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Volume 84, Issue 1
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XIV. Change of the Inhibitory Activity of Bowman-Birk Inhibitor upon Replacements of the α-Chymotrypsin Reactive Site Serine Residue by Other Amino AcidsShoji ODANI, Tokuji IKENAKA1978 Volume 84 Issue 1 Pages 1-9
Published: July 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAll the known reactive site P1' positions (notation of Schechter and Berger, 1967) of legume double-headed proteinase inhibitors are occupied by serine residues. To investigate the role of this serine residue, P1' serine (residue 44) at the chymotrypsin-reactive site of Bowman-Birk inhibitor was replaced with other amino acids by the method of Kowalski and Laskowski, Jr. (1976), using limited proteolysis with chymotrypsin, removal of the serine residue by the Edman degradation method and condensation with activated amino acid esters. By this method six inhibitor derivatives having a Ser, Ala, Thr, Val, Leu, or Gly residue at position 44 were prepared. α-Chymotrypsin inhibitory activity was retained in this order, i.e. the Ser44 derivative was the most active and the G1y44 derivative was essentially inactive to α-chymotrypsin.
Gly44-inhibitor prepared by periodate oxidation of Ser44 followed by transamination gave the same result. These results suggest that at least an α-methyl or methylene (methine) group in the P1' amino acid residue is indispensable for the chymotrypsin inhibitory activity. Bulky side chains in this position appeared to decrease the activity, although a hydroxymethyl side chain was most effective. It is evident that P1' serine requires conservation for the effective manifestation of the inhibitory activity of the legume double-headed inhibitors.View full abstractDownload PDF (603K) -
Kazuo YOSHIZAKI1978 Volume 84 Issue 1 Pages 11-18
Published: July 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS31P-nuclear magnetic resonance was applied to living muscles of bullfrogs, and the time courses of metabolic changes of ATP, creatine phosphate, inorganic phosphate, and sugar phosphates were studied under anaerobic and aerobic conditions. A decrease in creatine phosphate was observed in the resting muscle under anaerobic conditions with a concomitant decrease in the intracellular pH, while the ATP level remained constant. With the use of 2, 4-dinitro-1-fluorobenzene and iodoacetic acid, ATP disappeared quickly. When the resting muscle was perfused with oxygen-saturated glucose-Ringer's solution, the amount of creatine phosphate increased gradually. These findings indicate that anaerobic glycolysis is insufficient for even the resting energy consumption whereas oxidative phosphorylation is sufficient. The effects of tetanic stimulation on living muscles were also studied. When glycolysis and oxidative phosphorylation were suppressed, the intracellular energy store was depleted by the tetanic contraction. Anaerobic glycolysis produced rapid recovery of the energy store level, although it was insufficient to reach the initial level. Aerobic oxidative phosphorylation produced sufficient energy to reach the initial level, and this level was never exceeded. This finding suggests the existence of a regulatory mechanism for the energy store level.View full abstractDownload PDF (567K) -
Roichi ITOH, Takeshi NISHINO, Chikako USAMI, Keizo TSUSHIMA1978 Volume 84 Issue 1 Pages 19-26
Published: July 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA specific rabbit antiserum was prepared against chicken liver xanthine dehydrogenase [EC 1. 2. 1. 37] and used to investigate changes in the amount of the enzyme protein in the liver during dietary adaptation. When the enzyme activity was decreased by the administration of a low protein diet, quantitative immunoprecipitation and Laurell electroimmunoassay showed not only a decrease in the amount of the enzyme antigen but also a reduction of the apparent molecular activity of the enzyme. This was confirmed by the finding that the activity-flavin ratio and the activity-molybdenum ratio of the enzyme were lowered in chickens adapted to the low protein diet.
The relative rate of synthesis of xanthine dehydrogenase, expressed as radioactivity incorporated into the enzyme protein divided by radioactivity incorporated into total cell homogenate protein, was measured by pulse-labeling the liver protein with L-[4, 5-3H]leucine. Xanthine dehydrogenase was isolated by quantitative immunoprecipitation followed by electrophoresis of the dissolved immunoprecipitate on sodium dodecyl sulfate-polyacrylamide gel. The relative rate of synthesis increased 6- to 9-fold, while the enzyme activity increased 10- to 30-fold in vivo following the administration of a high protein diet. The rate of degradation of xanthine dehydrogenase was higher in chickens adapted to the low protein diet than in those adapted to the high protein diet, the apparent half lives being 1 day and 3 days, respectively. Although some limitations and assumptions were present in the techniques used in these experiments, these results suggest that the increase in the hepatic xanthine dehydrogenase content in the chickens adapted to the high protein diet was mainly due to a rise in the rate of enzyme synthesis and, to a minor degree, to a decrease in the rate of enzyme degradation.View full abstractDownload PDF (1227K) -
II. Purification and Properties of a -GlucosidaseToshiya HIRAYAMA, Sigeo HORIE, Hideo NAGAYAMA, Kazuo MATSUDA1978 Volume 84 Issue 1 Pages 27-37
Published: July 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThree components (GA, GB-1, and GB-2) of β-glucosidase were detected in the culture filtrate of Pyricularia oryzae grown in a cellulose or cellulose derivative medium. Among them, GB-1 was induced most strongly.
