Purification of the Mr 22,000 calcium-binding protein (sorcin) associated with multidrug resistance and its detection with monoclonal antibodies.
A low molecular weight cytoplasmic protein (Mr 19,000-22,000) has been reported to be overexpressed in some multidrug-resistant cells. We have found that a cytoplasmic protein with a molecular weight of 22,000 is highly expressed in the human myelogenous leukemia K562 cells resistant to Adriamycin (K562/ADM). The Mr 22,000 protein was shown to be one of the major calcium-binding proteins in the cytoplasmic extract from K562/ADM cells. The protein was purified to apparent homogeneity from K562/ADM cells using a four-step procedure including ammonium sulfate fractionation, anion-exchange chromatography, and gel filtration. 1.5 mg of the Mr 22,000 protein was purified from 3.0 x 10(9) of K562/ADM cells. The protein was acidic (pI 5.3) and exists as a homodimer (Mr 44,000) as revealed by gel filtration and sucrose density-gradient centrifugation. The purified protein appeared as a single band (Mr 22,000) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence or absence of reducing agents, suggesting that the homodimer was generated by noncovalent linkage. Monoclonal antibodies specific to the Mr 22,000 protein were raised by in vitro immunization with purified protein or by in vivo immunization with the crude membrane fraction of K562/ADM. These antibodies were used as probes for the detection of the protein. We have surveyed the expression of the Mr 22,000 protein in various multidrug-resistant and -sensitive cell lines, and found that the overexpression of the protein is not a sufficient nor a necessary condition for the acquisition of the multidrug-resistant phenotype.
Hamada
H
H
Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo.
Okochi
E
E
Oh-hara
T
T
Tsuruo
T
T
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000302:isolation & purification* / Q000378:metabolism; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D003078:Colchicine / Q000494:pharmacology; D006224:Cricetinae; D004317:Doxorubicin / Q000494:pharmacology; D004351:Drug Resistance; D006801:Humans; D051379:Mice; D008970:Molecular Weight; D009363:Neoplasm Proteins / Q000302:isolation & purification*; D009369:Neoplasms; D014750:Vincristine / Q000494:pharmacology
D002135:Calcium-Binding Proteins; D009363:Neoplasm Proteins; C503113:SRI protein, human; C503114:Sri protein, mouse; D014750:Vincristine; D004317:Doxorubicin; D003078:Colchicine; D002118:Calcium