The Journal of Biochemistry
Online ISSN : 1756-2651
Print ISSN : 0021-924X
79 巻, 6 号
選択された号の論文の26件中1~26を表示しています
  • Takayuki KITAGAWA, Keizo INOUE, Shoshichi NOJIMA
    1976 年 79 巻 6 号 p. 1123-1133
    発行日: 1976/06/25
    公開日: 2008/11/18
    ジャーナル フリー
    Liposomes have been prepared with lysolecithin (1-acyl-sn-3-glycerylphosphorylcholine), egg lecithin (3-sn-phosphatidylcholine), dicetyl phosphate, and cholesterol. The ability to function as a barrier to the diffusion of glucose marker and the sensitivities of the liposomes to hypotonic treatment and other reagents which modified the permeability were examined. Generally, lysolecithin incorporation decreased the effectiveness of the membranes as a barrier to glucose and made the membranes more “osmotically fragile.” Cholesterol incorporation counteracted the effect of incorporated lysolecithin. The more cholesterol incorporated into liposomes, the more lysolecthin could be incorporated into the membrane without loss of function as a barrier. With more than 50 mole% of cholesterol, lysolecithin alone could form membranes which were practically impermeable to glucose. The hemolytic activity of lysolecithin was affected by mixing with various lecithins or cholesterol. Liposomes containing lysolecithin, which have the ability to trap glucose marker, showed poor hemolytic activity, while lipid micelles with lysolecithin (which could trap little glucose) showed almost the same hemolytic activity as lysolecithin itself. There seems to be a close correlation between hemolytic activity and barrier function of lipid micelles.
  • Takayuki KITAGAWA, Keizo INOUE, Shoshichi NOJIMA
    1976 年 79 巻 6 号 p. 1135-1145
    発行日: 1976/06/25
    公開日: 2008/11/18
    ジャーナル フリー
    The diffusion of glucose from phospholipid membranes has been measured in the presence of serum albumin or methylated serum albumin.
    At neutral pH, serum albumin enhanced the rate at which glucose diffused from liposomes containing more than a certain amount of lysolecithin. Net charge of the membrane is not important for the reaction, since positively charged membranes containing stearylamine showed almost the same reactivity as negatively charged liposomes containing dicetyl phosphate.
    Carboxylmethylated albumin showed enhancement of the diffusion rate of glucose from negatively but not from positively charged liposomes. The amount of methylated albumin required to affect liposomes was much smaller than the amount of albumin required to damage liposomes containing lysolecithin.
    Cholesterol incorporation suppressed the sensitivity of liposomes to both proteins, albumin and methylated albumin. The effect of temperature and fatty acid composition of phospholipids on the sensitivity of liposomes to proteins suggests the importance of the fluidity of the membrane, especially in the case of methylated albumin.
  • Takayuki KITAGAWA, Keizo INOUE, Shoshichi NOJIMA
    1976 年 79 巻 6 号 p. 1147-1155
    発行日: 1976/06/25
    公開日: 2008/11/18
    ジャーナル フリー
    Permeability properties of multilamellar liposomes prepared from synthetic, saturated short-chain (C8, C10, C12) lecithins (3-sn-phosphatidyl choline) were studied. Dioctanoyllecithin, didecanoyllecithin, and dilauroyllecithin form “stable” bilayers which are practically impermeable to glucose when prepared with proportions of more than 1.2, 0.75, and 0.6 of cholesterol (molar ratio to phospholipid), respectively. Dioctanoyllecithin liposomes were rather leaky above 30°, even when a proportion of 1.5 of cholesterol (molar ratio to lecithin) was incorporated. Judging from their sensitivities to temperature, Triton X-100, serum albumin, and other reagents, short-chain lecithin bilayers show characteristic properties, different from those of both saturated long-chain lecithins (dipalmitoyllecithin and dimyristoyllecithin) and lecithins having unsaturated fatty acid residues (egg lecithin and dioleoyllecithin.).
  • Hajime HIRATA, Nobuhito SONE, Masasuke YOSHIDA, Yasuo KAGAWA
    1976 年 79 巻 6 号 p. 1157-1166
    発行日: 1976/06/25
    公開日: 2008/11/18
    ジャーナル フリー
    1. Thermostable membrane vesicles which were capable of active transport of alanine dependent on either respiration or an artificial membrane potential were isolated from the thermophilic aerobic bacterium PS3.
