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Volume 79 (1976)
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- Issue 4 Pages 693-
- Issue 3 Pages 455-
- Issue 2 Pages 237-
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Volume 79, Issue 2
Displaying 1-26 of 26 articles from this issue
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Kiyoshi IKEDA, Kozo HAMAGUCHI1976 Volume 79 Issue 2 Pages 237-247
Published: February 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe pH dependence of the binding of a dye, Biebrich Scarlet, to hen egg-white lysozyme [EC 3.2.1.17] was studied at ionic strength 0.3 and 25° by following circular dichroic (CD) bands originating from the bound dye. This binding involved one of the catalytic groups, Glu 35.
The effect of the binding of N-acetylglucosamine (G1cNAc), its dimer or trimer on the binding of this dye was also studied at pH 7.5 by measuring changes in the CD bands of the dye bound to lysozyme. It was shown that there are two sites for simultaneous binding of these saccharides in the lysozyme molecule. The stronger binding of the saccharide was noncompetitive and the weaker binding was competitive with the dye binding. The binding constants for the stronger binding site (the upper portion of the lysozyme cleft) were in good agreement with those previously determined by following changes in the tryptophyl CD bands of lysozyme. The binding constants to the weaker site were about 1.1×104, 5×102, and 5 M-1 for the trimer, dimer, and monomer of GlcNAc, respectively. Assuming that the trimer, dimer, and monomer occupy subsites D, E, and F; E and F; and E, respectively, the unitary free energies of saccharide binding were estimated to be about -1.9, -3.3, and -2.7 kcal/mole for D, E, and F, respectively.View full abstractDownload PDF (805K) -
Masaaki KURODA, Koscak MARUYAMA1976 Volume 79 Issue 2 Pages 249-257
Published: February 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe repolymerizability of native thin filaments of rabbit skeletal muscle was examined under various conditions. The native thin filaments usually did not repolymerize after depolymerization, but it was found that the process was only inhibited at the nucleation step of the G-F transformation of actin. The inhibited state was released by the addition of sonicated F-actin fragments as seeds or by direct sonication.
An attempt to isolate the actin moiety from native thin filaments showed that the isolated actin was very similar to Straub-type G-actin2 in its polymerizability. However, a protein factor which inhibits the polymerization of G-actin was obtained by ammonium sulfate fractionation of the depolymerized native thin filaments. Thus, the apparent lack of repolymerizability of native thin filaments was found to be due to the action of this factor on the polymerization of G-actin.View full abstractDownload PDF (2510K) -
XI. Re-evaluation of the Transition of ATPase Activity during the Initial PhaseMichihiro SUMIDA, Tohru KANAZAWA, Yuji TONOMURA1976 Volume 79 Issue 2 Pages 259-264
Published: February 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSWe previously reported (J. Biochem. 70, 95-123 (1971)) that the time course of P1 liberation in the reaction of Ca2+, Mg2+-dependent ATPase [EC 3.6.1.3] of fragmented sarcoplasmic reticulum (SR) consists of a lag. phase, a burst phase, and a steady phase. We also showed that the rate constant, kd, of decomposition of the phosphorylated intermediate (E_??_P) decreases during the initial phase, and suggested that the burst phase is due to transition of the kd value. Recently, Froehlich and Taylor (J. Biol. Chem. 250, 2013-2021 (1975)) claimed that the P1 burst is caused by the formation of an acid-labile intermediate containing phosphate (E•P) formed by rapid hydrolysis of E_??_P. In the present study, the transition of the kd value during the initial phase was measured precisely, and the results showed that the burst phase is due to a transition in the kd value, not to the existence of E•P. The main results obtained were as follows:
1. After the SR had been phosphorylated with [γ-32p]ATP in the presence of Mg2+ and Ca2+ ions, further phosphorylation was stopped by the addition of EGTA. The concentration of E_??_32P then decreased exponentially with time.
2. The first-order rate constant, kd, of decomposition of E_??_32P after adding EGTA decreased with increase in the interval, t, between the start of E_??_32P formation and the time of adding EGTA. The value of kd was given by
kd=(kd, initial-kd, steady)×exp(-ktr•t)+kd, steady,
kd, initial/kd, steady=7.3, and ktr=0.5sect-1, where kd, initial and kd, steady are the rate constant of E_??_P decomposition immediately after starting the reaction and that in the steady state, respectively. The value of ktr is the rate constant for transition in the kd value.
