Pressure-Induced Frequency Lineshifts in the nu2 Band of Ammonia: An Experimental Test of the Rydberg-Ritz Principle

The hemolytic lectin, CEL-III, is a Ca2+-dependent, galactose/N-acetylgalactosamine-specific lectin purified from the marine invertebrate, Cucumaria echinata (Holothuroidea). After binding to specific carbohydrates on the erythrocyte surface, CEL-III forms ion-permeable pores by oligomerizing in the membrane, which leads to colloid osmotic rupture of the cells. When incubated with liposomes composed of total lipids from the human erythrocyte membrane, CEL-III efficiently induced the leakage of carboxyfluorescein (CF) trapped in the vesicles, suggesting the presence of its receptor in the membrane lipids. The rate of CF-leakage increased with increasing temperature, although the hemolytic activity of CEL-III had been found to be much higher at lower temperatures (around 10 degrees C). Identification of the receptor for CEL-III was performed by examining the ability of individual lipids from human erythrocytes to induce CF-leakage from DOPC-liposomes. As a result, the most effective receptor was found to be lactosyl ceramide (LacCer), while globoside (Gb4Cer) also showed slight induction of CF-leakage. On the other hand, a binding assay involving CEL-III-horseradish peroxidase conjugate indicated that CEL-III exhibits similar affinity for LacCer and Gb4Cer, suggesting that the structure or length of the carbohydrate portion of sphingoglycolipids is also relevant as to their ability to induce CF-leakage in addition to their affinity. Electron micrographs of CEL-III-treated liposomes revealed that CEL-III induced considerable morphological changes in the vesicles, while a clearly distinguishable oligomeric structure of the protein was not observed.     

Use of retinoic acid as a fluorescent probe to study conformational change of the albumin molecule

The binding of retinoic acid to serum albumin induces quenching of the protein fluorescence when it is excited at 280 nm, on the other hand the bound ligand acquires intrinsic fluorescence. Albumin has two kinds of binding sites for retinoic acid with an affinity constant of 10(5) M-1 and 10(4) M-1 respectively. The binding is entropically driven and produces a conformational change at the environment of the albumin tryptophan residues. This change was described by an equilibrium constant assuming two conformational states of the albumin tryptophan residues. Retinoic acid binds to the albumin fatty acid binding sites, producing a perturbation in the warfarin and benzodiazepine binding sites of this protein.     

Introduction of a tryptophan reporter group into the ATP binding motif of the Escherichia coli UvrB protein for the study of nucleotide binding and conformational dynamics

The DNA-dependent ATPase activity of UvrB is required to support preincision steps in nucleotide excision repair in Escherichia coli. This activity is, however, cryptic. Elicited in nucleotide excision repair by association with the UvrA protein, it may also be unmasked by a specific proteolysis eliminating the C-terminal domain of UvrB (generating UvrB*). We introduced fluorescent reporter groups (tryptophan replacing Phe47 or Asn51) into the ATP binding motif of UvrB, without significant alteration of behavior, to study both nucleotide binding and those conformational changes expected to be essential to function. The inserted tryptophans occupy moderately hydrophobic, although potentially heterogeneous, environments as evidenced by fluorescence emission and time-resolved decay characteristics, yet are accessible to the diffusible quencher acrylamide. Activation, via specific proteolysis, is accompanied by conformational change at the ATP binding site, with multiple changes in emission spectra and a greater shielding of the tryptophans from diffusible quencher. Titration of tryptophan fluorescence with ATP has revealed that, although catalytically incompetent, UvrB can bind ATP and bind with an affinity equal to that of the active UvrB* form (Kd of approximately 1 mM). The ATP binding site of UvrB is therefore functional and accessible, suggesting that conformational change either brings amino acid residues into proper alignment for catalysis and/or enables response to effector DNA.     

