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Volume 84 (1978)
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Volume 84, Issue 6
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1978 Volume 84 Issue 6 Pages 1323
Published: 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDownload PDF (13K) -
Mamoru HASHINOTSUME, Kazuya HIGASHINO, Toshikazu HADA, Yuichi YAMAMURA1978 Volume 84 Issue 6 Pages 1325-1333
Published: December 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRat serum carboxylesterase [carboxylic ester hydrolase, EC 3.1.1.1] was purified by ammonium sulfate precipitation, chromatography on DEAE-cellulose, QAE Sephadex A-50 and brushite, and gel filtration on Sephadex G-200. This purified enzyme was shown to be a single protein band on slab gel electrophoresis and its final specific activity was 49.5 units/mg protein. This enzyme was very sensitive to diisopropylfluorophosphate (DFP) but not to p-chloro-mercuribenzoate (PCMB), eserine, o-iodosobenzoate, NaF or ethylenediamine tetraacetic acid (EDTA). The pH profile of the reactions catalysed by this enzyme showed broad opti-mum between pH 6.0 and 8.8. The activity of purified enzyme was not affected by Ca2+ Mg2+, Mn2+, Cu2+, Zn2+, Ni2+, Co2+, and Cd2+ at 1mM concentration. The molecular weight measured by gel filtration was approximately 84, 000 and the isoelectric point was 4.4. The enzymatic properties were not changed by neuraminidase treatment with regard to heat stability, pH optimum, sensitivity to metal ions and inhibitors, and Km values for p-nitrophenylesters of different acyl C-chain length.View full abstractDownload PDF (1540K) -
Yukio IKEHARA, Kenji MANSHO, Keitaro KATO1978 Volume 84 Issue 6 Pages 1335-1338
Published: December 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA single injection of colchicine, 1mg/kg body weight, remarkably increased the activity of alkaline phosphatase in rat livers, followed by its increase in serum. Combination of bile duct ligation with colchicine administration had an additional effect on the alkaline phosphatase level above those induced by the individual treatments alone. Treatment with cycloheximide significantly inhibited the inducible effect of colchicine on the enzyme suggesting that protein synthesis is prerequisite to the elevation of the alkaline phosphatase caused by colchicine treatment.View full abstractDownload PDF (322K) -
Masami TAKAHASHI, Yuji TONOMURA1978 Volume 84 Issue 6 Pages 1339-1355
Published: December 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe binding properties of dynein arms to the A- and B-tubules of outer doublets of cilia from Tetrahymena pyriformis were examined, with the following results:
1. When 30s dynein purified from Tetrahymena cilia was added to doublets deficient in dynein arms, it bound to both A- and B-tubules almost equally and formed arms along the edges. The overall length of arms bound to the A-tubule was 22±3nm, and that of arms bound to the B-tubule was 24±3nm. Each arm bound to the A- and B-tubules was pointed toward the base at angles of 55°±7° and 48°±7°, respectively. In the presence of sufficient amounts of dynein, the arms along the A- and B-tubules were located at intervals of 22.8±1.5nm and 22.5±1.7nm, respectively.
2. On adding ATP, only the arms bound to the B-tubule were dissociated from the doublet decorated with arms on both sides. The dissociated arms rebound themselves to the B-tubule after hydrolysis of the ATP. When several doublets decorated with arms along both A- and B-tubules were arrayed side by side, the interdoublet spacing increased from 14±2nm to 17±2nm on addition of ATP.
3. The turbidity of a suspension of trypsin [EC 3.4.21.4]-treated axonemes decreased rapidly on addition of ATP, then recovered partially. Observations by dark-field microscopy and electron microscopy showed that the doublets which had slid out from the axonemes on ATP addition formed large aggregates after hydrolysis of the ATP. The dynein arms were also solubilized from the axonemes upon addition of ATP, and rebound themselves to the B-tubule after hydrolysis of the added ATP.
4. The double-reciprocal plot for the ATPase [EC 3.6.1.3] activity of the trypsin-treated axonemes against ATP concentration was composed of two straight lines, from which the Km values were estimated to be 1.0 and 12.7 μM. The dependence of the decrease in turbidity of the axonemal suspension on ATP concentration indicated that the binding of ATP to sites with an apparent dissociation constant of 1μM induced dissociation of the arms from the B-tubule.View full abstractDownload PDF (6650K) -
Hiroshi AKANUMA, Makoto YAMASAKI1978 Volume 84 Issue 6 Pages 1357-1362
Published: December 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA novel method for the use of CM-Sephadex in affinity chromatography is described. This method involves a carbodiimide-mediated synthesis of a lactone derivative, which is subsequently hydrazinolyzed to CM-Sephadex hydrazide. More than 90% of the carboxyl groups was converted to the hydrazide form by this method without any discernible degrada-tion of the Sephadex beads. The hydrazide beads were further converted to the azide form and coupled with D-arginine and D-phenylalanine derivatives. These Sephadex derivatives contained more than 0.5 mmol of each amino acid per g dry beads and were found to be effective and specific adsorbents for carboxypeptidase B. This CM-Sephadex hydrazide should be useful in affinity chromatography because the resulting specific adsorbents involve stable amide coupling linkages and retain the physical properties of their precursor, Sephadex, which are favorable for column operation.View full abstractDownload PDF (499K) -
II. Further Characterization and Studies on Mode of Hydrolysis of Physiologically Active PeptidesMotoharu HAYASHI1978 Volume 84 Issue 6 Pages 1363-1372
Published: December 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA large-scale purification of monkey brain arylamidase was carried out. Amino acid analyses indicate that the enzyme is rich in acidic amino acids and is poor in cystine. The amino terminal residue was determined to be alanine by dansylation. The enzyme was activated by sulfhydryl compounds. Dithiothreitol was more effective than β-mercaptoethanol. Bestatin competitively inhibited the enzyme activity and the K1 value was calculated to be 2.5×10-7M, which was of the same order as that of puromycin. The inhibitions by puromycin and bestatin were reversible. The enzyme hydrolyzed di-, tri-, and oligopeptides including physiologically active peptides. Of physiologically active peptides, enkephalins and Met-Lys-bradykinin, which possess a neutral amino acid at the N-terminal position, were more rapidly hydrolyzed by the enzyme. Peptides such as LH-RH and TRH, which possess a pyrrolidonecarboxylyl group at the N-terminal position, and substance P and bradykinin, which possess a proline residue adjacent to the N-terminal residue, were not hydrolyzed by the enzyme. The Km values for various peptides indicate that the enzyme has higher affinity for oligopeptides than di- and tripeptides. The aminopeptidase activity of the enzyme was also competitively inhibited by puromycin and bestatin. Analyses of the hydrolysis products of various peptides by the dansylation method indicate that the enzyme has both kinin-converting activity and angiotensinase activity.View full abstractDownload PDF (748K) -
Makoto KAGEYAMA1978 Volume 84 Issue 6 Pages 1373-1379
Published: December 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effect of pyocin R1 on the glucose metabolism of sensitive Pseudomonas cells was investigated. Upon treatment with pyocin R1, although the rate of O2 uptake of the sensitive cells for glucose or gluconate was not very much affected at first, the final level of O2 uptake was greatly reduced. When 2-oxogluconate was used as a substrate, O2 uptake was immediately halted by pyocin. By determining the amounts of glucose, gluconate, and 2-oxogluconate before and after the reaction and the amount of O2 consumed, it was concluded that glucose was exclusively metabolized via the following pathway with quantitative accumulation of 2-oxogluconate after pyocin treatment.
