Ochratoxin A-binding proteins in rat organs and plasma and in different cell lines of the kidney.
In order to detect cellular proteins which bind the mycotoxin ochratoxin A (OTA) we coupled OTA covalently to horseradish peroxidase (HRP). The peroxidase activity of the conjugate was used to detect these proteins in Western (ligand) blot analysis. Only signals caused by OTA binding to proteins were viewable. HRP alone detected no proteins and OTA-HRP binding could be inhibited by free OTA. Several proteins from the rat intestine, liver, spleen, and kidney were detected by OTA. Also rat plasma proteins bind OTA which confirms previous findings. In all renal cell lines investigated (MDCK-C11, OK, LLC-PK1, IHKE, and SKPT) there are several proteins which bind OTA. Comparison of the PonceauS stain on the nitrocellulose sheet with the signal obtained from OTA-HRP unveiled proteins with high specific OTA binding. Especially, proteins with molecular masses between 55 and 60 kDa, 40 and 45 kDa and 25 and 30 kDa showed OTA binding in all samples. OTA was partially displaced by aspartame and phenylalanine from some but not all proteins. Binding to cytosolic and organellar proteins was comparable in all investigated cell lines. In the OK cell organellar compartment a 62 kDa protein is preferentially detected by OTA-HRP although virtually no protein band is detectable. In conclusion we have found a method to clearly detect proteins which bind OTA. With this new method we proved that OTA has the potential to bind to several proteins yet specific binding differs dramatically. Thus, highly specific binding of OTA possibly makes certain proteins a preferential target of OTA toxicity. Furthermore, binding contributes to intracellular accumulation of OTA, thus leading to a prolonged half life in the mammalian body and emphasises the toxic potential of this fungal metabolite.
Schwerdt
G
G
Physiologisches Institut, Universität Würzburg, Germany. phio037@mail.uni-wuerzburg.de
Freudinger
R
R
Silbernagl
S
S
Gekle
M
M
D000818:Animals; D001218:Aspartame / Q000494:pharmacology; D001667:Binding, Competitive / Q000187:drug effects; D001798:Blood Proteins / Q000378:metabolism*; D002460:Cell Line; D007668:Kidney / Q000166:cytology / Q000378:metabolism*; D009183:Mycotoxins / Q000378:metabolism; D009793:Ochratoxins / Q000378:metabolism*; D010649:Phenylalanine / Q000494:pharmacology; D011485:Protein Binding / Q000187:drug effects; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D013347:Subcellular Fractions / Q000378:metabolism
D001798:Blood Proteins; D009183:Mycotoxins; D009793:Ochratoxins; D011506:Proteins; C025589:ochratoxin A; D010649:Phenylalanine; D001218:Aspartame