Selective localization of a Golgi apparatus acid phosphatase isoenzyme in bone using pyridoxal-5'-phosphate.
Substrates commonly used for localizing bone Golgi apparatus (GA) acid phosphatase (AcPase), e.g., beta-glycerophosphate, p-nitrophenylphosphate, cytidine-5'-monophosphate, and di(dicyclohexylammonium)-2-napthylthiolphosphate, give strong staining not only of GA but also of lysosomes. Thiamine pyrophosphate and inosine-5'-monophosphate--substrates that give good GA staining in some soft tissues--give only lysosomal staining in bone. No previously used substrate or substrate-effector combination has selectively localized the GA acid phosphatase in bone. This article describes results using a new AcPase medium having pyridoxal-5'-phosphate (PLP) as substrate. In bone this medium produced strong staining of the osteoblast GA, but relatively little staining of lysosomes, including lysosomes in osteoclasts. The weak lysosomal staining was almost totally eliminated, without affecting the GA reaction, by pretreating the tissue in 0.3% NH3 solution. Conversely, elevated ionic strength of the substrate medium eliminated the GA reaction, while having little effect on lysosomal staining. The GA enzyme was very sensitive to 1 mM tartrate whereas the lysosomal enzyme was not. These differences suggest the presence of distinct isoenzymes in the two locations. The distribution of osteoblasts with stained GA coincided with the distribution of strongest alkaline phosphatase activity and rapid bone mineralization, supporting previous suggestions that osteoblast GA AcPase is involved in the processing of one or more newly synthesized bone matrix components.
D000135:Acid Phosphatase / Q000032:analysis*; D000818:Animals; D006056:Golgi Apparatus / Q000201:enzymology*; D006651:Histocytochemistry; D007527:Isoenzymes / Q000032:analysis*; D008247:Lysosomes / Q000201:enzymology; D009994:Osmolar Concentration; D010006:Osteoblasts / Q000201:enzymology*; D010011:Osteocytes / Q000201:enzymology; D011732:Pyridoxal Phosphate; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D051381:Rats; D013644:Tartrates / Q000494:pharmacology
D007527:Isoenzymes; D000644:Quaternary Ammonium Compounds; D013644:Tartrates; D011732:Pyridoxal Phosphate; D000135:Acid Phosphatase