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Volume 68 (1970)
- Issue 6 Pages 763-
- Issue 5 Pages 603-
- Issue 4 Pages 427-
- Issue 3 Pages 255-
- Issue 2 Pages 137-
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Volume 68, Issue 5
Displaying 1-19 of 19 articles from this issue
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Ryuichi KATO, Atsushi TAKAHASHI, Yoshihito OMORI1970 Volume 68 Issue 5 Pages 603-612
Published: November 25, 1970
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSProgesterone hydroxylation activity of liver microsomes was investigated in male and female rats under altered states of thyroid function. Treatment with thyroxine markedly decreased the activity of progesterone hydroxylation by liver microsomes in male rats, but did not significantly alter the activity in female rats. In contrast, the conversion of progesterone to Δ4-reduced metabolites was increased by treatment of male rats with thyroxine. Thyroidectomy also markedly decreased the progesterone hydroxylating activity in male rats, but did not decrease in female rats. The content of P-450 per microsomal protein and the magnitude of progesterone-induced spectral change per microsomal protein and per P-450 were decreased in thyroxine-treated male rats, but similar changes were not observed in thyroidectomized male rats.
From these results it is conceivale that the progesterone hydroxylating activity may be reduced through the decrease in the content of P-450 and the substrate in-teraction with P-450 in liver microsomes from thyroxine-treated male rats, whereas in those from thyroidectomized rats, the hydroxylating activity may be reduced through the decrease in the reduction of P-450 binding to the substrate.
These results indicated that the effects of thyroxine treatment and thyroidectomy on the progesterone hydroxylating activity in liver microsomes of male and female rats were similar to those on the activities of aminopyrine N-demethylation and hexobarbital hydroxylation which are regulated by androgen. On the basis of these results, mechanism(s) of decrease in the progesterone hydroxylating activity in thyroxine-treated and thyroidectomized male rats is discussed.View full abstractDownload PDF (796K) -
Ryuichi KATO, Akira TAKANAKA, Atsushi TAKAHASHI1970 Volume 68 Issue 5 Pages 613-623
Published: November 25, 1970
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe administration of thyroxine to male rats decreased the content of P-450 and the magnitude of spectral change of P-450 induced by various drugs in hepatic microsomes.
The magnitudes of spectral changes per P-450 induced by hexobarbital and aminopyrine were decreased, whereas those induced by aniline and zoxazolamine were not significantly affected.
In contrast to the results observed with male rats, the magnitudes of the spectral changes induced by hexobarbital and aminopyrine were not significantly decreased in microsomes from thyroxine-treated female rats.
These results suggest that the binding capacity of P-450 with hexobarbital and aminopyrine was decreased by thyroxine probably through an impairment of androgen action to increase the binding capacity of P-450. The decrease in the binding capac-ity of P-450 with hexobarbital and aminopyrine is assumed to be a responsible factor for the decrease in the activities of hexobarbital hydroxylation and aminopyrine N-demethylation.
Since the ratios of the hydroxylating activities of the drugs to the magnitudes of spectral changes were increased in microsomes from thyroxine-treated rats, the increase in the rate of the reduction of P-450-drug complex has been suggested.
Thyroidectomy did not affect the content of P-450, but decreased the binding capacity of P-450 with hexobarbital and aminopyrine in the males.
Since the ratios of the hydroxylating activities of the drugs to the magnitudes of spectral changes were decreased in microsomes from thyroidectomized rats, the decrease in the rate of the reduction of P-450-drug complex has been suggested.
The Km (Michaelis constant) values and Ks (spectral dissociation constant) values for hexobarbital were increased in microsomes from thyroxine-treated male rats, whereas the Km and Ks values for aniline were not affected.