Purified GB-1 was homogeneous on polyacrylamide gel electrophoresis and showed an approximately 1, 400-fold increase of specific activity over the starting material. The molecular weight was determined to be 240, 000 by sodium dodecyl sulfate-gel electrophoresis. A similar value was also obtained by sucrose density gradient centrifugation. The enzyme contained a high proportion of acidic amino acids and mannose, and the isoelectric point of the enzyme was pH 4.15.
The enzyme had a pH optimum of 5.5 and a temperature optimum at 55°C. -Glucosidase activity was inhibited by Mn2+, Cu2+, Hg2+, p-chloromercuribenzoate, and glucono-δ-lactone. The enzyme split off glucose units one by one from the nonreducing ends of not only β-glucooligosaccharides but also some β-glucans, such as carboxymethylcellulose, laminaran, pustulan, and zeagallan. The affinity for cello- and laminari-oligosaccharides tended to increase in parallel with the chain length.View full abstractDownload PDF (708K) -
Takao NAKAMURA, Ichiro MATSUOKA1978 Volume 84 Issue 1 Pages 39-46
Published: July 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe construction of an adiabatic microcalorimeter using a rapid-response microthermistor (t1/2=50ms) as a detector is described. The apparatus uses an air-tight 5ml Dewar flask as the reaction cell, and permits simultaneous recording of temperature change and oxygen consumption in a reaction medium at a full-scale sensitivity of<0.01° and stability of<0.0003°/ 60s in the measurement of temperature change. Reactant solution could be added to the reaction medium in the cell at any time during the course of a reaction. The apparatus was calibrated by means of the reaction of HCl with NaOH, and those catalyzed by peroxidase and glucose oxidase. Time courses of heat production and oxygen consumption by rat liver mitochondria in various states of respiration with succinate or glutamate as a substrate were recorded. In respiration uncoupled by 2, 4-dinitrophenol (DNP), the apparent enthalpy change per gram atom of oxygen consumed (ΔHa) was in agreement with the heat of combustion of these substrates. In state 4 respiration ΔHa was reduced to 54 or 51% of that in DNP-uncoupled respiration with succinate or glutamate as a substrate, respectively. In state 3 respiration ΔHa was further reduced. On adding DNP to mitochondria after exhaustion of oxygen, heat production was observed in anaerobiosis which was quantitatively in agreement with that expected from the reaction of hydrolysis of ATP, which had been formed in the preceding state 3 respiration, caused by DNP-stimulated ATPase activity of mitochondria.View full abstractDownload PDF (556K) -
Akira KISHIMOTO, Terutoshi MORI, Yoshimi TAKAI, Yasutomi NISHIZUKA1978 Volume 84 Issue 1 Pages 47-53
Published: July 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA cyclic nucleotide-independent protein kinase, which was produced from its proenzyme upon limited proteolysis by a Ca2+-dependent protease (Takai, Y., Yamamoto, M., Inoue, M., Kishimoto, A., & Nishizuka, Y. (1977) Biochem. Biophys. Res. Commun. 77, 542-550), showed an ability to phosphorylate not only muscle glycogen phosphorylase kinase but also glycogen synthase, resulting in activation and inactivation of the respective enzymes, although the protein kinase was less active than adenosine 3': 5'-monophosphate (cyclic AMP)-dependent protein kinase toward glycogen synthase. Available evidence indicates that this new protein kinase shows pleiotropic functions apparently similar to those described for cyclic AMP-dependent protein kinase. Nevertheless, these protein kinases were clearly distinguishable from each other in their response to cyclic nucleotides and susceptibility to protein inhibitor.View full abstractDownload PDF (503K) -
Ryo TAGUCHI, Hiroh IKEZAWA1978 Volume 84 Issue 1 Pages 55-63
Published: July 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe hydrolytic action of phospholipase C and sphingomyelinase on right-side-out and inside-out sealed ghosts prepared from sheep, horse, and human erythrocytes was examined. Phospholipase C from Bacillus cereus acts on phospholipids of right-side-out ghosts of sheep, horse, and human red cells, in spite of its inability to hydrolyze phospholipids in these intact erythrocytes. Phospholipase C from Pseudomonas aureofaciens also acts on sheep right-side-out ghosts in spite of its inability to hydrolyze phospholipids in intact sheep erythrocytes. As regards sealed ghosts of sheep erythrocytes, an asymmetric distribution of phospholipids was suggested: sphingomyelin was present mainly on the outer surface of right-side-out ghosts and on the inner surface of inside-out ghosts, while phosphatidylethanolamine was present mainly on the inner surface of right-side-out ghosts and on the outer surface of inside-out ghosts. However, the possibility of translocation of phospholipids during preparation was not completely excluded in our experiments.
