Nonaqueous fractionation of HeLa cells in glycols

Abstract

A further modification of Behren's method of nonaqueous cell fractionation, using glycols as media for homogenization and centrifugation, was presented. HeLa cells were frozen in melting Freon-12 (CCl₂F₂), dried under vacuum at −30°C, sonicated in hexylene glycol at −35°C, and centrifuged through either propylene glycol or a mixture of the two glycols at −40°C. The centrifugation yielded a nuclear pellet and a cytoplasmic supernatant. The supernatant was recentrifuged at −10°C, yielding a cytoplasmic pellet. The success of the method depended on the temperature-dependent viscosities of the glycols and on the aggregation properties of cell structures in cold glycols. The allowed ranges of low temperatures were critical but not difficult to use; methods are given for sonication and for centrifugation. The two pellet fractions together contained 90% or more of cellular proteins and nucleic acids. Distribution of [³H]uridine-labeled nucleic acids showed that the first pellet (nuclei) contained over 95% of the nuclear markers, DNA, and ribosomal RNA precursors, plus about 10% of the cytoplasmic marker, 18 S ribosomal RNA. The cytoplasmic pellet contained less than 5% of the nuclear markers. Two enzyme activities were tested; DNA polymerase, a mostly soluble nuclear marker frequently eluted in aqueous fractionation, and lactate dehydrogenase, a cytoplasmic marker. The two enzymes each lost activity in propylene glycol but not in a mixture of 90% hexylene glycol and 10% propylene glycol, so the glycol mixture was used as a centrifugation medium when studying enzymes. The glycol mixture somtimes gave more cytoplasmic material, up to 20% of the 18 S ribosomal RNA, in the nuclear pellet. The fractionation showed, as expected, that DNA polymerase activity was 95% nuclear and lactate dehydrogenase activity was more than 68% cytoplasmic. The concentration of cytoplasmic material afforded by the glycol method allowed the detection of a small amount (approx. 5%) of DNA polymerase activity not associated with nuclei. The chief reason for use of the glycol method instead of other methods of cell fractionation is that easily solubilized cellular material can be recovered in concentrated pellet form in the appropriate nuclear or cytoplasmic fraction.