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21.
We show the unexpectedly important role of the protein environment in the primary step of the photoreaction of the yellow protein after light illumination. The driving force of the trans-to-cis isomerization reaction was analyzed by a computational method. The force was separated into two different components: the term due to the protein-chromophore interaction and the intrinsic term of the chromophore itself. As a result, we found that the contribution from the interaction term was much greater than that coming from the intrinsic term. This accounts for the efficiency of the isomerization reaction in the protein environment in contrast to that in solution environments. We then analyzed the relaxation process of the chromophore on the excited-state energy surface and compared the process in the protein environment and that in a vacuum. Based on this analysis, we found that the bond-selectivity of the isomerization reaction also comes from the interaction between the chromophore and the protein environment. 相似文献
22.
Ishikura M Hagiwara K Takishita K Haga M Iwai K Maruyama T 《Marine biotechnology (New York, N.Y.)》2004,6(4):378-385
Recent molecular biological studies have revealed that some photosymbiotic invertebrates dwelling in coral reefs host several genetically different dinoflagellates, Symbiodinium species, as symbionts. However, little is known about the difference in physiologic characteristics among these symbionts living in a single host, because some Symbiodinium strains are difficult to culture in vitro. To isolate some of these Symbiodinium strains, we have developed an agar culture medium plate containing antibiotics and a giant clam tissue homogenate. Using-this medium we isolated two new Symbiodinium strains from two molluscan hosts, Tridacna crocea and Pteraeolidia ianthina, each of which hosted two different Symbiodinium strains belonging to Symbiodinium C and D, respectively. The tissue homogenate was essential for the growth of Symbiodinium D. Although it was not essential for the growth of Symbiodinium C, it did stimulate the initial growth. For the isolation of some Symbiodinium strains, isolation medium containing host homogenate is effective. 相似文献
23.
Molecular characterization of mammalian dicarbonyl/L-xylulose reductase and its localization in kidney 总被引:6,自引:0,他引:6
Nakagawa J Ishikura S Asami J Isaji T Usami N Hara A Sakurai T Tsuritani K Oda K Takahashi M Yoshimoto M Otsuka N Kitamura K 《The Journal of biological chemistry》2002,277(20):17883-17891
In this report, we first cloned a cDNA for a protein that is highly expressed in mouse kidney and then isolated its counterparts in human, rat hamster, and guinea pig by polymerase chain reaction-based cloning. The cDNAs of the five species encoded polypeptides of 244 amino acids, which shared more than 85% identity with each other and showed high identity with a human sperm 34-kDa protein, P34H, as well as a murine lung-specific carbonyl reductase of the short-chain dehydrogenase/reductase superfamily. In particular, the human protein is identical to P34H, except for one amino acid substitution. The purified recombinant proteins of the five species were about 100-kDa homotetramers with NADPH-linked reductase activity for alpha-dicarbonyl compounds, catalyzed the oxidoreduction between xylitol and l-xylulose, and were inhibited competitively by n-butyric acid. Therefore, the proteins are designated as dicarbonyl/l-xylulose reductases (DCXRs). The substrate specificity and kinetic constants of DCXRs for dicarbonyl compounds and sugars are similar to those of mammalian diacetyl reductase and l-xylulose reductase, respectively, and the identity of the DCXRs with these two enzymes was demonstrated by their co-purification from hamster and guinea pig livers and by protein sequencing of the hepatic enzymes. Both DCXR and its mRNA are highly expressed in kidney and liver of human and rodent tissues, and the protein was localized primarily to the inner membranes of the proximal renal tubules in murine kidneys. The results imply that P34H and diacetyl reductase (EC ) are identical to l-xylulose reductase (EC ), which is involved in the uronate cycle of glucose metabolism, and the unique localization of the enzyme in kidney suggests that it has a role other than in general carbohydrate metabolism. 相似文献
24.
