title: Methylisocyanate and actin polymerization: the in vitro effects of carbamylation.
abstract: Uremia has been implicated in cataractogenesis due to protein carbamylation by cyanate derived from urea. The present study was designed to directly identify the effects of carbamylation on actin polymerization and the possible contribution to cataract formation. The susceptibility of actin to carbamylation is expected because of the 19 lysines distributed along its length. The lysines of actin were selectively carbamylated by methylisocyanate (MIC) at pH 8.0 and 4 degrees C and actin polymerization assayed by high-shear viscometry, fluorescence and transmission electron microscopy. Our results provide evidence that non-enzymatic carbamylation of the lysine residues prevents the polymerization of actin. In addition, this carbamylated actin inhibited the polymerization of nascent, unmodified actin. High-shear viscosity measurements demonstrated decreased initial apparent rates and decreased steady-states (final specific viscosities) of polymerization. Fluorescence measurements showed decreased relative intensities of fluorescence versus control and confirmed the inhibitory effects of carbamylation by MIC on the steady state of F-actin. Transmission electron microscopy (TEM) showed the presence of disorganized filaments when carbamylated actin was added to polymerizing unmodified actin. Our results suggest that carbamylation of actin can cause a loss of ordered filament structure and shape of the lens fiber cell, thus predisposing it to cataract development.
authors.affiliation: Department of Ophthalmology, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark 07103-2757.
authors.forename: C L
authors.initials: CL
authors.lastname: Kuckel
authors.forename: B W
authors.initials: BW
authors.lastname: Lubit
authors.forename: P K
authors.initials: PK
authors.lastname: Lambooy
authors.forename: P N
authors.initials: PN
authors.lastname: Farnsworth
mesh_terms: D000199:Actins / Q000737:chemistry* / Q000302:isolation & purification; D000818:Animals; D001737:Biuret; D002021:Buffers; D003485:Cyanates / Q000737:chemistry* / Q000494:pharmacology; D017953:Isocyanates; D008239:Lysine / Q000737:chemistry; D008854:Microscopy, Electron; D009132:Muscles / Q000737:chemistry; D011108:Polymers; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D014783:Viscosity
chemical_list: D000199:Actins; D002021:Buffers; D003485:Cyanates; D017953:Isocyanates; D011108:Polymers; D001737:Biuret; C008461:methyl isocyanate; D008239:Lysine