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1.
Oncogene ; 36(22): 3137-3148, 2017 06 01.
Article in English | MEDLINE | ID: mdl-27941877

ABSTRACT

A well-studied RNA-binding protein Hu Antigen-R (HuR), controls post-transcriptional gene regulation and undergoes stress-activated caspase-3 dependent cleavage in cancer cells. The cleavage products of HuR are known to promote cell death; however, the underlying molecular mechanisms facilitating caspase-3 activation and HuR cleavage remains unknown. Here, we show that HuR cleavage associated with active caspase-3 in oral cancer cells treated with ionizing radiation and chemotherapeutic drug, paclitaxel. We determined that oral cancer cells overexpressing cyclooxygenase-2 (COX-2) limited the cleavage of caspase-3 and HuR, which reduced the rate of cell death in paclitaxel resistant oral cancer cells. Specific inhibition of COX-2 by celecoxib, promoted apoptosis through activation of caspase-3 and cleavage of HuR in paclitaxel-resistant oral cancer cells, both in vitro and in vivo. In addition, oral cancer cells overexpressing cellular HuR increased the half-life of COX-2 mRNA, promoted COX-2 protein expression and exhibited enhanced tumor growth in vivo in comparison with cells expressing a cleavable form of HuR. Finally, our ribonucleoprotein immunoprecipitation and sequencing (RIP-seq) analyses of HuR in oral cancer cells treated with ionizing radiation (IR), determined that HuR cleavage product-1 (HuR-CP1) bound and promoted the expression of mRNAs encoding proteins involved in apoptosis. Our results indicated that, cellular non-cleavable HuR controls COX-2 mRNA expression and enzymatic activity. In addition, overexpressed COX-2 protein repressed the cleavage of caspase-3 and HuR to promote drug resistance and tumor growth. Altogether, our observations support the use of the COX-2 inhibitor celecoxib, in combination with paclitaxel, for the management of paclitaxel resistant oral cancer cells.


Subject(s)
Carcinoma, Squamous Cell/genetics , Caspase 3/metabolism , Cyclooxygenase 2/genetics , Mouth Neoplasms/genetics , RNA-Binding Proteins/metabolism , Carcinoma, Squamous Cell/pathology , Humans , Mouth Neoplasms/pathology
2.
Biochemistry ; 47(1): 339-47, 2008 Jan 08.
Article in English | MEDLINE | ID: mdl-18081321

ABSTRACT

Aquaporin 0 (AQP0), also known as major intrinsic protein of lens, is the most abundant membrane protein in the lens and it undergoes a host of C-terminally directed posttranslational modifications. The C-terminal region containing the major phosphorylation sites is a putative calmodulin-binding site, and calmodulin has been shown to regulate AQP0 water permeability. The purpose of the present study was to elucidate the role of AQP0 phosphorylation on calmodulin binding. AQP0 C-terminal peptides were synthesized with and without serine phosphorylation on S231 and S235, and the ability of these peptides to bind dansyl-labeled calmodulin and the calcium dependence of the interaction was assessed using a fluorescence binding assay. The AQP0 C-terminal phosphorylated peptides were found to have 20-50-fold lower affinities for calmodulin than the unphosphorylated peptide. Chemical cross-linking studies revealed specific sites of AQP0-calmodulin interaction that are significantly reduced by AQP0 phosphorylation. These data suggest that AQP0 C-terminal phosphorylation affects calmodulin binding in vivo and has a role in regulation of AQP0 function.


Subject(s)
Aquaporins/chemistry , Aquaporins/metabolism , Calmodulin/chemistry , Calmodulin/metabolism , Eye Proteins/chemistry , Eye Proteins/metabolism , Membrane Glycoproteins/chemistry , Membrane Glycoproteins/metabolism , Amino Acid Sequence , Aquaporins/genetics , Calmodulin/genetics , Eye Proteins/genetics , Membrane Glycoproteins/genetics , Models, Molecular , Molecular Sequence Data , Phosphorylation , Protein Binding , Protein Structure, Secondary , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Spectrometry, Fluorescence , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Trypsin/metabolism
3.
Vis Neurosci ; 18(3): 393-9, 2001.
Article in English | MEDLINE | ID: mdl-11497415

