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oai:www.ideals.illinois.edu:2142/22853 2023-07-10 col_2142_5131 col_2142_14795 com_2142_5130 com_2142_14794 com_2142_14793 com_2142_8903

Jonas, Ana Ji, Yong 2011-05-07T13:53:40Z 2011-05-07T13:53:40Z 10000-01-01 1996 Limited proteolysis was used to study the domain structure and to produce a large N-terminal fragment of human apolipoprotein A-I (apoA-I). Digestion of reconstituted high density lipoprotein (rHDL) prepared with apoA-I and dipalmitoyl phosphatidylcholine (DPPC) or palmitoyloleoyl PC (POPC) by chymotrypsin, trypsin, elastase, or subtilisin generated a major fragment of $\sim$22 kDa. Under milder conditions proteolysis of lipid-free apoA-I produced a fragment of similar size. The fragments shared the same N-terminus as intact apoA-I and the chymotryptic fragment had a molecular weight of 22,384 as determined by mass spectrometry. Thus the fragment consists of the N-terminal 192 residues of apoA-I. In aqueous solution the fragment, apoA-I(1-192), had a similar $\alpha$-helical content to apoA-I ($\sim$52%) but existed only as monomers and dimers. ApoA-I(1-192) lysed dimyristoyl PC (DMPC) liposomes slowly compared with apoA-I, but did form rHDL complexes with POPC or DPPC when prepared by the sodium cholate dialysis method. The isolated apoA-I(1-192) rHDLs activated lecithin cholesterol acyltransferase (LCAT), promoted cholesterol efflux from rat heptoma cells, and bound tightly to HeLa cells, indicating that the C-terminal 51 residues of apoA-I are crucial for self-association and initial lipid-binding but are not required for specific LCAT activation, cholesterol efflux or cell surface binding. A highly efficient system was developed for overexpressing mature apoA-I and two C-terminal deletion mutants, apoA-I(1-164) and apoA-I(1-186), in E. coli. The recombinant apoA-I constituted more than 15% of the total cell protein. ApoA-I(1-164) was produced to a much lower level ($\sim$2-fold) than the other two proteins, suggesting that the C-terminal domain of apoA-I protects the protein from degradation. Essentially pure ($>$95%) apoA-I(1-164) and apoA-I(1-186) were obtained by reverse phase HPLC, giving a final yield for apoA-I(1-164) of $\sim$2 mg/liter culture. ApoA-I(1-164) formed more dimers than monomers and had a lower $\alpha$-helical content than apoA-I(1-192); its Trp residues were more exposed to the aqueous environment. ApoA-I(1-164) exhibited decreased lipid-binding ability, indicating that the region between 165 and 192 is involved in lipid-protein interactions. Nonetheless, apoA-I(1-164) bound lipids cooperatively and formed POPC rHDL of distinct and defined sizes when prepared by the sodium cholate method. Made available in DSpace on 2011-05-07T13:53:40Z (GMT). No. of bitstreams: 2 license.txt: 4922 bytes, checksum: 910b249b4beec47e7ab768910c8f966f (MD5) 9702551.pdf: 4935507 bytes, checksum: 9ae3315318d73864281184ba050b4216 (MD5) Previous issue date: 1996 Item marked as restricted to the 'UIUC Users [automated]' Group (id=2) by Howard Ding (hding2@illinois.edu) on 2011-05-07T15:00:28Z Item is restricted indefinitely. Restriction data tranferred 2014-07-01T11:28:36-05:00 Original Data Group with Access UIUC Users [automated] Release Date: none Reason: ETDs are only available to UIUC Users without author permission ETDs are only available to UIUC Users without author permission U of I Only 9780591087949 AAI9702551 (UMI)AAI9702551 http://hdl.handle.net/2142/22853 eng Copyright 1996 Ji, Yong Biology, Molecular Chemistry, Biochemistry Health Sciences, General Structural and functional studies of the C-terminal domain of human apolipoprotein A-I: Limited proteolysis and deletion mutagenesis text Biochemistry Biochemistry University of Illinois at Urbana-Champaign Dissertation Ph.D.