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61.
In order to investigate the kinetics and mechanism of the removal of zinc ions from bovine carbonic anhydrase [EC 4.2.1.1] (BCA), several chelating agents with various stability constants were used to remove zinc from BCA. The second-order rate constants (kaap) of zinc removal from BCA were found to be in the following order; 2,6-pyridinedicarboxylic acid greater than 2-pyridinecarboxylic acid greater than 2,4-pyridinedicarboxylic acid greater than 2,3-pyridinedicarboxylic acid greater than or approximately 1,10-phenanthroline greater than or approximately 5-methyl-1,10-phenanthroline greater than 2,2'-bipyridine. With similar chelating agents the greater the stability constant, the faster was the rate of removal of zinc ions from BCA. With EDTA, trans-1,2-cyclohexanediaminetetraacetic acid, and nitrilotriacetic acid, the rate of zinc ion removal from the native enzyme was governed by the rate of spontaneous dissociation of zinc enzyme. The rate constants for the removal of zinc ions from BCA were governed by the affinity of the chelating agents for the metal ion and the conformation of the chelating agents. Based on these findings, reaction pathways for various chelating agents are proposed. 相似文献
62.
Quantitative study of the cytochrome c acting in the photosyntheticsystem of the blue-green alga Anabaena variabilis (M-2) wasdone with membrane fragments and intact cells. Membrane fragments highly active in the NADP+-Hill reaction(above 200 µmoles/mg chl.a;-hr) retained photoresponsivecytochrome c equal only one-tenth that of P700, while the plastocyanincontent was almost equal to that of P700. The cytochrome contentin intact cells was a little larger than that in membrane fragmentson the chlorophyll a basis. However, the values relative toP700 (1/9) and plastocyanin (1/10) were identical with thosein membrane fragments. The content was also far smaller thanthat of reaction center II's (1/6). If the cytochrome mediatesall electrons from reaction center II, the cytochrome oxidation-reductionshould have a rate constant of 2.4?102 sec1 which isone order above of the rate constant of the cytochrome reduction(2.3 to 3.5?101sec1). These quantitative relationshipsindicate that in Anabaena variabilis (M-2), c-type cytochrome,either cytochrome f or algal cytochrome c, cannot function inthe main electron flow between two reaction centers. (Received September 8, 1978; ) 相似文献
63.
In the search for the photoreceptor in photocontrolled phycoerythrinformation, photoreversible absorption changes of chromoproteinsin vivo and in vitro were studied with the blue-green alga Tolypothrixtenuis. Neither intact cells nor crude extracts of soluble proteinsshowed any significant absorption changes which were reversiblyinduced by green and red light. However, the photoresponse wasobservable when the crude protein extracts were treated withthe chaotropic reagent guanidine-HCl (0.4 M, for 1 hr in thedark). Isolated phycocyanin and allophycocyanin also showedthe same photoresponse after the guanidine-HCl treatment. Thedifference spectrum (green minus red) of guanidine-HCl-treatedphycocyanin was almost identical with that shown by phycochromea of Bj?rn and Bj?rn (3), and the allophycocyanin showed thesame difference spectrum as those of phycochrome c of Bj?rnand Bj?rn and the photoreversible pigment isolated by Scheibe(7). Urea at a concentration higher than 1 M or alkaline incubation(pH 8.5) also showed the same effect. The results were interpretedas indicating that phycocyanin and allophycocyanin obtain theability for photoresponsiveness when their protein conformation,probably around the chromophore site, is modified. (Received October 30, 1978; ) 相似文献
64.
