PMSF. The following results were obtained.
When normal rats received an intraperitoneal injection of PMSF (10mg per 100g body weight), nuclear serine proteases were inhibited almost completely for at least 90min. PMSF did not affect the synthesis of proteins and RNAs of ribosomes and other subcellular fractions.
The effects of PMSF treatment in vivo on the degradation of newly synthesized ribosomal proteins and histones in regenerating rat liver pretreated with a low dose of actinomycin D, which preferentially inhibited rRNA synthesis, were examined by using the double-isotope method. It was found that PMSF treatment did not affect their degradation. On the other hand, administration of E-64, a thiol protease inhibitor, to partially hepatectomized rats inhibited the degradation of those proteins markedly. From these results, it is concluded that the nuclear thiol protease, but not serine proteases, is preferentially involved in the degradation of newly synthesized ribosomal proteins and histones which are not associated with rRNA and DNA, respectively.View full abstractDownload PDF (577K) -
Nobuko MARUYA, Atsuo HIWATASHI, Yoshiyuki ICHIKAWA, Toshio YAMANO1983 Volume 93 Issue 5 Pages 1239-1247
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA renal ferredoxin was purified from bovine renal mitochondria to electrophoretic purity. The molecular weight of the renal ferredoxin was estimated by gel filtration and SDS-polyacrylamide gel electrophoresis to be 12, 500 and 13, 000, respectively. The optical absorption spectrum of renal ferredoxin in the oxidized form showed two peaks at 416 and 457 nm in the visible region, and the EPR absorption spectrum showed peaks at gx=gy=1.94 and gz=2.02 in the reduced form at 13 K. These spectra were typical of the 2S-2Fe type ferredoxins. Dissimilarities were recognized in the amino acid composition and isoelectric point between bovine renal ferredoxin and bovine adrenodoxin, but not in the optical, magnetic, and immunochemical properties.
The reconstitution of 25-hydroxyvitamin D3-1α-hydroxylase system was performed with the three components of NADPH-adrenodoxin reductase from bovine adrenal mitochondria, renal ferredoxin, and cytochrome P-451α from bovine renal mitochondria. The results showed that the renal ferredoxin was essential for the 1α-hydroxylase activity of 25-hydroxyvitamin D3.View full abstractDownload PDF (1988K) -
Toshihiko HIRANO, Haruki YAMADA, Toshio MIYAZAKI1983 Volume 93 Issue 5 Pages 1249-1257
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe carbohydrate requirement for cell adhesion of aggregation-competent cells of Dictyostelium discoideum has been examined by use of a selective glycosylation inhibitor of N-glycosyl protein, tunicamycin (TM). TM completely inhibited EDTA-stable cell adhesion and glycosylation of some membrane glycoproteins in aggregation-competent cells of D. discoideum (Yamada, H., et al. (1982) J. Biochem. 92, 399-406). The present study showed that the inhibition of EDTA-stable cell adhesion by TM was prevented significantly when the cells were treated with TM in the presence of a protease inhibitor, leupeptin (LP), whereas the inhibition of glycosylation by TM was not prevented. The cell extract of aggregation-competent cells contained acid proteases, and LP strongly inhibited acid protease from D. discoideum in vitro. On analysis by SDS-polyacrylamide gel electrophoresis (PAGE), many protein bands present in the membrane fraction of control cells disappeared or decreased on TM treatment of the cells in the absence of LP, however, some of these proteins were restored when the cells were treated with TM in the presence of LP. These results strongly support an idea that EDTA-stable cell adhesion characteristic to aggregation-competent cells is mediated by glycoproteins with asparaginelinked carbohydrate. However, the requirement for the carbohydrate moiety of the glycoprotein in cell adhesion appears to be indirect in that it acts to protect the protein moiety from proteolytic degradation.View full abstractDownload PDF (1899K) -
Hiromichi KUMAGAI, Hikoichi SAKAI1983 Volume 93 Issue 5 Pages 1259-1269
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA protein which binds to both tubulin and tubulin polymer was isolated from porcine brains. This protein has a molecular weight of 35, 000 on SDS-polyacrylamide gel electrophoresis (designated as 35 K protein). The 35 K protein was purified through several steps of purification including ammonium sulfate fractionation, Sephadex G-100 gel filtration column chromatography, microtubule protein-agarose gel affinity column chromatography and phosphocellulose column chromatography. The 35 K protein caused pronounced enhancement of the turbidity increase produced by tubulin polymerization in the presence of DMSO, but did not have the ability to initiate polymerization of pure tubulin in the absence of DMSO. It was demonstrated that 35 K protein co-sediments with tubulin polymer in a concentration-dependent manner. Electron microscopic observation revealed the formation of bundles of tubulin polymer. Since the effect of 35 K protein was coupled with tubulin polymerization, 35 K protein did not cause the turbidity increase under conditions where tubulin polymerization was inhibited by Ca2+, or colchicine. The 35 K protein adsorbed on tubulin-Sepharose 4 B was eluted by the addition of 2mM ATP. ATP was shown to inhibit the interaction of 35 K protein with tubulin dimer or polymer. The 35 K protein was finally identified as glyceraldehyde 3-phosphate dehydrogenase from properties such as mobility on SDS-polyacrylamide gel electrophoresis, cleavage pattern on limited proteolysis, ability to bind to tubulin, and so on.View full abstractDownload PDF (3890K) -
Hiroshi MORII, Yuji TONOMURA1983 Volume 93 Issue 5 Pages 1271-1285
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSFragmented sarcoplasmic reticulum (FSR) from rabbit skeletal muscle was passively loaded with 45Ca2+. Its Ca2+-induced Ca2+ release was measured in the presence of 0.1M KCl and 5mM MgCl2 at 0°C by Millipore filtration. The following results were obtained.
