1 Department of Neurochemistry, New York State Institute for Basic Research in Developmental Disabilities, Staten Island, New York 10314. 2 Department of Biochemistry, Faculty of Medicine, National Uni- versity of Singapore, 10 Kent Ridge Crescent, Singapore 119260. 3 Department of Pathophysiology, Tongji Medical College, HUST, Wuhan 430030, P.R. China. 4 Department of Geriatric Medicine, Karolinska Institutet B56-K, S-141 86 Huddinge, Sweden. Multiple Forms of Phosphatase from Human Brain: Isolation and Partial Characterization of Affi-Gel Blue Nonbinding Phosphatase Activities Li Yao Cheng, 1,2 Jian-Zhi Wang, 1,3 Cheng-Xin Gong, 1 Jin-Jing Pei, 1,4 Tanweer Zaidi, 1 Inge Grundke-Iqbal, 1 and Khalid Iqbal 1,5 (Accepted March 12, 2001) Phosphatases extracted from a human brain were resolved into two main groups, namely affi- gel blue-binding phosphatases and affi-gel blue-nonbinding phosphatases. Affi-gel blue bind- ing phosphatases were further separated into four different phosphatase activities, designated P1–P4, and described previously (1). In the present study we describe the affi-gel blue-non- binding phosphatases which were separated into seven different phosphatase activities, desig- nated P5–P11 by poly-(L-lysine)-agarose and aminohexyl Sepharose 4B chromatographies. These seven phosphatase activities were active toward nonprotein phosphoester. P7–P11 and to some extent P5 could also dephosphorylate a phosphoprotein. They displayed different enzyme kinetics. On the basis of activity peak, the apparent molecular mass as estimated by Sephadex G-200 column chromatography for P5 was 49 kDa; P6, 32 kDa; P7, 150 kDa; P8, 250 kDa; P9, 165 kDa; P10, 90 kDa and P11, 165 kDa. Immunoblot analysis indicated that P8–P11 may be- long to PP2B family, whereas P7 may associate with PP2A. The phosphatases P7–P11 were found to be effective in the dephosphorylation of Alzheimer’s disease abnormally hyperphos- phorylated tau. The resulting dephosphorylated tau regained its activity in promoting the micro- tubule assembly, suggesting that P7–P11 might regulate the phosphorylation of tau protein in the brain. KEY WORDS: Protein phosphatases; human brain; microtubule assembly; microtubule-associated protein tau; Alzheimer’s disease. 425 0364-3190/01/0400–0425$19.50/0 © 2001 Plenum Publishing Corporation neurons in the brain (2–4). The major protein subunit of PHF is abnormally hyperphosphorylated tau protein (5–8). Tau protein from adult human brain consists of six isoforms due to alternative mRNA splicing (9–10). In normal brain, tau protein facilitates tubulin poly- merization and stabilizes microtubule assembly. The integrity of microtubule system is essential for neu- ronal functions in the transport of vital materials be- tween its cell body and synapses. Tau isolated from INTRODUCTION One of the distinct characteristics of Alzheimer’s disease (AD) is the presence of neurofibrillary tangles (NFT) of paired helical filaments (PHF) in the selected Neurochemical Research, Vol. 26, No. 4, 2001, pp. 425–438 5 Address reprint requests to: Khalid Iqbal, Ph.D., Chairman, De- partment of Neurochemistry, NYS Institute for Basic Research, 1050 Forest Hill Road, Staten Island, New York 10314-6399. Tel: (718) 494-5259; Fax: (718) 494-1080; E-mail: kiqbal@mail. con2.com