    2. Uptake of alanine was dependent on the oxidation of ascorbate-phenazine metho-sulfate or on generated or exogenous NADH, but succinate and malate failed to drive the uptake. The optimum temperature for respiration-driven uptake of alanine was 45 to 60°.
    3. Potassium ion-loaded vesicles were prepared by incubating vesicles at 55° in 0.5M potassium phosphate. The addition of valinomycin elicited rapid and transient uptake of alanine under the test conditions. Uptake of alanine in response to valinomycin was progressively enhanced by the addition of dicylohexylcarbodiimide, but was completely abolished in the presence of a proton conductor or synthetic permeable cation. The effect of dicyclohexylcarbodiimide was dependent on its concentration and was maximal at a concentration of 0.4mM.
    4. The proton permeability of membrane vesicles was reduced by the addition of dicyclohexylcarbodiimide. A small but significant difference was found in the initial rates of proton uptake in the presence of dicyclohexylcarbodiimide with and without alanine. The results suggest that protons alanine are transported simultaneously in a stoichiometric ratio of 1:1.
    5. The uptake of alanine was also driven by a pH gradient induced by an instantaneous pH drop in a suspension of alkali-loaded vesicles. Thus, alanine accumulation was driven not only by an electrical potential but also by a pH gradient.
    6. Addition of ATP resulted in the inhibition of alanine uptake dependent on artificial membrane potential. ATP hydrolysis by membrane ATPase created a membrane potential which was inside-positive, and this might decrease the effective membrane potential (generated by K+ efflux mediated by valinomycin) available to drive alanine uptake.
  • XVII. On the Existence and Nature of Substrate Amino Acids Bound to Purified Branched Chain Amino Acid-binding Proteins of Escherichia coli
    Hiroshi AMANUMA, Jindow ITOH, Yasuhiro ANRAKU
    1976 年 79 巻 6 号 p. 1167-1182
    発行日: 1976/06/25
    公開日: 2008/11/18
    ジャーナル フリー
    The substrate binding reactions of purified, homogeneous leucine-isoleucine-valine-threonine binding protein (LIVT-binding protein) and leucine specific binding protein (Ls-binding protein) of Escherichia coli were extensively examined by an equilibrium dialysis method using radioactive substrates. The concave downward Lineweaver-Burk plots for the reactions were obtained and they showed that the maximal number of binding sites per molecule of each protein was 1. It was also found that the specific binding activity of LIVT-binding protein was significantly altered by the protein concentration.
    Dansylation with dansyl chloride and subsequent thin layer chromatography of the purified LIVT-binding protein revealed the existence of isoleucine, and leucine andjor valine, bound to the protein noncovalently, The total amount of these amino acids was about equimolar to that of the protein. These bound amino acids were found to be exchanged with an added substrate amino acid when the protein was dialyzed against buffer containing one of the substrate amino acids of this protein. Using the protein containing radioactive isoleucine as a bound substrate, the exchange reaction was shown to occur rapidly and stoichiometrically. Release of these bound amino acids from the protein solution was found to occur very slowly in the absence of the exchange reaction on dialysis or during gel filtration. The affinity of the bound amino acids for the protein was measured using protein depleted of the bound substrates by treatment with 6 M urea. Substrate binding reactions of the depleted protein showed Michaelis-Menten type kinetics with dissociation constants of 1-2×10-7M and nearly 1 as the maximal number of binding sites.
    These findings indicated that the substrate binding reaction of LIVT-binding protein is a simple association-dissociation reaction with dissociation constants of around 1×10-7M for its substrates, and that owing to these small dissociation constants the purified protein can retain about an equimolar amount of its substrates which are in a dynamic equilibrium with the protein. Based on these findings and theoretical considerations, a rationale explaining multiphasic binding kinetics produced by binding protein having a small dissociation constant for substrate and a single binding site in the molecule was presented. Similar conclusions were made about Ls-binding protein in which a small amount of leucine was detected by dansylation.
    Purification and properties of the third binding protein for branched chain amino acids of E. coli were described.
  • Satoko ISEMURA, Tokuji IKENAKA
    1976 年 79 巻 6 号 p. 1183-1196
    発行日: 1976/06/25
    公開日: 2008/11/18
    ジャーナル フリー
    Rat serum albumin was cleaved into seven peptides by cyanogen bromide treatment followed by reduction and carboxymethylation, The amino acid composition and the N-terminus of each peptide and the amino acid sequences of four peptides were determined by conventional methods. An alignment of these peptides in the original protein was proposed in the light of the established sequences of bovine and human plasma albumin.
