Electrophoresis 1982,3, 151-157Polynucleotide-polyacrylamide gel electrophoresi! 15 1 monomorphic. Detailed discussion of these genetic variations will be presented elsewhere (H. Czikeli, in preparation). This work was supported in part by grant No. 4059 from the ,,Fonds zur Forderung der wissenschaftlichen Forschung".Intracellular deoxyribonucleases (DNases) in human peripheral blood leukocytes have been identified and characterized by polynucleotide-polyacrylamide gel electrophoresis (PPAGE). This technique combines the protein separating powers of polyacrylamide gel electrophoresis with the sensitivity of detection characteristic of an enzyme assay. Visualization of nuclease activity is made possible by the inclusion of high molecular weight DNA substrate within the sieving portion of a narrow polyacrylamide gel. No sodium dodecyl sulfate, urea, or other protein denaturants are used in this procedure. However, electrophoresis of the contents of up to lo6 cells/gel is accomplished using conditions under which the enzymes are inactive. The positions of DNases are then revealed by incubation of the gel in an appropriate buffer followed by staining for intact DNA. Colorless regions correspond to the presence of enzyme. Used as a pattern recognition tool, PPAGE revealed that leukocytes from normal donors contained lower levels of certain activities than did cells from donors with chronic myelogenous leukemia. The rapid simultaneous characterization of multiple activities is made possible by simply using different buffer compositions in the incubation step. For example, ionic strength and pH optima may be found directly for activities present in unpurified human intracellular contents. The reproducibility of this method permits comparison of individual activities by using Ferguson plot analysis to detect differences or similarities among enzymes. These analyses revealed that DNase activities in human peripheral blood leukocytes fell into a minimum of two families. Modifications of overall effective charge probably account for microheterogeneity. Abbreviations: AGIEF: A~~~~~ gel isoelectric focusing; ~~-1 1 1 : A,,tithrombin 111; PI: Isoelectric point(s); AGCIE: Agarose gel crossed immunoelectrophoresis.EDTA-plasma (containing 5~ Na3-EDTA), citrated plasma (blood: 3.8 % sodium citrate = 9:l) and serum Samples were obtained from healthy Japanese people and stored at -80 "c until used. Desialylation of plasma or purified AT-I11 were 0 Verlag Chemie GmbH, D-6940 Weinheim, 1982 0173-0835/82/0306-0157~2.50/0

Individual native nuclease activities from human leucocytes are separated by using two-dimensional gel electrophoresis in an apparatus that allows the simultaneous running of 28 gels. Proteins are separated by isoelectric focusing in a disc gel, followed by electrophoresis into a slab gel containing DNA. Protein denaturants are avoided in the second dimension by the use of a running pH well above the optimal pH for DNAase (deoxyribonuclease) activity. Electrophoresed gels are incubated in appropriate buffers to activate nuclease activity. After staining for intact DNA, the positions of active enzymes, unobscured by the presence of other proteins, are revealed as colourless spots in a reddish-purple field. The technique is easy to use and is sensitive to 50pg of DNAase I. Versatility is provided by the use of either acidic or basic electrophoresis running buffers and by the use of specific gel incubation conditions to reveal different sets of enzyme activities. Two DNAases active at pH 7.4 in the presence of Mg2+ and Ca2+, and sixteen DNAases active at acidic pH and not requiring metals, are detected. Treatment of the human enzymes with specific glycosidases reveals that many of the human DNAases are glycoproteins containing negatively charged moieties and may be derived from modification of parent activities.

Native DNAase (deoxyribonuclease) activities derived from mouse peritoneal cavity and peripheral blood components were separated, detected, and characterized by electrophoresis into polyacrylamide gels containing DNA, followed by incubation of the gels, and staining of the substrate to reveal only the DNAase activities. Resident peritoneal macrophages contained 12 DNAase-II-like activities that were characteristic of that cell type, whereas lymphocytes and granulocytes each contained five DNAases. Induction of inflammation by peritoneal injection of thioglycollate resulted in changes in macrophage DNAase expression, including: increased total DNAase activity, a decrease in the number of activities from 12 to 11, increased activity of a specific subset of the enzymes, and a change in the apparent size of a specific subset of the enzymes. Electrophoretic and enzymic properties and sensitivity to endo-beta-N-acetylglucosaminidase H indicated that the macrophage activities probably represented charge variants of one or two parent peptide chains.