Kentaro Hozumi

Conformation of nucleoplasmin and its interaction with DNA-protamine complex as a simple model of fish sperm nuclei.

Nucleoplasmin was isolated from Xenopus laevis eggs and purified by an improved method using an open column. Its conformation was investigated spectrophotometrically by UV, CD and fluorescence. It was shown that alpha-helix content of nucleoplasmin was 30-40%, and one of the two tryptophan residues in nucleoplasmin located in the hydrophobic surroundings and the other in the relatively hydrophilic surroundings. The isolated nucleoplasmin was found to decondense sperm nuclei of salmon also, suggesting a possibility of the existence of nucleoplasmin-like protein in fish as well. Collapse of the protamine (salmine)-DNA complex as a simple model for fish sperm nuclei by nucleoplasmin was directly observed by measuring OD320 of aqueous protamine-DNA mixtures. This is a molecular level observation for the removal of protamine from DNA-protamine complex.

Mechanism of salmon sperm decondensation by nucleoplasmin.

Removal of protamine from DNA-protamine (salmine, protamine from salmon sperm) complexes by nucleoplasmin was examined and compared with that of poly-L-glutamic acid (PLGA) using turbidity and ethidium bromide (EB) treatment methods. When nucleoplasmin or PLGA was added to a DNA-protamine complex solution, turbidity was decreased and the amount of EB intercalated into DNA was increased. These results suggest that nucleoplasmin and PLGA can remove protamine from DNA-protamine complexes. The effect of nucleoplasmin was more potent than that of PLGA. Direct interaction of nucleoplasmin with protamine was confirmed by mixing experiments using circular dichroism (CD) and fluorescence spectroscopies. Results suggest that nucleoplasmin is bound to protamine in a 1:1 ratio and that Trp126 is located near a hydrophilic region containing a polyglutamic acid tract of nucleoplasmin which was obviously influenced by its binding with protamine. It would appear that the polyglutamic acid tract in nucleoplasmin plays a critical role for binding with protamine.

The role of nucleoplasmin — molecular chaperone protein — in the fertilization process

Nucleoplasmin is the most abundant protein in the Xenopus laevis oocyte nucleus. It is able to assemble nucleosomes by binding histones and transferring them to DNA in vitro, and the first protein named as molecular chaperone [1]. It is an acidic, thermostable protein and forms a stable pentamer. Recently, it was reported that nucleoplasmin removed sperm nuclear binding proteins, protamines, from the sperm nuclei of Xenopus laevis and converted into nucelosome in vitro. Thus nucleoplasmin plays a leading role in the whole fertilization process and may act as both of an assembly and a disassembly factors for remodeling of sperm chromatin in vivo [2, 3]. We have studied the interaction of nucleoplasmin with DNA-salmine complex as a simple model of salmon sperm nuclei in a molecular level, and reported that a poly glutamic acid tract in the carboxyl terminus side of nucleoplasmin is mainly related to the removal of protamine from sperm nuclei [4]. In the present study, the interaction of nucleoplasmin with histone octamer, fractionated histones and also with DNA were investigated by fluorescence to obtain the further information on the mechanism of the conversion into nucleosome by nucleoplasmin.