Potential Calcium-binding Sites Research Articles

A finely tuned interplay between calcium binding, ionic strength and pH modulates conformational and oligomerization equilibria in the Respiratory Syncytial Virus Matrix (M) protein

As in most enveloped RNA viruses, the Respiratory Syncytial Virus Matrix (RSV-M) protein plays key roles in viral assembly and uncoating. It also plays non-structural roles related to transcription modulation through nucleo-cytoplasmic shuttling and nucleic acid binding ability. We dissected the structural and conformational changes underlying the switch between multiple functionalities, identifying Ca2+ binding as a key factor. To this end, we tackled the analysis of M's conformational stability and equilibria. While in silico calculations predict two potential calcium binding sites per protomer, purified RSV-M dimer contains only one strongly bound calcium ion per protomer. Incubation of RSV-M in the presence of excess Ca2+ leads to an increase in the thermal stability, confirming additional Ca2+ binding sites. Moreover, mild denaturant concentrations trigger the formation of higher order oligomers which are otherwise prevented under Ca2+ saturation conditions, in line with the stabilizing effect of the additional low affinity binding site. On the other hand, Ca2+ removal by chelation at pH 7.0 causes a substantial decrease in the thermal stability leading to the formation of amorphous, spherical-like aggregates, as assessed by TEM. Even though the Ca2+ content modulates RSV-M oligomerization propensity, it does affect its weak RNA binding ability. RSV-M undergoes a substantial conformational change at pHs 4.0 to 5.0 that results in the exposure of hydrophobic surfaces, an increase beta sheet content but burial of tryptophan residues. While low ionic strength promotes dimer dissociation at pH 4.0, physiological concentrations of NaCl lead to the formation of soluble oligomers smaller than 400 kDa at pH 4.0 or insoluble aggregates with tubular morphology at pH 5.0, supporting a fine tuning by pH. Furthermore, the dissociation constants estimated for the low- and high affinity calcium binding sites are 13 μM and 58 nM, respectively, suggesting an intracellular calcium sensing mechanism of RSV-M upon infection.We uncover a finely tuned interplay between calcium binding, ionic strength, and pH changes compatible with the different cellular compartments where M plays key roles, revealing diverse conformational equilibria, oligomerization, and high order structures, required to stabilize the virion particle by a layer of molecules positioned between the membrane and the nucleocapsid.

Characterisation of a novel cold-adapted calcium-activated transglutaminase: implications for medicine and food processing.

Transglutaminases are a family of enzymes that catalyse the cross‐linking of proteins by forming covalent bonds between lysine and glutamine residues in various polypeptides. Cross‐linking reactions are involved in blood clots, skin formation, embryogenesis and apoptosis. Clinically, these enzymes appear to be implicated in neurodegenerative diseases, tumours and coeliac diseases. Transglutaminases have great potential for use in the food industry because of their ability to cross‐link proteins that are not normally linked. Here, a gene coding for transglutaminase from Atlantic cod was cloned into a bacterial expression vector and used to transform protein expression in a strain of Escherichia coli. The successful expression of recombinant transglutaminase protein from Atlantic cod (AcTG‐1) as a soluble protein upon induction at low temperature was confirmed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, immunoblotting and mass spectrometry analysis. Biochemical characterisation demonstrated that the transglutaminase was active between 0 and 65 °C, but was completely inactivated after 20‐min incubation at 70 °C. Interestingly, the enzyme displayed cold‐adapted features, such as temperature instability combined with high catalytic efficiency at low temperatures (8–16 °C). In addition, the enzyme had optimal activity at 50 °C, a new feature for a cold‐adapted enzyme. AcTG‐1 was active in the pH range from 6 to 9, with an optimum at pH 8, and required 5 mm calcium for maximum activity. Potential calcium‐binding sites in the enzyme were predictable, making the enzyme an appropriate model for studying structure–function relationships in the calcium‐dependent transglutaminase family. In vitro gel analysis revealed that transglutaminase cross‐linked casein, collagen and gelatin. The binding of fish fillets in the presence of recombinant AcTG‐1 provided further macroscopic proof for the potential application of AcTG‐1 as a biological cross‐linker in the food industry. Once binding occurred, fish fillets withstood further processing such as frying, boiling, freeze‐thawing and chilling. The low‐temperature activity and new enzymatic properties of AcTG‐1 appear to offer advantages over commercially available enzymatic glues in the food industry.

