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Enhancement of calcium sensitivity of lipocortin I in phospholipid binding induced by limited proteolysis and phosphorylation at the amino terminus as analyzed by phospholipid affinity column chromatography.

Authors
Type
Published Article
Journal
The Journal of biological chemistry
Publication Date
Volume
264
Issue
12
Pages
6948–6955
Identifiers
PMID: 2540167
Source
Medline
License
Unknown

Abstract

A phospholipid column was prepared by coating siliconized porous glass beads with phospholipids. The analysis of the Ca2+ requirement of lipocortin I and its derivatives in the binding to phospholipids was carried out with this column. The Ca2+ concentration required for 50% binding to the phospholipid column at room temperature was about 30 microM for lipocortin I, while that was reduced to 15 microM when lipocortin I was phosphorylated by the epidermal growth factor receptor/kinase, and a further reduction in the Ca2+ requirement was observed with proteolytic cleavage at the N-terminal region. Cathepsin D and calpain I (low calcium-requiring form of calcium-activated neutral protease) rapidly cleaved human placental lipocortin I at Trp-12 and Lys-26, respectively. These N-terminal-truncated proteins required only 5 microM Ca2+ for 50% binding to the phospholipid column. This enhancement of Ca2+ sensitivity by limited proteolysis was also observed for porcine lung lipocortin I. Essentially the same results were obtained when the Ca2+ sensitivities of the modified lipocortins I were analyzed using dispersed phospholipid vesicles instead of the phospholipid affinity column. Equilibrium dialysis indicated that the release of the N-terminal region markedly increased the affinity of lipocortin I for Ca2+ in the presence of phosphatidylserine, without any appreciable change of the number of Ca2+-binding sites. Limited proteolysis by endogenous proteases such as calpain may be an important regulatory mechanism for the Ca2+ sensitivity of lipocortin I in phospholipid binding.

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