Affinity For Calcium Ions Research Articles

Hydroxyapatite coating on stainless steel pre-coated with bovine serum albumin at ambient conditions

A biomimetic process for coating of nanosized hydroxyapatite on stainless steel, which capitalises the dual nature of the protein bovine serum albumin in both metal binding and a strong affinity for calcium ions, has been developed. The novelty of the process lies in pre-conditioning the metallic surface using the above protein prior to its mineralization with hydroxyapatite at ambient conditions. The microporous morphology of these coatings may provide favourable solubility and resorbability as desired by many orthopaedic and orthodontic applications.

A Novel Isoform of the Secretory Pathway Ca2+,Mn2+-ATPase, hSPCA2, Has Unusual Properties and Is Expressed in the Brain

Unlike lower eukaryotes, mammalian genomes have a second gene, ATP2C2, encoding a putative member of the family of secretory pathway Ca2+,Mn(2+)-ATPases, SPCA2. Human SPCA2 shares 64% amino acid identity with the protein defective in Hailey Hailey disease, hSPCA1. We show that human SPCA2 (hSPCA2) has a more limited tissue distribution than hSPCA1, with prominent protein expression in brain and testis. In primary neuronal cells, endogenous SPCA2 has a highly punctate distribution that overlaps with vesicles derived from the trans-Golgi network and is thus different from the compact perinuclear distribution of hSPCA1 seen in keratinocytes and nonpolarized cells. Heterologous expression in a yeast strain lacking endogenous Ca2+ pumps reveals further functional differences from hSPCA1. Although the Mn(2+)-specific phenotype of hSPCA2 is similar to that of hSPCA1, Ca2+ ions are transported with much poorer affinity, resulting in only weak complementation of Ca(2+)-specific yeast phenotypes. These observations suggest that SPCA2 may have a more specialized role in mammalian cells, possibly in cellular detoxification of Mn2+ ions, similar to that in yeast. We point to the close links between manganese neurotoxicity and Parkinsonism that would predict an important physiological role for SPCA2 in the brain.

Conversion of Human α-lactalbumin to an Apo-like State in the Complexes with Basic Poly-Amino Acids: Toward Understanding of the Molecular Mechanism of Antitumor Action of HAMLET

It was recently shown that alpha-lactalbumin associated with oleic acid (HAMLET) interacts with core histones thereby triggering apoptosis of tumor cells (J. Biol. Chem. 2003, 278, 42131). In previous work, we revealed that monomeric alpha-lactalbumin in the absence of fatty acids can also interact with histones and, moreover, with basic poly-amino acids (poly-Lys and poly-Arg) that represent simple models of histone proteins (Biochemistry 2004, 43, 5575). Association of alpha-lactalbumin with histone or poly-Lys(Arg) essentially changes its properties. In the present work, the character of the changes in structural properties and conformational stability of alpha-lactalbumin in the complex with poly-Lys(Arg) has been studied in detail by steady-state fluorescence, circular dichroism, and differential scanning calorimetry. Complex formation strongly depends on ionic strength, confirming its electrostatic nature. Experiments with the poly-amino acids of various molecular masses demonstrated a direct proportionality between the number of alpha-lactalbumin molecules bound per poly-Lys(Arg) and the surface area of the poly-amino acid random coil. The binding of the poly-amino acids to Ca2+-saturated human alpha-lactalbumin decreases its thermal stability down to the level of its free apo-form and decreases Ca2+-affinity by 4 orders of magnitude. The conformational state of alpha-lactalbumin in a complex with poly-Lys(Arg), named alpha-LActalbumin Modified by Poly-Amino acid (LAMPA), differs from all other alpha-lactalbumin states characterized to date, representing an apo-like (molten globule-like) state with substantially decreased affinity for calcium ion. The requirement for efficient conversion of alpha-lactalbumin to the LAMPA state is a poly-Lys(Arg) chain consisting of several tens of amino acid residues.

