Cytochemistry of Golgi fractions prepared from rat liver.
Cytochemical tests for several marker enzymes were applied to liver tissue and to the three Golgi fractions (GF(1), GF(2), GF(3)) separated by the procedure of Ehrenreich et al. from liver homogenates of alcohol-treated rats. 5'-Nucleotidase (AMPase) reaction product was found in all three fractions but in different locations: It occurred along the inside of the membrane of VLDL-filled vacuoles in GF(1) and GF(2), and along the outside of the cisternal membranes in GF(3). In the latter it was restricted to the dilated cisternal rims and was absent from the cisternal centers. The AMPase activity found in the fractions by biochemical assay is therefore indigenous to Golgi components and is not due to contamination by plasma membrane. Acid phosphatase (AcPase) reaction product was detected within lysosomal contaminants in GF(1) and within many VLDL-filled vacuoles in GF(1) and GF(2), indicating that AcPase activity is due not only to contaminating lysosomes, but also to enzyme indigenous to Golgi secretory vacuoles. G-6-Pase reaction product was present in GF(3) and within contaminating endoplasmic reticulum fragments, but not in other fractions. Thiamine pyrophosphatase (TPPase) was localized to some of the VLDL-filled vacuoles and cisternae in GF(1) and GF(2), and was not found in the cisternae in GF(3). The results demonstrate the usefulness of cytochemical methods in monitoring the fractionation procedure: They have (a) allowed a reliable identification of contaminants, (b) made possible a distinction between indigenous and contaminating activities, and (c) shown, primarily by the results of the TPPase test, that the procedure achieves a meaningful subfractionation of Golgi elements, with GF(1) and GF(3), representing primarily trans-Golgi elements from the secretory Golgi face, and GF(3) consisting largely of cis-Golgi components from the opposite face.
D000135:Acid Phosphatase / Q000032:analysis; D000249:Adenosine Monophosphate; D000818:Animals; D002458:Cell Fractionation; D002499:Centrifugation, Density Gradient; D000431:Ethanol / Q000494:pharmacology; D005952:Glucose-6-Phosphatase / Q000032:analysis; D006056:Golgi Apparatus / Q000187:drug effects / Q000201:enzymology*; D006651:Histocytochemistry; D008079:Lipoproteins, VLDL / Q000032:analysis; D008099:Liver / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D008247:Lysosomes / Q000032:analysis; D008297:Male; D008854:Microscopy, Electron; D008930:Mitochondria, Liver / Q000032:analysis; D009708:Nucleotidases / Q000032:analysis; D011755:Pyrophosphatases / Q000032:analysis; D051381:Rats; D013835:Thiamine Pyrophosphate
D008079:Lipoproteins, VLDL; D000431:Ethanol; D000249:Adenosine Monophosphate; D009708:Nucleotidases; D000135:Acid Phosphatase; D005952:Glucose-6-Phosphatase; D011755:Pyrophosphatases; D013835:Thiamine Pyrophosphate