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PubMed1994

An ELISA for detecting anti-Plasmodium spp. antibodies in African black-footed penguins (Spheniscus demersus).

An enzyme-linked immunosorbent assay (ELISA) utilizing 3 Plasmodium falciparum antigens, R32tet32, P.F.R27, and crude red blood cell extract, was developed for the detection of circulating anti-Plasmodium relictum or anti-Plasmodium elongatum antibodies in sera from naturally infected adult African black-footed penguins (Spheniscus demersus) at The Baltimore Zoo, Maryland. A concentration of 2.0 micrograms/ml of each antigen was optimal in terms of specificity, sensitivity, and test speed. It was possible to detect anti-Plasmodium spp. antibodies at a dilution of 10(-4.11). Low absorbance values (less than 0.050) of nonspecific background were observed. The binding efficacy of anti-penguin IgG coupled to alkaline phosphatase to antibodies in the penguin sera was significantly higher than the binding efficacy of anti-chicken IgG. All penguins, bled in the winter time, in controlled mosquito-free conditions had anti-Plasmodium spp. antibodies reactive with P. falciparum antigens. The penguins showed age-dependent variation in antibody levels. There was a decrease in antibody titration units that was significantly correlated with the number of outdoor exposure years experienced by the birds, despite the season-comparable epizootiologic conditions in their summer open-air habitat. We concluded that the decrease of anti-malarial antibodies could be explained by an antibody-mediated equilibrium of immunity in naturally immunized birds harboring endothelial-stage parasites. The ELISA described is sensitive, and it requires a minimal amount of equipment to collect the blood samples. The assay can be used for detecting and monitoring levels of anti-Plasmodium spp. antibodies in selected groups of penguins.

Analysis of Variance↗
PubMed2006

Age- and gender-related differences in ischemia/reperfusion injury and cardioprotection: effects of diazoxide.

BACKGROUND: Recent studies have demonstrated that aging is associated with reduced tolerance to ischemia and that the aged (not senescent) female heart has greater susceptibility to ischemia as compared with the aged male heart. Previously, we have shown that ischemia can be modulated with cardioplegia in the male heart; however, efficacy in the female heart was unknown. METHODS: In this study, male and female mature (15 to 20 weeks) aged (>32 months) rabbit hearts (n = 134) were subjected to Langendorff perfusion. Control hearts were perfused for 180 minutes. Global ischemia hearts received 30 minutes of equilibrium, 30 minutes of global ischemia, and 120 minutes of reperfusion. Cardioplegia +/- diazoxide was infused separately, 5 minutes before global ischemia. RESULTS: Global ischemia significantly decreased postischemic functional recovery and significantly increased infarct size in the mature and aged male and female heart (p < 0.05 versus control). The effects of global ischemia were significantly exacerbated (p < 0.05) in the aged heart as compared with the mature heart. Cardioplegia +/- diazoxide significantly increased postischemic functional recovery and significantly decreased infarct size in mature male and female hearts, but these effects were significantly decreased in the aged heart (p < 0.05) and in the aged female as compared with the aged male heart. CONCLUSIONS: Postischemic functional recovery and infarct size are affected by age but not by gender. The cardioprotection afforded by cardioplegia is affected by age and gender with a strong age-by-gender interaction for end-diastolic pressure and infarct size. Our results indicate that currently optimized cardioplegia protocols effective in the male heart are not as efficacious in the aged female heart.

Aging↗
PubMed2000

Interaction of different oligomeric states of hexameric DNA-helicase RepA with single-stranded DNA studied by analytical ultracentrifugation.