Structure of Human GIVD Cytosolic Phospholipase A2 Reveals Insights into Substrate Recognition

Cytosolic phospholipases A2 (cPLA2s) consist of a family of calcium-sensitive enzymes that function to generate lipid second messengers through hydrolysis of membrane-associated glycerophospholipids. The GIVD cPLA2 (cPLA2δ) is a potential drug target for developing a selective therapeutic agent for the treatment of psoriasis. Here, we present two X-ray structures of human cPLA2δ, capturing an apo state, and in complex with a substrate-like inhibitor. Comparison of the apo and inhibitor-bound structures reveals conformational changes in a flexible cap that allows the substrate to access the relatively buried active site, providing new insight into the mechanism for substrate recognition. The cPLA2δ structure reveals an unexpected second C2 domain that was previously unrecognized from sequence alignments, placing cPLA2δ into the class of membrane-associated proteins that contain a tandem pair of C2 domains. Furthermore, our structures elucidate novel inter-domain interactions and define three potential calcium-binding sites that are likely important for regulation and activation of enzymatic activity. These findings provide novel insights into the molecular mechanisms governing cPLA2's function in signal transduction.

Calcium negatively regulates meprin β activity and attenuates substrate cleavage.

The meprin β metalloproteinase is an important enzyme in extracellular matrix turnover, inflammation, and neurodegeneration in humans and mice. Previous studies showed a diminished cleavage of certain meprin β substrates in the presence of calcium, although the mechanism was not clear. With the help of a specific fluorogenic peptide assay and the human amyloid precursor protein as substrate, we demonstrated that the influence of calcium is most likely a direct effect on human meprin β itself. Analyzing the crystal structures of pro- and mature meprin β helped to identify a cluster of negatively charged amino acids forming a potential calcium binding site. Mutation of 2 of these residues (D204A and D245A) led to severe differences in proteolytic activity and cellular localization of meprin β. D245A was almost completely inactive and largely stored into intracellular vesicles, indicating severe misfolding of the protein. Astonishingly, D204A was not transported to the cell surface, but exhibited strong β-secretase activity, resulting in massive accumulation of Aβ-peptides. This could be explained by constitutive maturation of this meprin β mutant already in the early secretory pathway. We hypothesize that lacking D204 abrogates the capability of binding calcium in the catalytic domain, an important step for proper folding of the propeptide and subsequent inhibition of the protease. This is supported by the inhibition constant of calcium for meprin β (inhibitory constant 50 = 11 mM), which resembles the physiologic concentrations found in the endoplasmic reticulum. For instance, it was shown for amyotrophic lateral sclerosis that a loss of calcium in the endoplasmic reticulum leads to the misfolding of calcium-dependent proteins, which might also be relevant for proper function of meprin β.

A fibrinogen-related protein (TfFREP2) gene involving in the immune response of Trachidermus fasciatus against Vibrio anguillarum

Fibrinogen-related proteins play important roles in the immune responses. We have obtained a cDNA encoding a novel fibrinogen-related protein from roughskin sculpin Trachidermus fasciatus (T. fasciatus) and named it as TfFREP2. The N and C terminus of TfFREP2 contain a putative 21-amino acid signal peptide and a typical 217-amino acid fibrinogen-like domain, which is conserved in all fibrinogen-related proteins. TfFREP2 has three glycosylation sites and two potential calcium-binding sites that are possibly involved in calcium coordination. The results of tissue specific checking showed that the mRNA and protein of TfFREP2 were particularly abundant in skin and gill among all the tested tissues. TfFREP2 mRNA and protein expression changed significantly after being challenged by Vibrio anguillarum pathogen in those immune-barrier tissues, such as skin and gill. Furthermore, recombinant TfFREP2 is able to agglutinate and bind V. anguillarum in the presence of calcium ion. The above results suggest that TfFREP2 might be involved in the host defense of fish against V. anguillarum infection.