Contrary to our expectation, phospholipases C from Bacillus cereus and Pseudomonas aureofaciens, which can act on pure phosphatidylcholine and phosphatidylethanolamine, but not on sphingomyelin, attacked phosphatidylethanolamine of horse right-side-out ghosts more easily than that of horse inside-out ghosts. Furthermore, latency of inside-out ghosts was not affected even by the extensive hydrolysis of phospholipids, while latency of right-side-out ghosts was rapidly reduced by the actions of phospholipase C and sphingomyelinase. The physical state of phospholipids on inside-out ghosts may be more complicated than that on right-side-out ghosts. Total activity of NADH-cytochrome c oxidoreductase was depressed by hydrolysis of phospholipids on ghosts and became more sensitive to inhibition by saponin.View full abstractDownload PDF (666K) -
Selective Cleavage of Petide Bonds by a Serine Protease from the Muscle Layer of Rat Small IntestineKeiko KOBAYASHI, Nobuhiko KATUNUMA1978 Volume 84 Issue 1 Pages 65-74
Published: July 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe kinetic constants of a serine protease from the muscle layer of rat small intestine for three ester substrates were compared with those reported for bovine chymotrypsin A. The Km values for acetyl-tyrosine ethyl ester and acetyl-phenylalanine ethyl ester were very similar to those of chymotrypsin A, but the catalytic activity per mol of serine protease was only one fiftieth as high as that of chymotrypsin A.
The selectivity of action of the serine protease from the muscle layer of small intestine was examined using various polypeptide substrates, such as glucagon, oxidized insulin B chain, luteinizing hormone-releasing hormone, and neurotensin. Among the peptides bonds cleaved in these substrates, the most susceptible bonds were Tyr-Leu, Trp-Leu, Phe-Phe, Tyr-Ile, and Tyr-Gly, while Phe-Tyr and Pro-Arg-Arg-Pro were less susceptible. However, unlike the chymotrypsin group, when the amino acid on the carboxyl side of tyrosine, tryptophan or phenylalanine was serine, threonine or glutamic acid, these peptide bonds were not susceptible to the protease under the experimental conditions. These results suggest that the specificity of the serine protease from the muscle layer of small intestine is that of the chymotrypsin group, but differs from that of chymotrypsin A or C.View full abstractDownload PDF (730K) -
Toshinobu SUZAKI, Hikoichi SAKAI, Sachiko ENDO, Ichiro KIMURA, Yoshino ...1978 Volume 84 Issue 1 Pages 75-81
Published: July 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMicrotubule (MT) assembly from one-cycle porcine brain tubulin was analyzed by viscometry, turbidimetry, sedimentation and electron microscopy in the presence of various kinds of anions and different amounts of GTP. Using K or Na salts, the effectiveness for increasing the viscosity of tubulin solution was in the order glutamate, fluoride, acetate>chloride>bromide>iodide. The magnitude of tubulin polymerization was measured as a function of the K glutamate concentration by the sedimentation method. The amount of assembled MT's was saturated even at a glutamate concentration of 0.05M and the tubulin-MT equilibrium remained stationary until the glutamate concentration was increased to 0.9M. On the other hand, the specific viscosity increased with increasing glutamate concentration from 0.05 to 0.35M. Furthermore, at GTP concentrations ranging from 0.4 to 1.4mm, tubulin solution containing 0.1M glutamate exhibited a stable and constant plateau value of turbidity. However, simultaneous viscosity measurements indicated that the viscosity increase was dependent on the GTP concentration. The maximum viscosity attained at 1.4mm GTP was twice that attained at 0.4mm GTP. Viscometry again did not accurately reflect the mass concentration of the assembled MT's. After the viscosity of a tubulin solution with low GTP content had reached a plateau, additional GTP caused the viscosity to increase to twice the original level without any change in turbidity. The apparent GTPase activity of the tubulin solution was 0.002μmol P1 released/mg/min, which caused a significant amount of GTP to be hydrolyzed during a long incubation. These results suggested that the changes in the viscosity of MT suspension without any change in the mass concentration reflect changes in the flexibility of MT's, probably caused by reversible loosening of a part of the MT surface lattice depending on the concentrations of glutamate and GTP.View full abstractDownload PDF (527K) -
Toshio MURAKAMI, Takashi MURACHI1978 Volume 84 Issue 1 Pages 83-91
