Preservation and separation of endomembrane marker enzyme activity in potato leaf homogenates

To minimize rapid browning and membrane degradation of crude microsomes, leaves of Solanum tuberosum (cv. Kennebec and cv. Katahdin) were initially homogenized in the presence of various inhibitors of polyphenol oxidase, phospholipase, and protease activity. To obtain and maintain marker enzyme activities used to identify plasma membranes, Golgi membranes, and endoplasmic reticulum, it was necessary to homogenize young leaves in the presence of sulfhydryls at pH 7.8. Further separation of these membranes, as determined by distribution of total activities of marker enzymes in linear sucrose density gradients, indicated a relatively pure plasma membrane fraction (1.15 g/cm3) free from contamination by thylakoids (1.19 g/cm3) and other endomembrane components. However, the distribution of specific activities across the gradient revealed that plasma membranes isolated from green tissue may be contaminated by Golgi membranes and not necessarily by plastid membranes.