Ultrastructural localization of Tartrate‐resistant acid phosphatase (purple acid phosphatase) activity in chicken cartilage and bone

Abstract

Tartrate-resistant acid adenosine triphosphatase activity at pH 6.5, using a lead-salt method, was localized at light and electron microscopic levels in cartilage and bone matrices, osteoclasts, and chondroclasts. Cartilage matrix staining occurred after vascular invasion of the growth plate. In osteoclasts, activity was present in lysosomes, extracellular ruffled border channels, and the underlying cartilage and bone matrices. Staining artifacts occurred at lower pH levels (pH 5.4, 5.0). Adenosine diphosphate, p-nitrophenylphosphate, thiamine pyrophosphate, and alpha-naphthylphosphate also acted as substrates; but no activity was observed when adenosine monophosphate, adenylate-(beta, gamma-methylene) diphosphate, and beta-glycerophosphate were used. The activity was inhibited by NaF, dithionite, and a high concentration of p-chloromercuribenzoic acid, and activated by simultaneous addition of FeCl2 and ascorbic acid, as has been shown in biochemical studies. These histochemical results support the view that the adenosine triphosphate hydrolyzing activity at pH 6.5 is due to tartrate-resistant acid phosphatase (TRAP). There were some differences in ultrastructural localization between TRAP and tartrate-sensitive acid phosphatase (TSAP) activities in osteoclasts: TSAP activity was more intense in lysosomes and Golgi complexes and TRAP was stronger in the cartilage and bone matrices. It is suggested, therefore, that most of TRAP is in an inactive form in cells and is activated when secreted.

Acid phosphatases

“…60 This last observation is in contrast to Reinholt and colleagues 55 and Fukushima et al 61 At these sites, TRAP was colocalised with the organic products of bone degradation that had been released from bone matrix during resorption and endocytosed into the osteoclast cells. The model was put forward that TRAP containing vesicles from the biosynthetic pathway fuse with transcytotic vesicles transporting matrix degradation products from the ruffled border to the FSD of osteoclasts.…”

Tartrate-resistant acid phosphatase activity and glutathione levels are modulated during hFOB 1.19 osteoblastic differentiation

“…TRAP is synthesized by different cells from the monohistiocytic lineage. It is also found in placenta, macrophages and leukocytes, but it is most significant expression occurs in osteoclasts, what renders this enzyme a marker of bone resorption (Miyazaki et al 2003;Fukushima et al 1991;Habermann et al 2007). In the interior of the cells, TRAP is found mainly in lysosomes, and, secondarily, in the cytoplasm or in the extracellular matrix.…”

A Histochemical Study of Tartrate-Resistant Acid Phosphatase Activity in Rat Osteoblasts.

“…The concentration of L(+)-tartaric acid was confined to 10 mM because a higher concentration (50 mM used for the azo-dye method) caused precipitation of the incubation medium in the lead-salt method. The incubation medium was adjusted to pH 6.0 to avoid staining artifacts that may occur at lower pH (5.0 or 5.8), as described by Fukushima et al [8]. Substratefree medium was used as a control.…”

“…Approximately 2 im thick sections were cut, floated on water droplets on slides, and allowed to air dry at 10°C overnight, then examined for TRAP activity. Since the azo-dye method and the leadsalt method can be utilized for histochemical detection of TRAP at the light microscopic level [8], both methods were employed to evaluate TRAP reactivity in this study.…”