glucose 1/2O2 → gluconate 1/2O2 → 2-oxogluconate
The possible mechanism of this change is discussed.View full abstractDownload PDF (472K) -
Kinetic Mechanism of UDP-Glucose: Glycogen 4-α-GlucosyltransferaseHidenari TAKAHARA, Kazuo MATSUDA1978 Volume 84 Issue 6 Pages 1381-1387
Published: December 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe kinetic mechanism of glycogen synthase [UDP-glucose: glycogen 4-α-glucosyltransferase, EC 2.4.1.11], glucose-6-P-dependent form, from Neurospora crassa has been investigated by initial velocity experiments and studies with inhibitors in the presence of sufficient levels of glucose-6-P. The rate equation was different from those of common two-substrate systems because one of the substrates, glycogen, is also a product. The reaction rates were determined by varying the concentration of one of the substrates while keeping that of the other constant. Double-reciprocal plots of initial velocity measurements were linear and showed converging line patterns. UDP was found to act competitively when the substrate UDP-glucose was varied, but noncompetitively when glycogen was varied. On the basis of these results, it is concluded that glycogen synthase, glucose-6-P-dependent form, from N. crassa has a rapid equilibrium random Bi-Bi mechanism. Rate constant and dissociation constants for each step of this mechanism were estimated.View full abstractDownload PDF (446K) -
Michio DOHI, Hiroshi INOUYE, Akihiko KIKUCHI, Luigi GORINI1978 Volume 84 Issue 6 Pages 1389-1399
Published: December 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn in vitro system for the synthesis of ornithine transcarbamylase (OTCase) was established using iS-30 extract from E. coli MDS6-2(λ) and DNA of a λ transducing phage carrying argI and argF genes. This in vitro synthesis was completely dependent on the addition of DNA, and was sensitive to chloramphenicol and rifampicin.
Radioisotopic analysis confirmed that the synthesized enzyme catalyzes the carbamylation of ornithine to citrulline. In the in vitro system the repression and derepression of OTCase synthesis could be observed by mixing iS-30 extracts prepared from argR+ and argR- cells. A remarkable maturation effect could be observed for the FFF enzyme, but not for the III enzyme.
This system is considered to reflect the in vivo situation, and should therefore be useful for investigations on the regulation of OTCase synthesis in vivo.View full abstractDownload PDF (756K) -
Michio DOHI, Akihiko KIKUCHI, Luigi GORINI1978 Volume 84 Issue 6 Pages 1401-1409
Published: December 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe regulation profiles of OTCase (argF, argI) synthesis in vitro were investigated by using the in vitro system described in the accompanying paper. Addition of 2.6mM arginine, crude repressor and partially purified repressor to the in vitro system demonstrated that, λdargF-DNA-directed OTCase-FFF synthesis is more sensitive to the repressor than λpargI-DNA-directed OTCase-III synthesis. The effects of some low-molecular substances on FFF and III syntheses were investigated; guanosine 3'-diphosphate 5'-diphosphate (ppGpp) stimulated both syntheses while cAMP and guanosine 5'-tetraphosphate (Gpppp) were not effective on III synthesis and were slightly inhibitory for FFF synthesis. These substances had no effect on the maturation of the enzyme or on the activity of the enzyme, FFF or III, synthesized.
We suggest that argF- and argI-genes are regulated in a slightly different fashion and that the operator-promotor regions are not completely identical for these two genes.View full abstractDownload PDF (653K) -
Yasuo SUZUKI, Makoto MATSUMOTO1978 Volume 84 Issue 6 Pages 1411-1422
Published: December 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA new type of soluble phospholipase A1 [EC 3.1.1.32] was found in the supernatant fraction (cytosol) obtained by centrifugation at 105, 000×g for 90min of homogenates in 0.25M sucrose of FL cells and several other cultured cells (L-132 cells, Vero cells, and BEK-1 cells). Phospholipase A1 in FL cell cytosol showed activity only in the presence of a non-ionic detergent, Triton X-100, or certain phospholipids such as phosphatidylinositol, cardiolipin, phosphatidylserine, phosphatidic acid, phosphatidylethanolamine, and lysophosphatidylcholine, among which phosphatidylinositol was the most active stimulator of the activity.
This soluble enzyme in FL cells acted optimally at acidic pH like lysosomal acid phospholipases A1 and A2, which were recovered in the soluble subfraction of the lysosomal fraction. The acid phospholipase A1 in the cytosol was shown to be different from the lysosomal phospholipase A1 on the following counts: (1) the cytosol enzyme showed activity on the addition of phospholipids or Triton X-100, whereas lysosomal phospholipase A1 was present in the active form even in the absence of these activators; (2) no release of the lysosomal acid phosphatase and Triton X-100-independent phospholipase A1 or A2 into the cytosol occurred during cell fractionation; (3) the heat stabilities of these two enzymes as well as the effects of detergents on them were different.
Similar Triton X-100-dependent enzyme activity was also found in the cytosol of several rat tissues.View full abstractDownload PDF (926K) -
Increase in the Concentrations of Ornithine and Acetylglutamate in Rat Liver in Response to Urea Synthesis Stimulated by the Injection of an Ammonium SaltTakeyori SAHEKI, Tomoichi OHKUBO, Tsunehiko KATSUNUMA1978 Volume 84 Issue 6 Pages 1423-1430
Published: December 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. Intraperitoneal injection of ammonium chloride or ammonium bicarbonate, 2 mmol/kg body weight, caused an increase in the concentrations of ammonia (maximum at 5min) and urea (maximum at 10min) in the livers of rats fed on laboratory chow.
2. The increase in the concentration of urea was accompanied by about 200%, 100%, and 40% increases in the concentrations of acetylglutamate, ornithine, and citrulline, respectively.
3. Overnight starvation before ammonium chloride injection did not affect the results.
4. In the livers of rats fed on a high protein diet, the ornithine level was increased to a similar extent in response to ammonium salt injection but that of acetylglutamate was not, and the concentrations of these amino acids and urea remained at the highest levels.