Hetero Interaction with an Amino Acid Globally Enhances Cooperative Activation of CaSR in Response to Extracellular Signaling

Calcium sensing receptor (CaSR), along other members of the family C G protein-coupled receptors (GPCRs), play very important roles in responding to changes in the extracellular calcium concentrations and in circulating levels of amino acids and integrating these extracellular signals into alterations in intracellular signaling pathways. However, detailed structure properties of the CaSR which are necessary to characterize the mechanism of its physiological function are still unrevealed. We have reported several potential calcium-binding sites located within the CaSR's extracellular domain using our developed computational algorithms. In the present study, we first report the differential effects of several disease-related mutations located at the predicted calcium binding sites on the inhibition and activation of intracellular calcium responses using single cell imaging. Mutating to different residues at two locations near the hinge region of the ECD could lead to either significantly lose of function of the receptor or gain of function (switch function mutations). Amino acid binding results in differential rescue effect in altering intracellular calcium responses, especially calcium oscillations.We have further probe the effect of mutation and amino acid binding on the correlation motion, cooperativity, and synergistic activation using mammalian expressed and purified extracellular domain (ECD) of CaSR with various spectroscopic methods including the tryptophan fluorescence titration, circular dichroism (CD) and nuclear magnetic resonance (NMR) as well as molecular dynamic simulation. These results provide important implications for our understanding of how the CaSR integrates information about these two completely different classes of agonists--an inorganic divalent cation, and another hand, a nutrient-- how the receptor senses these agonists in healthy and diseased states.

Disease Related Mutations Adjacent to Predicted Multiple Ca2+ Binding Sites of Ca2+-Sensing Receptor Altering Intracellular Ca2+ Oscillations via Extracellular Calcium and Amino Acid Signaling

Calcium sensing receptor (CaSR), along other members of the family C G protein-coupled receptors (GPCRs), play very important roles in responding to changes in the extracellular calcium concentrations and in circulating levels of amino acids and integrating these extracellular signals into alterations in intracellular signaling pathways. We have reported several potential calcium-binding sites located within the CaSR's extracellular domain using our developed computational algorithms. In the present study, we first report the differential effects of several disease-related mutations located at the predicted calcium binding sites on the inhibition and activation of intracellular calcium responses using single cell imaging. Interestingly, mutating to different residues at two locations near the hinge region of the ECD could lead to either significantly lose of function of the receptor or gain of function (switch function mutations). Amino acid binding results in differential rescue effect in altering intracellular calcium responses, especially calcium oscillations. We further analyzed the effect of mutation and amino acid binding on the correlation motion, cooperativity, and synergistic activation use computational methods. These results provide important implications for our understanding of how the CaSR integrates information about these two completely different classes of agonists--an inorganic divalent cation, and another hand, a nutrient-- how the receptor senses these agonists in healthy and diseased states.

Differential Activation of Intracellular Calcium Oscillations by Multiple Calcium and L-Phenylalanine Binding Sites Located at the Extracellular Domain of Calcium-Sensing-Receptor

Calcium sensing receptor (CaSR), along other members of the family C G protein-coupled receptors (GPCRs), play very important roles in responding to changes in the extracellular calcium concentrations and in circulating levels of amino acids and integrating these extracellular signals into alterations in intracellular signaling pathways. We have reported several potential calcium-binding sites located within the CaSR's extracellular domain using our developed computational algorithms based on geometric factors and surface electrostatic potentials. In the present study, we first report the differential effects of several disease-related mutations located at the predicted calcium binding sites on the inhibition and activation of intracellular calcium responses using both a cell population assay and single cell imaging. We then identify a potential amino acid binding site using computational methods and site-directed mutagenesis. The effect of amino acid binding in altering intracellular calcium responses, especially calcium oscillations, and its synergistic interaction with the effects of extracellular calcium on these parameters are also investigated. A common activation mechanism for CaSR and other family C GPCRs, such as mGluR1 by extracellular calcium and amino acid is been proposed. These results could have important implications for our understanding of how the CaSR integrates information about these two completely different classes of agonists--one an inorganic divalent cation, the other a nutrient--and how the receptor senses these agonists in health and in disease states.

The binding of HIV-1 gp41 membrane proximal domain to its mucosal receptor, galactosyl ceramide, is structure-dependent

The peptide of HIV-1 envelope gp41 (a.a 628–683), referred to herein as P5, contains P1, a conserved galactose-specific lectin domain for binding the mucosal HIV-1-receptor, galactosyl ceramide (GalCer), as shown earlier, and a potential calcium-binding site (a.a 628–648). P1 contains contiguous epitopes recognized by the broadly neutralizing antibodies 2F5, 4E10, Z13. However, similar neutralizing antibodies could not be raised in animal model using immunogens based on these epitopes. We now show that the structure of both P5 and P1 peptides, as measured by circular dichroism, differs according to their environment: aqueous or lipidic, and as a function of calcium concentration. P5, but not P1, binds to calcium with a low binding affinity constant in the order of 2.5 × 10 4. Calcium binding results in a conformational change of P5, leading in turn to a decrease in affinity for GalCer. Hence, the affinity of the gp41-lectin site for the galactose harbored by the mucosal HIV-1 receptor GalCer is modulated by the peptide secondary and tertiary structure and the local environment. Therefore, definition of the conformation of this novel extended gp41 membrane proximal region, containing the conserved peptide P1 and the Ca 2+ binding site, could help designing an immunogen efficient at inducing neutralizing anti-HIV-1 antibodies.

Cloning of the gene and characterization of the enzymatic properties of the monomeric alkaline phosphatase (PhoX) fromPasteurella multocidastrain X-73

We have identified a new phoX gene encoding the monomeric alkaline phosphatase from Pasteurella multocida X-73. This gene was not found in the published genome sequence of Pasteurella multocida pm70. Characterization of the recombinant PhoX of Pasteurella multocida X-73 showed that it is a monomeric enzyme, activated by Ca(2+) and possibly secreted by the Tat pathway. These features distinguish phosphatases of the PhoX family from those of the PhoA family. All proteins of the PhoX family were found to contain a conserved motif that shares significant sequence homology with the calcium-binding site of a phosphotriesterase known as diisopropylfluorophosphatase. Site-directed mutagenesis revealed that D527 of PhoX might be the ligand bound to the catalytic calcium. This is the first report on identification of homologous sequences between PhoX and the phosphotriesterase and on the potential calcium-binding site of PhoX.

A3 Domain Residue Glu1829 Contributes to A2 Subunit Retention in Factor VIIIa.