Analytical ultracentrifugation was used to determine the molecular mass, M, of hexameric DNA-helicase RepA at pH 5.8 and 7.6. At pH 7.6, a molecular mass of 179.5+/-2.6 kDa was found, consistent with the known hexameric state of RepA, (RepA)(6). At pH 5.8, (RepA)(6) associates to form a dimer with a molecular mass of 366.2+/-4.1 kDa. Analytical ultracentrifugation was also applied to characterize the interaction of single-stranded DNA (ssDNA) with the two different oligomeric states of (RepA)(6) at pH 5.8 and 7.6. The dissociation constants, K(d), for the equilibrium binding of (dA)(30) to the (RepA)(6) dimer at pH 5.8 and to (RepA)(6) at pH 7.6 were determined at 10 degrees C in the presence of 0.5 mM ATPgammaS, 10 mM MgCl(2) and 60 mM NaCl as K(d5.8)=0.94+/-0.13 microM at pH 5.8 and K(d7. 6)=25.4+/-6.4 microM at pH 7.6. The stoichiometries, n, for the two complexes (dA)(30)/(RepA)(6) dimer and (dA)(30)/(RepA)(6) at pH 5.8 and 7.6 were calculated from the corresponding binding curves. At pH 5.8 one (dA)(30) molecule was bound per (RepA)(6) dimer, while at pH 7.6 one (dA)(30) molecule was bound to one (RepA)(6). Binding curves were compatible with a single ssDNA binding site present on the (RepA)(6) dimer and on (RepA)(6), respectively, with no indication of cooperativity. (RepA)(6) tends to form larger aggregates under acidic conditions (pH<6.0) which are optimal for ssDNA binding. In contrast, at pH 5.8 in the presence of 60 mM NaCl, only the (RepA)(6) dimer was observed both in the absence and presence of (dA)(30).

Biopolymers↗
PubMed2000

Cytochrome b562 variants: a library for examining redox potential evolution.

A general understanding of how cytochromes evolve within a fixed structure to optimize redox potential for specific bioenergetic processes does not exist. Toward this end, a library approach is used to investigate the range and distribution of redox potential which occurs when all sequence space available through mutation at two positions is examined within a fixed structural motif. Random mutation of Phe61 and Phe65 of cytochrome b562 (E. coli), and subsequent examination of a statistically significant sampling of this library, demonstrates that the redox potential can vary over 100 mV (>25% of the known accessible potential in native proteins with axial His-Met ligation) through mutation at these two positions. The redox potential of the wild-type protein occurs at an extremum of the distribution observed, indicating that Phe61 and Phe65 were most likely naturally selected to differentially stabilize the reduced state of the protein. At the other extremum, a compositionally conservative set of mutations (F61I, F65Y) leads to a 100 mV shift in the redox equilibrium toward the oxidized state. NMR analyses indicate that a charge-dipole interaction which results from mutation of phenylalanine to tyrosine at position 65 may be responsible.

Amino Acid Substitution↗
PubMed2000

Contribution of the hydrogen-bond network involving a tyrosine triad in the active site to the structure and function of a highly proficient ketosteroid isomerase from Pseudomonas putida biotype B.

Delta(5)-3-Ketosteroid isomerase from Pseudomonas putida biotype B is one of the most proficient enzymes catalyzing an allylic isomerization reaction at rates comparable to the diffusion limit. The hydrogen-bond network (Asp99... Wat504...Tyr14...Tyr55...Tyr30) which links the two catalytic residues, Tyr14 and Asp99, to Tyr30, Tyr55, and a water molecule in the highly apolar active site has been characterized in an effort to identify its roles in function and stability. The DeltaG(U)(H2O) determined from equilibrium unfolding experiments reveals that the elimination of the hydroxyl group of Tyr14 or Tyr55 or the replacement of Asp99 with leucine results in a loss of conformational stability of 3.5-4.4 kcal/mol, suggesting that the hydrogen bonds of Tyr14, Tyr55, and Asp99 contribute significantly to stability. While decreasing the stability by about 6.5-7.9 kcal/mol, the Y55F/D99L or Y30F/D99L double mutation also reduced activity significantly, exhibiting a synergistic effect on k(cat) relative to the respective single mutations. These results indicate that the hydrogen-bond network is important for both stability and function. Additionally, they suggest that Tyr14 cannot function efficiently alone without additional support from the hydrogen bonds of Tyr55 and Asp99. The crystal structure of Y55F as determined at 1.9 A resolution shows that Tyr14 OH undergoes an alteration in orientation to form a new hydrogen bond with Tyr30. This observation supports the role of Tyr55 OH in positioning Tyr14 properly to optimize the hydrogen bond between Tyr14 and C3-O of the steroid substrate. No significant structural changes were observed in the crystal structures of Y30F and Y30F/Y55F, which allowed us to estimate approximately the interaction energies mediated by the hydrogen bonds Tyr30...Tyr55 and Tyr14...Tyr55. Taken together, our results demonstrate that the hydrogen-bond network provides the structural support that is needed for the enzyme to maintain the active-site geometry optimized for both function and stability.