5. In the livers of rats fed on a low protein diet, the concentration of urea did not change until 5 min after the injection and then increased gradually. The sum of the concentrations of ornithine and citrulline did not change significantly until 10min, while the concentration of acetylglutamate increased more quickly (maximum at 10min).
6. In perfused liver, the addition of ammonium salt to the perfusate caused an increase in the concentrations of acetylglutamate and citrulline as observed in vivo, while the concentration of ornithine, one-fifth of that in vivo, did not respond to the addition.
From these results, we conclude that ammonia, a direct substrate for urea synthesis, may control the concentrations of both ornithine and acetylglutamate in the liver, directly or indirectly.View full abstractDownload PDF (633K) -
Preparation of Calcium-Sensitive Myosin and Troponin-TropomyosinKunihiko KONNO1978 Volume 84 Issue 6 Pages 1431-1440
Published: December 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe Ca-regulatory system in squid mantle muscle was studied. The findings were as follows.
(a) Squid mantle myosin B (squid myosin B) was Ca-sensitive, and its Ca-sensitivity was unaffected by addition of a large amount of rabbit skeletal myosin (skeletal myosin) or rabbit skeletal F-actin (skeletal F-actin).
(b) Squid myosin was prepared from the mantle muscle. It showed a heavy chain component and two light chain components in the SDS-gel electrophoretic pattern: the molecular weights of the latter two were 17, 000 and 15, 000. Actomyosin reconstituted from squid myosin and skeletal (or squid) actin showed Ca-sensitivity in superprecipitation and Mg-ATPase assays. EDTA treatment had no effect on the Ca-sensitivity of squid myosin.
(c) Squid mantle actin (squid actin) was prepared by the method of Spudich and Watt. Hybrid actomyosin reconstituted by using the pure squid actin preparation with skeletal myosin showed no Ca-sensitivity in Mg-ATPase assay, whereas that reconstituted using crude squid actin showed marked Ca-sensitivity. The crude squid actin contained four protein components which were capable of associating with F-actin in 0.1M KCl, 1mM MgCl2 and 20mM Tris-maleate (pH 7.5).
(d) Native tropomyosin was prepared from squid mantle muscle, and it conferred Ca-sensitivity on skeletal actomyosin as well as on a hybrid actomyosin reconstituted from squid actin and skeletal myosin.
(e) Squid native tropomyosin was separated into troponin and tropomyosin fractions by placing it in 0.4M LiCl at pH 4.7. The troponin fraction was further purified by DEAE-cellulose chromatography. Squid troponin thus obtained was different in mobility from rabbit skeletal or carp dorsal troponin; three bands of squid troponin corresponded to molecular weights of 52, 000, 28, 000, and 24, 000 daltons. It could confer Ca-sensitivity in the presence of tropomyosin on skeletal actomyosin as well as on a hybrid reconstituted from squid actin and skeletal myosin.
(f) Squid myosin B, and two hybrid actomyosins were compared as regards Ca and Sr requirements for their Mg-ATPase activities. The myosin-linked regulatory system rather than the thin-filament-linked regulatory system was predominant in squid myosin B. Squid myosin B required higher Ca2+ and Sr2+ concentrations for Mg-ATPase activity; half-maximal activation of Mg-ATPase was obtained at 0.8μM Ca2+ and 28μM Sr2+ with skeletal myosin B, and at 2.5μM Ca2+ and 140μM Sr2+ with squid myosin B.View full abstractDownload PDF (2425K) -
XI. Interaction of 8-Substituted FMNH2 with LuciferaseTakahide WATANABE, Kunio MATSUI, Sabu KASAI, Takao NAKAMURA1978 Volume 84 Issue 6 Pages 1441-1446
Published: December 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe interaction of bacterial luciferase from Photobacterium phosphoreum with reduced flavin was investigated using various 8-substituted FMNH2 analogs. Flavins tested were FMNH2 and FMNH2 substituted at the 8 position with HO-, CH3O-, C2H5O-, Cl-, Br-, I-, H2N-, (CH3)HN-, and (CH3)2N. 8-CH3O-, C2H5O-, Cl-, and Br-FMNH, showed luminescent activity in the luciferase reaction with emission peaks at various wavelengths. 8-HO- and I-FMNH2 were competitive inhibitors toward FMNH2 in the luminescent reaction. 8-Amino analogs of FMNH2 showed no luminescent or inhibitory activity. The dissociation constant of the luciferase-FMNH2 analog complex was determined kinetically as a substrate or inhibitor constant. A contribution of the imino group at position 5 in the isoalloxazine ring to the FMNH2 binding to luciferase was suggested by a Hammett plot of the dissociation constants.View full abstractDownload PDF (450K) -
Takahisa SHINOMIYA, Chiyoko FUNAYAMA-MACHIDA, Tsuneko UCHIDA1978 Volume 84 Issue 6 Pages 1447-1451
Published: December 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAntibodies to a trinucleotide, ApApA, were prepared by injecting bovine serum albumin conjugated with ApApA into rabbits. The specificities of the antibodies were determined by estimating the inhibition of the binding of [14C]ApApA to the antibodies by various nonradioactive mono-, oligo-, and polynucleotides, using the ammonium sulfate precipitation method. The antibodies were found to react with ApApN sequences and oligoadenylic acids, but also reacted slightly with polyadenylic acid, RNA and DNA. Significant crossreactions were observed with other oligonucleotides containing adenosine.View full abstractDownload PDF (389K) -
Kayoko NAKAMURA, Koui TAKAHASHI, Shizuo WATANABE1978 Volume 84 Issue 6 Pages 1453-1458
Published: December 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMyosin-like protein and actin-like protein from E. coli formed filaments very similar in structure to those of myosin and actin from skeletal muscle. At 0.2M KCl, a large number of “thick filaments” of uniform size (about 0.6-0.7μm long and about 20 nm wide) was present. These thick filaments aggregated as the KCl concentration decreased to less than 0.2M. Filaments of actin-like protein were decorated with muscle heavy meromyosin, showing “arrowheads” The arrowhead structure disappeared in the presence of ATP.
A mixture of E. coli myosin-like protein and rabbit skeletal actin exhibited a gelation phenomenon on the addition of ATP. The phenomenon was reversible and showed ATP specificity. However, the gelation phenomenon was not observed with the mixture of E. coli actin-like protein and E. coli myosin-like protein.
