A CD4+ T cell line-secreted factor, growth promoting for normal and leukemic B cells, identified as thioredoxin.
In this study, a B cell growth stimulatory factor, constitutively secreted by a human CD4+ T cell hybridoma clone, MP6, has been purified and characterized. Serum-free 24 h culture media from MP6 cells were collected, concentrated by ultrafiltration and separated by gel chromatography. Fractions were analyzed for stimulatory activity using [3H]thymidine incorporation in normal and leukemic (B-CLL) B cells as target cells. Activity was present in a 12 kDa protein peak. Upon storage this lost activity indicating that the factor was sensitive to air oxidation, a well-known property of mammalian thioredoxins (Trxs). Treatment of the inactive fraction with dithiothreitol restored full activity. When culture medium was analyzed with a radioimmunoassay for human placenta Trx, the MP6 clone was shown to release 30-50 ng/ml per million cells during 24 h. The B cell stimulatory activity of the MP6 medium was removed by Sepharose-bound anti-human placenta Trx IgG and activity was recovered by elution from the antibodies. Furthermore, MP6 medium showed Trx activity with NADPH and Trx reductase using an insulin disulfide reduction assay. Starting from 5 l of serum-free MP6 conditioned medium, the Trx was purified approximately 100,000-fold. After gel electrophoresis banding, the material was analyzed by peptide sequencing and a full length sequence of an 104 amino acid long protein was obtained. This Trx sequence was identical to the previously published sequence of human Trx from HTLV-1 transformed T cells, adult T cell leukemia-derived factor/Trx.(ABSTRACT TRUNCATED AT 250 WORDS)
Rosen
A
A
Department of Cell Biology, Faculty of Health Sciences, University of Linköping, Sweden.
Lundman
P
P
Carlsson
M
M
Bhavani
K
K
Srinivasa
B R
BR
Kjellström
G
G
Nilsson
K
K
Holmgren
A
A
D000595:Amino Acid Sequence; D000906:Antibodies; D001402:B-Lymphocytes / Q000187:drug effects / Q000502:physiology*; D015496:CD4-Positive T-Lymphocytes / Q000378:metabolism*; D002460:Cell Line; D002474:Cell-Free System; D002846:Chromatography, Affinity; D002999:Clone Cells; D017077:Culture Media, Conditioned / Q000032:analysis; D016207:Cytokines / Q000032:analysis; D004357:Drug Synergism; D006133:Growth Substances / Q000096:biosynthesis / Q000302:isolation & purification* / Q000494:pharmacology; D006801:Humans; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000378:metabolism; D008969:Molecular Sequence Data; D011863:Radioimmunoassay; D013879:Thioredoxins / Q000276:immunology / Q000302:isolation & purification*
D000906:Antibodies; D017077:Culture Media, Conditioned; D016207:Cytokines; D006133:Growth Substances; D013879:Thioredoxins