Preparation and purification

Spectrin is extracted from hemoglobin-free erythrocyte membranes by incubating it at 37^oC in 5-10 volume of extraction buffer (0.1mM EDTA, 0.5mM 2-mercaptoethanol, 0.2mM DFP, pH 9.0). The membranes are removed by centrifugation. The suprenatant includes primarily spectrin and actin. Spectrin hetrodimer (aplha and beta subunits) is purified by Sepharose CL-4B gel chromatography. Spectrin dimer dissociates into alpha and beta subunits at alkaline pH (> pH 10.5) or in the presence of 3M Urea. Both subunits can be separated by Mono-Q ion-exchange chromatography [JBC 267:14775-82 (92), Biochem. 37:272-80 (98)]

FAD-dependent, mitochondrial glycerol-3-phosphate dehydrogenase (EC 1.1.99.5) is located in the outer surface of the inner mitochondrial membrane and exists as a single subunit. Mammalian enzyme shows strong homology to yeast and bacterial FAD-dependent glycerol phosphate dehydrogenase and has two EF-hands at its C-terminus unlike yeast and bacterial enzyme [JBC 269:14363 (94)].

Allograft inflammatory factor-1 (AIF1) is "a cytokine inducible, tissue-specific transcript transiently expressed in response to vascular trauma." [BBRC 228:29 (96)]. It has 143 residues with two EF-hands. Homologs of the first fifty residues have not been identified. The first domains of AIF1 and of GPD are most similar. The arrangement of possible calcium binding ligands (S-G-G---S--D) of the second EF-hand is unusual.

Preparation and purification

Osteonectin was extracted from bone with 0.5M EDTA, then purified by using gel filtration and polyanion anion-exchange fast protein liquid chromatography [Biochem. J. 253:139-51 (88)]. BM40 was purified from basement-memebrane-producing mouse EHS tumor [EJB 161:405-64(86)]. SPARC is a major product of mouse embryo parietal endoderm [EMBO J. 5:1465-72 (86)]. SPARC wa purified from primary Sertoli cell enriched culture medium by sequential anion-exchange, gel-permeation, C4 reversed phase, and diphenyl reversed phase HPLC [BBRC 167:1393-9 (90)].

Physical characteristics

BM40 binds calcium and several types of collagen at the EF-hand and the alpha-helical domains in C-terminal half of the molecule. It also binds plasminogen. Am 80 kDa protein purified from bone cross-links to BM40 with disulfide [BJ 330:1423-31 (98)].

Covalent modification

N-linked glycosylation in BM40 is observed. There are two potential N-glycosylation site in human osteonectin but only one site (Asn99-X-Thr-101) is glycosylated [JBC 270:23212-7 (95)].

QR1, named from the cDNA, QR1, cloned from the neuroretina of quail, Coturni. coturnix has 676 residues. Its C-terminal portion is similar to the extracellular glycoprotein, BM40. QR1 and BM40 are the only examples of (normally) extracellular EF-hands proteins. Transcription of QR1 takes place only during the late phase of retinal development and is shut off sharply at hatching. [PNAS 88:4503 (91)]].

A 542 residue protein of unknown function has been isolated from the nematode, Onchocerca volvulus, as well as its close homolog of 482 residues from Caenorhabditis elegans [Infec. Immun. 8:3491-501 (00)]. It has twelve EF-hands, eleven of which conform well to the consensus of EF-hand and appear to bind calcium. The fourth EF-hand is highly diverged; however, of all EF-hand domains it most closely resembles EF12 #2. The eleven interdomain spacings are all six residues; this pattern strengthens the interpretation that the 41 (6 + 29 + 6) residues between EF-hands 3 and 5 harbors an aberrant domain. In a dendrogram the six ODD domains cluster together and are congruent with the six EVEN domains. Further domains 1(2) & 7(8), 3(4) & 9(10), and 5(6) & 11(12) most closely resemble one another, reflecting four cycles of gene duplication and fusion. It is surely significant that the O. volvulus and the C. elegans proteins align well, with no deletions, over the 414 residues (12 * 29 + 11 * 6) residues encompassing the twelve EF-hands but show many deletions in the N-terminal and C-terminal domains. The six pairs of EF-hands might be arranged helically and undergo a cooperative change in conformation upon binding calcium.

