Vitamin K dependent in vitro production of prothrombin

During prothrombin biosynthesis, glutamyl residues in prothrombin precursor proteins are carboxylated to gamma-carboxyglutamyl residues by a vitamin K dependent carboxylase. Calcium-dependent and calcium-independent rat prothrombin antibody subpopulations have been produced and utilized to study the liver microsomal precursors of prothrombin that accumulate when vitamin K action is blocked. A substantial portion of the precursor pool accumulating in the vitamin K deficient or warfarin-treated rat will react with a Ca2+-dependent antibody at high calcium concentration and appears to be partially carboxylated. During in vitro incubation in the presence of vitamin K, the fraction of the precursor pool which is tightly bound to the microsomal membrane appears to be the preferred substrate for the vitamin K dependent carboxylation. A small amount of completely carboxylated rather than a large amount of partially carboxylated products are produced during these incubations. Treatment with a Sepharose-bound prothrombin antibody demonstrated that about 20-25% of the total carboxylated microsomal protein precursor pool consists of prothrombin precursors. This treatment removes an equal amount of total carboxylase activity, and the enzyme is active in this carboxylase precursor-antibody complex.     

Beta-hydroxyaspartic acid in the first epidermal growth factor-like domain of protein C. Its role in Ca2+ binding and biological activity

Protein C is a vitamin K-dependent regulator of blood coagulation. It has beta-hydroxyaspartic acid in position 71 which is in the first of its two domains that are homologous to epidermal growth factor (EGF). This region has recently been demonstrated to have a Ca2+ binding site with a Kd of approximately 100 microM. Recombinant human protein C, expressed in mammalian tissue culture, had full biological activity and contained beta-hydroxyaspartic acid. Furthermore, it had a Ca2+-dependent epitope in the EGF-like domain, recognized by a monoclonal antibody. In contrast, a mutant recombinant human protein C in which beta-hydroxyaspartic acid had been replaced with glutamic acid in position 71 did not have the Ca2+-dependent epitope, and its biological activity was reduced to about 10% of normal. Fab' fragments of this antibody inhibited the anticoagulant activity of plasma-derived activated protein C, apparently by interfering with the interaction between activated protein C and its cofactor, protein S. The latter contains four tandemly arranged EGF homology domains. We propose that beta-hydroxyaspartic acid is directly involved in Ca2+ binding in protein C and in related proteins and that protein C interacts with protein S by means of its EGF homology regions.     

The vitamin K-dependent bone protein is accumulated within cultured osteosarcoma cells in the presence of the vitamin K antagonist warfarin

Osteosarcoma cells grown in normal culture medium secrete bone gamma-carboxyglutamic acid protein (BGP, osteocalcin) which is identical with BGP purified from the bone matrix. Two tests indicate that the secreted medium protein contains the full complement of three gamma-carboxyglutamate residues present on BGP purified from the bone matrix. First, the secreted protein from ROS 17/2 and bone matrix BGP have identical isoelectric points (pI = 4.0). Second, they have identical hydroxyapatite binding behavior. If warfarin is added to the culture medium, the secreted protein has a higher isoelectric point (pI = 4.6) and a lower affinity for hydroxyapatite characteristic of thermally decarboxylated or non-gamma-carboxylated BGP. The observed shift in isoelectric point of secreted BGP after warfarin treatment from pH 4.0 to 4.6 is also reflected in the presence of pI = 4.1 and pI = 4.6 species intracellularly. These isoelectric species correspond to fully carboxylated BGP and noncarboxylated BGP, which are in the process of secretion. Addition of 10 micrograms/ml of warfarin causes a specific 47% reduction in secretion rate of BGP, while at the same time, the intracellular BGP concentration increases 3-fold. These phenomena appear related to the interruption by warfarin of the normal sequence of processing of precursor BGP proteins, as a new, immunoreactive species with a higher isoelectric pH not present in control cells appears to be responsible for the increased intracellular antigen within warfarin-treated cells. Our results show that vitamin K-dependent processing is important for normal secretion of BGP from the cell.     

