ABSTRACT
In previous research, we observed that tubulin can be found in three fractions within erythrocytes, i.e., attached to the membrane, as a soluble fraction, or as part of a structure that can be sedimented by centrifugation. Given that its differential distribution within these fractions may alter several hemorheological properties, such as erythrocyte deformability, the present work studied how this distribution is in turn affected by Ca2+, another key player in the regulation of erythrocyte cytoskeleton stability. The effect of Ca2+ on some hemorheological parameters was also assessed. The results showed that when Ca2+ concentrations increased in the cell, whether by the addition of ionophore A23187, by specific plasma membrane Ca2 + _ATPase (PMCA) inhibition, or due to arterial hypertension, tubulin translocate to the membrane, erythrocyte deformability decreased, and phosphatidylserine exposure increased. Moreover, increased Ca2+ was associated with an inverse correlation in the distribution of tubulin and spectrin, another important cytoskeleton protein. Based on these findings, we propose the existence of a mechanism of action through which higher Ca2+ concentrations in erythrocytes trigger the migration of tubulin to the membrane, a phenomenon that results in alterations of rheological and molecular aspects of the membrane itself, as well as of the integrity of the cytoskeleton. (AU)
Subject(s)
Humans , Erythrocytes/metabolism , Tubulina/metabolism , Calcium/metabolism , Cell Membrane/metabolism , Cytoskeleton/metabolism , Erythrocyte Deformability/physiologyABSTRACT
The fact that glycerol preserves microtubules from depolymerizing in vitro, and that some ions such as Ca(II) and Mg(II), regulate the assembly-disassembly process of these structures, induced us to study the effect of several sugars, glycols and metal ions on solubility and colchicine affinity of tubulin in rat brain homogenates, and of purified microtubular protein. Inhibition of colchicine binding was significant with glycerol, polyethylene glycol 1000 (PEG-2) and the ions A1(III), Co(II), Ni(II), while compounds structurally related to glycero (glucose and sucrose) did not inhibition it. Mannitol, instead, increased the activity a 47% over control. Apparently the presence of some compounds in brain homogenates [PEG-2 (1000) and NI (II)] favored tubulin sedimentation when these latterwere centrifuged at 100,000 x g for 150 min at 20 degrees C, but the form in which tubulin becomes aggregated in the pellet is unknown. Nickel ion madeinsoluble microtubular protein of homogenates and the purified one by more than 90% without causing significant inhibition of the colchicine binding. The sediment containing nickel-treated two cycles purified microtubular protein observed with the electron microscope did not present microtubules, but it revealed the presence of irregular, wavy and streteched structures, but it revealed the presence of irregular, wavy and stretched structures bearing highly dense dotted material. The sediments became soluble in phosphate-glutamate buffer (pH 6.8) and, when incubated in polymerizing conditions, gave rise to microtubules undistinguishable from those prepared with untreated purified protein (AU)
Subject(s)
Animals , Female , Rats , Brain Chemistry , Carbohydrates/pharmacology , Cations/pharmacology , Colchicine/metabolism , Glycols/pharmacology , Nickel/pharmacology , Tubulina/metabolism , Aluminum/pharmacology , Cobalt/pharmacology , Fixatives/pharmacology , Microtubules/drug effects , Nerve Tissue Proteins/metabolism , Polymers , Chemical Precipitation , Solubility , Protein Binding/drug effectsABSTRACT
The fact that glycerol preserves microtubules from depolymerizing in vitro, and that some ions such as Ca(II) and Mg(II), regulate the assembly-disassembly process of these structures, induced us to study the effect of several sugars, glycols and metal ions on solubility and colchicine affinity of tubulin in rat brain homogenates, and of purified microtubular protein. Inhibition of colchicine binding was significant with glycerol, polyethylene glycol 1000 (PEG-2) and the ions A1(III), Co(II), Ni(II), while compounds structurally related to glycero (glucose and sucrose) did not inhibition it. Mannitol, instead, increased the activity a 47% over control. Apparently the presence of some compounds in brain homogenates [PEG-2 (1000) and NI (II)] favored tubulin sedimentation when these latterwere centrifuged at 100,000 x g for 150 min at 20 degrees C, but the form in which tubulin becomes aggregated in the pellet is unknown. Nickel ion madeinsoluble microtubular protein of homogenates and the purified one by more than 90% without causing significant inhibition of the colchicine binding. The sediment containing nickel-treated two cycles purified microtubular protein observed with the electron microscope did not present microtubules, but it revealed the presence of irregular, wavy and streteched structures, but it revealed the presence of irregular, wavy and stretched structures bearing highly dense dotted material. The sediments became soluble in phosphate-glutamate buffer (pH 6.8) and, when incubated in polymerizing conditions, gave rise to microtubules undistinguishable from those prepared with untreated purified protein
Subject(s)
Animals , Female , Rats , Carbohydrates/pharmacology , Cations/pharmacology , Colchicine/metabolism , Glycols/pharmacology , Nickel/pharmacology , Brain Chemistry , Tubulina/metabolism , Aluminum/pharmacology , Chemical Precipitation , Cobalt/pharmacology , Fixatives/pharmacology , Protein Binding , Microtubules , Polymers , Nerve Tissue Proteins/metabolism , SolubilityABSTRACT
El análisis morfométrico aplicado a fibras preterminales de cerebro y a células de Sertoli de testículo del sapo Bufo arenarum mostraron que los microtúbos desaparecen en un 75% y 92% respectivamente cuando los animales son enfriados a 1-2-C por 90 minuots. Las estimaciones hechas en cuerpos neuronales de cerebro y en otras células del testículo mostraron los mismos resultados. Trabajos previos de los autores (López y Bertini, 1982) mostraron que el grado de polimerización de tubulina (porcentaje de tubulina insoluble) en sapos enfriados, solo decreció en un 40%. Se postula que el elevado porcentaje de tubulina insolubre en animales enfriados es decreció en un 40%. Se postula que el elevado porcentaje de tubulina insoluble en animales enfriados es debido a la existencia de un pool de tubulina no estructurada en microtúbulos, unida a estructuras membranosas en sitios de alta afinidad y/o a oligómeros de tubulina no reconocible como microtúbulos por microscopía electrónica (AU)
Subject(s)
Animals , Male , Cold Temperature/adverse effects , Microtubules/ultrastructure , Tubulina/metabolism , Cerebrum/ultrastructure , Testis/ultrastructureABSTRACT
El análisis morfométrico aplicado a fibras preterminales de cerebro y a células de Sertoli de testículo del sapo Bufo arenarum mostraron que los microtúbos desaparecen en un 75% y 92% respectivamente cuando los animales son enfriados a 1-2-C por 90 minuots. Las estimaciones hechas en cuerpos neuronales de cerebro y en otras células del testículo mostraron los mismos resultados. Trabajos previos de los autores (López y Bertini, 1982) mostraron que el grado de polimerización de tubulina (porcentaje de tubulina insoluble) en sapos enfriados, solo decreció en un 40%. Se postula que el elevado porcentaje de tubulina insolubre en animales enfriados es decreció en un 40%. Se postula que el elevado porcentaje de tubulina insoluble en animales enfriados es debido a la existencia de un pool de tubulina no estructurada en microtúbulos, unida a estructuras membranosas en sitios de alta afinidad y/o a oligómeros de tubulina no reconocible como microtúbulos por microscopía electrónica
