BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS Pages 1207-1211
Vol. 186, No. 3, 1992 August 14, 1992
NEUROCALCIN,
A NOVEL
WITH
EF-HAND
THREE
RETINAL
AMACRINE
CALCIUM BINDING PROTEIN DOMAINS, EXPRESSED IN CELLS AND GANGLION CELLS
Akio Nakano, Motomu Terasawa, Masato Watanabe, Nobuteru Usuda +, Takashi Morita +, and Hiroyoshi Hidaka* Department of Pharmacology, Nagoya University School of Medicine, Tsurumai 65, Showa-ku, Nagoya 466, Japan +Department of Anatomy, Shinshu University School of Medicine, Asahi, Matsumoto 390, Japan Received June 26, 1992
Summary: Neurocalcin (molecular weight 23,000 and 24,000) is a newly identified Ca 2+ binding protein with three EF-hand domains and has a strong amino acid sequence homology with visinin and recoverin (Terasawa, M., Nakano, A., Kobayashi, R., and Hidaka, H. J. Biol. Chem. In press). We produced antibody against neurocalcin. Immunoblotting showed the presence of neurocalcin in bovine retina as well as brain, suggesting that neurocalcin was a neuron specific Ca 2+ binding protein. Immunohistochemistry revealed the expression of neurocalcin in retinal amacrine cells and ganglion cells but not in the photoreceptor layer. This distribution of neurocalcin was quite different from that of visinin and recoverin. Our results suggest that neurocalcin may play an important role in a Ca2÷ signal pathway of the nervous system. ~ ~992 Academic
Press,
Inc.
It is generally accepted that intracellular Ca 2+ binding proteins play a critical and central role in various biological events as an initial step of the Ca2+ messenger system. A class of Ca 2÷ binding proteins exhibits general structural principles, the so-called EFhand structures (1). Calmodulin is present in most cells, but most other Ca 2+ binding proteins are expressed in a cell-type specific manner and their functions and target proteins remain unknown. Calmodulin antagonists, such as W-7, are useful tools for elucidating the physiological function of calmodulin and other Ca2+ binding proteins (2, 3). We reported earlier that W-7 interacted with calmodulin in a Ca 2÷ dependent manner and inhibited * To whom correspondence should be addressed. The abbreviations used are: EGTA, glycoletberdiaminetetraacetic acid; kDa, kilodalton; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; Tricine, Tris(hydroxymethyl)methylglycine; W-7, N-(6-aminohexyl-5-chloro-l-naphthalene sulfonamide); W-77, (S)-p-(2-aminoethyloxy)-N-[2-(4-benzyloxycalbonylpiperazinyl)1-(p-methyloxybenzyl)]-N-methylbenzenesulfonamide dihydrochloride.
1207
0006-291X/92 $4.00 Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 186, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Ca2+-calmodulin dependent enzymes such as cyclic nucleotide phosphodiesterase (4) and myosin light chain kinase (5). W-7 coupled sepharose column can be used for purification of S-100 protein from bovine tissues (2). A novel Ca 2÷ binding protein which we term neurocalcin was purified by affinity chromatography on a newly synthesized calmodulin antagonist, W-77, coupled sepharose column from bovine brain (6). Neurocalcin composed of 23 kDa and 24 kDa polypeptides and had three EF-hand domains. In this report, we first produced antibody against neurocalcin. Immunoblotting showed that neurocalcin was a neuron specific Ca 2+ binding protein.