A conserved epitope on several human vitamin K-dependent proteins. Location of the antigenic site and influence of metal ions on antibody binding

A murine monoclonal antibody (designated H-11) produced by injecting mice with purified human protein C was found to bind several human vitamin K-dependent proteins. Using a solid-phase competitive radioimmunoassay with antibody immobilized onto microtiter plates, binding of 125I-labeled protein C to the antibody was inhibited by increasing amounts of protein C, prothrombin, and Factors X and VII over a concentration range of 1 X 10(-8) to 1 X 10(-6) M. Other vitamin K-dependent proteins including Factor IX and protein S did not inhibit or inhibited only at the highest concentration binding of radiolabeled protein C to the immobilized antibody. Chemical treatment of prothrombin with a variety of agents including denaturation by sodium dodecyl sulfate, reduction with mercaptoethanol followed by carboxymethylation with iodoacetic acid, citraconylation of lysine residues, removal of metal ion with EDTA, or heat decarboxylation did not destroy the antigenic site recognized by the antibody as measured by immunoblotting of prothrombin or prothrombin derivative immobilized onto nitrocellulose. Immunoblotting of purified vitamin K-dependent polypeptides with the monoclonal antibody following sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretic transfer to nitrocellulose indicated that the antigenic site was found on the light chains of protein C and Factor X. Chymotrypsin digestion of prothrombin and isolation on QAE-Sephadex of the peptide representing amino-terminal residues 1-44 of prothrombin further localized the antigenic site recognized by the monoclonal antibody to the highly conserved gamma-carboxyglutamic acid-containing domain. The exact location of the antigenic determinant for antibody H-11 was established using synthetic peptides. Antibody H-11 bound specifically to synthetic peptides corresponding to residues 1-12 of Factor VII and 1-22 of protein C. Comparison of protein sequences of bovine and human vitamin K-dependent proteins suggests that the sequence Phe-Leu-Glu-Glu-Xaa-Arg/Lys is required for antibody binding. The glutamic acid residues in this peptide segment are the first 2 gamma-carboxyglutamic acid residues near the amino-terminal end in the native proteins. Increasing concentrations of Ca2+, Mg2+, or Mn2+ partially inhibited binding of 125I-protein C to the antibody in a solid-phase assay system with half-maximal binding observed at divalent metal ion concentrations of 2, 4, and 0.6 mM, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

Proteolytic formation and properties of a fragment of protein C containing the gamma-carboxyglutamic acid rich domain and the EGF-like region

The function of the epidermal growth factor (EGF) like domains in the vitamin K dependent plasma proteins is largely unknown. In order to elucidate the function of these domains in protein C, we have devised a method to isolate the EGF-like region from the light chain connected to the NH2-terminal region, containing the gamma-carboxyglutamic acid (Gla) residues. This was accomplished by tryptic cleavage of protein C that had been reversibly modified with citraconic anhydride to prevent cleavage at the lysine residue (in position 43) that is located between the two regions. The isolated fragment consists of residues 1-143 from the light chain of protein C connected by a disulfide bond to residues 108-131 from the heavy chain. Upon Ca2+ binding to the isolated Gla-EGF fragment from bovine protein C, the tryptophan fluorescence emission was quenched in a manner indicating binding to at least two classes of binding sites. These were presumably the Gla-independent Ca2(+)-binding site located in the EGF-like region and the lower affinity sites in the Gla region. A comparison with the tryptophan fluorescence quenching that occurred upon Ca2+ binding to the separately isolated EGF-like and Gla regions suggested that the EGF-like region influenced the structure and Ca2+ binding of the Gla region. The isolated Gla-EGF fragment functioned as an inhibitor of the anticoagulant effect of activated protein C in a clotting assay, whereas no inhibition was observed with either the Gla region or the EGF-like region.     