Amino Acid Substitution↗
PubMed2002

The search for bishomoaromatic semibullvalenes and barbaralanes: computational evidence of their identification by UV/Vis and IR spectroscopy and prediction of the existence of a blue bishomoaromatic semibullvalene.

Time-dependent B3LYP/6-31G calculations have been performed at the optimized C(2) or C(2v) geometries of several substituted semibullvalenes (1(deloc)) and barbaralanes (2(deloc)), to compare the computed vertical electronic excitation energies with the temperature-dependent, long-wavelength absorptions that have been observed in the UV/vis spectra of some of these compounds by Quast and co-workers. The excellent agreement between the calculated vertical excitation energies and the observed values of lambda(max) provides strong support for the identification of the bishomoaromatic species 1(deloc) and 2(deloc) as the source of these absorptions. Furthermore, the CN stretching frequencies, computed for the C(2) geometry of 1,5-dimethyl-2,6-dicyano-4,8-diphenylsemibullvalene (1f(deloc)), fit the low-frequency absorptions seen in the IR spectrum of 1f, thus furnishing independent evidence that bishomoaromatic geometries of semibullvalenes have, in fact, been observed spectroscopically. B3LYP/6-31G calculations predict that 2,6-dicyano-4,8-diphenylsemibullvalene 1c has a C(2) equilibrium geometry (1c(deloc)) and that the long-wavelength UV/vis absorption (lambda(max) = 585 nm) and CN stretching frequencies (2192 and 2194 cm(-1)) computed for 1c(deloc) should serve to identify this bishomoaromatic semibullvalene when it is synthesized.

Journal Article↗
PubMed2004

Franck-Condon simulation of the single-vibronic-level emission spectra of HPCl/DPCl and the chemiluminescence spectrum of HPCl, including anharmonicity.

Restricted-spin coupled-cluster single-double plus perturbative triple excitation [RCCSD(T)] potential energy functions (PEFs) were calculated for the X (2)A" and A (2)A' states of HPCl employing the augmented correlation-consistent polarized-valence-quadruple-zeta (aug-cc-pVQZ) basis set. Further geometry optimization calculations were carried out on both electronic states of HPCl at the RCCSD(T) level with all electron and quasirelativistic effective core potential basis sets of better than the aug-cc-pVQZ quality, and also including some core electrons, in order to obtain more reliable geometrical parameters and relative electronic energy of the two states. Anharmonic vibrational wave functions of the two states of HPCl and DPCl, and Franck-Condon (FC) factors of the A (2)A'-X (2)A" transition were computed employing the RCCSD(T)/aug-cc-pVQZ PEFs. Calculated FC factors with allowance for Duschinsky rotation and anharmonicity were used to simulate the single-vibronic-level (SVL) emission spectra of HPCl and DPCl reported by Brandon et al. [J. Chem. Phys. 119, 2037 (2003)] and the chemiluminescence spectrum reported by Bramwell et al. [Chem. Phys. Lett. 331, 483 (2000)]. Comparison between simulated and observed SVL emission spectra gives the experimentally derived equilibrium geometry of the A (2)A' state of HPCl of r(e)(PCl) = 2.0035 +/- 0.0015 A, theta(e) = 116.08 +/- 0.60 degrees, and r(e)(HP) = 1.4063+/-0.0015 A via the iterative Franck-Condon analysis procedure. Comparison between simulated and observed chemiluminescence spectra confirms that the vibrational population distribution of the A (2)A' state of HPCl is non-Boltzmann, as proposed by Baraille et al. [Chem. Phys. 289, 263 (2003)].

