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PubMed2003

Molecular phenotyping of a trinucleotide repeat (D5S373) experimental conditions.

The aim of this study is to assess the utility of the STR D5S373 in human identification. PCR amplification and electrophoretic separation were optimized in order to achieve unambiguous phenotyping. We concluded that primer concentration and annealing temperature are the main factors affecting the specificity of PCR. In our population survey including three human major groups (Europe, Sub-Saharan Africa, and Asia), up to six alleles and six interalleles have been found ranging in size from 86 to 101 bp. The phenotypes were determined using horizontal polyacrylamide gel electrophoresis, a technique which has turned out to be suitable for separating fragments as close as 1 bp. In each population, the genotype frequencies conformed to the expectations of genetic equilibrium. Sequence studies were carried out to make the allele nomenclature fit to ISFH recommendations. Results from our population analysis of D5S373 show clear differences in allelic frequency patterns among the three major human groups examined. Human identification parameters estimated from our study are similar to those obtained for other STRs currently used in DNA testing.

Alleles↗
PubMed1994

Purification and partial characterization of 3-hydroxyisobutyryl-coenzyme A hydrolase of rat liver.

An unusual feature of valine catabolism is a reaction in which an intermediate of its catabolic pathway, (S)-3-hydroxyisobutyryl-CoA, is hydrolyzed to give the free acid and CoA-SH. The enzyme responsible for this reaction, 3-hydroxyisobutyryl-CoA hydrolase (EC 3.1.2.4), was purified 7200-fold from rat liver in this study. The purified enzyme consists of a single polypeptide with an M(r) of 36,000 in the native and denatured forms. The hydrolase is highly specific for (S)-3-hydroxyisobutyryl-CoA and 3-hydroxypropionyl-CoA (Km, 6 and 25 microM, respectively) with optimal activity around pH 8. The turnover rate of the enzyme for (S)-3-hydroxyisobutyryl-CoA is 270 s-1, which is high relative to other enzymes of the valine pathway. Likewise, activity of the enzyme expressed on a wet weight basis is also very high in the major tissues of the rat. These findings suggest that rapid destruction of (S)-3-hydroxyisobutyryl-CoA produced during valine catabolism is physiologically important. We propose that the need for a mechanism to protect cells against the toxic effects of methacrylyl-CoA, which is maintained in equilibrium with (S)-3-hydroxyisobutyryl-CoA by crotonase, explains why valine catabolism involves this enzyme and why its tissue activity is so high.

Acyl Coenzyme A↗
PubMed2003

Use of high concentration contrast media (HCCM): principles and rationale--body CT.

Numerous complex pharmacokinetic interrelationships affect the use of contrast media for computed tomography (CT) imaging. The volume, concentration, and rate of injection, all affect the degree of enhancement that is achieved with an injection of contrast material. In addition, the injection technique, whether the contrast is infused with a constant injection rate (uniphasic injection) or whether the rate is altered during the injection (multiphasic injection) also affect the magnitude and duration of contrast enhancement. In body CT imaging, the liver poses unique challenges in managing the use of intravenous contrast material because of its dual blood supply and the need to complete imaging before equilibrium occurs between the intravascular and extravascular compartments. The magnitude of hepatic enhancement that is ultimately achieved is related primarily to the amount of iodinated contrast material that accumulates in the extravascular space within the target organ, independent of the speed of the CT scanner. The key determinant of the onset of the equilibrium phase is the injection duration. Given that a high injection flow rate (4-5 ml/s) is desirable for arterial phase imaging, the injection duration is maintained with use of an appropriate contrast volume. Thus, modifications of total iodine dose are best done with alterations in contrast concentration. The magnitude of arterial enhancement that is achieved is related to both the concentration and rate of contrast administration. The speed of the scanner determines its ability to record image data during the most advantageous time period, the peak of arterial enhancement. Thus, rapid imaging is particularly advantageous for optimal contrast use in CT angiography as well as in multiphasic imaging of the parenchymal organs.