Calcium-dependent alpha-helical structure in osteocalcin

Osteocalcin is an abundant Ca2+-binding protein of bone containing three residues of vitamin K dependent gamma-carboxyglutamic acid (Gla) among its 49 (human, monkey, cow) or 50 (chicken) amino acids. Gla side chains participate directly in the binding of Ca2+ ions and the adsorption of osteocalcin to hydroxylapatite (HA) surfaces in vivo and in vitro. Osteocalcin exhibits a major conformational change when Ca2+ is bound. Metal-free chicken osteocalcin is a random coil with only 8% of its residues in the alpha helix as revealed by circular dichroism. In the presence of physiological levels of Ca2+, 38% of the protein adopts the alpha-helical conformation with a transition midpoint at 0.75 mM Ca2+ in a rapid, reversible fashion which (1) requires an intact disulfide bridge, (2) is proportionally diminished when Gla residues are decarboxylated to Glu, (3) is insensitive to 1.5 m NaCl, and (4) can be mimicked by other cations. Tyr fluorescence, UV difference spectra, and Tyr reactivity to tetranitromethane corroborate the conformational change. Homologous monkey osteocalcin also exhibits Ca2+-dependent structure. Integration of predictive calculations from osteocalcin sequence has yielded a structural model for the protein, the dominant features of which include two opposing alpha-helical domains of 9-12 residues each, connected by a bea turn and stabilized by the Cys23-Cys29 disulfide bond. Cation binding permits realization of the full alph a-helical potential by partial neutralization of high anionic charge in the helical domains. Periodic Gla occurrence at positions 17, 21, and 24 has been strongly conserved throughout evolution and places all Gla side chains on the same face of one alpha helix spaced at intervals of approximately 5.4 A, closely paralleling the interatomic separation of Ca2+ in the HA lattice. Helical osteocalcin has greatly increased affinity for HA; thus, the Ca2+-induced structural transition may perform an informational role related to bone metabolism.     

The interaction of a Ca2+-dependent monoclonal antibody with the protein C activation peptide region. Evidence for obligatory Ca2+ binding to both antigen and antibody

Protein C undergoes Ca2+-induced conformational changes required for activation by the thrombin-thrombomodulin complex. A Ca2+-dependent monoclonal antibody (HPC4) that blocks protein C activation was used to study conformational changes near the activation site in protein C. The half-maximal Ca2+ dependence was similar for protein C and gamma-carboxy-glutamic acid-domainless protein C for binding to HPC4 (205 +/- 23 and 110 +/- 29 microM Ca2+, respectively), activation rates (214 +/- 22 and 210 +/- 37 microM), and intrinsic fluorescence of gamma-carboxyglutamic acid-domainless protein C (176 +/- 34 microM). Protein C heavy chain binding to HPC4 was half-maximal at 36 microM Ca2+, although neither the heavy chain nor HPC4 separately bound Ca2+ with high affinity. The epitope was lost when the activation peptide was released. A synthetic peptide, P (6-17), which spans the activation site, exhibited Ca2+-dependent binding to HPC4 (half-maximal binding = 6 microM Ca2+). Thus, each decrease in antigen structure resulted in a reduced Ca2+ requirement for binding to HPC4. Tb3+ and Ca2+ binding studies demonstrated a Ca2+-binding site in HPC4 required for high affinity antigen binding. These studies provide the first direct evidence for a Ca2+-induced conformational change in the activation region of a vitamin K-dependent zymogen. Furthermore, Ca2+ binding to HPC4 is required for antigen binding. The multiple roles of Ca2+ described may be useful in interpretation of other metal-dependent antibody/antigen interactions.     

Identification of amino acids in the gamma-carboxylation recognition site on the propeptide of prothrombin

A gamma-carboxylation recognition site on the propeptide of the vitamin K-dependent blood coagulation proteins directs the carboxylation of glutamic acid residues by binding to the vitamin K-dependent carboxylase. To determine residues that define this site, we evaluated the effect of mutation of certain residues in the prothrombin propeptide on the extent of carboxylation. The prothrombin cDNA modified by site-specific mutagenesis was expressed in Chinese hamster ovary cells using a system that yields functional fully carboxylated prothrombin. The cell supernatants containing recombinant prothrombin were evaluated for the extent of gamma-carboxylation by immunoassay. Conformation-specific anti-prothrombin:Ca(II)-specific antibodies measure native completely carboxylated prothrombin; anti-prothrombin:total antibodies measure all forms of prothrombin, regardless of gamma-carboxyglutamic acid content. Mutation of His-18 to Gly, Val-17 to Ser, Leu-15 to Gly or Asp, or Ala-10 to Asp was associated with a partial (30-65%) inhibition of gamma-carboxylation. Mutation of Ala-14 to Ser or Ser-8 to Val did not inhibit gamma-carboxylation. From this and earlier work, residues whose mutation leads to a significant impairment of carboxylation include His-18, Val-17, Phe-16, Leu-15, and Ala-10. Residues whose mutation does not alter the carboxylation recognition site include Ala-14, Ser-8, Arg-4, and Arg-1. To determine the size of the recognition site, the in vitro carboxylation of propeptide-containing synthetic peptides was compared. A 28-residue peptide, based upon residues -18 to +10 of prothrombin, and a 54-residue peptide, based upon residues -18 to +36 of prothrombin, were carboxylated by partially purified bovine carboxylase with similar Km values of 2-5 microM. These results indicate that the gamma-carboxyglutamic acid-rich region of prothrombin makes a minimal contribution to carboxylase binding. A molecular surface of about five amino acids located within the propeptide appears to define the carboxylation recognition site on the precursor forms of the vitamin K-dependent proteins.     