LPS consists of eight EF-hand domains. Domains 1 and 5, domains 2 and 6, domains 3 and 7, and domains 4 and 8 are similar. LPS evolved by a recent duplication of a four-domain precursor. These four pairs of domains are similar to the four domains of SPEC; however, they are not the closest neighbours. The postulate that LPS evolved by gene duplication and fusion from the ancestor of SPEC is strengthened by the similar distribution of introns in the two halves of LPS and in SPEC [6516]. The function of LPS is not known.

CLBN is found in several epithelial tissues, and in many neurons. Its synthesis is induced by vitamin D and is associated with calcium absorption in intestine. However, it is not congruent with ICBP, sometimes referred to as calbindin D9k. Calretinin is found mainly in a variety of neurons. It is also found in developing rat ovary. Calretinin is similar to CLBN and is placed in the same subfamily; however, the functions of both proteins remain unknown.

Domains 1, 3 & 5 and domains 2, 4 & 6 are more closely related. Interdomains 1,2 and 3,4 and 5,6 are similar and are inferred to be homologous. CLBN appears to have evolved by two duplications and fusions from a two domain precursor. The six domains, individually synthesized, aggregate in pairwise combination to approximate the spectral properties of the intact CLBN [Prot. Sci. 6:2385 (97)].

Preparation and purification

CLBN is characterized by its increased electrophoretic mobility in nondenaturating gels in the presence of EDTA. It is heat stable; however, heat treatment can be circumvented by ammonium sulfate precipitation. CLBN is further purified by the calcium-dependent elution on DEAE cellulose [1726, 5252].

Calretinin is partially purified from brain homogenate by precipitation in 60-80% saturated ammonium sulfate. Further purification is carried out by combination of ion-exchange chromatography and gel-filtration chromatography [6067].

Physical characteristics

CLBN and calretinin have six EF-hand domains. Both proteins can bind four equivalents of calcium [276].

The gene product of eps15 (EP15) encodes a protein of 897 residues. The first domain of 282 residues contains a candidate phosphorylation site as well as six putative EF-hands, two of which are inferred to bind calcium. EP15 is phosphorylated on tyrosine following EGFR activation by EGF in vivo, or directly by EGFR in vitro [Mol. Cell. Biol. 13:5814 (93)].

Ciliary reversal in Tetrahymena pyriformis is calcium-dependent. TCBP is inferred to be the calcium-binding protein that regulates ciliary reversal [5267]. TCBP-23 is 207 and TCBP-25 218 residues long. Although they are only 35% identical in amino acid sequence, their eDNA sequences are 49% identical [6472]. Domains 1 and 3 and domains 2 and 4 more closely resemble one another and interdomains 1,2 and 3,4 are similar. The origin of the inferred two-domain precursor is obscure.

Preparation and purification

TCBP is purified from Tetrahymena cilia extracts by heat treatment, ammonium sulfate fractionation, DEAE-cellulose ion-exchange chromatography, and hydroxyapatite column chromatography. TCBP is also purified by calcium-dependent binding to phenyl-Sepharose 4B [5267, 6472].

Physical characteristics

TCBP is inferred to function in ciliary reversal. It shows a calcium-dependent change in electrophoretic mobility shift [5267].

P26olf (P26) was prepared from the olfactory epithelium of Rana catesberiana [BBRC 251:860-7 (98)]. P26 has 217 residues. The two putative EF-hands in the first half of P26 are most similar to the two in the second half; as in TCBP this reflects a recent gene duplication. EF-hand 3 is separated from EF-hand 2 by 36 residues that are 60% identical to the C-terminal twenty residues of a rabbitt S100. The expectation value of 0.97 indicates that the alignment is not significant for the entire database but this similarity with another EF-hand protein is interesting. None of the EF-hands are inferred to bind calcium in canonical conformation. However, P26 is purified by elution from phenyl Sepharose (following initial binding in 1.0 mM [Ca²⁺]) with EGTA. It is possible that domains 1 and 3 have an unconventional coordination as found in domains 1 of S100, ICBP, and HYFL.