3. The value of kd was also obtained from the time course of E_??_32P decomposition after adding a large amount of unlabelled ATP to stop E_??_32P formation. The kd value thus obtained was the same as the value obtained by stopping E_??_P formation with EGTA.
4. The observed time course of P1 liberation after adding ATP to the SR was interpreted quantitatively in terms of the observed time course of E_??_P formation and the value of kd given above.
Furthermore, we previously showed that when ADP was added to E_??_P, ATP was formed in an amount equal to the amount of decrease in E_??_P. Therefore, the possibility that in the forward reaction of SR-ATPase an appreciable amount of E•P exists in equilibrium with E_??_P is excluded.View full abstractDownload PDF (449K) -
I. Decreases of β-Glycosidase Activities in Diabetic Rat KidneyHisako FUSHIMI, Seiichiro TARUI1976 Volume 79 Issue 2 Pages 265-270
Published: February 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSDeposition of PAS2-positive materials and thickening of the basement membrane in vascular lesions are characteristic findings in diabetes mellitus, suggesting altered metabolism of glycoprotein. Changes in the activities of the glycosidases, β-N-acetylglucosaminidase [EC 3.2.1.30], R-glucuronidase [EC 3.2.1.31], Q-galactosidase [EC 3.2.1.23], and β-glucosidase [EC 3.2.1.21] were measured in various organs and the serum of diabetic rats. The activities of the first three enzymes listed above were found to be much reduced in the kidney but increased in the serum. The decreased activities of β-glycosidases in the kidney may be one of the factors responsible for the pathogenesis of microangiopathy.View full abstractDownload PDF (422K) -
II. An Insulin-dependent Isozyme of β-N-AcetylglucosaminidaseHisako FUSHIMI, Seiichiro TARUI1976 Volume 79 Issue 2 Pages 271-275
Published: February 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPreviously we reported that β-glycosidase activities were markedly decreased in the kidney but increased in the serum of diabetic rats. To examine these changes, the isozymes of β-N-acetylglucosaminidase [EC 3.2.1.30] of rats were examined by DEAE-cellulose column chromatography. At least 3 major isozymes were found in both the kidney and liver. The main isozyme was type II isozyme in normal rat kidney and type III in normal rat liver. The activity of the type II isozyme in the kidney was markedly lowered when the total activity was decreased in diabetes and its normal activity was restored on insulin treatment, in parallel with increase in the total activity in diabetes. No significant change was found in the chromatographic pattern of isozymes in the liver in diabetes. In diabetic rat serum, the increase of total activity was found to be due to increase of type I and II isozymes.View full abstractDownload PDF (387K) -
Development of Enzyme Activity and Effects of AntibioticsHiroshi HYODO1976 Volume 79 Issue 2 Pages 277-282
Published: February 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSPhenylalanine ammonia-lyase [EC 4.3.1.5] activity increased rapidly after a 3-hr lag period in potato tuber (Solanum tuberosum L. cv. May Queen) disks incubated in a suitable medium in the dark at 25°. The activity reached a maximum after incubation for about 40 hr. The effects of actinomycin D, 6-methylpurine, cycloheximide, chloramphenicol, and mitomycin C on the induction of phenylalanine ammonia-lyase were investigated during incubation of the disks. Actinomycin D, 6-methylpurine, and cycloheximide all inhibited the formation of phenylalanine ammonia-lyase, though cycloheximide was the most effective at low concentrations. Application of actinomycin D for the initial lag period (3 hr) caused strong inhibition; however, if it was supplied later it did not inhibit but actually increased phenylalanine ammonialyase formation. In contrast, cycloheximide was effective over most of the incubation period. Chloramphenicol and mitomycin C did not inhibit phenylalanine ammonialyase induction, but markedly stimulated it. Light was not an essential factor for phenylalanine ammonia-lyase induction in the wounded tissue.View full abstractDownload PDF (474K) -
Kiyoshi NOSE1976 Volume 79 Issue 2 Pages 283-288
Published: February 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAlkaline phosphatase [EC 3.1.3.1] was purified about 250-fold from rat kidney, and its enzymological properties were studied.
Kidney homogenate was extracted with n-butanol, passed through Sephadex G-200 and chromatographed on a DEAE-cellulose column. The peak from the DEAE-cellulose column was subjected to isoelectric focusing, and the alkaline phosphatase activity was separated into two peaks. The molecular weights of alkaline phosphatase in these peaks were 4.8×104 and 1.0×105, as determined by SDS-polyacryl-amide gel electrophoresis.