ABSTRACT

The visual pigment from the ultraviolet (UV) cone photoreceptor of the tiger salamander has been cloned, expressed, and characterized. The cDNA contains a full-length open reading frame encoding 347 amino acids. The phylogenetic analysis indicates that the highest sequence homology is to the visual pigments in the S group. The UV opsin was tagged at the carboxy-terminus with the sequence for the 1D4 epitope. This fusion opsin was expressed in COS-1 cells, regenerated with 11-cis retinal (A1) and immuno-purified, yielding a pigment with an absorbance maximum (lambdamax) of 356 nm which is blue shifted from the absorption of retinal itself. The transducin activation assay demonstrated that this pigment is able to activate rod transducin in a light-dependent manner. Regeneration with 11-cis 3,4-dehydroretinal (A2) yielded a pigment with a lambdamax of 360 nm, only 4 nm red shifted from that of the A1 pigment, while bovine rhodopsin generated with A2 showed a 16-nm red shift from the corresponding A1 pigment. These results demonstrate that the trend for a shorter wavelength pigment to have a smaller shift of lambdamax between the A1 and A2 pigments also fits UV pigments. We hypothesize that the small red shift with A2 could be due to a twist in the chromophore that essentially isolates the ring double bond(s) from conjugation with the rest of the polyene chain.


Subject(s)
Retinal Cone Photoreceptor Cells/metabolism , Retinal Pigments/genetics , Ultraviolet Rays , Urodela/genetics , Amino Acid Sequence/genetics , Animals , Base Sequence/genetics , COS Cells , Cloning, Molecular , DNA, Complementary/genetics , Gene Expression , Light , Molecular Sequence Data , Retinal Pigments/physiology , Rod Opsins/genetics , Sequence Homology , Transducin/physiology
4.
Biochem Mol Biol Int ; 47(5): 773-80, 1999 May.
Article in English | MEDLINE | ID: mdl-10365248

ABSTRACT

The structure of bacteriorhodopsin (bR) has been probed by a large number of experimental methods. In earlier work distance constraints measured from the 1BRD Brookhaven structure (1, 2) were used to guide site-directed mutagenesis/affinity labeling experiments (3-5). In the present study we report on the use of limited molecular dynamics (MD) investigations of the same bR/affinity label system. We show here that the chiral center introduced when 4-bromo-all-trans retinal is synthesized produces variable impact on potential crosslinking. Our MD analysis suggests the following ranking of binding site mutants in order of reactivity: R118C > S118C >> S121C > R141C >> S141C >>> R121C, R138C, S138C. Chirality appears to have limited effect for the M118C mutants but shows more dramatic impact for the T121C and S141C mutants. These results are in excellent agreement with the experimental observations and offer encouragement that MD can be a useful component of experimental design with considerable predictive power.


Subject(s)
Bacteriorhodopsins/chemistry , Cysteine/physiology , Retinaldehyde/analogs & derivatives , Bacterial Proteins/chemistry , Cross-Linking Reagents/pharmacology , Models, Molecular , Mutagenesis , Retinaldehyde/pharmacology
5.
J Gen Virol ; 79 ( Pt 11): 2687-94, 1998 Nov.
Article in English | MEDLINE | ID: mdl-9820144

ABSTRACT

Combined androgen and oestrogen treatment of male or female Syrian hamsters results via an unknown mechanism in the formation of leiomyosarcomas in the reproductive tract. We have examined the possibility that retroviral gene expression may play a role in tumorigenesis. Evidence of virus-like particles in epididymis and seminal fluid is shown in electron micrographs. We identified expressed retroviral sequences by using RT-PCR to amplify a conserved retroviral reverse transcriptase coding region in RNA isolated from epididymis, testis, clarified seminal fluid and uterus. Phylogenetic analysis allowed us to classify the sequences into two distinct groups: (1) mammalian type-C viruses, having similarity to Moloney murine leukaemia virus, feline leukaemia virus and gibbon ape leukaemia virus amongst others; (2) a mixed ABCD group containing, for example, Chinese hamster and murine intracisternal A-particle virus sequences, mouse mammary tumour virus and human and simian retroviral sequences. The presence of putative full-length retrovirus related to mammalian type-C viruses in the epididymis and uterus was confirmed by Northern blot analysis. However, steroid treatment did not alter retroviral RNA levels in the epididymis or in a uterine tumour relative to untreated uterus. In summary, Syrian hamster reproductive tissues were found to express unique retroviral sequences; however, their role, if any, in hormonal carcinogenesis remains unresolved.