1. The amounts of Ca2+-induced Ca2+ release from heavy SR, light SR, and unfractionated SR were 80, 20, and 60% of the amounts of preloaded Ca2+, respectively. Therefore, the experiments were carried out with unfractionated FSR. 2. The Ca2+induced Ca2+ release from FSR was inhibited by procaine, but unaffected by caffeine and trifluoperazine. The rate of Ca2+ release decreased markedly with decreasing pH. 3. Various adenine nucleotides (ATP, AMPPNP, ADP, AMP) accelerated the Ca2+ release, and the accelerating effect was reversible. CTP had no effect on the release, but inhibited the accelerating effect of AMPPNP. 4. In the presence of 15 μM external free Ca2+, the final amount of the Ca2+ release was unaffected. The rate of Ca2+ release was markedly increased by AMP; the dependence of the rate on AMP concentration followed a Michaelis-Menten type equation with a Hill coefficient of 1 and an apparent affinity for AMP of about 2mM. 5. In the presence of AMP, the amount of Ca2+ released increased, while the relative rate was unaffected by increasing the external Ca2+ concentration. The final amount released increased from 0 to 60% of the amount of preloaded Ca2+ by increasing the free Ca2+ concentration from 0.06 to 0.24 μM. The effect of external Ca2+ on the release was reversible. 6. The ratio between the amount of preloaded Ca2+ and that of Ca2+ release was independent of the Ca2+ concentration used for preloading. Furthermore, the dependence of the final amount of Ca2+-induced Ca2- release on external Ca2+ was unaffected by internal Ca 2+.
The results suggest that most of the FSR vesicles have no or only one channel for the Ca 2+-induced Ca2+ release, that the channel has active and inactive forms and the equilibrium between them shifts to the active form when adenine nucleotide binds to the channel with a Hill coefficient of I, and that the gate opens in an allor-none fashion when the external Ca2+ concentration exceeds the threshold. The amount of Ca2+ release per unit time (the Ca2+ passes through the open gate of the active channel) is proportional to the concentration of internal Ca2+.View full abstractDownload PDF (1121K) -
Isolation and Characterization of Nucleases from a Clinical Isolate of Serratia marcescens kums 3958Kensuke YONEMURA, Koki MATSUMOTO, Hiroshi MAEDA1983 Volume 93 Issue 5 Pages 1287-1295
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo new extracellular nucleases, nucleases SM, and SM2, were purified from the culture fluid of S. mareescens kums 3958, a fresh clinical isolate. The purification was carried out by the following steps; ammonium sulfate precipitation, and DEAEcellulose and Sephadex G-100 column chromatography. At the final step, nucleases SM1 and SM2 were purified about 3, 700- and 1, 000-fold, respectively. They were free from phosphomonoesterase and phosphodiesterase activities. The pIs were 8.1 and 7.5 for nucleases SM1 and SM2, respectively. The molecular weight was estimated to be 35, 000 for both enzymes by SDS-polyacrylamide disc gel electrophoresis. The results of amino acid analyses showed that both the threonine and serine contents were higher in nuclease SM2 than in SM1. Furthermore, nuclease SM1 was more stable than nuclease SM, at 4°C. The other properties of the two enzymes were similar; pH optimum (8.0), Mg2+ or Mn2+ for activation, and inhibition by chemical reagents such as EDTA and pyrophosphate. No significant difference was found in base specificity between nucleases SM1 and SM2. Both enzymes specifically degraded double-stranded homopolymers, especially poly (I) poly (C), as well as yeast RNA and calf thymus DNA. They hardly degraded, however, single-stranded homopolymers such as poly(dA), poly (G), and poly (U).View full abstractDownload PDF (1225K) -
Shin-ichi NAKAYAMA, Youko NAGASHIMA, Masamatsu HOSHINO, Akihiko MORIYA ...1983 Volume 93 Issue 5 Pages 1297-1304
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo investigate the active site structures of porcine pepsin and Rhizopus chirnensis acid protease (RAP), spin label techniques were applied for these enzymes.