Anti-serum against alkaline phosphatase from rat kidney was prepared, and was shown to neutralize the activity from kidney, liver or bone, but not that from intestine.View full abstractDownload PDF (384K) -
I. Tyrosinase-catalyzed Modification of AsparaginaseMasanobu TOKUSHIGE, Fumio MORIYA1976 Volume 79 Issue 2 Pages 289-292
Published: February 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSAsparaginase [EC 3.5.1.1] of Escherichia coli, an anti-tumor enzyme, was inactivated in a time-dependent fashion by mushroom tyrosinase [EC 1.14.18.1]. The inactivation did not proceed, however, when heat-inactivated tyrosinase was used. Exclusion of the atmospheric oxygen or addition of diethyldithiocarbamate, a copper selective chelating agent, prevented the inactivation. The difference absorption spectrum of tyrosinase-inactivated asparaginase versus intact asparaginase exhibited the appearance of marked absorption peaks at 300 and 350nm. These results indicate that the tyrosyl residue(s) of asparaginase, which is essential for the activity is enzymatically modified by tyrosinase.View full abstractDownload PDF (279K) -
Masahiko ENDO, Zensaku YOSIZAWA1976 Volume 79 Issue 2 Pages 293-298
Published: February 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe endometrial scrapings obtained from the uteri of estrogen-treated rabbits were incubated with N-acetyl-D-[1-3H] glucosamine and [35S] sulfate, and then the incubation medium (M-Fr) was separated from the tissue. The tissue was subsequently homogenized exhaustively in 0.25M sucrose, and the insoluble residue (R-Fr) was separated. The supernatant at 8, 500×g for 10min of the homogenate was subjected to subcellular fractionation by discontinuous sucrose gradient ultracentrifugation, and a thiamine pyrophosphatase-rich fraction (G-Fr) was obtained. Complex carbohydrates were then separated from M-Fr, R-Fr, and G-Fr. The radioactivities incorporated into these complex carbohydrates suggested that sulfated glycoprotein synthesized in G-Fr was secreted into M-Fr.
In order to confirm the above observation, labelled sulfated glycoprotein was isolated from the incubation medium. Subsequently, N-acetylglucosamine sulfate doubly labelled with 3H and 35S was separated from an acid hydrolyzate of the sulfated glycoprotein. The results indicated that N-acetyl-D-[1-3H] glucosamine was incorporated into N-acetylglucosamine residues and [35S]sulfate into sulfates located most probably at the 6-position of N-acetylglucosamine residues of sulfated glycoprotein.View full abstractDownload PDF (500K) -
I. Quantitative Separation of Trace Amounts of Alditols from Mixtures with a Large Excess of MonosaccharidesHaruki YAMAGUCHI, Shuichi INAMURA, Kiyoshi MAKINO1976 Volume 79 Issue 2 Pages 299-303
Published: February 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSA method has been devised for the quantitative separation of trace amounts of alditols from mixtures with large amounts of monosaccharides, using a strongly basic ion-exchange resin.
Ten kinds of common reducing monosaccharides used were strongly retained by a very basic anion-exchange resin in the hydroxyl form, whereas the corresponding alditols showed no significant affinity for the basic resin. Model studies showed excellent recoveries of alditol from known mixtures of an alditol and the corresponding aldose at ratios in the range from 1:1×103 to 1:1×104 (by weight). Application of this procedure to dextrans after reduction and hydrolysis resulted in quantitative separation of the terminal alditol.View full abstractDownload PDF (428K) -
Akiko HIROSE, Jun-ichi KUMAGAI, Kazutomo IMAHORI1976 Volume 79 Issue 2 Pages 305-311
Published: February 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSIt has recently been found that so-called native colicin E3, which has been used for studies of its mode of action, is a complex of two kinds of proteins. The complex could be dissociated into the two components in SDS. These components were isolated by gel filtration in 1% SDS followed by treatment with Dowex-2 to remove bound SDS. One component, characterized by its low molecular weight, prevented colicin E3-induced inhibition of poly(U)-dependent protein synthesis and was designated as immunity substance. The other component (protein A), which was of high molecular weight, had 100-fold higher in vitro ribosome-inactivating activity than native colicin E3, but had lower bacteriocidal activity.