Factor VIII circulates as a heterodimer composed of a heavy chain (HC) and light chain (LC). Thrombin converts factor VIII into the active cofactor, factor VIIIa, by cleaving HC into A1 and A2 subunits. While the A1 subunit maintains a stable interaction with the LC-derived A3C1C2 subunit, the A2 subunit is weakly associated in the trimer and this low affinity interaction accounts for the instability of factor VIIIa activity. Although the majority of binding energy for A2 retention is thought to be derived from its interaction with the A1 subunit, the factor VIII structure model based on homology with ceruloplasmin suggests a binding interface between A2 and A3 domains. While exploring potential calcium binding sites in LC by Ala scanning mutagenesis of acidic amino acids, we encountered one mutant (factor VIII:E1829A) that exhibited an ~10-fold increase in the apparent decay rate of factor VIIIa activity compared with the wild type control, as monitored by a factor Xa generation assay (see figure). This decay rate was significantly reduced by the addition of exogenous A2 subunit. In addition, the activity of factor VIII:E1829A measured by one-stage clotting and two-stage factor Xa generation assays were ~20% and ~10% of wild type factor VIII, respectively, consistent with an assay discrepancy due to increased lability of the mutant factor VIIIa. Affinity values for A1 and A2 subunits for wild type and E1829 A3C1C2 subunits were assessed using subunits purified from the factor VIII proteins. To measure A1 subunit affinity for A3C1C2, wild type or E1829A A3C1C2 subunit (10 nM) was reacted with A1 subunit (0–200 nM), and subsequently factor VIIIa activity from the reconstituted A1/A3C1C2 plus excess A2 subunit (400 nM) was measured by factor Xa generation assay. To measure A2 subunit affinity for A3C1C2, wild type or E1829A A3C1C2 subunit (1 μM) and A1 subunit (100 nM) were reacted and then mixed with A2 (0–1 μM) and regenerated cofactor activity was determined. A1 binding affinity for E1829A-A3C1C2 was comparable to the wild type A3C1C2 (Kd = 20.1 ± 3.4 nM and 15.3 ± 3.7 nM, respectively) while A2 binding affinity was reduced (Kd = 385 ± 54 nM for E1829A, 153 ± 34 nM for wild type). Furthermore, examination of the hemophilia A database shows the mutation of Glu1829 to Gly as a mild phenotype consistent with a functional role for this acidic residue. These results suggest that residue E1829 in the A3 domain contributes to A2 subunit retention following activation of the procofactor, possibly by mediating a binding interaction between the two subunits. [Display omitted]

Essential Role of B-helix Calcium Binding Sites in Annexin V-Membrane Binding

Crystal structures of annexin V have shown up to 10 bound calcium ions in three different types of binding sites, but previous work concluded that only one of these sites accounted for nearly all of the membrane binding affinity of the molecule. In this study we mutated residues contributing to potential calcium binding sites in the AB and B helices in each of the four domains (eight sites in total) and in DE helices in the first, second, and third domains (three sites in total). We measured the affinity of each protein for phospholipid vesicles and cell membranes by quantitative calcium titration under low occupancy conditions (< 1% saturation of available membrane binding sites). Affinity was calculated from the midpoint and slope of the calcium titration curve and the concentration of membrane binding sites. The results showed that all four AB sites were essential for high affinity binding, as were three of the four B sites (in domains 1, 2, and 3); the DE site in the first domain made a slight contribution to affinity. Multisite mutants showed that each domain contributed additively and independently to binding affinity; in contrast, AB and B sites within the same domain were interdependent. The number of functionally important sites identified was consistent with the Hill coefficient observed in calcium titrations. This study shows an essential and previously unappreciated role for B-helix calcium binding sites in the membrane binding of annexins and indicates that all four domains of the molecule are required for maximum membrane binding affinity.

Functional properties of recombinant factor V mutated in a potential calcium-binding site.

Activated coagulation factor V (FVa) is a cofactor of activated factor X (FXa) in prothrombin activation. FVa is composed of a light chain (LC) and a heavy chain (HC) that are noncovalently associated in a calcium-dependent manner. We constructed a recombinant FV Asp111Asn/Asp112Asn mutant (rFV-NN) to abolish calcium binding to a potential calcium-binding site in FVa in order to study the specific role of these residues in the expression of FVa activity. Whereas thrombin-activated recombinant FV wild type (rFV-wt) presented with stable FVa activity, incubation of rFV-NN with thrombin resulted in a temporary increase in FVa activity, which was rapidly lost upon prolonged incubation. Loss of FVa activity was most likely due to dissociation of HC and LC since, upon chromatography of rFVa-NN on a SP-Sepharose column, the HC did not bind significantly to the resin whereas the LC bound and could be eluted at high ionic strength. In contrast, rFVa-wt adhered to the column, and both the HC and LC coeluted at high ionic strength. In the presence of phospholipid vesicles, the loss of rFVa-NN activity was partially prevented by FXa, active site inhibited FXa, and prothombin in a dose-dependent manner. We conclude that the introduced amino acid substitutions result in a loss of the high-affinity (calcium-dependent) interaction of the HC and LC of FVa. We propose that the introduced substitutions disrupt the calcium-binding site in FV, thereby yielding a FV molecule that rapidly loses activity following thrombin-catalyzed activation most likely via dissociation of the HC and LC.