Preparation and purification

P26Olf was extracted from frog olfactory epithelium. It was purified by calcium-dependent binding to phenyl Sepahrose, DEAE Sepharose CL-4B column chromatography, gel filtration HPLC and Mono-Q ion-exchange column chromatography [BBRC 251:860-7 (98)].

Physical characteristics

Phospholipase C (PLC) [EC 3.1.4.3] hydrolyzes phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5-trisphosphate and diacylglycerol which activate IP3 receptor calcium channel and protein kinase C, respectively. It has an absolute requirement for calcium and is activated by guanine nucleotide binding proteins (PLC-beta), or by receptor tyrosine kinases (PLC-gamma), or by transglutaminase II (PLC-delta). All three vertebrate isoforms of PLC have a plekstrin homolog domain, followed by four EF-hands, a triosephosphate isomerase-like beta-barrel catalytic domain, and a C2 domain [56]. Although calcium is absolutely required for activity, it is bound to the catalytic domain. None of the four EF-hands have canonical calcium binding loops nor is Ca²⁺ bound in the crystal structure [Nature 380:595 (96)].

Two similar proteins, CBP1 and CBP2, have been identified in Dictyostelium discoideum [FEBS Lett. 382:198 (96)]. Both contain four EF-hands and bind calcium in gel overlay experiments. Expression of CBP1 and CBP2 is increased during development.

PFS is membrane associated and the major calcium binding protein in Plasmodium falciparum [6409]. PFS is 343 residues long and has six EF-hands, all of which are inferred to bind calcium. The N-terminus has a secretory signal sequence (3-20). The C-terminal hydrophobic domain 345-359 may signal transfer to a glycosylphosphatidylinositol anchor. The C-terminal IDEL motif indicates that PFS is found within the endoplasmic reticulum [Mol. Biochem. Parasitol. 89:283 (97)].

Preparation and purification

PFS is purified by two-dimensional gel electrophoresis [6409].

CDPK is CAM-independent. It is found in root and shoot tissues of soybean (Glycine max) and oats (Arena sativa). Immunocytological localization indicates association with the microfilament system. Oat CDPK associates with plasma membranes. The 328-residue kinase domain at N-terminus is homologous to CAM kinase II. The four EF-hands at the C-terminus are all inferred to bind calcium. A pseudosubstrate sequence and a CAM-binding motif are found at the junction region between the kinase domain and the CAM-like domain. A membrane ATPase is one of the substrates of CDPK [BBA 1350:109 (97)].

Preparation and purification

CDPK is purified from soybean by DEAE-cellulose ion-exchange chromatography, phenyl-Sepharose hydrophobic interaction chromatography, and Sephadex G-100 gel chromatography. The final purification is achieved by chromatography on Cibacron-blue Sepharose [5279].

Physical characteristics

Proteolytic cleavage sites

CDPK from oats is normally present as a calcium and lipid-dependent kinase that is capable of associating with plasma membrane. By immunological analysis, CDPK is present as Mr = 61000 (major) and Mr=79000 (minor) bands. Both forms are calcium- and lipid-dependent and are associated with plasma membrane. Soluble calcium-dependent lipid-independent kinase is also purified from oats; it is supposed that this is a proteolytic degradation product generated during purification [4181].

Plasmodium falciparum has a gene that encodes an inferred protein kinase. The C-terminus contains four EF-hands, all of which probably bind calcium. The DNA that encodes PFPK contains four introns: one between domains 1 and 2 and one each within domains 2, 3, and 4 [6527]. It has not been characterized.

PFPK is 524 long and is encoded by the PfCPK gene from P. falciparum. Residues 56-325 encode a region homologous to protein kinases and to CDPK, a protein kinase from plant, with which it shares ~40% sequence identity. The catalytic domains are homologous; however the four EF-hands of PFPK are not congruent with CDPK nor are they congruent with CTER, as is CDPK. The two chimeric kinases probably acquired their respective four EF-hands by different duplication and splicing events.

SPEC mRNAs begin to accumulate 20 h after fertilization in ectoderm cells of the sea urchin embryo [JMB 202:417 (88)]. A 15 kDa protein in eggs of sea urchin, Hemicentrotus pulcherrintus, also belongs to this subfamily. This protein is localized in nuclei of fertilized eggs and in the mitotic apparatus of dividing eggs. The antibody against this protein arrests cell division; hence, SPEC is inferred to play an important role in mitosis of sea urchin egg. SPEC is about 150 residues long and contains four EF-hands. .