Since the Km and Ks values for hexobarbital are clearly dependent on androgen, these results again suggest the impairment of androgen action in thyroxine-treated male rats.View full abstractDownload PDF (893K) -
II. Preparation and Some Properties of Mitochondria from MyceliaMasao KAWAKITA1970 Volume 68 Issue 5 Pages 625-631
Published: November 25, 1970
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMitochondria were isolated from young mycelia of Aspergillus orvzae. A set of spring-loaded, counter-rotating rollers of stainless steel were used to disrupt cell walls. The mitochondrial preparation oxidized several intermediates of the tricarboxylic acid cycle and showed distinct respiratory control. Oxygen uptake was strongly in-hibited by cyanide and antimycin A. Rotenone or Amytal inhibited oxidation of NAD-linked substrates only incompletely even at higher concentrations than those required for complete inhibition of the oxidation by mammalian mitochondria. NAD-linked substrates produced larger P/O ratios than succinate. Site I phosphorylation seems to occur in contrast to the case with mitochondria from Saceharomyces. 2, 4-Dinitrophenol uncoupled phosphorylation from respiration at a concentration of 10-4M. Tri-n-butyl-tin chloride depressed state 3 respiration to the level of state 4 res-piration with concomitant lowering of the P/O ratio. Inhibition of state 3 respira-tion by this reagent was released by 2, 4-dinitrophenol.View full abstractDownload PDF (1179K) -
Isamu SHIIO, Hachiro OZAKI1970 Volume 68 Issue 5 Pages 633-647
Published: November 25, 1970
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSGlutamate dehydrogenase [EC 1. 4. 1. 4] was sonically extraced from Brevibacterium flavum, a glutamate-producing bacterium, and purified 65-fold. The enzyme was specific for NADP(H), and showed homotropic interactions with respect to α-keto-glutarate and L-glutamate. The enzyme showed two peaks of activity in the chro-matogram obtained by Sephadex G-200 gel-filtration. However, when the gel filtra-tion was carried out with the buffer containing 0. 1M KCl, only one peak corresponding to a smaller molecular weight species was observed. In the presence of KCl, the enzyme did not exhibit the homotropic interactions described above and was markedly activated at lower concentrations of α-ketoglutarate and glutamate. The result of kinetic analysis indicated that the enzymatic reaction proceeded in a sequential order of addition of substrates. In the forward reaction (glutamate formation), NADPH, α-ketoglutarate, and ammonium ion react in this order with the enzyme, and glutamate and then NADP+ is released from the enzyme. The Michaelis and dissociation constants for these five reactants were determined. The enzyme was inhibited by the reaction products, glutamate in the forward reaction and ammonium ion and α-ketoglutrate in the reverse reaction, but not by other TCA**-cycle inter-mediates and amino acids. The control of activity by this product inhibition was interpreted as a mechanism of biological regulation of glutamate synthesis and deg-radation.View full abstractDownload PDF (1011K) -
Tatsuhiko YAGI1970 Volume 68 Issue 5 Pages 649-657
Published: November 25, 1970
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSParticulate hydrogenase [EC 1. 12. 2. 1] of Desulfovibrio vulgaris has been solubilized and purified to a nearly homogeneous state. Its isoelectric point was about 6.25. It contained about 8 atoms Fe per mole.
This enzyme is specific to cytochrome c3 as an electron carrier in the H2 evolu-tion and H2 consumption reactions. pKfor ferrocytochrome c3 in the H2 evolution was 4.15. In this reaction, reduced methyl viologen could replace ferrocytochrome c3 to a limited extent, but the reaction with this artificial electron carrier was stim-ulated by cytochrome c3. “pKm” for this stimulating effect of cytochrome c3 was 5.90. The enzyme had, thus, two Kms for ferrocytochrome c3 in the H2 evolution reaction.
In the H2 consumption reaction, Km for H2 was less than 8 mmHg. The enzyme catalyzed the hydrogen-deuterium exchange reaction in the absence of cytochrome c3. This reaction was also stimulated by cytochrome c3.