Immunoaffinity purification of protein C by using conformation-specific monoclonal antibodies to protein C-calcium ion complex

We isolated protein C from a barium citrate-adsorbed fresh plasma and human factor IX concentrate by immunoaffinity chromatography on a column of Sepharose coupled with monoclonal antibodies to protein C. The antibodies used were conformation-specific monoclonal antibodies to the calcium-induced structure of protein C. Protein C was bound to antibodies coupled with Sepharose in the presence of calcium ions and was eluted with EDTA. This immunopurification resulted in a 13,000-fold purification of the fully functional zymogen from plasma. The immunoaffinity-isolated protein C was found to have higher amounts of single-chain protein C than conventionally isolated protein C when analyzed by sodium dodecyl sulfate-polyacrylamide gels under reduced conditions. The factor IX concentrate was applied to this Ca2+-dependent antibody JTC-3-immobilized Sepharose in the presence of 5 mM CaCl2, and protein C with its gamma-carboxyglutamic acid (Gla) domain intact was firstly bound to this column and then eluted by metal chelation with EDTA. When flow-through fractions were applied again in the presence of Ca2+ to this column, modified protein C which had lost its N-terminal 42-residue peptide was weakly bound to this column. It was eluted in the absence of Ca2+. However, only a low percentage of modified protein C was detectable by an enzyme-linked immunosorbent assay using Ca2+-dependent monoclonal antibody JTC-3 and peroxidase-labeled immunopurified polyclonal antibody. These results indicate that factor IX concentrate has both Gla-domain-intact and Gla-domainless protein C. Moreover, it suggests that Ca2+-dependent monoclonal antibody JTC-3 may recognize the coupled conformational change of protein C induced by the combined effect of Ca2+ binding to the Gla domain and to other parts of protein C.     

Involvement of EF hand motifs in the Ca(2+)-dependent binding of the pleckstrin homology domain to phosphoinositides.

The pleckstrin homology (PH) domains of phospholipase C (PLC)-delta1 and a related catalytically inactive protein, p130, both bind inositol phosphates and inositol lipids. The binding to phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] by PLC-delta1 is proposed to be the critical interaction required for membrane localization to where the substrate resides; it is also required for the Ca(2+)-dependent activation of PLC-delta1 observed in the permeabilized cells. In the proximity of the PH domain, both PLC-delta1 and p130 possess the EF-hand domain, containing classical motifs implicated in calcium binding. Therefore, in the present study we examined whether the binding of the PH domain to PtdIns(4,5)P2 is regulated by changes in free Ca2+ concentration within the physiological range. A Ca2+ dependent increase in the binding to PtdIns(4,5)P2 was observed with a full-length PLC-delta1, while the isolated PH domain did not show any Ca2+ dependence. However, the connection of the EF-hand motifs to the PH domain restored the Ca2+ dependent increase in binding, even in the absence of the C2 domain. The p130 protein showed similar properties to PLC-delta1, and the EF-hand motifs were again required for the PH domain to exhibit a Ca2+ dependent increase in the binding to PtdIns(4,5)P2. The isolated PH domains from several other proteins which have been demonstrated to bind PtdIns(4,5)P2 showed no Ca2+ dependent enhancement of binding. However, when present within a chimera also containing PLC-delta1 EF-hand motifs, the Ca2+ dependent binding was again observed. These results suggest that the binding of Ca2+ to the EF-hand motifs can modulate binding to PtdIns(4,5)P2 mediated by the PH domain.     