  • Yoshifumi MATSUDA, Hiroshi MORIYA, Chiaki MORIWAKI, Yukio FUJIMOTO, Mi ...
    1976 年 79 巻 6 号 p. 1197-1200
    発行日: 1976/06/25
    公開日: 2008/11/18
    ジャーナル フリー
    The fluorometric method of Nash for determination of methanol was applied in assay of kallikrein-like arginine esterases using N-α-tosyl-L-arginine methyl ester (TAME) as substrate. The results obtained using this method corresponded well with those obtained in the usual colorimetric methods using hydroxamate, chromotropic acid, and 3-methyl-2-benzothiazolone hydrazone and the present method was more sensitive than these colorimetric methods.
  • The Amino Acid Sequence of a Glycopeptide Portion (Fragment 1) Following the C-Terminus of the Bradykinin Moiety
    Yong Nam HAN, Hisao KATO, Sadaaki IWANAGA, Tomoji SUZUKI
    1976 年 79 巻 6 号 p. 1201-1222
    発行日: 1976/06/25
    公開日: 2008/11/18
    ジャーナル フリー
    On incubation of bovine plasma high-molecular-weight (HMW) kininogen with purified plasma kallikrein [EC 3.4.21.8], a large glycopeptide fragment and the vasoactive peptide, bradykinin, were initially liberated ; the former, named fragment 1.2, was subsequently cleaved into fragment 1 (glycopeptide) and the previously established fragment 2 (histidine-rich peptide). The isolated fragment 1.2 contained a total of 108 to 110 amino acid residues and carbohydrates, and the amino-terminal sequence Ser-Val-Gln was established. The other fragment, fragment 1, consisted of a total of 69 amino acid residues with serine and arginine (and lysine) at the amino and carboxyl termini, respectively. It contained eleven residues each of histidine and glycine, together with an oligosaccharide chain consisting of galactosamine, hexose and sialic acid. The complete amino acid sequence of fragment 1 was determined by Edman degradation and standard enzymatic and chemical techniques. These results established the following sequence:
    H-S_??_er-Val-Gln-Val-Met-Lys-Thr-Glu-Gly-S_??_r-Thr-_??_-Val-Ser (CHO)-_??_-Pro-His-Ser-Ala-M_??_t-Ser-Pro-Val-Gln-Asp-Glu-Glu-Arg-Asp-S_??_r-Gly-Lys-Glu-Gln-Gly-Pro-Thr-His-Gly-H_??_s-Gly-Trp-Asp-His-Gly-Lys-Gln-Lys-Gln-Ile-Lys-L_??_u-His-Gly-Leu-Gly-Leu-Gly-His-Lys-His-Lys-His-L_??_s-His-Asp-Gln-His-Gly-His-His-_??_OH
  • Fumiko YAMADA, Noriko TAKAHASHI, Takashi MURACHI
    1976 年 79 巻 6 号 p. 1223-1234
    発行日: 1976/06/25
    公開日: 2008/11/18
    ジャーナル フリー
    Fruit bromelain FA2, the main proteinase component of the juice of pineapple fruit, has been purified and characterized.
    1. Efficient extraction of this enzyme from the crude material was possible using “Cellulosin AP, ” a microbial polysaccharidase preparation containing cellulase, hemi-cellulase, and pectinase. The enzyme was purified mainly by successive applications of anion-exchange chromatography, yielding an apparently homogeneous protein as judged by several physical, chemical, and immunochemical criteria. Properties of FA2 include : molecular weight, 31, 000; isoelectric point, pH 4.6; absorbance at 280 nm of a 1% solution at pH 7.0 per cm, 19.2.
    2. FA2 gave only alanine phenylthiohydantoin upon amino-terminal group analysis by the Edman procedure. Stepwise degradation yielded the amino-terminal sequence Ala-Val-Pro-Gln-Ser-He-Asp-Trp-Arg-Asp-Tyr-Gly-Ala. The amino acid composition of FA2 was not markedly different from that of stem bromelain, except for a much smaller lysine content and a smaller alanine content relative to glycine in FA2. FA2 contained neither amino sugars nor neutral carbohydrates as determined by several methods, so FA2 is not a glycoprotein.
