Isolation of a Golgi apparatus-rich fraction from rat liver. II. Enzymatic characterization and comparison with other cell fractions.
Enzymatic activities associated with Golgi apparatus-, endoplasmic reticulum-, plasma membrane-, mitochondria-, and microbody-rich cell fractions isolated from rat liver were determined and used as a basis for estimating fraction purity. Succinic dehydrogenase and cytochrome oxidase (mitochondria) activities were low in the Golgi apparatus-rich fraction. On the basis of glucose-6-phosphatase (endoplasmic reticulum) and 5'-nucleotidase (plasma membrane) activities, the Golgi apparatus-rich fraction obtained directly from sucrose gradients was estimated to contain no more than 10% endoplasmic reticulum- and 11% plasma membrane-derived material. Total protein contribution of endoplasmic reticulum, mitochondria, plasma membrane, microbodies (uric acid oxidase), and lysosomes (acid phosphatase) to the Golgi apparatus-rich fraction was estimated to be no more than 20-30% and decreased to less than 10% with further washing. The results show that purified Golgi apparatus fractions isolated routinely may exceed 80% Golgi apparatus-derived material. Nucleoside di- and triphosphatase activities were enriched 2-3-fold in the Golgi apparatus fraction relative to the total homogenate, and of a total of more than 25 enzyme-substrate combinations reported, only thiamine pyrophosphatase showed a significantly greater enrichment.
D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D002474:Cell-Free System; D002499:Centrifugation, Density Gradient; D000242:Cyclic AMP / Q000032:analysis; D003576:Electron Transport Complex IV / Q000032:analysis; D004721:Endoplasmic Reticulum / Q000201:enzymology; D005952:Glucose-6-Phosphatase / Q000032:analysis; D006056:Golgi Apparatus / Q000201:enzymology*; D006652:Histological Techniques; D008099:Liver / Q000166:cytology* / Q000201:enzymology*; D008297:Male; D008930:Mitochondria, Liver / Q000201:enzymology; D010744:Phosphoric Monoester Hydrolases / Q000032:analysis*; D051381:Rats; D013385:Succinate Dehydrogenase / Q000032:analysis; D000637:Transaminases / Q000032:analysis*
D000242:Cyclic AMP; D013385:Succinate Dehydrogenase; D003576:Electron Transport Complex IV; D000637:Transaminases; D010744:Phosphoric Monoester Hydrolases; D005952:Glucose-6-Phosphatase