Journal Article↗
PubMed1979

Valproic acid: in vitro plasma protein binding and interaction with phenytoin.

Because valproic acid (VPA) is highly bound to plasma protein, several variables affecting binding will significantly alter the quantity of free drug which is pharmacologically active. Therefore, total VPA plasma concentrations do not reflect the therapeutic strength of the drug in tissue. We have performed equilibrium dialysis and ultrafiltration studies of VPA binding to plasma protein. The converging data in these in vitro studies indicate a clinically significant alteration in the percent of free VPA when total drug concentration exceeds 80 micrograms/ml. Saturation of drug binding sites probably occurs in this range. At 20--60 micrograms/ml VPA there is 5% free drug, with a significant increase to 8% free at 80 micrograms/ml; free drug increases to over 20% at 145 micrograms/ml total VPA. Human plasma, which is low in albumin, has twice the quantity of free VPA as normal plasma (10 versus 5% free). The clinical evidence of interaction between VPA and phenytoin is confirmed in vitro by the increase in the free fraction of both drugs. VPA binding decreases by 3--6%, while phenytoin binding decreases 5--6% as both drugs reach high plasma concentrations. When appropriate, laboratory reports should be available defining concentration of free drug in plasma for optimal interpretation of drug concetrations relative to clinical effects.

Blood Proteins↗
PubMed1985

Analysis of methadone disposition in the pregnant rat by means of a physiological flow model.

A physiological flow model simulating the pregnant rat is constructed for methadone. The model includes brain, fetal, hepatic, intestinal, muscular, pulmonar, and renal tissues. Since methadone kinetics may provide valuable information for optimal therapy, an attempt is made to describe methadone kinetics in brain and other tissues simultaneously. The concentration-time profiles of methadone in various tissues after an i.v. bolus dose of 2 mg/kg are reasonably described by the model. The role of the different organs in the disposition of methadone is further explored by simulations. It is found that methadone is initially sequestered in lung tissues immediately after intravenous administration. Therefore, both venous and arterial blood pools are included in the model. Rapid uptake then takes place into vascular-rich organs, including kidneys, liver, and muscle, followed by redistribution into less penetrable organs, such as brain, fetal, and intestinal tissues. Data indicate that diffusional resistance governs the transfer of drug into brain, fetal, and intestinal tissues. Simulations suggest that muscular tissues play an important role in the rat and in man, becoming the major methadone reservoir. The tissue-to-blood partition coefficients derived from equilibrium conditions in this study are generally higher than those reported hitherto. The model is scaled up to a human to investigate whether it can be used to predict the concentration of methadone in different organs after a certain dose. Volume of distribution (Vdss) and biological half-life are consistent with earlier findings in man. The study is done by means of the GC-MS method with selected ion-monitoring where deuterated methadone is used as an internal standard.

Animals↗
PubMed1999

The role of cytochrome b559 and tyrosineD in protection against photoinhibition during in vivo photoactivation of photosystem II

