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Results for “Functional characterization”

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PubMed1993

Sequence and functional characterization of a third inositol trisphosphate receptor subtype, IP3R-3, expressed in pancreatic islets, kidney, gastrointestinal tract, and other tissues.

Inositol 1,4,5-trisphosphate (IP3) functions as a second messenger for many neurotransmitters, hormones and growth factors. It causes the release of Ca2+ from intracellular stores by binding to specific receptors that are coupled to Ca2+ channels. Recent studies have shown that there is a family of IP3 receptors, and the complete sequences of two members of this family and partial sequences of two others have been reported. We have determined the complete sequence of a third IP3 receptor, designated IP3R-3, and characterized its pharmacological properties and sites of expression. Rat IP3R-3 is 2670 amino acids in size, has 62 and 64% identity with IP3R-1 and IP3R-2, and is predicted to have a similar structure including a region of eight potential membrane-spanning segments at its COOH terminus, which presumably functions as a Ca2+ channel. Expression of recombinant rat IP3R-3 in COS-7 cells showed that it bound IP3 as well as inositol 1,3,4,5-tetrakisphosphate and inositol hexakisphosphate. Immunohistocytochemical studies of cells expressing recombinant IP3R-3 indicated that it has a preferential cellular distribution in the endoplasmic reticulum. RNA and protein blotting studies indicate that IP3R-3 is expressed in a number of different cultured cell lines including insulin-secreting RINm5F cells. The IP3R-3 is also expressed in adult pancreatic islets, kidney, gastrointestinal tract, and brain. Reverse transcriptase-polymerase chain reaction amplification of IP3R-1, -2, and -3 mRNAs in adult rat pancreatic islets indicated that IP3R-3 was the predominant subtype expressed in this tissue and thus may be responsible for mediating the effects of IP3 on insulin secretion.

Amino Acid Sequence↗

PubMed2003

Functional characterization of Drosophila sialyltransferase.

Sialylation is an important carbohydrate modification of glycoconjugates in the deuterostome lineage of animals. By contrast, the evidence for sialylation in protostomes has been scarce and somewhat controversial. In the present study, we characterize a Drosophila sialyltransferase gene, thus providing experimental evidence for the presence of sialylation in protostomes. This gene encodes a functional alpha2-6-sialyltransferase (SiaT) that is closely related to the vertebrate ST6Gal sialyltransferase family, indicating an ancient evolutionary origin for this family. Characterization of recombinant, purified Drosophila SiaT revealed a novel acceptor specificity as it exhibits highest activity toward GalNAcbeta1-4GlcNAc carbohydrate structures at the non-reducing termini of oligosaccharides and glycoprotein glycans. Oligosaccharides are preferred over glycoproteins as acceptors, and no activity toward glycolipid acceptors was detected. Recombinant Drosophila SiaT expressed in cultured insect cells possesses in vivo and in vitro autosialylation activity toward beta-linked GalNAc termini of its own N-linked glycans, thus representing the first example of a sialylated insect glycoconjugate. In situ hybridization revealed that Drosophila SiaT is expressed during embryonic development in a tissue- and stage-specific fashion, with elevated expression in a subset of cells within the central nervous system. The identification of a SiaT in Drosophila provides a new evolutionary perspective for considering the diverse functions of sialylation and, through the powerful genetic tools available in this system, a means of elucidating functions for sialylation in protostomes.

Amino Acid Sequence↗

PubMed2000

Functional characterization of glycosylation-deficient human P-glycoprotein using a vaccinia virus expression system.