These results provide compelling evidence that the E. coli myosin-like protein and actin-like protein we isolated are essentially identical to myosin and actin, respectively.View full abstractDownload PDF (1989K) -
Analysis by a New Serum Lipase Assay Method (The BALB-DTNB Method) in Combination with Gel-Filtration and Iso-Electrofocusing TechniquesShigeru KUROOKA, Tsugio KITAMURA1978 Volume 84 Issue 6 Pages 1459-1466
Published: December 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSVery low levels of lipase can easily be measured by a new serum lipase assay method (the BALB-DTNB method), using BAL-tributyrate (BALB) as a substrate, 5, 5'-dithiobis(2-nitrobenzoic acid) as a chromogenic SH reagent, phenylmethylsulfonylfluoride as an inhibitor of esterases and sodium dodecyl sulfate as a surfactant. The BALB-DTNB method has a higher sensitivity than the conventional serum lipase assay methods, and proved useful for analyzing the properties of serum lipases in combination with gel-filtration on a Sephacryl S 200 column and isoelectrofocusing in an Ampholine column. Serum samples containing high levels of lipases from patients with pancreatic diseases or patients in whom the pancreatic exocrine gland had been stimulated by injecting caerulein and secretin were analyzed by these methods. The lipolytic profiles obtained indicated the presence of a lipase with an estimated molecular weight of 46, 000 and isoelectric points of 7.4, 6.8, or/and 6.4. A lipase with properties similar to those of the serum lipase was found to be present in human pancreatic juice.View full abstractDownload PDF (641K) -
Noriko TAKAHASHI, Hina NISHIBE1978 Volume 84 Issue 6 Pages 1467-1473
Published: December 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA new type of glycopeptidase hydrolyzing β-aspartylglycosylamine linkages was partially purified from almond emulsin by chromatography on Sephadex G-200 and DE 52. The enzyme degraded stem bromelain glycopeptide, Asn-Asn(Man3, Xyl1, Fuc1, GlcNAc2)-Glu-Ser-Ser, to yield equimolar amounts of intact oligosaccharide, peptide (Asn-Asp-Glu-Ser-Ser), and ammonia. The Km value for the stem bromelain glycopeptide was 4mM, and the optimum pH was 5.2. The enzyme was markedly inhibited by 10mM Cu2+, Fe3+, and Zn2+. Thiol inhibitors and actinomycete protease inhibitors had no effect.
The glycopeptides used as substrates were prepared from stem bromelain, ovalbumin or ovotransferrin. The enzyme hydrolyzed glycopeptides with 3-11 amino acid residues, whereas it did not hydrolyze glycopeptides with 1-2 amino acid residues. Furthermore, Asn-oligosaccharide was not inhibitory to the enzyme.View full abstractDownload PDF (526K) -
Mikio KATO, Takachika AZUMA, Takashi ISOBE, Kozo HAMAGUCHI1978 Volume 84 Issue 6 Pages 1475-1483
Published: December 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe formation of interchain disulfide bonds from partially reduced Bence Jones protein (Nag, type λ) and Fab(t) fragments of IgGl myeloma proteins was studied in the presence of various disulfide reagents. The results could be well explained in terms of the scheme proposed previously (Kishida et al. (1976) J. Biochem. 79, 91-105). In this scheme, it was assumed that two kinds of intermediate, which form mixed disulfides with either of the paired thiol groups, are produced. For type λ Bence Jones proteins, only one of the two intermediates can form the inter L-L disulfide bond. The fraction of the intermediate having the ability to form the inter L-L disulfide bond was estimated to be 72%. of the total Nag protein and was the same irrespective of the kind of disulfide reagent examined. For Fab(t), on the other hand, both intermediates equally can form the inter Fd-L disulfide bond. On the basis of the results with cystamine, it was shown that the formation of an inter Fd-L disulfide bond from the intermediate proceeds about 100 times as rapidly as that of an inter L-L disulfide bond.View full abstractDownload PDF (627K) -
Hiroyuki OHNO, Yukiko MORIKAWA, Fumio HIRATA1978 Volume 84 Issue 6 Pages 1485-1494
Published: December 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe NAD+-linked 15-hydroxyprostaglandin dehydrogenase (PGDH) of swine lung was purified to a high specific activity by affinity chromatographies on prostaglandin (PG) and NAD+-Sepharose. The affinities of the enzyme for various synthetic analogues of PGA, E, F, and I and their inhibitory effects on the enzymatic reaction were examined. The modification of the alkyl side chain of PG, particularly at C-15 or C-16, reduced the affinity of the enzyme for these PG analogues. Furthermore, 14-methyl-13, 14-dihydro-PGE1 and 16cyclopentyl-w-trinor-15-epi-PGE2 were potent inhibitors of PGDH.View full abstractDownload PDF (576K) -
Properties of the Enzyme in Pupal Fat BodyIsao MORISHIMA1978 Volume 84 Issue 6 Pages 1495-1500
Published: December 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAdenylate cyclase was assayed in a sonicated preparation of silkworm pupal fat body. The adenylate cyclase was found mostly in the particulate fraction. The activity depended upon either Mg2+ or Mn2+, and the degree of stimulation by Mn2+ was 2 times greater than that by Mg2+ compared at the saturating concentrations. In the presence of Mg2+, the enzyme was inhibited by both EGTA and high concentrations of Ca2+, showing biphasical response to Ca2+ The enzyme was stimulated several-fold by NaF. The enzyme exhibited typical Michaelis-Menten kinetics and Km values were 0.13mM for MgATP and 0.086mM for MnATP.View full abstractDownload PDF (430K) -
Harun K. M. YUSUF, John W. T. DICKERSON1978 Volume 84 Issue 6 Pages 1501-1506
Published: December 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effect of undernutrition was studied on the content and composition of the gangliosides of forebrain, brain stem, and cerebellum of Wistar rats. Undernourishment from before birth upto 21 days of age did not produce any significant change in the content of gangliosides in any brain area, but when continued upto 121 days it resulted in a significant decrease of the lipids in each of these brain regions. At this age the different ganglioside fractions were, however, affected differently in the different brain areas. Thus the accumulation of the ganglioside GD1a was most affected in the forebrain, of ganglioside GD1b in the brain stem, and of ganglioside GT1 in the cerebellum. These were the largest fractions of total gangliosides in the respective brain areas at 121 days of age. The results suggest that the brain may be vulnerable to undernourishment at any time when a biochemical process in development of the tissue is in progress.View full abstractDownload PDF (452K) -
Seiichi KAWASHIMA, Chikahiko EGUCHI, Kazutomo IMAHORI1978 Volume 84 Issue 6 Pages 1507-1511
Published: December 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTo identify the residues modified by radioactive N6-p-bromoacetaminobenzyladenosine-5'-phosphate in phosphorylase b, the peptic peptides of the modified enzyme were purified. Two radioactive peptides were isolated, each of which had a cysteine residue modified. The cysteine residue involved in the AMP site was determined to be cysteine-317 and the other reactive cysteine residue was cysteine-171.View full abstractDownload PDF (356K) -
An UncA Mutant (uncA401) with Defective α SubunitHiroshi KANAZAWA, Setsuo SAITO, Masamitsu FUTAI1978 Volume 84 Issue 6 Pages 1513-1517
Published: December 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSInactive coupling factor ATPase (F1) was prepared from an uncoupled mutant (uncA401) of Escherichia coli. Reconstitution of ATPase activity was observed when α subunit from wild-type F1 was added to the dissociated inactive F1 and the mixture was dialyzed against buffer containing ATP and Mg2+. ATPase was also reconstituted when the mixture of α subunit (wild type) and crude extract from the mutant was dialyzed against the same buffer. These results indicate that the mutant is defective in α subunit, suggesting that the uncA401 locus carries the structural gene for α subunit, and that this polypeptide plays an essential role in ATPase activity in F1, molecule.View full abstractDownload PDF (905K) -
Toichiro HOSOYA, Yukifumi NAGAI, Tohru INAGAKI, Miki HAYASHI1978 Volume 84 Issue 6 Pages 1519-1528
Published: December 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCastration resulted in a rapid decrease in the activity of RNA polymerase I (or A) of isolated nuclei of rat prostates. The decrease was mainly ascribed to the diminution in the number of in vivo initiated RNA chains. The “free form” activity of the enzyme, however, which was estimated by the use of exogenous template and actinomycin D, increased 24 h after castration, then dropped rapidly.