Comparison of spin labeled porcine pepsin and RAP suggested that the active site cleft of porcine pepsin was narrower at the top, but wider at the bottom than that of RAP. Addition of pepstatin restricted the motion of the labeled nitroxide radicals. Under alkaline conditions, the enzymes changed their conformation discontinuously and irreversibly to open the active site clefts and to lose the binding ability for pepstatin. The denaturation points of both the enzymes were determined to be pH 6. 2.View full abstractDownload PDF (579K) -
Koichi SUZUKI1983 Volume 93 Issue 5 Pages 1305-1312
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe reaction of calcium activated neutral protease (CANP) with an epoxysuccinyl derivative (E64c) and iodoacetic acid (IAA) was examined in the presence of Ca2+, which specifically accelerates the rate of inactivation by these inhibitors. 1. E64c and IAA (100-fold molar excess) each inactivated CANP within a few minutes at O°C. Upon inactivation, I mol of E64c or IAA was incorporated into CANP. The reaction of CANP with E64c or IAA was optimal at pH 7.5-8.0, where CANP shows the maximum enzyme activity.
2. CANP modified with E64c or IAA lost one of the 3 SH groups (class II SH groups) which were exposed at the surface by addition of Cant. No conformation change of CANP was observed as a result of the modifications.
3. Leupeptin inhibited the modifications. A total of 1 mol of E64c and IAA was incorporated into CANP by successive labeling of CANP with E64c and IAA.
It was concluded from these results that E64c and IAA specifically react with the same class II SH group, which is regarded as the active site of CANP.View full abstractDownload PDF (595K) -
Hirokazu KOYAMA1983 Volume 93 Issue 5 Pages 1313-1319
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSL-Phenylalanine oxidase, purified to homogeneity from Pseudomonas sp. P-501, had a molecular weight of about 140, 000 and consisted of two subunits identical in molecular weight (about 68, 000). The sedimentation coefficient (s020, w) of the enzyme was determined to be 8.18 S by ultracentrifugation. The enzyme showed absorption maxima at 276, 390, and 466 nm and a shoulder around 490 nm and contained 2 mol of FAD per mol of enzyme.
Oxygen-18 supplied as molecular oxygen was incorporated into the carbonyl group of α-phenylacetamide formed by the enzymic oxidation of L-phenylalanine. Michaelis constants of the enzyme were 1.07×10-2mM for L-phenylalanine and 1.82mM for oxygen. Maximum activities in oxidation and oxygenation (catalyzed simultaneously by the enzyme) were observed at different pHs and different temperatures. Several metal ions inhibited the oxidase activity preferentially.View full abstractDownload PDF (545K) -
Tatsuhisa SEGAWA, Shintaro SUGAI1983 Volume 93 Issue 5 Pages 1321-1328
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBovine, human, and goat α-lactalbumins prepared by the ordinary methods were found to contain 1.1-1.3 atoms of Ca per protein molecule. Removal of Ca2+ was shown to destabilize the tertiary structures in the three proteins. The three apoproteins were indicated to change in the conformation by heat from the native-like to the unfolded state.
Degree of restoration of the native tertiary structure in 5mM Tris-HCl and 0.1mM EDTA at pH 7.2 and 25°C by addition of Ca2+ was determined from change in CD ellipticity at 270 nm, and the apparent binding constant of Ca2+ was analyzed to be 2.5×108 (bovine), 3.0×108 (human), and 2.8×108M-1 (goat). Also, value of the binding constant of Ca2+ to the native-like apoform was estimated from the apparent binding constant and equilibrium constant of the conformational change of the apoform.
The binding properties of Mn2+, Mg2+, and Zn2+ to the bovine protein at neutral pH are also discussed.View full abstractDownload PDF (592K) -
Nobuhito SONE, Yasuo KAGAWA, Yutaka ORII1983 Volume 93 Issue 5 Pages 1329-1336
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. The effects of aeration on the growth and cytochrome patterns of thermophilic bacterium PS3 were studied; bacteria grown with strong aeration synthesized cytochromes c, b, and αα3 while those grown with low aeration, showing non-exponential growth, synthesized higher amounts of cytochromes c and b including o, and a lower amount of cytochrome α (α3). The CO-difference spectra indicated that the terminal oxidase was cytochrome αα3 for high aeration conditions and the cytochrome o for low aeration conditions.
2. Cytochrome o can be solubilized by Triton X-100 from the membrane fraction of bacteria grown under oxygen-limited conditions. The carbon monooxide complex of cytochrome o, obtained by exposing this extract to CO, was photolyzed and the subsequent rebinding of CO was analyzed; it followed first order kinetics with a rate constant of around 8 s-1 at 25°C. At liquid nitrogen temperature, CO-rebinding did not occur.
3. The CO-difference spectrum of purified cytochrome oxidase sample from the bacteria grown with strong aeration (Sone, N., et al. (1979) FEBS Lett. 106, 39-42) revealed the presence of a small amount of a cytochrome o-like pigment besides cytochrome αα3. Analysis of the CO complexes of these chromophores showed rate constants of 29-30 s-1 for cytochrome αα3 and 35-42 s-1 for the o-like pigment, indicating that the cytochrome o-like pigment contaminating the purified cytochrome oxidase preparation was not typical cytochrome o.View full abstractDownload PDF (572K) -
Masakazu SUGAHARA1983 Volume 93 Issue 5 Pages 1337-1342
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIt was found that 4-aminobutyraldehyde (ABAL) is a precursor convertible to γ-aminobutyric acid (GABA) in vivo. [2, 3-3H] ABAL was synthesized from [2, 3-3H] putrescine. After the subcutaneous administration of [3H]ABAL at the dose of 1 μmol/g body weight, [3H] GABA was produced in the mouse brain in an amount of about 350 nmol/g brain in 10min. After oral administration of [3H] ABAL at the dose of 2 μmol/g body weight, [3H] GABA was also produced in the brain in an amount of about 530 nmol/g brain in 30min. It seems that peripherally administered ABAL penetrates the blood-brain barrier into the central nervous system and is rapidly metabolized to GABA in the brain.View full abstractDownload PDF (439K) -
Kiyoshi IKEDA, Kyozo HAYASHI1983 Volume 93 Issue 5 Pages 1343-1351
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSpH-dependence of the binding constant of Ca2+ to β-bungarotoxin was studied at 25°C and ionic strength 0.2 by the tryptophyl fluorescence and ANS-fluorescence methods. The results were compared with those of other phospholipases A2.
The binding constant at neutral pH value was in good agreement with that reported by Abe et al. ((1977) Eur. J. Biochem. 80, 1-12). The pK value of an ionizable group was perturbed from 7.25 to 5.90 by the Ca2+ binding. Competition with the Ca2+ binding was observed in the protonation of another group with a pK value of 5.05. The former group was assigned as His 48 in the A chain of the protein and the latter as Asp 49 to which Ca2+ ion can coordinate. No participation of the α-amino group was observed. The pK value (7.25) of His 48, determined in the present experiment, was very close to that (6.9) estimated from the pH-dependence of the chemical reaction of p-bromophenacyl bromide with His 48 (Kondo et al. (1978) J. Biochem. 84, 1291-1300).
