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Volume 87 (1980)
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- Issue 5 Pages 1261-
- Issue 4 Pages 1003-
- Issue 3 Pages 679-
- Issue 2 Pages 369-
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Volume 87, Issue 2
Displaying 1-38 of 38 articles from this issue
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II. Ca2+ Binding of Aorta LeiotoniniMasato HIRATA, Takashi MIKAWA, Yoshiaki NONOMURA, Setsuro EBASHI1980 Volume 87 Issue 2 Pages 369-378
Published: February 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe ability of leiotonin and actin of bovine aorta smooth muscle to form a tightly bound complex (Hirata et al. (1977) J. Biochem. 82, 1973-1976) was utilized to measure Ca2+ and Sr2+ binding to aortic leiotonin, and the physiological significance of this binding was investigated:
1) The leiotonin-actin complex showed strong affinity for Ca2+ whereas the actin filament without leiotonin showed weak affinity, binding Ca2+ only at relatively high Ca 2+ concentrations. When leiotonin C was removed from the leiotonin-actin complex, the Ca binding capacity of the complex fell to the level of the actin filament, and the original level was restored by the addition of chicken gizzard leiotonin C. Thus, leiotonin C was shown to be responsible for the Ca binding of the aortic thin filament at low Ca2+ concentrations.
2) The ability of the actin complex to bind Ca2+ mentioned above paralleled its ability to induce superprecipitation together with myosin.
3) The Ca2+ sensitivity of myosin light chain phosphorylation coincided with that of superprecipitation of myosin B, but the Sr2+ sensitivity of the phosphorylation was dissociated from that of superprecipitation, the phosphorylation being several times more sensitive to Sr2+ than superprecipitation. This supports the view that phosphorylation is not involved in the superprecipitation of aortic myosin B.
4) Modulator protein (calmodulin) could replace the function of leiotonin C in producing superprecipitation. However, the Sr2+ sensitivity of the superprecipitation of a reconstituted system containing modulator protein was in good accord with that of phosphorylation of this system, but was several times greater than that of the superprecipitation of a reconstituted system containing leiotonin C. It was, therefore, concluded that the Ca2+binding protein operating under physiological conditions is not modulator protein, but leiotonin.View full abstractDownload PDF (1366K) -
Nature of the Prosthetic Group of MyeloperoxidaseTakeshi ODAJIMA1980 Volume 87 Issue 2 Pages 379-391
Published: February 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe absorption spectra of alkaline pyridine hemochrome of myeloperoxidase in its native, acid, and modified forms were similar to those of heme a, and the molar extinction coefficient of myeloperoxidase heme was very similar to that of heme a, assuming that myeloperoxidase contains only one heme. The anaerobic titration of myeloperoxidase with dithionite showed that one electron was consumed per molecule of the enzyme for its conversion to its reduced form. The EPR spectrum of myeloperoxidase indicated that the enzyme contains both highspin heme and non-heme iron. Carbonyl reagents, such as borohydride, hydrazine, and benzhydrazide, reacted with myeloperoxidase, causing blue shifts in its absorption spectrum. The heme was labeled with a tritium of boro[3H]hydride, suggesting that the reagents reacted with a formyl group on the porphyrin ring of the myeloperoxidase heme.
When hydrazine was added to cyanide complex I of myeloperoxidase the complex was converted to the hydrazine-enzyme compound. Myeloperoxidase reacted with bisulfite to form a compound with an absorption spectrum similar to that of cyanide complex I. Boro-hydride-treated myeloperoxidase formed only one cyanide complex, while the native enzyme formed two different cyanide complexes, I (Kd =0.3, μM) and II (approximate Kd =0.1mM) The EPR spectrum indicated that cyanide complex I of myeloperoxidase still contained highspin heme. The results suggested that cyanide complex I and the bisulfite compound of myeloperoxidase were adducts between the nucleophilic reagents and the formyl group of myeloperoxidase heme.
Based on these results, we concluded that one of the two iron atoms in a myeloperoxidase molecule exists in a formyl-heme moiety similar to heme α and the other exists as a non-heme iron.View full abstractDownload PDF (786K) -
Hiroyuki WATABE, Joan I. GITLIN, David GITLIN1980 Volume 87 Issue 2 Pages 393-397
Published: February 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA Cell membrane fraction from term human placenta was prepared by homogenization and ultracentrifugation. The fraction was found to bind both human IgG and human serum albumin. Maximal specific binding occurred at pH 5.2, and the amount of binding was dependent upon incubation time, temperature, buffer, and ionic strength. The binding of human serum albumin was inhibited by preincubation with H-IgG but the reverse did. not happen.View full abstractDownload PDF (315K) -
Sachio GOTO1980 Volume 87 Issue 2 Pages 399-406
Published: February 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe trypsin-catalyzed hydrolysis of a synthetic ester substrate, dansyl-n-arginine methyl ester (D-DAME), was followed by the entire progression curve method. This ester substrate, which is a D-enantiomer of arginine, is hydrolyzed to the extent of nearly 100 % without causing substrate activation. The experiments were performed by changing either the pH or the initial substrate concentration. The double-reciprocal plot of the velocity of hydrolysis against the residual substrate concentration showed good linearity. As regards the dependency of this straight line on pH at a constant initial substrate concentration, both the slope and the intercept on the 1/v axis decreased with decrease of hydrogen ion concentration in the pH range below 8 and the lines intersected at a point in the second quadrant. At constant pH the slope of the double-reciprocal plot between the velocity of hydrolysis and the residual substrate concentration increased linearly with increase of the initial substrate concentration. The results obtained cannot be interpreted simply in terms of the three-step mechanism for the trypsinand chymotrypsin-catalyzed hydrolyses of ester substrates. In particular, it is significant that the catalytic rate constant obtained for the overall enzymatic reaction is very similar to the rate constant of conformational change of the enzyme molecule due to the formation of enzyme-substrate complex in the trypsin-catalyzed hydrolyses of L-and D-DAME.View full abstractDownload PDF (509K) -
Kimiko OHTANI, Satoaki SHIBATA, Akira MISAKI1980 Volume 87 Issue 2 Pages 407-416
Published: February 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA lectin purified from the Tora-bean (Phaseolus vulgaris) by affinity chromatography with Con-A Sepharose was shown to be a glycoprotein containing 7.8 % neutral sugars (D-mannose, N-acetyl-D-glucosamine, L-fucose, and D-xylose, in a molar ratio of 9.6 : 2.0 : 0.6 : 0.7). Its molecular weight was 130, 000, as estimated by exclusion gel chromatography, and SDS gel electrophoresis showed that it consists of four subunits of molecular weight 32, 000.