In vivo photoactivation of Photosystem II was studied in the FUD39 mutant strain of the green alga Chlamydomonas reinhardtii which lacks the 23 kDa protein subunit involved in water oxidation. Dark grown cells, devoid of oxygen evolution, were illuminated at 0.8 μE m-2s-1 light intensity which promotes optimal activation of oxygen evolution, or at 17 μE m-2s-1, where photoactivation compete with deleterious photodamage. The involvement of the two redox active cofactors tyrosineD and cytochrome b559 during the photoactivation process, was investigated by EPR spectroscopy. TyrosineD on the D2 reaction center protein functions as auxiliary electron donor to the primary donor P+680 during the first minutes of photoactivation at 0.8 μE m-2s-1 (compare with Rova et al., Biochemistry, 37 (1998) 11039-11045.). Here we show that also cytochrome b559 was rapidly oxidized during the first 10 min of photoactivation with a similar rate to tyrosineD. This implies that both cytochrome b559 and tyrosineD may function as auxiliary electron donors to P+680 and/or the oxidized tyrosine&z.ccirf;Z on the D1 protein, to avoid photoinhibition before successful photoactivation was accomplished. As the catalytic water-oxidation successively became activated, TyrosineD remained oxidized while cytochrome b559 became rereduced to the equilibrium level that was observed prior to photoactivation. At 17 μE m-2s-1 light intensity, where photoinhibition competes significantly with photoactivation, tyrosineD was very rapidly completely oxidized, after which the amount of oxidized tyrosineD decreased due to photoinhibition. In contrast, cytochrome b559 became reduced during the first 2 min of photoactivation at 17 μE m-2s-1. After this, it was reoxidized, returning to the equilibrium level within 10 min. Thus, during in vivo photoactivation in high-light cytochrome b559 serves two functions. Initially, it probably oxidizes the reduced primary acceptor pheophytin, thereby relieving the acceptor side of reductive pressure, and later on it serves as auxiliary electron donor, preventing donor-side photoinhibition.

Journal Article↗
PubMed1981

Steroid-protein interactions. Human corticosteroid binding globulin: some physicochemical properties and binding specificity.

Reducing agents (dithiothreitol and beta-mercaptoethanol) significantly decrease the affinity constants of the human corticosteroid-binding globulin (CBG)-cortisol complex in proportion to their concentration; the resulting Ka values are more consistent than those obtained in the absence of the reductants. The effect is reversible. The equilibrium association constants of the CBG complexes with cortisol and progesterone show a relatively broad pH maximum between pH 8 and 11. In this pH range, cortisol was found to be bound more strongly than progesterone; this relationship is reversed around pH 6. The van't Hoff plot of the temperature effect on Ka of the CBG-cortisol complex (4-41 degrees C) exhibits a nonlinear, possibly biphasic temperature dependency. The shape of the van't Hoff plot was similar in the presence of mercaptoethanol. The association of cortisol and progesterone to human CBG at 4 and 37 degrees C is enthalpy driven, compensating for the unfavorable change in entropy. Studies with 47 steroids served to elucidate the influence on binding affinity of polar and nonpolar groups and other structural alterations. The contribution of specific structural changes in the steroid molecule to the free energy of binding can be calculated from the results. Important structures for optimal binding are the 20-oxo group, a 10 beta-methyl group, and a double bond at the 4 position. A complementary image of the binding site with respect to the nature of binding at various locations is proposed.

Animals↗
PubMed1993

Quantification of myocardial muscarinic receptors with PET in humans.

The potential for noninvasive quantification of myocardial muscarinic receptors using PET data, a mathematical model, multi-injection protocols and 11C-labeled methylquinuclidinyl benzilate (MQNB) as a radioligand was previously demonstrated in dogs. The present study examines the possibility of optimizing the experimental protocol to make this approach suitable for human studies. For six normal subjects, the protocol included three injections: a tracer injection, followed 30 min later by an injection of an excess of unlabeled MQNB (displacement) and then 30 min later by a simultaneous injection of unlabeled and labeled MQNB (coinjection). The model input function was estimated from the PET data corresponding to the left ventricular cavity. This protocol enables a separate evaluation of all parameters of a ligand-receptor model which includes three compartments and seven parameters. The complexity of this three-injection protocol, however, appears to be inconvenient for clinical use. A simplified two-injection protocol (tracer injection and coinjection) was evaluated in five other normal subjects and the results were compared to those obtained with the three-injection protocol. In regions of interest over the left ventricle, the mean value of the receptor concentration B'max and the equilibrium dissociation constant Kd were 26 +/- 7 pmole/ml tissue and 2.0 +/- 0.5 pmole/ml tissue, respectively. The possible existence of nonspecific binding was studied in two subjects using a double-displacement protocol. The corresponding rate constant was found to be very low (0.03 min-1).

