Characterization, cell-free synthesis and processing of apolipoprotein A-I of rat high-density lipoproteins

• DOI: 10.1021/bi00512a017
• Publication Date: 2005-03-08T07:16:37Z
• Authors: Ming-Huey Lin-Su; Yen-Chiu Lin-Lee; William A. Bradley; Lawrence Chan
• Keywords: Male; 0301 basic medicine; Immunodiffusion; Apolipoprotein A-I; Cell-Free System; Rats; 03 medical and health sciences; Apolipoproteins; Liver; Protein Biosynthesis; Animals; Amino Acid Sequence; RNA, Messenger; Amino Acids; Lipoproteins, HDL

Rat apolipoprotein A-I (apoA-I) was isolated from delipidated high-density lipoproteins by sequential chromatography on Sephacryl S-200 and Sephadex G-150 columns in guanidine buffer. The purified protein had an apparent Mr of 27 000 and was homogeneous by NaDodSO4 and urea gel electrophoresis. Its amino acid composition was similar to that previously reported by Swaney et al. [Swaney, J. B., Wraithwaite, F., & Eder, H. G. (1977) Biochemistry 16, 271-278]. Microsequencing yielded an N-terminal sequence of Asp-Glu-Pro-Pro-Val-(Ser)-Glu-. Rabbit antisera were generated against the purified rat apoA-I and were shown to be monospecific against the protein by immunodiffusion and immunoelectrophoresis. Total poly(A) RNA was isolated from the rat liver by extraction in guanidine hydrochloride buffer and oligo(dT)-cellulose chromatography. In vitro translation of the RNA was performed in both wheat germ and nuclease-treated reticulocyte lysate systems, using [35S]Met as the radioactive amino acid precursor. Immunoreactive 35S- labeled apoA-I synthesized in vitro was precipitated by a rabbit antirat apoA-I serum. It was analyzed on an NaDodSO4- acrylamide slab gel and visualized by fluorography. The in vitro product was found to have an apparent Mr of 28 500, being larger than the authentic plasma protein by approximately 1500 daltons. When translation was performed in the presence of dog pancreatic microsomal membranes, the immunoprecipitable material was cotranslationally cleaved to a product identical in size (Mr 27 000) with plasma apoA-I. Thus, we have synthesized in vitro a putative precursor to rat apoA-I, designated preapoA-I. The preapoA-I has been processed in a cell-free system to its mature plasma counterpart by the addition of exogenous microsomal membranes.