Colicin E3 was reconstituted from the two isolated protein components. The reconstituted colicin E3, when compared with protein A, showed a decrease in in vitro activity (inhibition of poly(U)-dependent protein synthesis), but had higher bacteriocidal activity in vivo. Thus complex formation of protein A with immunity substance should play an important role in the bacteriocidal action, but protein A itself might inactivate ribosomes in the interior of the sensitive cells.View full abstractDownload PDF (1440K) -
Yoshiyuki TAMURA, Kazuko OKAMURA, Akihiro OTSUKA, Setsuro FUJII1976 Volume 79 Issue 2 Pages 313-319
Published: February 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSC1r was isolated from human serum by DEAE-cellulose column chromatography in the presence of EDTA. The isolated C1r did not hydrolyze Nα-acetyl-L-arginine methyl ester, unless activated by brief treatment with trypsin [EC 3. 4. 21. 4]. On the column, the C1 esterase inhibitor activity was found to coincide with Clr but not C1s (another subcomponent of the first component).
Cl_??_ was isolated from the euglobulin fraction of human serum by DEAF-cellulose column chromatography. On Sephadex G-200 column chromatography, C1r was eluted in the void volume, whereas C1_??_ was eluted in a position corresponding to a molecular weight of 140, 000-160, 000. The results indicate that, on activation, C1r was converted to an enzyme of lower molecular weight.
Cl_??_ was converted to inactivated Cl_??_ by addition of pseudoglobulin and the latter was isolated by DEAF-cellulose column chromatography. The inactivated C1_??_ was found to have a larger molecular weight than C1_??_.
Trypsin, which activated Clr to C1_??_, completely inactivated the C1 esterase inhibitor activity of the C1r fraction.
From the above evidence it is concluded that C1r is a complex of C1 esterase inhibitor and C1_??_.View full abstractDownload PDF (545K) -
II. Structural Changes Induced by Denaturants and Their ReversibilityTaro OGISO, Masahide AOYAMA, Mihoko WATANABE, Yoshio KATO1976 Volume 79 Issue 2 Pages 321-328
Published: February 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSNo change in the activity of a trypsin inhibitor from barley was observed on treatment with heat and denaturants such as urea and guanidine hydrochloride. Thus, the conformational properties of the inhibitor were investigated. CD spectra of the native inhibitor were analysed by a curve-fitting technique using the data for poly-L-lysine. The results indicate that the inhibitor contains 41% α-helix, 25% β-structure, and 34% unordered conformation. In the presence of 2-8M urea or 3M guanidine hydrochloride, the CD spectra were almost unchanged. On treatment at 100° for 10min in 8M urea the depth of the trough at 200-240nm decreased considerably, while both 6M guanidine hydrochloride and 6M guanidine hydrochloride at 100° reduced the molar ellipticity of the negative band at 222nm by about two-thirds. When inhibitor treated in 6M guanidine hydrochloride at 100° was diluted 10- or 30-fold, the conformation was significantly reformed. The CD spectra at alkaline pH showed that the inhibitor also has resistance to alkali. As a result of difference spectrum studies, it was shown that the inhibitor had peaks at 294 and 285nm and troughs at 290 and 280nm, due to denaturation, in strong denaturing media, 8M urea at 100°C and 6M guanidine hydrochloride. The positive and negative peaks, however, immediately disappeared on removal of the denaturant. From spectrophotometric titrations, the phenolic hydroxyl groups were found to be ionized above pH 10.5. Four tyrosine residues are rapidly titrated and the last one was ionized above pH 12.5 with partial inactivation. Cleavage of the disulfide bridges in the inhibitor induced a very marked decrease in the value of [θ]222, leading to complete loss of the inhibitory activity. A possible interpretation of these data is that the inhibitor contains a rigid disulfide loop, and the disulfide bridges contribute to the structural stability and reversibility of the conformational changes of the protein.View full abstractDownload PDF (621K) -
Toshiharu HASE, Keishiro WADA, Hiroshi MATSUBARA1976 Volume 79 Issue 2 Pages 329-343