P-glycoprotein (P-gp), the product of human MDR1 gene, which functions as an ATP-dependent drug efflux pump, is N-linked glycosylated at asparagine residues 91, 94, and 99 located within the first extracellular loop. We report here the biochemical characterization of glycosylation-deficient (Gly(-)) P-gp using a vaccinia virus based transient expression system. The staining of HeLa cells expressing Gly(-) P-gp (91, 94, and 99N-->Q), with P-gp specific monoclonal antibodies, MRK-16, UIC2 and 4E3 revealed a 40 to 50% lower cell-surface expression of mutant P-gp compared to the wild-type protein. The transport function of Gly(-) P-gp, assessed using a variety of fluorescent compounds indicated that the substrate specificity of the pump was not affected by the lack of glycosylation. Additional mutants, Gly(-) D (91, 94, 99N-->D) and Gly(-) Delta (91, 94, 99 N deleted) were generated to verify that the reduced cell surface expression, as well as total expression, were not a result of the glutamine substitutions. Gly(-) D and Gly(-) Delta Pgps were also expressed to the same level as the Gly(-) mutant protein. (35)S-Methionine/cysteine pulse-chase studies revealed a reduced incorporation of (35)S-methionine/cysteine in full length Gly(-) P-gp compared to wild-type protein, but the half-life ( approximately 3 hr) of mutant P-gp was essentially unaltered. Since treatment with proteasome inhibitors (MG-132, lactacystin) increased only the intracellular level of nascent, mutant P-gp, the decreased incorporation of (35)S-methionine/cysteine in Gly(-) P-gp appears to be due to degradation of improperly folded mutant protein by the proteasome and endoplasmic reticulum-associated proteases. These results demonstrate that the unglycosylated protein, although expressed at lower levels at the cell surface, is functional and suitable for structural studies.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2000

Functional characterization of the enhancer blocking element of the sea urchin early histone gene cluster reveals insulator properties and three essential cis-acting sequences.

Insulator elements can be functionally identified by their ability to shield promoters from regulators in a position-dependent manner or their ability to protect adjacent transgenes from position effects. We have previously reported the identification of a 265 bp sns DNA fragment at the 3' end of the sea urchin H2A early histone gene that blocked expression of a reporter gene in transgenic embryos when placed between the enhancer and the promoter. Here we show that sns interferes with enhancer-promoter interaction in a directional manner. When sns is placed between the H2A modulator and the inducible tet operator, the modulator is barred from interaction with the basal promoter. However, the tet activator (tTA) can still activate the promoter, even in the presence of sns, demonstrating that sns does not interfere with activity of a downstream enhancer. In addition, the H2A modulator can still drive expression of a divergently oriented transcription unit, suggesting that sns does not inhibit binding of transcription factor(s) to the enhancer. To identify cis-acting sequence elements within sns which are responsible for insulator activity, we have performed in vitro DNase I footprinting and EMSA analysis, and in vivo functional assays by microinjection into sea urchin embryos. We have identified three binding sites for protein complexes: a palindrome, a direct repeat, and a C+T sequence that corresponds to seven GAGA motifs on the transcribed strand. Insulator function requires all three cis-acting elements. Based on these results, we conclude that sns displays properties similar to the best characterized insulators and suggest that directional blocking of enhancer-activated transcription by sns depends on the assembly of distinct DNA-protein complexes.

Animals↗

PubMed2003

The type 1 CD10/neutral endopeptidase 24.11 promoter: functional characterization of the 5'-untranslated region.

The cell surface zinc metalloproteinase CD10/neutral endopeptidase 24.11 (NEP) is expressed on normal and malignant lymphoid progenitors, granulocytes and a variety of epithelial cells. Because CD10/NEP functions as part of a regulatory loop that controls local concentrations of peptide substrates and associated peptide-mediated signal transduction, its role in each tissue is different depending on the availability of substrate. To characterize further how this widely distributed molecule is regulated differentially in each tissue, we analysed the major type 2 CD10/NEP promoter and found three functionally important transcription factor binding sites, one of which was identical to CCAAT-binding transcription factor/nuclear transcription factor Y. In this report, we analyse the type 1 CD10/NEP promoter and found a functionally important transcription factor binding site in the 5'-untranslated region. The results of the competition and supershift experiments demonstrated that the functionally important transcription factor was identical to Sp1. Our results suggest that ubiquitously expressed Sp1 may play an important role in differentiation stage-specific regulation of CD10/NEP expression in lymphoid lineage.

5' Untranslated Regions↗

PubMed2004

Functional characterization of recombinant batroxobin, a snake venom thrombin-like enzyme, expressed from Pichia pastoris.