No significant isotope effect was observed in the H2 evolution and H2 consump-tion reactions catalyzed by hydrogenase.View full abstractDownload PDF (1387K) -
XI. Modification of the Single Arginine Residue in Ribonuclease T1 by Phenylglyoxal and GlyoxalKenji TAKAHASHI1970 Volume 68 Issue 5 Pages 659-664
Published: November 25, 1970
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. In order to obtain some information on the role of the single arginine residue at position 77 in the enzymatic activity of ribonuclease T, [EC 2. 7. 7. 26], the enzyme was allowed to react at pH 8.0 and 25°C with phenyiglyoxal and glyoxal, reagents known to modify arginine residues in proteins fairly specifically. When the enzyme (0.16% solution) was treated with a 760-fold molar excess of phenyiglyoxal, activity toward both RNA and 2', 3'-cyclic guanylic acid was lost in parallel. Under this condition the half life was about 4 hr. A similar inactivation occurred when the enzyme was treated with glyoxal. In this case, however, the activity toward RNA was lost somewhat faster than that toward 2', 3'-cyclic guanylate. Inactivation was slower in the presence of 3'-guanylic acid.
2. Amino acid analyses of acid hydrolysates of the inactivated proteins showed that the arginine residue and one alanine residue (presumably the amino-terminal residue) had been lost and that the loss of activity almost paralleled the loss of the arginine residue. The reactivity of the active site glutamic acid-58 toward iodoacetate was also decreased almost in parallel with the loss of the arginine residue by reac-tion with phenyiglyoxal.
3. These lines of evidence seem to indicate that the single arginine residue in ribonuclease T1 is present at or near the active center of the enzyme and may be part of the binding site or involved in building the architecture of the active center of the enzyme.View full abstractDownload PDF (398K) -
X. Functions of Two Subfragments, S-1, of the Myosin MoleculeYutaro HAYASHI, Yuji TONOMURA1970 Volume 68 Issue 5 Pages 665-680
Published: November 25, 1970
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe yield of S-1*** from tryptic digests of HMM was 65%. The s20, W value of S-1 was 5.03S at a concentration of 14.2 mg per ml. S-1 decomposed into smaller com-ponents at a rate of about 7% of total material per day in neutral salt solution at 0°C. A quarter as much degradation occurred on addition of 0.07M sucrose. The Ca2+-ATPase [EC 3. 6. 1. 3] activity of S-1 decreased in proportion to increase in the amount of smaller components. In 4.6-5M guanidine-HC1 and in alkaline (pH 11-11.5) solution, S-1 showed a broad sedimentation pattern with s20, w values of the peak of 1.1 and 1.5 S, respectively. The pattern of elution from Sephadex G-200 and G-100 showed that S-1 was degraded into small components in 5M guanidine-HCl or at pH 11.
The ratio of the ATPase activities of myosin (M. W. 4.8×105), HMM (3.4×105) and S-1 (1.2×105) on a molar basis was 1:1:0.5. This ratio was independent of the modifiers used. When myosin was subjected to carboxamidomethylation under the conditions where one specific cysteine residue was completely modified with IAA, the Ca2+ and Mg2+-ATPase activities of myosin increased 11.7 and 9.7 fold, respec-tively, but the EDTA-ATPase activity decreased by 76%. HMM and S-1 were pre-pared from this modified myosin. The extents of activation and inhibition of ATPase activities by IAA during their production of these fragments remained almost con-stant.
The amount of the initial stoichiometric burst of Pi-liberation per mole of S-1 was 0.55 to 0.6 mole, which was half of those of myosin and HMM. The rate of initial rapid Pi-liberation was independent of the ATP concentration when the latter was lower than 0.6 mole per mole of S-1, but at concentrations above this, it in-creased with increase in ATP concentration. The amount of initial rapid Pi-libera-tion increased linearly with the ATP concentration until the amount of added ATP reached about 0.6 mole per mole of S-1, and at higher ATP concentrations remained constant at this value.View full abstractDownload PDF (2487K) -
Takeshi KATO, Toshio SHIMADA1970 Volume 68 Issue 5 Pages 681-687
Published: November 25, 1970
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA procedure for isolation and crystallization of ferritin from dolphin spleen is described and some physicochemical properties of ferritin are also reported.