The lectin reacts with various glycoproteins, i.e., blood group substances, human parotid salivary glycoprotein, fetuin, and bovine submaxillary mucin. Divalent cations, such as Ca2+ Mn2+, and Mg2+, appear to stimulate its reactivity. Inhibition tests using the glycopeptide fragment from fetuin and some oligosaccharides, as well as the binding test with 14C-N-acetyl-lactosamine suggest that the sequence of D-galactose, N-acetyl-D-glucosamine, and n-mannose residues in the carbohydrate chain of fetuin is essential for binding.View full abstractDownload PDF (1285K) -
An Application of “Inverse Substrates” to Mechanistic Studies of the EnzymeKazutaka TANIZAWA, Yoichi KASABA, Yuichi KANAOKA1980 Volume 87 Issue 2 Pages 417-427
Published: February 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTrypsin [EC 3. 4. 21. 4]-catalyzed hydrolysis of “inverse substrates” was investigated kinetically. “Inverse substrates” for trypsin are specific substrates in which the arrangement of the sitespecific group is reversed compared to that of the normal substrate, e. g., a cationic center is included in the leaving group instead of being in the acyl moiety (Tanizawa, K., Kasaba, Y., & Kanaoka, Y. (1977) J. Am. Chem. Soc. 99, 4485-4488). Acyl enzyme intermediates formed specifically from these substrates are advantageous for the mechanistic analysis of trypsin action, since the cationic group liberated from the acyl moiety can no longer exhibit specific interaction with the enzyme binding site in the subsequent deacylation stage.
Remarkable rate acceleration at the deacylation step was observed on adding amidinium or ammonium compounds. The effects of the size of the acyl moiety and the charged molecule on the acceleration were examined. Latent properties of 1-butylamine as an activator were found in the present study. Based on these observations, it is suggested that a cationic molecule which can be well accommodated together with the acyl group within the active center cleft causes rate enhancement, with associated conformational changes.View full abstractDownload PDF (746K) -
Atsushi TAKEDA, Tsuneo MIYAHARA, Akira HACHIMORI, Tatsuya SAMEJIMA1980 Volume 87 Issue 2 Pages 429-439
Published: February 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effects of thiol compounds on the conformation of porcine erythrocyte catalase were examined. The thiol compounds showed two types of reactivity with the catalase in terms of changes in absorption spectra. One is characterized by the appearance of a new absorption maximum at 595 nm; this was seen with 2-mercaptoethanol (designated as inactive catalase type I). The other is characterized by new maxima at 535 and 570 nm, and this was seen with reduced glutathione, dithiothreitol, cysteine, and cysteamine (inactive catalase type II). The thiol compounds caused gradual inactivation of catalase, correlating with the enhancement of the absorption maximum at 595 nm or 570 nm. Removal of excess thiol reagents from the reaction mixtures caused partial recovery of activity, which was more marked with inactive catalase type II. Similar reversibility was observed in the absorption, CD and MCD spectra, whereas reversibility was not observed for inactive catalase type I. The MCD spectra suggested conversion of heme groups from a high to a low spin state on incubation with thiols, e.g., reduced glutathione, leading to inactive catalase type II. α-Helical conformation of the polypeptide backbone and titratable free SH groups in the catalase molecule were unaffected by all these thiol treatments. It is suggested that “active oxygen” which may be produced on incubation of catalase with thiol compounds, was responsible for the formation of inactive catalase type II.View full abstractDownload PDF (774K) -
I. Estimation of the Activities in the Cells Grown on Various CompoundsMinoru MORIKAWA, Katsura IZUI, Masaaki TAGUCHI, Hirohiko KATSUKI1980 Volume 87 Issue 2 Pages 441-449
Published: February 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIntracellular concentrations of phosphoenolpyruvate (PEP) and five kinds of allosteric effectors (acetyl-CoA, fructose 1, 6-bisphosphate, GTP, L-aspartate, and L-malate) of PEP carboxylase were measured in E. coli cells grown on various compounds as a carbon source. Based on the data obtained, reaction systems which contained a definite concentration of the enzyme and the ligands at the concentrations found in vivo were constructed and the enzyme activities were measured. The ratio of each activity thus obtained to the maximal activity attainable with the same concentration of enzyme and saturating concentrations of the activators was estimated. For the cells grown on glucose, glycerol, or lactate, the extent of exhibition of the enzyme activity was 2-15% of the maximal activity. For the cells grown on acetate or oleate, the extent was 1-3 %. For the cells grown on succinate, L-aspartate, L-malate, or glucose plus L-aspartate, the extent was less than 0.4%. Consideration of the data obtained in the present studies, together with those obtained in our previous studies on the enzyme level (Teraoka, H. et al. (1970) J. Biochem. 67, 567-575), showed that the control of the enzyme reaction in vivo is considerably different from that expected from the in vitro experiments, and that deficiencies of “coarse control” are covered by a “fine control.”View full abstractDownload PDF (649K) -
Takashi MANABE, Nobuhiro TAKAHASHI, Kiyotsugu KOJIMA, Tomotaka SHINODA ...1980 Volume 87 Issue 2 Pages 451-464
Published: February 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHuman sera from myeloma patients were subjected to two-dimensional electrophoresis. Heterogeneity in molecular weight and in isoelectric point of the myeloma proteins were demonstrated on the protein map. The patterns of five major immunoglobulin classes differed from each other, implying that the five classes of myeloma immunoglobulin can be distinguished by examining the two-dimensional electrophoretic patterns. Sugar content analysis suggested that differences in sialic acid content may be one of the origins of the heterogeneity of myeloma proteins observed in isoelectric focusing.View full abstractDownload PDF (3637K) -
Effects of Calcium Ions on the Activation of Gluconeogenesis by Norepinephrine in Perfused Rat LiverTsukasa SUGANO, Masakazu SHIOTA, Hiroaki KHONO, Masakazu SHIMADA, Nozo ...1980 Volume 87 Issue 2 Pages 465-472
Published: February 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effects of calcium ions on the activation of gluconeogenesis by norepinephrine were studied in the perfused rat liver. The norepinephrine-mediated enhancement of gluconeogenesis was accompanied by a transient increase in the reduction level of pyridine nucleotides. There was a positive correlation between the increases in the rate of glucose production and the reduction level of pyridine nucleotides induced by norepinephrine. These changes were “dose-dependent” with respect to the norepinephrine concentration from 5 nM to 1 μM and were mimicked by phenylephrine. The omission of calcium from the perfusate did not modify the basal rate of gluconeogenesis from lactate and pyruvate or the increased rate of glucose production induced by glucagon and isoproterenol. The extents of the responses to nor-epinephrine and phenylephrine, however, were decreased markedly in liver perfused with a calcium-free medium and/or with phentolamine. Infusion of calcium into the calcium-deficient liver caused an abrupt elevation of glucose production together with a transient reduction of pyridine nucleotides, and the original extent of the response to norepinephrine was recovered. The data presented provide evidence indicating that stimulation of gluco-neogenesis by norepinephrine is mediated through an α-adrenergic and calcium-dependent mechanism in which redox changes of mitochondrial pyridine nucleotides are involved.View full abstractDownload PDF (526K) -
Gilbu SOE, Nozomu NISHI, Tomisaburo KAKUNO, Jinpei YAMASHITA, Takekazu ...1980 Volume 87 Issue 2 Pages 473-481
Published: February 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIt is known in the case of the photosynthetic bacterium Rhodospirillum rubrum that the ATPase solubilized from acetone powder of chromatophores shows activity in the presence of Ca2+, but not in the presence of Mg2+ (Ca2+-ATPase), whereas chromatophores show ATPase activity in the presence of either cation. The present study deals with the purification and identification of the factor capable of converting Ca2+-ATPase into Mg2+-ATPase (conversion factor) present in chromatophores.
1. The conversion factor was effectively solubilized from chromatophores in 50% acetone, and purified by chromatography on columns of silica gel and Sephadex LH-20.
2. The purified conversion factor was found to contain unsaturated fatty acids (C16:1 and C18:1) by infrared spectroscopy, proton magnetic resonance spectroscopy, thin layer chro-matography, and gas liquid chromatography. The molar ratio of the C16:1 to the C18:1 acids was approximately 3 : 2.
3. Activity for the conversion of Ca2+-ATPase into Mg2+-ATPase (conversion activity) was investigated among authentic fatty acids (C14 to C20). All unsaturated fatty acids other than elaidic acid (C18:1 Δ9, trans) showed conversion activity. The activity with palmitoleic acid (C16:1 Δ9) was highest, being about 6 times as high as the activity with vaccenic acid (C18:1 Δ11) or oleic acid (C18:1 Δ9).
4. Under optimum conditions, the conversion activity with purified conversion factor was the same as that with palmitoleic acid, indicating that the naturally occurring conversion factor is palmitoleic acid.View full abstractDownload PDF (505K) -
Kaoru OMICHI, Nobuko NAGURA, Tokuji IKENAKA1980 Volume 87 Issue 2 Pages 483-489
Published: February 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe reactive site of Streptomyces subtilisin inhibitor (SSI) was investigated by chymotryptic cleavage of the reactive site peptide bond and its resynthesis by inhibitor-subtilisin complex formation. The results show that the reactive site peptide bond of the inhibitor is Met(73)-Val(74) and that subtilisin specifically cleaved the Leu(6)-Tyr(7) bond of SSI upon complex formation.View full abstractDownload PDF (990K) -
Masasuke YOSHIDA, Koki OHNO, Yasuaki TAKEUCHI1980 Volume 87 Issue 2 Pages 491-495
Published: February 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBacteriorhodopsin from Halobacterium halobium was stable and active in high concentrations of guanidine hydrochloride. However the decay of the intermediate with absorption maximum at 407 nm and the uptake of protons from the medium were remarkably delayed. At alkaline pH values, all the molecules of bacteriorhodopsin existed as the 407 nm intermediate in the light, and one proton per bacteriorhodopsin molecule was released.View full abstractDownload PDF (314K) -
Yasuhito NAKAGAWA, Shoshichi NOJIMA, Keizo INOUE1980 Volume 87 Issue 2 Pages 497-502
Published: February 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe transfer of various steroids and α-tocopherol between liposomes was examined. The transfer rate of cholest-4-en-3-one and epicholesterol between egg yolk phosphatidylcholine liposomes was much higher than that of cholesterol. Rapid transfer was also noted for cop-rostanol and 5-cholesten-3β-ol-7-one, while the transfer of cholestanol, which has a 3β-hydroxyl residue and a planar nucleus structure, was similar to that of cholesterol. These results suggest that the mode of interaction between steroid and phospholipid may be an im-portant factor controlling the rate of transfer.
It was found that α-tocopherol could also be transferred between liposomes. The transfer rate of α-tocopherol was dependent on the temperature and the phospholipid composition.View full abstractDownload PDF (473K) -
Shigeko ARAKI, Yuichi KOMAI, Mei SATAKE1980 Volume 87 Issue 2 Pages 503-510
Published: February 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA novel sphingophosphonolipid was isolated from the skin of a marine gastropod, Aplysia kurodai. It was composed of one mol each of sphingosine base, fatty acid, glucose, galacto-samine, and an unknown sugar and two mol each of galactose and aminophosphonic acid. The unknown sugar was identified as 3-O-methylgalactose by GC-mass spectrometry of the alditol acetate derivative and by GLC of the trimethylsilyl methylglycoside derivative. The aminophosphonic acid was identified as 2-aminoethylphosphonic acid by TLC of the free acid and the dinitrophenyl (DNP-) compound and by GLC of the O-di-trimethylsilyl-N-isothio-cyanate compound. Thus, a ceramide bis(2-aminoethylphosphono)-pentaoside structure having an oligosaccharide chain consisting of one mol each of glucose, 3-O-methylgalactose and galactosamine and two mol of galactose was proposed.View full abstractDownload PDF (1410K) -
Kenji YAMAMOTO, Osamu KAMATA, Nobuo KATSUDA, Keitaro KATO1980 Volume 87 Issue 2 Pages 511-516
Published: February 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe immunological properties of acid proteinases from rat spleen, two types of cathepsin D and a cathepsin E-like enzyme, were examined. The rabbit antiserum was prepared against the major form of cathepsin D (cathepsin D-I) from rat spleen. The antiserum quantitatively precipitated the enzyme activity from the purified cathepsin D-1 preparation. On immuno-diffusion analysis, the antiserum showed an identical reaction with the minor form of cathepsin D (cathepsin D-II) from rat spleen. Immunoelectrophoresis showed that the precipitin line with cathepsin D-II ran somewhat faster to the anode than that with cathepsin D-I. The cathepsin E-like acid proteinase was neither precipitated nor inhibited by the antiserum to cathepsin D-I, indicating that the cathepsin E-like enzyme is different from cathepsin D. Im-munological gel diffusion with the antiserum indicated that rat spleen cathepsin D was im-munologically identical with cathepsin D obtained from rat brain, thymus, lungs, heart, liver, kidneys, and adrenals.View full abstractDownload PDF (1582K) -
Kikuo OGATA, Kazuo TERAO, Toshio UCHIUMI1980 Volume 87 Issue 2 Pages 517-524
Published: February 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe GTP- and ATP-hydrolyzing activities of the rat liver 5S RNA-L5 (according to the proposed common nomenclature (1)) protein complex designated as 5S RNP were stimulated by pig liver elongation factor 2 (EF-2) plus aminoacyl-tRNA, both of which were required for the stimulation. The stimulative effect of aminoacyl-tRNA on GTP hydrolysis by the complete system containing 5S RNP and EF-2 was dependent on the concentration of aminoacyl-tRNA. Aminoacyl-tRNA also stimulated ATP hydrolysis by the complete system but did not stimulate the ATP hydrolysis by 5S RNP alone or EF-2 alone. While deacylated tRNA stimulated the ATPase activity of the complete system, it had no effect on the GTPase activity. Preincubated tRNA lacking the 3'-terminal CCA moiety had little effect on the GTPase and ATPase activities of the complete system. Fusidic acid inhibited the GTPase and the ATPase activities of the complete system with and without aminoacyl-tRNA, although the extent of the inhibition was larger in the presence of aminoacyl-tRNA.
These results suggest that 5S RNP may be a component of the GTPase center of rat liver 60S subunits.View full abstractDownload PDF (530K) -
Hiroshi NAKAMURA, Anthony N. MARTONOSI1980 Volume 87 Issue 2 Pages 525-534
Published: February 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe influence of phospholipid environment upon the mobility of spin labels covalently bound to the Ca2+-transport ATPase (ATP phosphohydrolase [EC 3. 6. 1. 3]) was studied by electron spin resonance spectroscopy in native and reconstituted sarcoplasmic reticulum membranes. Fragmented sarcoplasmic reticulum of rabbit skeletal muscle was covalently labeled with maleimide spin-labels of different chain length or with 4-(2-iodoacetamido)-2, 2, 6, 6-tetramethyl-piperidinooxyl, and the phospholipids were exchanged for dipalmitoylphosphatidylcholine or dioleoylphosphatidylcholine.
With short-chain maleimide or iodoacetamide spin labels, the spectrum of the protein-bound label reflected the change in microenvironment caused by replacement of endogenous phospholipids with dipalmitoylphosphatidylcholine as a decrease in mobility. In contrast, after labeling with long-chain maleimide derivatives, there were no noticeable differences in the spectra before and after substitution with dipalmitoylphosphatidylcholine. Replacement of endogenous phospholipids with dioleoylphosphatidylcholine did not affect the spectra.
The data indicate that increased viscosity in the environment of Ca2+-transport ATPase produced by replacement of sarcoplasmic reticulum lipids with dipalmitoylphosphatidylcholine reduces the mobility of short-chain maleimide spin labels covalently attached to the Ca2+-transport ATPase polypeptide.View full abstractDownload PDF (690K) -
Isamu MATSUMOTO, Nobuko SENO, Anne M. GOLOVTCHENKOMATSUMOTO, Toshiaki ...1980 Volume 87 Issue 2 Pages 535-540
Published: February 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe have found a simple procedure to convert epoxy-activated agarose into amino derivatives using ammonia solution. The amino derivatives of agarose were succinylated with succinic anhydride and then activated with N-hydroxysuccinimide according to the method of Cuat-recasas and Parikh.
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Coupling of the ligand to the activated agarose (shown above) was performed under mild conditions. The adsorbent thus obtained has no charged group in the linkage region between the ligand and agarose, thus reducing the nonspecific adsorption, and the bonds formed (ether and amide bonds) are stable even in an alkaline medium.