Adult↗
PubMed2000

Steady-state kinetic mechanism of recombinant avocado ACC oxidase: initial velocity and inhibitor studies.

The gaseous plant hormone ethylene modulates a wide range of biological processes, including fruit ripening. It is synthesized by the ascorbate-dependent oxidation of 1-aminocyclopropyl-1-carboxylate (ACC), a reaction catalyzed by ACC oxidase. Recombinant avocado (Persea americana) ACC oxidase was expressed in Escherichia coli and purified in milligram quantities, resulting in high levels of ACC oxidase protein and enzyme activity. An optimized assay for the purified enzyme was developed that takes into account the inherent complexities of the assay system. Fe(II) and ascorbic acid form a binary complex that is not the true substrate for the reaction and enhances the degree of ascorbic acid substrate inhibition. The K(d) value for Fe(II) (40 nM, free species) and the K(m)'s for ascorbic acid (2.1 mM), ACC (62 microM), and O(2) (4 microM) were determined. Fe(II) and ACC exhibit substrate inhibition, and a second metal binding site is suggested. Initial velocity measurements and inhibitor studies were used to resolve the kinetic mechanism through the final substrate binding step. Fe(II) binding is followed by either ascorbate or ACC binding, with ascorbate being preferred. This is followed by the ordered addition of molecular oxygen and the last substrate, leading to the formation of the catalytically competent complex. Both Fe(II) and O(2) are in thermodynamic equilibrium with their enzyme forms. The binding of a second molecule of ascorbic acid or ACC leads to significant substrate inhibition. ACC and ascorbate analogues were used to confirm the kinetic mechanism and to identify important determinants of substrate binding.

Amino Acid Oxidoreductases↗
PubMed2003

Selection of genetic markers for association analyses, using linkage disequilibrium and haplotypes.

The genotyping of closely spaced single-nucleotide polymorphism (SNP) markers frequently yields highly correlated data, owing to extensive linkage disequilibrium (LD) between markers. The extent of LD varies widely across the genome and drives the number of frequent haplotypes observed in small regions. Several studies have illustrated the possibility that LD or haplotype data could be used to select a subset of SNPs that optimize the information retained in a genomic region while reducing the genotyping effort and simplifying the analysis. We propose a method based on the spectral decomposition of the matrices of pairwise LD between markers, and we select markers on the basis of their contributions to the total genetic variation. We also modify Clayton's "haplotype tagging SNP" selection method, which utilizes haplotype information. For both methods, we propose sliding window-based algorithms that allow the methods to be applied to large chromosomal regions. Our procedures require genotype information about a small number of individuals for an initial set of SNPs and selection of an optimum subset of SNPs that could be efficiently genotyped on larger numbers of samples while retaining most of the genetic variation in samples. We identify suitable parameter combinations for the procedures, and we show that a sample size of 50-100 individuals achieves consistent results in studies of simulated data sets in linkage equilibrium and LD. When applied to experimental data sets, both procedures were similarly effective at reducing the genotyping requirement while maintaining the genetic information content throughout the regions. We also show that haplotype-association results that Hosking et al. obtained near CYP2D6 were almost identical before and after marker selection.

Alleles↗
PubMed2005

The high-affinity maltose/trehalose ABC transporter in the extremely thermophilic bacterium Thermus thermophilus HB27 also recognizes sucrose and palatinose.