The binding constant of Ca2+, KEM=6.76×103M-1, when all the ionizable groups participating in the Ca2+ binding are completely deprotonated, was found to be very similar to those for all the phospholipases A2 reported previously, indicating similarity in the conformation of the Ca2+ binding site. Inhibitory metal ions, Ba2+ and Sr2+, exhibited binding constants similar to that of Ca2+ at neutral pH value.View full abstractDownload PDF (642K) -
Takashi TERAMOTO, Hiromasa TOJO, Toshio YAMANO, Mitsuhiro OKAMOTO1983 Volume 93 Issue 5 Pages 1353-1360
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIntracellular phospholipase A2 was purified to homogeneity from rat spleen. The enzyme was efficiently concentrated by precipitation with trichloroacetic acid and purified by sequential use of column chromatographies on DEAE-Sepharose, octyl-Sepharose and Bio-Gel P-30. The positional specificity of the enzyme for an acylester bond at the sn-2-position of phospholipids was established. The purified enzyme has a pH optimum ranging from 8.0 to 10.5 and the molecular weight of the enzyme was estimated to be 14, 700-14, 800 by sodium dodecyl sulfate polyacrylamide gel electrophoresis and by gel filtration with a TSK-GEL G 2000 SW column. The purified enzyme requires Ca2+ for activity. The activity was not enhanced by the presence of purified calmodulin.View full abstractDownload PDF (1138K) -
Nobuhiro HARADA, Tsuneo OMURA1983 Volume 93 Issue 5 Pages 1361-1373
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe syntheses of two different molecular species of cytochrome P-450, P-450 (PB), and P-450 (MC), were examined using normal and drug-treated rats, and the rate of synthesis was correlated with the drug-induced increase of the amounts in the liver microsomes of treated animals. Phenobarbital (PB) and 3-methylcholanthrene (MC) were used as inducers. The syntheses of cytochrome b5 and NADPH-cytochrome P-450 reductase (fpT) were also examined. In vivo incorporation of L-[3H]leucine indicated a large increase of P-450 (PB) synthesis in the livers of PB-treated animals, which reached a plateau value about 18-fold higher than the control level at 12h. The synthesis of fpT was also stimulated by PB showing a peak value of 3 times the control at 12h after PB administration, but it returned to the control level afterwards. On the other hand, the syntheses of P-450 (MC) and cytochrome b5 did not change at all. Similarly, MC administration selectively induced the synthesis of P-450 (MC), which was about 24 times the control at 6h, whereas those of P-450 (PB), cytochrome b5, and fpT were not affected by MC. Analysis of nascent peptides and in vitro translation of polysomes and total liver RNA prepared from control and drug-treated animals were also carried out, and the results of these in vitro experiments confirmed the observations in in vivo incorporation studies. It was found that both free and membrane-bound ribosomes participate in the syntheses of P-450 (PB) and P-450 (MC) in the case of control animals. PB and MC induced the syntheses of P-450 (PB) and P-450 (MC) by bound ribosomes but not by free ribosomes, and the contribution of bound ribosomes to the syntheses of these two species of cytochrome P-450 was predominant in the case of drugtreated animals. The molecular sizes of in vitro synthesized P-450 (PB) and P-450 (MC) were the same as those of authentic samples prepared from liver microsomes.View full abstractDownload PDF (1960K) -
Hiroyuki SADANO, Tsuneo OMURA1983 Volume 93 Issue 5 Pages 1375-1383
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe turnover of two forms of cytochrome P-450, phenobarbital (PB) inducible form (P-450 (PB)) and 3-methylcholanthrene inducible form (P-450 (MC)), as well as NADPH-cytochrome P-450 reductase (fpT) and cytochrome b5, in rat liver was studied. 14C-Labeled DL-leucine, sodium bicarbonate, or δ-aminolevulinic acid was given to normal and inducer-treated rats, and the subsequent decay of the radioactivities was measured to calculate the half-lives of the microsomal proteins. The half-lives of the proteins of P-450 (PB), P-450 (MC), fpT, and cytochrome b5 in the liver of normal rats were 30, 15, 50, and 80h, respectively, with [14C]leucine, and 25, 15, 35, and 50h, respectively, with sodium [14C]biearbonate. These data indicate that the two forms of cytochrome P-450 turn over at different rates, and their half-lives are shorter than those of the other components of the microsomal monooxygenase system. The use of radioactive bicarbonate showed that the observed turnover rates of P-450 (PB) and P-450 (MC) were little affected whereas those of fpT and cytochrome b5 which turn over relatively slowly, were more significantly affected by the re-utilization of the amino acid when radioactive leucine was used. The half-lives of