Materials and Methods
Materials. Bovine brain and retina were obtained from a local slaughterhouse. Neurocalcin was purified from bovine brain with W-77 coupled sepharose 6B column followed by C18 reverse phase column (6). All other reagents were of analytical grade. Analytical procedures. Tricine-SDS-PAGE was performed according to the method of Sch~igger and von Jagow (7). Protein concentration was determined by the method of Bradford (8) using bovine serum albumine as a standard. Production of antibody against neurocalcin. Polyclonal antibody was raised in rabbits by injecting subcutaneously 0.5 mg of purified neurocalcin with complete Freund's adjuvant four times at 2-week intervals. The rabbits were bled 7 days after the last injection. Production of antibody was checked by immunoblots, using bovine brain crude extracts as the antigen. Iramunoblotting. Proteins separated by SDS-PAGE were transfered to a nitrocellulose membrane (0.2 tam pore size; Schleicher & Schuell, Dassel, Germany) in 48 mM Tris, 39 mM glycine, 1.3 mM SDS, and 20 % (vol/vol) methanol at 15 V for 30 min (9). For immunodetection, the procedure of Burnette (10) was followed, except that second antibody was horseradish peroxidase-linked anti-rabbit IgG (Medical & Biological Lab., Nagoya, Japan). Antigen-antibody complex was visualized by reacting the bound peroxidase with diaminobenzidine and peroxidase. Immunohistochemistry. Bovine cerebellum and retina were fixed by 4 % paraformaldehyde/0.1 M sodium phosphate, pH 7.4, for 3 h. After washing in 0.1 M lysine/0.15 M NaCI/0.1 M sodium phosphate, pH 7.4, for 1 h, they were immersed in graded sucrose solutions: 10, 15, and 20 % for 10 h each. They were frozen in a mixture of dry ice and n-hexane and sectioned at 6 ~tm using a cryostat, and placed on slide glass. The sections were exposed to 10 % normal horse serum in Phosphate-buffered saline (PBS) for 30 min, and then incubated with antibody against neurocalcin diluted from 1:1000 to 1:3000 in PBS for 2 h. After being washed in PBS, they were processed for avidin-biotinylated peroxidase complex staining (Vector Laboratories Inc., Burlingame, CA). Reaction product was developed by incubation in diaminobenzidine and peroxidase in 50 mM Tris buffer, pH 7.5.
Results and Discussion
Bovine brain was homogenized in 4 volumes of 40 mM Tris-HCl, pH 7.5, containing 2 mM EGTA and centrifuged at 20,000 x g for 30 min. The supernatant fluid was separated by SDS-PAGE and transfered to nitrocellulose membranes. The blots were incubated with antibody against neurocalcin diluted 1:2000 and immunoreactive bands were visualized with 500-fold diluted second antibody. Fig. 1 shows the antibody was reactive with neurocalcin and no other proteins in the brain homogenates cross-reacted with the antibody. Extracts from various bovine tissues with 2 mM EGTA-containing buffer were separated by SDS-PAGE and blotted to nitrocellulose membranes to determine the tissue 1208
Vol. 186, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A kDa ~i~i~ili~i 97 - - ~ 66 ~ 43 - i~ 31 ~ ii ~ --
B
kDa 97~
iiiiii~411~iiiiii~ iii~ii~iiiii!iiiiiiii!iil i~i:iiiiiiiiiiiiii!iiiiii ~i)'!~i':;i'iiiiiiiiii iii
43-31~ 22--
14~
!iiiii,iiiiiiiiiiiiiii!
iiiiiiiiiiiiiiiiiii!ii~!iiiiiiiiiiiiiiiiiii~i~i~ a
b
c
d
e
Fig. 1. I m m u n o s p e c i f i c i t y of a n t i b o d y against n e u r o c a l c i n . EGTA extracts of bovine brain were separated by SDS-PAGE and blotted to a nitrocellulose membrane. The membrane was then treated with 2000-fold diluted antibody against neurocalcin and visualized by immunoblots. (A) Coomassie stained gel. (B) Immune gel replicate using antibody against neurocalcin. Lane a and c, 30 ~tg of brain homogenates; lane b and d, 1 ~tg of neurocalcin. Fig. 2. Tissue d i s t r i b u t i o n o f n e u r o c a l c i n . Various bovine tissues were homogenized in 2 mM EGTA-containing buffer and centrifuged at 20,000 x g for 30 min. The supernatant fluid was separated by SDS-PAGE (30 Ixg protein/lane) and transferred to nitrocellulose membranes. Blotting was performed with antibody against neurocalcin diluted 1:2000. Lane a, cerebrum; lane b, cerebellum; lane c, brain stem; lane d, spinal cord; lane e, retina. Molecular-mass markers stained with amido black are shown at left.