We have studied the transport of trehalose and maltose in the thernophilic bacterium Thermus thermophilus HB27, which grows optimally in the range of 70 to 75 degrees C. The K(m) values at 70 degrees C were 109 nM for trehalose and 114 nM for maltose; also, a high K(m) (424 nM) was found for the uptake of sucrose. Competition studies showed that a single transporter recognizes trehalose, maltose, and sucrose, while d-galactose, d-fucose, l-rhamnose, l-arabinose, and d-mannose were not competitive inhibitors. In the recently published genome of T. thermophilus HB27, two gene clusters designated malEFG1 (TTC1627 to -1629) and malEFG2 (TTC1288 to -1286) and two monocistronic genes designated malK1 (TTC0211) and malK2 (TTC0611) are annotated as trehalose/maltose and maltose/maltodextrin transport systems, respectively. To find out whether any of these systems is responsible for the transport of trehalose, the malE1 and malE2 genes, lacking the sequence encoding the signal peptides, were expressed in Escherichia coli. The binding activity of pure recombinant proteins was analyzed by equilibrium dialysis. MalE1 was able to bind maltose, trehalose, and sucrose but not glucose or maltotetraose (K(d) values of 103, 67, and 401 nM, respectively). Mutants with disruptions in either malF1 or malK1 were unable to grow on maltose, trehalose, sucrose, or palatinose, whereas mutants with disruption in malK2 or malF2 showed no growth defect on any of these sugars. Therefore, malEFG1 encodes the binding protein and the two transmembrane subunits of the trehalose/maltose/sucrose/palatinose ABC transporter, and malK1 encodes the ATP-binding subunit of this transporter. Despite the presence of an efficient transporter for trehalose, this compound was not used by HB27 for osmoprotection. MalE1 and MalE2 exhibited extremely high thermal stability: melting temperatures of 90 degrees C for MalE1 and 105 degrees C for MalE2 in the presence of 2.3 M guanidinium chloride. The latter protein did not bind any of the sugars examined and is not implicated in a maltose/maltodextrin transport system. This work demonstrates that malEFG1 and malK1 constitute the high-affinity ABC transport system of T. thermophilus HB27 for trehalose, maltose, sucrose, and palatinose.

ATP-Binding Cassette Transporters↗
PubMed1990

Calbindin-D28K, a 1 alpha,25-dihydroxyvitamin D3-induced calcium-binding protein, binds five or six Ca2+ ions with high affinity.

Calbindin-D28K is a 1 alpha,25-dihydroxyvitamin D3-dependent protein that belongs to the superfamily of high affinity calcium-binding proteins which includes parvalbumin, calmodulin, and troponin C. All of these proteins bind Ca2+ ligands by an alpha-helix-loop-alpha-helix domain that is termed an EF-hand. Calbindin-D28K has been reported previously to have four high affinity Ca2(+)-binding sites (KD less than 10(-7)) as quantitated by equilibrium dialysis. With the determination of the amino acid sequence, it was clear that there are in fact six apparent EF-hand domains, although the Ca2(+)-binding functionality of the two additional domains was unclear. It was of interest to quantitate the Ca2(+)-binding ability of chick intestinal calbindin-D28K utilizing several different Ca2+ titration methods that cover a range of macroscopic binding constants for weak or strong Ca2+ sites. Titrations with the Ca2+ chelator dibromo-1,2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (5,5'-Br2BAPTA), a Ca2+ selective electrode, and as followed by 1H NMR, which measure KD values of 10(-6)-10(-8) M, 10(-4)-10(-7) and 10(-3)-10(-5) M, respectively, gave no evidence for the presence of weak Ca2(+)-binding sites. However, Ca2+ titration of the fluorescent Ca2+ chelator Quin 2 in the presence of calbindin-D28K yielded a least squares fit optimal for 5.7 +/- 0.8 Ca2(+)-binding sites with macroscopic dissociation constants around 10(-8) M. The binding of Ca2+ by calbindin was found to be cooperative with at least two of the sites exhibiting positive cooperativity.

Aminoquinolines↗
PubMed2006

Versatile interactions of the antimicrobial peptide novispirin with detergents and lipids.