Lens culinaris hemagglutinin-Sepharose 4B prepared by this method was successfully used for the affinity chromatography of solubilized human red blood cell membrane in deter-gent solution and was stable when elution was performed with borate buffer, pH 9.8View full abstractDownload PDF (1043K) -
I. Purification and Properties of Cytochrome b-561 from Enteromorpha proliferaYasutomo SUGIMURA, Fuminori YOSHIZAKI, Jun KATAGIRI, Chiharu HORIUCHI1980 Volume 87 Issue 2 Pages 541-547
Published: February 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCytochrome b-561 (Enteromorpha prolifera) was extracted from a green alga, E. prolifera, by immersion of the dried thalli in phosphate buffer solution. Purification was carried out by ammonium sulfate fractionation, DEAE-cellulose and DEAE-Sephadex column chromatographies, Bio-Gel gel filtration, and hydroxyapatite column chromatography. The reduced form of the cytochrome exhibited absorption maxima at 561 (α), 530.5 (β), and 428.5 nm (γ), and the oxidized form at 530.5, 417 (γ), and 275 nm. The α-band of the reduced form was sym-metric without any shoulder. The pyridine hemochrome showed absorption maxima at 556, 524, and 418 nm. The cytochrome does not combine with carbon monoxide or cyanide. The cytochrome showed little peroxidase activity. The cytochrome is oxidized by ferricyanide and reduced by cysteine, ascorbate, and hydrosulfite. Ferrocyanide and hydroquinone do not completely reduce it. Autoxidation of the cytochrome was found to be very slow. The midpoint potential (Em) of the cytochrome was determined by equilibration with the ferro- and ferri-EDTA system to be +0.23 volt at pH 7.0 and 25°C. The molecular weight of the cyto-chrome, estimated by Sephadex gel filtration, was 67×103.View full abstractDownload PDF (520K) -
Yasuhiko OZAKI, Keishiro WADA, Toshiharu HASE, Hiroshi MATSUBARA, Tosh ...1980 Volume 87 Issue 2 Pages 549-555
Published: February 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA purothionin homolog was isolated from barley flour and purified by CM-52 column chromatography. It showed potent lethal activity towards brewer's yeast and its complete amino acid sequence was determined to be as follows. Lys-Ser-Cys-Cys-Arg-Ser-Thr-Leu-Gly-Arg-Asn-Cys-Tyr-Asn-Leu-Cys-Arg-Val-Arg-Gl y-Ala-Gln-Lys-Leu-Cys-Ala-Gly-Val-Cys-Arg-Cys-Lys-Leu-Thr-Ser-Ser-Gly-Lys-Cys-Pro-Thr-Gly-Phe-Pro-Lys. It thus consists of 45 amino acid residues with 8 cysteines. The number of amino acid residues and the positions of the 8 cysteines are identical with those of wheat purothionins. There is a high degree of homology in the primary structures of these proteins.View full abstractDownload PDF (417K) -
Toshio GOTOH, Seiko KAMADA1980 Volume 87 Issue 2 Pages 557-562
Published: February 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSSodium dodecyl sulfate (SDS)-gel electrophoresis of erythrocruorin from the polychaete Tylorrhynchus heterochaetus revealed the presence of four subunits with molecular weights of 12, 000, 22, 000, 23, 500, and 54, 000 in a molar ratio of 6:1:2:3, respectively. The largest subunit dissociates into polypeptide chains of 13, 500 molecular weight in the presence of mercaptoethanol, whereas each of the other subunits consists of a single polypeptide chain. Hemochromogen determination gave a minimum molecular weight of 26, 500 per mol of heme group.
A model of the subunit structure of the erythrocruorin molecule is proposed, composed of 144 subunits (252 polypeptide chains) possessing 144 heme groups. The molecular weight of the erythrocruorin was calculated to be 3.636×106View full abstractDownload PDF (978K) -
Hiroshi NAKATANI, Keiko KITAGISHI, Keitaro HIROMI1980 Volume 87 Issue 2 Pages 563-571
Published: February 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBinding between four kinds of acid proteinases (pepsin, acid proteinase from Cladosporium, and acid proteinases A and B from Aspergillus niger) and aliphatic alcohols, which are competitive inhibitors of acid proteinases, was studied in terms of the absorbance changes of zinc(II)-pyridine-2-azo p-dimethylaniline (Zn-PAD) complex, a catalytic site probe for acid proteinases. Binding between three of the enzymes (pepsin, acid proteinase from Cladosporiuin, and acid proteinase B from Aspergillus niger) and n-amyl alcohol was studied in terms of the fluorescence changes of 2 p-toluidinylnaphthalene-6-sulfonate (TNS), a probe for hydrophobic sites of proteins. It was found that five alcohols (methanol, ethanol, n-propanol, n-butanol, and n-amyl alcohol) affected the absorption spectra of the Zn-PAD-enzyme complexes, and n-amyl alcohol affected the fluorescence spectra of the enzyme-TNS complexes. The results are consistent with the view that the binding of alcohols with the enzymes in a molar ratio of 1:1 induces the spectral change. These results suggest that the acid proteinases studied have a hydrophobic binding site for alcohols near the catalytic site. The logarithm of the apparent dissociation constant between the acid proteinases and alcohols(logK1) decreased linearly with the increase of the chain length of the alcohols. The standard Gibbs free energy change for the binding between pepsin and methylene groups of alcohol was estimated to be about 2.5 kJ/mol (0.6 kcal/mol) per methylene group (at 25°C, pH 5.0).View full abstractDownload PDF (592K) -
Keiko KITAGISHI, Hiroshi NAKATANI, Keitaro HIROMI1980 Volume 87 Issue 2 Pages 573-579
Published: February 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe fluorescence of pepsin-bound 2-p-toluidinylnaphthalene-6-sulfonate (TNS) decreases upon the binding of Streptomyces pepsin inhibitor (SPI) with the enzyme. Equilibrium dialysis experiments showed this decrease to arise from the release of TNS from pepsin due to the binding of SPI in a molar ratio of 1 : 1.