A novel calcium-binding peptide from the cuticle of the crayfish, Procambarus clarkii

A novel peptide named calcification-associated peptide (CAP)-2 was isolated from the exoskeleton of the crayfish, Procambarus clarkii. CAP-2 consists of 65 amino acid residues and has a 44% sequence identity with CAP-1 characterized previously. It has a chitin-binding domain observed in many arthropod cuticle proteins. CAP-2 showed inhibitory activity on calcium carbonate precipitation and chitin-binding ability. A CAP-2 cDNA was cloned using RT-PCR and RACE and the open reading frame encoded a precursor peptide consisting of a signal peptide and CAP-2. RT-PCR revealed that CAP-2 mRNA was exclusively expressed in the epidermal tissue during the postmolt stage, the site and stage being associated with calcification. Calcium-binding assay using recombinant CAP-2 revealed that this peptide had affinity for calcium ions with a K d value of about 1 mM. All these results suggest that CAP-2 serves as a nucleator or a regulator in the calcification of the exoskeleton.

Relationship Between Fragmentation of Myofibrils and Liberation of Phospholipids from Z‐Disks Induced by Calcium Ions at 0.1 mM: Mechanism of Tenderization of Pork and Beef during Postmortem Aging

ABSTRACT We studied the myofibril fragmentation using porcine and bovine myofibrils to determine the mechanism by which myofibrils are fragmented during postmortem aging of meat. Both myofibril fragmentation and liberation of phospholipids from Z‐disks were maximally induced by calcium ions at 0.1 mM. These phenomena showed identical dependencies on pH and temperature in the presence of calcium ions at 0.1 mM. All phospholipids in Z‐disks, except for lysophosphatidylethanolamine, had affinity for calcium ions. Therefore, we concluded that liberation of calcium‐phospholipid compounds from Z‐disks causes weakening of the Z‐disk structure, resulting in the myofibril fragmentation during postmortem aging of meat.

Effect of Reversible Cross-linker, N,N‘-Bis(acryloyl)cystamine, on Calcium Ion Adsorption by Imprinted Gels

Imprinted gels incorporating two different breakable cross-linkers, a PbMAA2 complex and a disulfide (S−S) bond, were prepared by radical polymerization. After the lead ions were removed by washing, these gels showed a high affinity for calcium ions. Breakage and subsequent reconnection of the S−S bonds in the absence of Ca2+ decreases the Ca2+ binding affinity of the gel. This indicates that random reconnection of the S−S bonds produces a frustration in the adsorption of Ca2+ by the carboxyl groups. However, if the S−S bonds were reconnected in the presence of Ca2+ and the Ca2+ was subsequently removed (the post-imprinting technique), the resulting gels showed a higher binding affinity for Ca2+. This indicates that the post-imprinting technique creates a more favorable conformation for Ca2+ binding in the polymer network. We interpret our data to mean that “memory” of target-binding sites was encoded effectively into the polymer network by the initial imprinting technique and then enhanced by the post-im...

A New Approach To Design Imprinted Polymer Gels without Using a Template

With the aim of developing polymeric gels sensitive to external stimuli and able to reversibly adsorb and release divalent ions, we prepared copolymer gels of N-isopropylacrylamide (NIPA) and N,N‘-cystaminebis(acrylamide) (BAC) weakly cross-linked with N,N‘-methylenebis(acrylamide) (BIS). After polymerization, the −S−S− bonds of BAC mers were broken and oxidized to form pairs of sulfonic groups. The juxtaposition of two anionic groups favors the interaction with a divalent cation. Calcium adsorption experiments showed that the gels prepared in this way memorized the position of the pairs of sulfonic groups after swelling and reshrinking. This “imprinting” effect was tested for several concentrations of adsorbing groups and permanent cross-linkers. The control gels prepared with 2-acrylamido-2-methylpropanesulfonic acid (AMPS), where sulfonic groups were randomly distributed, showed difficulty in forming pairs. Their affinity for calcium ions decayed exponentially as a function of cross-linker concentration. In contrast, the affinity of the imprinted gels was much greater than that of random gels and did not decrease with BIS, showing that memorization has been achieved.

Calcium binding to an elastic portion of connectin/titin filaments.