A thrombin-like enzyme of Bothrops atrox moojeni venom, batroxobin, specifically cleaves fibrinogen alpha chain, resulting in the formation of non-crosslinked fibrin clots. The cDNA encoding batroxobin was cloned, expressed in Pichia pastoris and the molecular function of purified recombinant protein was also characterized. The recombinant batroxobin had an apparent molecular weight of 33 kDa by SDS-PAGE analysis and biochemical activities similar to those of native batroxobin. The purified recombinant protein strongly converted fibrinogen into fibrin clot in vitro, and shortened bleeding time and whole blood coagulation time in vivo. However, it did not make any considerable alterations on other blood coagulation factors. Several lines of experimental evidence in this study suggest that the recombinant batroxobin is a potent pro-coagulant agent.

Amino Acid Sequence↗

PubMed2006

Functional characterization of an orphan nuclear receptor, Rev-ErbAalpha, in chondrocytes and its potential role in osteoarthritis.

OBJECTIVE: To evaluate the expression and function of the orphan nuclear receptor Rev-ErbAalpha in articular cartilage and to investigate its role in osteoarthritis (OA). METHODS: Expression of Rev-ErbAalpha was analyzed at both the messenger RNA and protein levels in human and bovine articular cartilage and chondrocytes by real-time polymerase chain reaction (TaqMan) and immunocytochemical techniques. The effects of cartilage catabolic and anabolic agents on the expression of Rev-ErbAalpha were evaluated by TaqMan analysis. Overexpression was achieved by either adenoviral transduction or treatment with a peroxisome proliferator-activated receptor alpha agonist, whereas expression was suppressed by antisense oligonucleotides. RESULTS: Among the 48 known nuclear receptors, Rev-ErbAalpha was found to be the most highly expressed in OA cartilage. It is known to function as a transcription repressor. Treatment of articular chondrocytes with known catabolic agents resulted in the induction of Rev-ErbAalpha, whereas stimulation with anabolic agents led to a decrease in expression. Overexpression of the nuclear receptor was associated with an increase in the expression of matrix-degrading enzymes such as matrix metalloproteinase 13 and aggrecanase. In contrast, a decrease in Rev-ErbAalpha expression led to a concomitant reduction in the activity of matrix-degrading enzymes. CONCLUSION: This study is the first to demonstrate that Rev-ErbAalpha is highly expressed in OA articular chondrocytes and that its expression is modulated by known cartilage catabolic and anabolic stimuli. We also demonstrated that modulation of Rev-ErbAalpha expression in chondrocytes may be a novel means of regulating the expression and production of multiple matrix-degrading enzymes. These observations suggest that Rev-ErbAalpha may be a novel therapeutic target for OA.

ADAM Proteins↗

PubMed1997

Structural and functional characterization of homo-oligomeric complexes of alpha and beta chaperonin subunits from the hyperthermophilic archaeum Thermococcus strain KS-1.

To elucidate the function of group II chaperonin, the gene for the chaperonin from the hyperthermophilic archaeum Thermococcus strain KS-1 was cloned and sequenced. Two distinct genes coding for chaperonin subunits, designated alpha and beta, were obtained, and their deduced amino acid sequences are highly homologous to those of group II chaperonins from other sources. The alpha and beta subunits were individually expressed in Escherichia coli. Both of the recombinant subunits assemble to constitute the homo-oligomeric double-ring complexes, which are prone to form large aggregates. The alpha aggregate is dissociated into the typical chaperonin ring complex by incubation in buffer containing 15% (v/v) methanol, while the beta aggregate cannot be dissociated. At high temperature, both of the recombinant complexes have weak ATPase activities. They are able to arrest refolding of a chemically denatured thermophilic enzyme in the absence of ATP, and refolding is resumed when ATP is supplemented. These results suggest that homo-oligomeric complexes of the archaeal chaperonin have activity.

3-Isopropylmalate Dehydrogenase↗

PubMed2001

Structural and functional characterization of the mouse fatty acid translocase promoter: activation during adipose differentiation.