    3. By labeling the reactive cysteine residue (CYS) with [14C]iodoacetate, the following partial amino acid sequence has been determined.
    Asn-Glx-Asn-Pro-Cys-Gly-Ala-CYS
  • A Rapid Formation of Lysine-derived Crosslinks by Chick Embryo Aorta
    Masamitsu MIYOSHI, Masao KANAMORI, Joel ROSENBLOOM
    1976 年 79 巻 6 号 p. 1235-1243
    発行日: 1976/06/25
    公開日: 2008/11/18
    ジャーナル フリー
    Aortas of 13-day-old chick embryo were lebeled for 0.5 hr with [14C]Iysine and sub-jected to a serial extraction after chase for 1-24 hr with [12C]lysine. Substantial radioactivity was found in insoluble elastin after 3 hr chase. The effect of β-amino-propionitrile on lebeling with [14C]lysine was also examined. Each fraction was hydrolyzed and applied to a short column on an amino acid analyzer. Radioactivity was found in desmosine and isodesmosine of insoluble elastin as early as 1 hr after the beginning of chase. The radioactivity increased rapidly at 2 hr and very slowly thereafter. A large count, which was separated into five peaks on a long column, was observed in other lysine derivatives at 2 hr and increased steadily up to 24 hr, while the lysine count decreased from 1:0.5 to 1:6 against lysine derivatives and from 1:0.04 to 1:0.9 against quarter-desmosine after 24 hr. The oxidation of lysine residues incorporated during the 0.5 hr pulse was almost completed during the first 1 hr of chase, and these oxidized residues were incorporated into crosslinks during the following 1 hr. It is suggested that poorly crosslinked elastin accumulated in the soluble fractions. The presence of crosslinking derived from lysine residues was also indicated in the microfibril fraction.
  • Effects of Preincubation in the Cold on Polyuridylic Acid-dependent Polyphenylalanine Synthesis at High Temperature
    Yoshiko OHNO-IWASHITA, Tairo OSHIMA, Kazutomo IMAHORI
    1976 年 79 巻 6 号 p. 1245-1252
    発行日: 1976/06/25
    公開日: 2008/11/18
    ジャーナル フリー
    1. It was found that preincubation of the reaction mixture in the cold enhanced polyuridylic acid-directed polyphenylalanine synthesis by a cell-free extract of Thermus thermophilus HB8 at high temperature.
    2. The effect of preincubation was most marked at 10-25° in the presence of 20mM Mg2+, Preincubation at 65° failed to stimulate the incorporation.
    3. The presence of phenylalanyl-tRNA, polyuridylic acid, and ribosomes was essential for preincubation in the cold to be effective.
    4. A ternary complex of amino acyl-tRNA, polyuridylic acid, and a ribosome formed at low temperature was isolated by CPG-10 column chromatography; the isolated complex initiated polyphenylalanine synthesis effectively at high temperature.
    5. The amount of the ternary complex formed depends on the preincubation time and the concentration of Mg2+. Since the amount of the complex correlated positively to the rate of polyphenylalanine synthesis at high temperature, the effectiveness of preincubation in the cold is presumably due to the formation of the ternary complex of phenylalanyl-tRNA, polyuridylic acid, and a ribosome.
  • Keiichi UEMURA, Atsushi HARA, Tamotsu TAKETOMI
    1976 年 79 巻 6 号 p. 1253-1261
    発行日: 1976/06/25
    公開日: 2008/11/18
    ジャーナル フリー
    Various sphingolipids were chemically modified in their sphingosine base by ozonolysis and reduction. The derivatives obtained from Forssman globoside, globoside I, galactosyl ceramide, and sphingomyelin were purified and all were found to be hemolytic. The presence of cholesterol could inhibit this activity.
    The simultaneous cleavage at a double bond in the fatty acid as well as in the sphingosine of Forssman globoside resulted in the formation of a more polar compound with no detectable hemolytic activity. The haptenic reactivity was retained after ozonolysis and reduction of Forssman globoside, as shown by precipitin line formation in agar gel with appropriate antibodies, The results indicate that this modification procedure may be useful in studies of the physiological and immunological properties of sphingolipids.
  • Susumu ITO, Taro NAKAMURA, Yoshitomo EGUCHI
    1976 年 79 巻 6 号 p. 1263-1272
    発行日: 1976/06/25
    公開日: 2008/11/18
    ジャーナル フリー
    Methioninase of Pseudornonas putida was purified to homogeneity, as judged by polyacrylamide gel electrophoresis, with a specific activity 270-fold higher than that of the crude extract.