the proteins of P-450 (PB), P-450 (MC), fpT, and cytochrome b5 in the liver of PB-treated rats were 20, 20, 25, and 30 h, respectively, with radioactive bicarbonate. Apparently, PB has no stabilizing effect on the turnover of the microsomal proteins examined. The turnover of fpT and cytochrome b, was rather stimulated by PB treatment and the half-lives became closer to those of P-450 (PB) and P-450 (MC), indicating a possible simultaneous degradation of microsomal membrane constituents in the liver of PB-treated rats. The half-lives of the heme of P-450 (PB), P-450 (MC), and cytochrome b5 in the liver of normal rats were 15, 15, and 40h, respectively, when radioactive δ-aminolevulinic acid was used. The heme of P-450 (PB) was apparently dissociable and turned over faster than its protein moiety, whereas the heme and protein portions of P-450 (MC) and cytochrome b5 turned over at almost identical rates.View full abstractDownload PDF (703K) -
Hiroshi MATSUBARA, Katsura INOUE, Toshiharu HASE, Hisahide HIURA, Tomi ...1983 Volume 93 Issue 5 Pages 1385-1390
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe amino acid sequence of an [8Fe-8S] ferredoxin isolated from the culture medium of Rhodospirillum rubrum, a photosynthetic purple non-sulfur bacterium, was determined by a combination of various conventional procedures. The sequence was A-Y-K-I-E-E-T-C-I-S-C-G-A-C-A-A-E-C-P-V-N-A-I-E-Q-G-D-T-I-F- V-V-N-A-D-T-C-I-D-C-G-N-C-A-N-V-C-P-V-G-A-P-V-A-E (55 amino acid residues). It lacked methionine, leucine, histidine, arginine, and tryptophan. The molecular weight was calculated to be 5, 568 excluding iron and sulfur atoms. The distribution of 8 cysteine residues was exactly the same as that of clostridia)-type ferredoxin, suggesting retention of the duplication of the bacterial ancestral ferredoxin gene. The extracellular ferredoxin of R. rubrum was compared with other ferredoxins observed in closely related photosynthetic bacteria and the evolutionary significance of this ferredoxin is discussed.View full abstractDownload PDF (445K) -
Ken-ichi AMANO, Edgar RIBI, John L. CANTRELL1983 Volume 93 Issue 5 Pages 1391-1399
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSEndotoxic glycolipids extracted from the polysaccharide heptose-less Re mutant of Salmonella typhimurium were hydrolyzed with alkaline and acid reagents. Treatment with hydroxylamine caused the liberation of all O-ester linked fatty acids and resulted in abrogation of the toxicity (lethality to chick embryos) and ability to regress tumors (line-10 tumors in strain 2 guinea pigs). Treatment with dilute sodium hydroxide caused partial removal of O-ester linked fatty acids without loss of these activities. Toxicity and tumor-regressive potency were retained after removal of 2-keto-3-deoxyoctonate (KDO) by exposing the glycolipids to sodium acetate solution at pH 4.5. The majority of the glycolipids of the endotoxic extracts were rendered non-toxic but retained antitumor activity when hydrolyzed with boiling 0.1 N hydrochloric acid, which split KDO and glycosidic phosphate from the glycolipid molecules. Non-toxic glycolipid fractions possessing antitumor activity were separated from the acid hydrolysate by means of preparative thin layer chromatography. It was concluded that glycosidic bound phosphate and at least a portion of the fatty acids of the lipid A moiety are essential for toxicity, but that this phosphate is not an essential structural feature for tumor-regression activity.View full abstractDownload PDF (1198K) -
Kei-ichi KAMISANGO, Hideji FUJII, Hiroyuki OKUMURA, Ikuo SAIKI, Yoshio ...1983 Volume 93 Issue 5 Pages 1401-1409
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn immunologically active teichoic acid component was isolated from the cell wall of Listeria monocytogenes strain EGD. The teichoic acid component, accounting for about 20% of the weight of cell wall, contained N-acetylglucosamine, rhamnose, ribitol, and phosphorus in a molar ratio of 0.95:1.0:0.97:0.98. The molecular weight of the teichoic acid chain was about 120, 000 as analyzed by gel filtration. The probable structure was deduced from the results of methylation analysis, Smith degradation, and proton magnetic resonance spectrometry of the teichoic acid, together with the characterization of fragments obtained by treatment with hydro-fluoric acid, as follows:
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Inhibition testing with monosaccharide and fragments obtained from HF treatment of Listeria teichoic acid in the quantitative precipitin reaction suggested that the rhamnose residue is a major antigenic determinant.View full abstractDownload PDF (715K) -
Hidenari TAKAHARA, Hyogo SINOHARA1983 Volume 93 Issue 5 Pages 1411-1419
