Different recognition by peroxisome proliferator structures in rat peroxisomal induction: application of sandwich ELISA using monoclonal antibody against rat peroxisomes.
A novel assay for a peroxisomal beta-oxidation enzyme by sandwich ELISA using a monoclonal antibody (RPX-5) against purified rat liver peroxisomes was developed. Immunoblot analysis revealed that RPX-5 recognized a 78 Kd protein, which is a peroxisomal bifunctional enzyme (PBE) in the beta-oxidation pathway. Immunoprecipitation by RPX-5 and the resulting reduction of PBE activity were dependent on RPX-5 concentrations. Sandwich ELISA using RPX-5 could be used to assay PBE in the range of 30 to 2000 ng protein/ml. In rat hepatocyte cultures, the PBE amount by this assay correlated well with PBE activity, with correlation coefficients of 0.965. Studying the mechanisms of peroxisomal induction, patterns of peroxisomal induction were examined by co-treatment of rat hepatocytes with various peroxisome proliferators (PxPs). Treatment with clofibrate and bezafibrate resulted in neither an additive nor synergistic effect on PBE level. On the other hand, co-treatment with either bezafibrate-Wy-14,643 or clofibrate-MEHP(mono(2-ethylhexyl)phthalate) both resulted in an additive effect. From these results, it is suggested that PxPs of the fibrate group may exert their functions via a common process, and non-fibrate PxPs via a different process in hepatocytes. The cognition site for peroxisome proliferators, therefore, might not involve a single site for inducing peroxisomal enzymes.
Kawano
H
H
Faculty of Pharmaceutical Sciences, Kobe Gakuin Universiy, Japan.
Nishi
F
F
Kamitani
Y
Y
Ochi
H
H
Miyake
M
M
Mayumi
T
T
Hama
T
T
D015094:3-Hydroxyacyl CoA Dehydrogenases / Q000276:immunology / Q000378:metabolism; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis; D002478:Cells, Cultured; D004746:Enoyl-CoA Hydratase / Q000276:immunology / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D007535:Isomerases; D015227:Lipid Peroxidation; D008099:Liver / Q000201:enzymology / Q000378:metabolism; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D009097:Multienzyme Complexes / Q000276:immunology / Q000378:metabolism; D063994:Peroxisomal Bifunctional Enzyme; D020025:Peroxisome Proliferators / Q000494:pharmacology*; D020675:Peroxisomes / Q000201:enzymology / Q000276:immunology / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000378:metabolism
D000911:Antibodies, Monoclonal; D009097:Multienzyme Complexes; D020025:Peroxisome Proliferators; D018160:Receptors, Cytoplasmic and Nuclear; D014157:Transcription Factors; D015094:3-Hydroxyacyl CoA Dehydrogenases; C576254:Ehhadh protein, mouse; D004746:Enoyl-CoA Hydratase; D063994:Peroxisomal Bifunctional Enzyme; D007535:Isomerases