    1. The purified enzyme had an S20, w of 8.37, a molecular weight of 160, 000, and an isoelectric point of 5.6.
    2. A break in the Arrhenius plot was observed at 40° and the activation energies below and above this temperature were 15.5 and 2.97 kcal per mole, respectively.
    3. In addition to L-methionine, various S-substituted derivatives of homocysteine and cysteine could serve as substrates. D-Methionine, 2-oxo-4-methylthiobutanoate, and related non sulfur-containing amino acids were inert. Equimolar formation of α-ketobutyrate and CH3SH was observed with methionine as a substrate. 4. In addition to the protein peak at 278nm, two absorption maxima were observed at 345 and 430nm at pH 7.5. Hydroxylamine removed the enzyme-bound pyridoxal phosphate, resulting in almost complete resolution with the concomitant disappearance of both peaks. Reconstruction of the treated enzyme could be achieved by addition of the cofactor; the Km value was calculated to be 0.37μM. 5. The reported purified enzyme should be designated as L-methionine methanethiollyase (deaminating).
  • Minoru HAMADA, Tadayasu HIRAOKA, Kichiko KOIKE, Kyoto OGASAHARA, Tamot ...
    1976 年 79 巻 6 号 p. 1273-1285
    発行日: 1976/06/25
    公開日: 2008/11/18
    ジャーナル フリー
    Pyruvate dehydrogenase [EC 1.2.4.1] was separated from the pyruvate dehydrogenase complex and its molecular weight was estimated to be about 150, 000 by sedimentation equilibrium methods. The enzyme was dissociated into two subunits (α and β), with estimated molecular weights of 41, 000 (α) and 36, 000 (β), respectively, by polyacryl-amide gel electrophoresis in sodium dodecyl sulfate. The subunits were separated by phosphocellulose column chromatography and their chemical properties were examined. The subunit structure of the pyruvate dehydrogenase was assigned as α2β2. The content of right-handed a-helix in the enzyme molecule was estimated to be about 29 and 28% by optical rotatory dispersion and by circular dichroism, respectively.
    The enzyme contained no thiamine-PP, and its dehydrogenase activity was completely dependent on added thiamine-PP and partially dependent on added Mg2+ and Ca2+. The Km value of pyruvate dehydrogenase for thiamine diphosphate was estimated to be 6.5×10-5M in the presence of Mg2+ or Ca2+. The enzyme showed highly specific activity for thiamine-PP dependent oxidation of both pyruvate and α-ketobutyrate, but it also showed some activity with α-ketovalerate, α-ketoisocaproate, and α-ketoisovalerate. The pyruvate dehydrogenase activity was strongly inhibited by bivalent heavy metal ions and by sulfhydryl inhibitors; and the enzyme molecule contained 27 moles of 5, 5'-dithiobis(2-nitrobenzoic acid)-reactive sulfhydryl groups and a total of 36 moles of sulfhydryl groups. The inhibitory effect of p-chloromercuri-benzoate was prevented by preincubating the enzyme with thiamine-PP plus pyruvate. The structure of pyruvate dehydrogenase necessary for formation of the complex is also reported.
  • Koichi SUZUKI, Ken HIBINO, Kazutomo IMAHORI
    1976 年 79 巻 6 号 p. 1287-1295
    発行日: 1976/06/25
    公開日: 2008/11/18
    ジャーナル フリー
    Conditions for the hybridization of glyceraldehyde-3-phosphate dehydrogenase (GPD) [EC 1.2.1.12] in the presence of dilute borate were examined with horseshoe crab and rabbit GPDs. Hybridization was strongly dependent upon pH, borate concen-tration, and temperature. The optimum medium for hybridization was 10mM Tris-HCl-1mM 2-mercaptoethanol-1mM EDTA containing 10-20mM borate, pH 8.5-9.0. Hybridization was performed by incubation of two
    electrophoretically distinct GDPs at 30° for 3-15 hr in the above medium at a protein concentration of 1-2mg/ml.
    The time course of hybridization was analyzed under the optimized conditions. Symmetrical A2B2-type hybrid appeared only 5min after incubation for 1 hr.
