Purification of rabbit and human serum paraoxonase.
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.
Furlong
C E
CE
Division of Medical Genetics, School of Medicine, University of Washington, Seattle 98195.
Richter
R J
RJ
Chapline
C
C
Crabb
J W
JW
D000595:Amino Acid Sequence; D000818:Animals; D043303:Aryldialkylphosphatase; D002847:Chromatography, Agarose; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004789:Enzyme Activation; D006801:Humans; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010744:Phosphoric Monoester Hydrolases / Q000097:blood* / Q000737:chemistry / Q000302:isolation & purification*; D011817:Rabbits; D013194:Staining and Labeling; D013379:Substrate Specificity
D010744:Phosphoric Monoester Hydrolases; D043303:Aryldialkylphosphatase