distribution of neurocalcin. Immunoreactive bands were detected in bovine cerebrum, cerebellum, brain stem, spinal cord, and retina (Fig. 2). Neurocalcin was not detected in bovine lung, heart, liver, spleen, pancreas, intestine, adipose tissue, and testis (data not shown). Fig. 3 shows the localization of neurocalcin in bovine cerebellar cortex. The molecular layer and granular layer were stained moderately, while Purkinje cell layer was stained
3P
Fie. 3. I m m u n o h i s t o c h e m i c a l localization of neurocalcin in b o v i n e cerebellum. The molecular layer and granular layer were moderately stained, whereas
Purkinje cell layer was weakly immunoreactive. P, Purkinje cell layer. Bar = 150 ~n. 1209
Vol. 186, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ON
2IN
Fig. 4. Immunohistochemical localization of neurocalcin in bovine retina. Amacrine cells and ganglion cells were stained strongly but the photoreceptor layer was negative. G, ganglion cell; IN, inner nuclear layer; ON, outer nuclear layer. Bar = 60 ~tm.
weakly, Immunohistochemical analysis also revealed the expression of neurocalcin in retinal amacrine cells and ganglion cells but not in the photoreceptor layer (Fig. 4). Recently, using a drug affinity chromatographic technique, we identified members of the S-100 protein family, calcyclin from rabbit lung (11), calvasculin from bovine aorta (12), and calgizzarin from chicken gizzard and rabbit lung (13). Neurocalcin was purified from bovine brain using a newly synthesized compound, W-77, coupled sepharose column. Most Ca 2÷ binding proteins with EF-hand have two or four domains. Palvalbumin, visinin, and recoverin are members of the three EF-hands group. Visinin is a 24 kDa retinal cone specific Ca 2+ binding protein (14). Recoverin is a 26 kDa retinal rod and cone specific Ca 2÷ binding protein and activates guanylate cyclase (15). Neurocalcin was a new member of the three EF-hands group and had a strong amino acid sequence homology with visinin and recoverin (6,16). We first produced antibody against neurocalcin. Immunoblotting showed the presence of neurocalcin in bovine central nervous tissues and retina and suggested that neurocalcin was a neuron specific Ca 2+ binding protein. Immunohistochemical analysis revealed that neurocalcin was expressed in retinal amacrine cells and ganglion cells but not in the photoreceptor layer. This distribution of neurocalcin was quite different from that of visinin and recoverin. Four isoproteins of neurocalcin were partially separated by C18 reverse phase column (6) and their amino acid sequences were distinct but highly homologous with the deduced amino acid sequences from the cDNA which we reported earlier (16). This suggested that neurocalcin had multiple isoproteins in brain. The antibody against neurocalcin was reactive with all isoproteins (data not shown), and it remains to be elucidated whether there is a selective expression of isoproteins, at least in cerebellum and retina. Neurocalcin, visinin, and recoverin are members of the three EF-hands group and share an extensive homology with each other. Visinin and recoverin are retinal photoreceptor specific Ca 2+ binding proteins, while neurocalcin is a neuron specific Ca 2÷ binding protein. Since proteins with homologous amino acid sequences usually have similar functions, neurocalcin may play an important role in the nervous system. 1210
