Purification of rabbit and human serum paraoxonase

Abstract

Rabbit serum paraoxonase/arylesterase has been purified to homogeneity by Cibacron Blue-agarose chromatography, gel filtration, DEAE-Trisacryl M chromatography, and preparative SDS gel electrophoresis. Renaturation (Copeland et al., 1982) and activity staining of the enzyme resolved by SDS gel electrophoresis allowed for identification and purification of paraoxonase. Two bands of active enzyme were purified by this procedure (35,000 and 38,000). Enzyme electroeluted from the preparative gels was reanalyzed by analytical SDS gel electrophoresis, and two higher molecular weight bands (43,000 and 48,000) were observed in addition to the original bands. This suggested that repeat electrophoresis resulted in an unfolding or other modification and slower migration of some of the purified protein. The lower mobility bands stained weakly for paraoxonase activity in preparative gels. Bands of each molecular weight species were electroblotted onto PVDF membranes and sequenced. The gas-phase sequence analysis showed that both the active bands and apparent molecular weight bands had identical amino-terminal sequences. Amino acid analysis of the four electrophoretic components from PVDF membranes also indicated compositional similarity. The amino-terminal sequences are typical of the leader sequences of secreted proteins. Human serum paraoxonase was purified by a similar procedure, and ten residues of the amino terminus were sequenced by gas-phase procedures. One amino acid difference between the first ten residues of human and rabbit was observed.

Facilitated replacement of Kupffer cells expressing a paraoxonase-1 transgene is essential for ameliorating atherosclerosis in mice

“…These findings are consistent with previous studies showing that PON1 expressed by Chinese hamster ovary cells is retained within the plasma membrane and inefficiently secreted (30). The lack of cleavage of the N-terminal signal sequence of endogenous PON1 may be responsible for its inefficient secretion (34)(35)(36). Additional studies showed that after an 18-h incubation of peritoneal macrophages obtained from PON1-Tg mice, no PON1 protein or enzyme activity was secreted (i.e., detected in culture medium).…”

“…These findings support experiments showing that PON1 protein and enzyme activity are retained in the microsomal membrane fraction of peritoneal macrophages ( Fig. 1 B and C) (34)(35)(36). Consistent with the results of others (23,24), consumption of the cholesterolenriched atherogenic diet caused a Ϸ30% decrease in plasma activities of PON1 (Fig.…”

Purification of bovine serum paraoxonase and its immobilization on Eupergit C 250 L by covalent attachment

“…Some purification studies indicate differences in the migration of PON bands in SDS-PAGE 24 . Furlong et al 31 demonstrated two PON bands purified from rabbit serum. Sequence analyses have indicated five potential N-glycosylation sites in rabbit PON and four in human PON 32 .…”

Adenovirus-mediated human paraoxonase1 gene transfer to provide protection against the toxicity of the organophosphorus pesticide toxicant diazoxon

“…The staining for PON1 activity was adopted from methods described before. 39,40 The staining solution contained 100 ml of 50 mM Tris-Cl, pH 7.4, 10 mM calcium chloride, 50 mg of b-naphthylacetate dissolved in 1 ml of ethanol and 50 mg of solid Fast Blue RR. Undissolved naphthylacetate and Fast Blue RR are present, but do not interfere with color development.…”