Angiography↗
PubMed2005

Enhanced coagulation for satisfying the arsenic maximum contaminant level under variable and uncertain conditions.

This study evaluated the effects of influent variability and model parameter uncertainty when utilizing enhanced coagulation modification to bring existing treatment plants into compliance with a stricter arsenic regulation. Enhanced coagulation modification options include: (1) increased ferric chloride dose, (2) addition of an acid dose, and (3) a combination of the individual options. Arsenic removal is described by adsorption to hydrous ferric oxide with a surface complexation model and subsequent removal through sedimentation and filtration. The least-cost modification for reliably satisfying the arsenic regulation is determined using an optimization algorithm that explicitly includes variability and uncertainty. The ferric chloride only modification is always the least-cost treatment modification. The ferric chloride and acid modification could be the least-cost option when considering waste handling processes due to a tradeoff between modification cost and sludge production. By inclusion of variability and uncertainty, the relative importance of individual parameter distributions for determining whether the arsenic regulation is reliably satisfied is assessed. Influent arsenic concentration variability is always critical, while variability in the influent pH and sulfate concentrations and uncertainty in the filter removal efficiency and equilibrium adsorption constant for the triple bond Fe(s)OHCa2+ surface species are critical or important, depending on influent conditions.

Adsorption↗
PubMed2001

Clusters containing open-shell molecules: minimum-energy structures and low-lying isomers of ArnCH (X 2 pi), n = 1 to 15.

The size evolution of the equilibrium structures of open-shell ArnCH (X 2 pi) Van der Waals clusters is investigated for n = 1 to 15. We describe a method for combining pair potentials for Ar-CH and Ar-Ar interactions to obtain potential energy surfaces for ArnCH clusters. For each cluster size considered, the global and a few energetically close local minima are calculated using simulated annealing followed by a direct minimization scheme. Ar2CH is found to have an unusually stable planar structure, which persists as a motif in larger ArnCH clusters and has a strong effect on their optimal geometries. The lowest-energy isomers of ArnCH with n = 3 to 11 have all Ar atoms in a shell around CH. The only exception is Ar4CH, where the fully solvated isomer is 3 cm-1 higher in energy than the optimal isomer with CH bound to the surface of the Ar4 tetrahedron. For n = 7 to 11, the minimum-energy structure of ArnCH derives from the global minimum of the Arn + 1 cluster, by replacing the Ar atom at the bottom of the pentagonal bipyramid with CH. The lowest-energy structure of Ar12CH is that of the optimal icosahedral Ar13 cluster, with CH replacing one of the Ar atoms on the cluster surface. This structure supports the proposition based on the spectroscopic data, that for ArnCH clusters with about 10 to 50 Ar atoms CH resides on the surface of Arn.

Journal Article↗
PubMed2004

Resonance Raman spectra of uracil based on Kramers-Kronig relations using time-dependent density functional calculations and multireference perturbation theory.

The use of time-dependent density functional calculations for the optimization of excited-state structures and the subsequent calculation of resonance Raman intensities within the transform-theory framework is compared to calculations of Hartree-Fock/configuration interaction singles-type (CIS). The transform theory of resonance Raman scattering is based on Kramers-Kronig relations between polarizability tensor components and the optical absorption. Stationary points for the two lowest excited singlet states of uracil are optimized and characterized by means of numerical differentiation of analytical excited-state gradients. It is shown that the effect of electron correlation leads to substantial modifications of the relative intensities. Calculations of vibrational frequencies for ground and excited states are carried out, which show that the neglect of Duschinsky mixing and the assumption of equal wave numbers for ground and excited state are not in all cases good approximations. We also compare the transform-theory resonance Raman intensities with those obtained within a simple approximation from excited-state gradients at the ground-state equilibrium position, and find that they are in qualitative agreement in the case of CIS, but show some important differences in calculations based on density functional theory. Since the results from CIS calculations are in better agreement with experiment, we also present approximate resonance Raman spectra obtained using excited-state gradients from multireference perturbation theory calculations, which confirm the CIS gradients.

