Different tartrate sensitivity and pH optimum for two isoenzymes of acid phosphatase in osteoclasts. An electron-microscopic enzyme-cytochemical study

Toshitaka Akisaka, Gus Permana, Hiroyuki Kawaguchi, Yoshio Shigenaga

Research output: Contribution to journalArticlepeer-review

26 Citations (Scopus)

Abstract

By differentiation of substrate specificity, pH optimum range, and sensitivity to various inhibitors, 2 isoenzymes of acid phosphatase in bone cells have been studied at the electron-microscopic level. When p-nitrophenyl phosphate was used for the substrate, the demonstrable enzyme activity was affected by neither tartrate nor sodium fluoride. The reaction product, when incubated at pH 5-6, was detected in all sites along the pathway for the biosynthesis of acid phosphatase in the osteoclast, including the perinuclear space, cisternae of the endoplasmic reticulum, Golgi complex, various vesicles, and vacuoles. In the osteoclasts attached to bone, the enzymatic activity was demonstrated at the extracellular ruffled border and on the eroded bone surface. Reaction products became confined to lysosomes and extracellular ruffled border when incubated at pH 6-7. Unattached osteoclasts showed a similar intracytoplasmic localization of enzyme as the attached ones, except for the absence of the extracellular enzyme activity. The mononuclear, immature type of osteoclast also resembled the mature osteoclast in terms of enzymatic localization. Except for the osteoclasts, the acid p-nitrophenyl phosphatase activity was restricted to lysosomal vesicles in various bone cells, monocytes, and macrophages. Such activity was inhibited by adding 50 mM tartrate to the p-nitrophenyl phosphate medium. When β-glycerophosphate or p-nitrocatechol sulfate was the substrate, most of the reaction product was localized intracellularly. Unlike the acid p-nitrophenyl phosphatase, the acid β-glycerophosphatase or arylsulfatase activity in osteoclasts and other bone cells was inhibited completely by 10 mM tartrate or 10 mM sodium fluoride. Even preincubation of 100 mM tartrate in the buffer inhibited β-glycerophosphatase activity completely, but p-nitrophenyl phosphatase activity was inhibited incompletely. Consequently, our results suggest that acid p-nitrophenyl phosphatase is a useful cytochemical marker for identification of the osteoclast family at electron-microscopic levels of resolution.

Original languageEnglish
Pages (from-to)69-76
Number of pages8
JournalCell And Tissue Research
Volume255
Issue number1
DOIs
Publication statusPublished - 1 Jan 1989

Keywords

  • Acid phosphatase
  • Chick
  • Osteoclasts
  • Tartrate resistant p-NPPase
  • Ultracytochemistry

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