Vol. 128, No. 2, 1985 8lOCHEMlCAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS April 30, 1985 Pages 705-712 SEPARATION OF RABBIT LIVER LATENT AND SPONTANEOUSLY ACTIVE PHOSPHORYLASE PHOSPHATASES BY CHROMATOGRAPHY ON HEPARIN- SEPHAROSE Ferenc ErdBdi, Csilla Csortos, Gyijrgy Bot and P&l Gergely Institute of Medical Chemistry, University School of Medicine Debrecen, Bern t&r 18/B, ~-4026, Hungary Received March 4, 1985 SUMMARY: Latent and spontaneously active forms of phosphorylase phosphatase were separated by heparin-Sepharose chromatography of rabbit liver extract. polycation (histone Hl, The latent enzyme had an absolute polybrene) requirement for the activity assayed with phosphorylase a and phosphorylase kinase substrates. Ethanol treatment resulted Tn the activation of both phosphatases by dissociating of 150-180 kDa holoenzymes to 33-38 kDa catalytic subunits as judged by gel filtration. The latent and spontane- ously active phosphatases were differentiated according to their abilities to dephosphorylate the a and the B subunits of phos- phorylase kinase and sensitivities to inhibition by inhibitor-2 or heparin, and were classified as type-2A and type-l phospha- tases, respectively. 0 1985 Academic Press, Inc. The protein phosphatases can be grouped into two classes. Type-l phosphatases are rather specific for the dephosphorylation of the B subunit of phosphorylase kinase and are inhibited by heat-stable proteins, termed inhibitor-l and inhibitor-2. Type-2 phosphatases preferentially dephosphorylate the c1 subunit of phosphorylase kinase and are insensitive to inhibition by heat- -stable inhibitors (1). The existence of apparently "latent" phosphatases in skeletal muscle and aortic smooth muscle has been also demonstrated and their activities are stimulated by histone Hl, protamine and polylysine (2-5). Heparin can also distinguish between the type-l and type-2A phosphatases as heat-stable inhibitors do (6). Polybrene, a heparin antagonist polycationic macromolecule, prevents phosphatase-1 from the inhibition caused by either heparin or heat-stable inhibitors (7). Heparin-Sepharose chromatography 0006-291X/85 $1.50 705 Copyright 0 I985 by Academic Press, Inc. Ail rights of reproduction in any form reserved.