Calcium Binding Properties of γ-Crystallin: CALCIUM ION BINDS AT THE GREEK KEY βγ-CRYSTALLIN FOLD

The beta- and gamma-crystallins are closely related lens proteins that are members of the betagamma-crystallin superfamily, which also include many non-lens members. Although beta-crystallin is known to be a calcium-binding protein, this property has not been reported in gamma-crystallin. We have studied the calcium binding properties of gamma-crystallin, and we show that it binds 4 mol eq of calcium with a dissociation constant of 90 microm. It also binds the calcium-mimic spectral probes, terbium and Stains-all. Calcium binding does not significantly influence protein secondary and tertiary structures. We present evidence that the Greek key crystallin fold is the site for calcium ion binding in gamma-crystallin. Peptides corresponding to Greek key motif of gamma-crystallin (42 residues) and their mutants were synthesized and studied for calcium binding. These peptides adopt beta-sheet conformation and form aggregates producing beta-sandwich. Our results with peptides show that, in Greek key motif, the amino acid adjacent to the conserved aromatic corner in the "a" strand and three amino acids of the "d" strand participate in calcium binding. We suggest that the betagamma superfamily represents a novel class of calcium-binding proteins with the Greek key betagamma-crystallin fold as potential calcium-binding sites. These results are of significance in understanding the mechanism of calcium homeostasis in the lens.

Three-dimensional model of the SHBG-like region of anticoagulant protein S: New structure-function insights

Protein S (PS) is a vitamin K-dependent glycoprotein that consists of several modules including a C-terminal sex hormone-binding globulin (SHBG)-like domain that has been subdivided into two laminin LG-type domains. The SHBG-like region of PS is known to bind to a complement regulator molecule, C4b-binding protein (C4BP), coagulation factor Va (FVa) and receptor tyrosine kinases. Inherited PS deficiency has been associated with thromboembolic disease. Yet, study of the mechanisms by which the SHBG-like region of PS serves its essential functions has so far been hampered because of the lack of structural information. Recently, the three-dimensional (3D) structure of LG domains from plasma SHBG, laminin and neurexin have been reported and were found related to the pentraxin family. We used these X-ray structures to build homology models of the SHBG-like region of human PS. We then analyzed previously reported experimental/clinical data in the light of the predicted structures. A potential calcium-binding site is found in the first LG domain of PS and D292 could play a role in this process. This region is close to the interface between the two LG domains and is also surrounded by segments that have been suggested by synthetic peptide studies to be important for C4BP or FVa binding. The 39 point mutations linked to PS deficiencies or reported as neutral variants were rationalized in the 3D structure. Proteins 2001;43:203-216.

Histoplasma acquisition of calcium and expression of CBP1 during intracellular parasitism.

A highly adapted parasite of macrophages, the yeast phase of Histoplasma capsulatum, survives and proliferates within phagolysosomes, while the mycelial phase exists only as a saprophyte in the soil. We have shown previously that these two phases of Histoplasma differ in their calcium requirements for growth and in the production of a released calcium-binding protein (CBP). Cloning and sequencing the CBP1 gene revealed two introns, a putative signal peptide and potential calcium-binding sites. We also evaluated CBP1 expression by reverse transcription-polymerase chain reaction (RT-PCR) of yeasts grown in broth culture and within two host cell types, a macrophage-like cell line and respiratory epithelial cells. H. capsulatum yeasts expressed CBP1 in all of these settings. Splenocytes from mice immunized with H. capsulatum yeasts responded to purified CBP in proliferation assays, providing evidence for the production of CBP during the infection of mammalian hosts. In addition, after H. capsulatum yeasts were subjected to a calcium-free shock, exogenously added CBP allowed yeasts to incorporate more calcium than yeasts incubated without added CBP. These results suggest that CBP may function to provide yeasts with calcium when they are in a low-calcium environment, such as the phagolysosomal compartment within macrophages.