Published: July 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe conformational changes in a hemoglobin with modified heme have been studied, employing its susceptibility to proteases as an index. The heme-modified derivatives, choleglobin and verdohemoglobin, were obtained by ascorbic acid-coupled oxidation of human oxyhemoglobin. Tryptic hydrolysis showed that choleglobin and verdohemoglobin are approximately 10 times and 15 times more susceptible to the protease, respectively, than oxyhemoglobin, but that the rate of hydrolysis of verdohemoglobin is less than one-third of that of a globin freed from heme. A considerable enhancement of proteolytic susceptibility after ascorbic acidcoupled oxidation was also observed in the case of monomeric myoglobin, while no change in susceptibility occurred in the case of cytochrome c, in which the heme remained intact after the ascorbic acid treatment. Similar results were obtained with α-chymotrypsin and papain. These observations have led us to conclude that the oxidation of the heme group in a heme protein induces some conformational changes in its protein moiety, causing a marked enhancement of proteolytic susceptibility. Circular dichroism and gel filtration measurements demonstrated that these conformational changes in choleglobin and verdohemoglobin are not as drastic as those involving removal of heme or dissociation into dimers and monomers, or unfolding of the backbone structure, but are more localized changes occurring on the surface of the molecule.View full abstractDownload PDF (642K) -
Kanefusa KATO, Yasuhisa HARUYAMA, Yoshitaka HAMAGUCHI, Eiji ISHIKAWA1978 Volume 84 Issue 1 Pages 93-102
Published: July 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSGuinea pig anti-porcine insulin was determined quantitatively by three enzyme-linked procedures. (Anti-insulin immunoglobulins in the antiserum used were mostly IgG.) Procedure a: Anti-insulin to be assayed was bound to insulin immobilized on a small piece of silicone rubber and anti-insulin IgG bound onto the solid phase was allowed to react with Fab' of rabbit anti-guinea pig IgG conjugated with β-D-galactosidase from Escherichia coli. Procedure b: Anti-insulin was trapped by insulin immobilized on silicone rubber and allowed to react with insulin conjugated with β-D-galactosidase. Procedure c: IgG in anti-insulin serum was trapped by rabbit anti-guinea pig IgG antibody (IgG) immobilized on silicone rubber and anti-insulin trapped was allowed to react with insulin conjugated with β-D-galactosidase. A linear dose-response relation of β-D-galactosidase activity bound onto the solid phase to the dilution of anti-insulin serum with a buffer was observed between 3×105-fold and 9×104-fold dilutions by Procedures a and c and between 3×105-fold and 3×102-fold dilutions by Procedure b. Non-specific adsorption of normal guinea pig IgG onto silicone rubber resulted in an excessively high blank in the assay by Procedure a when the dilution of the antiserum with buffer was less than 9×104-fold. The binding of insulin-β-D-galactosidase conjugate to the solid phase in the assay by Procedure b was significantly inhibited by unknown factor(s) in normal serum when the dilution of the serum in the assay mixture was less than 100-fold. The amounts of serum that could be used in Procedure c was limited (to 150, μl of 9×104-fold diluted serum) by the amount of anti-guinea pig IgG antibody immobilized on the solid phase. Because of these limitations, the maximal dilution of antiserum with normal serum that still gave a linear dose-response relation was 300-, 10, 000-, and 1, 000-fold in Procedures a, b, and c, respectively.View full abstractDownload PDF (756K) -
Fumiya OBATA, Rume SAKAI, Hiroyuki SHIOKAWA1978 Volume 84 Issue 1 Pages 103-109
Published: July 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSConcanavalin A (Con A) consisting solely of intact subunits, i.e., fragment-free Con A, was prepared by the following procedure. Con A obtained by a modification of the method of Cunningham et al. (Abe, Y. & Ishii, S. (1974) Seikagaku (in Japanese) 46, 559) was dialyzed against 1 mm EDTA and chromatographed on a CM-cellulose column at pH 7.1 in the presence of 1mm EDTA. The chromatography gave three major peaks, F1, F2, and F3. Fragment-free Con A was obtained in the peak eluted last (F3). This Con A gave a single band on polyacrylamide gel electrophoresis (PAGE) in sodium dodecyl sulfate (SDS), but gave a minor band as well as the major band in PAGE at pH 4.5. The minor band was removed by gel filtration on a Bio-Gel P-150 column. By our method, fragment-free Con A can be prepared even from jack bean meal containing a large proportion of fragment-containing Con A.