Published: February 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe amino acid sequence of the major ferredoxin component isolated from a bluegreen alga, Aphanothece sacrum, has been fully determined. Chymotryptic and tryptic peptides of carboxymethyl-ferredoxin and chymotryptic peptides of oxidized ferredoxin were prepared and their sequences were analyzed. Together with previous work on the amino terminal sequence, established with a sequence analyzer, these peptide sequences permitted the complete amino acid sequence of this ferredoxin to be deduced. It was composed of 96 amino acid residues with five cysteine residues. The cysteine residue at the 18th position usually found in other chloroplast-type ferredoxins was replaced by a valine residue and the cysteine residue at the 85th position was common with that in Scenedesmus ferredoxin. The sequence was Ala-Ser-Tyr-Lys-Val-Thr-Leu-Lys-Thr-Pro Asp-Gly-Asp-Asn-Val-Ile-Thr-Val-Pro-Asp-Asp-Glu-Tyr-Ile-Leu-Asp- Val-Ala-Glu-Glu-Glu-Gly-Leu-Asp-Leu-Pro-Tyr-Ser-Cys-Arg-Ala-Gly-Ala-Cys-Ser-Thr-Cys-Ala-Gly-Lys-Leu-Val-Ser-Gly-Pro- Ala-Pro- Asp- Glu-Asp- Gln-Ser-Phe-Leu-Asp- Asp- Asp- Gin-Ile-Gin- Ala-Gly-Tyr-Ile-Leu-Thr-Cys-Val -Ala-Tyr-Pro-Thr-Gly-Asp-Cys-Val-Ile-Glu-Thr-His-Lys-Glu-Glu-Ala-Leu-Tyr. Calculation of the numbers of amino acid differences among chloroplast-type ferredoxins indicates that the Aphanothece ferredoxin is far divergent not only from higher plant ferredoxins but also from Spirulina ferredoxins, blue-green algal ferredoxins. Some comments on the evolution of ferredoxins are presented.View full abstractDownload PDF (1057K) -
Masaru OTANI, Hirose YAMAUCHI, Toshio MEGURO, Shyuichi KITAZAWA, Susum ...1976 Volume 79 Issue 2 Pages 345-352
Published: February 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSCalcitonin was extracted from the pericardium and esophagus of eel in quantities sufficient to permit purification and chemical characterization. Homogeneous calcitonin could be isolated by a six-step fractionation starting from acetone powder of the organs.
The fractionation procedure consisted of acid extraction, gel filtration on Sephadex G-75, chromatography on SP-Sephadex C-25, gel filtration on Sephadex G-50, chroma-tography on carboxymethylcellulose, and gel filtration on Sephadex G-50. Fractionation of the hormone was monitored by assay of its biological activity and from its behaviour on thin layer chromatography and polyacrylamide gel disc electrophoresis. The hormone contained 32 amino acid residues, like calcitonins from other species of animals, but its amino acid composition was different from those of previously characterized hormones. Eel calcitonin possessed almost the same, or higher, biological activity as the salmon or chicken hormone, which show the highest specific activity among calcitonins so far isolated.View full abstractDownload PDF (588K) -
Toshiharu NODA, Kozo NARITA1976 Volume 79 Issue 2 Pages 353-359
Published: February 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe primary structure of eel calcitonin, containing 32 amino acid resudues, was elucidated using the reduced and aminoethylated derivative. Manual Edman degradation was employed from the N-terminus to the 31st residue in an average yield of about 90% and the C-terminal prolineamide was characterized as its dansyl derivative. The following amino acid sequence was deduced for eel calcitonin : Cys-Ser-Asn-Leu-Ser-Thr-Cys-Val-Leu-Gly-Lys-Leu-Ser- Gln- G lu-Leu-His- Lys-Leu-GI n-Thr-Tyr-Pro-Arg-Thr-Asp-Val-Gly-Ala-Gly-Thr-Pro-NH2. Among the 32 amino acids of eel calcitonin only three amino acid replacements were observed, at the 26th, 27th, and 29th positions, in comparison with salmon calcitonin I, which is known to possess the highest hormonal activity among calcitonins so far isolated from various sources in a homogeneous state.View full abstractDownload PDF (536K) -
Yuzo SHIOI, Ken-ichiro TAKAMIYA, Mitsuo NISHIMURA1976 Volume 79 Issue 2 Pages 361-371
Published: February 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSNAD+ reductase of the green photosynthetic bacterium Prosthecochloris aestuarii was isolated and purified by ammonium sulfate fractionation, DEAE-cellulose column chromatography, and Sephadex G-200 gel filtration.
This enzyme is an FAD-containing flavoprotein and has absorption maxima at 485 (shoulder), 452, 411, and 385nm (the 411nm band is due to cytochrome). The molecular weight of the enzyme as determined by gel filtration using Sephadex G-200 is 119, 000. The enzyme catalyzes the reduction of NAD+ and NADP+ by photoreduced spinach ferredoxin or reduced benzyl viologen. It also catalyzes the reduction of cytochromes and dyes such as benzyl viologen and 2, 6-dichloroindophenol (DCIP) with NADH or NADPH as the electron donor. The reduction of cytochrome c-555(550) of this organism is accelerated by the addition of cofactors such as menadione. In these reactions, the enzyme is more specific for NAD+ or NADH than for NADP+ or NADPH. Reduction of cytochrome c or dyes catalyzed by the enzyme is strongly inhibited by rotenone or amytal, but not by antimycin A or o-phenanthroline.
