Mouse lymphocyte enzymatic markers. A rapid method for achieving selective solubilization and efficient recovery of the membrane-bound 5'-nucleotidase.
A simple method is described for achieving a good recovery and a partial purification of the membrane-bound 5'-nucleotidase (5'-N) from mouse lymphocytes. The experimental procedure is based upon plasma membrane isolation on polycationic beads and selective solubilization of the enzyme activity from bead-bound plasma membranes. With this method, more than 95% of the 5'-N activity detectable in the whole cell homogenates can be routinely recovered in a single fraction showing a 5'-N specific activity which is at least 60 times higher than that found in the crude homogenate. This method also provides a complete separation of 5'-N from the membrane-bound alkaline phosphatase (AP), as well as from any other interfering non-specific phosphatase. Since this method is rapid and highly reproducible even when small amounts of lymphocytes are available, it may be useful for detecting changes in 5'-N activity in the different T- and B-lymphocyte subpopulations.
D015720:5'-Nucleotidase; D000469:Alkaline Phosphatase / Q000032:analysis; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007770:L-Lactate Dehydrogenase / Q000032:analysis; D008214:Lymphocytes / Q000201:enzymology*; D008722:Methods; D051379:Mice; D008810:Mice, Inbred C57BL; D009708:Nucleotidases / Q000032:analysis / Q000302:isolation & purification*; D017830:Octoxynol; D011092:Polyethylene Glycols / Q000494:pharmacology; D012995:Solubility; D013154:Spleen / Q000201:enzymology
D011092:Polyethylene Glycols; D017830:Octoxynol; D007770:L-Lactate Dehydrogenase; D009708:Nucleotidases; D000469:Alkaline Phosphatase; D015720:5'-Nucleotidase