The administration of testosterone to castrated rats caused an increase in the activities of both RNA polymerases I and II (or B), which started 2 h and reached the maximum 12 h after the administration. No initial rise in RNA polymerase II activity was observed during the first 2 h.
The administration of cycloheximide to normal rats caused a very rapid decrease of the activity of template-bound RNA polymerase I of isolated prostatic nuclei (t1/2=1.7 h), while the “free form” activity of the enzyme did not appreciably change until 3 h. The androgenstimulated increase in the “engaged form” of the RNA polymerase I of isolated nuclei was completely abolished by the administration of cycloheximide 60min before killing.
Based on the results obtained, the role of protein(s) with a rapid turn-over which is/are androgen-dependent and presumably participating in the control of preribosomal RNA synthesis is discussed.View full abstractDownload PDF (758K) -
Hiroshi SUZUKI, Hirofumi ONISHI, Koui TAKAHASHI, Shizuo WATANABE1978 Volume 84 Issue 6 Pages 1529-1542
Published: December 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIn our previous study (Onishi, H., Suzuki, H., Nakamura, K., Takahashi, K., and Watanabe S. J. Biochem. 83, 835-847, 1978), we found it to be characteristic of chicken gizzard myosit that thick filaments of gizzard myosin are readily disassembled by a stoichiometric amoun of ATP (3 mol of ATP per mol of myosin), and that the ATPase activity of gizzard myosil in the ATP-disassembled state is much lower than that of gizzard myosin disassembled by a high concentration of KCl. We now report the following findings: (1) Thick filament; of (unphosphorylated) gizzard myosin can be in a bipolar structure or in a non-polar structure, depending on the method of preparing the thick filaments. (2) Thick filament, of (unphosphorylated) gizzard myosin in either the bioplar or the non-polar structure are readily disassembled by ATP. (3) Addition of rabbit skeletal C-protein does not confe ATP resistance on thick filaments of (unphosphorylated) gizzard myosin. (4) (Unphospho. rylated) gizzard myosin in the ATP-disassembled state is in a dimeric form as determined by ultracentrifugation. Moreover, 0.2M KCl-dissociated gizzard myosin in monomeric forte is converted to a dimeric form by ATP. (5) The Mg-ATPase activity of (unphosphorylated) gizzard myosin is much lower in its dimeric form (less than one-tenth) than in its monomeric form. The activity depression observed around 0.15M KCl is therefore due to the formation of myosin dimers. (6) Skeletal L-meromyosin can increase the very low activity of (unphosphorylated) gizzard myosin ATPase at low ionic strength (0.13 M KCl) by forming ATPresistant hybrid filaments with (unphosphorylated) gizzard myosin, preventing the formation of myosin dimers. (7) Gizzard myosin in which one of the light-chain components is phosphorylated by myosin light-chain kinase can form thick filaments which are resistant to the disassembling action of ATP. (8) Even in the presence of ATP, thick filaments of phosphorylated gizzard myosin do not disassembled into myosin dimers. Accordingly, the ATPase activity of phosphorylated gizzard myosin does not show activity depression at low ionic strength.View full abstractDownload PDF (4817K) -
A Model of Dissimilatory Nitrite ReductaseYutaka ORII, Hideo SHIMADA1978 Volume 84 Issue 6 Pages 1543-1552
Published: December 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe reaction of bovine heart ferrocytochrome c with nitrite was studied under various conditions. The reaction product was ferricytochrome c at around pH 5, whereas at around pH 3 it was Compound I, characterized by twin peaks at 529 and 563 nm of equal intensity. However, ferrocytochrome c decreased obeying first-order kinetics over the pH range examined, irrespective of the presence or absence of molecular oxygen. The apparent first-order rate constant was proportional to the square of the nitrite concentration at pH 4.4 and it increased as the pH was lowered. At pH 3 the reaction was so rapid that it had to be followed by stopped-flow and rapid-scanning techniques. The apparent rate constant at this pH was found to increase linearly with the nitrite concentration. Based on these results the active species of nitrite was concluded to be dinitrogen trioxide at pH 4.4 and nitrosonium ion, NO+, at pH 3.
Compound II was formed by reaction of ferrocytochrome c and NO gas at acidic and alkaline pH values. The absorption peaks were at 533 and 563 nm at pH 3, and at 538 and 567 nm at pH 12.9. This compound was also formed by reducing Compound I with reduc-tants. Compound I prepared from ferricytochrome c and NO was stable below pH 6. However, appreciable absorption peaks for ferrocytochrome c appeared between pH 8 and 10, because Compound I was dissociated into ferrocytochrome c and NO+, and because ferrocytochrome c thus formed reacted with NO very slowly in this pH region. Saccharomyces ferricytochrome c under NO gas behaved differently from mammalian cytochrome, indicating the significance of the nature of the heme environment in determining the reactivity. Only at extreme pH values was Compound II formed exclusively and persisted.
A model system for dissimilatory nitrite reductase was constructed by using bovine heart cytochrome c, nitrite and NADH plus PMS at pH 3.3, and a scheme involving cyclic turnover of ferrocytochrome c, Compound I and Compound II is presented, with kinetic parameters.View full abstractDownload PDF (708K) -
The pH-Dependent Reactions of Pseudomonas aeruginosa Nitrite Reductase with Nitric Oxide and NitriteHideo SHIMADA, Yutaka ORII1978 Volume 84 Issue 6 Pages 1553-1558
Published: December 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSOxidized Pseudomonas nitrite reductase (Ps.NiR) reacted with nitric oxide (NO) forming mostly Comp. I at the heme c and heme d moieties in the acidic pH region (<pH 6). Above pH 6 the reduced heroes were predominant as a result of the dissociation of the initially formed Comp. I with simultaneous release of NO+. However, Comp. II was formed at heme d to some extent between pH 6 and 7. Comp. II (d) was also formed by direct reaction of reduced Ps.NiR with NO below pH 7, whereas Comp. II (c) appeared only below pH 6. These results could be explained as being due to a higher affinity of the reduced heme d than of the reduced heme c toward NO. In the pH region higher than neutral the reduced hemes remained unattacked except at extreme alkaline pH.