The results of stopped-flow kinetic study on the concentration dependence of the apparent rate constant for the binding reaction between pepsin and SPI using the fluorescence decrease of TNS as a probe were consistent with the interpretation that the binding reaction consists of at least two steps, including a fast bimolecular step followed by a slow unimolecular step. It was found that a fluorescence decrease occurred in both steps. The rate-pH profile suggested the participation of some ionizable group(s) (possibly a carboxyl group) in the binding.View full abstractDownload PDF (503K) -
II. Conformational Change around the S2 Thiol Group Related to the Essential Intermediate of ATP HydrolysisTsuneo KAMEYAMA1980 Volume 87 Issue 2 Pages 581-586
Published: February 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effect of F-actin on the conformation around a specific thiol group, S2, in heavy meromyosin was investigated. The extent of the change was estimated from the residual activity of Ca2+ ATPase after modifying the thiol with N-ethylmaleimide (NEM). Experiments were carried out with a modified heavy meromyosin (HMM*), in which S1 had been blocked with NEM, to observe the reactivity of S2 alone.
1. F-Actin markedly increased the ATP-induced conformational change around S2, but did not affect the adenylyl imidodiphosphate (AMPPNP)-induced change, and markedly supressed the adenylyl methylenediphosphate (AMPPCP)-induced change.
2. The initial burst of HMM ATPase was retained after the modification of S1, Replacement of Mg2+ with Mn2+ in the medium reduced the cooperative action of F-actin and ATP with concomitant loss of the initial burst.
3. Nevertheless, F-actin was capable of activating the steady-state ATPase activity of HMM* even in the presence of Mn2+
4. The degree of activation by F-actin of the ATP-induced increase in the reactivity of S2 did not parallel that of the steady-state ATP splitting, when the KCI concentration of the medium was varied.
The results indicate that the actin-induced local conformational change in the S2 region is related to an energized state of the myosin molecule caused by Mg2+-ATP, but is apparently not related to the actin-activated steady-state ATPase activity.View full abstractDownload PDF (396K) -
III. Reactivity of S2 Thiol and DTNB-Reactive Thiols of Porcine Cardiac MyosinTsuneo KAMEYAMA, Mariko KOMATSU, Takamitsu SEKINE1980 Volume 87 Issue 2 Pages 587-592
Published: February 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe reactivity of thiol groups in cardiac myosin obtained from porcine ventricles was compared with that in rabbit skeletal myosin.
1. The specific thiol group, S2, of cardiac myosin became reactive to N-ethylmaleimide (NEM) upon the addition of Mg2+-ATP or Mg2+-ADP, as in the case of skeletal myosin, although to a lesser degree. Enhancement of the reactivity of S2 resulting from cooperative interaction between F-actin and Mg2+-ATP, but not Mg2+-ADP, which was first found in skeletal myosin, was also observed in cardiac myosin.
2. The total number of 5, 5'-dithiobis(2-nitrobenzoate) (DTNB)-reactive thiols decreased in the presence of Mg2+-ADP and decreased further in the presence of Mg2+-ATP for both myosins, in contrast to the concomitant increase in the reactivity of S2. The decrement was smaller in cardiac myosin than in skeletal myosin.
3. The DTNB-reactive thiols were classified into three groups with respect to their reactivity. The third or most slowly reacting thiol group, was found to be remarkably susceptible to Ca2+ at physiological concentrations (10-8-10-6 M) in the presence of Mg2+-ADP for cardiac myosin, but in striking contrast, in the presence of Mg2+-ATP for skeletal myosin. This suggests that the nature of the predominant and relatively long-lived intermediate of myosin ATPase, the structure of which is sensitive to Ca2+, may be different in cardiac and skeletal myosins.View full abstractDownload PDF (443K) -
VI. Effect of ADP on the Reactivity of Thiol Groups Other than S2Keiichi YAMAMOTO, Takamitsu SEKINE1980 Volume 87 Issue 2 Pages 593-599
Published: February 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSExperiments were carried out to explore the location of thiol groups which became less reactive when S2 became more reactive in the presence of ADP. Myosin was first treated with N-ethylmaleimide to block S1 and then treated with a fluorogenic thiol reagent, N-(7-dimethyl amino-4-methylcoumarinyl) maleimide (DACM), in the presence or absence of ADP. From the distribution of DACM in the two kinds of DACM-treated myosin, it was found that thiol groups of all the light chains and of the 50 K fragment of subfragment-1 heavy chain became less reactive to DACM in the presence of ADP. Since it is known that the ADP binding site and S2 are located in the 26 K and 21 K fragments of subfragment-1 heavy chain, respectively, our results suggest that the 50 K, 26 K, and 21 K fragments of subfragment-1 heavy chain and light chains are closely connected with each other in the subfragment-1.View full abstractDownload PDF (2343K) -
Kunihiro UENISHI, Wayne E. CRISS, Shiro KAKIUCHI1980 Volume 87 Issue 2 Pages 601-607
Published: February 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe activities of cyclic nucleotide phosphodiesterase [EC 3. 1. 4. 17] and levels of Ca2+-dependent modulator protein in rapidly growing (3924 A) and slowly growing (9633 and 7794 A) Morris hepatomas were compared with those in normal or host liver. Hydrolysis of cyclic GMP in the supernatants of tumor homogenates was more stimulated than that in supernatants of normal liver homogenates by Ca2+ and modulator protein: 3.39-fold (3924 A), 2.20-fold (9633), and 1.26-fold (normal liver) stimulations were seen. Ca2+-activatable phosphodiesterase, which has been found in a variety of nontumor tissues, was identified in extracts of hepatomas by gel filtration chromatography. The greater effect of Ca2+ and modulator protein on tumor extracts was due to elevated levels of this enzyme with concomitant reductions in the levels of other types of phosphodiesterase in the hepatoma extracts. 3924 A showed a greater change in isozyme pattern than 9633 or 7794 A.