Vitamin K-dependent carboxylation. In vitro modification of synthetic peptides containing the gamma-carboxylation recognition site

Synthetic peptides including the gamma-carboxylation recognition site and acidic amino acids were compared as substrates for vitamin K-dependent gamma-carboxylation by bovine liver carboxylase. The 28-residue proPT28 (proprothrombin -18 to +10) and proFIX28 (pro-Factor IX -18 to +10) were carboxylated with a Km of 3 microM. The Vmax of proPT28 was 2-3 times greater than that of proFIX28. An analog of proFIX28 that contained the prothrombin propeptide had a Vmax 2-3-fold greater than an analog of proPT28 that contained the Factor IX propeptide. proFIX28/RS-1, based upon Factor IX Cambridge, proFIX28/RQ-4, based upon Factor IX Oxford 3, and proFIX28 had equivalent Km and Vmax values. Analogs of proPT28 containing Ala6-Glu7 or Glu6-Ala7 were carboxylated at equivalent rates. A peptide containing Asp6-Asp7 was carboxylated at a rate of about 1% of that of Glu carboxylation. Carboxylation of peptides containing Asp6-Glu7 and Glu6-Asp7 yielded results identical with peptides containing Ala6-Glu7 and Glu6-Ala7. Carboxymethylcysteine was not carboxylated when substituted for Glu6 in a peptide containing Asp7. These results indicate that the prothrombin propeptide is more efficient in the carboxylation process than is the Factor IX propeptide, but that both propeptides direct carboxylation; the gamma-carboxylation recognition site does not include residues -4 and -1; aspartic acid and carboxymethylcysteine are poor substrates for the carboxylase, but aspartic acid does not inhibit the carboxylation of adjacent glutamic acids.     

Ca2+-dependent K+ permeability of heart sarcolemmal vesicles. Modulation by cAMP-dependent protein kinase activity and by calmodulin

The Ca2+-dependent K+ permeability of heart sarcolemma vesicles was measured by following the transmembrane movement of the charge compensating tetraphenylborate anion. The increase in vesicles permeability induced by Ca2+ is lost when membrane proteins are dephosphorylated by an endogenous protein phosphatase and is restored by a phosphorylation process catalysed by a cAMP-dependent protein kinase. The calmodulin antagonist R 24571 lowers the Ca2+-dependent K+ permeability by decreasing the Ca2+ affinity of the K+ transporting system.     

Developmental and Functional Studies of Parvalbumin and Calbindin D28K in Hypothalamic Neurons Grown in Serum-Free Medium

The Ca2+-binding proteins parvalbumin (Mr = 12K) and calbindin D28K [previously designated vitamin D-dependent Ca2+-binding protein (Mr = 28K)] are neuronal markers, but their functional roles in mammalian brain are unknown. The expression of these two proteins was studied by immunocytochemical methods in serum-free cultures of hypothalamic cells from 16-day-old fetal mice. Parvalbumin is first detected in all immature neurons, but during differentiation, the number of parvalbumin-immunoreactive neurons greatly declines to a level reminiscent of that observed in vivo, where only a subpopulation of neurons stains for parvalbumin. In contrast, calbindin D28K was expressed throughout the period investigated only in a distinct subpopulation of neurons. Depolarization of fully differentiated hypothalamic neurons in culture resulted in a dramatic decrease of parvalbumin immunoreactivity but not of calbindin D28K immunoreactivity. The parvalbumin staining was restored on repolarization. Because the anti-parvalbumin serum seems to recognize only the metal-bound form of parvalbumin, the loss of immunoreactivity may signal a release of Ca2+ from intracellular parvalbumin during depolarization of the cells. We suggest that parvalbumin might be involved in Ca2+-dependent processes associated with neurotransmitter release.     