Conformational changes of the insulin receptor upon insulin binding and activation as monitored by fluorescence spectroscopy

We have characterized the changes in intrinsic fluorescence that the insulin receptor undergoes upon ligand binding and autophosphorylation. The binding of insulin to its receptor results in an increase in the receptor's fluorescence intensity, emission energy and anisotropy. We monitored the time course of the anisotropy change, and these data, coupled with studies monitoring the energy transfer from insulin receptor tryptophan donors to a fluorescent-labeled insulin, allowed us to conclude that the change in anisotropy is due to a conformational change in the receptor induced by hormone binding. Since insulin association is very fast, the time course also allowed us to estimate the slower rate of formation of this conformationally-altered state. The time course of receptor autophosphorylation was measured under similar conditions and was found to be similar to the ligand-induced anisotropy time course. The simultaneous use of two fluorescent-labeled insulin analogs also allowed us to assess the maximum distance between the two hormones bound to the receptor. Addition of ATP produces a large, seemingly instantaneous increase in anisotropy. Our observation that ATP binds to the insulin receptor in the presence and absence of insulin supports the idea that the conformational change produced by insulin binding increases the rate of autophosphorylation rather than increases ATP affinity. A suggested model for these changes is presented.     

Deconvolution of the fluorescence emission spectrum of human antithrombin and identification of the tryptophan residues that are responsive to heparin binding

Heparin causes an allosterically transmitted conformational change in the reactive center loop of antithrombin and a 40% enhancement of tryptophan fluorescence. We have expressed four human antithrombins containing single Trp --> Phe mutations and determined that the fluorescence of antithrombin is a linear combination of the four tryptophans. The contributions to the spectrum of native antithrombin at 340 nm were 8% for Trp-49, 10% for Trp-189, 19% for Trp-225, and 63% for Trp-307. Trp-225 and Trp-307 accounted for the majority of the heparin-induced fluorescence enhancement, contributing 37 and 36%, respectively. Trp-49 and Trp-225 underwent spectral shifts of 15 nm to blue and 5 nm to red, respectively, in the antithrombin-heparin complex. The blue shift for Trp-49 is consistent with partial burial by contact with heparin, whereas the red shift for Trp-225 and large enhancement probably result from increased solvent access upon heparin-induced displacement of the contact residue Ser-380. The enhancement for Trp-307 may result from the heparin-induced movement of helix H seen in the crystal structure. The time-resolved fluorescence properties of individual tryptophans of wild-type antithrombin were also determined using the four variants and showed that Trp-225 and Trp-307 experienced the largest change in lifetime upon heparin binding, providing support for the steady-state fluorescence deconvolution.     

Simple test to evaluate the risk of urinary calcium stone formation

The pH dependence of the association of apo trp repressor with the series of ligands, tryptophan, tryptamine, indole propionic acid (IPA), and trans-beta-indole acrylic acid (IAA), has been studied using fluorescence titrations and isothermal titration microcalorimetry (ITC). The purpose of such a comparison of ligands and the pH dependency studies is to reveal the role played by the side-chain functional groups in the energetics of the binding of the ligands to the protein. We find that, whereas the binding of tryptamine and IPA have essentially no pH dependence between pH 6 and 10, the binding of tryptophan and IAA depends on pH. For IAA, the affinity drops between pH 6 and 10, consistent with a shift in pKa of some group on the protein from a value of pKa 7.4 to 7.9 upon binding of this ligand. The affinity of IAA also drops below pH 5, but shows saturable binding at pH 2-3, where the protein has previously been found to exist as a partially folded monomeric state. For tryptophan, the pH dependence data indicate that the equilibrium is complicated. We present a model to describe the data in which the alpha-ammonium group of tryptophan has its pKa shifted upward upon binding (i.e. preferential binding of the protonated form of this functional group) and in which the pKa of an unknown group on the protein also has its pKa increased.     