Alpha-connectin/titin-1 exists as an elastic filament that links a thick filament with the Z-disk, keeping thick filaments centered within the sarcomere during force generation. We have shown that the connectin filament has an affinity for calcium ions and its binding site(s) is restricted to the beta-connectin/titin-2 portion. We now report the localization and the characterization of calcium-binding sites on beta-connectin. Purified beta-connectin was digested by trypsin into 1700- and 400-kDa fragments. which were then subjected to fluorescence calcium-binding assays. The 400-kDa fragment possesses calcium-binding activity; the binding constant was 1.0 x 10(7) M(-1) and the molar ratio of bound calcium ions to the 400-kDa fragment reached a maximum of 12 at a free calcium ion concentration of approximately 1.0 microM. Antibodies against the 400-kDa fragment formed a sharp dense stripe at the boundary of the A and the I bands, indicating that the calcium-binding domain constitutes the N-terminal region of beta-connectin, that is, the elastic portion of connectin filaments. Furthermore, we estimated the N-terminal location of beta-connectin of various origins (n = 26). Myofibrils were treated with a solution containing 0.1 mM CaCl2 and 70 microM leupeptin to split connectin filaments into beta-connectin and a subfragment, and chain weights of these polypeptides were estimated according to their mobility in 2% polyacrylamide slab gels. The subfragment exhibited a similar chain weight of 1200+/-33 kDa (mean+/-SD), while alpha- and beta-connectins were variable in size according to their origin. These results suggest that the apparent length of the 1200-kDa subfragment portion is almost constant in all instances, about 0.34 microm at the slack condition, therefore that the C-terminus of the 1200-kDa subfragment, that is, the N-terminus of the calcium-binding domain, is at the N2 line region of parent filaments in situ. Because the secondary structure of the 400-kDa fragment was changed by the binding of calcium ions, connectin filaments could be expected to alter their elasticity during the contraction-relaxation cycle of skeletal muscle.

Calcium Affinity of Regulatory Sites in Skeletal Troponin-C Is Attenuated by N-Cap Mutations of Helix C

Site-directed mutagenesis was used to make amino acid substitutions at position 54 of skeletal troponin C, testing a relationship between the stability of helix C and calcium ion affinity at regulatory sites in the protein. Normally, threonine at position 54 is the first helical residue, or N-cap, of the C helix; where helices C and D, and the loop between, comprise binding site II. Mutations were made in the context of a previously described phenylalanine 29→tryptophan (F29W) variant (Trigo-Gonzalez et al., Biochemistry 31, 7009–7015 (1992)), which allows binding events to be monitored through changes in the intrinsic fluorescence of the protein. N-Cap substitutions at position 54 were shown to attenuate the calcium affinity of regulatory sites in the N-terminal domain. Calcium affinities diminished according to the series T54 T54S > T54A > T54V > T54G with dissociation constants of 1.36 × 10−6, 1.36 × 10−6, 2.09 × 10−6, 2.28 × 10−6, and 4.24 × 10−6 M, respectively. The steady state binding of calcium to proteins in the mutant series was seen to be monophasic and cooperative. Calcium off-rates were measured by stopped flow fluorescence and in every instance two transitions were observed. The rate constant of the first transition, corresponding to ∼99% of the change in fluorescence, was between 900 ± 20 and 1470 ± 100 s−1, whereas the rate constant of the second transitions was between 94 ± 9 and 130 ± 23 s−1. The significance of two transitions remains unclear, though both rate constants occur on a time scale consistent with the regulation of contraction.

Sequential calcium binding to the regulatory domain of calcium vector protein reveals functional asymmetry and a novel mode of structural rearrangement.

Calcium vector protein (CaVP) from amphioxus is a two-domain, calcium-binding protein (18.3 kDa) of the calmodulin superfamily. Only two of the four EF-hand motifs (sites III and IV) have a significant binding affinity for calcium ions. We determined the solution structure of the domain containing these active sites (C-CaVP: W81-S161), in the Ca(2+)-saturated state, using NMR spectroscopy and restrained molecular dynamics. The tertiary structure is similar to other Ca(2+)-binding domains containing a pair of EF-hand motifs. The apo state has spectroscopic and thermodynamic characteristics of a molten globule, with conserved secondary structure but highly fluctuating tertiary organization. Titration of C-CaVP with Ca(2+) revealed a stepwise ion binding, with a stable equilibrium intermediate in which only site III binds a calcium ion. Despite a highly fluctuating structure of the free site IV, the calcium-bound site III has a persistent structure, with similar secondary elements but different interhelix angle and hydrophobic packing relative to the fully calcium-saturated state.

Identification of a calcium binding site in Staphylococcus hyicus lipase: generation of calcium-independent variants.