Subject(s)
Epididymis/virology , Genome, Viral , RNA, Viral/genetics , Retroviridae/isolation & purification , Uterus/virology , Amino Acid Sequence , Animals , Base Sequence , Cats , Cricetinae , Female , Humans , Male , Mesocricetus , Mice , Molecular Sequence Data , Phylogeny , Retroviridae/genetics , Sequence Analysis
6.
Mol Vis ; 4: 10, 1998 Jul 15.
Article in English | MEDLINE | ID: mdl-9675215

ABSTRACT

PURPOSE: Salamander retinas are known to contain at least three cone pigments and two rod pigments. The purpose of this study was to clone and characterize the visual pigments from salamander cones. METHODS: cDNA fragments of cone pigments were amplified from a salamander retina cDNA library by PCR using a pair of primers with consensus for visual pigments. These fragments were cloned and used as probes for library-screening. The full-length cDNAs were isolated from the retinal library using the cloned PCR products as probes. DNA sequences were determined by the dideoxynucleotide chain termination method. RESULTS: Two pigment cDNAs were cloned and sequenced from the salamander library. The global GenBank search showed that they do not match any existing sequences but have significant sequence similarity to visual pigments. One of the pigment cDNAs showed a high sequence homology with red cone pigments from other species and thus, was designated as a red cone opsin. The other pigment was designated as a blue cone opsin as it is most homologous to the chicken and goldfish blue cone pigments. Both cDNAs contain a full-length coding region encoding 365 amino acids in the red and 363 amino acids in the blue cone pigment. Hydropathy analysis predicted that both pigments could form seven hydrophobic transmembrane helices. Both pigments retain the key amino acid residues critical for maintaining the structure and function of opsins and have similar G-protein interaction sequences which differ from that of rod opsin. Phylogenetic analysis indicates that the red opsin belongs to the L group and the blue opsin belongs to the M1 group of visual pigments. CONCLUSIONS: The salamander red and blue cone pigments share high sequence homology with the cone pigments of other species.


Subject(s)
Retinal Cone Photoreceptor Cells/chemistry , Rod Opsins/chemistry , Rod Opsins/genetics , Urodela/genetics , Amino Acid Sequence , Animals , Base Sequence , Blotting, Southern , Chickens/genetics , Cloning, Molecular , Goldfish/genetics , Molecular Sequence Data , Phylogeny , Polymerase Chain Reaction , Protein Structure, Secondary , Sequence Analysis, DNA , Sequence Homology
7.
Invest Ophthalmol Vis Sci ; 37(9): 1907-13, 1996 Aug.
Article in English | MEDLINE | ID: mdl-8759361

ABSTRACT

PURPOSE: Salamander photoreceptor cells have been used widely as models in vision research. However, the salamander opsin genes had not been cloned. The purpose of this study was to clone a salamander rhodopsin and to determine its primary structure and cell type-specific expression. METHODS: Using salamander retina RNA as a template and Xenopus rhodopsin-specific oligonucleotides as primers, reverse transcription and polymerase chain reaction (RT-PCR) were used to amplify and clone a rhodopsin cDNA fragment. This fragment was used as a probe to isolate a full-length cDNA of the rhodopsin from a cDNA library of salamander retina. The dideoxynucleotide chain termination method was used to determine the nucleotide sequence. Single rod and cone cells were isolated by micromanipulation, and the absorbance spectra of the rod outer segments were measured with a photon-counting microspectrophotometer. Individual rod and cone cells were lysed for RT-PCR and Southern blot analysis to detect cell-specific expression of this gene. RESULTS: A 1.2 kb rhodopsin cDNA containing the full-length coding region of rhodopsin has been cloned and sequenced from the larval tiger salamander, Ambystoma tigrinum. This cDNA encodes 354 amino acids that, by hydropathy profile, could form seven transmembrane domains characteristic of other rhodopsins. Sequence identity was found with other amphibian rhodopsins at the nucleic acid (82% to 83%) and the amino acid (88% to 89%) levels. Key amino acids critical for structure and function of rhodopsin have been retained. The mRNA of this rhodopsin was identified in red rod cells (lambda max 506 nm). No expression of the gene was detected in cone cells. CONCLUSIONS: The cloned rhodopsin is a newly isolated member of the G protein-coupled receptor superfamily. This protein is expressed in rods but not in cones.