Effect of high pressure on the association of melittin to membranes

To determine the underlying basis for the sensitivity of peripheral peptides to lipid packing, we monitored the change in association of melittin to different membranes under hydrostatic pressure by fluorescence polarization and by fluorescence intensity in the presence of aqueous quenchers. Association to lysophosphatidylcholine micelles or to membranes composed of dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, palmitoyloleoylphosphatidylcholine, or dioleoylphosphatidylcholine was found to be stable from 1 to 2000 atm. Similar results were obtained using multilamellar vesicles, small unilamellar vesicles, or large unilamellar vesicles. Thus, the increase in lipid chain packing induced by pressure does not alter the association of bound complexes. This result indicates similar compressibilities of the peptide and the head group binding region. Increasing the ionic strength to increase the charge of the free peptide also resulted in a pressure-insensitive complex showing that the hydration does not change upon binding. This conclusion is substantiated by a lack of van't Hoff delta H to dioleoylphosphatidylcholine large unilamellar vesicles. To gain a more molecular picture of these associations, the rotational properties of the tryptophan side chain of bound melittin as a function of lipid packing was also studied. These data indicate subtle differences in peptide orientation in different lipids.     

Tryptophan substitutions surrounding the nucleotide in catalytic sites of F1-ATPase

Novel tryptophan substitutions, surrounding the nucleotide bound in catalytic sites, were introduced into Escherichia coli F1-ATPase. The mutant enzymes were purified and studied by fluorescence spectroscopy. One cluster of Trp substitutions, consisting of beta-Trp-404, beta-Trp-410, beta-Asp-158 (lining the adenine-binding pocket), and beta-Trp-153 (close to the alpha/beta-phosphates), showed the same fluorescence responses to MgADP, MgAMPPNP, and MgATP and the same nucleotide binding pattern with MgADP and MgAMPPNP, with one site of higher and two sites of lower affinity. Therefore, in absence of catalytic turnover (and of gamma-subunit rotation), sites 2 and 3 appeared similar in affinity, and the region of the catalytic site sensed by these Trp substitutions did not change conformation with different nucleotides. In contrast, alpha-Trp-291 and beta-Trp-297, both close to the gamma-phosphate, showed very different fluorescence responses to MgADP versus MgAMPPNP, and in these cases the response was due exclusively or predominantly to nucleotide binding at the first, high-affinity catalytic site, thus allowing specific detection of this site. Titration with MgATP showed that the high-affinity site was present under conditions of steady-state, Vmax MgATP hydrolysis.     

Fluorescence quenching and time-resolved fluorescence studies on Momordica charantia (bitter gourd) seed lectin

Chemical modification studies implicated tryptophan (Trp) residues in the sugar binding activity of Momordica charantia lectin (MCL) [Mazumdar, T., Gaur, N. & Surolia, A. (1981) Eur. J. Biochem. 113, 463-470]. In the present study, the accessibility and environment of Trp residues in MCL were investigated by intrinsic fluorescence quenching and time-resolved fluorescence. The emission lamda max of native MCL in the absence as well as in the presence of 0.1 M lactose was around 335 nm, which shifted to 365 nm in the presence of 8 M urea, suggesting that the Trp residues which are predominantly buried in the hydrophobic core of the native lectin get exposed to the aqueous environment upon denaturation. At a quencher concentration of 0.5 M, the extent of quenching observed for the native MCL with acrylamide, I- and Cs+ was 46%, 17% and 12%, respectively. In the presence of 0.1 M lactose this quenching was smaller, suggesting that the sugar ligand provides a partial protection to the Trp residues. In time-resolved fluorescence measurements, the decay curves could be fitted well to a biexponential function with the estimated life times 0.92 ns and 4.64 ns for the native protein and 1.15 ns and 5.1 ns in the presence of 0.1 M lactose. All these results are consistent with the involvement of Trp residues in the sugar-binding activity of MCL.     

Optical spectroscopy of nicotinoprotein alcohol dehydrogenase from Amycolatopsis methanolica: a comparison with horse liver alcohol dehydrogenase and UDP-galactose epimerase