    Hybridization of GPDs from 7 different species was examined under the optimal conditions. Hybridization was detected with rabbit-horseshoe crab, yeast-rabbit, yeast-chicken, and chicken-horseshoe crab combinations.
    Subunit-subunit interaction, the mechanism of hybridization, and the structure of GPD are discussed based on the results obtained.
  • Osamu SUZUKI, Etsuko NOGUCHI, Kunio YAGI
    1976 年 79 巻 6 号 p. 1297-1299
    発行日: 1976/06/25
    公開日: 2008/11/18
    ジャーナル フリー
    A simple fluorometric assay for monoamine oxidase (MAO) [EC 1.4.3.4] activity towards β-phenylethylamine (PEA) was devised. The procedure consists in measuring the disappearance of PEA fluorometrically. The disappearance of PEA was completely inhibited by pargyline, a potent inhibitor of MAO. MAO activity for PEA was linear with 10mg to 100mg of liver tissue in 3ml of reaction mixture for up to 90min of incubation. Using this method, the Vmax values and the apparent Km values of MAO for PEA in several rat tissues were determined, and compared with those for benzylamine and 5-hydroxytryptamine (5-HT).
  • Mikihiko KOBAYASHI, Kazuo MATSUDA
    1976 年 79 巻 6 号 p. 1301-1308
    発行日: 1976/06/25
    公開日: 2008/11/18
    ジャーナル フリー
    Dextransucrase [EC 2.4.1. 5] activity from cell-free culture supernatant of Leuconostoc mesenteroides NRRL B-1299 was purified by (NH4)2SO4 fractionation, adsorption on hydroxyapatite, chromatography on DEAE-cellulose and gel filtration on Sephadex G-75. The extracellular enzyme was separated into two principal forms, enzymes I and N, and the latter was shown to be an aggregated form of the protomer, enzyme I. Enzymes I and N were both electrophoretically homogeneous and their relative activities reached 820 and 647 times that of the culture supernatant, respectively. On sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis, enzyme N dissociated into the protomer enzyme I, with a molecular weight of 48, 000. Enzyme I was gradually converted into enzyme N upon aging, and this conversion was stimulated in the presence of NaCl. The optimum pH and temperature of enzyme I activity were pH 6.0 and 40°, respectively, while those of enzyme N were pH 5.5 and 35°. The Km values of enzymes I and N were 13.9 and 13.1mM, respectively. Ca2+, Mg2+, Fe2+, and Co2+ stimulated the activity of enzyme N, and EDTA showed a potent inhibitory effect on this enzyme. Moreover, the activity of enzyme N was more effectively stimulated by exogenous dextrans as compared with enzyme I.
  • Lysozyme and Chymotrypsinogen
    Taro IZUMI, Hideo INOUE
    1976 年 79 巻 6 号 p. 1309-1321
    発行日: 1976/06/25
    公開日: 2008/11/18
    ジャーナル フリー
    Ultraviolet difference absorption spectra produced by ethylene glycol were measured for hen lysozyme [EC 3.2.1.17] and bovine chymotrypsinogen. N-Acetyl-L-tryptophan-amide and N-acetyl-L-tyrosinamide were employed as model compounds for tryptophyl and tyrosyl residues, respectively, and their ultraviolet difference spectra were also measured as a function of ethylene glycol concentration. By comparison of the slopes of plots of molar difference extinction coefficients (Δε) versus ethylene glycol concentration for the proteins with those of the model compounds at peak positions (291-293 and 284-287nm) in the difference spectra, the average number of tyrosyl as well as tryptophyl residues in exposed states could be estimated. The results gave 2.7 tryptophyl and 1.9 tyrosyl residues exposed for lysozyme at pH 2.1 and 2.6 tryptophyl and 3.4 tyrosyl residues exposed for chymotrypsinogen at pH 5.4. The somewhat higher tyrosyl exposure of chymotrypsinogen, compared with the findings from spectrophotometric titration and chemical modification, was not unexpected, because Δε285 was larger than Δε292, and the situation is discussed with reference to preferential interaction of ethylene glycol with the tyrosyl residues and/or side chains in the vicinity of the chromophore in the protein. The procedure employed in the present work seems to be suitable for estimation of the average number of exposed tryptophyl and tyrosyl residues in tryptophan-rich proteins.
