Isolation and complete covalent structure of liver microsomal paraoxonase

Abstract

Paraoxonase (PON1) is a serum esterase exclusively associated with high-density lipoproteins; it might confer protection against coronary artery disease by destroying pro-inflammatory oxidized lipids in oxidized low-density lipoproteins. Here I show that rabbit liver microsomes contain a PON analogue (MsPON) and report the isolation and complete covalent structure of MsPON. In detergent-solubilized microsomes, MsPON co-purifies with the microsomal triacylglycerol transfer protein (MTP) complex. MsPON was separated from the complex and purified to homogeneity under non-denaturing conditions. Automated sequence analysis of intact MsPON and peptides obtained from enzymic and chemical cleavages led to the elucidation of the complete covalent structure of MsPON. The protein is a single polypeptide consisting of 350 residues. The sequence of rabbit liver microsomal MsPON is 60% identical with that of rabbit serum PON1, and 84% identical with the sequence predicted by a human cDNA of unknown function, designated PON3. MsPON has a hydrophobic segment at the N-terminus that might serve to anchor the protein to the microsomal membrane or to the MTP complex. Unlike in the serum enzyme, two potential N-glycan acceptor sites in MsPON are not glycosylated. An absence of N-glycans was also indicated in the rabbit liver MTP. MsPON has a single free cysteine residue at position 38 and a disulphide bond between Cys-279 and Cys-348. The microsomal enzyme lacks three residues at the N-terminus that are present in the serum protein. MsPON lacks four residues at the C-terminus that are present in the rabbit serum protein but absent from human serum PON1. On the basis of the observation that MsPON displays a high degree of similarity with serum PON1, it is proposed that MsPON might have a function related to that of PON1 in serum high-density lipoprotein complexes.

Purification and kinetic properties of rabbit liver paraoxonase 1

“…2B). In previous studies, purified PON1 has been shown to be a doublet within 37-48 kDa, depending on the glycosylation state of the enzyme [28,31,36,37,43]. In our study, rabbit liver PON1 enzyme was purified to homogeneity using a new purification approach with a better yield than other studies in the literature.…”

“…Size exclusion chromatography has not been generally used as the first step of PON1 purification [24][25][26][30][31][32][33][34][35][36][37]. However, this initial step was required to eliminate a higher percentage of proteins present in the extract.…”

“…Since liver PON1 is an essentially microsomal enzyme, the purification consists of a multistep procedure. To date, several studies with rat and mouse liver PON1 defined solubilization and subcellular distribution of the enzyme but there are very few studies describing the purification of PON1 from rabbit liver [26][27][28][29][30][31]36]. In these studies, usually more than three chromatographic steps are used including ion exchange and hydroxyapatite non-specific affinity chromatographies.…”

Molecular characterization and expression analysis of the porcine paraoxonase 3 (PON3) gene

“…2). Earlier work performed on the rabbit PON3 protein identified Cys 42 as a free cysteine, whereas a disulphide bond was assigned between residues Cys 283 and Cys 352 (Ozols, 1999). In contrast, Cys 283 was identified as the free cysteine in human PON1 protein (Sorenson et al, 1995) and this residue was found to be essential for protection against LDL oxidation (Aviram et al, 1998) but not for PON arylesterase activity (Sorenson et al, 1995).…”

Tandem purification of two HDL-associated partner proteins in human plasma, paraoxonase (PON1) and phosphate binding protein (HPBP) using hydroxyapatite chromatography

“…Hydroxyapatite chromatography has previously been successfully used as the first step for purification of microsomal rat liver PON1 [19,25] and PON3 [26], rabbit liver microsomal PON1 [27]. Besides, it was also used as the final step to purify variants of mammalian recombinant PON1 (issued from a synthetic construct from rabbit, mouse, rat, and human PON1 genes) expressed in E. coli [7].…”