The NADH absorbance spectrum of nicotinoprotein (NADH-containing) alcohol dehydrogenase from Amycolatopsis methanolica has a maximum at 326 nm. Reduced enzyme-bound pyridine dinucleotide could be reversibly oxidized by acetaldehyde. The fluorescence excitation spectrum for NADH bound to the enzyme has a maximum at 325 nm. Upon excitation at 290 nm, energy transfer from tryptophan to enzyme-bound NADH was negligible. The fluorescence emission spectrum (excitation at 325 nm) for NADH bound to the enzyme has a maximum at 422 nm. The fluorescence intensity is enhanced by a factor of 3 upon binding of isobutyramide (Kd = 59 microM). Isobutyramide acts as competitive inhibitor (Ki = 46 microM) with respect to the electron acceptor NDMA (N,N-dimethyl-p-nitrosoaniline), which binds to the enzyme containing the reduced cofactor. The nonreactive substrate analogue trifluoroethanol acts as a competitive inhibitor with respect to the substrate ethanol (Ki = 1.6 microM), which binds to the enzyme containing the oxidized cofactor. Far-UV circular dichroism spectra of the enzyme containing NADH and the enzyme containing NAD+ were identical, indicating that no major conformational changes occur upon oxidation or reduction of the cofactor. Near-UV circular dichroism spectra of NADH bound to the enzyme have a minimum at 323 nm (Deltaepsilon = -8.6 M-1 cm-1). The fluorescence anisotropy decay of enzyme-bound NADH showed no rotational freedom of the NADH cofactor. This implies a rigid environment as well as lack of motion of the fluorophore. The average fluorescence lifetime of NADH bound to the enzyme is 0.29 ns at 20 degreesC and could be resolved into at least three components (in the range 0.13-0.96 ns). Upon binding of isobutyramide to the enzyme-containing NADH, the average excited-state lifetime increased to 1.02 ns and could be resolved into two components (0.37 and 1.11 ns). The optical spectra of NADH bound to nicotinoprotein alcohol dehydrogenase have blue-shifted maxima compared to other NADH-dehydrogenase complexes, but comparable to that observed for NADH bound to horse liver alcohol dehydrogenase. The fluorescence lifetime of NADH bound to the nicotinoprotein is very short compared to enzyme-bound NADH complexes, also compared to NADH bound to horse liver alcohol dehydrogenase. The cofactor-protein interaction in the nicotinoprotein alcohol dehydrogenase active site is more rigid and apolar than that in horse liver alcohol dehydrogenase. The optical properties of NADH bound to nicotinoprotein alcohol dehydrogenase differ considerably from NADH (tightly) bound to UDP-galactose epimerase from Escherichia coli. This indicates that although both enzymes have NAD(H) as nonexchangeable cofactor, the NADH binding sites are quite different.     

Quantitation of methionyl peptides in nanomole quantities by a fluorometric method

A quantitative and highly specific method to determine low concentrations of methionyl peptides, which do not contain tryptophan or cysteine residues, has been developed. The method is based on the stoichiometry and selectivity of N-chlorosuccinimide (NCS) towards methionine and N-acetyltryptophan. N-Chlorosuccinimide reacts with N-acetyltryptophan in a 1:1 ratio to produce the N-acetyl-2-oxindolealanine--a derivative essentially devoid of fluorescence. The decrease in fluorescence intensity is approximately linear with respect to the NCS concentration. Preincubation of NCS with methionine or methionyl peptide consumes a stoichiometric amount of the reagent and the unreacted NCS is quantitated by the decrease in fluorescence intensity resulting upon incubation of the mixture with 1 eq of N-acetyltryptophan. Less than 1 nmol of methionyl peptide can be accurately quantitated by this method.     

Fluorescence-energy transfer in human estradiol 17 beta-dehydrogenase-NADPH complex and studies on the coenzyme binding,

Fluorescence spectroscopy was used to examine the interaction between human estradiol 17 beta-dehydrogenase (estrogenic 17 beta-hydroxysteroid dehydrogenase, 17 beta-HSD) and the cofactor NADPH. After the binding of NADPH to the enzyme, there was an emission enhancement at 436 nm following an excitation at 295 nm, as compared to the cofactor alone. This phenomenon was attributed to a radiationless transfer of excitation energy from 17 beta-HSD to the enzyme-bound cofactor. The distance of 2.69 nm, between the bound NADPH and the sole tryptophan residue (Trp46) within one subunit, has been determined using fluorescence energy transfer. This result coincides very well with the same distance, recently calculated from the crystallographic coordinates obtained by Ghosh et al. [Ghosh, D., Pletnev, V. Z., Zhu, D.-W., Wawrzak, Z., Duax, W. L., Pangborn, W., Labrie, F. & Lin, S.-X. (1995) Structure 3, 503-513]. Compared to free NADPH, the fluorescence emission of enzyme-bound NADPH was increased in intensity and its maximum blue-shifted from 457 nm to 436 nm. Binding of NADPH to 17 beta-HSD was studied by fluorescence titration. The enzyme binds two molecules of NADPH with a Kd = 0.73 +/- 0.2 microM. The dissociation constant was further confirmed by the method of coenzyme protection against cold inactivation of the enzyme. The binding was little altered in the presence of estradiol-17 beta. The environment of tryptophan residues on the surface of the enzyme is discussed.     