Preparation and purification

The Hernicentrotus protein is purified from eggs by using DEAE-cellulose ion-exchange chromatography, hydroxyapatite chromatography, and Sephadex G-50 gel chromatography [5221].

Physical characteristics

The Hemicentrotus protein binds calcium, and shows calcium-dependent electrophoretic mobility change. SPEC does not activate cAMP-phosphodiesterase, and does not bind to fluperazine-Sepharose in the presence of calcium [5221].

p24 thyroid protein (TPP), or calcyphosine, consists of four EF-hands. Domains 3 and 4 resemble one another most closely. This deviation from the ODD, EVEN pattern may reflect a recent duplication. The function of TPP is unknown. The synthesis and phosphorylation of p24 is upregulated by thyrotropin (TSH) and cyclic AMP agonists. Its synthesis is downregulated by dedifferentiating agents such as epidermal growth factor and 12-O-tetradecanoylphorbol-l3-acetate [6320]. TPP has 189 residues in four EF-hand domains. Another member of the subfamily has been found in lobster and crayfish muscle [EJB 227:97 (95)].

Preparation and purification

p24 was identified by two-dimensional gel electrophoresis of dog thyroid cell proteins [6320]. CCBP-23, a crayfish homolog of TPP, was purified from muscle tissue extract by using calcium-dependent hydrophobic -interaction chromatography on a pheny Sepharose CL-4B column. Further purification was performed by FPLC anion-exchange chromatography on a Mono-Q column [EJB 227:97 (95)].

Physical characteristics

p24 is phosphorylated in intact cells in response to TSH. The putative phosphorylation site is Ser40, which is in the first calcium-binding loop at position -Y. The fourth domain contains an inserted Glu after Ser at the Y vertex. The putative calcium coordination might be either the sequence SEDTE or DSTDE, similar to the calcium binding of domain 3, DSDTD. More unique is the insertion of 17 residues --- EFQDYYSGVSASMDTDE --- instead of the Glu at position -Z. Whether this insertion results in an F helix five turns longer and/or in altered calcium affinity is unknown [6320]. The affinity and stoichiometry of calcium binding has not been determined. pI of CCBP-23 is 5.4. A disulfide-linked homodimer was formed in the absence of reducing agents [EJB 227:97 (95)].

Covalent modification

N-terminal Ser of CCBP-23 is acetylated [EJB 227:97 (95)].

1F8 is 211 residues long, TB17 is 233 residues long and both consist of four EF-hands, all of which are inferred to bind calcium.

Physical characteristics

Sarcoplasm calcium binding protein (SARC) binds three Ca²⁺ or Mg²⁺ ions competitively. It is found both as a monomer and as a dimer; it may function as a calcium buffer. Neither the sequences of its four EF-hands nor of the three interdomain linkers closely resemble other EF-hands or linkers. Crystal structures are available of SARC from the sandworm, Nereis diversicolor, [JBC 266:652 (91)] and from amphioxus, Branchiostoma lanceolatum, [JMB 229:461 (93)]. Although the sequences share only 22% overall identity, the main chain traces of the two are nearly superimposable. All four domains have Ca²⁺ bound. The hydrophobic faces of lobes 1,2 and 3,4 contact one another.

Preparation and purification

SARC is extracted from muscle in the presence of calcium and protease inhibitors. The extract is fractionated by ammonium sulfate fractionation, Sephadex G-100 gel chromatography and DEAE-cellulose ion-exchange chromatography in the presence of calcium. Finally, it is purified by DEAE-cellulose ion-exchange chromatography in the presence of EDTA [287].

Physical characteristics

SARCs exist as both monomers and dimers. The relative and absolute affinities for both calcium and magnesium vary significantly with the type of SARC [224].

