Purification and primary structure of Capl, an S-100-related calcium-binding protein isolated from bovine retina.
Calcium protein placental homolog (Capl) is an S-100-related calcium-binding protein selectively expressed in cell lines that have been induced to grow or differentiate. In addition, the expression of Capl correlates with the induction of the metastatic phenotype in tumor cell lines and the transformation of normal cells by activated oncogenes or chemical carcinogens. Although not previously associated with the nervous system, in this study, Capl was purified from bovine neural retina by a combination of phenyl-Sepharose and organomercurial chromatography. The complete amino acid sequence of bovine Capl was established primarily by Edman degradation of peptides generated by cleavage of methionyl, lysyl, glutamyl, and aspartyl bonds. NH2-terminal methionyl and aspartyl peptides were analyzed by tandem mass spectrometry, which provided the sequence of the first 8 residues and identified the NH2-terminal blocking group as an acetyl moiety. The molecular mass of the intact protein determined by electrospray mass spectrometry (M(r) = 11,716.75 +/- 0.42) and the calculated molecular mass deduced from the amino acid composition (M(r) = 11,718) were in agreement, thus supporting the accuracy of the sequence assignment. Capl isolated from the retina was shown to be indistinguishable by mass and immunochemical properties from its counterpart in the bovine aorta, which previously was the only source of purified Capl. Northern analysis using cloned Capl cDNA revealed that Capl mRNA is present not only in the retina but the choroid as well. Further support for choroidal localization came from immunohistochemical experiments using specific anti-Capl antibodies. The physiological significance of Capl in ocular tissues and the aorta is discussed.
Polans
A S
AS
R. S. Dow Neurological Sciences Institute, Good Samaritan Hospital, Portland, Oregon.
Palczewski
K
K
Asson-Batres
M A
MA
Ohguro
H
H
Witkowska
D
D
Haley
T L
TL
Baizer
L
L
Crabb
J W
JW
D000595:Amino Acid Sequence; D000818:Animals; D001011:Aorta / Q000378:metabolism; D015152:Blotting, Northern; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002417:Cattle; D002829:Choroid / Q000378:metabolism; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008969:Molecular Sequence Data; D012160:Retina / Q000378:metabolism*; D000071999:S100 Calcium-Binding Protein A4; D009418:S100 Proteins / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D002135:Calcium-Binding Proteins; D000071999:S100 Calcium-Binding Protein A4; D009418:S100 Proteins; C499413:S100A4 protein, Bos taurus; C077591:S100A4 protein, human