In the above CM-cellulose chromatography, a period of dialysis of Con A against a buffer containing EDTA was very important for the preparation of fragment-free Con A. When Con A was dialyzed overnight, the protein could be fractionated as described above. However, Con A which had been dialyzed for a week was not adsorbed at all on the column. In the presence of metals (Ca2+ and Mn2+), Con A was adsorbed on the column equilibrated with a metal-containing buffer and eluted as a single peak. Results in the three kinds of chromatography suggest that negatively charged Con A is more demetalized than positively charged Con A, that fragment-containing Con A loses bound metals more rapidly than fragment-free Con A, and that Con A is demetalized in discrete step(s).View full abstractDownload PDF (1586K) -
Yutaka KIRINO, Taka'aki OHKUMA, Hiroshi SHIMIZU1978 Volume 84 Issue 1 Pages 111-115
Published: July 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe technique of saturation transfer electron spin resonance has been applied to study the rotational diffusion of spin-labeled Ca2+, Mg2+-dependent ATPase molecules in the membranes of sarcoplasmic reticulum vesicles. Comparison of the present data with those for spin-labeled hemoglobin undergoing isotropic rotation leads to a value of 2×10-4s for the apparent rotational correlation time at 20°C for the membrane-bound protein. Consideration of the anisotropy of the Brownian rotation of the membrane-bound ATPase suggests that the true correlation time for the expected axial rotation may be somewhat smaller than the apparent value. An Arrhenius plot of the rotational motion shows a break, which is interpreted as indicating the occurrence of a conformational change of the ATPase molecule at about 15°C.View full abstractDownload PDF (384K) -
I. Different Effects of Cadmium Administered In Vivo to Adult, Young, and Ethionine-Fed RatsNobuhiro SATO, Takenobu KAMADA, Toshihiko SUEMATSU, Motoaki SHICHIRI, ...1978 Volume 84 Issue 1 Pages 117-125
Published: July 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe changes in liver mitochondrial respiratory activities and cytochrome concentrations were investigated when cadmium chloride was administered orally to adult, young, and ethionine-fed rats. Following a seven-day administration of 30ppm cadmium in drinking water, adult rats showed no change, while young rats and ethionine-fed rats exhibited a marked increase in mitochondrial respiration with concomitant decrease of respiratory control index and P/O ratio. The concentrations of cytochromes aa3, b, and c+c1 in liver mitochondria were unchanged in adult rats, but increased significantly in ethionine-fed rats. In young rats receiving cadmium the liver mitochondrial protein increased with a slight change in the cytochrome concentration in mitochondria. It was further found that in adult rats a higher concentration (300ppm) of cadmium in drinking water was toxic to the liver mitochondrial functions. Thus, the effect of oral administration of cadmium on the liver mitochondrial function depends on the condition of the animals.View full abstractDownload PDF (599K) -
II. Protective Effect of Hepatic Soluble Fraction against Cadmium-Induced Mitochondrial DysfunctionNobuhiro SATO, Takenobu KAMADA, Toshihiko SUEMATSU, Hiroshi ABE, Fujiy ...1978 Volume 84 Issue 1 Pages 127-133
Published: July 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCadmium chloride (Cd) stimulated state 4 respiration at a concentration as low as 0.3nmol/mg protein of mitochondria (ca. 0.3μm) and completely inhibited respiration at a concentration of 10nmol/mg protein of mitochondria (ca. 10μM) in isolated rat liver mitochondria. A spectrophotometric study has suggested that one of the interaction sites of Cd in mitochondria is located between the cytochromes b and the substrates, possibly in the flavin region. The sensitivity of mitochondrial respiration to Cd was almost the same in preparations from both adult and young rat livers. It was found that the soluble fraction prepared from the adult rat liver had a protective effect against the Cd-induced inhibition of mitochondrial respiration when the fraction was incubated in the mitochondria) respiratory assay medium. However, the protective effect of the souble fraction prepared from young rat liver was much smaller than that of the fraction obtained from adult rat liver. It is thus suggested that the interaction of Cd with liver soluble protein(s) is important for minimizing the Cd toxicity to mitochondria, and that differences in the protective effect of the liver soluble fraction might be responsible for the different sensitivities to Cd toxicity of adult and young rat livers, which was reported in the preceding paper (Sato, N. et al. (1978) J. Biochem. 84, 117-125). The protective effect of the liver soluble fraction prepared from young rat (31 days old) became more apparent upon prior treatment of the young rats with spironolactone (0.12g/kg body weight).View full abstractDownload PDF (534K) -
Kaoru MIYAZAKI, Hideaki HAGIWARA, Yosinobu NAGAO, Yuhsi MATUO, Takekaz ...1978 Volume 84 Issue 1 Pages 135-143
Published: July 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. The total contents of histones and non-histone proteins in the nuclei prepared in the presence of diisopropytfluorophosphate from five kinds of normal tissues and Rhodamine sarcoma of rats were determined, and the species and relative contents of the individual proteins were analyzed by SDS-polyacrylamide gel electrophoresis.
