Structural and biochemical characterization of calhepatin, an S100-like calcium-binding protein from the liver of lungfish (Lepidosiren paradoxa).
We report the biochemical characterization of calhepatin, a calcium-binding protein of the S100 family, isolated from lungfish (Lepidosiren paradoxa) liver. The primary structure, determined by Edman degradation and MS/MS, shows that the sequence identities with the other members of the family are lower than those between S100 proteins from different species. Calhepatin is composed of 75 residues and has a molecular mass of 8670 Da. It is smaller than calbindin D(9k) (78 residues), the smallest S100 described so far. Sequence analysis and molecular modelling predict the two EF-hand motifs characteristic of the S100 family. Metal-binding properties were studied by a direct 45Ca2+-binding assay and by fluorescence titration. Calhepatin binds Ca2+ and Cu2+ but not Zn2+. Cu2+ binding does not change the affinity of calhepatin for Ca2+. Calhepatin undergoes a conformational change upon Ca2+ binding as shown by the increase in its intrinsic fluorescence intensity and lambda(max), the decrease in the apo-calhepatin hydrodynamic volume, and the Ca2+-dependent binding of the protein to phenyl-Superose. Like most S100 proteins, calhepatin tends to form noncovalently associated dimers. These data suggest that calhepatin is probably involved in Ca2+-signal transduction.
Instituto de Química y Fisicoquímica Biológicas, Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires, Argentina.
Santiago M
SM
Di Pietro
José A
JA
Santomé
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D020013:Calcium Signaling; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D019281:Dimerization; D019143:Evolution, Molecular; D005399:Fishes / Q000378:metabolism*; D008099:Liver / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009928:Organ Specificity; D011487:Protein Conformation; D009418:S100 Proteins / Q000737:chemistry; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D013050:Spectrometry, Fluorescence
D002135:Calcium-Binding Proteins; D009418:S100 Proteins; D002118:Calcium