CALCIUM-ACTIVATED PROTEASES IN THE BOVINE PARATHYROID-GLAND - POTENTIAL ROLE IN DEGRADATION OF PARATHYROID-HORMONE TO PEPTIDE-FRAGMENTS

Our studies suggest that protein kinase C is involved in low calcium ( Ca2+)-stimulated secretion of parathyroid hormone (PTH) but not direct ly in high Ca2+-stimulated intracellular degradation of PTH to secrete d carboxyl-terminal fragments (C-PTH), an important component of Ca2+- regulated PTH secretion. The present study was undertaken to determine the presence of calcium-activated proteases, 84 kDa (micro)-calpain a nd 80 kDa (milli)-calpain, in the bovine parathyroid, and whether they could degrade PTH to C-terminal fragments. Immunocytochemistry of bov ine parathyroid tissue using antibodies raised against bovine heart mi cro- and milli-calpain detected both isoforms of calpain. Western blot ting of total bovine parathyroid cell protein prepared from primary ce ll cultures confirmed the presence of both isoforms of calpain, demons trated by specific milli- and micro-calpain bands. Purified bovine PTH (bPTH) was incubated in vitro with human erythrocyte micro-calpain an d the cleavage products were separated by reverse-phase HPLC. Eluant f ractions were assayed with an RIA with equimolar sensitivity to C-PTH and bPTH, and peak areas integrated. Micro-calpain produced a C-PTH pe ak from bPTH which co-eluted with the major C-PTH secreted by parathyr oid cells in culture. C-PTH production by micro-calpain, expressed as per cent area under the curve, increased from 0% in the absence of eit her micro-calpain or Ca2+, to 71.5% when a 5:1 molar ratio of bPTH to calpain was used. Amino acid sequencing and analysis of the immunoreac tive PTH cleavage products indicated the presence of two fragments of bPTH in the C-PTH peak, bPTH(47-84) and BPTH-(69-84). In summary, both isoforms of calpain are present in the bovine parathyroid and calpain s may play a role in the Ca2+-dependent degradation of PTH to secreted C-terminal fragments.