The histochemical distribution of the thiol proteases cathepsin B and dipeptidyl peptidase I and the serine protease dipeptidyl peptidase II was examined in rat bone and joint using amino acid derivatives of 4-methoxy-2-naphthylamine (MNA). The liberated MNA was then visualized by simultaneous coupling with fast blue B. Cathepsin B was examined with CBZ-Arg-Arg-MNA, dipeptidyl peptidase I (DPP I) with Gly-Arg- or Pro-Arg-MNA, and dipeptidyl peptidase II (DPP II) with Lys-ALA- or Lys-Pro-MNA. Bright red reaction product indicative of proteolytic activity was observed in most cell types associated with bone and its surrounding connective tissues, including osteocytes, osteoblasts, chondrocytes, chondroblasts, fibroblasts, and macrophages. Surprisingly, protease activity in osteoclasts could not be established with certainty, and it was concluded that these enzymes are either absent, present in very low amounts, or secreted as soon as they are synthesized rather than stored within the cell. The cells of the resting zone of the growth plate were intensely reactive for DPP II but were only moderately reactive for cathepsin B and DPP I. The reverse was true of the proliferating and hypertrophic layers. The protease activity observed in bone, cartilage, tendon, ligament, and synovium would be expected to contribute significantly to normal protein metabolism as well as to pathological destruction in these tissues.
The histochemical demonstration of acid phosphatase activities against phosphoethanolamine (PEA), phosphorylcholine (PC), and D-ephedrine phosphate (DEP) are reported for a variety of rat tissues and are compared to acid beta-glycerophosphatase (beta GPase) activity. Intense acid beta GPase activity was demonstrated in all tissues examined. However, liver, kidney, intestine, spleen and bone marrow cells failed to exhibit any enzyme activity against PEA, PC, or DEP. In addition, significant differences in the hydrolysis of these substrates were noted among the tissues that did demonstrate activity (bone, tooth, oral mucosa, sebaceous gland, and prostate gland). These observations suggest that PEA, PC, and DEP are more specific substrates for acid phosphatase than beta GP and permit the differential localization of several distinct acid phosphatase isoenzymes.
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