Identification of a 37 kDa tacrolimus, sirolimus and cyclosporine binding immunophilin possessing glyceraldehyde 3-phosphate dehydrogenase activity isolated from the Jurkat T cell line.
OBJECTIVE
The isolation and partial characterization of a 37 kDa minor immunophilin from the Jurkat cell line which binds to cyclosporine (CsA), Tacrolimus (FK506) and Sirolimus (RAPA).


METHODS
Using standard protein purification steps including isoelectric focusing and cation exchange chromatography, we have isolated and purified to homogeneity a minor immunophilin from the Jurkat cell line which has a molecular weight of 37 kDa. Binding properties for immunosuppressive drugs CsA, FK506 and RAPA were assessed by Scatchard and displacement analysis. The amino acid analysis and the protein sequences were also studied.


RESULTS
The immunophilin was purified to homogeneity and the molecular weight corresponds to 37 kDa. Saturation experiments using 3Hdihydro FK506 gave a Kd of 4.5 nM and the Bmax of 117 nmol/mg protein. Displacement studies using 3Hdihydro FK506 and RAPA gave a Kd of 0.8 nM. For CsA binding, the protein showed somewhat less avid binding. The amino acid composition was in close agreement with the amino acid composition of uracil DNA glycosylase which corresponds to part of the monomer of glyceraldehyde 3 phosphate dehydrogenase (G3PD). Protein digestion gave at least 3 peptides. The primary sequence of the first of these matched 7 of 8 residues of human liver nuclear uracil DNA glycosylase. The 37 kDa immunophilin was found to have G3PD activity not inhibited by FK506.


CONCLUSIONS
The amino acid analysis, protein sequences, binding properties and G3PD activity indicate that this 37 kDa immunophilin is different from any other known immunophilins.
Murthy
J N
JN
Department of Laboratory Medicine, Children's National Medical Center, Washington, DC 20010, USA.
Goodyear
N
N
Soldin
S J
SJ
D000276:Adjuvants, Immunologic; D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification*; D002852:Chromatography, Ion Exchange; D016572:Cyclosporine / Q000737:chemistry; D045647:DNA Glycosylases; D004591:Electrophoresis, Polyacrylamide Gel; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000737:chemistry; D006801:Humans; D019169:Jurkat Cells / Q000737:chemistry; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases / Q000737:chemistry; D011090:Polyenes / Q000737:chemistry; D020123:Sirolimus; D016559:Tacrolimus / Q000737:chemistry; D051981:Uracil-DNA Glycosidase
D000276:Adjuvants, Immunologic; D002352:Carrier Proteins; D011090:Polyenes; D016572:Cyclosporine; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; D051981:Uracil-DNA Glycosidase; D020123:Sirolimus; D016559:Tacrolimus