In this study we have identified the presence of a high-affinity binding site for calcium in the lipase from Staphylococcus hyicus. By means of isothermal titration calorimetry we showed that the enzyme binds one calcium per molecule of enzyme with a dissociation constant of 55 microM. The residual activity of the apoenzyme compared to the activity in the presence of calcium ions varies from 65% at 10 degreesC to nearly zero at 40 degreesC. On the basis of primary sequence alignment with other staphylococcal lipases and the lipases from Bacillus thermocatenulatus and from Pseudomonas glumae in combination with site-directed mutagenesis, aspartates 354 and 357 could be identified as calcium ligands. Kinetic measurements with the D357E variant showed that replacement of Asp357 by a glutamate decreased the affinity for calcium ions 30-fold. Introduction of a lysine, an asparagine, or an alanine at position 357 and of a lysine or an asparagine at position 354 resulted in calcium-independent variants. Isothermal titration calorimetry confirmed the loss of calcium binding. Although the D357K, D357N, and D357A variants did not bind calcium, at room temperature they were nearly as active as wild-type lipase in the presence of calcium, but at elevated temperatures these calcium-independent lipases showed a reduced activity. Over the whole temperature range the activities of the D354K and D354N variants are significantly lower than wild-type enzyme in the presence of calcium and are comparable to the activity of the wild-type apoenzyme. Our results show that binding of calcium is important for the structural stabilization of staphylococcal lipases (and possibly other lipases) and that it is possible to engineer calcium-independent variants on the basis of limited structural homology with another lipase.

Six decades of dentinogenesis research. Historical and prospective views on phosphophoryn and dentin sialoprotein.

The molecular basis underlying the mineralization process associated with the conversion of predentin to dentin is poorly understood. What is clear is that a unique set of non-collagenous proteins (NCPs) participate in the nucleation process and in hydroxyapatite growth during dentin formation. Phosphophoryn (PP), the most abundant NCP in dentin, is secreted by odontoblasts and appears at the mineralization front. Dentin sialoprotein (DSP), another NCP, also appears at the mineralization front, but only accounts for 5-8% of the weight of dentin NCPs. Functionally, PP is believed to be directly involved in tile nucleation process by virtue of its ability to bind to collagen type I, and its high affinity for calcium ions. Based on the analysis of the putative rat PP amino acid sequence, this latter activity is believed due to the highly phosphorylated character of PP, which results from the dual actions of casein kinases I and II at selected domains within PP. The precise role of DSP is currently unknown. In situ studies demonstrate that DSP is substantially expressed in odontoblasts and transiently expressed in preameloblasts. However, no information is currently available to directly explain DSP's role in mineralization. Genetically, we and others have now identified a novel DSP-PP bicistronic mammalian transcriptional unit, suggesting that the functional roles of these two NCPs may also be tightly coupled with respect to dentinogenesis. Certainly, further exciting studies are now needed to explain how this DSP-PP transcriptional unit is finally expressed: whether DSP and PP associate with one another, or with collagen at the mineralization front: and how selective mutations in either gene may influence dentin mineralization.

Fluorescence Detection of Calcium-Binding Proteins with Quinoline Ca-Indicator Quin2

We have established a fluorescence method to detect calcium-binding proteins by making use of the quinoline Ca indicator quin2. Authentic calcium-binding proteins were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis and then electrophoretically transferred onto polyvinylidene difluoride membranes. Transfers were incubated with nonradioactive calcium ions, then with quin2 to detect the calcium-binding proteins as fluorescent bands by illumination with UV light. Calmodulin and parvalbumin of EF hand conformation calcium-binding type were clearly identified. Quin2 distinguished smooth muscle α-actinin from skeletal muscle α-actinin; the former was faintly fluorescent, having a low affinity for calcium ions. In whole myofibril preparations from skeletal muscles, troponin-C, connectin (titin), and nebulin were intensely fluorescent, being shown to have calcium-binding ability. The fluorescence method is an accurate, safe, and simple procedure to detect the binding of calcium ions to proteins following electrophoresis. The overlay technique described can be completed within 15 h and detects as little as 38 ng/well of troponin-C in the starting sample.