The ferritin could only be obtained by treatment with 50% acetone (final concentration) in combination with the original method of Granick.
The average iron and phosphorus contents of the ferritin are 20.8% and 0.012%, respectively.
The molecular size of ferritin and the diameter of the iron micell, measured from electron-microphotographs, are smaller than those of horse ferritin. These values decrease in the order : horse>dolphin>tuna fish.
The isoelectric point of this ferritin is pH 4. 8.
No significant differences were observed between horse and dolphin spleen ferritins in the following properties : the absorption spectra in the infra-red and ultra-violet regions, the iron releasing curve, the absence of detectable N-terminal amino acid, and the behaviors on Tisclius or acrylamide-gel electrophoresis.
Dolphin ferritin, like those of horse and tuna fish, contains much aspartic acid, giutamic acid and leucine, However it contains more proline and less isoleucine than horse ferritin.View full abstractDownload PDF (2325K) -
Toshisuke KAWASAKI, Ikuo YAMASHINA1970 Volume 68 Issue 5 Pages 689-699
Published: November 25, 1970
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. Kinetic studies of the incorporation of D-glucosamine-1-14C into microsomal subfractions of rat liver revealed that C-fraction (0.26% DOC extract) contained the precursors of plasma glycoproteins in addition to membrane glycoproteins, whereas M-fraction (0.5% DOC extract) was mainly composed of membrane glycoproteins.
2. Two decay slopes were obtained for the membrane glycoproteins during a period from 24 hr to 183 hr after the isotope injection. Half-lives of the slower phases esti-mated from the slopes after 75 hr were 58-94 hr, the values being about the same as those obtained by labelling proteins with amino acid. The existence of two decay slopes which was not observed using amino acid to label the proteins suggested that the carbohydrate and polypeptide moieties of glycoproteins turned over independently, in an earlier phase of metabolism.
3. D-Glucosamine-1-14C was incorporated into glycoproteins exclusively as glucosamine and sialic acid. The specific activity of the protein bound glucosamine was higher than that of sialic acid during the first 1.5 hr, but thereafter the ratio of specific activity of sialic acid to hexosamine increased gradually, reaching values of 2.6-2.9 for C-fraction and 1.7-1.8 for M-fraction after 24 hr. These facts indicated the metabolic heterogeneity of the carbohydrate chains of microsomal glycoproteins.View full abstractDownload PDF (865K) -
Isamu SHIIO, Ryuichi MIYAJIMA, Konosuke SANO1970 Volume 68 Issue 5 Pages 701-710
Published: November 25, 1970
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAspartate kinases [ATP: L-aspartate 4-phosphotransferase, EC 2. 7. 2. 4] were partially purified from Brevibacterium flavum and its mutant, which is resistant to L-threonine plus S-(2-aminoethyl)-L-cysteine (AEC), * a lysine analogue, and produces a large amount of L-lysine, and their properties were compared with each other.
Both enzymes showed a homotropic interaction of the substrate, aspartate, which disappeared in the presence of the activator, threonine or ammonium sulfate. The degree of activation by ammonium sulfate was larger with the parental enzyme than with the mutant enzyme.
In the presence of ammonium sulfate, double reciprocal plots of the reaction rate against one substrate concentration at various fixed concentrations of another sub-strate were linear and met at a point with both enzymes. Moreover, ADP, one of the reaction products inhibited the enzymes competitively to both substrates, aspartate and ATP, suggesting rapid equilibrium random Bi Bi mechanism for both enzymatic reactions. Kms for aspartate and ATP were similar with both enzymes.
Threonine slightly activated the mutant enzyme but partially competitively in-hibited the parental enzyme in the presence of ammonium sulfate, while, in the absence of the salt, it was an activator for both enzymes. Gel filtration experiments showed dimer formation by the addition of threonine in the presence of ammonium sulfate. Regardless of the presence of ammonium sulfate, lysine inhibited both en-zymes to the same degree at a high concentration.
