Detection of adult T-cell leukemia-derived factor/human thioredoxin in human serum.
Adult T-cell leukemia (ATL)-derived factor (ADF), originally defined as an inducer of interleukin-2 receptor/alpha-chain (IL-2R/p55) of human T-lymphotropic virus type I (HTLV-I) positive T cells, is a human homologue of redox-active coenzyme thioredoxin (Trx) of Escherichia coli. In this study, an enzymatic assay system based on the dithiol-dependent insulin-reducing activity of ADF/Trx was established (insulin-reducing assay) to determine the amount of ADF/Trx in human serum using NADPH and Trx reductase purified from human placenta. Insulin-reducing activity was detected in all of the serum samples from healthy volunteers (n = 30) screened by this assay, with a mean +/- SD of 10.9 +/- 2.4 U/l. This mean value corresponds with the concentration of 223 ng recombinant ADF/Trx (rADF/Trx)/ml. Human serum is known to contain several redox-active proteins with ADF/Trx motifs. To differentiate the contribution of these proteins and ADF/Trx to the insulin-reducing activity, the anti-rADF/Trx monoclonal antibody (mAb)-conjugated affinity column-depleted sera obtained from an identical source was used for analysis. The affinity column-depleted sera demonstrated a loss of over 99% of the original activity, while control column depleted sera lost less than 4%. Furthermore, the amount of affinity-purified ADF/Trx molecules eluted from the same column almost corresponded with the amount estimated by the insulin-reducing activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Kitaoka
Y
Y
Department of Biological Responses, Institute for Virus Research, Kyoto, Japan.
Sorachi
K
K
Nakamura
H
H
Masutani
H
H
Mitsui
A
A
Kobayashi
F
F
Mori
T
T
Yodoi
J
J
D000328:Adult; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D002846:Chromatography, Affinity; D016207:Cytokines; D005260:Female; D006801:Humans; D015151:Immunoblotting; D007328:Insulin / Q000737:chemistry; D008297:Male; D008969:Molecular Sequence Data; D009363:Neoplasm Proteins / Q000097:blood*; D010084:Oxidation-Reduction; D013880:Thioredoxin-Disulfide Reductase; D013879:Thioredoxins / Q000097:blood*
D000911:Antibodies, Monoclonal; D016207:Cytokines; D007328:Insulin; D009363:Neoplasm Proteins; C055533:adult T cell leukemia-derived factor; D013879:Thioredoxins; D013880:Thioredoxin-Disulfide Reductase