AEQ is about 189 residues long; numerous isoforms are found in one organism. The easily recognized domains are numbered 1, 3, and 4. Residues 44-107 might correspond to a fourth EF-hand diverged beyond recognition. A similar ambiguity exists for PPTS. Aequorin (AEQ) (EC 1.13.12.5 renilla-luciferin 2-monooxygenase) is a calcium-dependent photoprotein that oxidizes luciferin (coelenterazine) and thereby produces the luminescence of the marine coelenterate, Aequoria victoria [Biochem. 26:1326 (87)]. In the anthozoan, Renilla reniformis, calcium induced bioluminescence involves two protein, luciferase and luciferin binding protein (LBP). LBP has four EF-hands but lacks the luciferase activity of aequorin [FEBS Lett. 268:287 (90)].

Preparation and purification

Aequorin is purified from Aequorea by ammonium sulfate fractionation (25-75% of saturation), Sephadex G-50 gel filtration, QAE-Sephadex A-50 ion-exchange chromatography, a second Sephadex G-50 gel filtration, and DEAE-Sephadex A-50 ion-exchange chromatography [5151]. The luciferin-binding protein is purified from Renilla by aluminum hydroxide gel adsorption, DEAE-cellulose column chromatography, Sephadex G-75 gel chromatography, DEAE-Biogel ion-exchange chromatography, p-benzyloxyaniline Sepharose column chromatography, and Sephadex G-75 gel chromatography [5160].

Physical characteristics

The luminescence reaction of luciferin (colenterazine) requires molecular oxgen. Aequorin binds both celeterazine and molecular oxygen as a chromophore. After the binding of three equivalents of calcium to aequorin, coelenterazine is oxidized to coelentramide by the bound oxygen. This yields light (l~ max = 470 nm), CO2 and a blue fluorescent protein that consists of apoaequorin and ceolenteramide [5291]. Celenterazine has been proposed to exist in peroxidized form. It was revealed by X-ray crystallography that aequorin is a globular molecule containing a hydrophobic core cavity that accommodates the ligand coelenterazine-2-hydroperoxiside. The overall conformation is most similar to SARC [Nature 405:372-376 (00)]. Several hydrogen bonds stabilizes the binding of celentrazine-2-hydroperoxiside including the Y184-H169-W173 triad which interacts the hydroperoxiside coupled to celetrazine. AEQ has only a few residues N- and C-terminal to the four EF-hand domains. The triad is in the C-terminal extention. When calcium binds at either or both EF-hands 1 and 4, helix orientation of these EF-hands would change which induces the disruption of hydrogen bond network including Y184-H169-W173. No longer stabilized, the peroxiside is free to initiate the light-emitting reaction [Nature 405:372-376 (00)]. Renilla contains a luciferase, which catalyses the oxidation of luciferin in the presence of dissolved oxygen producing oxiluciferin and C02. This is the classical luciferin-luciferase reaction. Luciferase purified in the presence of EDTA is not calcium sensitive; it can oxidize free luciferin. The calcium-triggered luciferin-binding protein restores calcium sensitivity to bioluminescence. The luciferin-binding protein, like aequorin, contains three calcium-binding EF-hand domains. A region of about 40 residues between EF-hands 1 and 2 is of uncertain evolutionary origin and may be involved in binding luciferin. In the presence of calcium this bound luciferin is presented to the luciferase in a reactive state [5160].

Protein phosphatase (PPTS) (EC 3.1.3.16) is encoded by the rdgC gene of D. melanogaster. PPTS is 661 residues long and has a phosphoprotein phosphatase domain at its N-terminus that is 30% identical with the catalytic domains of type 1, 2A and 2B serine/threonine protein phosphatases. Three EF-hands are easily recognized and indicated in table 1 as 1, 3 & 4 [6446]. Nominal EF-hand 2, occupying 56 residues between domains 1 and 3, may be a far diverged EF-hand paired with domain 1, another example of guilt by association.

The cDNA clone, Hra32, corresponds to a RNA transcript that accumulates in Phaseolus vulgaris (jack bean) during a hypersensitive reaction [Mol. Plant Microbe Interact. 12:712 (99)]. The encoded protein, H32, of 161 residues, has four EF-hands, all of which are predicted to bind calcium.

