Purification and characterization of cytoplasmic protamine messenger ribonucleoprotein particles from rainbow trout testis cells.
Poly(A)-containing protamine messenger ribonucleoprotein particles [poly(A+) pmRNP particles] have been isolated from the polysomal and free cytoplasmic subcellular fractions of trout testis cells by a two-step isolation procedure. Ethylenediaminetetraacetic acid (EDTA) treated particles from both cytoplasmic fractions were first fractionated by sucrose gradient centrifugation and the putative pmRNP particles localized by utilizing 3H-labeled protamine complementary DNA (pcDNA) probes. In addition, particles present in these fractions were characterized by their translational activity in the heterologous, rabbit reticulocyte cell-free system and the protein components of crude mRNP complexes analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoesis. The final purification step involved affinity chromatography of pooled gradient fractions on oligo(dT)-cellulose from which intact pmRNP could be eluted with distilled water at 40 degrees C. Highly purified particles from both polysomal and free cytoplasmic fractions prepared by this procedure had buoyant densities of 1.35-1.37 g/cm3 in CsCl or a protein content of approximately 82%. Particles isolated from EDTA-dissociated polysomes were actively translated in vitro, while their free cytoplasmic counterparts were not. High salt washed pmRNP particles or the RNA extracted from pmRNP preparations, however, directed the synthesis of trout protamines in this system. A model of the activation of stored pmRNP particles in vitro and in vivo is presented.
D000818:Animals; D002499:Centrifugation, Density Gradient; D003593:Cytoplasm / Q000032:analysis; D004492:Edetic Acid; D004591:Electrophoresis, Polyacrylamide Gel; D008297:Male; D008970:Molecular Weight; D011061:Poly A / Q000302:isolation & purification* / Q000378:metabolism; D011479:Protamines / Q000096:biosynthesis*; D012333:RNA, Messenger / Q000302:isolation & purification* / Q000378:metabolism; D013737:Testis / Q000032:analysis*; D014337:Trout
D011479:Protamines; D012333:RNA, Messenger; D011061:Poly A; D004492:Edetic Acid