Micellar acid-base potentiometric titrations of weak acidic and/or insoluble drugs

A lectin-induced orientation change of a helical glycopeptide in lipid bilayer membranes was studied. Glycopeptides composed of hydrophobic nona-(G8) and pentapeptide (G4) with a fluorescent probe at the N-terminal and a lactose unit at the C-terminal were synthesized. The glycopeptides were incorporated into lipid bilayer membranes with the lactose unit exposed to the aqueous phase and the peptide chain buried in the membrane. G8 takes a partially helical structure in the membrane, while G4 an irregular structure. Upon binding of lectin to G8 held in the membrane of DPPC liposome, enhancement of fluorescence intensity of the N-terminal anthryl group, reduction of fluorescence quenching of the anthryl group with acrylamide, and increase of CF-leakage from the DPPC liposome were observed. G8', which lacks the O-anthryrlmethylserine residue from G8, formed a voltage-dependent ion channel in BLM experiments. The frequency of single current fluctuations induced by G8' incorporation increased with addition of lectin. These results indicate that the peptide segment of G8 prefers taking a more perpendicular orientation to the membrane upon association with lectin.     

Spectroscopic characterization of arrestin interactions with competitive ligands: study of heparin and phytic acid binding

A combination of intrinsic fluorescence and circular dichroic (CD) spectroscopy has been used to characterize the complexes formed between bovine retinal arrestin and heparin or phytic acid, two ligands that are known to mimic the structural changes in arrestin attending receptor binding. No changes in the CD spectra were observed upon ligand binding, nor did the degree of tryptophan fluorescence quenching change significantly in the complexes. These data argue against any large-scale changes in protein secondary or tertiary structure accompanying ligand binding. The change in tyrosine fluorescence intensity was used to determine the dissociation constants for the heparin and phytic acid complexes of arrestin. The only change observed was a saturable diminution of tyrosine fluorescence signal from the protein. For both ligands, the data suggest two distinct binding interactions with the protein--a high--affinity interaction with Kd between 200 and 300 nM, and a lower affinity interaction with Kd between 2 and 8 microM. Study of collisional quenching of tyrosine fluorescence in free arrestin and the ligand-replete complexes indicates that 10 of the 14 tyrosine residues of the protein are solvent-exposed in the free protein; this value drops to between 5 and 6 solvent-exposed residues in the high-affinity complexes of the two ligands. These data suggest that ligand binding leads to direct occlusion of between 4 and 5 tyrosine residues on the solvent-exposed surface of the protein, but not to any large-scale changes in protein structure. The large activation energy previously reported to be associated with arrestin-receptor interactions may therefore reflect localized movements of the N- and C-termini of arrestin, which are proposed to interact in the free protein through electrostatic interactions. Binding of the anionic ligands heparin, phytic acid, or phosphorylated rhodopsin may compete with the C-terminus of arrestin for these electrostatic interactions, thus allowing the C-terminus to swing out of the binding region.     