    The effects of ethylene glycol on the circular dichroism spectra of lysozyme at pH 2.1 and chymotrypsinogen at pH 5.4 were also investigated. At high ethylene glycol concentrations, both proteins were found to undergo conformational changes in the direction of more ordered structures, presumably more helical for lysozyme and more β-structured for chymotrypsinogen.
  • Shoichi NAKASHIMA, Nobuaki TAKE, Hirohito HAYASHI, Masanori MAZAKI
    1976 年 79 巻 6 号 p. 1323-1330
    発行日: 1976/06/25
    公開日: 2008/11/18
    ジャーナル フリー
    Mice primed with chemically modified bacterial α-amylase (BaA) [EC 3.2.1.1 α-amylase, B. subtilis], which was neither cross-reactive with anti-BαA antibody nor able to induce a humoral anti-BαA antibody response, developed enhanced responses to a subsequent challenge with native BaA (Nakashima et al. (1974) J. Biochem. 76, 349-357). The present studies were designed to examine the relationship of priming doses of BaA derivatives to the level of enhancement of the helper activity.
    Increasing the priming dose of modified antigens resulted in a greater degree of helper cell response until the maximal level of enhancement was reached. When injections for priming and challenge were given intraperitoneally, priming doses of D-BαA, M-BαA, and RM-BαA required for the maximal enhancement of helper activity were about 15, 50, and 15μg, respectively. Further increase in the priming dose, conversely, resulted in suppressin of the enhanced helper activity, irrespective of whether the time interval between priming and challenge was 10 or 28 days. Suppression of the enhanced helper activity upon excessive dose priming with modified BαA derivatives was not specific for the anti-BαA antibody response. On the basis of these results it is suggested that this phenomenon of suppression might be partly accounted for by the regulatory mechanism functioning in antigenic competition.
  • C. R. MIDDAUGH, R. D. MacELROY
    1976 年 79 巻 6 号 p. 1331-1344
    発行日: 1976/06/25
    公開日: 2008/11/18
    ジャーナル フリー
    The enzyme ribose-5-phosphate isomerase [EC 5.3.1.6] was partially purified from a mesophilic organism, Thiobacillus thioparus, and from an extreme thermophile, Bacillus caldolyticus. The stability and kinetics of the two enzymes were compared with regard to temperature in the presence of a series of neutral salts and alcohols. The thermal stability of both enzymes was altered such that the salts (NH4)2S04, NaCl, KCl, and LiCl increased stability, while LiBr, CaC12, methanol, ethanol, and 1-propanol decreased stability. Ethylene glycol had little effect on the mesophilic enzyme, but increased the stability of the thermophilic protein.
    The kinetics of both enzymes were also affected by the salts and alcohols, and Arrhenius plots of two kinetic parameters, Km and Vmax, displayed discontinuities, or sharp changes in slope, at characteristic temperatures, TD. Neutral salts and alcohols altered the temperature of discontinuity in a sequence similar to that observed in studies of thermal stability. It is suggested that the slope change is due to temperature-dependent alterations in the enzymes at specific, but undefined, loci at the active site, although no evidence is offered for the absence of a larger conformation change in the entire enzyme.
  • Naranjan S. DHALLA, Madhu B. ANAND, James A. C. HARROW
    1976 年 79 巻 6 号 p. 1345-1350
    発行日: 1976/06/25
    公開日: 2008/11/18
    ジャーナル フリー
    Rat heart sarcolemma prepared by the hypotonic shock-LiBr treatment method was found to bind calcium by a concentration-dependent and saturable process. The calcium binding values at 50μM and 1.25mM Ca2+ concentrations were about 30 and 250nmoles/mg protein, respectively. Both Mg2+ and ATP inhibited calcium binding and no evidence for energy-linked calcium binding with sarcolemma was found. On the other hand, maximal ATP hydrolysis by heart sarcolemma was seen at 4mM Mg2+ or Ca2+. The Ca2+-ATPase [EC 3.6.1.3] activity was depressed by the presence of Mg2+ or excess ATP. Low concentrations (10-100μM) of Ca2+ failed to stimulate ATP hydrolysis in the presence of various concentrations of Mg-ATP. These results indicate the absence of a “calcium pump” mechanism in the heart sarcolemmal membrane preparation employed in this study.