A Theory of the Effects of the Field-Dependent Affinity of Calcium Ions on the Lamellar Phase of Dipalmitoyl Lecithin

The behavior of dipalmitoyl phosphatidylcholine bilayers in CaCl2aqueous solution found by Inokoet al.(Biochim. Biophys. Acta413, 24 (1975)) is analyzed, with four states I–IV appearing with increasing concentrations of CaCl2from 0 mMto 1M.The present theory takes into account the field-dependent association constantKof Ca2+ions to the membrane surface, which was derived from a theoretical investigation (Y. Izumitani,J. Colloid Interface Sci.166,143 (1994)) of the electrostatic cation–dipole interaction. The present theory is based on the DLVO theory of colloid stability, which was also used in the analysis of this phenomena by Ohshima and Mitsui (J. Colloid Interface Sci.63,525 (1978)), but in the present theory, the transition of states I → II is conditioned by the membrane flexibility and the retardation effects are taken into account in the van der Waals interaction in the lamellar structure. The discrepancy between the observed values for the repeat distance of the lamellar phase and the theoretical predictions of Ohshima's theory can be fairly well explained by the present theory with state III at 10–100 mMCaCl2concentration. The undulation effect is neglected in our treatment but the membrane flexibility can qualitatively explain the hysteresis in the I–II transition.

Glutamate substitution in repeat IV alters divalent and monovalent cation permeation in the heart Ca2+ channel

In voltage-gated ion channels, residues responsible for ion selectivity were identified in the pore-lining SS1-SS2 segments. Negatively charged glutamate residues (E393, E736, E1145, and E1446) found in each of the four repeats of the alpha 1C subunit were identified as the major determinant of selectivity in Ca2+ channels. Neutralization of glutamate residues by glutamine in repeat I (E393Q), repeat III (E1145Q), and repeat IV (E1446Q) decreased the channel affinity for calcium ions 10-fold from the wild-type channel. In contrast, neutralization of glutamate residues in repeat II failed to significantly alter Ca2+ affinity. Likewise, mutation of neighboring residues in E1149K and D1450N did not affect the channel affinity, further supporting the unique role of glutamate residues E1145 in repeat III and E1446 in repeat IV in determining Ca2+ selectivity. Conservative mutations E1145D and E1446D preserved high-affinity Ca2+ binding, which suggests that the interaction between Ca2+ and the pore ligand sites is predominantly electrostatic and involves charge neutralization. Mutational analysis of E1446 showed additionally that polar residues could achieve higher Ca2+ affinity than small hydrophobic residues could. The role of high-affinity calcium binding sites in channel permeation was investigated at the single-channel level. Neutralization of glutamate residue in repeats I, II, and III did not affect single-channel properties measured with 115 mM BaCl2. However, mutation of the high-affinity binding site E1446 was found to significantly affect the single-channel conductance for Ba2+ and Li+, providing strong evidence that E1446 is located in the narrow region of the channel outer mouth. Side-chain substitutions at 1446 in repeat IV were used to probe the nature of divalent cation-ligand interaction and monovalent cation-ligand interaction in the calcium channel pore. Monovalent permeation was found to be inversely proportional to the volume of the side chain at position 1446, with small neutral residues such as alanine and glycine producing higher Li+ currents than the wild-type channel. This suggests that steric hindrance is a major determinant for monovalent cation conductance. Divalent permeation was more complex. Ba2+ single-channel conductance decreased when small neutral residues such as glycine were replaced by bulkier ones such as glutamine. However, negatively charged amino acids produced single-channel conductance higher than predicted from the size of their side chain. Hence, negatively charged residues at position 1446 in repeat IV are required for divalent cation permeation.

Engineering Magnesium Selectivity in the Helix–Loop–Helix Calcium-Binding Motif

Engineering magnesium selectivity into the helix–loop–helix (hlh) cation binding site is relatively unstudied in the calmodulin superfamily of calcium-regulated proteins, which include parvalbumin, oncomodulin, troponin C, calbindin, and calmodulin. Studies using a 33-residue synthetic peptide model of thehlhcation binding motif have indicated that magnesium will induce structural change in those peptide motifs containing three or four acid residues in chelating positions with a single-acid-pair on the Z-axis. Decreasing the cation binding cavity size in Z-axis acid-paired motifs through replacement of chelating residues in the +Z or −X metal ion coordinating positions in the loop region by glutamic acid has been successful in decreasing the calcium ion affinity. The same changes did not create or enhance magnesium binding in the 33-residue modelhlhcation binding motif.

Conformational changes of bovine bone osteonectin induced by interaction with calcium.

