Isolation of proliferation factor of immature T-cell clone in concanavalin A-stimulated splenocyte culture supernatant.
An athymic mouse-derived immature T-cell clone, N-9F, was not maintained by interleukin-2 alone but required another soluble factor, contained in concanavalin A-stimulated rat splenocyte culture supernatant, namely T cell growth factor (TCGF), for its proliferation. An N-9F-proliferation factor (NPF) was isolated in a pure form from TCGF. N-9F cells and immature thymocytes proliferated in the presence of NPF at 10-11-10-8 g/ml in a dose-dependent manner, but adult thymocytes were not stimulated by NPF. NPF increased DNA synthesis of N-9F. NPF increased CD4 and CD8 double negative thymocytes and CD8 single positive thymocytes in fetal thymus organ culture. A hamster anti-NPF antiserum possessing the capacity to neutralize N-9F proliferation activity of NPF decreased double negative thymocytes. The amino-terminal amino acid sequence of NPF was identified to be Ser-Leu-Pro-Cys-Asp-Ile-Cys-Lys-Thr-Val-Val-Thr-Glu-Ala-Cys-Asn-Leu-Leu-Lys-Asp- and was identical to that of rat saposin A. The apparent molecular weight of NPF, 16000, was comparable to that of saposin A. A rabbit anti-mouse recombinant His-tag (mrH)-saposin A antibody recognized a 16000 MW molecule in TCGF. A Hitrap-saposin A antibody column bound NPF and pulled down the NPF activity in TCGF. Thus, NPF in TCGF was a saposin A-like protein possessing the capacity for growth and differentiation of immature thymocytes.
Kohama
Yasuhiro
Y
Department of Immunology, Graduate School of Pharmaceutical Sciences, Osaka University, Osaka, Japan. kohama@phs.osaka-u.ac.jp
Shinoda
Shinji
S
Hagihara
Katsunobu
K
Hashimoto
Takeshi
T
Yamaguchi
Asuka
A
Nakamura
Akihiro
A
Tsuchiya
Takahiro
T
Tsujikawa
Kazutake
K
Yamamoto
Hiroshi
H
D000595:Amino Acid Sequence; D000818:Animals; D002455:Cell Division / Q000276:immunology; D002478:Cells, Cultured; D002999:Clone Cells / Q000276:immunology; D003208:Concanavalin A / Q000276:immunology; D006023:Glycoproteins / Q000276:immunology; D006133:Growth Substances / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D007106:Immune Sera / Q000276:immunology; D007376:Interleukin-2 / Q000276:immunology; D008213:Lymphocyte Activation / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D008819:Mice, Nude; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011817:Rabbits; D049231:Saposins; D013154:Spleen / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology*
D006023:Glycoproteins; D006133:Growth Substances; D007106:Immune Sera; D007376:Interleukin-2; D049231:Saposins; D003208:Concanavalin A