Carotenoid photobleaching induced by photosystem II action wasstudied using membrane fragments of the blue-green alga Anabaenavariabilis. Special attention was paid to the action of O2. Carotenoid photobleaching elicited by carbonyl cyanide m-chlorophenylhydrazone(CCCP) depended on O2. However, the addition of H2O2, sodiumsilicotungstate or potassium ferricyanide (Ferri), an electronacceptor for reaction center II action, removed the O2-dependency.These results indicate that O2 acts as the electron acceptorfor this reaction. When both CGCP and Ferri were present, a short illumination(0.25 sec) caused a rapid photobleaching followed by a slowrecovery in the subsequent dark period. The spectrum of theabsorption decrease in the light was identical with that ofthe absorption increase in the subsequent dark, indicating thata reversible process is involved in the carotenoid photobleaching.The size in the dark recovery relative to the light bleachingbecame larger under anaerobic conditions and smaller under higherpartial pressure of O2. The reuslts were interpreted as indicatingthat O2 does not function in the primary process including areversible bleaching step, but is involved in the slow and irreversiblebleaching process. (Received April 3, 1978; ) 相似文献
65.
Ovalbumin, which contains one intrachain disulfide bond and four cysteine sulfhydryls, was reduced with dithiothreitol under non-denaturing conditions, and its conformation and stability were compared with those of the disulfide-bonded form. The CD spectrum in the far-UV region revealed that the overall conformation of the reduced form is similar to that of the disulfide-bonded one. Likewise, the inaccessibility to trypsin and the non-reactivity of the four cysteine sulfhydryls, exhibited by the native disulfide-bonded ovalbumin, were still retained in the disulfide-reduced form. Thus, the reduced ovalbumin appeared to substantially take the native-like conformation. However, the near-UV CD spectrum slightly differed between the native and disulfide-reduced forms. Protein alkylation with a fluorescent dye and subsequent sequence analysis showed that the two sulfhydryls (Cys73 and Cys120) originating from the disulfide bond are highly reactive in the reduced form. Furthermore, upon proteolysis with subtilisin, the N-terminal side of Cys73 was cleaved in the reduced form, but not in the disulfide-bonded one. Upon heat denaturation, the transition temperature of the reduced form was lower, by 6.8 degrees C, than that of the disulfide-bonded one. Thus, we concluded that ovalbumin has a native-like conformation in its disulfide-reduced form, but that the local conformation of the reduced form fluctuates more than that of the disulfide-bonded one. Such local destabilization may be related to the decreased stability against heat denaturation. 相似文献
66.
The biodegradative threonine deaminase from Escherichia coli is activated allosterically by AMP. To identify the residues interacting with the phosphate group of AMP at the binding site, we used the affinity labeling reagent, adenosine diphosphopyridoxal (AP2-PL). In the absence of AMP, the enzyme formed the Schiff base with AP2-PL and Scatchard plot analysis showed a biphasic pattern, the respective Kd values for the high- and low-affinity binding phases being 20 and 110 microM. The former value is comparable to the Kd value of the enzyme for AMP. In the presence of AMP, the Schiff base formation was greatly reduced. Although the maximal activating effect of adenosine diphosphopyridoxine, a non-reactive derivative of AP2-PL, was about 13% of that of AMP, the half-saturation concentration was almost the same. These findings suggest that AP2-PL specifically labeled the lysyl residue(s) at the AMP-binding site of the enzyme. To identify the labeled residue(s), we reduced the modified enzyme with sodium borohydride, then cleaved it with cyanogen bromide and Achromobacter lyticus protease I. Reverse-phase HPLC was used to isolate two labeled peptides from the digest. Their amino acid compositions and sequences showed that Lys-111 and Lys-113 were labeled. We conclude that these two lysyl residues are located around the phosphate group of AMP at the allosteric regulation site of the enzyme. 相似文献
67.
