Biochemical and calmodulin binding properties of estrogen receptor binding cyclophilin expressed in Escherichia coli.
Bovine estrogen receptor binding cyclophilin (ERBC), a cyclophilin component of the unactivated estrogen receptor, has been efficiently expressed in Escherichia coli as a fusion with glutathione S-transferase (GST) and purified by single-step chromatography on glutathione-agarose. Thrombin cleavage from GST allowed the isolation of purified, recombinant ERBC. The fusion protein, GST-ERBC, and recombinant ERBC were both characterised for peptidyl prolyl cis-trans isomerase activity. With N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide as substrate, GST-ERBC demonstrated a kcat/KM value of 5.1 x 10(5) M-1s-1 at 5 degrees C. The isomerase activity was inhibited by cyclosporin A with an IC50 value of 1030 nM. These values indicate that ERBC has a decreased catalytic efficiency and sensitivity to cyclosporin A relative to human cyclophilin. Retention of the GST-ERBC fusion protein on calmodulin-agarose in the presence of Ca2+ and subsequent elution with EGTA has provided evidence that ERBC is a calmodulin-binding protein.
Ratajczak
T
T
Department of Endocrinology and Diabetes, Sir Charles Gairdner Hospital, Nedlands, Australia.
Carrello
A
A
Minchin
R F
RF
D000591:Amino Acid Isomerases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D002147:Calmodulin / Q000378:metabolism*; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D002853:Chromatography, Liquid; D003001:Cloning, Molecular; D016572:Cyclosporine / Q000494:pharmacology; D004926:Escherichia coli; D005982:Glutathione Transferase / Q000235:genetics; D008969:Molecular Sequence Data; D019696:Peptidylprolyl Isomerase; D011960:Receptors, Estrogen / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D002147:Calmodulin; D002352:Carrier Proteins; D011960:Receptors, Estrogen; D011993:Recombinant Fusion Proteins; D016572:Cyclosporine; D005982:Glutathione Transferase; D000591:Amino Acid Isomerases; D019696:Peptidylprolyl Isomerase