When sodium nitrite was added anaerobically to the reduced enzyme in the presence of NADH and PMS, Comp. II (c) appeared even at (and below) pH 7.2, whereas above that pH the reduced heme was predominant. Consequently it was concluded that under these turnover conditions Comp. II (c) was formed actually from Comp. I (c) by accepting an electron, but not by the direct reaction of reduced heme c with NO. The same mechanism could also explain the behavior of the heme d moiety, thus being in accord with the reaction scheme for Ps.NiR proposed previously (l).View full abstractDownload PDF (472K) -
Hitoshi UENO1978 Volume 84 Issue 6 Pages 1559-1565
Published: December 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSTwo large fragments derived from rabbit skeletal muscle α-tropomyosin were prepared by cyanogen-bromide cleavage. The two fragments, CnlA (residues 11 to 127) and CnlB (residues 142 to 281), were fractionated on a QAE-Sephadex A50 column in the absence of urea.
CnlA alone or CnlB alone showed no capacity to bind to troponin. But the combinations of CnlB with the N-chain and of CnlB with the s-fragment both showed a definite capacity to bind to troponin. The N-chain (residues 1 to 189) and the s-fragment (tentatively assigned as a mixture of residues 13 to 149 and 13 to 128) were obtained by cleavage at Cys 190 and tryptic digestion of α-tropomyosin, respectively.
CnlA alone, CnlB alone, their combination, and the combination of CnlB with the N-chain or the s-fragment, had no capacity to regulate actin-myosin interaction, as examined by retardation of superprecipitation rate in the presence of troponin.
The possible interaction between six fragments of α-tropomyosin, namely, CnlA, CnlB, the N-chain, the C-chain, the s-fragment, and the p-fragment (particularly the cross-combination of the polypeptide chains from the different kinds of fragments), was investigated by dissolving several pairs of fragments in 8M urea, then removing urea slowly by dialysis to promote the combination of the polypeptide chains. The samples thus treated were applied to gel electrophoresis. New bands due to association products were observed with the following combinations: the N-chain and CnlA, the N-chain and the s-fragment, and CnlB and the pfragment. On the other hand, the mixtures of one of the N-terminal fragments (CnlA, the N-chain, and the s-fragment) and one of the C-terminal ones (CnlB, the C-chain, and the pfragment) showed similar patterns to the sum of the original fragments without new bands.
The results obtained here support our earlier propose that troponin-binding regions in the molecular structure of tropomyosin are located at two regions, one near the 150th residue of the N-terminal side and the other near residues from Cys 190 to an unknown site in the Cterminal side. Thus, it is inferred that there is no biochemical homology between the N and the C-terminal portions of the tropomyosin molecule.View full abstractDownload PDF (956K) -
IV. Effects of Various Treatments In Vivo on the Golgi ApparatusYukinobu HINO, Akira ASANO, Ryo SATO1978 Volume 84 Issue 6 Pages 1567-1572
Published: December 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe effects of various treatments in vivo on the intracellular contents of the Golgi apparatus and microsomes (endoplasmic reticulum) in rat liver were studied. Partial hepatectomy increased the content of Golgi apparatus. Laparotomy also increased the content of Golgi apparatus, but to a lesser extent than partial hepatectomy. In contrast, the content of micro-somes remained unchanged by these treatments. On the other hand, the plasma seromucoid content was markedly increased by laparotomy, but unchanged by partial hepatectomy. Papain administration also caused an increase in the content of Golgi apparatus. The contents of both organelles were increased by the injection of phenobarbital. These results indicate that the control mechanisms of proliferation of Golgi apparatus are different from those of endoplasmic reticulum. These findings are discussed in relation to the functions of the Golgi apparatus, and it is suggested that the major function of the organelle at a given time is determined by the major metabolic demands at that time.View full abstractDownload PDF (464K) -
Shoji TAJIMA, Keiichi ENOMOTO, Ryo SATO1978 Volume 84 Issue 6 Pages 1573-1586
Published: December 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRabbit cytochrome b5 was incorporated into single-walled phosphatidylcholine liposomes, and the cytochrome b5-liposome complex thus formed was digested with trypsin. Protein chemical characterization indicated that the main products formed were 1) a hydrophilic (heme-con-taining) fragment of the cytochrome corresponding to the sequence consisting of the masked NH2-terminus through residue 88, 2) a hydrophobic peptide which spans residue 91 to the COOH-terminus (residue 133), and 3) a dipeptide, Beryl-lysine, derived from residues 89 and 90. The hydrophobic peptide was obtained in the form of its complex with liposomes. It was concluded that trypsin cleaved rather specifically the peptide bonds between residues 88 and 89 (Arg-Ser) and between residues 90 and 91 (Lys-Leu). Tryptic digestion of free, un-bound cytochrome b5 also resulted in the cleavage of the same peptide bonds. These results are not consistent with the proposal of Visser et al. (Visser, L., Robinson, N.C., & Tanford, C. (1975) Biochemistry 14, 1194-1199) that the hydrophilic and hydrophobic domains of cyto-chrome b5 are connected to each other by a link peptide consisting of some 15 amino acid resi-dues and that this link peptide can be cut out by the action of trypsin. The circular dichroism spectrum of intact cytochrome b5 or its complex with liposomes in the far-ultraviolet region was closely similar to the sum of the spectra of the hydrophilic fragment and the hydrophobic peptide (or its complex with liposomes). This indicates that the tryptic cleavage of the cytochrome molecule does not induce any significant changes in the conformations of the hydrophilic and hydrophobic moieties of the molecule and thus provides further evidence that the three-dimensional structures of the two domains are independent of each other.View full abstractDownload PDF (2363K) -
Hiroshi MUNAKATA, Zensaku YOSIZAWA1978 Volume 84 Issue 6 Pages 1587-1592
Published: December 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSComplex saccharides were extracted with 0.9% NaC1 at 0°C from rabbit small intestine, then fractionated with cetylpyridinium chloride (CPC), followed by DEAE-Sephadex A-25 column chromatography and by GEON-zone electrophoresis. An acidic glycoprotein (Fr. A-1) thus obtained was shown to be homogeneous by electrophoreses on cellulose acetate mem-brane and on sodium dodecyl sulfate (SDS)-polyacrylamide gel, as well as by gel filtration on Sepharose 4B. It contained 40.0% protein, 52.7% carbohydrate, and 1.6%. sulfate. The principal sugars in the sulfated glycoprotein were galactose, glucosamine, galactosamine, and sialic acid. Small quantities of mannose, L-fucose, and glucose were also present. Glutamic acid, threonine, aspartic acid, alanine, serine, leucine, proline, glycine, valine, and histidine were the major amino acids of the protein moiety. Small amounts of other basic amino acids, sulfur-containing amino acids and aromatic amino acids were also present.View full abstractDownload PDF (430K) -
IX. Isolation and Amino Acid Sequences of the Peptides Containing the Active Site Aspartyl Residues Reactive with Diazoacetyl-DL-norleucine Methyl Ester and 1, 2-Epoxy-3-(p-nitrophenoxy)Shin NAKAMURA, Kenji TAKAHASHI1978 Volume 84 Issue 6 Pages 1593-1600
Published: December 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA single peptide containing an active-site residue reactive with diazoacetyl-DL-norleucine methyl ester (DAN) was isolated from a peptic digest of Rhizopus chinensis acid protease which had been inactivated by stoichiometric reaction with DAN in the presence of cupric ions. The amino acid sequence of the peptide (D-III) was determined to be Asp-Thr-Gly-Thr-Thr-Leu. This sequence is highly homologous with those around the DAN-reactive aspartyl residues in pepsin [EC 3.4.23.1], chymosin (rennin) [EC 3.4.23.4], and penicillopepsin [EC 3.4.23.7].