Roles of His-79 and Tyr-180 of D-xylose/dihydrodiol dehydrogenase in catalytic function 总被引:2,自引:0,他引:2
Asada Y Aoki S Ishikura S Usami N Hara A 《Biochemical and biophysical research communications》2000,278(2):333-337
Mammalian dimeric dihydrodiol dehydrogenase is identical with d-xylose dehydrogenase and belongs to a protein family with prokaryotic proteins including glucose-fructose oxidoreductase. Of the conserved residues in this family, either His-79 or Tyr-180 of d-xylose/dihydrodiol dehydrogenase has been proposed to be involved in the catalytic function. Site-directed mutagenesis was used to examine the roles of the two residues of the monkey enzyme. A mutant, Y180F, was almost inactive, but, similarly to the wild-type enzyme, exhibited high affinity for NADP(H) and fluorescence energy transfer upon binding of NADPH. The H79Q mutation had kinetically largest effects on K(d) (>7-fold increase) and K(m) (>25-fold increase) for NADP(H), and eliminated the fluorescence energy transfer. Interestingly, the dehydrogenase activity of this mutant was potently inhibited with a 190-fold increase in the K(m) for NADP(+) by high ionic strength, which activated the activity of the wild-type enzyme. These results suggest a critical role of Tyr-180 in the catalytic function of this class of enzymes, in addition to functions of His-79 in the coenzyme binding and chemical steps of the reaction. 相似文献
25.
Soichiro Sakamoto Hiroshi Kawabata Taro Masuda Tatsuki Uchiyama Chisaki Mizumoto Katsuyuki Ohmori H. Phillip Koeffler Norimitsu Kadowaki Akifumi Takaori-Kondo 《PloS one》2015,10(10)
Ferritin is an iron-storage protein composed of different ratios of 24 light (L) and heavy (H) subunits. The serum level of ferritin is a clinical marker of the body’s iron level. Transferrin receptor (TFR)1 is the receptor not only for transferrin but also for H-ferritin, but how it binds two different ligands and the blood cell types that preferentially incorporate H-ferritin remain unknown. To address these questions, we investigated hematopoietic cell-specific ferritin uptake by flow cytometry. Alexa Fluor 488-labeled H-ferritin was preferentially incorporated by erythroid cells among various hematopoietic cell lines examined, and was almost exclusively incorporated by bone marrow erythroblasts among human primary hematopoietic cells of various lineages. H-ferritin uptake by erythroid cells was strongly inhibited by unlabeled H-ferritin but was only partially inhibited by a large excess of holo-transferrin. On the other hand, internalization of labeled holo-transferrin by these cells was not inhibited by H-ferritin. Chinese hamster ovary cells lacking functional endogenous TFR1 but expressing human TFR1 with a mutated RGD sequence, which is required for transferrin binding, efficiently incorporated H-ferritin, indicating that TFR1 has distinct binding sites for H-ferritin and holo-transferrin. H-ferritin uptake by these cells required a threshold level of cell surface TFR1 expression, whereas there was no threshold for holo-transferrin uptake. The requirement for a threshold level of TFR1 expression can explain why among primary human hematopoietic cells, only erythroblasts efficiently take up H-ferritin. 相似文献
26.
Ishiguro H Kishimoto T Furuya M Nagai Y Watanabe T Ishikura H 《Cancer immunology, immunotherapy : CII》2005,54(12):1191-1199
Tumor-derived cytokines, such as interleukin (IL)-6, function in the context of tumor-to-host interactions, and their functions in immune-compromised hosts need to be addressed in the light of ever- increasing number of patients under immunosuppression. We studied the effects, in immune-comprised animals, of tumor-derived IL-6 on tumor growth using an experimental tumor vaccination model. Murine mammary carcinoma FM3A clone 25 (CL25) cells, which neither produce IL-6 nor express IL-6 receptors, were used. cDNA for murine IL-6 (mIL-6) was introduced to the CL25 cells, resulting in a high-producer (mIL-6H) clone. In the severe combined immune-deficient (SCID) mice, the inoculation 3 weeks earlier of mIL-6H to a dorsal flank site suppressed the growth of the CL25 cells at the opposite flank site; a tumor-derived IL-6-mediated vaccination effect occurred. In the T-cell-deficient nude mice, the inoculations 4 weeks earlier of mIL-6H suppressed the growth of CL25, but the simultaneous inoculation of these transfectants did not affect the growth of CL25. Reducing the number of inoculated transfectants or a shorter vaccination period obscured the suppressive effect. The amounts of circulating tumor-reactive immunoglobulin did not correlate with the suppressive effect. The subcutaneous injection of the anti-CD40 antibody generated a further suppression of tumor growth in the mIL-6H-inoculated, but not in the mock-inoculated, T-cell-deficient mice. In the immune-competent hosts, a suppressive effect was not observed. Natural killer (NK) activity was augmented in the spleen of mIL-6H-inoculated scid mice. This study indicated a possible vaccination effect with tumor-derived IL-6 in immune-compromised hosts. 相似文献
27.
