RESUMO
The relation between the activity of the sperm-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDS) and the motility of sperms was investigated. It was found that the mean value of GAPDS activity in sperm samples with low motility is 2.5-3-fold lower than that in samples with high motility. Sperm motility was shown to diminish in the presence of superoxide anion, hydroxyl radical, and hydrogen peroxide. The decrease in sperm motility in the presence of hydrogen peroxide was proportional to the concentration of the oxidant and correlated with the decrease in GAPDS activity (r = 0.96). Based on the literature data on the importance of GAPDS for the motility of sperms together with the presented observations, it was concluded that the decrease in the sperm motility in the presence of reactive oxygen species is due to the oxidation of GAPDS and inhibition of glycolysis.
Assuntos
Gliceraldeído-3-Fosfato Desidrogenases , Motilidade dos Espermatozoides , Espermatozoides/enzimologia , Animais , Gliceraldeído-3-Fosfato Desidrogenases/deficiência , Gliceraldeído-3-Fosfato Desidrogenases/fisiologia , Glicólise/efeitos dos fármacos , Cavalos , Humanos , Peróxido de Hidrogênio/farmacologia , Masculino , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Motilidade dos Espermatozoides/efeitos dos fármacos , Motilidade dos Espermatozoides/fisiologia , Superóxidos/metabolismoRESUMO
Sperm-specific glyceraldehyde-3-phosphate dehydrogenase (GAPDS) is bound to the fibrous sheath of the sperm flagellum through the hydrophobic N-terminal domain of the enzyme molecule. Expression of human GAPDS in E.coli cells yields inactive and insoluble protein. Presumably, the N-terminal domain prevents correct folding of the full-length recombinant enzyme. To obtain GAPDS in a soluble and active form, a recombinant enzyme lacking in 68 amino acids of the N-terminal domain (dN-GAPDS) was expressed in E.coli cells. Purified dN-GAPDS was shown to be a protein of 9.3 nm in diameter (by dynamic light scattering), which is close to the size of the muscle tetrameric glyceraldehyde-3-phosphate dehydrogenase (8.6 nm). The catalytic properties of the protein differed a little from those of the muscle glyceraldehyde-3-phoshate dehydrogenase. However, compared to muscle glyceraldehyde-3-phoshate dehydrogenase, dN-GAPDS exhibited enhanced thermostability (the transition midpoints values are 60.8 and 67.4°C, respectively) and was much more resistant towards action of guanidine hydrochloride (inactivation constants are 2.45±0.018 and 0.118 ± 0.008 min(-1), respectively). The enhanced stability of dN-GAPDS is likely to be related to some specific features of the GAPDS structure compared to that of the muscle enzyme: 1) reduced number of solvent-exposed salt bridges; 2) 2 additional buried salt bridges; and 3) 6 additional proline residues in GAPDS meeting the "proline rule". It is assumed that high stability of the sperm-specific GAPDS is of importance for the efficiency of fertilization.
Assuntos
Gliceraldeído-3-Fosfato Desidrogenases/metabolismo , Proteínas Recombinantes/metabolismo , Espermatozoides/enzimologia , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Sítios de Ligação/genética , Biocatálise , Estabilidade Enzimática , Escherichia coli/genética , Gliceraldeído-3-Fosfato Desidrogenases/química , Gliceraldeído-3-Fosfato Desidrogenases/genética , Temperatura Alta , Humanos , Concentração de Íons de Hidrogênio , Cinética , Masculino , Modelos Moleculares , Dados de Sequência Molecular , Músculos/enzimologia , Mutação , Prolina/química , Prolina/genética , Prolina/metabolismo , Desnaturação Proteica , Estrutura Terciária de Proteína , Coelhos , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Homologia de Sequência de AminoácidosRESUMO
Glyceraldehyde-3-phosphate dehydrogenase (GAPDs) was purified from human sperms and properties of the enzyme were investigated. After sonication of sperms, the most part of GAPDs is associated with the insoluble cell fraction. Trypsin treatment results in the cleavage of part of the N-terminal domain of the enzyme yielding a soluble fragment that was purified by hydrophobic chromatography on Phenyl-Sepharose. The isolated fragment was shown to be a tetramer with molecular weight of approximately 150 kD (according to Blue Native PAGE) and composed of subunits of 40 kD (according to SDS-PAGE). The specific activity of the isolated fragment reached 374 U/mg. It is supposed that GAPDs exists in sperms as the tetrameric molecule bound to the fibrous sheath of the flagellum through the N-terminus of one or two subunits. Comparative study of the amino acid sequences of mammalian GAPDs revealed conservative cysteine residues (C21, C94, and C150) that are specific for the sperm isoenzyme and absent in the somatic isoenzyme. Residue C21 can be involved in the formation of the disulfide bond between the N-terminal domain of GAPDs and fibrous sheath proteins.