Journal Article↗
PubMed1995

Histidine tagging both allows convenient single-step purification of bovine rhodopsin and exerts ionic strength-dependent effects on its photochemistry.

For rapid single-step purification of recombinant rhodopsin, a baculovirus expression vector was constructed containing the bovine opsin coding sequence extended at the 3'-end by a short sequence encoding six histidine residues. Recombinant baculovirus-infected Spodoptera frugiperda cells produce bovine opsin carrying a C-terminal histidine tag (v-opshis6x). The presence of this tag was confirmed by immunoblot analysis. Incubation with 11-cis-retinal produced a photosensitive pigment (v-Rhohis6x) at a level of 15-20 pmol/10(6) cells. The histidine tag was exploited to purify v-Rhohis6x via immobilized metal affinity chromatography. Optimized immobilized metal affinity chromatography yielded a binding capacity of > or = 35 nmol of v-Rhohis6x per ml of resin and purification factors up to 500. Best samples were at least 85% pure, with an average purity of 70% (A280 nm/A500 nm = 2.5 +/- 0.4, n = 7). Remaining contamination was largely removed upon reconstitution into lipids, yielding rhodopsin proteoliposomes with a purity over 95%. Spectral analysis of v-Rhohis6x showed a small but significant red shift (501 +/- 1 nm) compared to wild type rhodopsin (498 +/- 1 nm). The pK alpha of the Meta I<==>Meta II equilibrium in v-Rhohis6x is down-shifted from 7.3 to 6.4 resulting in a significant shift at pH 6.5 toward the Meta I photointermediate. Both effects are reversed upon increasing the ionic strength. FT-IR analysis of the Rho-->Meta II transition shows that the corresponding structural changes are identical in wild type and v-Rhohis6x.

Amino Acid Sequence↗
PubMed2004

Transcriptional Repressor CopR: use of SELEX to study the copR operator indicates that evolution was directed at maximal binding affinity.

CopR is one of the two copy number control elements of the streptococcal plasmid pIP501. It represses transcription of the repR mRNA encoding the essential replication initiator protein about 10- to 20-fold by binding to its operator region upstream of the repR promoter pII. CopR binds at two consecutive sites in the major groove of the DNA that share the consensus motif 5'-CGTG. Previously, the minimal operator was narrowed down to 17 bp, and equilibrium dissociation constants for DNA binding and dimerization were determined to be 0.4 nM and 1.4 microM, respectively. In this work, we used a SELEX procedure to study copR operator sequences of different lengths in combination with electrophoretic mobility shift assays of mutated copR operators as well as copy number determinations to assess the sequence requirements for CopR binding. The results suggest that in vivo evolution was directed at maximal binding affinity. Three simultaneous nucleotide exchanges outside the bases directly contacted by CopR only slightly affected CopR binding in vitro or copy numbers in vivo. Furthermore, the optimal spacer sequence was found to comprise 7 bp, to be AT rich, and to need an A/T and a T at the 3' positions, whereas broad variations in the sequences flanking the minimal 17-bp operator were well tolerated.

3' Flanking Region↗
PubMed1987

A review of why and how we age: a defense of multifactorial aging.

