TY - JOUR
T1 - Chromogranin A
T2 - Osmotically active fragments and their susceptibility to proteolysis during lysis of the bovine chromaffin granules
AU - Helle, K. B.
AU - Reed, R. K.
AU - Ehrhart, M.
AU - Aunis, D.
AU - Hogue Angeletti, R.
PY - 1990
Y1 - 1990
N2 - Osmotically active fragments of chromogranin A (Chr A) were studied in lysates from bovine chromaffin granules (CG) disrupted in the presence or absence of inhibitors of endogenous proteolytic activities. The effects of various methods of lysis were examined by micro-osmometry, PAGE-SDS electrophoretic techniques and immunoblots with polyclonal anti-Chr A sera. Osmotically active 'small' Chr A fragments (below 30 kDa) were conspicuous in lysates containing cocktails of leupeptin, pepstatin A, pHMB, PMSF and aprotinin. The osmotically inactive native Chr A in the 68-100 kDa range and the osmotically active fragments below 47 kDa were degraded in lysates at neutral or acid pH in the absence of inhibitors. However, degradation of the native Chr A and intermediates below 47 kDa could be prevented by extraction directly from intact CG, notably in cold or boiling distilled water. On the other hand, the main product after large-scale extraction of CG in 1 M acetic acid (pH 1.9, 100°C) was a novel, osmotically active fragment (22 kDa), immunostaining only for the N-terminal sequence (Chr A1-40). The heat-stable fraction (M(r,n))̄ 23 kDa) exhibited concentration-independent colloid osmotic pressures even in the absence of phosphate, a property which may distinguish this N-terminal-containing fragment from the larger intermediates, probably containing the pancreastatin sequence, and other regions at the C-terminal side of the prohormone molecule. The functional roles of these osmotically active intermediates in the processing of Chr A are not yet known.
AB - Osmotically active fragments of chromogranin A (Chr A) were studied in lysates from bovine chromaffin granules (CG) disrupted in the presence or absence of inhibitors of endogenous proteolytic activities. The effects of various methods of lysis were examined by micro-osmometry, PAGE-SDS electrophoretic techniques and immunoblots with polyclonal anti-Chr A sera. Osmotically active 'small' Chr A fragments (below 30 kDa) were conspicuous in lysates containing cocktails of leupeptin, pepstatin A, pHMB, PMSF and aprotinin. The osmotically inactive native Chr A in the 68-100 kDa range and the osmotically active fragments below 47 kDa were degraded in lysates at neutral or acid pH in the absence of inhibitors. However, degradation of the native Chr A and intermediates below 47 kDa could be prevented by extraction directly from intact CG, notably in cold or boiling distilled water. On the other hand, the main product after large-scale extraction of CG in 1 M acetic acid (pH 1.9, 100°C) was a novel, osmotically active fragment (22 kDa), immunostaining only for the N-terminal sequence (Chr A1-40). The heat-stable fraction (M(r,n))̄ 23 kDa) exhibited concentration-independent colloid osmotic pressures even in the absence of phosphate, a property which may distinguish this N-terminal-containing fragment from the larger intermediates, probably containing the pancreastatin sequence, and other regions at the C-terminal side of the prohormone molecule. The functional roles of these osmotically active intermediates in the processing of Chr A are not yet known.
KW - Chr A
KW - chromogranins
KW - immunoactive fragments
KW - limited proteolysis
KW - osmotic behaviour
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U2 - 10.1111/j.1748-1716.1990.tb08885.x
DO - 10.1111/j.1748-1716.1990.tb08885.x
M3 - Article
C2 - 2191538
AN - SCOPUS:0025319373
SN - 0001-6772
VL - 138
SP - 565
EP - 574
JO - Acta Physiologica Scandinavica
JF - Acta Physiologica Scandinavica
IS - 4
ER -