Acid hydrolases in HeLa cells: comparison of methods for light microscopy.
To distinguish lysosome populations of HeLa cells, acid phosphatase, beta-glucuronidase, arylsulfatase and esterase were demonstrated using various substrates and couplers with different fixations, pHs and inhibitors. The substrates chosen were for acid phosphatase, naphthol AS-BI phosphate with fast red violet LB at pH 4.6; for beta-glucuronidase, naphthol AS-BI beta-D-glucuronide with fast red violet LB at pH 4.4; for arylsulfatase, p-nitrocatechol sulfate, with lead as the capturing ion, at pH 4.8 and 5.6; and for esterase, naphthol AS-D acetate with fast blue BB at pH 6.5. In the azo-dye methods, the coupling was always simultaneous and results were satisfactory with unfixed cells. For optimal demonstration of arylsulfatase, cells were fixed in glutaraldehyde in 0.1 M cacodylate buffer pH 7.2, 2% for 24 hr or 6.25% for 2 hr, and washed for 1-9 days in 0.1 M veronal acetate buffer pH 7.2, 7.5% with respect to sucrose. Two groups of lysosomes were distinguished. One comprised small bodies, probably primary lysosomes, which lay in a cluster near the nucleus. They had quite stable membranes and were mostly acid phosphatase-positive. They sometimes contained beta-glucuronidase or esterase, but rarely arylsulfatase. The other group included all the acid hydrolase-positive bodies scattered throughout the rest of the cytoplasm. They were mostly larger, with more labile membranes, and contained beta-glucuronidase, esterase or arylsulfatase, but rarely acid phosphatase.
Watt
M E
ME
Oral Biology Group, University of Glasgow Dental School, United Kingdom.
D000135:Acid Phosphatase / Q000032:analysis; D001192:Arylsulfatases / Q000032:analysis; D004396:Coloring Agents; D004791:Enzyme Inhibitors; D004950:Esterases / Q000032:analysis*; D005966:Glucuronidase / Q000032:analysis; D006367:HeLa Cells; D006651:Histocytochemistry; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008853:Microscopy / Q000295:instrumentation / Q000379:methods; D013379:Substrate Specificity
D004396:Coloring Agents; D004791:Enzyme Inhibitors; D004950:Esterases; D000135:Acid Phosphatase; D001192:Arylsulfatases; D005966:Glucuronidase