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.
Subject(s)
Actins/chemistry , Cyanates/chemistry , Isocyanates , Actins/isolation & purification , Animals , Biuret , Buffers , Cyanates/pharmacology , Lysine/chemistry , Microscopy, Electron , Muscles/chemistry , Polymers , Rabbits , Spectrometry, Fluorescence , ViscosityABSTRACT
Several methods (fluorescence, high- and low-shear viscosity, and electron microscopy) have been applied to measure the effects of lysozyme on actin polymerization. Under our conditions, at pH 8.0 and 20 degrees C, lysozyme is predominantly dimeric and its major effect is to inhibit the steady-state polymerization of actin. Those actin filaments formed in the presence of lysozyme are significantly shortened with recurrent amorphous densities along the filament length. However, at pH 6.4 and 37 degrees C, lysozyme is monomeric and actin filament cross-linking is observed. We reasoned that in hen egg white lysozyme the tripeptide L-arginyl-glycyl-aspartate (RGD), a sequence capable of mimicking a portion of the receptor sites of extracellular matrix proteins, might be important in lysozyme self-association and, therefore, actin-lysozyme interaction. The presence of RGD in the lysozyme-actin polymerizing solutions at pH 8.0 and 20 degrees C caused an inhibition of the dimeric lysozyme effects, while RGD alone had no effects on actin polymerization. Therefore, RGD most likely binds to a complementary RGD sequence on lysozyme and alters its ability to interact with actin and modify polymerization.
Subject(s)
Actin Cytoskeleton/metabolism , Actins/metabolism , Muramidase/metabolism , Polymers , Actin Cytoskeleton/ultrastructure , Actins/ultrastructure , Amino Acid Sequence , Animals , Fluorescence , Fluorescent Dyes , Iodoacetamide/analogs & derivatives , Molecular Sequence Data , Oligopeptides/pharmacology , Rabbits , Surface Properties , ViscosityABSTRACT
The relationship between the metabolic gradient within the ocular lens and its cellular and molecular organization is discussed. The lens shares a number of organizational similarities with other stratified ectodermal tissues. All of these tissues were avascular and therefore, dependent upon the simple diffusion of nutrients from the surrounding medium. They contain non-metabolizing cell layers which are continually produced by an irreversible maturation process. This process is not random aging but a well orchestrated sequence of events dependent upon the physico-chemical properties of specialized proteins typical of each tissue. In the lens, these proteins are the crystallins. The signal for this maturation is decreased metabolism in conjunction with cell dehydration. Metabolism of these maturing cells is decreased by limited nutrient penetration because of barriers to nutrient diffusion and the high rate of utilization of nutrients by the more metabolically active cells near the basement membrane. Additionally, the regulation of anaerobic metabolism is dependent upon the maintenance and dissolution of an organized array of enzymes and the effects of dehydration, i.e. excluded volume effects and modified macromolecular organization. In the lens, the absence of metabolism during the maturation of the inner fiber cell layers is as important as the presence of active metabolism where differentiation and active protein synthesis occur. Therefore, any stress that disrupts tissue organization will have a detrimental effect on lens integrity.
Subject(s)
Lens, Crystalline/metabolism , Aging/metabolism , Animals , Crystallins/metabolism , Diffusion , Extracellular Space/metabolism , Glycolysis , Intercellular Junctions/metabolismABSTRACT
Based on recent findings from our laboratories, we propose the working hypothesis that the macromolecular organization of the outer cortex is dependent upon a functional link which occurs between actin, gamma-crystallin and metabolism. This hypothesis is rooted in the following observations: (1) actin polymerization is energy-dependent; (2) exogenously added gamma-crystallin modifies the rate, extent and pattern of actin polymerization in vitro; (3) the metabolite L-alpha-glycerol phosphate appears to bind to gamma-crystallin, and (4) glycolytic enzymes occur in an organized array on actin filaments.