Part 1: Longevity is optimized such that reproduction is maximized. Williams (Evolution, 11 (1957) 398-411) proposed pleiotropic genes with beneficial effects during youth and harmful effects at older ages. Because of environmental death (e.g. predation, disease, accidents), even a small increase in younger reproduction could outweigh a large harmful effect at older ages. Guthrie (Perspect. Biol. Med., 12 (1969) 313-324) and Kirkwood (Nature, 270 (1977) 301-304; New Sci., 81 (1979) 1040-1042; Physiological Ecology: An Evolutionary Approach, Blackwell, Oxford, 1981, pp. 165-189; Hum. Genet., 60 (1982) 101-121; Proc. R. Soc. Lond., B205 (1979) 531-546; Handbook of the Biology of Aging, 2nd Edn., Von Nostrand Reinhold New York, 1985, pp. 27-44) proposed that additional longevity requires a further investment of resources when young, thereby reducing the resources available for reproduction when young. The gene(s) controlling this partitioning of resources between younger and older reproduction are a good example of Williams's pleiotropic genes. Population biology provides a great deal of evidence of a tradeoff between younger and older reproduction. A "marginal longevity theorem" is proposed which states that for a population in equilibrium with its environment a marginal change in any gene affecting longevity should cause equal and opposite marginal changes in younger and older expected reproduction. Senescence is not irrelevant in the wild; rather, the amount of senescence in the wild results from a balance between its marginal costs to older reproduction and its associated marginal benefits in younger reproduction. Part 2: The wide variety of damage prevention processes in the body are subject to the problem of diminishing returns. Consequently, a broad spectrum of damage occurs in the body, varying in frequency, harmfulness, and ease of repair. The types of damage which are prevented or repaired tend to be more frequent, harmful, and easily prevented or repaired. In contrast, aging damage (which accumulates) consists of a large number of different types of damage which (when considered separately) are relatively infrequent, less harmful, and/or more difficult to repair. Only when these types of damage accumulate to become very numerous do they (when considered collectively) become significant. Since the selective advantages associated with senescence apply to all parts of the body, primary aging damage occurs in all tissues, cells, and subcellular organelles. The distribution of metabolic resources among the various damage repair and prevention processes is optimized.(ABSTRACT TRUNCATED AT 400 WORDS)

Aging↗
PubMed1996

A graphic analysis of the biomechanics of the massless bi-articular chain. application to the proximal bi-articular chain of the human finger.

In this paper a model is presented which visualises the biomechanical functioning of the loaded and unloaded theree-tendon bi-articular chain. This model allows to graphically determine in any position of the chain (i) the exact ranges of loads which can be sustained by the different motors; (ii) the motors forces; (iii) the feasibility of the (unloaded) equilibrium; and (iv) the conditions for the good controllability of the bi-articular chain. These results are applied to the proximal three-motor bi-articular chain of the human finger, when controlled by the superficial flexor, interosseus, and extensor only. It is shown that (i) the anatomic position of the superficial flexor and extensor is a prime determinant in the good functioning of this chain; (ii) the proximal bi-articular chain of the human finger can well sustain certain flexion loads but is structurally weak for extension loads; and (iii) the chain is not optimally controllable.

Algorithms↗
PubMed1983

Use of protein A to remove immunoglobulins from serum in hybridoma culture media.

The levels of protein A-reactive immunoglobulin (PA-Ig) in foetal bovine serum were measured in commercial batches. For tissue culture media incorporating 10% foetal bovine serum, the levels of bovine PA-Ig were of a similar order to those of mouse monoclonal antibodies produced by hybridomas grown in such media. The equilibrium constants were calculated for the binding to protein A-Sepharose of a number of mouse monoclonal antibodies, and of PA-Ig in foetal bovine serum and normal mouse serum. The average affinity of the mouse PA-Ig was 10 times higher than that of the bovine PA-Ig, suggesting that the two could be separated by affinity chromatography on protein A-Sepharose. The mouse monoclonal antibodies, however, displayed a range of affinity 1.5-100 times that of the bovine PA-Ig, indicating that such separation could not be generally applied. The optimal technique involved removing PA-Ig from bovine serum before its inclusion in the culture medium and then purifying the monoclonal antibody on a second protein A-Sepharose column.

Animals↗
PubMed2000

Tert.-butylcarbamoylquinine as chiral ion-pair agent in non-aqueous enantioselective capillary electrophoresis applying the partial filling technique.