Vol. 186, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
References
1. Kretsinger, R.H. (1980) CRC Crit. Rev. Bit)chem. 8, 119-174. 2, Endo, T., Tanaka, T., Isobe, T., Kasai, H., Okuyama, T., and Hidaka, H. (1981) J. Biol. Chem. 256, 12485-12489. 3. Hidaka, H., and Tanaka, T. (1985) In Calmodulin Antagonists and Cellular Physiology (H. Hidaka, and D.J. Hartshorne, Eds.), pp. 13-23. Academic Press, Orlando, FL. 4. Hidaka, H., Yamaki, T., Asano, M., and Totsuka, T. (1978) Blood Vessels 15, 55-64. 5. Hidaka, H., Yamaki, T., Naka, M., Tanaka, T., Hayashi, H., and Kobayashi, R. (1980) Mol. Pharmacol. 17, 66-72. 6. Terasawa, M., Nakano, A., Kobayashi, R., and Hidaka, H. (1992) J. Biol. Chem. In press. 7. Sch~igger, H., and yon Jagow, G. (1987) Anal. Biochem. 166, 368-379. 8. Bradford, M.M. (1976) Anal. Biochem. 72, 248-254. 9. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. USA. 76, 4350-4354. 10. Burnette, W.N. (1981) Anal. Biochem. 112, 195-203. 11. Tokumitsu, H., Kobayashi, R., and Hidaka, H. (1991) Arch. Biochem. Biophys. 288, 202-207. 12. Watanabe, Y., Kobayashi, R., Ishikawa, T., and Hidaka, H. (1992) Arch. Biochem. Biophys. 292, 563-569. 13. Todoroki, H., Kobayashi, R., Watanabe, M., Minami, H., and Hidaka, H. (1991) J. Biol. Chem. 266, 18668-18673. 14. Yamagata, K., Goto, K., Kuo, C-H., Kondo, H., and Miki, N. (1990) Neuron 2, 469-476. 15. Dizhoor, A.M., Ray, S., Kumar, S., Niemi, G., Spencer, M., BroUey, D., Walsh, K.A., Philipov, P.P., Hurley, J.B., and Stryer, L. (1991) Science 251, 915-918. 16. Okazaki, K., Watanabe, M., Ando, Y., Hagiwara, M., Terasawa, M., and Hidaka, H. (1992) Biochem. Biophys. Res. Commun. 185, 147-153.
1211
Vol. 186, No. 3, 1992 August 14, 1992
NEUROCALCIN,
A NOVEL
WITH
EF-HAND
THREE
RETINAL
AMACRINE
CALCIUM BINDING PROTEIN DOMAINS, EXPRESSED IN CELLS AND GANGLION CELLS
Akio Nakano, Motomu Terasawa, Masato Watanabe, Nobuteru Usuda +, Takashi Morita +, and Hiroyoshi Hidaka* Department of Pharmacology, Nagoya University School of Medicine, Tsurumai 65, Showa-ku, Nagoya 466, Japan +Department of Anatomy, Shinshu University School of Medicine, Asahi, Matsumoto 390, Japan Received June 26, 1992
Summary: Neurocalcin (molecular weight 23,000 and 24,000) is a newly identified Ca 2+ binding protein with three EF-hand domains and has a strong amino acid sequence homology with visinin and recoverin (Terasawa, M., Nakano, A., Kobayashi, R., and Hidaka, H. J. Biol. Chem. In press). We produced antibody against neurocalcin. Immunoblotting showed the presence of neurocalcin in bovine retina as well as brain, suggesting that neurocalcin was a neuron specific Ca 2+ binding protein. Immunohistochemistry revealed the expression of neurocalcin in retinal amacrine cells and ganglion cells but not in the photoreceptor layer. This distribution of neurocalcin was quite different from that of visinin and recoverin. Our results suggest that neurocalcin may play an important role in a Ca2÷ signal pathway of the nervous system. ~ ~992 Academic
Press,
Inc.