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSContrapsin and α-1-antitrypsin have been recently characterized as major protease inhibitors in mouse plasma (Takahara, H. & Sinohara, H. (1982) J. Biol. Chess. 257, 2438-2446). We have studied the effects of the two inhibitors upon various serine proteases prepared from mouse tissues. Trypsin, plasmin and trypsin-like proteases of the submaxillary gland were inhibited by contrapsin but not by α-1-antitrypsin. On the other hand, chymotrypsin, elastase, and thrombin were inactivated by α-1-antitrypsin but not by contrapsin. Thus, their inhibitory spectra did not overlap each other in spite of their broad specificities. The inhibition of trypsin, chymotrypsin, and elastase was rapid and stoichiometric, whereas the inhibition of the other proteases was relatively slow. Contrapsin accounted for almost the total capacities of mouse plasma to inhibit both trypsin and submaxillary gland trypsin-like proteases, whereas α-1-antitrypsin was responsible for nearly all the capacities of plasma to inhibit both chymotrypsin and elastase.View full abstractDownload PDF (718K) -
Akio OHSHIMA, Hiroshi NARITA, Makoto KITO1983 Volume 93 Issue 5 Pages 1421-1425
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAlkaline phosphatase was entrapped in reverse micelles formed in n-heptane with surfactants (bis(2-ethylhexyl)sodium sulfosuccinate, phosphatidylcholine, phos-phatidylethanolamine, or phosphatidic acid) and water. The entrapped enzyme could express its activity in the reverse micelles of the above surfactants only when the substrate, p-nitrophenylphosphate, was within the reverse micelles of bis (2-ethylhexyl) sodium sulfosuccinate. The optimum pH of activity in the reverse micelles was higher by about one pH unit than that determined in bulk water. Regardless of water content (mol water/mol surfactant=10 or 14.8), the Km value was about 30mM, while Vmax at the higher water content (14.8) was 2-4 times greater than that at 10. Activation energy of the reaction depended on the kind of reverse micelles. Differences in carbon chain length of solvents showed no effect on the kinetic properties of the enzyme.View full abstractDownload PDF (403K) -
III. Evidence that the Amino Terminus of the Heavy Subunit Is the Membrane Binding SegmentYoshiko MATSUDA, Akihiko TSUJI, Nobuhiko KATUNUMA1983 Volume 93 Issue 5 Pages 1427-1433
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSγ-Glutamyltranspeptidase solubilized with Triton X-100 from the membrane fraction of rat kidney was previously shown to contain a hydrophobic domain that anchors the enzyme to the membrane, and which may be localized in the aminoterminal portion of the heavy subunit. The light subunit does not associate with the membrane [Tsuji et al. (1980) J. Biochem. 87, 1567-1571]. In the current work, the membrane binding portion of γ-glutamyltranspeptidase was obtained by treating Triton-solubilized enzyme with papain. This treatment resulted in solubilization of about 8.8% of the protein mass of the Triton-solubilized γ-glutamyltranspeptidase from the heavy subunit. The product split from the heavy subunit had a molecular weight of 6, 000 judging from its amino acid composition. The residual enzyme showed no loss of catalytic activity but became hydrophilic. The fragment removed from γ-glutamyltranspeptidase by papain treatment was not particularly hydro-phobic and contained carbohydrate. The partial amino-terminal sequences and carboxyl-terminal amino acids of the heavy and light subunits of γ-glutamyltrans-peptidase solubilized with Triton X-100 and papain were determined. The light subunits of γ-glutamyltranspeptidase solubilized with Triton X-100 and papain contained the same sequence, Thr-Ala-(X)-Leu, as an amino-terminal portion, but that of the heavy subunit of the form solubilized with Triton X-100 contained the sequence Met-Lys-Asn-Arg-Phe-Leu-Val-Leu-Gly-Leu-Val-Ala-Val-Val-Leu-Val-Phe-Val-Ile-Ile-Gly- and the sequence of the hydrophobic domain resembled that of the signal peptide which contributes to directing newly synthesized secretory protein to associate with the membrane of the endoplasmic reticulum. The papain-solubilized form had a completely different amino-terminal sequence, Gly-Pro-Pro-Leu-. The carboxyl-terminal amino acids of the subunits of the two forms were determined by carboxypeptidase digestion; that of both light subunits was found to be phenylalanine, and that of both heavy subunits to be tyrosine. These results indicate that the amino-terminal region of the heavy subunit of γ-glutamyltranspeptidase is removed by proteolysis with papain and that it is composed of a hydrophobic domain and a hydrophilic domain. The hydrophobic domain contains the aminoterminus of the heavy subunit, includes about 20 hydrophobic amino acids and contributes to anchorage of the enzyme to the membrane. The hydrophilic domain following the hydrophobic domain probably contains carbohydrate.View full abstractDownload PDF (607K) -