Erythrocruorin of Glossoscolex paulistus (Oligochaeta, Glossoscolecidae): modulation of oxygen affinity by specific antibodies

1) The Soret region absorption spectrum of erythrocruorin (ERC) obtained from Glossoscolex paulistus, shows that oxy-ERC has a maximum absorption peak at 416 nm while the deoxy-ERC from has a maximum at 427 nm. 2) In the presence of a specific antiserum (anti-ERC) and of anti-ERC immunoglobulin G raised in rabbits, there is a deviation to low wavelengths in the maximum absorption peak of deoxy-ERC while for the oxy form a red-shift is noticed. These shifts accompanied an increased affinity of the hemeprotein for oxygen, possibly because of changes in the overall macromolecular conformation. 3) A decrease in the oxygen affinity of erythrocruorin is observed when large amounts of non-specific serum are used. The same effect is observed in the presence of serum albumin, probably as a result of non-specific binding between the albumin and erythrocruorin. 4) The fluorimetric titration of erythrocruorin with anti-ERC Fab fragments results in a decrease in the intrinsic tryptophan fluorescence of the hemeprotein, a response indicative of a modification in the ERC's quaternary structure.     

Probing the regulatory domain interface of D-3-phosphoglycerate dehydrogenase with engineered tryptophan residues

D-3-Phosphoglycerate dehydrogenase from Escherichia coli is a homotetrameric enzyme which is allosterically regulated by the end product of its pathway, L-serine. The enzyme binds 4 L-serine molecules at two interfaces formed by the noncovalent association of the regulatory domains. The two domains that comprise each interface are related by an approximately 180 degrees axis of symmetry, and two serine molecules bind at each interface by forming a hydrogen bond network between the domains. A model has been proposed that suggests that serine functions by drawing adjacent domains together and that this in turn translates a conformational change to the active site. A tryptophan residue has been engineered into the helices flanking the regulatory interfaces that displays significant quenching in response to serine binding. Residues on the adjacent subunit appear to be primarily responsible for the tryptophan quenching and thus support the hypothesis that serine binding leads to an increase in the proximity between residues on neighboring subunits. Serine binding studies show that this quenching, as well as inhibition of enzymatic activity, are essentially complete when only two of the four serine binding sites are occupied. The requirement for only one serine per interface is consistent with the notion that the interface is formed by relatively rigid domains and that hydrogen bonding at only a single site is all that is required to substantially close the interface. The fluorescence quenching in response to L-serine binding generally correlates with enzymatic inhibition, but there appears to be a slight lag in inhibition relative to quenching at low serine concentrations. The observed fluorescence quenching of residues in the regulatory domains of D-3-phosphoglycerate dehydrogenase provide the first direct evidence for a conformational change in response to effector binding and provide a means to monitor the first step in the allosteric mechanism.     

Spectroscopic characterization of a high-affinity calmodulin-target peptide hybrid molecule

We describe the properties of a hybrid protein comprising the full length of the Xenopus laevis calmodulin sequence, followed by a pentapeptide linker (GGGGS), and residues 3-26 of M13, the calmodulin binding region of skeletal muscle myosin light chain kinase. The properties of the hybrid protein are compared with those of the complex formed between Drosophila calmodulin and a peptide corresponding to residues 1-18 of the M13 sequence. The addition of calcium to the hybrid protein produces pronounced changes in the near- and far-UV CD spectra, in the fluorescence emission spectrum of the single tryptophan residue at position 4 in the M13 sequence, and in the accessibility of this tryptophan residue to acrylamide quenching. These changes are consistent with the tryptophan residue being immobilized in a hydrophobic environment and with the hybrid protein adopting a more alpha-helical structure when calcium is bound. The increased alpha-helicity derives from changes in both the calmodulin and peptide regions of the hybrid protein. Changes in the circular dichroism and fluorescence properties of the hybrid protein as a function of the calcium to hybrid protein ratio are consistent with the fact that these changes parallel the cooperative binding of all four calcium ions. The hybrid protein shows greatly increased affinity (>250-fold) for calcium compared with calmodulin itself. Macroscopic calcium binding constants (K(1)-K(4)) were determined from calcium titrations performed in the presence of the calcium chelator Quin 2. Values for log(K(1)K(2)) and log(K(3)K(4)) were determined to be 15.4 +/- 0.2 and 15.59 +/- 0.22 (20 degrees C). The corresponding values for Drosophila calmodulin alone are 11.65 +/- 0.15 and 9.66 +/- 0.25. Consistent with this increased affinity for calcium stopped-flow kinetic studies suggest that the dissociation rate for the N-terminal calcium ions is reduced to at least 0.77 s(-1), compared with approximately 700 s(-1) for Drosophila calmodulin in the absence of peptide. This hybrid protein illustrates the principle whereby the binding of a peptide sequence covalently attached to calmodulin can enhance the average calcium affinity by more than 2 orders of magnitude. Conversely, the target sequence in the hybrid protein undergoes a calcium-induced conformational change to bind to the calmodulin in a conformation very similar to that of the corresponding dissociable target sequence binding to calmodulin, but with a greatly enhanced affinity due to its physical proximity to the binding site. This avoidance of the energetic penalty of dissociation may be a key contributory factor in determining the high affinity and specificity of the complex multiple interactions involved in recognition of biological targets by calmodulin.     