EFH5 is the product of the EF-hand 5 gene originally identified in Trypanosoma brucei (TbEFH5) and recently in T. cruzi (TcCUB) and in Leishmania tarentolae (LtEFH5). The inferred calcium-binding ability for the domains of the TbEFH5 product is -++-, and for both TcCUB and LtEFH5 Whether EFH5 performs the same function in the three organisms remains to be seen. The TbEFH5 gene is transcribed as a polycistronic precursor RNA. It follows the CAM gene C by 111 bp and precedes ubiquitin-EP52/l by 116 bp and is inferred to be under the control of a single distant upstream promoter [6513, 6106]. The TbEFH5 gene is transcribed as a polycistronic precursor RNA. It follows the CAM gene C by 111 bp and precedes ubiquitin-EP52/l by 116bp [6513, 6106]. EFH5 is inferred to be under the control of a single distant upstream promoter in T. brucei, T. cruzi, and L. tarentolae.

CVP protein is inferred to be involved in stimulus-contraction coupling in Amphioxus; however, its exact function remains unknown.

CVP is 161 residues long and consists of four EF-hand domains. It is unusual among the EF-hand proteins, which are all found in the cytosol, in having a disulfide bond. Cys16 (1.2) and Cys78 (2.26) are near one another in the canonical lobe. In the model [Prot. Engineering 4:23 (90)] of CVP, based on the crystal structures of CAM and of TNC, a target (-helix is bound between the two lobes of CVP and the disulfide bond linkingalpha-helices E1 and F2 is accommodated with no steric interference.

Preparation and purification

CVP is purified from Amphioxus muscle by heat treatment, ion-exchange chromatography and gel chromatography [5170].

Physical characteristics

CVP has four EF-hand domains and binds two Ca²⁺ ions noncooperatively. CVP interacts with an endogenous 36 kDa protein in a calcium dependent manner [5170]. Domains 3 and 4 are inferred to bind calcium. There are N-trimethyllysines at positions 3.7 and 3.28.

PM129 clone from A. thaliana (PMAT) cDNA, enriched for plasma membrane associated proteins, contains a single continuous open reading frame [Plant Physiol. 102:1059 (93)]. The 37 residues before the first of four EF-hand domains may comprise a distinct domain. Each of the linkers 1,2 2,3 and 3,4 is eight residues long. There are three Cys residues in the fourth EF-hand and one in the 3,4 linker; a disulfide bond could be formed without distorting the canonical EF-hand. It is strange that domains 1 and 2 of PMAT most closely resemble one another and that domain 4 of PMAT closely resembles domains 1 and 3 of CVP.

LAV1 cDNA was identified as a plasmodial stage specific mRNA of Physarum polycephalurn [3755]. The 220-residue domain at the N-terminus has no known homolog, which is alpha helical. The four EF-hands at the C-terminus are inferred to bind calcium. LAV1 cDNA encodes a 355 amino acid protein. There are four EF-hand domains at the Cterminus. LAV may not be congruent with any other EF-hand protein, but interdomains 1,2 and 3,4 resemble 1,2 and 3,4 of CTER. Linker 2,3 consists of a sole Leu. This places severe constraints on the spatial relationship between the N-terminal lobe and the C-terminal lobe. The overall conformation of LAV1 EF-hand domain is similar to CALP whose linker 2, 3 also consists of only one amino acid. The function of this protein is unknown and no homolog has been identified for the residues at the N-terminus.

Physical characteristics

Recombinant LAV1 protein (CBP40) binds 4 atoms of calcium ions per molecule with pCa (1/2) = 6.5. When N-terminal alpha helical domain was removed, the affinity for calcium decreased to pCa (1/2) = 4.6 [Biochem. 39:3827-34 (00)].

CMSE is the only known EF-hand protein from prokaryotes [6455]. It has not been characterized. CMSE is 177 residues long and has four EF-hand domains.

The entire genome of the poxvirus (Entomopoxvirinae) of the North American migratory grasshopper, Melanoplus sanguinipes, is 236 kbp and contains 267 open reading frames, of which 107 are similar to previously described genes [J. Virol. 73:533 (99)]. One gene encodes MSV, 140 residues. MSV has four EF-hands, the first two of which are inferred to bind calcium. It is the only known EF-hand protein from a virus.

Preparation and purification

PARV is purified by a three-step procedure involving ammonium sulfate fractionation, Sephadex G-75 gel filtration, and DEAE-cellulose ion-exchange chromatography [5274]. Oncomodulin is purified by heat treatment, ammonium sulfate fractionation and DEAE cellulose ion-exchange chromatography [3167].

