Yokoyama N, Kuno T, Furuyama S, Wang J H
Department of Physiology, Nihon University School of Dentistry at Matsudo, Chiba, Japan.
Mol Cell Biochem. 1994 Mar 30;132(2):101-8. doi: 10.1007/BF00926918.
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)
人类、小鼠和大鼠脑钙调神经磷酸酶的分子克隆表明，该酶的α亚基存在两个基因。大鼠脑文库中α和β片段的α和β已在细菌中表达，以产生特异性的抗钙调神经磷酸酶α和抗钙调神经磷酸酶β抗体（久野等人，《神经化学杂志》58:1643 - 1651，1992年）。选择性mRNA剪接产生了额外的钙调神经磷酸酶同工酶，其中一些含有ATVEAIEADE插入序列。本研究中制备了针对该插入序列合成肽的抗体。先前从牛脑中纯化的三种钙调神经磷酸酶同工酶（BPI、BPII、BPIII）（横山和王，《生物化学杂志》266:14822 - 14829，1991年），与细菌表达的大鼠α和β片段一起，用两种钙调神经磷酸酶α亚基单克隆抗体VJ6和VD3、大鼠抗钙调神经磷酸酶α和抗钙调神经磷酸酶β特异性多克隆抗体以及插入肽抗体进行了分析。牛脑钙调神经磷酸酶同工酶BPI和BPIII与抗钙调神经磷酸酶α和抗钙调神经磷酸酶β抗体均发生反应。虽然BPII与抗钙调神经磷酸酶α抗体反应，但不与抗钙调神经磷酸酶β抗体反应，它在对单克隆抗体的免疫反应性方面与表达的α片段不同。结果表明，牛脑钙调神经磷酸酶同工酶不能简单地归类为源自α或β基因产物。（摘要截短于250字）