Oxidation of apolipoprotein(a) inhibits kringle-associated lysine binding: the loss of intrinsic protein fluorescence suggests a role for tryptophan residues in the lysine binding site

Lipoprotein(a) [Lp(a)] is a low-density lipoprotein complex consisting of apolipoprotein(a) [apo(a)] disulfide-linked to apolipoprotein B-100. Lp(a) has been implicated in atherogenesis and thrombosis through the lysine binding site (LBS) affinity of its kringle domains. We have examined the oxidative effect of 2,2'-azobis-(amidinopropane) HCl (AAPH), a mild hydrophilic free radical initiator, upon the ability of Lp(a) and recombinant apo(a), r-apo(a), to bind through their LBS domains. AAPH treatment caused a time-dependent decrease in the number of functional Lp(a) or r-apo(a) molecules capable of binding to fibrin or lysine-Sepharose and in the intrinsic protein fluorescence of both Lp(a) and r-apo(a). The presence of a lysine analogue during the reaction prevented the loss of lysine binding and provided a partial protection from the loss of tryptophan fluorescence. The partial protection of fluorescence by lysine analogues was observed in other kringle-containing proteins, but not in proteins lacking kringles. No significant aggregation, fragmentation, or change in conformation of Lp(a) or r-apo(a) was observed as assessed by native or SDS-PAGE, light scattering, retention of antigenicity, and protein fluorescence emission spectra. Our results suggest that AAPH destroys amino acids in the kringles of apo(a) that are essential for lysine binding, including one or more tryptophan residues. The present study, therefore, raises the possibility that the biological roles of Lp(a) may be mediated by its state of oxidation, especially in light of our previous study showing that the reductive properties of sulfhydryl-containing compounds increase the LBS affinity of Lp(a) for fibrin.     

An aquaporin-2 water channel mutant which causes autosomal dominant nephrogenic diabetes insipidus is retained in the Golgi complex

Calcyclin (CaCY) is a member of the S100 subfamily of helix-loop-helix (EF-hand) calcium-binding proteins. Human CaCY was overexpressed in Escherichia coli and purified with an overall yield of 40 mg/l culture. Ca2+ and Zn2+ binding properties of CaCY were examined with respect to the oxidation state of the single Cys residue at position 3. CaCY with the SH group either reduced, blocked or oxidized stays as a dimer as shown by analytical ultracentrifugation. Upon binding of Ca2+, CaCY exhibits 30% enhancement of the Tyr fluorescence, the apparent binding constant (Ka) being 2.8-5.8x10(4) M(-1). Oxidized CaCY binds Ca2+ approximately twice as weakly than its reduced form. The affinity for Ca2+ is increased in the presence of caldesmon, which could be a potential target molecule. Fully reduced CaCY binds Zn2+ with an affinity of at least 1.0x10(7) M(-1). As compared to Ca2+, Zn2+ binding results in a three times greater enhancement of the Tyr fluorescence. Saturation occurs at a Zn2+/CaCY ratio of 2:1. The reactivity of Cys3 is reduced by Zn2+ binding, although oxidized CaCY still binds Zn2+. On the basis of the effects of thiol-directed labels on the affinities for Ca2+ and Zn2+, the fluorescence changes accompanying the binding, and the CaCY reactivity with a hydrophobic probe, it was concluded that the two cations bind to CaCY at different sites: Ca2+ binds probably at the EF-hand type sites, whereas binding of at least one Zn2+ ion involves the Cys residue, and results in a different structural change.