In contrast to the parental enzyme, concerted inhibition by lysine plus threonine was not observed with the mutant enzyme. Furthermore, a simultaneous addition of threonine diminished the inhibitory effect of lysine.
Isoleucine only slightly activated the mutant enzyme, while about twice activa-tion was observed with the parental enzyme.
In conclusion, a genetic alteration occurred in aspartate kinase of an analogue-resistant mutant affected all tested actions of the allosteric effectors, threonine, iso-leucine, and ammonium sulfate but not those of the substrates and the competitive inhibitor, lysine.
Effect of AEC was similar to those of lysine with both enzymes. Thus, the specific growth inhibition of the parental strain by this analogue plus threonine which was reversed by the addition of lysine, seems to be caused by the concerted inhibition of the aspartate kinase. The resistance of the mutant to these amino acids as well as lysine overproduction in the mutant can be well explained by the lack of the concerted inhibition in the mutant enzyme.View full abstractDownload PDF (683K) -
Shoji OHKUMA, Den'ichi MIZUNO1970 Volume 68 Issue 5 Pages 711-716
Published: November 25, 1970
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS(1) The turnover of ribosomal RNA (r-RNA) in mouse peritoneal macrophages was studied in a cell culture system, where macrophages did not multiply but remained alive in vitro for at least a week.
Macrophages were incubated with 3H-uridine for 24 hr to allow uniform labeling of RNA's. They were then transferred to fresh medium containing non-radioactive uridine and chased for 4 days. The specific activity of RNA decreased logarithmi-cally with time, the half-life being about 48 (36-70) hr. The cellular RNA content remained constant and the synthetic activity of RNA did not change greatly during this time. This result shows that there is a turnover of the RNA.
(2) The RNA species undergoing metabolic turnover were found to he mainly ribosomal RNA's by sucrose density gradient centrifugation.View full abstractDownload PDF (469K) -
VI. Synthesis of Phenyl 2'-O-Methyl-α-maltoside and an Enzymatic InvestigationHitoshi ARITA, Yoshio MATSUSHIMA1970 Volume 68 Issue 5 Pages 717-722
Published: November 25, 1970
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMethylation of 2, 3, 2', 3', 4'-penta-O-acetyl-l, 6-anhydro-β-maltose with methyl iodide and silver oxide gave a mixture of mono-0-methyl derivatives of maltose, and the position of methoxyl group was proved by gas-liquid chromatography to be 2', 3', or 4'. The ratio of each O-methyl isomer was found to be approximately 2:1:4.
1, 6 anhydro-ring was cleaved by acetolysis and glycosidation was performed by fusion with phenol and ZnCl2. The mixture of O-methylated phenyl-α-maltoside (IVa, b, c) was treated with Taka-amylase A [EC 3. 2. 1. 1], and the derivative survived was isolated by gel filtration. This compound was proved to be phenyl 2'-O-methyl-a-maltoside, and was found to be completely immune to the enzyme. It showed no inhibitory action on the enzymatic hydrolysis of phenyl-α-maltoside.View full abstractDownload PDF (416K) -
Sumihiro HASE, Yoshio MATSUSHIMA1970 Volume 68 Issue 5 Pages 723-730
Published: November 25, 1970
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe present paper describes the isolation of a glucose containing polysaccharide fromm the cell walls of Micrococcus lysodeikticus by lysozyme [EC 3. 2. 1. 17] digestion fol-lowed by ECTEOLA-cellulose chromatography, acetylation, diborane reduction and deacetylation. The results of periodate oxidation of the polysaccharide indicate that the N-acetylmannosaminuronic acid residues in the original polysaccharide are linked through their 4-positions provided that the residues are situated at the internal posi-tion of the polysaccharide chain.View full abstractDownload PDF (547K) -
Takashi MATSUBARA, Yoshihiro TOCHINO1970 Volume 68 Issue 5 Pages 731-736
Published: November 25, 1970
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSLiver mitochondria were prepared from normal, adrenalectomized, thyroidectomized or hypophysectomized rats and the respiratory activities were measured polarogra-phically. The oxidative phosphorylation (P/O ratio) and the respiratory control were not greatly disturbed in the hormone-deficient rats, although the respiratory rates, both in states 3 and 4, were markedly decreased in the thyroidectomized and hvpo-physectomized rats. By in vivo administration of alloxan to normal rats, the mito-chondrial content of non-esterified fatty acids was extremely increased and uncoupling of respiration was elicited, whereas the administration of alloxan to the hormone-deficient rats produced neither hyperlipemia nor fatty liver even in the presence of hyperglycemia, and the free fatty acid content of the liver mitochondria was within the normal range. The respiratory patterns of mitochondria from the hormone-de-ficient rats were normal even after the administration of alloxan. The efficiency of oxidative phosphorylation and the ratio of respiratory control of mitochondria from these animals were also not altered by the administration of alloxan in vivo.
