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 directly in high Ca(2+)-stimulated intracellular degradation of PTH to secreted carboxyl-terminal fragments (C-PTH), an important component of Ca(2+)-regulated PTH secretion. The present study was undertaken to determine the presence of calcium-activated proteases, 84 kDa (micro)-calpain and 80 kDa (milli)-calpain, in the bovine parathyroid, and whether they could degrade PTH to C-terminal fragments. Immunocytochemistry of bovine parathyroid tissue using antibodies raised against bovine heart micro- and milli-calpain detected both isoforms of calpain. Western blotting of total bovine parathyroid cell protein prepared from primary cell cultures confirmed the presence of both isoforms of calpain, demonstrated by specific milli- and micro-calpain bands. Purified bovine PTH (bPTH) was incubated in vitro with human erythrocyte micro-calpain and the cleavage products were separated by reverse-phase HPLC. Eluant fractions were assayed with an RIA with equimolar sensitivity to C-PTH and bPTH, and peak areas integrated. Micro-calpain produced a C-PTH peak from bPTH which co-eluted with the major C-PTH secreted by parathyroid cells in culture. C-PTH production by micro-calpain, expressed as per cent area under the curve, increased from 0% in the absence of either 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 immunoreactive PTH cleavage products indicated the presence of two fragments of bPTH in the C-PTH peak, bPTH47-48 and bPTH69-84. In summary, both isoforms of calpain are present in the bovine parathyroid and calpains may play a role in the Ca(2+)-dependent degradation of PTH to secreted C-terminal fragments.
Waton
P H
PH
Department of Medicine, University of Calgary, Alberta, Canada.
Mortimer
S T
ST
Wang
K K
KK
Croall
D E
DE
Hanley
D A
DA
D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002154:Calpain / Q000378:metabolism; D002417:Cattle; D010450:Endopeptidases / Q000378:metabolism*; D004789:Enzyme Activation; D006801:Humans; D006868:Hydrolysis; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D010280:Parathyroid Glands / Q000201:enzymology*; D010281:Parathyroid Hormone / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism
D010281:Parathyroid Hormone; D010446:Peptide Fragments; D010450:Endopeptidases; D002154:Calpain; D002118:Calcium