Effect of calcium on the stability of the platelet membrane glycoprotein IIb-IIIa complex

Platelet membrane glycoproteins IIb and IIIa form a Ca2+-dependent heterodimer complex that contains binding sites for fibrinogen, von Willebrand factor, and fibronectin following platelet stimulation. We have studied the effect of Ca2+ on the stability of the IIb-IIIa complex using a IIb-IIIa complex-specific monoclonal antibody A2A9 to detect the presence of the complexes. Soluble IIb and IIIa interacted with A2A9-Sepharose only in the presence of Ca2+ with 50% IIb-IIIa binding requiring 0.4 microM Ca2+. In contrast, at 25 degrees C 125I-A2A9 binding to intact unstimulated platelets suspended in buffers containing EDTA or ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid was independent of the presence of Ca2+. However, the effect of Ca2+ chelators on 125I-A2A9 binding varied with temperature. At 37 degrees C, 125I-A2A9 binding to intact platelets became Ca2+-dependent with 50% binding requiring 0.4 microM Ca2+. This effect of temperature was not due to a change in platelet membrane fluidity because enrichment or depletion of platelet membrane cholesterol did not influence antibody binding. But, 125I-A2A9 binding to intact platelets at 25 degrees C did become Ca2+-dependent when the pH was increased above 7.4. Thus, at 1 nM Ca2+ and 25 degrees C, 50% antibody binding occurred at pH 9.0. Our studies demonstrate that Ca2+-dependent IIb-IIIa complexes are present on unstimulated platelets and that the Ca2+ binding sites responsible for the stability of these complexes are located on the external platelet surface. Our experiments also suggest that changes in platelet cytosolic Ca2+ do not regulate the formation of IIb-IIIa complexes.     

Characteristics of the Ca2+ pump and Ca2+-ATPase in the plasma membrane of rat myometrium.

A plasma membrane-enriched fraction from rat myometrium shows ATP-Mg2+-dependent active calcium uptake which is independent of the presence of oxalate and is abolished by the Ca2+ ionophore A23187. Ca2+ loaded into vesicles via the ATP-dependent Ca2+ uptake was released by extravesicular Na+. This showed that the Na+/Ca2+ exchange and the Ca2+ uptake were both occurring in plasma membrane vesicles. In a medium containing KCl, vanadate readily inhibited the Ca2+ uptake (K1/2 5 microM); when sucrose replaced KCl, 400 microM-vanadate was required for half inhibition. Only a slight stimulation of the calcium pump by calmodulin was observed in untreated membrane vesicles. Extraction of endogenous calmodulin from the membranes by EGTA decreased the activity and Ca2+ affinity of the calcium pump; both activity and affinity were fully restored by adding back calmodulin or by limited proteolysis. A monoclonal antibody (JA3) directed against the human erythrocyte Ca2+ pump reacted with the 140 kDa Ca2+-pump protein of the myometrial plasma membrane. The Ca2+-ATPase activity of these membranes is not specific for ATP, and is not inhibited by mercurial agents, whereas Ca2+ uptake has the opposite properties. Ca2+-ATPase activity is also over 100 times that of calcium transport; it appears that the ATPase responsible for transport is largely masked by the presence of another Ca2+-ATPase of unknown function. Measurements of total Ca2+-ATPase activity are, therefore, probably not directly relevant to the question of intracellular Ca2+ control.     

Periostin, a member of a novel family of vitamin K-dependent proteins, is expressed by mesenchymal stromal cells

The modification of glutamic acid residues to gamma-carboxyglutamic acid (Gla) is a post-translational modification catalyzed by the vitamin K-dependent enzyme gamma-glutamylcarboxylase. Despite ubiquitous expression of the gamma-carboxylation machinery in mammalian tissues, only 12 Gla-containing proteins have so far been identified in humans. Because bone tissue is the second most abundant source of Gla-containing proteins after the liver, we sought to identify Gla proteins secreted by bone marrow-derived mesenchymal stromal cells (MSCs). We used a proteomics approach to screen the secretome of MSCs with a combination of two-dimensional gel electrophoresis and tandem mass spectrometry. The most abundant Gla-containing protein secreted by MSCs was identified as periostin, a previously unrecognized gamma-carboxylated protein. In silico amino acid sequence analysis of periostin demonstrated the presence of four consensus gamma-carboxylase recognition sites embedded within fasciclin-like protein domains. The carboxylation of periostin was confirmed by immunoprecipitation and purification of the recombinant protein. Carboxylation of periostin could be inhibited by warfarin in MSCs, demonstrating its dependence on the presence of vitamin K. We were able to demonstrate localization of carboxylated periostin to bone nodules formed by MSCs in vitro, suggesting a role in extracellular matrix mineralization. Our data also show that another fasciclin I-like protein, betaig-h3, contains Gla. In conclusion, periostin is a member of a novel vitamin K-dependent gamma-carboxylated protein family characterized by the presence of fasciclin domains. Furthermore, carboxylated periostin is produced by bone-derived cells of mesenchymal lineage and is abundantly found in mineralized bone nodules in vitro.     

