Decreased heterogeneity of CS histone variants after hydrolysis of the ADP-ribose moiety.
Sea urchin CS histone variants are electrophoretically heterogeneous when analyzed in two dimensional polyacrylamide gels (2D-PAGE). Previous results suggested that this heterogeneity is due to the poly (ADP-ribosylation) of these proteins. Consequently, native CS histone variants were subjected to different treatments to remove the ADP-ribose moiety. The incubation in 1 M hydroxylamine was not effective in eliminating the polymers of ADP-ribose from CS variants, and the treatment with sodium hydroxide was deleterious to the proteins. In contrast, the ADP-ribose moiety was successfully removed from the CS variants by incubation with phosphodiesterase (PDE). To eliminate contamination of CS histone variants with PDE extract, the enzyme was covalently bound to Sepharose 4B prior to its utilization. Treatment of native CS histone variants with this immobilized phosphodiesterase removed around 85% of the total ADP-ribose moiety from these proteins. After S-PDE treatment the complex electrophoretic pattern of CS histone variants in 2-D PAGE decreases to five major fractions. From these results we conclude that the electrophoretic heterogeneity of native CS histone variants is mainly due to the extent to which five main CS histone variants are poly(ADP)-ribosylated).
Imschenetzky
M
M
Department of Molecular Biology, Universidad de Concepción, Chile.
Morín
V
V
Carvajal
N
N
Montecino
M
M
Puchi
M
M
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D002970:Cleavage Stage, Ovum; D003619:Dansyl Compounds / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D005456:Fluorescent Dyes; D014644:Genetic Variation; D006031:Glycosylation / Q000187:drug effects; D006657:Histones / Q000378:metabolism*; D006834:Hydrazines / Q000494:pharmacology; D006868:Hydrolysis; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D008297:Male; D010504:Periodic Acid / Q000494:pharmacology; D043264:Phosphodiesterase I; D010727:Phosphoric Diester Hydrolases / Q000494:pharmacology; D011064:Poly Adenosine Diphosphate Ribose / Q000032:analysis; D012617:Sea Urchins; D012972:Sodium Hydroxide / Q000494:pharmacology
D003619:Dansyl Compounds; D005456:Fluorescent Dyes; D006657:Histones; D006834:Hydrazines; D006898:Hydroxylamines; D010504:Periodic Acid; D000246:Adenosine Diphosphate Ribose; D011064:Poly Adenosine Diphosphate Ribose; D019811:Hydroxylamine; C027913:dansyl hydrazine; D012972:Sodium Hydroxide; D010727:Phosphoric Diester Hydrolases; D043264:Phosphodiesterase I