Novispirin G-10 is an 18-residue designed cationic peptide derived from the N-terminal part of an antimicrobial peptide from sheep. This derivative is more specific for bacteria than the parent peptide. We have analyzed Novispirin's interactions with various amphipathic molecules and find that a remarkably wide variety of conditions induce alpha-helical structure. Optimal structure induction by lipids occurs when the vesicles contain 40-80% anionic lipid, while pure anionic lipid vesicles induce aggregation. SDS also forms aggregates with Novispirin at submicellar concentrations but induces alpha-helical structures above the cmc. Both types of aggregates contain significant amounts of beta-sheet structure, highlighting the peptide's structural versatility. The cationic detergent LTAC has a relatively strong affinity for the cationic peptide despite the peptide's net positive charge of +7 at physiological pH and total lack of negatively charged side chains. Zwitterionic and nonionic detergents induce alpha-helical structures at several hundred millimolar detergent. We have solved the peptide structure in SDS and LTAB by NMR and find subtle differences compared to the structure in TFE, which we ascribe to the interaction with an amphiphilic environment. Novispirin is largely buried in the SDS-micelle, whereas it does not enter the LTAC-micelle but merely forms a dynamic equilibrium between surface-bound and nonbound Novispirin. Thus, electrostatic repulsion can be overruled by relatively high-detergent concentrations or by deprotonating a single critical side chain, despite the fact that Novispirin's ability to bind to amphiphiles and form alpha-helical structure is sensitive to the electrostatics of the amphiphilic environment. This emphasizes the versatility of cationic antimicrobial peptides' interactions with amphiphiles.

Antimicrobial Cationic Peptides↗
PubMed1986

A novel dipeptidyl peptidase II from the porcine ovary. Purification and characterization of a lysosomal serine protease showing enhanced specificity for prolyl bonds.

A variant form of dipeptidyl peptidase II (DPP II), initially reported under the name of "DPP V" (Eisenhauer, D. A., and McDonald, J. K. (1982) Fed. Proc. 41, 507), was detected in aqueous extracts of porcine ovaries on the basis of a markedly enhanced action on prolyl bonds. This porcine form of DPP II, which was most sensitively assayed on Phe-Pro-2-naphthylamide (Phe-Pro-NNap), was purified 1400-fold to a specific activity of 28 mumol/min/mg of protein (pH 6.0, 37 degrees C) from an aqueous extract of hog ovaries taken during pregnancy, when ovarian levels of DPP II are some 3- to 8-fold higher. Purification involved ammonium sulfate fractionation, molecular exclusion chromatography, chromatofocusing, affinity chromatography on concanavalin A-Sepharose 4B, and high performance ion exchange chromatography. The purified enzyme, which was apparently electrophoretically homogeneous at pH 3, 7, and 8.8 (pI = 5.0), was shown to be an Mr = 110,000 glycoprotein containing about 2% carbohydrate (primarily mannose) and less than 1% sialic acid, and to consist of two noncovalently linked Mr = 54,000 subunits. A serine catalytic mechanism was supported by inhibitor studies and by common mobilities seen during electrophoresis for (histochemically detected) Phe-Pro-arylamidase activity and the [14C]diisopropyl fluorophosphate-labeled enzyme. In the standard fluorometric assay at 37 degrees C, Phe-Pro-NNap (0.2 mM) was hydrolyzed optimally at pH 6.0 (Km = 45 microM; kcat = 54 s-1). In comparison to the rate seen on Lys-Ala-NNap, the usual DI P II assay substrate, rates seen on Phe-Pro-, Lys-Pro-, and Arg-Pro-NNap were about 8-, 4-, and 2-fold higher, respectively. No action occurred on N-blocked derivatives or on Pro-NNap. Action on oligopeptides appeared to be limited to tripeptides, in particular those containing proline or alanine in the P1 position, i.e. Phe-Pro-Ala (100%), Lys-Ala-Ala (35%), Ala-Ala-Ala (33%), and Gly-Pro-Ala (26%). Only a trace of activity was seen on Phe-Pro-Ala-Ala, and none on Z-Phe-Pro-Ala or Ala-Ala-Ala-OMe. Evidence for the lysosomal localization of DPP II included sedimentability and latency and its distribution, coincident with acid phosphatase, in two distinct isopycnic regions following equilibrium density centrifugation. Lysosomal heterogeneity was suggested by this dual isopycnic banding.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