It is suggested that NAD+ photoreduction in this organism takes place via direct electron flow with the mediation of ferredoxin and flavoprotein enzyme, as in Chlorobium and green plants.View full abstractDownload PDF (824K) -
Jun'ichi HASE, Kyoichi KOBASHI, Noboru NAKAI, Satomi ONOSAKA1976 Volume 79 Issue 2 Pages 373-380
Published: February 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSRetinol-binding protein (RBP) was purified from fresh urine of patients suffering from
“Itai-Itai” disease. The purified preparation contained two types of apo-RBP (Apo I and II) in equal amounts as major components (about 85% of the total RBP). The corresponding two retinol-binding forms (Holo I and II) were present as minor components (about 15% of the total).
Retinol-binding complex was produced by mixing an n-heptane solution of retinol with an aqueous solution of the purified apo-RBP preparation; the molar ratio of retinol to the protein in the reconstituted complex was 0.8, which is close to the equimolar ratio of retinol to RBP obtained from human plasma. Investigations of the binding of apo-RBP with various terpenoids revealed that an isoprene structure possessing conjugated double bonds is necessary for binding, which is strengthened by the presence of a β-ionone ring. Carboxylic acids tested were bound to apo-RBP with high affinity, possibly due to ionic binding of the carboxylic anion with the protein, supported by hydrophobic residues of the compounds. A carbonyl group promoted binding in the case of low molecular compounds, but was less effective in higher molecular compounds. A hydroxyl group was less effective for binding in small molecules, but was markedly effective in the case of retinol binding. The higher affinity of retinol with apo-RBP was probably due to the hydroxyl group, enhanced by the hydrophobic nature of the isoprene moiety. Retinol acetate and methyl retinoate did not bind to RBP at all, which appears to exclude the possibility that the hydrophobicity alone is responsible for the binding.View full abstractDownload PDF (612K) -
Teruo IWASAKI, Toshifumi KIYOHARA, Mitsuyoshi YOSHIKAWA1976 Volume 79 Issue 2 Pages 381-391
Published: February 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe complete amino acid sequence of an active fragment of potato proteinase inhibitor IIa has been established by the Edman degradation procedure and the carboxypeptidase technique. Sequence analyses were carried out on the reduced and carboxymethylated active fragment and its Cryptic peptides. To aid in the alignment of some tryptic peptides, the partial sequences of two fragments obtained by selective tryptic cleavage of the reactive site peptide bond of inhibitor IIa at acidic pH, with subsequent reduction and carboxymethylation, were also analyzed. The active fragment consisted of 45 amino acid residues including 6 half-cystine residues. Degradation of the intact active fragment by subtilisin [EC 3.4.21.14] at pH 6.5 yielded 3 cystine-containing peptides. Sequence analyses of these peptides revealed that the 3 disulfide linkages were located between Cys(10) and Cys(24), Cys(14) and Cys(35), and Cys(20) and Cys(43). The reactive site peptide bond of inhibitor IIa, a Lys-Ser bond, was located between positions 32 and 33 of the active fragment. The overall sequence of the active fragment was quite different from those of potato chymotrypsin inhibitor I (subunit A) and potato carboxypeptidase inhibitor.View full abstractDownload PDF (770K) -
Hitoshi KONDO, Hiroshi NAKATANI, Keitaro HIROMI1976 Volume 79 Issue 2 Pages 393-405
Published: February 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. It was found that 6-p-toluidinylnaphthalene-2-sulfonate (TNS) showed pronounced fluorescence enhancement when it was added to α-, β-, and γ-cyclodextrin solutions. 2. The following results were obtained by quantitative study of the interactions of three kinds of cyclodextrins with TNS by following TNS fluorescence at pH 5.3 and 25°.
i) α-Cyclodextrin forms a 1:1 complex with TNS. ii) β- and γ-Cyclodextrins form 1:1 and also 2:1 complexes; in the latter two cyclodextrin molecules bind to one TNS molecule. iii) The dissociation constants of cyclodextrin-TNS complexes were determined to be 54.9mM for α-cyclodextrin, 0.65mM for -cyclodextrin and 0.66mM for γ-cyclodextrin in the 1:1 complex, and the secondary dissociation constants in the 2:1 complex were 71.4mM for β-cyclodextrin and 32.6mM for r-cyclodextrin. iv) The apparent quantum yields of cyclodextrin-TNS complexes were also determined, using quinine sulfate as a standard, to be 0.040 for α-cyclodextrin, 0.024 for β-cyclodextrin, and 0.004 for γ-cyclodextrin in the 1:1 complex, and the apparent quantum yields attributable to secondary binding in the 2:1 complex were 0.136 for β-cyclodextrin and 0.074 for γ-cyclodextrin. v) Pronounced TNS fluorescence enhancement seems to be due to the formation of inclusion complexes of cyclodextrins with TNS.
