Abstract
Human lipocortin I is a 38.5-kDa phospholipase A2 inhibitor that has been produced in Escherichia coli in large quantities by recombinant DNA technology (Wallner, B.P., Mattaliano, R.J., Hession, C., Cate, R. L., Tizard, R., Sinclair, L.K., Foeller, C., Chow, E.P., Browning, J.L., Ramachandran, K.L., and Pepinsky, R.B. (1986) Nature 320, 77-80). To localize the region within the protein responsible for its inhibitory activity, we generated a series of fragments of the recombinant product by limited proteolysis with elastase and characterized their structure by sequencing and peptide mapping. Five active fragments have been analyzed in detail. The smallest is an 18-kDa fragment derived from the amino-terminal half of lipocortin. Three of the larger fragments contain this region. The fifth fragment is missing 83 amino acids from the amino terminus. A region common to all the active fragments (amino acid residues 97-178) is 70% homologous with the corresponding region from a second member of the lipocortin family which recently was cloned (Huang, K-S., Wallner, B.P., Mattaliano, R.J., Tizard, R., Burne, C., Frey, A., Hession, C., McGray, P., Sinclair, L.K., Chow, E.P., Browning, J.L., Ramachandran, K.L., Tang, J., Smart, J.E., and Pepinsky, R.B. (1986) Cell 46, 191-199) and thus presumably is important for activity. In addition to inhibitory fragments, we have isolated a 3-kDa proteolytic fragment from the amino terminus of lipocortin I that contains the known phosphorylation site for protein-tyrosine kinases. Because of sequence homology of the 3-kDa fragment with biologically active synthetic peptides from pp60v-src and middle T antigen, its release by proteases may represent an important part of the activity of lipocortin.
Highlights
To localize the re- quences in the lipocortin-like proteins suggests that they may gion within the protein responsible for its inhibitory activity, we generated a series of fragments of the recombinant product by limited proteolysis withelastase and characterized their structure by sequencing and peptidemapping
B. (1986)Cell 46,191-199) and presum- acids near the amino terminus. This is an ably is important for activityI.n addition to inhibitory exposed region that is sensitive to proteolysis (Ref. 31; see fragments, we haveisolated a 3-kDa proteolytic frag- below) and that can be phosphorylated by protein-tyrosine ment fromtheaminoterminus of lipocortin I that and by protein-serine-threonine kinases [31,32,33]
Becauseof sequence homology of the 3-kDa fragment witbhiologically active syntheticpeptides from pp60’” and middle T antigen, its release by proteases may represent an important part of the activity of lipocortin
Summary
Samples were subjected to SDS-PAGE and FIG. 1. The sample was elution buffer (0.1% SDS, 0.2 M Tris-HC1, pH 7.5, 5 mM EDTA, 5 mM DTT, and0.1 mg/ml BSA) was added to each slice, and thegels were crushed with a glass rod. Four volumes of -20 "C acetone were added, and the samples were stored for 30 min in a dry ice/ethanol bath. The remainder of the digest was used for generating protein for activity measurements In these analyses, fragments were visualized with KCl, eluted from the gel, renatured, and assayed. After 20 min, 1 ml of Fragments were separated by preparative SDS-PAGE, electroeluted renaturation buffer (160 mM Tris-HC1, pH 7.9, 1.6 mM DTT, 2 mM EDTA, 0.8 mg/ml BSA, and 20% glycerol) was added. For digestion of electroeluted samples, preparations first were treated with 4 volumes of -20 "C acetonferaagnmdent incubated for 30 min in a dry ice/ethanol bath.
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