It is generally accepted that intracellular Ca 2+ binding proteins play a critical and central role in various biological events as an initial step of the Ca2+ messenger system. A class of Ca 2÷ binding proteins exhibits general structural principles, the so-called EFhand structures (1). Calmodulin is present in most cells, but most other Ca 2+ binding proteins are expressed in a cell-type specific manner and their functions and target proteins remain unknown. Calmodulin antagonists, such as W-7, are useful tools for elucidating the physiological function of calmodulin and other Ca2+ binding proteins (2, 3). We reported earlier that W-7 interacted with calmodulin in a Ca 2÷ dependent manner and inhibited * To whom correspondence should be addressed. The abbreviations used are: EGTA, glycoletberdiaminetetraacetic acid; kDa, kilodalton; PAGE, polyacrylamide gel electrophoresis; SDS, sodium dodecyl sulfate; Tricine, Tris(hydroxymethyl)methylglycine; W-7, N-(6-aminohexyl-5-chloro-l-naphthalene sulfonamide); W-77, (S)-p-(2-aminoethyloxy)-N-[2-(4-benzyloxycalbonylpiperazinyl)1-(p-methyloxybenzyl)]-N-methylbenzenesulfonamide dihydrochloride.
1207
0006-291X/92 $4.00 Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
Vol. 186, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Ca2+-calmodulin dependent enzymes such as cyclic nucleotide phosphodiesterase (4) and myosin light chain kinase (5). W-7 coupled sepharose column can be used for purification of S-100 protein from bovine tissues (2). A novel Ca 2÷ binding protein which we term neurocalcin was purified by affinity chromatography on a newly synthesized calmodulin antagonist, W-77, coupled sepharose column from bovine brain (6). Neurocalcin composed of 23 kDa and 24 kDa polypeptides and had three EF-hand domains. In this report, we first produced antibody against neurocalcin. Immunoblotting showed that neurocalcin was a neuron specific Ca 2+ binding protein.
Materials and Methods
Materials. Bovine brain and retina were obtained from a local slaughterhouse. Neurocalcin was purified from bovine brain with W-77 coupled sepharose 6B column followed by C18 reverse phase column (6). All other reagents were of analytical grade. Analytical procedures. Tricine-SDS-PAGE was performed according to the method of Sch~igger and von Jagow (7). Protein concentration was determined by the method of Bradford (8) using bovine serum albumine as a standard. Production of antibody against neurocalcin. Polyclonal antibody was raised in rabbits by injecting subcutaneously 0.5 mg of purified neurocalcin with complete Freund's adjuvant four times at 2-week intervals. The rabbits were bled 7 days after the last injection. Production of antibody was checked by immunoblots, using bovine brain crude extracts as the antigen. Iramunoblotting. Proteins separated by SDS-PAGE were transfered to a nitrocellulose membrane (0.2 tam pore size; Schleicher & Schuell, Dassel, Germany) in 48 mM Tris, 39 mM glycine, 1.3 mM SDS, and 20 % (vol/vol) methanol at 15 V for 30 min (9). For immunodetection, the procedure of Burnette (10) was followed, except that second antibody was horseradish peroxidase-linked anti-rabbit IgG (Medical & Biological Lab., Nagoya, Japan). Antigen-antibody complex was visualized by reacting the bound peroxidase with diaminobenzidine and peroxidase. Immunohistochemistry. Bovine cerebellum and retina were fixed by 4 % paraformaldehyde/0.1 M sodium phosphate, pH 7.4, for 3 h. After washing in 0.1 M lysine/0.15 M NaCI/0.1 M sodium phosphate, pH 7.4, for 1 h, they were immersed in graded sucrose solutions: 10, 15, and 20 % for 10 h each. They were frozen in a mixture of dry ice and n-hexane and sectioned at 6 ~tm using a cryostat, and placed on slide glass. The sections were exposed to 10 % normal horse serum in Phosphate-buffered saline (PBS) for 30 min, and then incubated with antibody against neurocalcin diluted from 1:1000 to 1:3000 in PBS for 2 h. After being washed in PBS, they were processed for avidin-biotinylated peroxidase complex staining (Vector Laboratories Inc., Burlingame, CA). Reaction product was developed by incubation in diaminobenzidine and peroxidase in 50 mM Tris buffer, pH 7.5.
Results and Discussion
Bovine brain was homogenized in 4 volumes of 40 mM Tris-HCl, pH 7.5, containing 2 mM EGTA and centrifuged at 20,000 x g for 30 min. The supernatant fluid was separated by SDS-PAGE and transfered to nitrocellulose membranes. The blots were incubated with antibody against neurocalcin diluted 1:2000 and immunoreactive bands were visualized with 500-fold diluted second antibody. Fig. 1 shows the antibody was reactive with neurocalcin and no other proteins in the brain homogenates cross-reacted with the antibody. Extracts from various bovine tissues with 2 mM EGTA-containing buffer were separated by SDS-PAGE and blotted to nitrocellulose membranes to determine the tissue 1208
Vol. 186, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
A kDa ~i~i~ili~i 97 - - ~ 66 ~ 43 - i~ 31 ~ ii ~ --
B
kDa 97~
iiiiii~411~iiiiii~ iii~ii~iiiii!iiiiiiii!iil i~i:iiiiiiiiiiiiii!iiiiii ~i)'!~i':;i'iiiiiiiiii iii
43-31~ 22--
14~
!iiiii,iiiiiiiiiiiiiii!
iiiiiiiiiiiiiiiiiii!ii~!iiiiiiiiiiiiiiiiiii~i~i~ a
b
c
d
e
Fig. 1. I m m u n o s p e c i f i c i t y of a n t i b o d y against n e u r o c a l c i n . EGTA extracts of bovine brain were separated by SDS-PAGE and blotted to a nitrocellulose membrane. The membrane was then treated with 2000-fold diluted antibody against neurocalcin and visualized by immunoblots. (A) Coomassie stained gel. (B) Immune gel replicate using antibody against neurocalcin. Lane a and c, 30 ~tg of brain homogenates; lane b and d, 1 ~tg of neurocalcin. Fig. 2. Tissue d i s t r i b u t i o n o f n e u r o c a l c i n . Various bovine tissues were homogenized in 2 mM EGTA-containing buffer and centrifuged at 20,000 x g for 30 min. The supernatant fluid was separated by SDS-PAGE (30 Ixg protein/lane) and transferred to nitrocellulose membranes. Blotting was performed with antibody against neurocalcin diluted 1:2000. Lane a, cerebrum; lane b, cerebellum; lane c, brain stem; lane d, spinal cord; lane e, retina. Molecular-mass markers stained with amido black are shown at left.
distribution of neurocalcin. Immunoreactive bands were detected in bovine cerebrum, cerebellum, brain stem, spinal cord, and retina (Fig. 2). Neurocalcin was not detected in bovine lung, heart, liver, spleen, pancreas, intestine, adipose tissue, and testis (data not shown). Fig. 3 shows the localization of neurocalcin in bovine cerebellar cortex. The molecular layer and granular layer were stained moderately, while Purkinje cell layer was stained
3P
Fie. 3. I m m u n o h i s t o c h e m i c a l localization of neurocalcin in b o v i n e cerebellum. The molecular layer and granular layer were moderately stained, whereas
Purkinje cell layer was weakly immunoreactive. P, Purkinje cell layer. Bar = 150 ~n. 1209
Vol. 186, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
ON
2IN
Fig. 4. Immunohistochemical localization of neurocalcin in bovine retina. Amacrine cells and ganglion cells were stained strongly but the photoreceptor layer was negative. G, ganglion cell; IN, inner nuclear layer; ON, outer nuclear layer. Bar = 60 ~tm.
