BIOCHEMICAL AND CYTOCHEMICAL STUDY OF TRIMETAPHOSPHATASE ACTIVITY IN MAMMALIAN TISSUES

• Published in: ACTA HISTOCHEMICA ET CYTOCHEMICA Vol. 25; no. 1-2; pp. 53 - 59
• Main Authors: SEGUCHI, HARUMICHI, KOBAYASHI, TOSHIIHIRO, OKADA, TERUHIKO, ZHANG, SHU-XIN, NISHIYAMA, SHOJI, SAZ, EVA GARCIA DEL
• Format: Journal Article
• Language: English
• Published: Kyoto JAPAN SOCIETY OF HISTOCHEMISTRY AND CYTOCHEMISTRY 1992; Japan Society of Histochemistry and Cytochemistry
• Subjects: Analytical, structural and metabolic biochemistry; Biological and medical sciences; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Hydrolases; Tissue; Vertebrata; Mammalia; Enzymatic activity; Trimetaphosphatase; Enzyme
• ISSN: 0044-5991; 1347-5800
• DOI: 10.1267/ahc.25.53

Trimetaphosphatase (TMPase) was biochemically and cytochemically investigated using rat and guinea pig tissues. TMPase was partially purified, and its activity was visualized and measured by the method of Doty et al. (J. Histochem. Cytochem. 25: 1381, 1977), using a dot-blot apparatus. TMPase was salted out in fractions of 60-80% ammonium sulfate. TMPase activity was observed in early protein fractions of Sephadex G-100 column. The molecular weight and pI of the partially purified TMPase were 130KD and 6.1, respectively. Electrophoretically, TMPase and acid phosphatase (ACPase) activities were observed in different bands. The present results clearly demonstrated that TMPase and ACPase are two different proteins. Cytochemically, the TMPase activity was elucidated using an improved method which employs cerium salt as capture agent, and the results were compared with those of the lead-based method. The incubation medium of the cerium-based method contained 20 mM acetate buffer, pH 3.9, 2 mM cerium chloride, 1 mM trimetaphosphate, 5% sucrose, and 0.00015% Triton X-100. The localization of the TMPase activity differed from that of ACPase in all tissues employed. TMPase activity was observed mainly in tubular structures. Using the cerium-based method, nonspecific precipitates were considerably reduced as compared with the lead-based method.