3. The pronounced fluorescence enhancement of TNS accompanying the formation of cyclodextrin-TNS inclusion complexes was effectively utilized to selectively monitor the rate of β-cyclodextrin hydrolysis by Taka-amylase A [EC 3.2.1.1] at pH 5.3 and 25°. The Michaelis constant Km and molecular activity ko in the hydrolysis of β-cyclodextrin catalyzed by Taka-amylase A were determined to be 10.0±0.5mM and 224.1±11.1min-1, respectively. Furthermore, this method was applied to the kinetic study of inhibition by maltose and α-cyclodextrin of the Taka-amylase A-catalyzed hydrolysis of β-cyclodextrin.View full abstractDownload PDF (986K) -
I. Turnover Rate of PhosphatidylinositolKeizo WAKU, Yusuo NAKAZAWA, Wataru MORI1976 Volume 79 Issue 2 Pages 407-411
Published: February 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESS1. Radioactive precursors, 32P1 [1-14C] glycerol, and [1-14C] acetate, were individually injected into the peritoneal cavity of mice bearing Ehrlich ascites tumor, and the rates of incorporation into phospholipid fractions of Ehrlich ascites tumor cells were estimated. Although no distinct difference in specific activities was observed between phosphatidylinositol and other phospholipid classes as regards the incorporation of [1-14C]acetate or [1-14C]glycerol, a higher rate of incorporation of 32P1 into phosphatidyl-inositol was observed. The specific activity of phosphatidylinositol reached more than ten times that of phosphatidylcholine in the first hour.
2. The radioactivities incorporated into the phospholipids of Ehrlich ascites tumor cells and liver were estimated after simultaneous injection of 32P1, and [2-3H]inositol. The incorporation of 32P, into phosphatidylinositol of liver was similar in specific activity to those of other phospholipids. The ratio (3H/32P1) of phosphatidylinositol decreased only slightly in the ascites tumor cells, while an appreciable decrease of the ratio was observed in the liver during the first 3 hr.
3. These results suggest that phosphatidylinositol synthesis through pathways other than de novo synthesis is rapid in ascites tumor cells.View full abstractDownload PDF (397K) -
Takaaki KOBAYASHI, Kazuhiko KAJI, Hikoichi SAKAI1976 Volume 79 Issue 2 Pages 413-418
Published: February 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSNucleoside diphosphate kinase [EC 2.7.4.6] of sperm flagella and Tetrahymena cilia is detected mostly in the outer fiber fraction, suggesting an association of the enzyme with the outer fiber microtubules. The enzyme does not catalyze transphosphorylation between microtubule-bound GDP and exogenous ATP. Even when undulatory movement of glycerinated sperm is induced by MgATP, no phosphorylation is detected in the outer fiber fraction. These facts do not appear to support the hypothesis that the phosphorylation of microtubule-bound GDP is involved in the mechanism of fiagellar movement.View full abstractDownload PDF (432K) -
Akio INOUE, Yuji TONOMURA1976 Volume 79 Issue 2 Pages 419-434
Published: February 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSH-Meromyosin (HMM) was digested with insoluble papain [EC 3.4.22.2]. Neither the size of the initial burst of P, liberation (0.5 mole/mole of myosin head) nor the Mg2+-ATPase [EC 3.6.1.3] activity of HMM in the steady state was affected by this treatment. Acto-S-1 was obtained by mixing F-actin with HMM digested with insoluble papain (HMM-S-1).
The size of the initial burst of P1 liberation of acto-S-1 was 0.35 mole/mole of S-1 at an ATP concentration of 0.5 mole/mole of S-1, and 0.5 mole/mole of S-1 at ATP concentrations above 1 mole/mole of S-1.