Calcium binding domains and calcium-induced conformational transition of SPARC/BM-40/osteonectin, an extracellular glycoprotein expressed in mineralized and nonmineralized tissues

SPARC, BM-40, and osteonectin are identical or very closely related extracellular proteins of apparent Mr 43,000 (Mr 33,000 predicted from sequence). They were originally isolated from parietal endoderm cells, basement membrane producing tumors, and bone, respectively, but are rather widely distributed in various tissues. In view of the calcium binding activity reported for osteonectin, we analyzed the SPARC sequence and found two putative calcium binding domains. One is an N-terminal acidic region with clusters of glutamic acid residues. This region, although neither gamma-carboxylated nor homologous, resembles the gamma-carboxyglutamic acid (Gla) domain of vitamin K dependent proteins of the blood clotting system in charge density, size of negatively charged clusters, and linkage to the rest of the molecule by a cysteine-rich domain. The other region is an EF-hand calcium binding domain located near the C-terminus. A disulfide bond between the E and F helix is predicted from modeling the EF-hand structure with the known coordinates of intestinal calcium binding protein. The disulfide bridge apparently serves to stabilize the isolated calcium loop in the extracellular protein. As observed for cytoplasmic EF-hand-containing proteins and for Gla domain containing proteins, a major conformational transition is induced in BM-40 upon binding of several Ca2+ ions. This is accompanied by a 35% increase in alpha-helicity. A pronounced sigmoidicity of the dependence of the circular dichroism signal at 220 nm on calcium concentration indicates that the process is cooperative. In view of its properties, abundance, and wide distribution, it is proposed that SPARC/BM-40/osteonectin has a rather general regulatory function in calcium-dependent processes of the extracellular matrix.     

ATP Release, Adenosine Formation, and Modulation of Dynorphin and Glutamic Acid Release by Adenosine Analogues in Rat Hippocampal Mossy Fiber Synaptosomes

Using a hippocampal subcellular fraction enriched in mossy fiber synaptosomes, evidence was obtained indicating that adenosine derived from a presynaptic pool of ATP may modulate the release of prodynorphin-derived peptides. and glutamic acid from mossy fiber terminals. Synaptosomal ATP was released in a Ca2+-dependent manner by K+-induced depolarization. The rapid hydrolysis of extracellular [14C]ATP in the presence of intact mossy fiber synaptosomes resulted in the production of [14C]adenosine. Micromolar concentrations of a stable adenosine analogue, 2-chloroadenosine, inhibited the K+-stimulated release of both dynorphin B and dynorphin A(1-8). 2-Chloroadenosine failed to suppress the evoked release of glutamic acid, measured in these same superfusates, unless the mossy fiber synaptosomes were pretreated with D-aspartic acid to deplete the cytosolic, Ca2+-independent, pool of this acidic amino acid. In synaptosomes pretreated in this manner, release of the remaining Ca2+-dependent pool of glutamic acid was significantly inhibited by NiCl2, 2-chloroadenosine, 5'-N-ethylcarboxamidoadenosine, cyclohexyladenosine, and R(-)-N6(2-phenylisopropyl)adenosine, but not by ATP. 2-Chloroadenosine-induced inhibition was reversed when the external CaCl2 concentration was raised from 1.8 mM to 6 mM. 8-Phenyltheophylline, an adenosine receptor antagonist, effectively blocked the inhibitory effects of 2-chloroadenosine on mossy fiber synaptosomes and significantly enhanced the K+-evoked release of both glutamic acid and dynorphin A(1-8) when added alone to the superfusion medium. These results support the proposition that depolarized hippocampal mossy fiber synaptosomes release endogenous ATP and are capable of forming adenosine from extracellular ATP, and that endogenous adenosine may act at a presynaptic site to inhibit the further release of glutamic acid and the prodynorphin-derived peptides.     