The potential of tert.-butylcarbamoylquinine as chiral selector (SO) added to a non-aqueous background electrolyte for the capillary electrophoretic separation of the enantiomers of N-derivatized amino acids (selectands, SAs) is evaluated. Separation is based on different ion-pair formation equilibrium constants of (R) and (S) enantiomers of the negatively charged chiral analytes with the positively charged quinine-derived chiral SO and on mobility differences of free and complexed SAs, so that differences in the overall migration behavior of the SA enantiomers result. To suppress problems associated with the high UV absorption of the chiral SO and thus the high detector background in the 'total filling technique', the 'partial filling technique' has been adopted. Several parameters including filling time and length of SO zone, respectively, SO concentration, type of background electrolyte, have been evaluated. Using such an optimized method, for example, (R) and (S) enantiomers of 2,4-dinitrophenyl (DNP)-protected proline could be separated with alpha=1.08, R(S)=6.60, and N=130,000 theoretical plates within 15 min. Similar alpha values, resolution, and efficiencies were observed for other DNP-protected, as well as for diverse, N-derivatized amino acids like N-benzoyl, N-9-fluorenylmethoxycarbonyl, N-3,5-dinitrobenzyloxycarbonyl amino acids. A repeatability study clearly validated the robustness of the method and revealed its practical applicability.

Anions↗
PubMed1991

Pharmacokinetics of amrinone during cardiac surgery.

Amrinone is a nonglycosidic noncatecholamine with both vasodilator and positive inotropic effects that may be administered to patients undergoing cardiac surgery. As an initial step toward elucidating the optimal dosage of amrinone for cardiac surgical patients we studied the pharmacokinetics of amrinone during and after cardiac surgery requiring cardiopulmonary bypass. The study population comprised 35 adult patients, each receiving a single dose of amrinone (0.75, 1.5, 2.0, or 2.5 mg/kg) administered into the venous reservoir near the end of cardiopulmonary bypass. Additionally, 15 of the 35 patients also received intravenous infusions of either 5 or 10 micrograms.kg-1.min-1. Arterial blood was sampled over the next 22 h, and plasma concentrations of amrinone were determined by high-performance liquid chromatography. Protein binding of amrinone, assayed by equilibrium dialysis, was 21.6 +/- 2.5%. The decay of amrinone concentrations in plasma over time was fit to a biexponential equation by nonlinear least-squares regression. The manufacturer's recommended dose of 0.75 mg/kg followed by an infusion of 10 micrograms.kg-1.min-1 was inadequate to maintain the plasma concentration within the therapeutic range based on the pharmacodynamics of amrinone in patients with chronic congestive heart failure. This was due to significant redistribution of amrinone in the body after the loading dose. To maintain a therapeutic plasma concentration of 1.5-2.0 micrograms/ml, a larger loading dose or a supplemental loading dose as well as a continuous infusion is required.

Amrinone↗
PubMed1995

Prediction of protein displacement by simplified immobilized metal ion affinity chromatographic model.

A simplified theoretical model was developed to predict complex behavior in protein displacement systems of immobilized metal ion affinity chromatography (IMAC). In this paper, experimental techniques for estimating equilibrium parameters were developed. The equilibrium parameters obtained were used to simulate displacement processes and to investigate the effect of mobile phase modifier (MPM) gradients in the IMAC separating systems. In the induced MPM gradient conditions, the solute movement analysis was able to predict the displacement separation well characterized by the intersections of the operation line under the effective displacer concentrations. The theoretical and experimental results demonstrate the utility of the IMAC model in predicting induced mobile phase modifier gradients and complex behavior in metal affinity displacement chromatography. Furthermore, the development of a rapid method for obtaining displacement profiles facilitates methods development and optimization of IMAC displacement separations.

Animals↗
PubMed1997

Effects of NaCl on the linkages between O2 binding and subunit assembly in human hemoglobin: titration of the quaternary enhancement effect.