The concentrations of modulator protein in the soluble fraction from hepatomas were about 2.6 times (3924A) and 1.4 times (9633) that in the same fraction from normal liver. However, its concentrations in the particulate fraction of the hepatomas were about one-fifth (3924 A) and one-half (9633) of that in the same fraction of normal liver. Consequently, the sums of the amounts of modulator protein in the two fractions of tumors were similar to that in normal liver. It is suggested that the Ca 2+/modulator protein system may be involved in the control of cell proliferation.View full abstractDownload PDF (528K) -
Masao KAWAKITA, Kimiko YASUOKA, Yoshito KAZIRO1980 Volume 87 Issue 2 Pages 609-617
Published: February 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRabbit sarcoplasmic reticulum vesicles were treated with N-ethylmaleimide (NEM) at pH 7.0. At 1.5mM NEM, only 4 SH groups permol of ATPase peptide were modified in 25min at 30°C. Two of these are essential for Ca2+ transport, one being involved in E-P formation (SHF), and the other in its decomposition (SHD), whereas the other two are apparently nonessential (SHN and SHN'). SHN was modified first, followed by SHD, SHN', and SHF, in this order. Modification of SHD was accompanied by the loss of Ca2+-transport activity, while E-P forming activity survived until the least reactive one (SHF) was modified. At a lower NEM concentration (4×10-5M) SHN could be selectively modified without loss of the enzyme activity.
SHF could be protected by adenyl-5'-yl-imidodiphosphate (AMP-P(NH)P) in the presence of Ca2+ ions, whereas SHD was not. SHD was distinctly less reactive in the absence of Ca2+ (<10-7 M) than in its presence. Changes in the reactivity of these SH groups may be related to conformational changes of the ATPase molecule induced by the binding of Ca2+ and ATP.View full abstractDownload PDF (700K) -
Mitsuyoshi YOSHIKAWA, Toshifumi KIYOHARA, Teruo IWASAKI, Makoto KAWATA ...1980 Volume 87 Issue 2 Pages 619-627
Published: February 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSProteinase inhibitor II' from adzuki beans was subjected to peptic digestion. One of the resulting fragments, which inhibited chymotrypsin but not trypsin, was composed of 27 amino acid residues. The fragment was confirmed to be derived from the chymotrypsin-inhibitory domain of the original inhibitor. Another fragment, which inhibited trypsin only, contained 38 amino acid residues and consisted of two peptide chains. One of them, consisting of 25 amino acid residues, corresponded to the original reactive site region for trypsin. These fragments were also obtained from inhibitor II by peptic digestion. These findings confirm that these inhibitors, which do not inhibit chymotrypsin and trypsin simultaneously, have separate and independent domains for the inhibition of each enzyme. The active fragments are homologous in chemical structures with the two fragments from soybean Bowman-Birk proteinase inhibitor. However, unlike the fragments from Bowman-Birk inhibitor, our chymotrypsin-inhibitory fragment was a potent inhibitor of the enzyme and was as resistant as the intact inhibitor to the attack of excess chymotrypsin. The trypsin-inhibitory fragment had a lower anti-tryptic action than the original inhibitor and was gradually inactivated by trypsin. These differences between our fragments and those of the Bowman-Birk inhibitor are probably a result of the replacement of a few amino acid residues in the reactive site regions.View full abstractDownload PDF (518K) -
Toshitaka NAKAMURA, Yutaka NAGAI1980 Volume 87 Issue 2 Pages 629-637
Published: February 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDevelopmental changes in the synthesis of glycosaminoglycans and collagen in embryonic chick skins have been studied by labeling the materials with D-[1-3H]-glucosamine and L-[2, 33H]-proline, respectively, in culture.
In day 8 to day 20 embryos and 2-day-old chicks, hyaluronic acid was predominantly synthesized, with chondroitin sulfate, dermatan sulfate, and heparan sulfate as minor products.
The syntheses of all these glycosaminoglycan components increased on days 14-16, when collagen synthesis also increased rapidly. The tissue deposition of hyaluronic acid and chon-droitin sulfate, however, decreased continuously, while that of dermatan sulfate increased steadily. These results suggest that increased rates of turnover of hyaluronic acid and chon-droitin sulfate are essential to initiate remodelling of the tissue.
Rapid increases in the synthesis and tissue deposition of hyaluronic acid occurred from day 20 to the newly hatched stage. This may be a response of the embryo to the coming change of environment from in ovo to ex ovo by carrying a large quantity of bound water in the tissue space. Type I collagen accounted for 68-85% of the newly synthesized collagens throughout the stages examined. Type III collagen was detectable from earlier stages (0.2-1.0%), and started to increase on day 14, but then increased sharply from day 17 to the newly hatched stage (8.6%) in parallel with an increased rate of collagen deposition in the tissue.
Syntheses of type I trimer collagen and type X and Y chains with properties similar to those of αB and αA were also confirmed at all the stages examined, though the levels of the latter two components were only a few percent of the total collagen.View full abstractDownload PDF (1177K) -
Sumiko HAMANAKA(néeYASUE), Shizuo HANDA, Jun INOUE, Atsuhiko HA ...1980 Volume 87 Issue 2 Pages 639-643
Published: February 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe ganglioside patterns of erythrocytes from individual horses and cattle were examined. Variations in the ganglioside patterns were found in both horses and cattle.
In the erythrocytes of most horses examined, NeuGc-Gal-Glc-ceramide (NeuGc-GM3) was predominant with some 4-O-Ac-NeuGc-Glc-ceramide (4-O-Ac-NeuGc-GM3), but 3 of 25 horses examined had only NeuGc-GM3 with no 4-O-Ac-NeuGc-GM3.