Fatty acid translocase (FAT/CD36) is a cell-surface glycoprotein that functions as a receptor/transporter for long-chain fatty acids (LCFAs), and interacts with other protein and lipid ligands. FAT/CD36 is expressed by various cell types, including platelets, monocytes/macrophages and endothelial cells, and tissues with an active LCFA metabolism, such as adipose, small intestine and heart. FAT/CD36 expression is induced during adipose cell differentiation and is transcriptionally up-regulated by LCFAs and thiazolidinediones in pre-adipocytes via a peroxisome-proliferator-activated receptor (PPAR)-mediated process. We isolated and analysed the murine FAT/CD36 promoter employing C(2)C(12)N cells directed to differentiate to either adipose or muscle. Transient transfection studies revealed that the 309 bp upstream from the start of exon 1 confer adipose specific activity. Sequence analysis of this DNA fragment revealed the presence of two imperfect direct repeat-1 elements. Electrophoretic mobility-shift assay demonstrated that these elements were peroxisome-proliferator-responsive elements (PPREs). Mutagenesis and transfection experiments indicated that both PPREs co-operate to drive strong promoter activity in adipose cells. We conclude that murine FAT/CD36 expression in adipose tissue is dependent upon transcriptional activation via PPARs through binding to two PPREs located at -245 to -233 bp and -120 to -108 bp from the transcription start site.

Adipocytes↗

PubMed2000

Production and functional characterization of two mouse/human chimeric antibodies with specificity for the tumor-associated Tn-antigen.

In this work, we have constructed two functional mouse/human chimeric antibodies (IgMkappa and IgG1kappa isotypes) by inserting genomic DNA fragments encoding VH and Vkappa variable regions of the murine monoclonal antibody IgMK-83D4 into mammalian expression vectors containing human mu, gamma1, and kappa constant exons, and by transfecting them into the nonsecreting mouse myeloma X-63 cell line. In previous works, we have demonstrated that 83D4 murine mAb reacts with Tn determinant (GalNAcalpha-O-Ser/Thr) expressed in 90% of breast, ovary, and colon carcinomas. Both expressed chimeric antibodies were purified from the transfected cell line supernatant by affinity chromatography, and their reactivities against Tn antigen were confirmed by ELISA on asialo ovine submaxilar mucin and immunofluorescence studies on MCF-7 breast carcinoma cell line. We have demonstrated by gel filtration chromatography, that the principal secreted forms were monomers for IgG1kappa and pentamers for IgMkappa. The binding affinities of these chimeric antibodies against synthetic Tn glycopeptides, were evaluated by surface plasmon resonance showing an affinity constant similar to that of 83D4 native antibody for IgMkappa and a lower affinity constant for IgG1kappa chimeric antibody. On the other hand, the replacement of mouse C regions with human C regions confers both chimeric antibodies the ability to activate human complement. These mouse/human chimeric antibodies should be much less immunogenic and could play an important role in the lysis of tumor cell expressing Tn-antigen. Therefore, these anti-Tn chimeric antibodies could be considered as potential tools for human in vivo studies.

Animals↗

PubMed2004

Functional characterization of human natural killer cells responding to Mycobacterium bovis bacille Calmette-Guérin.

The kinetics of activation and induction of several effector functions of human natural killer (NK) cells in response to Mycobacterium bovis bacille Calmette-Guérin (BCG) were investigated. Owing to the central role of monocytes/macrophages (MM) in the initiation and maintenance of the immune response to pathogens, two different experimental culture conditions were analysed. In the first, monocyte-depleted nylon wool non-adherent (NW) cells from healthy donors were stimulated with autologous MM preinfected with BCG (intracellular BCG). In the second, the NW cells were directly incubated with BCG, which was therefore extracellular. In the presence of MM, CD4+ T lymphocytes were the cell subset mainly expressing the activation marker, CD25, and proliferating with a peak after 7 days of culture. In contrast, in response to extracellular BCG, the peak of the proliferative response was observed after 6 days of stimulation, and CD56+ CD3- cells (NK cells) were the cell subset preferentially involved. Such proliferation of NK cells did not require a prior sensitization to mycobacterial antigens, and appeared to be dependent upon contact between cell populations and bacteria. Following stimulation with extracellular BCG, the majority of interferon-gamma (IFN-gamma)-producing cells were NK cells, with a peak IFN-gamma production at 24-30 hr. Interleukin (IL)-2 and IL-4 were not detectable in NK cells or in CD3+ T lymphocytes at any time tested. IL-12 was not detectable in the culture supernatant of NW cells stimulated with extracellular BCG. Compared to the non-stimulated NW cells, the NW cells incubated for 16-20 hr with BCG induced the highest levels of expression of apoptotic/death marker on the NK-sensitive K562 cell line. BCG also induced expression of the activation marker, CD25, and proliferation, IFN-gamma production and cytotoxic activity, on negatively selected CD56+ CD3- cells. Altogether, the results of this study demonstrate that extracellular mycobacteria activate several NK-cell functions and suggest a possible alternative mechanism of NK-cell activation as the first line of defence against mycobacterial infections.

