Cloning and expression of a cDNA encoding a new neurocalcin isoform (neurocalcin alpha) from bovine brain.
Neurocalcin (NC), a neuron-specific EF-hand Ca2+-binding protein, purified from bovine brain [Terasawa, Nakano, Kobayashi and Hidaka (1992) J. Biol. Chem. 267, 19596-19599] contains multiple isoforms. We previously cloned NCdelta from bovine brain and showed high expression in neuronal tissues [Okazaki, Watanabe, Ando, Hagiwara, Terasawa and Hidaka (1992) Biochem. Biophys. Res. Commun. 185, 147-153]. We report here the molecular cloning and expression of a cDNA encoding bovine brain NCalpha. The translated bovine protein is 191 amino acids long and shares 69.1% of its amino acid sequence with NCdelta. Recombinant NCalpha migrates as a single 23 kDa band and exhibits a Ca2+-dependent mobility shift on SDS/PAGE. Analysis of fluorescence emission spectra showed the Ca2+-induced peak at 337 nm. Interestingly, the mobility shift and the fluorescence intensity at 337 nm were larger for NCalpha than for NCdelta. In Ca2+-overlay experiments, however, the apparent affinity of NCalpha for 45Ca2+ was similar to that of NCdelta. Immunohistochemical analysis revealed NCalpha expression in the granular layer of the rat cerebellar cortex whereas NCdelta was found in the Purkinje cell layer. In the rat olfactory bulb, NCalpha was located in external tufted cells, and NCdelta was found in the periglomerular cells. These data demonstrate that NC isoforms differ in their tissue distribution and conformational changes induced by Ca2+ binding. Thus differential regulation of the two NC isoforms may be involved in control of neuron function.
Kato
M
M
Department of Pharmacology, Nagoya University School of Medicine, Tsurumai 65, Showa-ku, Nagoya 466, Japan.
Watanabe
Y
Y
Iino
S
S
Takaoka
Y
Y
Kobayashi
S
S
Haga
T
T
Hidaka
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000166:cytology / Q000378:metabolism*; D002118:Calcium / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D002417:Cattle; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry*; D051596:Neurocalcin; D010766:Phosphorylation; D011485:Protein Binding / Q000502:physiology; D011487:Protein Conformation / Q000187:drug effects; D044169:Receptors, Calcium-Sensing; D011994:Recombinant Proteins / Q000737:chemistry; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry; D051552:beta-Adrenergic Receptor Kinases
D002135:Calcium-Binding Proteins; D009419:Nerve Tissue Proteins; D051596:Neurocalcin; D044169:Receptors, Calcium-Sensing; D011994:Recombinant Proteins; D014364:Tryptophan; D017868:Cyclic AMP-Dependent Protein Kinases; D051552:beta-Adrenergic Receptor Kinases; D002118:Calcium