Subject(s)
Retinal Rod Photoreceptor Cells/metabolism , Rhodopsin/biosynthesis , Rhodopsin/genetics , Ambystoma , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , DNA Primers , DNA, Complementary , Gene Expression , Models, Structural , Molecular Sequence Data , Oligonucleotide Probes , Organ Specificity , Polymerase Chain Reaction , Protein Structure, Secondary , Rana pipiens , Retina/metabolism , Rhodopsin/chemistry , Xenopus laevis
8.
Biochemistry ; 33(38): 11624-30, 1994 Sep 27.
Article in English | MEDLINE | ID: mdl-7918376

ABSTRACT

The position of the chromophore within bacteriorhodopsin has been identified by cross-linking a cysteine group, introduced by site-specific mutagenesis, with a chromophore suitably derivatized with an active leaving group. Since bacteriorhodopsin has no cysteines, a site-specific cysteine mutant will contain only one free sulfhydryl group capable of reacting with the retinal analog. Met118, Thr121, and Ser141 were selected to be mutated to cysteine. No pigment absorbing in the visible region was obtained for the Ser141Cys mutant. The Met118Cys and Thr121Cys mutants have similar absorption maxima, proton pumping efficiencies and photocycles to those of the wild-type pigment. 4-Bromoretinal, in which the reactive allylic halide readily undergoes nucleophilic displacement, was used as the reactive chromophore. Pigments were obtained on reaction of all-trans-4-bromoretinal with the apoproteins of Met118Cys, Thr121Cys, and wild-type bacteriorhodopsin (lambda max = 464-470 nm). Analysis of the denatured pigments on SDS-polyacrylamide gels showed incorporation of tritiated chromophore into the Met118Cys mutant but not into the wild-type or Th4121Cys pigments. Met118Cys apoprotein which was preincubated with the cysteine-specific reagent N-ethylmaleimide formed a pigment with 4-bromoretinal but no cross-linking was observed, providing evidence that the cross-linking of the chromophore is to the cysteine at 118. We conclude that Met118 is positioned in the chromophore binding pocket, proximal to the C-4 position of cyclohexyl ring of retinal.


Subject(s)
Bacteriorhodopsins/metabolism , Halobacterium salinarum/metabolism , Retinaldehyde/analogs & derivatives , Retinaldehyde/metabolism , Affinity Labels , Amino Acid Sequence , Bacteriorhodopsins/chemistry , Bacteriorhodopsins/genetics , Binding Sites/genetics , Cross-Linking Reagents , Cysteine/chemistry , Cysteine/genetics , Escherichia coli/genetics , Halobacterium salinarum/genetics , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Proton Pumps , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Retinaldehyde/chemistry , Sequence Analysis, DNA
9.
Biochem Mol Biol Int ; 32(5): 933-9, 1994 Apr.
Article in English | MEDLINE | ID: mdl-8069243

ABSTRACT

Three successful photoaffinity labeling experiments of bacteriorhodopsin (bR) have been reported that used photoactivatible analogs of retinal to label the retinal binding site of the protein. Using molecular modeling techniques, the information about the retinal binding site derived from these studies is compared to the retinal binding site as defined by Henderson et al. (1990) using electron diffraction data. This comparison suggests some limitations to the use of photoaffinity labeling experiments for the determination of high resolution structural information.


