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Immunological approach to identify Calmodulin-stimulated phosphatase isozymes from bovine brain

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Abstract

Molecular cloning of human, mouse and rat brain CaM-stimulated phosphatase has suggested the existence of two genes for the α subunit of the enzymes. Aα and Aβ fragments of Aα and Aβ from rat brain library have been expressed in bacteria to produce specific anti-calcineurin Aα and anti-calcineurin Aβ antibodies (Kunoet al., J Neurochem 58: 1643–1651, 1992). Alternative mRNA splicing gives rise to additional calcineurin isozymes with some containing an insertion sequence of ATVEAIEADE. Antibody against synthetic peptide of this insertion sequence has been raised in this study. Three CaM-stimulated phosphatase isozymes previously purified from bovine brain (BPI, BPII, BPIII) (Yokoyama & Wang, J Biol Chem 266: 14822–14829, 1991), along with the bacterially expressed rat Aα and Aβ fragments, were analyzed by two calcineurin α subunit monoclonal antibodies VJ6 and VD3, the rat anti-calcineurin Aα and anti-calcineurin Aβ specific polyclonal antibodies, and the insertion peptide antibody. The bovine brain CaM-stimulated phosphatase isozymes BPI and BPIII reacted with both anti-calcineurin Aα and anti-calcineurin Aβ antibodies. While BPII reacted with anti-calcineurin Aα but not anti-calcineurin Aβ antibody, it differed from the expressed Aα fragment in immunoreactivity towards the monoclonal antibodies. The results show that the bovine brain CaM-stimulated phosphatase isozymes cannot be simply categorized as derived from Aα or Aβ genes products. On the other hand, on the basis of immunoreactivity toward the insertion antibody, the isozymes can be readily classified into those containing the insertion sequence (BPI, LPI) and those without the inserting (BPII, BPIII, LPII, LPIII).

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Abbreviations

CaM:

calmodulin

SDS:

sodium dodecyl sulfate

BP:

brain peak

LP:

lung peak

PAGE:

polyacrylamide gel electrophoresis

EGTA:

[ethylenebis(oxyethylenenitrilo)]-tetra acetic acid

cDNA:

complementary DNA

mAb:

monoclonal antibody

NBT:

nitro blue tetrazolium chloride

BCIP:

5-bromo 4-chloro-3-indolyl phosphate

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Authors and Affiliations

  1. Department of Physiology, Nihon University School of Dentistry at Matsudo, 2-870-1, Sakaecho-nishi, 271, Matsudo, Chiba, Japan

    Noriko Yokoyama & Shunsuke Furuyama

  2. Department of Pharmacology, Kobe University School of Medicine, 650, Kobe, Japan

    Takayoshi Kuno

  3. Department of Medical Biochemistry, MRC Group in Signal Transduction, University of Calgary, T2N 4N1, Calgary, Alberta, Canada

    Jerry H. Wang

Authors
  1. Noriko Yokoyama
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  2. Takayoshi Kuno
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  3. Shunsuke Furuyama
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  4. Jerry H. Wang
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Yokoyama, N., Kuno, T., Furuyama, S. et al. Immunological approach to identify Calmodulin-stimulated phosphatase isozymes from bovine brain. Mol Cell Biochem 132, 101–108 (1994). https://doi.org/10.1007/BF00926918

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  • DOI: https://doi.org/10.1007/BF00926918

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