2. In all kinds of tissues examined, the histones were similar in total content, species and relative content.
3. The total content of non-histone proteins varied with the kind of tissue as follows: Rhodamine sarcoma_??_brain ≥ liver_??_kidney_??_spleen ≥ thymus. In each kind of tissue examined, a few species of non-histone proteins were specific, whereas the remaining species were common. However, the relative contents of the individual species varied with the kind of tissue. Most of the tissue-specific non-histone proteins had molecular weights ranging between approximately 30, 000 and 40, 000 daltons.
4. When Rhodamine sarcoma grew on the back of the animals, the total content of non-histone proteins, but not that of histones, decreased appreciably in the liver. In other tissues, the decrease was insignificant. The decrease in the total content of non-histone proteins in the liver was mostly due to a decrease in a non-histone protein of approximately 52, 000 daltons and six non-histone proteins between approximately 30, 000 and 40, 000 daltons.
5. The tissue-specific distribution of non-histone proteins in all the tissues examined, and its change in the liver with the growth of Rhodamine sarcoma were not caused by proteases present in the nuclei.View full abstractDownload PDF (632K) -
Masaki NAKANE, Toshinori IDE, Kaijiro ANZAI, Susumu OHARA, Toshiwo AND ...1978 Volume 84 Issue 1 Pages 145-157
Published: July 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSUpon gentle lysis of exponentially growing mouse carcinoma cells FM3A by sodium dodecyl sulfate, DNA was released as a “DNA-protein complex” in a folded conformation. No histones could be detected in the DNA-protein complex. The proteins bound to DNA were found to be composed of several kinds of nonhistone proteins with a molecular weight range of 50, 000 to 60, 000; they appear to play a key role in stabilizing and maintaining the compact and folded structure of the complex. Removal of the proteins by Pronase or 2-mercaptoethanol produced a more relaxed structure sedimenting about half as fast as the original complex in a neutral sucrose gradient.
DNA in the folded complex is supercoiled, as indicated by the characteristic biphasic response of its sedimentation rate to increasing concentration of various intercalating agents, actinomycin D, ethidium bromide and acriflavine, with which the cells were treated before lysis. Pronase- or 2-mercaptoethanol-treated relaxed DNA still possessed the characteristic of closed-circular structure as judged from its response to intercalating agents. Nicking with γ-ray or 4NQO broke these superhelical turns and relaxed the folded complex to slower sedimenting forms equivalent to the relaxed DNA obtained on treatment with Pronase or 2-mercaptoethanol. Viscometric observations of DNA-protein complex were consistent with the above results.
A tentative model for the structure of this DNA-protein complex is proposed in which supercoiled DNA is folded into loops by several kinds of nonhistone proteins. Autoradiographic examination of the complex appeared to support this model.View full abstractDownload PDF (1661K) -
Noriyuki KOHASHI, Kenji YAMAGUCHI, Yu HOSOKAWA, Yoshihiko KORI, Osamu ...1978 Volume 84 Issue 1 Pages 159-168
Published: July 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA 20-fold increase in cysteine dioxygenase activity in the liver was found in intact rats fed on a 40% protein diet compared with rats fed on a 2.1% protein diet. The hepatic enzyme activity of rats fed on a 20% protein diet were only one-tenth of that of rats fed on the 40% protein diet. This suggests that cysteine dioxygenase in the liver is a regulatory enzyme in the cysteine degradative pathway under normal nutritional conditions.
The hepatic cysteine contents exhibited no significant change in rats fed on 2-20% protein diets but those in rats fed on a 30% protein diet were 2.5-fold those in rats fed on the 20 protein diet. The hepatic taurine contents were not changed by feeding 2-30% protein diets but were increased to 3 times those of rats fed on the 20Y. protein diet by feeding the 40 protein diet. The hepatic contents of both cysteine and taurine were not further increased by feeding diets of higher protein contents. On the other hand, urinary taurine excretion increased proportionally to the dietary protein levels.
The hepatic cysteine dioxygenase activities of adrenalectomized rats did not respond to the increase of dietary protein. The cysteine levels in the livers of adrenalectomized rats fed on a 75% protein diet were nevertheless increased to 0.32μmol/g liver weight, whereas those of intact rats fed on the same diets were 0.19μmol/g liver weight.
The inclusion of only cysteine or methionine of the amino acids so far tested in the 2% protein diet caused the hepatic cysteine dioxygenase activity to increase to the level of rats fed on the 75% protein diet.
The hepatic level of cysteine desulfhydrase, another cysteine-degrading enzyme, was much lower than that of cysteine dioxygenase and did not significantly respond to the dietary protein contents.