Subject(s)
Affinity Labels , Bacteriorhodopsins/chemistry , Light , Models, Molecular , Amino Acid Sequence , Evaluation Studies as Topic , Molecular Sequence Data , Molecular Structure , Protein Structure, Secondary
11.
Photochem Photobiol ; 56(6): 929-34, 1992 Dec.
Article in English | MEDLINE | ID: mdl-1492136

ABSTRACT

Halobacterium halobium, grown in a defined medium where tyrosine had been largely replaced with m-fluorotyrosine, biosynthetically produced purple membrane. Analysis of this membrane by high pressure liquid chromatography of phenylthiocarbamyl derivatized amino acids of membrane acid hydrolysates revealed that up to 50% of the tyrosine was present as the m-fluorotyrosine form. Yields of the purple membrane decreased as the level of incorporation increased. The experimental purple membrane showed a single 19F NMR resonance at -61.983 ppm (relative to trifluoroacetic acid). The bacteriorhodopsin (bR) in the purple membrane was normal as assayed by gel electrophoresis, isoelectric focusing, circular dichroic spectra, and UV-visible spectra. However, the fluorinated tyrosine bacteriorhodopsins at near neutral pH exhibited slightly slower rates of proton uptake and a slower M-state decay with biphasic kinetics reminiscent of alkaline solutions of bR (pH > 9). These results imply that the tyrosines in bacteriorhodopsin may play a role in the photoactivated proton translocation process of this pigment.


Subject(s)
Bacteriorhodopsins/biosynthesis , Bacteriorhodopsins/chemistry , Halobacterium salinarum/metabolism , Photochemistry , Tyrosine/analogs & derivatives , Tyrosine/metabolism
12.
Photochem Photobiol ; 56(2): 251-5, 1992 Aug.
Article in English | MEDLINE | ID: mdl-1502268

ABSTRACT

Retinol decomposes rapidly into a number of products, including its aldehyde form, retinal, when introduced into buffer in phospholipid vesicles or ethanol. Interphotoreceptor retinoid-binding protein at low concentrations is found to protect retinol from isomerization and oxidation. The addition of alpha-tocopherol to either liposomes or an ethanolic-buffer solution also prevents decomposition. Neither of these agents interferes with the successful regeneration of pigment with 9-cis retinal in rod outer segment preparations or the restoration of sensitivity by retinoids in isolated rod photoreceptors.


Subject(s)
Rod Cell Outer Segment/metabolism , Vitamin A/metabolism , Animals , Cattle , In Vitro Techniques , Isomerism , Oxidation-Reduction , Photochemistry , Retinol-Binding Proteins/pharmacology , Vitamin A/chemistry , Vitamin E/pharmacology
13.
J Biol Chem ; 266(21): 13752-8, 1991 Jul 25.
Article in English | MEDLINE | ID: mdl-1830308

ABSTRACT

The effects of changes in pH on the binding of agonists and antagonists to the human platelet thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor were determined. Competition binding studies were performed with the TXA2/PGH2 mimetic [1S-1 alpha,2 beta (5Z), 3 alpha(1E,3R*),4 alpha)]-7-[3-(3-hydroxy-4'-iodophenoxy)-1-buteny) 7-oxabicyclo-[2.2.1]-heptan-2-yl]-5-heptenoic acid ([125I]BOP). The pH optimum for binding of [125I] BOP to washed human platelets was broad with a range of pH 4-6 in contrast to that of the TXA2/PGH2 receptor antagonist 9,11-dimethyl-methano-11,12-methano-16-(3-iodo-4-hydroxyl)-13-aza-15 alpha,beta-omega-tetranorthromboxane A2 ([125I]PTA-OH) which was 7.4. Scatchard analysis of [125I]BOP binding in washed platelets at pH 7.4, 6.0, and 5.0 revealed an increase in affinity (Kd = 1.16 +/- 0.06, 0.64 +/- 0.09, and 0.48 +/- 0.05 nM, respectively) and an increase in the number of receptors (Bmax = 2807 +/- 415, 5397 +/- 636, and 7265 +/- 753 sites/platelet, respectively). The potency of I-BOP to induce shape change in washed platelets at pH 6.0 was also significantly increased from an EC50 value of 0.34 +/- 0.016 nM at pH 7.4 to 0.174 +/- 0.014 nM at pH 6.0 (n = 6, p less than 0.05). In contrast, the EC50 value for thrombin was unaffected by the change in pH. In competition binding studies with [125I]BOP, the affinity of the agonists U46619 and ONO11113 were increased at pH 6.0 compared to 7.4. In contrast, the affinity of the TXA2/PGH2 receptor antagonists I-PTA-OH, SQ29548, and L657925 were either decreased or unchanged at pH 6.0 compared to 7.4. Diethyl pyrocarbonate and N-bromosuccinimide, reagents used to modify histidine residues, reversed the increase in affinity of [125I]BOP at pH 6.0 to values equivalent to those at pH 7.4. In solubilized platelet membranes, the effects of NBS were blocked by coincubation with the TXA2/PGH2 mimetic U46619. The results suggest that agonist and antagonist binding characteristics are different for the TXA2/PGH2 receptor and that histidine residue(s) may play an important role in the binding of TXA2/PGH2 ligands to the receptor.