Purification and Characterization of a Calcium-Activated Neutral Protease from Monkey Cardiac MuscleKaoru HARA, Yoshikazu ICHIHARA, Kenji TAKAHASHI1983 Volume 93 Issue 5 Pages 1435-1445
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA calcium-activated neutral protease (CANP) was purified from monkey cardiac muscle by a method involving column chromatography on DEAE-cellulose, Sepharose CL-6B, DEAE-Sephacel, organomercurial-Sepharose 4B, and Sephadex G-150 in succession. This protease required both millimolar concentration of Ca2+- and the SH-group for activation, and it was maximally active around pH 8.0. It was strongly inhibited by thiol protease inhibitors such as iodoacetic acid, antipain, leupeptin, and epoxysuccinic acid derivatives. The molecular weight of this protease was estimated to be 110, 000 by gel filtration. Upon nondenaturing electrophoresis the purified protease gave two bands, both of which were active at millimolar concentration of Ca2+, indicating the existence of two forms of the protease. The less acidic band (form I CANP) contained two components with molecular weights of 74, 000 and 28, 000 and the more acidic one (form II CANP) contained components with molecular weights of 74, 000 and 26, 000. The protease was synergistically activated by Mn2+ and Ca2+ at a concentration where Mn2+ or Ca2+ alone was not effective. In the presence of millimolar level of Ca2+, limited autolysis reduced the Ca2+-requirement of this protease. The proteolysis of myo-fibrils by this protease resulted in the production of a component with a molecular weight of 30, 000 as well as various other higher and lower molecular weight peptide fragments.View full abstractDownload PDF (2527K) -
II. Microenvironments of Tryptophan ResiduesHitomi FUKUZAWA, Kenji AKI1983 Volume 93 Issue 5 Pages 1447-1453
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe tryptophan residues of two forms of pig heart lipoamide dehydrogenase (LD (I) and LD (II)) were investigated fluorometrically.
The tryptophan contents of LD (1) and LD (II) determined by the fluorescence method were 3 mol and 2 mol per mol of FAD, respectively. These values were in good agreement with those found by the MCD method.
The microenvironments of the tryptophan residues were investigated by fluo-rescence quenching titration with acrylamide. The tryptophan residues of both enzymes were in heterogeneous microenvironments, and CD spectra showed some differences between these microenvironments in the two enzymes. Energy transfer from tryptophan residues to bound FAD was equally efficient in the two enzymes.
It seems probable that the three tryptophan residues in LD (I) are all in different microenvironments, but that two of them are in microenvironments almost identical to those of the corresponding residues in LD (II).View full abstractDownload PDF (504K) -
Tairo OSHIMA, Seiko KAWAHATA1983 Volume 93 Issue 5 Pages 1455-1456
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA new pentaamine was extracted from an extreme thermophile, Thermus therinophilus strain HB8, and its chemical structure was determined to be 1, 16-diamino-4, 8, 12-triazahexadecane (see structure 1). A trivial name, homocaldopentamine, was proposed for the new naturally occurring polyamine.View full abstractDownload PDF (116K) -
Production and Characterization of a Monoclonal Antibody to Human Nervous System-Specific γγ EnolaseTomohiko HAYANO, Kanefusa KATO, Atsuko SHIMIZU, Yutaka ARIYOSHI, Yukio ...1983 Volume 93 Issue 5 Pages 1457-1460
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA mouse hybrid cell line producing an antibody to human nervous system-specific γγ enolase has been isolated by fusions between γγ-immunized mouse spleen cells and mouse myeloma cells (P3-NS-1/1-Ag4-1), followed by a screening procedure with an enzyme immunoassay. This particular cell line (El-G3) has secreted the antibody bearing γ2a/κ immunoglobulin chains. Specificity of the E1-G3 antibody was tested by immunoprecipitation of enolase activities with anti-mouse IgG, and by use of enzyme immunoassay systems for enolase isozymes which consisted of polyclonal rabbit antibodies. The E1-G3 antibody was found to be specific for the γ subunit of enolase, showing reactivities with human γγ and αγ enolases, and also with rat γγ enolase. However, the monoclonal antibody did not cross-react with the α or β subunit of human enolase.View full abstractDownload PDF (311K) -
1983 Volume 93 Issue 5 Pages 1461a
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDownload PDF (22K) -
1983 Volume 93 Issue 5 Pages 1461b
Published: 1983
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDownload PDF (22K)
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