On the other hand, two different peptides containing an active-site residue reactive with 1, 2-epoxy-3-(p-nitrophenoxy)propane (EPNP) were isolated from a peptic digest of the enzyme which had been inactivated by reaction with EPNP with incorporation of about 2 EPNP molecules per molecule of protein. One (EP-I) of the peptides was sequenced to be Asp-Thr-Gly-Ser-Ser-Asp. The amino acid sequence is highly homologous with those around the EPNP-reactive aspartyl residues in pepsin, and chymosin. The other peptide (EP-II) had the sequence Asp-Thr-Gly-Thr-Thr-Leu, which is identical with the peptide from the DAN-reactive site.
The amino-terminal aspartyl residue of each peptide was deduced to be the DAN- or EPNP- reactive site. These results indicate that the active site of Rhizopus chinensis acid protease involves two essential aspartyl residues, one reactive both with DAN in the presence of cupric ions and with EPNP, and the other reactive with EPNP.View full abstractDownload PDF (636K) -
Masao IWAMORI, Yoshitaka NAGAI1978 Volume 84 Issue 6 Pages 1601-1608
Published: December 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA novel monosialosyl ganglioside was isolated from human brain in a yield of 2.1nmol of the lipid-bound sialic acid per g of wet weight (0.09% of total lipid-bound sialic acid). The ganglioside was resistant to the action of Vibrio cholerae neuraminidase. By treatment with N-acetylhexosaminidase, methylation analysis, CrO3 oxidation, and partial acid hydrolysis, the structure of this ganglioside was determined to be;
Ga1NAc(β, 1-4)Gal(β, 1-3)GalNAc(β, 1-4)Gal(β, 1-4)Glc(β, 1-1)ceramide
(3-2 α) NeuNAc
The treatment of the ganglioside with 1N formic acid at 80°C for 2h produced mainly gangliotetraosyl ceramide, suggesting that the terminal N-acetylgalactosamine was more labile on formic acid treatment than galactose at nonreducing terminal.View full abstractDownload PDF (2702K) -
Tissue-Specific Distribution of N-Glycolylneuraminic Acid-Containing GM3Masao IWAMORI, Yoshitaka NAGAI1978 Volume 84 Issue 6 Pages 1609-1615
Published: December 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSMolecular species and concentrations of GM3 ganglioside in rabbit tissues, brain, thymus, lung, liver, stomach, intestine, kidney, testis, muscle, and erythrocytes were determined. The highest concentration was found in lung (431.7 nmol/g wet tissue). With the exceptions of brain, thymus, and erythrocytes, GM3 was the dominant ganglioside and comprised more than 50% of the total gangliosides. In brain and thymus, GM3 composed 0.76% and 17.28% of the total gangliosides, respectively. The sialic acid composition of GM3 was determined by mild methanolysis and neuraminidase treatment combined with either permethylation or trimethylsilylation. N-Glycolylneuraminic acid-containing GM3 was found in thymus, lung, kidney, and intestine in addition to N-acetylneuraminic acid-containing GM3, but N-acetyl-neuraminic acid was the sole sialic acid of GM3 in the other tissues. Sixty-four percent of the thymus GM3 consisted of N-glycolylneuraminic acid. High concentrations of C-20 sphingosine and stearic acid were characteristic for brain GM3 and the major long chain base of the other tissues was C-18 sphingosine. The fatty acid composition of GM3 varied in each tissue. The difference in mobility on a thin-layer plate was due to the difference in its molec-ular constitution, being derived from a combination of fatty acid, long chain base, and sialic acid.View full abstractDownload PDF (1831K) -
Yukio KATO, Heihachi NISHIHARA, Hideo INOUE, Eiichi GOHDA, Yoshiro TAK ...1978 Volume 84 Issue 6 Pages 1617-1623
Published: December 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe induction of hepatic ornithine decarboxylase [EC 4.1.1.17] is one of the earliest detectable events in liver regeneration. Administration of DL-α-hydrazino-δ-aminovaleric acid (DL-HAVA), a competitive inhibitor of ornithine decarboxylase, markedly inhibited the rise in the putrescine level and increase in DNA synthesis in rat liver after partial hepatectomy. DL-HAVA also depressed the rises in the activities of thymidine kinase [EC 2.7.1.21] and thy-midylate synthetase [EC 2.1.1.45] in regenerating rat liver, resulting in a large decrease in the hepatic deoxythymidine triphosphate (dTTP) pool that normally expands in response to the operation, though the induction of DNA polymerase [EC 2.7.7.7] was little affected by the inhibitor. These inhibitions by DL-HAVA were effectively reversed by the administration of putrescine, but not 1, 3-diaminopropane or cadaverine.