Subject(s)
Blood Platelets/metabolism , Bridged Bicyclo Compounds, Heterocyclic , Hydrazines/metabolism , Receptors, Prostaglandin/metabolism , Thromboxane A2/antagonists & inhibitors , 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid , Bridged Bicyclo Compounds/metabolism , Bromosuccinimide/chemistry , Carbazoles/metabolism , Cell Membrane/metabolism , Diethyl Pyrocarbonate/chemistry , Fatty Acids, Unsaturated/metabolism , Histidine/chemistry , Humans , Hydrogen-Ion Concentration , In Vitro Techniques , Kinetics , Ligands , Platelet Activation , Prostaglandin Endoperoxides, Synthetic/metabolism , Protein Binding , Receptors, Thromboxane , Receptors, Thromboxane A2, Prostaglandin H2 , Solubility , Thromboxane A2/analogs & derivatives , Thromboxane A2/metabolism
14.
Biochemistry ; 28(10): 4422-6, 1989 May 16.
Article in English | MEDLINE | ID: mdl-2765494

ABSTRACT

Protoheme-CO in aqueous solution does not exhibit a geminate ligand recombination reaction. Addition of a protein, either globin or serum albumin, to which heme binds strongly, leads to an observable geminate reaction in aqueous solution. The bimolecular kinetic data for the albumin-heme-CO complex show two stable components, one heme-like in rate and difference spectrum, and one hemoglobin-like. The geminate reaction correlates spectrally with the hemoglobin-like component.


Subject(s)
Carbon Monoxide/radiation effects , Heme/radiation effects , Serum Albumin/radiation effects , Binding Sites , Kinetics , Photolysis , Spectrophotometry
15.
Eur J Biochem ; 169(3): 611-5, 1987 Dec 15.
Article in English | MEDLINE | ID: mdl-3691509

ABSTRACT

Flash photolysis kinetics of carbon monoxide hemoglobin show a decrease in the fraction of ligand recombination occurring as geminate when the hemoglobin has fewer ligands bound. Fully saturated samples, normally referred to as R state, show approximately 50% geminate phase, while samples at low saturation (T state) show less than 3%. The latter result was obtained by photolysis of samples with a short delay after stopped flow of solutions of deoxy hemoglobin (Hb) and ligand. The decrease in the fraction of geminate phase was also observed using a double flash technique. The transient mixture of R and T states generated by flash photolysis of Hb-CO was probed with a weaker time-delayed photolysis pulse. The kinetics of both the geminate and bimolecular phases following the second pulse were measured. The fraction geminate signal was least at delays where the maximum proportion of liganded T state tetramer is expected. The biphasic bimolecular process is also an indicator of the allosteric state of Hb. The populations of R and T may be determined from the overall ligand recombination kinetics; however, the analysis is model-dependent. The fraction geminate reaction may provide a rapid measure of the amount of liganded hemes in the R and T states.


Subject(s)
Hemoglobins , Binding Sites , Heme , Hydrogen-Ion Concentration , Kinetics , Photolysis
16.
Biochemistry ; 25(23): 7591-6, 1986 Nov 18.
Article in English | MEDLINE | ID: mdl-3801435

ABSTRACT

Three allosteric states are required to describe the relaxation of (carbon monoxy) hemoglobin following flash photolysis. Combined absorbance and fluorescence probes were used. The absorbance signals consist of a component corresponding to ligand recombination and a component for the R-T transition. The fluorescence of 8-hydroxy-1,3,6-pyrenetrisulfonate (HPT), an analogue of 2,3-diphosphoglycerate, shows rates and amplitudes correlated with the absorbance transients. Measurements were made at pII 6, 6.5, and 7.0 at CO partial pressures of 0.1 and 1 atm. The fractional photolysis was varied in each case to change the initial distribution of the R states. Data show an immediate absorbance change due to ligand dissociation, while the changes in the ligand isosbestic and the fluorescence signals occur with time constants of 80 microseconds (at pH 6.5). The signals then show a biphasic return to equilibrium, characteristic of the allosteric system. The measurements provide three independent probes of the kinetics of the substates of hemoglobin. Although the ligand binding data can be generally represented by a two-state model, the fluorescence data require T states with different affinities for HPT.


