A simple method for the production of migration inhibitory factor by concanavalin A-stimulated lymphocytes

1977 ◽  
Vol 14 (2) ◽  
pp. 141-146 ◽  
Author(s):  
Edgar Pick ◽  
Philip Kotkes
2006 ◽  
Vol 26 (3) ◽  
pp. 346-351 ◽  
Author(s):  
Hiroaki Nakajima ◽  
Hitoshi Takagi ◽  
Norio Horiguchi ◽  
Mitsuo Toyoda ◽  
Daisuke Kanda ◽  
...  

1996 ◽  
Vol 316 (1) ◽  
pp. 285-293 ◽  
Author(s):  
Mark J. RAFTERY ◽  
Craig A. HARRISON ◽  
Paul ALEWOOD ◽  
Alun JONES ◽  
Carolyn L. GECZY

MRP14 (macrophage migration-inhibitory factor-related protein of molecular mass 14 kDa) is an S100 calcium binding protein constitutively expressed in human neutrophils which may be associated with cellular activation/inflammation. Murine MRP14 expression was up-regulated following concanavalin A activation of spleen cells, and the protein was isolated from conditioned medium in high yield (approx. 500 ng/ml). MRP14 had a mass of 12972±2 Da by electrospray ionization MS, whereas the theoretical mass derived from the cDNA sequence, after removal of the initiator Met, was 12918 Da, suggesting that the protein was post-translationally modified. We identified four post-translational modifications of MRP14: removal of the N-terminal Met, N-terminal acetylation, disulphide bond formation between Cys79 and Cys90, and 1-methylation of His106; the calculated mass was then 12971.8 Da. Methylation of His106 was further characterized after incubation of spleen cells with L-[methyl-3H]Met during concanavalin A stimulation. Sequential analysis of a peptide (obtained by digestion with Lys C) containing methylated His indicated that > 80% of the label in the cycle corresponded to His106, suggesting that the methyl residue was transferred from S-adenosyl-L-methionine. Comparison of the C18 reverse-phase HPLC retention times of phenylthiocarbamoyl derivatives of a hydrolysed digest peptide of MRP14 with those of standards confirmed methyl substitution on the 1-position of the imidazole ring. MRP14 bound more 65Zn2+ than the same amounts of the 10 kDa chemotactic protein (CP10) or S100β. Ca2+ decreased Zn2+ binding in S100β but it did not influence binding to MRP14, suggesting that the Zn2+ binding site was distinct from and independent of the two Ca2+ binding domains.


Sign in / Sign up

Export Citation Format

Share Document