Limited degradation of carbamoyl phosphate synthetase I by calcium-activated protease (calpain): electrophoretic evidence for removal of the C-terminal N-acetylglutamate regulatory domain.
The degradation of rat hepatic carbamoyl phosphate synthetase I (CPS) by calcium-activated thiol protease (calpain II) isolated from the same tissue was evaluated in vitro. Calpain was purified as a heterodimer containing subunits of 72-kDa (catalytic) and 29-kDa (regulatory). The identity of this protease as calpain II was confirmed by its dependence on calcium in the 2-4 mM range for maximal activity (525 microM calcium required for half-maximal activity) and reactivity with anti-calpain II antibody on Western blots. Calpain II was not activated (<10%) by Mg2+ or Mn2+. CPS degradation was monitored by discontinuous sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis (SDS-PAGE) and CPS fragments characterized with Western blotting with an anti-CPS antibody. Exposure of CPS (160-kDa) to calpain II resulted in the generation of single and limited degradation product of approximately 136-kDa. The smaller CPS fragment (approximately 24-kDa) appears unstable since it was not detected under these conditions. In contrast, the larger 136-kDa CPS fragment was quite stable despite extended incubation with calpain II (up to 60 min). Two-dimensional electrophoretic analysis (isoelectric focusing IEF/SDS-PAGE) revealed that the 136-kDa CPS fragment focused at more acidic isoelectric point (pI) than the parent molecule (pI range 5.95-6.35 vs 6.35-6.75, respectively). Based on the size and acidic pI shift of the degradation fragment, the calpain-susceptible site most likely involves removal of the positively-charged C-terminus of CPS. The potential significance of these findings to physiological regulation of CPS by calpain is discussed.
Makowski
G S
GS
Department of Laboratory Medicine, School of Medicine, University of Connecticut Health Center, Farmington 06030, USA.
Ramsby
M L
ML
D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D002154:Calpain / Q000302:isolation & purification / Q000378:metabolism*; D002222:Carbamoyl-Phosphate Synthase (Ammonia) / Q000378:metabolism*; D020134:Catalytic Domain; D003720:Densitometry; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D005971:Glutamates / Q000378:metabolism*; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D008099:Liver / Q000737:chemistry / Q000201:enzymology; D008297:Male; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry; D017434:Protein Structure, Tertiary; D051381:Rats
D005971:Glutamates; D010446:Peptide Fragments; D002154:Calpain; D002222:Carbamoyl-Phosphate Synthase (Ammonia); C016195:N-acetylglutamic acid; D002118:Calcium