  • II. Physicochemical Properties of Glycoprotein I
    Syoko KIDO, Masanobu JANADO, Hiroshi NUNOURA
    1976 年 79 巻 6 号 p. 1351-1356
    発行日: 1976/06/25
    公開日: 2008/11/18
    ジャーナル フリー
    Of the three major macromolecular components of the vitelline membrane of hen's egg, the lowest molecular weight component (previously designated component I) has been studied and its physicochemical properties clarified. The molecuar weight of this component is 27, 000 and its chemical composition is typical of a glycoprotein, consisting of protein (91%), total hexose (4.4%), hexosamine (glucosamine 2.3%; galactosamine 0.7%), and sialic acid (1.7%). Uronic acid was not found. The molar ratios of the constituent neutral sugars of this glycoprotein (GP-I) are as follows: fucose 3, mannose 5, galactose 5, glucose 1, and xylose 1. The amino acid profile shows a relatively high proportion of hydrophobic amino acids (39%), which may partly account for the insolubility of GP-I in water.
  • Takaaki KOBAYASHI, Takashi SIMIZU
    1976 年 79 巻 6 号 p. 1357-1364
    発行日: 1976/06/25
    公開日: 2008/11/18
    ジャーナル フリー
    Depolymerization of microtubules in the ATP-reassembly buffer permitted the preparation of GDPETNGTP. Incubation of this tubulin fraction at 35° with ATP induced the phosphorylation of E-site GDP into GTP, which was then dephosphorylated during microtubule assembly. Incubation of GDPETNGTP with phosphoenolpyruvate and pyruvate kinase [EC 2.7.1.40] also induced polymerization. Depolymerization of microtubules in the GTP-reassembly buffer yielded GTPETNGTP, which was capable of polymerizing into microtubules even in the absence of free GTP. In the presence of 4M glycerol, GDPETNGTP assembled into microtubules with no change in the bound nucleotides.
  • Shoji OHKUMA, Hiroshi NOGUCHI, Fumio AMANO, Den'ichi MIZUNO, Tomoyoshi ...
    1976 年 79 巻 6 号 p. 1365-1376
    発行日: 1976/06/25
    公開日: 2008/11/18
    ジャーナル フリー
    Apoferritin particles were found in mouse peritoneal macrophages cultured in vitro. They were found as 20S particles in the “ribosomal fraction” of macrophages labeled with L-[14C]glutamic acid. Possibilities that they were breakdown products of ribosomes or of other well-known contaminants of the ribosomal fraction were excluded because they did not incorporate [5-3H]uridine. They were resistant to RNase and were relatively resistant to detergent.
    The antibody against horse spleen apoferritin precipitated about 70% of the particles in the 20S region, judging by measurement of radioactivity. On in vitro incubation with Fell and suitable oxidizing agents the sedimentation coefficient of 80% of the 20S particles changed to about 60S, which corresponds to that of ferritin. SDS-polyacrylamide gel electrophoresis revealed the presence of subunit structures with the same molecular size as that of mouse liver apoferritin. Under the electron microscope, the particles appeared spherical with a relatively uniform diameter of about 130 A.
  • Yasuhiko MASUHO, Katsuhiko TOMIBE, Kimihiko MATSUZAWA, Tsuneo WATANABE ...
    1976 年 79 巻 6 号 p. 1377-1379
    発行日: 1976/06/25
    公開日: 2008/11/18
    ジャーナル フリー
    The conversion of the S-surfonate group in a peptide to a disulfide bond has been observed in vitro. This paper reports that the conversion of this group in human S-sulfonated γ-globulin (S-GG) appeared to occur in vivo judging from the change in molecular weight of S-GG observed in the presence of sodium dodecyl sulfate (SDS) and the restoration of hemolytic activity.
  • Tadashi YOSHIMOTO, Imao OKA, Daisuke TSURU
    1976 年 79 巻 6 号 p. 1381-1383
    発行日: 1976/06/25
    公開日: 2008/11/18
    ジャーナル フリー
    A method was developed for purification and crystallization of creatinase [creatine amidinohydrolase, EC 3.5.3.3] from Pseudomonas putida var. naraensis C-83. The purified preparation appeared homogeneous on disc electrophoresis and ultracentri-fugation and had a molecular weight of 94, 000. It was most active at pH 8 and stable between pH 6 and 8 for 24 hr at 37°. SDS-polyacrylamide gel electrophoresis indicated that the native enzyme was made up of two subunit monomers, the molecular weights of which were estimated to be 47, 000. Inhibition experiments suggested that a sulfhydryl group is located in or near the active site of the enzyme.
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