L-Xylulose reductase (XR) is a member of the short-chain dehydrogenase/reductase (SDR) superfamily. In this study we report the structure of the biological tetramer of human XR in complex with NADP(+) and a competitive inhibitor solved at 2.3 A resolution. A single subunit of human XR is formed by a centrally positioned, seven-stranded, parallel beta-sheet surrounded on either side by two arrays of three alpha-helices. Two helices located away from the main body of the protein form the variable substrate-binding cleft, while the dinucleotide coenzyme-binding motif is formed by a classical Rossmann fold. The tetrameric structure of XR, which is held together via salt bridges formed by the guanidino group of Arg203 from one monomer and the carboxylate group of the C-terminal residue Cys244 from the neighboring monomer, explains the ability of human XR to prevent the cold inactivation seen in the rodent forms of the enzyme. The orientations of Arg203 and Cys244 are maintained by a network of hydrogen bonds and main-chain interactions of Gln137, Glu238, Phe241, and Trp242. These interactions are similar to those defining the quaternary structure of the closely related carbonyl reductase from mouse lung. Molecular modeling and site-directed mutagenesis identified the active site residues His146 and Trp191 as forming essential contacts with inhibitors of XR. These results could provide a structural basis in the design of potent and specific inhibitors for human XR. 相似文献
28.
Goldar MM Nishie T Ishikura Y Fukuda T Takegawa K Kawamukai M 《Bioscience, biotechnology, and biochemistry》2005,69(7):1422-1426
The moc1/sds23 gene was isolated to induce sexual development of a sterile strain due to overexpression of adenylate cyclase in Schizosaccharomyces pombe. Here, we studied the functional conservation between moc1/sds23 and its two orthologs SDS23 and SDS24 in Saccharomyces cerevisiae. We observed that the temperature sensitivity, salt tolerance, cell morphology, and sterility of the Deltamoc1 mutant in S. pombe were recovered by expressing either S. cerevisiae SDS23 or SDS24. We found that deletion of both SDS23 and SDS24 resulted in the production of a large vacuole that was reversed by the expression of S. pombe moc1/sds23. In these ways we found that S. pombe Moc1/Sds23 and S. cerevisiae SDS23p or SDS24p are functional homologs. In addition we found that the Deltasds23 Deltasds24 diploid strain reduces cell separation in forming pseudohyphal-like growth in S. cerevisiae. Thus S. pombe moc1/sds23 and S. cerevisiae SDS23 or SDS24 are interchangeable with each other, but their disruptants are phenotypically dissimilar. 相似文献
29.
L-Gulonate 3-dehydrogenase (GDH) catalyzes the NAD(+)-linked dehydrogenation of L-gulonate into dehydro-L-gulonate in the uronate cycle. In this study, we isolated the enzyme and its cDNA from rabbit liver, and found that the cDNA is identical to that for rabbit lens lambda-crystallin except for lacking a codon for Glu(309). The same cDNA species, but not the lambda-crystallin cDNA with the codon for Glu(309), was detected in the lens, which showed the highest GDH activity among rabbit tissues. In addition, recombinant human lambda-crystallin that lacks Glu(309) displays enzymatic properties similar to rabbit GDH. These data indicate that GDH is recruited as lambda-crystallin without gene duplication. An outstanding feature of GDH is modulation of its activity by low concentrations of P(i), which decreases the catalytic efficiency in a dose dependent manner. P(i) also protects the enzyme against both thermal and urea denaturation. Kinetic analysis suggests that P(i) binds to both the free enzyme and its NAD(H)-complex in the sequential ordered mechanism. Furthermore, we examined the roles of Asp(36), Ser(124), His(145), Glu(157 )and Asn(196) in the catalytic function of rabbit GDH by site-directed mutagenesis. The D36R mutation leads to a switch in favor of NADP(H) specificity, suggesting an important role of Asp(36) in the coenzyme specificity. The S124A mutation decreases the catalytic efficiency 500-fold, and the H145Q, N196Q and N195D mutations result in inactive enzyme forms, although the E157Q mutation produces no large kinetic alteration. Thus, Ser(124), His(145) and Asn(196) may be critical for the catalytic function of GDH. 相似文献
30.
Regulator of complement activation (RCA) locus in chicken: identification of chicken RCA gene cluster and functional RCA proteins 总被引:2,自引:0,他引:2