Avian thymic hormone is purified from chicken thymus glands by heat treatment, acid precipitation, Sephadex chromatography and DEAE- or antibody affinity chromatography [6136].

Physical characteristics

Parvalbumin has two high-affinity Ca²⁺-Mg²⁺ sites. It binds two Mg²⁺ ions in resting muscle. After stimulation the substitution of Mg²⁺ by Ca²⁺ ions occurs more slowly than does the binding of calcium to the low-affinity sites of TNC. This explains why calcium can trigger contraction in the presence of a high concentration of high affinity Ca²⁺-Mg²⁺ sites of PARV in muscle cells. Relaxation occurs when Ca²⁺ diffuses from the low-affinity site of TNC to the high-affinity Ca²⁺-Mg²⁺ sites of PARV [311].

Oncomodulin is similar to parvalbumin in its amino acid sequence, but has different metal ion binding properties. It has one high-affinity Ca²⁺-Mg²⁺ site and one low-affinity Ca²⁺-specific site [286]. It has Asp at -Z domain 3; all other PARVs have Glu.

Avian thymic hormone has similar cation binding properties to those of parvalbumin [427].

Covalent modifications

The N-terminus of PARV is acetylated.

Ras guanyl nucleotide-releasing protein (GRP) has 795 residues and contains a homolog of a REM domain, a CDC25 domain, two EF-hands, and a diacylglycerol binding domain [Science 280:1082 (98)]. The two EF-hand loops are canonical and are expected to bind calcium. Helices E1 and F2 appear conventional. However, there are only 15 residues for helices F1 and E2. One or both of these helices is truncated and the relationship between the two EF-hands is unclear. Rat fibroblasts expressing the RasGRP encoding gene vector are transformed. Addition of a DAG analog causes sustained activation of Ras-Erk signaling and changes in cell morphology.

The PKD2 gene product (PKD) has 968 residues. Mutations in this gene in humans segregate with polycystic kidney disease [Science 272:1339 (96)]. The homolog, PKD1, constitutes a voltage-activated calcium (and sodium) channel in Caenorhabditis elegans. PKD has six membrane spanning helices. Two EF-hands follow the sixth transmembrane helix, a hundred residues from the C-terminus.

The ryanodine receptor (RYR) functions as a channel for release of calcium from endoplasmic reticulum of muscle and brain. The isoforms are ~5000 residues long and contain two EF-hands, the second of which is inferred to bind calcium. The channels open in the presence of ~10^-6 M Ca²⁺ (calcium induced calcium release) and closed at ~10^-3 M Ca²⁺. The EF-hands have low calcium affinity and have been suggested to function in closing the channels [Biochem. 37:4804 (98)].

CBL binds to phosphorylated tyrosine residues and functions as a negative regulator of many signaling pathways. The crystal structure [Nature 398:84 (99)] of CBL (minus residues 1 - 24) alone and in complex with a phosphotyrosine decapeptide that represents its binding site in ZAP-70 shows the pair of EF-hands wedged between a four helix bundle (4HB) and a divergent SH2 domain. The coordination of Ca²⁺ is unique as indicated in table 1 by "d". Although the residue at -X is Ser, the -X oxygen ligand comes from the carboxylate group of a Glu from the fourth helix of the 4HB. Although calcium is required for phosphopeptide binding, it interacts with the SH2 domain, not the pair of EF-hands. The pair of EF-hands may position the 4HB relative to the SH2 domain.

Calcium and integrin binding protein (CIB) binds specifically to the cytoplasmic domain of integrin (IIb of platelets [JBC 272:4651 (97)]. It has 191 residues and contains two EF-hands. No homolog has been identified for the first hundred residues.

Calsensin (SENS) is expressed in some the peripheral neurons of leech, Haemopsis sp., that fasciculate in a single axon tract. SENS has 83 residues and both EF-hands bind calcium [JCB 129:1355 (95)]. It co-purifies with a 200kDa protein, not yet characterized.

Groovin (GRV) (identical to kakapo) is encoded by grv (kak) and is expressed in tendon cells of D. melanogaster. GRV is essential for muscle dependent, tendon cell differentiation [JCB 143:1259 (98)]. GRV contains two EF-hands; both are inferred to bind calcium.