Novel purification strategy for human PON1 and inhibition of the activity by cephalosporin and aminoglikozide derived antibiotics.
Human serum paraoxonase (PON1, EC 3.1.8.1.) is a high-density lipid (HDL)-associated, calcium-dependent enzyme; its physiological substrates are not known. In this study, a new purification strategy for human PON1 enzyme was developed using two-step procedures, namely ammonium sulfate precipitation and sepharose-4B-l-tyrosine-1-napthylamine hydrophobic interaction chromatography. SDS-polyacrylamide gel electrophoresis of the enzyme indicates a single band with an apparent MW of 43 kDa. Overall purification rate of our method was found 227-fold. The V(max) and K(m) of the purified enzyme were determined 227.27 EU and 4.16 mM, respectively. The in vitro effects of commonly used antibiotics, namely gentamycin sulfate and cefazolin sodium was also investigated on the purified human serum PON1 enzyme and human liver PON1 enzyme from human hepatoma cell (HepG2). Gentamycin sulfate and cefazolin sodium caused a dose- and time-dependent decrease on PON1 activity in HepG2 cells. Moreover, gentamycin sulfate and cefazolin sodium were effective inhibitors on purified human serum PON1 activity with IC(50) of 0.887 and 0.0084 values, respectively. The kinetics of interaction of gentamycin sulfate and cefazolin sodium with the purified human serum PON1 indicated a different inhibition pattern. Cefazolin sodium showed a competitive inhibition with K(i) of 0.012+/-0.00065 mM. However, Gentamycin sulfate was inhibited in non-competitive manner with K(i) of 0.026+/-0.015. In order to determine the inhibition statue of these drugs on a living system, the effects of same antibiotics on PON1 enzyme activity of mouse serum PON1 and liver PON1 were investigated in vivo. Gentamycin sulfate (3.2 mg/kg) and cefazolin sodium (106.25 mg/kg) leads to the significant decrease in mouse serum PON1 after 2, 4, 6 h and 2, 4 h drug administration, respectively. Cefazolin sodium did not exhibit any inhibition effect for the liver PON1, in vivo.
Sinan
Selma
S
Department of Biology, Faculty of Art and Science, Balikesir University, 10100 Balikesir, Turkey. soznur@balikesir.edu.tr
Kockar
Feray
F
Arslan
Oktay
O
D000645:Ammonium Sulfate / Q000737:chemistry; D000818:Animals; D000900:Anti-Bacterial Agents / Q000008:administration & dosage / Q000494:pharmacology*; D043303:Aryldialkylphosphatase / Q000097:blood / Q000302:isolation & purification* / Q000378:metabolism; D002437:Cefazolin / Q000008:administration & dosage / Q000494:pharmacology; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects; D002511:Cephalosporins / Q000008:administration & dosage / Q000494:pharmacology*; D002845:Chromatography / Q000379:methods; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004791:Enzyme Inhibitors / Q000008:administration & dosage / Q000494:pharmacology; D005839:Gentamicins / Q000008:administration & dosage / Q000494:pharmacology*; D006801:Humans; D006868:Hydrolysis / Q000187:drug effects; D020128:Inhibitory Concentration 50; D007273:Injections, Intramuscular; D007700:Kinetics; D008099:Liver / Q000187:drug effects / Q000201:enzymology; D051379:Mice
D000900:Anti-Bacterial Agents; D002511:Cephalosporins; D004791:Enzyme Inhibitors; D005839:Gentamicins; D043303:Aryldialkylphosphatase; C468406:PON1 protein, human; D002437:Cefazolin; D000645:Ammonium Sulfate