These results seem to support the view that the disturbance of respiratory ac-tivity of liver mitochondria from diabetic rats may be caused by the elevation of the content of non-esterified fatty acids, and that the fatty acid content in turn may be regulated by the hormones secreted from the adrenal, thyroid and pituitary glands.View full abstractDownload PDF (426K) -
I. Gulonolactone-reducible Pigment as a Prosthetic Group of Gulonolactone OxidaseHidehiko NAKAGAWA, Akira ASANO1970 Volume 68 Issue 5 Pages 737-746
Published: November 25, 1970
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSGulonolactone oxidase [EC 1. 1. 3. 8] was effectively solubilized from rat liver micro-somes by the action of detergents but not with proteases. We established a new method of purification of the enzyme. After digestion with trypsin [EC 3. 4. 4. 4] which removed cytochrome b5 from the microsomes, the oxidase was solubilized by the treatment with Emasol 1130, a non-ionic detergent. The solubilized enzyme was further purified by DEAE-Sephadex A-50 and Sepharose 6 B column chromatog-raphy. Judging from the gel filtration, an apparent molecular weight of the enzyme was estimated to be about 100, 000. The presence of a gulonolactone-reducible pigment associated with enzyme activity was detected with partially purified preparation, which was almost free from cytochrome b5. The gulonolactone reduced minus oxidized difference spectrum of the enzyme had only one trough at 450-470mμ. L-Gulono-γ-lactone and L-galactono-γ-lactone were only specific substrates for the reduc-tion of the pigment. The specific content of the pigment increased in roughly parallel to the specific activity of the enzyme during the purification procedures. Based on above lines of evidence, we concluded that the pigment, which exhibits a difference spectrum by the reduction with gulonolactone, is a specific prosthetic group of gulonolactone oxidase.
The proposed possibility that the enzyme has two active sites is discussed with the results of the inhibition studies using PCMS and Na2S.View full abstractDownload PDF (1540K) -
Takeo YOSHINAGA, Katsura IZUI, Hirohiko KATSUKI1970 Volume 68 Issue 5 Pages 747-750
Published: November 25, 1970
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDownload PDF (893K) -
Tetsuo KATO, Michinori WAKI, Shuji MATSUURA, Nobuo IZUMIYA1970 Volume 68 Issue 5 Pages 751-753
Published: November 25, 1970
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDownload PDF (140K) -
I. Isolation of Type Specific PolysaccharideToshio MIYAZAKI, Toshiro YADOMAE, J. K. N JONES1970 Volume 68 Issue 5 Pages 755-758
Published: November 25, 1970
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDownload PDF (839K) -
Junzo KATO, Yachio ATSUMI, Machiko INABA1970 Volume 68 Issue 5 Pages 759-761
Published: November 25, 1970
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDownload PDF (180K)
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