Adult↗

PubMed1995

Functional characterization of human immunodeficiency virus type 1 nef genes in patients with divergent rates of disease progression.

We have studied the sequence and function of the human immunodeficiency virus type 1 (HIV-1) nef genes from nine patients with highly divergent rates of disease progression enrolled in a longitudinal study of HIV disease. Over an average of 7.8 years of follow-up, three patients had net positive changes in CD4+ T-cell counts, three patients had net negative changes in CD4+ T cells but did not develop AIDS, and three patients progressed to AIDS. The nef gene from each of these patients was amplified and cloned, and the sequence of 8 to 10 clones was determined. Only 2 of 88 (2.3%) nef genes recovered from these nine patients were grossly defective. Moreover, there was no relationship between the phylogeny of nef sequences and the corresponding rates of disease progression from these patients. Representative nef genes from all nine patients were tested for their abilities to downregulate cell surface CD4 in a transient-transfection assay. There was no correlation found between the functions of the nef genes from these patients and their corresponding rates of disease progression. We conclude that the nef gene is not a common mediator of the rate of HIV disease progression in natural infection.

Acquired Immunodeficiency Syndrome↗

PubMed1988

Phenotypic and functional characterization of human T cell clones.

The capacity of human peripheral blood-derived T cell clones to carry out a variety of functions was examined. T cell clones were generated by stimulating individual peripheral blood T cells with PHA by a procedure that yielded a growing clone from a mean of greater than 92% of the cultured cells. A total of 65 T cell clones (44 CD4+ and 21 CD8+) generated from two individual donors were examined for their functional capabilities. All T cell clones examined secreted IL-2, IFN-gamma, and lymphotoxin/tumor necrosis factor like activity when stimulated with immobilized mAb to the CD3 complex (64.1). When 54 additional T cell clones from a third donor were analyzed, all were found to produce IL-2. Upon activation with immobilized 64.1, all CD4+ clones and 91% of the CD8+ clones induced the generation of Ig-secreting cells from purified B cells. The CD8+ clones that did not serve as Th cells alone were able to augment the capacity of fresh CD4+ cells to generate Ig-secreting cells. Each of these clones was also found to effect MHC-unrestricted cytotoxicity upon activation with immobilized 64.1. The CD8+ clones were somewhat more effective killers than CD4+ clones, although there was considerable overlap. A total of 18 clones was analyzed for TCR beta-chain gene rearrangement. Of the clones exhibiting rearrangements of the beta-chain gene, 94% were found to have a single rearrangement pattern. Finally, the detailed phenotype of 15 (11 CD4+ and 4 CD8+) of these clones was examined. Variable numbers of cells of each of the clones expressed Ag identified by mAb 4B4 (CD29), Leu 8, Leu 15 (CD11b), and NKH1. Moreover, cells of 6 of 11 CD4+ clones and 4 of 4 CD8+ clones also expressed CD45R in addition to CD29; expression of CD45R and CD29 varied with the activation status of the clone. The current data demonstrate that nearly all of the T cell clones were able to accomplish each of the functions examined regardless of the surface phenotype. Inasmuch as the clones were generated using a technique that expanded more than 92% of the circulating T cells, the data imply that the progeny of the vast majority of T cells may have the inherent capacity to exert a wide array of functional activities.

Adult↗

PubMed2004

Functional characterization of muscarinic receptor subtypes in human duodenal secretion.