The erythrocytes of various breeds of cattle had a characteristic ganglioside pattern, but they could be divided into 4 types on the basis of the composition of their gangliosides.View full abstractDownload PDF (1476K) -
Susumu TSUNASAWA, Kiyoshi KAMITANI, Kozo NARITA1980 Volume 87 Issue 2 Pages 645-650
Published: February 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBy using the synthetic peptide ACTH1-24 as a model substrate, an enzyme that may be involved in the amino-terminal acetylation of certain proteins and growing nascent polypeptide chains has been found in hen's oviduct. It was partially purified by a four-step procedure comprising extraction from the homogenates, ammonium sulfate fractionation, chromatography on a column of QAE-Sephadex A-50, and gel filtration on a Sepharose 6B column. An enzyme preparation purified about 40-fold from the homogenates transferred the acetyl group from acetyl coenzyme A preferentially to the amino-terminal amino acids of several ACTH-related peptides at an optimum pH of around 7.2. This occurred to different extents depending on the peptide length and on the nature of the amino-terminal residue. The molecular weight of the enzyme was estimated to be approximately 250, 000 by gel filtration.View full abstractDownload PDF (892K) -
Purification and Enzymatic Characterization of Three EndoDNase Isoenzymes from Physarum polycephalumJaap H. WATERBORG, Charles M. A. KUYPER1980 Volume 87 Issue 2 Pages 651-661
Published: February 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThree alkaline DNases, A, B, and C, with preference for the digestion of double-stranded DNA (dsDNA) were partially purified from microplasmodia of Physarurn polycephalum. They were very similar but differed in their isoelectric points. These were pH 5.8 for DNase A, 7.1 for DNase B, and 9.1 for DNase C. All three enzymes consisted of a single polypeptide chain with a molecular weight of 16, 000 to 17, 000, which readily formed high molecular weight complexes with low enzyme activity. These complexes could be reversibly dissociated by urea, and DNase activity was quantitatively reactivated. The DNases hydrolyzed the substrate DNA by an endonucleolytic mechanism which gave 5'-phosphorylated products. Divalent cations, MnCl2 or MgCl2, were essential for enzyme activity at the optimum pH of approximately 8.5 and at low ionic strength. The optimal conditions of pH, buffer, divalent cations and ionic strength and the extent of inhibition by salt, phosphate ions or urea differed slightly but significantly between the different isoenzymes.View full abstractDownload PDF (1210K) -
Hiroyasu NISHIKAWA, Takanori FUJII, Seiji YAMADA, Kazuo YOSHIZAKI, Hir ...1980 Volume 87 Issue 2 Pages 663-666
Published: February 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS31P-NMR measurements of brain slices of guinea pig were successfully carried out for the first time with a new perfusion and spinning technique. The 31P-NMR spectrum showed resonance lines of phosphocreatine, fructose-6-phosphate, ATP, and inorganic phosphate. In the time course of the 31P-NMR spectra, the concentrations of phosphocreatine and fructose-6-phosphate, which were estimated from the initial spectrum, were 4.3 and 1.1 μmol/g of fresh tissue, respectively, and the estimates from the following ones showed that the concentration of phosphocreatine decreased to 2.7 μmol/g of fresh tissue while that of fructose-6-phosphate did not change significantly throughout the measurements.View full abstractDownload PDF (247K) -
Hiromichi KUMAGAI, Eisuke NISHIDA, Koichi ISHIGURO, Hiromu MUROFUSHI1980 Volume 87 Issue 2 Pages 667-670
Published: February 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe developed a simple new method for the isolation of calmodulin, a ubiquitous Ca2+-binding protein, by using tubulin-Sepharose 4 B column chromatography, and succeeded in rapid isolation of calmodulin from Tetrahymena pyriformis. The procedure was also shown to be successfully applicable to the isolation of calmodulins from starfish ovary, porcine brain, and monkey brain and, therefore, may be of general use for the rapid isolation of calmodulin.View full abstractDownload PDF (896K) -
Kosuke ICHIHARA, Ikuko KASAOKA, Emi KUSUNOSE, Masamichi KUSUNOSE1980 Volume 87 Issue 2 Pages 671-674
Published: February 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSBenzo(a)pyene hydroxylation activity was solubilized from rabbit intestinal mucosa microsomes and reconstituted with a cytochrome P-450 preparation obtained by fractionation with 6-amino-n-hexyl Sepharose 4 B, hydroxylapatite and CM-Sephadex C-50, and partially purified NADPH-cytochrome c reductase. Phosphatidylserine was required for the maximal activity, while phosphatidylcholine had no stimulatory effect. The carbon monoxide difference spectrum of the cytochrome P-450 fraction showed a maximum peak at 450nm. Although another cytochrome P-450 fraction was active for hexadecane hydroxylation, this fraction had little activity. The results indicate that more than one cytochrome P-450 exists in the intestinal mucosa microsomes.View full abstractDownload PDF (258K) -
Teruhiko TOYO-OKA, Junko OKAMOTO, Takeshi TANAKA, Tomoh MASAKI1980 Volume 87 Issue 2 Pages 675-678
Published: February 01, 1980
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAntiserum against purified chicken smooth muscle myosin was obtained from rabbits. The serum was specific for smooth muscle myosin and did not react with myosins from fast and slow skeletal muscle or cardiac muscle. Using this specific antibody, the location of smooth muscle myosin-like protein was studied by double antibody immunofluorescence microscopy with sections of chicken liver tissue. Marked fluorescence was demonstrated along the sinu-soids of hepatic tissue and the vascular walls. The findings in this study suggest that a myosinlike protein, which is immunologically similar to the myosin from smooth muscle but not to myosins from skeletal or cardiac muscle would exist on the sinusoidal surface of liver cells.View full abstractDownload PDF (732K)
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