Administration of 1, 3-diaminopropane, which inhibits the induction of ornithine decar-boxylase, caused metabolic alterations in regenerating rat liver similar to those produced by DL-HAVA. Injection of aminoguanidine, a potent inhibitor of diamine oxidase [EC 1.4.3.6], resulted in increased putrescine accumulation and then increased DNA synthesis in the regenerating liver. These results strongly suggest that putrescine synthesis is a prerequisite for liver regeneration and that polyamine(s) is involved in the synthesis of dTTP (and con-sequently DNA synthesis) in the liver after partial hepatectomy.View full abstractDownload PDF (547K) -
Sadaji TSUKAMOTO, Motonori OHNO1978 Volume 84 Issue 6 Pages 1625-1632
Published: December 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPapain [EC 3.4.22.2] polymerizes readily upon treatment with tetranitromethane (TNM) by forming intermolecular covalent linkages through its tyrosine residues (Tsukamoto, S. & Ohno, M. (1974) J. Biochem. 75, 1377-1380). Polymerization occurred optimally at pH 9.0 with S-sulfenylsulfonate papain. Circular dichroic spectra of polymerized papains showed a small change in ellipticity when compared with that of unmodified papain. Esterolytic activity of the modified enzyme toward benzoyl-L-arginine ethyl ester (BASE) was almost fully retained, at least up to the formation of hexamer, with an unchanged Km value. Spectrophotometric and amino acid analyses indicated that two or three tyrosine residues are involved in intermolecular crosslinks depending on the amount of TNM used. The tyrosine residues nitrated were identified as those at positions 61, 116, 103, and 4, the extent of nitration decreasing in this order. When activated papain was treated with increasing molar ratios of TNM, an essential sulfhydryl function was first oxidized and, at a 2-fold molar excess of the reagent, restoration of activity was no longer observed even after addition of dithiothreitol (DTT). The evidence obtained in the present study eliminates the possibility of inactivation due to nitration of a tryptophan residue, which had been suggested previously.View full abstractDownload PDF (559K) -
Takashi MIKAWA, Yoshiaki NONOMURA, Masato HIRATA, Setsuro EBASHI, Shir ...1978 Volume 84 Issue 6 Pages 1633-1636
Published: December 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAn acidic Ca-binding protein of about 18, 000 dalton, different from both modulator protein and troponin C, was found to be involved in the regulatory mechanism of smooth muscle contraction by the tropomyosin-leiotonin system.View full abstractDownload PDF (262K) -
Shigeo OHNO, Kazutomo IMAHORI1978 Volume 84 Issue 6 Pages 1637-1640
Published: December 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIt was confirmed by polyacrylamide gel electrophoresis that isolated 16S rRNA was cleaved by the active component (protein A) or the active fragment (T2A) of colicin E3. However, the degradation was random, in contrast with the specific cleavage observed in the interaction of colicin E3 with ribosomes. Furthermore, the active component and the active fragment had low activities, and far greater amounts of these materials were required for degradation of the isolated rRNA than for ribosome inactivation.
The degradation of rRNA cannot be due to contaminating ribonuclease(s), but is due to colicin E3 itself, because of the following facts. (1) Protein B of colicin E3, which specifi-cally inhibits the ribosome-inactivating activity of colicin E3, inhibited the degradation of rRNA. (2) Protein B of colicin E2, which inhibits the action of colicin E2 but not of colicin E3, failed to inhibit the degradation of rRNA. (3) The activity appeared in the peak of protein A or fragment T2A, respectively, when they were rechromatographed on Sephadex G-75.View full abstractDownload PDF (262K) -
Shigeo MURAKAWA, Takeshi TAKAHASHI1978 Volume 84 Issue 6 Pages 1641-1643
Published: December 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe presence and some properties of an NAD+ transport system were examined in PA5, a Mg, Ca-ATPase [EC 3.6.1.3]-defective mutant strain of Escherichia coli W2252. NAD+ uptake was stimulated by exogenous energy sources and dependent on external substrate concentrations with an apparent Km of about 25μM. Most of the radioactivity from [14C]-NAD+ accumulated in the cells was identified as NAD+. [14C]NAD+ uptake was competitively inhibited by unlabeled NAD+, NADP+, NMN+ or nicotinamide. Similar uptake activity was also observed in W2252.View full abstractDownload PDF (177K) -
II. Chelate Structure of Active CenterTomitake TSUKIHARA, Keiichi FUKUYAMA, Hiromasa TAHARA, Yukiteru KATSUB ...1978 Volume 84 Issue 6 Pages 1645-1647
Published: December 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA chloroplast-type ferredoxin from Spirulina platensis crystallized in an orthorhombic system, space group C2221, with cell dimensions a=62.32, b=28.51, and c=108.08 Å. The electron density map at 2.8 Å resolution was prepared by using the best phase angles determined by the single isomorphous replacement method coupled with the anomalous dispersion method. The chelating structure of the active center was revealed as follows. Of the six cysteinyl residues in the molecule, Cys 41, Cys 46, Cys 49, and Cys 79 are involved in the active center. Cys 41 and Cys 46 are coordinated to one iron atom, and Cys 49 and Cys 79 to the other iron atom. Only one of these cysteinyl residues, Cys 79, is comparatively apart from the other three in the amino acids sequence of the molecule, as found in the case of bacterial ferredoxin. It appears that the NH…S hydrogen bonds are around the active center, as in other non-heme iron sulfur proteins.View full abstractDownload PDF (202K) -
Tomisaburo KAKUNO, Hisahide HIURA, Jinpei YAMASHITA, Robert G. BARTSCH ...1978 Volume 84 Issue 6 Pages 1649-1651
Published: December 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSHydrogenase was easily solubilized from light-grown cells of R. rubrum with 10mM Na ethylenediaminetetraacetate. The enzyme thus obtained was so stable that loss of its activity was undetectable during storage at room temperature for 6 months under air atmosphere, provided that NaCl, KCl or CsCl was present at ≥0.7M.View full abstractDownload PDF (164K) -
Kunio YAGI, Nobuko OHISHI1978 Volume 84 Issue 6 Pages 1653-1655
Published: December 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe lifetime of the purple intermediate formed from D-amino acid oxidase [D-amino acid: O2 oxidoreductase (deaminating), EC 1.4.3.3] and a neutral D-amino acid under anaerobic conditions was measured with a series of neutral n-amino acids. The lifetime increases with increase in the number of carbons in the side chain of the amino acid up to 4-5 atoms and then decreases with further increase in the number of carbons. This suggests that the hydrophobicity of the alkyl group of the neutral D-amino acid determines the lifetime of the purple intermediate unless a steric effect occurs.View full abstractDownload PDF (168K) -
Iwao NISHIURA, Kazuyoshi TANAKA, Susumu YAMATO, Takashi MURACHI1978 Volume 84 Issue 6 Pages 1657-1659
Published: December 01, 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe occurrence of a novel and specific inhibitor of Ca2+-dependent neutral protease in rat liver has been demonstrated. The partially purified product is a heat-stable and acid-stable protein of an approximate molecular weight of 3×105, and readily inactivated by tryptic digestion. The inhibition of Ca2+-dependent protease is not a result of the binding of Ca2+ by the inhibitor.View full abstractDownload PDF (238K) -
1978 Volume 84 Issue 6 Pages 1661
Published: 1978
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDownload PDF (26K)
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