AIM: Acetylcholine (ACh) stimulates ion secretion in the small intestine and colon. The purpose of the present study was to characterize the ACh-induced electrogenic ion transport in human duodenum and determine the muscarinic receptor subtypes functionally involved. METHODS: Biopsies from the second part of duodenum were obtained from 28 patients during endoscopy. Biopsies were mounted in modified Ussing chambers with air-suction for measurements of short-circuit current by a previously validated technique. Short-circuit current was measured after application of chloride/bicarbonate transport inhibitors bumetanide, 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), diphenylamine-2-carboxylate (DPC), and acetazolamide. 4-diphenylacetoxy-N-methylpiperidine methiodide (4-DAMP) and two mamba toxins MT3 and MT7 were used to characterize the mAChR receptor subtypes involved. The effects of transport inhibitors and receptor antagonists were measured by comparing two consecutive responses of ACh on short-circuit current in the same biopsy specimen. RESULTS: Bumetanide and 4-DAMP significantly inhibited ACh-induced short-circuit current, whereas SITS, DPC, acetazolamide, mamba toxin MT3, and mamba toxin MT7 all failed to show any significant effect. CONCLUSION: In conclusion, our results indicate that muscarinic receptor subtype M3 acts as the main mediator of bumetanide-sensitive ACh-induced secretion in human duodenum.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗

PubMed2002

Functional characterization of coding polymorphisms in the human MDR1 gene using a vaccinia virus expression system.

The human MDR1-encoded transporter is a 170-kDa plasma membrane glycoprotein [P-glycoprotein (P-gp)] capable of binding and energy-dependent extrusion of structurally diverse organic compounds and drugs. P-gp seems to play a significant role in uptake, distribution, and excretion of many different drugs. To determine whether common polymorphic forms of P-gp are likely to alter function of P-gp, we characterized five known MDR1 coding polymorphisms (N21D, F103L, S400N, A893S, and A998T) using a vaccinia virus-based transient expression system. Cell surface expression of wild-type P-gp was time-dependent over a time course of 5.5 to 34.5 h; highest expression was obtained by 22 to 26.5 h after infection/transfection, indicating that a semiquantitative assay for P-gp expression levels was possible. HeLa cells stained with the P-gp specific monoclonal antibodies MRK-16 and Western blots probed with C219 revealed similar cell surface expression for the polymorphisms and for wild-type protein. Time-dependent P-gp pump function maximal at 22 h after infection/transfection was demonstrated for the following MDR1 fluorescence substrates: 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-pentanoic acid, succinimidyl ester (bodipy-FL)-verapamil, bodipy-FL-vinblastine, calcein-AM, bodipy-FL-prazosin, bisantrene, and bodipy-FL-forskolin, but not for daunorubicin. Transport studies of all tested substrates indicated that the substrate specificity of the pump was not substantially affected by any of the tested polymorphisms. Cell surface expression and function of double mutants including the more common polymorphisms (N21D-S400N, N21D-A893S, and S400N-A893S) showed no differences from wild-type. These results demonstrate that the common MDR1 coding polymorphisms result in P-gps with a cell surface distribution and function similar to wild-type P-gp.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2004

Genetic screening for melanocortin-4 receptor mutations in a cohort of Italian obese patients: description and functional characterization of a novel mutation.

Mutations in the human melanocortin-4 receptor (MC4-R) gene may account for up to 5.8% of morbid nonsyndromic obesity. We have screened 120 unrelated obese patients for variants of the MC4-R gene. Four heterozygous missense variants were detected, including two polymorphisms (Val(103)Ile and Ile(251)Leu) previously described in the literature. A novel heterozygous mutation (Glu(308)Lys) was detected in a 36-yr-old female patient. Compared with the wild-type receptor, cells expressing the mutated receptor showed a reduced stimulation of cAMP production and a reduction of radioactive alpha MSH binding. No segregation of the mutation with the obese phenotype could be demonstrated. A second, potentially pathogenic mutation (Ser(30)Phe) was detected in a 31-yr-old female patient. Functional analysis of the mutated receptor showed no change in the affinity to the natural ligand alpha MSH nor limited ability to stimulate cAMP production. Sixty lean subjects were also screened, and no additional variants of the MC4-R gene were observed, except for two individuals with the Val(103)Ile polymorphism. In conclusion, we have screened a population of Italian obese subjects for MC4-R variants, demonstrating a 1.7% prevalence of potentially pathogenic mutations. A novel heterozygous missense mutation (Glu(308)Lys) that impairs MC4-R functional activity in vitro was characterized.