X-Ray studies reveal lanthanide binding sites at the A/B 5 interface of E. coli heat labile enterotoxin

The crystal structure determination of heat labile enterotoxin (LT) bound to two different lanthanide ions, erbium and samarium, revealed two distinct ion binding sites in the interface of the A subunit and the B pentamer of the toxin. One of the interface sites is conserved in the very similar cholera toxin sequence. These sites may be potential calcium binding sites. Erbium and samarium binding causes a change in the structure of LT: a rotation of the A1 subunit of up to two degrees relative to the B pentamer.

Sequence of the pckA gene of Escherichia coli K-12: relevance to genetic and allosteric regulation and homology of E. coli phosphoenolpyruvate carboxykinase with the enzymes from Trypanosoma brucei and Saccharomyces cerevisiae

The sequence of the pckA gene coding for phosphoenolpyruvate carboxykinase in Escherichia coli K-12 and previous molecular weight determinations indicate that this allosteric enzyme is a monomer of Mr 51,316. The protein is homologous to ATP-dependent phosphoenolpyruvate carboxykinases from Trypanosoma brucei and Saccharomyces cerevisiae. A potential ATP binding site was conserved in all three sequences. A potential binding site for the allosteric activator, calcium, identified in the E. coli enzyme, was only partially conserved in T. brucei and S. cerevisiae, consistent with the observation that the enzymes from the latter organisms were not activated by calcium. The published sequence of the ompR and envZ genes from Salmonella typhimurium is followed by a partial sequence that is highly homologous to pckA from E. coli. The order of these genes and the direction of transcription of the presumptive S. typhimurium pckA gene are the same as those in E. coli. The potential calcium binding site of the E. coli enzyme is conserved in the partial predicted sequence of the S. typhimurium phosphoenolpyruvate carboxykinase, consistent with the observation that calcium activation of the S. typhimurium phosphoenolpyruvate carboxykinase is very similar to that observed for the E. coli enzyme. A pckA mRNA transcript was observed in stationary-phase cells but not in logarithmically growing cells. The mRNA start site was mapped relative to the sequence of the pckA structural gene.

Correction of the N-Terminal Sequences of the Human Plastin Isoforms by Using Anchored Polymerase Chain Reaction: Identification of a Potential Calcium-Binding Domain

Plastins are a family of at least three cytoplasmic protein isoforms that are expressed differentially between cells of the hematopoietic lineages and cells of solid tissues. Expression of the L-plastin isoform appears to be restricted to replicating blood cells, and the two T-plastin isoforms appear to be restricted to replicating cells of solid tissues. However, L-plastin is induced in many human solid tumor-derived cells. We used the anchored polymerase chain reaction technique to amplify and clone the missing 5' ends of plastin mRNAs. We found that both plastin isoforms contain a potential calcium binding site near the N terminus.

Correction of the N-terminal sequences of the human plastin isoforms by using anchored polymerase chain reaction: identification of a potential calcium-binding domain.

Plastins are a family of at least three cytoplasmic protein isoforms that are expressed differentially between cells of the hematopoietic lineages and cells of solid tissues. Expression of the L-plastin isoform appears to be restricted to replicating blood cells, and the two T-plastin isoforms appear to be restricted to replicating cells of solid tissues. However, L-plastin is induced in many human solid tumor-derived cells. We used the anchored polymerase chain reaction technique to amplify and clone the missing 5' ends of plastin mRNAs. We found that both plastin isoforms contain a potential calcium binding site near the N terminus.