Calmodulin-binding domains from isozymes of the plasma membrane Ca2+ pump have different regulatory properties.
Peptides C28R2 and C28R1A, representing the two main alternative classes of calmodulin-binding domains from the plasma membrane Ca2+ pump, were tested for their calmodulin-binding properties and for their capacity to interact with pump from which the calmodulin-binding domain had been removed by chymotryptic proteolysis. Peptide C28R2 was more effective in both capacities. Binding of peptide to calmodulin was measured by competition experiments. Such experiments indicated that Ki for C28R2 as an inhibitor of the pump-calmodulin interaction was 0.1 nM, whereas C28R1A had a Ki of 1 nM. Interaction of peptide with chymotryptically activated Ca2+ pump was measured by observing the inhibition by peptide of active Ca2+ transport into inside-out membrane vesicles at low Ca2+. Those experiments showed that C28R2 interacted relatively strongly (an IC50 of 1 microM), whereas C28R1A had an IC50 of 15 microM. The calmodulin-binding peptides had effects on both the K1/2 for Ca2+ and the Vmax of the proteolyzed pump. The effects on the K1/2 for Ca2+ were related to the net plus charge on the peptide, with the most positive peptides being most effective in competing with Ca2+. The substantial differences between C28R2 and C28R1A suggest that Ca2+ pumps containing calmodulin-binding domains like C28R1A have lower calmodulin affinities and higher activities in the absence of activator.
Enyedi
A
A
National Institute of Hematology and Blood Transfusion, Budapest, Hungary.
Filoteo
A G
AG
Gardos
G
G
Penniston
J T
JT
D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000378:metabolism; D002147:Calmodulin / Q000378:metabolism*; D002148:Calmodulin-Binding Proteins / Q000737:chemistry*; D002918:Chymotrypsin; D004912:Erythrocytes / Q000201:enzymology; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry
D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D010446:Peptide Fragments; D002918:Chymotrypsin; D000252:Calcium-Transporting ATPases; D002118:Calcium