Adolescent↗

PubMed1998

Functional characterization of Synechocystis sp. PCC 6803 delta psbU and delta psbV mutants reveals important roles of cytochrome c-550 in cyanobacterial oxygen evolution.

The functions of cytochrome c-550 and a 12 kDa protein in cyanobacterial oxygen evolution were studied with directed deletion mutants delta psbV and delta psbU of Synechocystis sp. PCC 6803, and the following results were obtained. (1) In contrast to the delta psbU mutant which is capable of autotrophic growth in the absence of Ca2+ or Cl- at a reduced rate, the delta psbV mutant lacking cytochrome c-550 could not grow at all without Ca2+ or Cl-. (2) The delta psbV mutant had a significantly reduced thermoluminescence emission intensity and flash oxygen yield, whereas the delta psbU mutant showed slight decreases in thermoluminescence intensity and flash oxygen yield, indicating corresponding decreases in the concentrations of O2-evolving centers in these mutants. (3) The delta psbV and delta psbU mutants exhibited elevated peak temperature for the thermoluminescence B- and Q-bands indicative of more stable S2 states. (4) The rise time of the O2 signal during the S3-[S4]-S0 transition was increased slightly in the delta psbV mutant but not in the delta psbU mutant. (5) The oxygen evolution was inactivated in the dark rapidly in the delta psbV mutant with a half-time of 28 min, but this did not happen in the delta psbU mutant. (6) Photoactivation of the oxygen-evolving complex after removal of the manganese cluster by hydroxylamine showed a higher quantum yield in the delta psbV mutant than in the delta psbU mutant or wild type. Taken together, these results indicated that cytochrome c-550 plays a substantial role in maintaining the stability and function of the manganese cluster in algal photosystem II, whereas the 12 kDa protein plays primarily a regulatory role in maintaining normal S-state transitions. These functional features of cytochrome c-550 and the 12 kDa protein were compared with those of the 23 and 17 kDa proteins in higher plant photosystem II and of the 33 kDa protein in both algal and plant photosystem II.

Bacterial Proteins↗

PubMed2001

Functional characterization of human organic anion transporting polypeptide B (OATP-B) in comparison with liver-specific OATP-C.

PURPOSE: To assess the functional characteristics of human organic anion transporter B (OATP-B) in comparison with those of the known, liver-specific OATP-C. METHODS: OATP-B or -C was expressed in HEK293 cells or Xenopus oocytes, and uptakes of estradiol-17beta-glucuronide and estrone-3-sulfate were measured using radiolabeled compounds. RESULTS: OATP-C transported both estrone-3-sulfate and estradiol-17beta-glucuronide, whereas OATP-B transported only the former. OATP-C-mediated uptake of estrone-3-sulfate exhibited biphasic saturation kinetics, whereas transports of estradiol-17beta-glucuronide by OATP-C and estrone-3-sulfate by OATP-B followed single-saturation kinetics. Inhibition kinetics showed that only the high-affinity site for estrone-3-sulfate on OATP-C was shared with glucuronide conjugates. Uptake of [3H]estrone-3-sulfate by OATP-B was inhibited by sulfate conjugates but not by glucuronide conjugates, whereas its uptake by OATP-C was inhibited by both types of conjugates. CONCLUSIONS: OATP-B accepted sulfate conjugates of steroids but not glucuronide conjugates, whereas OATP-C transported both types of steroid conjugates. Transport of estrone-3-sulfate by OATP-B and -C followed single- and biphasic-saturation kinetics, respectively, and the high-affinity site on OATP-C was the same as that for estradiol-17beta-glucuronide. Other OATPs, OATP-A and OATP-8, reportedly exhibit different preferences for steroid conjugates, and the specific recognition of sulfate conjugates seems to be unique to OATP-B.

Algorithms↗