In conclusion, cysteine dioxygenase plays an important role in the regulation of intra-cellular levels of both methionine and cysteine, and also glutathione, in rat liver.View full abstractDownload PDF (707K) -
Ken-ichi TSUTSUMI, Reiko TSUTSUMI-MAJIMA, Kensuke SHIMURA1978 Volume 84 Issue 1 Pages 169-177
Published: July 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA specific endonuclease involved in the processing of tRNA precursors was isolated and partially purified from the posterior silk gland of Bombyx mori, and designated as RNase P.Bmo. This enzyme was shown to catalyze the conversion of 4.5S precursor RNA to 4.1S RNA by trimming the 5'-additional segment from the precursor RNA. RNase P.Bmo required divalent cations, Mg2+ or Mn2+ In the presence of these divalent cations, K+ or NH4+ activated the RNase P.Bmo reaction. Optimum pH was observed around 8.0.
Ribosomal RNA's and mature tRNA from the silk gland were not cleaved by RNase P.Bmo. A 4.5S precursor RNA fraction containing formycin, an adenosine analog, was less susceptible to RNase P.Bmo than the normal one. These results indicate that RNase P.Bmo has a high substrate specificity.
An additional nuclease(s) was isolated. This activity was assumed to remove the extra 3'-segment of the 4.5S precursor RNA.View full abstractDownload PDF (608K) -
Takeshi MIZUNO, Makoto KAGEYAMA1978 Volume 84 Issue 1 Pages 179-191
Published: July 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA method is described for the preparation of outer and cytoplasmic membranes of Pseudomonas aeruginosa, and the outer membrane proteins characterized. Isolated outer and cytoplasmic membranes differed markedly in the content of 2-keto-3-deoxyoctonate (lipopolysaccharide) and phospholipid as well as in the localization of certain enzymes (NADH oxidase, succinate dehydrogenase, D-lactate dehydrogenase, malate dehydrogenase, and phospholipase), and also in the microscopic morphology. The outer membrane preparation showed activity neutralizing a certain bacteriocin or bacteriophages, whereas the cytoplasmic membrane preparation showed no neutralizing activity. The protein composition of membrane preparations from five different strains of P. aeruginosa {P14, M92 (PAO1), PACT, P15, and M2008 (PAT)} were determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. More than 50 protein bands were detected in the cytoplasmic membrane preparation. The protein compositions of outer membranes from the five different strains were very similar: at least 6 major bands were found (apparent molecular weights: Band D, 50, 000; band E, 45, 000; band F, 33, 000; bands G and H, 21, 000; and band I, 8, 000). The protein composition of outer membranes was affected by some physiological growth conditions. Some features of major outer membrane proteins were also studied. Band F showed anomalous migration on SDS polyacrylamide gel electrophoresis depending on the solubdizing conditions or pretreatment with TCA. Band I seemed to be a protein analogous to the lipoprotein which had been found in the outer membrane of Escherichia coli.View full abstractDownload PDF (4451K) -
Hiroshi NAKAJIMA, Koichi SUZUKI, Kazutomo IMAHORI1978 Volume 84 Issue 1 Pages 193-203
Published: July 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. Acetate kinase [EC 2. 7. 2. 1] from an thermophile, B. stearothermophilus, was purified and crystallized.
2. This enzyme was shown to be a tetramer of identical subunits which had a molecular weight of about 40, 000. Amino acid analysis showed no SH group. By analyzing the CD spectrum it was deduced that this enzyme is composed of 36% β-structure, 21% α-helix and 43% unordered structure.
3. This enzyme shared many common enzymatic properties with the counterpart from mesophiles, i.e. pH optimum, substrate specificity, requirement of metal ions and essential amino acid residues necessary for the catalytic activity. However, this enzyme was remarkably thermostable.
4. A plot of the reaction velocity against the concentration of acetate, ADP or acetyl phosphate gave a curve of the Michaelis-Menten type. However, such a plot against ATP gave a sigmoid curve, suggesting a homotropic allosteric nature of the enzyme.
