Effect of pancreatic lipase on supramolecular DNA complexes, nuclear matrix, and 70% ethanol-fixed cells is studied by elastoviscosimetry. It is shown that lipase induces DNA degradation not only in vitro but also in whole cells. Possible role of neutral lipids, in particular, DNA-bound diglycerides, in the arrangement of chromosomal DNA is discussed.
Key Words: supramolecular DNA complexes; nucleotides; eukaryotic cells, lipase, degradationIt has been previously shown that supramolecular DNA complexes (DNA-SC) isolated from various eukaryotic cells by mild phenol extraction apart from RNA and non-histone protein (1-2%) contain 0.12-1.2% (depending on cell type) DNA-bound lipids (neutral lipids dominate over phospholipids), which can be extracted by the method of Folch only by treating these complexes with DNase I [9,10]. Various experimental approaches (sedimentation, rotation viscosimetry, electron microscopy, and light scattering) have demonstrated [5,10,11] the resistance of DNA-SC to proteolytic enzymes and their susceptibility for lipophilic agents (Triton X-100, deoxycholate, 35% ethanol)and phospholipases C, A, and D, which induced degradation of the complex into fragments with a molecular weight of about l0 s D.Pancreatic lipase produces smaller degradation products (40-50• s D), which can be attributed to the presence of considerable amount of diglycerides (30% of neutral lipids in the complex [10]), a substrate of this enzyme.In the present study we demonstrate degradation of DNA-SC by lipase not only in solution, but also in whole cells and in nuclear matrix and discuss a possible role of neutral lipids (diglycerides) in the organization of chromosomal DNA.
MATERIALS AND METHODSUsing the method of phenol extraction we isolated two DNA fractions, water-soluble DNA-SC and water-insoluble DNA of phenol nuclear matrix (DNA-PNM) concentrated in the phenol--water interface, from various eukaryotic cells (rat thymus, erythrocytes, loach sperm, sarcoma-37, Zeidel ascitic hepatoma, leukemia L1210 and P388 cell lines). Isolation of DNA-SC and DNA-PNM as well as capillary elastoviscosimetry and rotation viscosimetry were described in details in our previous reports [10,11]. Effects of exogenous enzymes on whole cells were studied in cell suspension (2x107 cells/ml, initial concentration) fixed with 70% ethanol using standard cytofluorometric technique. In brief, 100 ~d-aliquot of ethanol-treated cell suspension was centrifuged (2 min, 3000g), the pellet was resuspended in 100 gl Hanks' solution, and 0.5% diethyl pyrocarbonate was added for inhibition of endogenous nucleases. The cell and DNA-PNM was lysed with a mixture containing 1 M NaC1, 0.05 M EDTA, and 0.25% Triton X-100, pH 8) at 24~ for 1 h (DNA-PNM) or 24 h (cells) as described previously [2,3,12]. Lipase from porcine pancreas (Serva) preincubated with phenylmethylsulfonyl fluoride (inhibition of proteolytic activity was experimentally verified), RNase-A (Merck) incubated at 80~ for 15 min, pronase P (Serva), bacterial endonuclease, phospholipase C from...
