Smooth muscle cells were dissociated from normal rabbit aorta by incubating the tissue in Hanks' solution containing elastase, collagenase, and hyaluronidase. The isolated cells contained significant amounts of the following acid hydrolases: N-acetyl-ß-glucosaminidase, N-acetyl-ß-galactosaminidase, ß-galactosidase, ß-glucuronidase, α-mannosidase, ß-glucosidase, acid phosphatase, and cathepsins C and D. The cells were disrupted and fractionated by isopycnic centrifugation on sucrose density gradients in the Beaufay automatic zonal rotor.
Lysosomes with a modal density of 1.16 were identified by the distribution of these acid hydrolases and by the latency of N-acetyl-ß-glucosaminidase and ß-galactosidase. Other particulate enzymes studied in these sucrose gradients included cytochrome oxidase and monoamine oxidase (mitochondria), 5'-nucleotidase and leucyl-ß-naphthylamidase (plasma membrane), and catalase (? peroxisome).
This microanalytical subcellular fractionation technique is applicable to the study of milligram quantities of many other tissues, both normal and pathological.