Oxygen binding by human hemoglobin (Hb) and the coupled reactions of dimer-tetramer assembly were studied over a range of NaCl concentrations (from 0.08 M to 1.4 M) at pH 7.4 and 21.5 degrees C. A strategy of multi-dimensional analysis was employed [G.K. Ackers and H.R. Halvorson, Proc. Natl. Acad. Sci. U.S.A., 91, (1974) 4312] to optimize the resolution of the contributions to cooperativity and their heterotropic salt linkages at each stoichiometric degree of O2 binding. A wide range of Hb concentration was utilized at each [NaCl] in which O2-linked subunit assembly reactions contributed significantly to the positions and shapes of the binding isotherms. Kinetic determinations yielded forward and reverse rate constants for assembly of the unligated species. Amplitudes for the assembly rate data had concentration dependences in agreement with the independently determined dimer-tetramer assembly constants of oxyhemoglobin. Concentration-dependent binding isotherms were analyzed, in combination with the kinetically determined equilibrium constants, to yield salt-linked components of cooperativity at the four stages of oxygenation. The principal results of this study were as follows. (i) Assembly of fully oxygenated Hb tetramers is opposed by NaCl: the dimer-to-tetramer equilibrium constant becomes two orders of magnitude less favorable over the [NaCl] range 0.08 M to 1.4 M. By contrast, for deoxy-Hb the assembly equilibrium constant is reduced only two-fold. (ii) Oxygen binding to dimers is non-cooperative over the entire salt range, whereas dimer affinity is slightly favored by increasing the NaCl concentration. (iii) Overall affinity of tetramers for O2 is opposed by NaCl, becoming an order of magnitude less favorable over the range employed. Most of this decrease occurs at the fourth binding step, which shows a large, salt-mediated quaternary enhancement effect; i.e., the assembly of dimers into tetramers at 0.08 M NaCl is accompanied by an eight-fold increase in O2 affinity. (iv) The quaternary enhancement effect at the last O2-binding step is titrated progressively by salt until it reaches a negligible value near the highest [NaCl] of this study. The lowest [NaCl] condition (0.08 M) elicits the greatest tetramer cooperativity with the largest maximal Hill coefficient and the greatest suppression of intermediates. Possible origins and mechanistic implications of these phenomena are considered.

Hemoglobins↗
PubMed2001

Relocalization in floppy free radicals: the OCNO and OCCHO isoelectronic series.

A survey of the XCNY and XCCHY radicals with X and Y = CH(2), NH, and O has been carried out by ab initio QCISD/6-311G(d,p) calculations to assess the impact of low-lying excited electronic states on the molecular dynamics. Multiple canonical structures may be drawn for each of these structural formulas, with the principal competition for most stable configuration between a (2)A' form with four electrons in a" orbitals and a (2)A" form with five a" electrons. Other low-lying configurations may include a 5a" state with nominally pentavalent nitrogen and a 6a" state. Optimized geometries and harmonic frequencies were evaluated for the lowest-energy minima on the potential energy surfaces. Localized unpaired electron density causes the 4a" state to be the most stable for (NH)CCHO and OCCHY, whereas allylic resonance stabilization favors the 5a" state for all other radicals in the set. For five of the 18 molecules studied, secondary minima (excluding conformers) are found within 30 kJ mol(-)(1) of the most stable state at the QCISD/6-311G(d,p) level, suggesting that photolysis or pyrolysis of parent compounds may result in multiple isomers of the resulting reactive intermediates. Predicted equilibrium geometries, approximate thermochemical quantities for dissociation of the central bond, and selected spectroscopic parameters are presented for all 18 structural formulas. Convergence tests were also performed for the glyoxallyl radical (OCCHO) to resolve discrepancies between single- and multireference post-SCF results. These tests find that extension of the MCSCF methods to include sigma bonding orbitals or virtual-orbital CI brings MCSCF relative energies into agreement with results from standard single-reference CI and CC methods. Relative configurational energies evaluated at Hartree-Fock levels routinely differ from post-SCF values by 30 kJ mol(-1) or more.