5. From the results of chemical modification it was deduced that an amino group and an imidazole group, at least, are involved in the active site of the enzyme.View full abstractDownload PDF (1382K) -
Hiroshi KIMURA, Susumu YAMATO, Takashi MURACHI1978 Volume 84 Issue 1 Pages 205-211
Published: July 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSNative human oxyhemoglobin, which has a rigid conformation resistant to proteases such as trypsin and subtilisin, could be hydrolyzed by these proteases at pH 7.0 after treatment with p-chloromercuribenzoate. The digestion curve of hemoglobin as a function of concentration of the mercurial was essentially parallel to the titration curve of hemoglobin with the mercurial, indicating that a relationship exists between susceptibility to proteases and modification of thiol groups of the protein. On the other hand, when myoglobin was used as a substrate, the degree of proteolysis did not increase after treatment with the mercurial. Circular dichroism measurements and gel-filtration experiments showed that the observed increase in susceptibility of hemoglobin to proteases was not due to a conformational change involving unfolding of α-helical structure, but was due to the dissociation of the tetrameric hemoglobin molecule into dimer and monomer after treatment with the mercurial.View full abstractDownload PDF (546K) -
III. Biosynthesis of NADPH-Cytochrome c Reductase and Cytochrome b5 by Loosely-Bound RibosomesTomoyuki HARANO, Tsuneo OMURA1978 Volume 84 Issue 1 Pages 213-223
Published: July 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMembrane-bound ribosomes were separated into two distinct classes (loosely-bound and tightly-bound ribosomes) by treatment with 0.6M KCl, 1mM puromycin, 0.05% DOC, or 10miss EDTA. It was also confirmed that any one of these reagents except for EDTA dissociated the same class of ribosomes from the membrane. A population of lighter microsomal vesicles was formed from rough microsomes upon the dissociation of loosely-bound ribosomes by treatment with these chemicals.
Rough microsomes were subfractionated into lighter and heavier fractions, L-rMs and H-rMs, by centrifugation using a discontinuous gradient of sucrose consisting of 1.3M, 1.5M, and 2.1M solutions. It was found that L-rMs was rich in loosely-bound ribosomes, whereas H-rMs contained a high proportion of tightly-bound ribosomes. It is likely that loosely-bound and tightly-bound ribosomes are heterogeneously distributed among rough microsomal vesicles.
Loosely-bound ribosomes and tightly-bound ribosomes synthesize different kinds of proteins. Two microsomal membrane proteins, NADPH-cytochrome c reductase and cytochrome b5, were exclusively synthesized by loosely-bound ribosomes, whereas serum albumin, which is a major component of the secretory proteins of hepatocytes, was synthesized only by tightly-bound ribosomes. Since the nascent peptides of NADPH-cytochrome c reductase and cytochrome b5 are released from bound ribosomes to the cytoplasmic surface of endoplasmic reticulum, while those of secretory proteins are discharged into the lumen across the membrane, the strength of the association between ribosomes and microsomal membrane seems to be correlated with the direction of release of nascent peptides.View full abstractDownload PDF (794K) -
I. Purification and CharacterizationShoichi ISHIURA, Hiromu MUROFUSHI, Koichi SUZUKI, Kazutomo IMAHORI1978 Volume 84 Issue 1 Pages 225-230
Published: July 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA calcium-activated neutral protease was purified 2, 700-fold over the crude extract from chicken skeletal muscle. The purified protease migrated as a single band on polyacrylamide gel electrophoresis with or without SDS. Its molecular weight was 80, 000 and pH optimum for activity was 7.7. The activity required strictly the presence of calcium (optimum concentration: 1.8mM) or strontium (optimum concentration: 10mM) ions. The protease was inhibited by leupeptin, which is known to be a strong inhibitor of papain, cathepsin B, trypsin, and plasmin.View full abstractDownload PDF (851K) -
Kanji FUJIKI, Akio FUKUDA, Yoshimi OKADA1978 Volume 84 Issue 1 Pages 231-234
Published: July 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe terminal bases of the viral ribonucleic acid from the Caulobacter ribonucleic acid phage φCp2 were examined. The viral ribonucleic acid contained guanosine triphosphate (pppG) at the 5'-terminus and adenosine (AOH) at the 3'-terminus.View full abstractDownload PDF (256K) -
Cristobal G. dos REMEDIOS, Darcy GILMOUR1978 Volume 84 Issue 1 Pages 235-238
Published: July 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSUsing glycerinated rabbit psoas myofibrils we demonstrate by electron microscopy that there is a filamentous material which remains after extraction of the fibrils with a modified Hasselbach-Schneider solution (containing 1.0M KCl) followed by extraction with 0.6M KI. The fibrils were visualized by conventional negative staining techniques using either 2% ammo-nium molybdate or 1% uranyl acetate. The appearance of the extracted fibrils together with their solubility properties strongly suggests that the extraction-resistant filamentous material is composed of connectin, an elastic protein recently characterized from muscle tissues.View full abstractDownload PDF (768K) -
Naozi TOYODA, Koscak MARUYAMA1978 Volume 84 Issue 1 Pages 239-241
Published: July 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA net-like structure extending through Z lines of myofibrils in frog cardiac muscle was clearly demonstrated under an electron microscope after the removal of myosin and actin. The diameter of the very thin filaments forming the nets was approximately 2nm; this width was thinner than that of the actin filament. The net structure is ascribed to connectin, an elastic protein of muscle.View full abstractDownload PDF (1191K) -
1978 Volume 84 Issue 1 Pages 243
Published: 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDownload PDF (21K)
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