Phenylglyoxal Reveals Phosphorylation-Dependent Difference in the Conformation of Acanthamoeba Myosin II Active Site M. Jolanta Redowicz 1 Department of Muscle Biochemistry, Nencki Institute of Experimental Biology, 3 Pasteur Street, 02-093 Warsaw, Poland Received July 10, 2000, and in revised form September 16, 2000 Acanthamoeba myosin II is regulated in an unique way by phosphorylation of three serine residues lo- cated within nonhelical tailpiece of the rod domain. Phosphorylation inhibits functions associated with the NH 2 -terminal motor domain, i.e., actin-activated activity and ability to move actin filaments. Number of data indicate functional communication between these distant domains. In this work, effect of modifi- cation of arginine residues with phenylglyoxal on the Ca 2 -ATPase activity and susceptibility to endopro- teinase ArgC cleavage of monomeric phospho- and de- phosphomyosin II has been investigated. Upon the phenylglyoxal treatment the activity of dephospho- myosin II was decreasing faster that the activity of phosphomyosin. The modification also affected the proteolytic fragmentation of phospho- and dephos- phomyosin II: the cleavage of heavy chain was further inhibited for phosphomyosin and enhanced for de- phosphomyosin with a concomitant exposure of an additional cleavage site within the head domain. No difference in the quantity of modified arginines was observed. These results indicate a difference between the conformation of active sites of phospho- and de- phosphomyosin II. © 2000 Academic Press Key Words: myosin; Acanthamoeba; conformation; ar- ginine modification; phosphorylation; phenyglyoxal. Functional activities of conventional myosin II from Acanthamoeba castellanii are regulated in an unique, unconventional way (1). Myosin II is composed of a pair of 171-kDa heavy chains and two pairs of 17.5-kDa and 17-kDa light chains. An NH 2 -terminal 90-kDa head domain, called also a motor domain, has the actin- binding and ATPase sites (2, 3). A COOH-terminal, 81 kDa, 90-nm-long rod domain participates in for- mation of 210- to 230-nm-long antiparallel minifila- ments comprised of eight myosin II monomers (4 – 6). The COOH-terminal 27 amino acid residues of each heavy chain form a nonhelical tailpiece containing three serine residues that can be phosphorylated by an endogenous myosin II heavy chain kinase (7, 8). Phos- phorylation negatively regulates myosin II functions associated with the head domain of filamentous myo- sin, i.e., inactivates its actin-dependent Mg 2+ -ATPase (10) and ability to move actin filaments in an in vitro motility assay (11). The question arises of how the phosphorylation of the COOH-terminal serine residues separated from the motor domain by the tail domain affects the activ- ity of Acanthamoeba myosin II. In contrast to the reg- ulation of certain other class-II myosins by light chain or/and heavy chain phosphorylation there is no evi- dence for this myosin isoform for direct head–tail in- teraction(s) and inhibition of filament formation (12). The data so far obtained for native myosin II or its recombinant wild-type and mutant rod preparations by means of various methods, including limited proteoly- sis (13, 14), in vitro motility assay (11), electric bire- fringence (15–17), differential scanning calorimetry (18, 19), or electron microscopy (5, 17), indicate func- tional communication between the head domain and COOH-terminal portion of the rod domain. However, the mechanism of this communication still remains to be elucidated. It is known that arginine residues are essential in the active sites of several enzymes that transfer phos- phorus containing groups (20). Decrease of the enzy- matic activity after phenylglyoxal treatment observed with skeletal, smooth, and cardiac myosins indicates 1 To whom correspondence and reprint requests should be ad- dressed. Fax: (48) 22-822-5342. E-mail: jolanta@nencki.gov.pl. 0003-9861/00 $35.00 413 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved. Archives of Biochemistry and Biophysics Vol. 384, No. 2, December 15, pp. 413– 417, 2000 doi:10.1006/abbi.2000.2126, available online at http://www.idealibrary.com on