weakly, Immunohistochemical analysis also revealed the expression of neurocalcin in retinal amacrine cells and ganglion cells but not in the photoreceptor layer (Fig. 4). Recently, using a drug affinity chromatographic technique, we identified members of the S-100 protein family, calcyclin from rabbit lung (11), calvasculin from bovine aorta (12), and calgizzarin from chicken gizzard and rabbit lung (13). Neurocalcin was purified from bovine brain using a newly synthesized compound, W-77, coupled sepharose column. Most Ca 2÷ binding proteins with EF-hand have two or four domains. Palvalbumin, visinin, and recoverin are members of the three EF-hands group. Visinin is a 24 kDa retinal cone specific Ca 2+ binding protein (14). Recoverin is a 26 kDa retinal rod and cone specific Ca 2÷ binding protein and activates guanylate cyclase (15). Neurocalcin was a new member of the three EF-hands group and had a strong amino acid sequence homology with visinin and recoverin (6,16). We first produced antibody against neurocalcin. Immunoblotting showed the presence of neurocalcin in bovine central nervous tissues and retina and suggested that neurocalcin was a neuron specific Ca 2+ binding protein. Immunohistochemical analysis revealed that neurocalcin was expressed in retinal amacrine cells and ganglion cells but not in the photoreceptor layer. This distribution of neurocalcin was quite different from that of visinin and recoverin. Four isoproteins of neurocalcin were partially separated by C18 reverse phase column (6) and their amino acid sequences were distinct but highly homologous with the deduced amino acid sequences from the cDNA which we reported earlier (16). This suggested that neurocalcin had multiple isoproteins in brain. The antibody against neurocalcin was reactive with all isoproteins (data not shown), and it remains to be elucidated whether there is a selective expression of isoproteins, at least in cerebellum and retina. Neurocalcin, visinin, and recoverin are members of the three EF-hands group and share an extensive homology with each other. Visinin and recoverin are retinal photoreceptor specific Ca 2+ binding proteins, while neurocalcin is a neuron specific Ca 2÷ binding protein. Since proteins with homologous amino acid sequences usually have similar functions, neurocalcin may play an important role in the nervous system. 1210
Vol. 186, No. 3, 1992
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
References
1. Kretsinger, R.H. (1980) CRC Crit. Rev. Bit)chem. 8, 119-174. 2, Endo, T., Tanaka, T., Isobe, T., Kasai, H., Okuyama, T., and Hidaka, H. (1981) J. Biol. Chem. 256, 12485-12489. 3. Hidaka, H., and Tanaka, T. (1985) In Calmodulin Antagonists and Cellular Physiology (H. Hidaka, and D.J. Hartshorne, Eds.), pp. 13-23. Academic Press, Orlando, FL. 4. Hidaka, H., Yamaki, T., Asano, M., and Totsuka, T. (1978) Blood Vessels 15, 55-64. 5. Hidaka, H., Yamaki, T., Naka, M., Tanaka, T., Hayashi, H., and Kobayashi, R. (1980) Mol. Pharmacol. 17, 66-72. 6. Terasawa, M., Nakano, A., Kobayashi, R., and Hidaka, H. (1992) J. Biol. Chem. In press. 7. Sch~igger, H., and yon Jagow, G. (1987) Anal. Biochem. 166, 368-379. 8. Bradford, M.M. (1976) Anal. Biochem. 72, 248-254. 9. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. USA. 76, 4350-4354. 10. Burnette, W.N. (1981) Anal. Biochem. 112, 195-203. 11. Tokumitsu, H., Kobayashi, R., and Hidaka, H. (1991) Arch. Biochem. Biophys. 288, 202-207. 12. Watanabe, Y., Kobayashi, R., Ishikawa, T., and Hidaka, H. (1992) Arch. Biochem. Biophys. 292, 563-569. 13. Todoroki, H., Kobayashi, R., Watanabe, M., Minami, H., and Hidaka, H. (1991) J. Biol. Chem. 266, 18668-18673. 14. Yamagata, K., Goto, K., Kuo, C-H., Kondo, H., and Miki, N. (1990) Neuron 2, 469-476. 15. Dizhoor, A.M., Ray, S., Kumar, S., Niemi, G., Spencer, M., BroUey, D., Walsh, K.A., Philipov, P.P., Hurley, J.B., and Stryer, L. (1991) Science 251, 915-918. 16. Okazaki, K., Watanabe, M., Ando, Y., Hagiwara, M., Terasawa, M., and Hidaka, H. (1992) Biochem. Biophys. Res. Commun. 185, 147-153.
1211