Journal Article↗
PubMed2001

Functional expression of recombinant type 1 ryanodine receptor in insect cells.

We have investigated the biochemical properties of the rabbit ryanodine receptor type 1 (RyR1) from skeletal muscle functionally expressed in insect sf 21 cells infected with recombinant baculovirus. Equilibrium [3H]ryanodine binding assays applied to total membrane fractions from sf 21 cells expressing recombinant RyR1 showed a non-hyperbolic saturation curve (Hill coefficient = 2.1). The [3H]ryanodine binding was enhanced by 1 mM AMP-PCP and 10 mM caffeine, whereas 10 mM Mg(2+) and 5 microM ruthenium red reduced the specific binding. The dependence of [3H]ryanodine binding on ionic strength showed positive cooperativity (Hill coefficient = 2.2) with a plateau at 1 M KCl. The recombinant RyR1 showed a bell-shaped [3H]ryanodine binding curve when free [Ca(2+)] was increased, with an optimal concentration around 100 microM.Confocal microscopy studies using the Ca(2+) ATPase selective inhibitor, thapsigargin coupled to fluorescein and ryanodine coupled to Texas red demonstrated that the recombinant RyR1 and the Ca(2+) ATPase co-localize to the same intracellular membrane. No significant RyR1 fluorescence was observed at the plasma membrane.Fluo-4-loaded sf 21 cells expressing recombinant RyR1 responded to activating-low ryanodine concentrations (100 nM) or caffeine (10 mM) with a sharp rise in intracellular Ca2 followed by a sustained phase, in contrast, sf 21 cells expressing the human bradykinin type 2 receptor did not respond to ryanodine or caffeine.These results demonstrate the expression of recombinant RyR1 in sf 21 cells with functional properties similar to what has been previously reported for native RyR1 in mammalian tissues, however, some differences were observed in [3H]ryanodine binding assays compared to native rabbit RyR1. Hence, the baculovirus expression system provides a generous source of protein to accomplish structure-function studies and an excellent model to assess functional properties of wild type and mutant RyR1.

Animals↗
PubMed1992

Characterization of gonadotropin-releasing hormone (GnRH) receptors in the ovary of common carp (Cyprinus carpio).

Gonadotropin-releasing hormone (GnRH) binding sites have been characterized in the fully mature common carp ovary, using an analog of salmon GnRH ([D-Arg6,Trp7,Leu8,Pro9-NEt]-GnRH; sGnRH-A) as a labeled ligand. Binding of sGnRH-A to carp follicular membrane preparation was found to be time-, temperature-, and pH-dependent. Optimal binding was achieved after 40 min of incubation at 4 degrees C at pH 7.6; binding was found to be unstable at room temperature. Binding of radioligand was a function of tissue concentration, with a linear correlation over the range of 8.0-40.0 micrograms membrane protein per tube. Incubation of membrane preparations with increasing levels of [125I]sGnRH-A revealed saturable binding at radioligand concentrations greater than 400 nM. The binding of [125I]sGnRH-A to the carp ovary was also found to be reversible; addition of unlabeled sGnRH-A (10(-6) M) after reaching equilibrium resulted in complete dissociation of [125I]sGnRH-A within 30 min, and the log dissociation plot indicated the existence of a single class of binding sites. Addition of unlabeled sGnRH-A displaced the bound [125I]sGnRH-A in a dose-related manner. Hill plot as well as Scatchard analysis suggested the presence of one class of high affinity GnRH binding sites. Bound [125I]sGnRH-A was also found to be displaceable by other GnRH peptides, including sGnRH ([Trp7,Leu8]-GnRH), cGnRH-II ([His5,Trp7,Tyr8]-GnRH) and a GnRH antagonist ([D-pGlu1,D-Phe2,D-PTrp3,6]-GnRH; GnRH-ANT) in a parallel fashion, indicating that these peptides bind to the same class of binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