To clarify calcium-induced conformational changes in bovine bone osteonectin, the protein was labeled with fluorescein isothiocyanate (FITC) in the presence and absence of calcium. By calcium titration using fluorescence spectrometry, it was demonstrated that FITC-osteonectin labeled in the presence of 2 mM CaCl2 showed a much higher affinity for calcium ions than did that labeled in the absence of calcium ions. The midpoint for completion of the increase in the intrinsic fluorescence (K0.5) of the two were 1 x 10(-7) M and 5 x 10(-7) M, respectively. By tryptic digestion and isolation of the fluorescent peptide of both FITC-osteonectins, the site of FITC-labeling was determined to be Lys174. Furthermore, it was found that the efficacy of labeling in this specific binding site was three times higher in the FITC-osteonectin labeled in the presence of 2 mM CaCl2 than in that labeled in the absence of calcium. The results indicate that in the presence of 2 mM CaCl2 the microenvironment around Lys174 of osteonectin was more open to modification than in the absence of calcium.

Calcium ionophore, A23187 and its amino acid complexes: spectroscopic and molecular modeling studies.

The circular dichroism, fluorescence, Nuclear Magnetic Resonance and BLM conductance studies indicate that A23187 forms a stable complex with amino acids at low ionophore concentrations (< 10(-4)M). However, A23187 prefers to be in a dimeric structure with no significant binding to amino acids, at concentrations higher than 10(-4)M. It was also observed that at lower concentrations, at which the amino acids bind to the ionophore, the affinity for calcium ions was several orders of magnitude lower than that at higher ionophore concentrations. We have also conducted molecular modeling studies to examine the structure of the A23187 dimer and its amino acid complexes. The results of these modeling studies strongly support our experimental results and validate the formation of a hydrogen bonded and energetically stable A23187 dimer and its amino acid complexes.

Increased calcium affinity of a fucosylated chondroitin sulfate from sea cucumber

Calcium binding and charge distribution on a fucosylated chondroitin sulfate and a standard chondroitin 6-sulfate have been studied using a metallochromic indicator and conductimetric titrations. The fucosylated chondroitin sulfate has a ≈5-fold greater affinity for calcium ions than the standard chondroitin 6-sulfate. Possibly, this increased affinity for calcium ions is due to the branches on the fucosylated chondroitin sulfate, since the calcium affinity of an unbranched, sulfated fucan is similar to that of the standard chondroitin 6-sulfate. More charged groups per disaccharide unit (and a shorter distance between these groups) also distinguish the fucosylated chondroitin sulfate from standard chondroitin 6-sulfate. Comparison between native and chemically modified (desulfated or carboxyl-reduced) polysaccharides suggests that the sulfate esters are responsible for the increased charge density of the fucosylated chondroitin sulfate and that the presence of the fucose branches does not alter the length of the repetitive units which compose the central core of chondroitin from sea cucumber. These results are consistent with the chemical studies of these two polysaccharides.

Influence of Six Mutations of the Protein C Gene on the Gla Domain Conformation and Calcium Affinity

The protein C Gla domain was studied in six families presenting a type II hereditary deficiency characterized by low activity in a coagulation assay and normal activity in an amidolytic assay. Five of these mutations, previously described by our group, affected Arg-5, Arg-1, Arg 229 and Ser 252. We report here the first natural Glu 7 to Asp mutation in a sixth family. We evaluated the binding of the mutated protein C to H11, a monoclonal antibody (mAb) known to recognize the sequence Phe4 to Arg9 of the Gla domain; the presence of calcium ions suppresses the recognition of this epitope by H11. Mutation of Arg229 to Gln and Ser252 to Asn did not modify the inhibition of protein C binding, whereas the Arg-1 to His mutation resulted in a loss of inhibition in the presence of CaCl2. This suggests that the protein C of this patient shows impaired carboxylation. The protein C from patients bearing the mutations Arg-5 to Trp, Arg-1 to Cys and Glu 7 to Asp bound poorly to H11 mAb, even in the absence of calcium ions. The calcium affinity of the Gla domain was studied by pseudo-affinity chromatography, in which protein C was successively eluted from a Mono Q column by CaCl2 10 mM and NaCl 0.6 M. Protein C from the patient bearing the Arg-5 to Asp mutation had a normal elution profile, suggesting that a modification of the propeptide cleavage site impairs the conformation of the Gla domain but not carboxylation.(ABSTRACT TRUNCATED AT 250 WORDS)