H Miyajima T Hirano S Hirose H Karasuyama K Okumura Z Ovary 《Journal of immunology (Baltimore, Md. : 1950)》1991,146(2):457-462
IgE production was obtained from B cells of BALB/c or nude mice when these cells were cultured with IL-4 plus LPS. IL-2 added to these cultures at the start (day 0), 1 or 2 days later completely suppressed the production of IgE. The production of IgG1 was also inhibited, but only if IL-2 was added on day 0. The production of other isotypes (IgM, IgG2a, IgG2b) was only slightly decreased by addition of IL-2. No suppression of IgE or IgG1 production was observed if monoclonal anti-IL-2 was added, whereas anti-IFN-gamma had no effect on the suppression of the production of these isotypes. The expression of CD23 on the third day of culture on B cells stimulated with LPS and IL-4 was markedly decreased when IL-2 was added to the cultures on day 0. Addition of monoclonal anti-IL-2 suppressed all effects produced by IL-2, whereas addition of anti-IFN-gamma had no effect. These results show that the suppression by IL-2, at least for the first signaling processes, are different from the suppression produced by IFN-gamma. 相似文献
68.
Inhibition of cholesterol absorption and synthesis in rats by sesamin 总被引:21,自引:0,他引:21
N Hirose T Inoue K Nishihara M Sugano K Akimoto S Shimizu H Yamada 《Journal of lipid research》1991,32(4):629-638
The effects of sesamin, a lignan from sesame oil, on various aspects of cholesterol metabolism were examined in rats maintained on various dietary regimens. When given at a dietary level of 0.5% for 4 weeks, sesamin reduced the concentration of serum and liver cholesterol significantly irrespective of the presence or absence of cholesterol in the diet, except for one experiment in which the purified diet free of cholesterol was given. On feeding sesamin, there was a decrease in lymphatic absorption of cholesterol accompanying an increase in fecal excretion of neutral, but not acidic, steroids, particularly when the cholesterol-enriched diet was given. Sesamin inhibited micellar solubility of cholesterol, but not bile acids, whereas it neither bound taurocholate nor affected the absorption of fatty acids. Only a marginal proportion (ca. 0.15%) of sesamin administered intragastrically was recovered in the lymph. There was a significant reduction in the activity of liver microsomal 3-hydroxy-3-methylglutaryl coenzyme A reductase after feeding sesamin, although the activity of hepatic cholesterol 7 alpha-hydroxylase, drug metabolizing enzymes, and alcohol dehydrogenase remained uninfluenced. Although the weight and phospholipid concentration of the liver increased unequivocally on feeding sesamin, the histological examination by microscopy showed no abnormality, and the activity of serum GOT and GPT remained unchanged. Since sesamin lowered both serum and liver cholesterol levels by inhibiting absorption and synthesis of cholesterol simultaneously, it deserves further study as a possible hypocholesterolemic agent of natural origin. 相似文献
69.
K Nakanishi K Nara H Hagiwara Y Aoyama H Ueno S Hirose 《European journal of biochemistry》1991,202(1):15-21
A cDNA clone encoding transglutaminase was isolated from a bovine-endothelial-cell cDNA library using oligonucleotide probes designed based on partial amino acid sequences of the purified protein. Sequencing of the cDNA insert revealed an open reading frame of 2061 bp coding for a protein of 687 amino acids. The sequence of bovine endothelial-cell transglutaminase was 88, 82, 80, 37, 37 and 37% identical with that of human endothelial, rat macrophage, guinea-pig liver, human and rat keratinocyte transglutaminases, and the human blood-coagulation factor XIIIa subunit, respectively. The cDNA clone was hybridized to a single mRNA species of 3.9 kb in the liver, lung, spleen and heart but not hybridized to RNA from the brain. Northern-blot analysis of mRNA from retinoid-treated cultured vascular endothelial cells revealed that retinoids were able to induce a large increase in the transglutaminase mRNA levels. 相似文献
70.
A monoclonal antibody (Z1H01) for an oligopeptide carrying an azobenzene group, was prepared under conditions where the azobenzene group is in the trans form. The antibody bound the hapten peptide effectively when the hapten peptide is in the trans form (K = 5 x 10(7) M-1), but the antibody released the hapten under irradiation with UV light where the hapten is in the cis form. The antibody bound the hapten again, when the hapten reverted to the trans form after irradiation with visible light. 相似文献