Decarboxylation of bovine prothrombin fragment 1 and prothrombin

Bovine prothrombin fragment 1 and prothrombin undergo decarboxylation of their gamma-carboxyglutamic acid residues when the lyophilized proteins are heated in vacuo at 110 degrees C for several hours. The fully decarboxylated fragment 1 product has lost its barium-binding ability as well as the calcium-binding function which causes fluorescence quenching in the presence of 2 mM Ca2+. There is no sign of secondary structure alteration in solution upon analysis by fluorescence emission and circular dichroic spectroscopy. A family of partially decarboxylated fragment 1 species generated by heating for shorter periods shows that the initial decrease in calcium-binding ability occurs almost twice as rapidly as the loss of gamma-carboxyglutamic acid. This is consistent with the idea that differential functions can be ascribed to the 10 gamma-carboxyglutamic acid residues in fragment 1, including both high- and low-affinity metal ion binding sites. Prothrombin itself also undergoes total decarboxylation without any apparent alteration in secondary structure. However, in this case the latent thrombin activity is progressively diminished during the heating process in terms of both clotting activity and hydrolysis of the amide substrate H-D-Phe-Pip-Arg-pNA. The present results indicate that in vitro decarboxylation of gamma-carboxyglutamic acid in dried proteins is useful for analyzing the detailed calcium-binding proteins of vitamin K dependent coagulation factors.     

The role of bound potassium ions in the hydrolysis of low concentrations of adenosine triphosphate by preparations of membrane fragments from ox brain cerebral cortex

1. The intrinsic Na(+), K(+), Mg(2+) and Ca(2+) contents of a preparation of membrane fragments from ox brain were determined by emission flame photometry. 2. Centrifugal washing of the preparation with imidazole-buffered EDTA solutions decreased the bound Na(+) from 90+/-20 to 24+/-12, the bound K(+) from 27+/-3 to 7+/-2, the bound Mg(2+) from 20+/-2 to 3+/-1 and the bound calcium from 8+/-1 to <1nmol/mg of protein. 3. The activities of the Na(+)+K(+)+Mg(2+)-stimulated adenosine triphosphatase and the Na(+)-dependent reaction forming bound phosphate were compared in the unwashed and washed preparations at an ATP concentration of 2.5mum (ATP/protein ratio 12.5pmol/mug). 4. The Na(+)-dependent hydrolysis of ATP as well as the plateau concentration of bound phosphate and the rate of dephosphorylation were decreased in the washed preparation. The time-course of formation and decline of bound phosphate was fully restored by the addition of 2.5mum-magnesium chloride and 2mum-potassium chloride. Addition of 2.5mum-magnesium chloride alone fully restored the plateau concentration of bound phosphate, but the rate of dephosphorylation was only slightly increased. Na(+)-dependent ATP hydrolysis was partly restored with 2.5mum-magnesium chloride; addition of K(+) in the range 2-10mum-potassium chloride then further restored hydrolysis but not to the control rate. 5. Pretreatment of the washed preparation at 0 degrees C with 0.5nmol of K(+)/mg of protein so that the final added K(+) in the reaction mixture was 0.1mum restored the Na(+)-dependent hydrolysis of ATP and the time-course of the reaction forming bound phosphate. 6. The binding of [(42)K]potassium chloride by the washed membrane preparation was examined. Binding in a solution containing 10nmol of K(+)/mg of protein was linear over a period of 20min and was inhibited by Na(+). Half-maximal inhibition of (42)K(+)-binding required a 100-fold excess of sodium chloride. 7. It was concluded (a) that a significant fraction of the apparent Na(+)-dependent hydrolysis of ATP observed in the unwashed preparation is due to activation by bound K(+) and Mg(2+) of the Na(+)+K(+)+Mg(2+)-stimulated adenosine triphosphatase system and (b) that the enzyme system is able to bind K(+) from a solution of 0.5mum-potassium chloride.