F-Actin (2mg/ml) was mixed with 3.4mg/ml (20μM) of HMM-S-1 in 50mM KCl, 2mM MgCl2, and 10mm Tris-HCl at pH 7.8 and 0°. After adding 10μM ATP (0.5 mole/mole of S-1) with 4mg/ml of pyruvate kinase [EC 2.7.1.40] and 12mm PEP, the reaction mixture was centrifuged at 1.6×105×g and 0° for 40-60min. The amounts of S-1 bound -to F-actin in the precipitate and of S-1 dissociated from F-actin in the supernatant were measured by SDS-gel electrophoresis. S-1 was divided equally between the supernatant and the precipitate, but the sizes of the initial bursts of P1 liberation of S-1 in the supernatant and the precipitate were about 0.7 and 0.3 mole/mole of S-1, respectively. When the separation procedures were repeated, the size of the initial burst of S-1 in the supernatant increased to 0.8 mole/mole of S-1, while that of S-1 in the precipitate decreased to 0.25 mole/mole of S-1.
Myosin was separated into two fractions, one bound to F-actin (precipitate) and the other dissociated from F-actin (supernatant), by centrifugation of actomyosin in the presence of various concentrations of ATP with an ATP-regenerating system at high ionic strength. At all ATP concentrations used, the myosin in the precipitate and supernatant gave the same sizes of initial burst (0.5 mole/mole of myosin head) as myosin before the separation.View full abstractDownload PDF (1180K) -
Tateo YAMANAKA1976 Volume 79 Issue 2 Pages 435-440
Published: February 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe ratio between the nitrite reductase and cytochrome oxidase activities of Pseudomonas aeruginosa nitrite reductase [EC 1.9.3.2] varies with kind of C-type cytochrome used as the electron donor. With cytochrome c-548, 554 (Micrococcus sp.), the nitrite reductase activity is greater than the cytochrome oxidase activity, while the former is smaller than the latter with cytochrome c-554 (Navicula pelliculosa). The aerobic oxidation catalyzed by this enzyme of denitrifying bacterial ferrocytochrome c is greatly accelerated on addition of nitrite, while that of the algal ferrocytochrome c is not affected or is even depressed by the salt. An accelerative effect of nitrite is generally observed with many kinds of C-type cytochromes which react with the enzyme very or fairly rapidly. The difference in the ratio of the two activities of the enzyme seems to arise according to whether or not nitrite affects the interaction of the C-type cytochrome with the enzyme.View full abstractDownload PDF (432K) -
Mayumi FUJIMOTO, Takaaki KAMEJI, Akira KANAYA, Hiroshi HAGIHIRA1976 Volume 79 Issue 2 Pages 441-449
Published: February 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSArginase [L-arginine amidinohydrolase EC 3.5.3.1] from rat small intestine was purified about 2, 200-fold and its properties were compared with those of the rat liver and kidney enzymes. Intestinal arginase was extremely labile on storage either at -10° or 4° and lost activity during purification unless 25mM L-valine was present.
The purified enzyme appeared to be homogeneous by disc electrophoresis and its molecular weight was estimated to be 120, 000 by Sephadex G-100 filtration. The enzyme was highly specific for L-arginine and showed maximal activity at pH 10.0 in the presence of Mn2+. The Km for L-arginine was 19mM and the activity was competitively inhibited by L-lysine, L-ornithine, L-valine, L-isoleucine, L-leucine, and L-proline, and non-competitively by L-tryptophan.
The enzyme was almost completely inactivated by treatment with EDTA at pH 4.5, then immediately returning it to pH 7.4, and assaying in the absence of Mn2+, but some activity was detected when it was assayed in the presence of Mn2+. Similar phenomena were observed with rat liver and kidney arginase, but the latter two enzymes dissociated into subunits on EDTA treatment at low pH, while intestinal arginase did not.View full abstractDownload PDF (1334K) -
Tamio NOGUCHI, Atunori KASHIWAGI, Takehiko TANAKA1976 Volume 79 Issue 2 Pages 451-454
Published: February 25, 1976
Released on J-STAGE: November 18, 2008
JOURNAL FREE ACCESSThe ornithine decarboxylase [EC 4.1.1.17] activities in the liver and spleen of tumorbearing mice increased remarkably, reaching a peak 4 to 6 days after inoculation of tumor cells. On the contrary, the enzyme activity in the kidney decreased during tumor growth and had almost disappeared on day 6 after tumor inoculation. Injection of cell-free tumor homogenate also raised the enzyme activities in the liver and spleen, but did not change the activity in the kidney. No increase in enzyme activity in the liver of mice was observed on injection of homogenates of normal tissues, such as liver, spleen, kidney, and muscle.View full abstractDownload PDF (283K)
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