Subject(s)
Hemoglobins/metabolism , Allosteric Regulation , Allosteric Site , Carboxyhemoglobin/metabolism , Humans , Kinetics , Models, Biological , Photolysis , Phytic Acid/blood , Protein Binding , Spectrometry, Fluorescence , Spectrophotometry
17.
Biochemistry ; 25(10): 2786-92, 1986 May 20.
Article in English | MEDLINE | ID: mdl-3718920

ABSTRACT

Recombination kinetics of CO to protoheme after laser photolysis have been measured vs. temperature and viscosity. A 25-ns laser pulse was focused on the sample to produce an excitation rate of 10(9)/s per heme. This temporarily populates the heme-CO state of dissociated pairs which either separate or recombine on a picosecond time scale in viscous glycerol-water solutions. From the equilibrium amplitude of the fraction dissociated during the laser pulse, the geminate recombination rate constant is calculated to be 3 X 10(9)/s. This rate coefficient is only weakly dependent on temperature or viscosity. As previously observed the fraction that escapes depends on the solvent viscosity [Marden, M. C. (1983) Ph.D. Thesis, University of Illinois-Urbana]. A model consisting of a single barrier plus diffusive escape is used to simulate the kinetics during and just after the flash.


Subject(s)
Carbon Monoxide , Heme , Ethylene Glycol , Ethylene Glycols , Glycerol , Kinetics , Lasers , Mathematics , Models, Biological , Photolysis
18.
Article in English | MEDLINE | ID: mdl-6150789

ABSTRACT

Hemoglobin (Hb) concentration, hematocrit (Hct), total organic phosphates (Ptot), nucleoside triphosphates (NTP), 2,3-diphosphoglycerate (DPG), and "inositol polyphosphates" (IPP) were measured in the erythrocytes of adult frogs at different times of day in winter and summer after acclimatization to 15 degrees C and an LD 12:12 photoperiod. The same measurements were also made on animals acclimated to LD 16:8 in summer. All of the measured parameters varied significantly at different times of the day and between seasons in animals acclimated to an LD 12:12 photoperiod. Summer animals acclimated to LD 16:8 showed significant diel cycles only in Hct and Hb.


Subject(s)
Acclimatization , Circadian Rhythm , Diphosphoglyceric Acids/blood , Erythrocytes/metabolism , Hemoglobins/metabolism , Inositol Phosphates/blood , Ribonucleotides/blood , Sugar Phosphates/blood , 2,3-Diphosphoglycerate , Animals , Hematocrit , Ranidae , Seasons
19.
Comp Biochem Physiol B ; 79(2): 195-201, 1984.
Article in English | MEDLINE | ID: mdl-6548941

ABSTRACT

An attempt was made to establish the binding of N-(2,4-diphosphobenzyl)-1-amino-5-naphthalenesulfonic acid, DIPANS, as an estimator of conformation in the carbonmonoxy (CO)-hemoglobins (Hbs) of several vertebrates. DIPANS failed to bind menhaden I, trout I or tuna Hbs which are ligand insensitive. Below a pH of 7.0, DIPANS bound menhaden II, Bufo, Xenopus, and human Hbs with a binding stoichiometry greater than one. The charge of the DIPANS molecule does not control its binding to these Hbs. The binding to human CO-Hb can not be due to Hb conformation. For Xenopus Hbs and menhaden II, conformation predominates DIPANS binding. The binding to CO-Hb of DIPANS, can not be unambiguously attributed to the Hb's quaternary conformation.


Subject(s)
Hemoglobins/metabolism , Naphthalenesulfonates/blood , Animals , Bufonidae , Carboxyhemoglobin/metabolism , Humans , Hydrogen-Ion Concentration , Kinetics , Protein Binding , Protein Conformation , Species Specificity , Trout , Tuna , Xenopus
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