Immunological approach to identify calmodulin-stimulated phosphatase isozymes from bovine brain.
Molecular cloning of human, mouse and rat brain CaM-stimulated phosphatase has suggested the existence of two genes for the alpha subunit of the enzymes. A alpha and A beta fragments of A alpha and A beta from rat brain library have been expressed in bacteria to produce specific anti-calcineurin A alpha and anti-calcineurin A beta antibodies (Kuno et 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 A alpha and A beta fragments, were analyzed by two calcineurin alpha subunit monoclonal antibodies VJ6 and VD3, the rat anti-calcineurin A alpha and anti-calcineurin A beta specific polyclonal antibodies, and the insertion peptide antibody. The bovine brain CaM-stimulated phosphatase isozymes BPI and BPIII reacted with both anti-calcineurin A alpha and anti-calcineurin A beta antibodies. While BPII reacted with anti-calcineurin A alpha but not anti-calcineurin A beta antibody, it differed from the expressed A alpha 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 A alpha or A beta genes products.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Physiology, Nihon University School of Dentistry at Matsudo, Chiba, Japan.
N
N
Yokoyama
T
T
Kuno
S
S
Furuyama
J H
JH
Wang
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001921:Brain / Q000201:enzymology*; D019703:Calcineurin; D002148:Calmodulin-Binding Proteins / Q000032:analysis*; D002417:Cattle; D003001:Cloning, Molecular; D015151:Immunoblotting; D007527:Isoenzymes / Q000032:analysis*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis; D010749:Phosphoprotein Phosphatases / Q000032:analysis*; D011233:Precipitin Tests; D051381:Rats; D013045:Species Specificity
D000911:Antibodies, Monoclonal; D002148:Calmodulin-Binding Proteins; D007527:Isoenzymes; D010446:Peptide Fragments; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases