Inhibition and partial reversal of the methylamine-induced conversion of "slow" to "fast" electrophoretic forms of human alpha 2-macroglobulin by modification of the thiols.
It has been shown previously [Van Leuven, F., Marynen, P., Cassiman, J. J., & Van den Berghe, H. (1982) Biochem. J. 203, 405-411] that 2,4-dinitrophenyl thiocyanate (DNPSCN) can block the conversion of "slow" to "fast" electrophoretic forms of human alpha 2-macroglobulin (alpha 2M) normally resulting from reaction of alpha 2M with methylamine. The kinetics of reaction of DNPSCN with alpha 2M in the presence of methylamine are examined here and shown to approximate pseudo first order, reflecting the rate-limiting reaction of alpha 2M with methylamine [Larsson, L. J., & Björk, I. (1984) Biochemistry 23, 2802-2807]. One mole of DNPS is liberated per mole of free thiol in alpha 2M, consistent with cyanylation of the thiol liberated upon scission of the internal thiol esters by methylamine. I3(-) can also react with the methylamine-generated thiol groups of alpha 2M with a stoichiometry consistent with conversion of the thiol to a sulfenyl iodide. Reaction of the thiol groups with either DNPSCN or I3(-) inhibits the conversion of alpha 2M from the "slow" to the "fast" electrophoretic form. Furthermore, DNPSCN added after the conformational change can partially reverse the change. A similar reversal can be effected by cyanylation, with NaCN, of methylamine-treated alpha 2M in which the liberated thiols have first been converted to mixed disulfides by reaction with dithiobis(nitrobenzoic acid). Differential scanning calorimetry shows nearly identical properties for the methylamine-treated "fast" form and the cyanylated "slow" form of alpha 2M.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Biochemistry, Vanderbilt University, School of Medicine, Nashville, Tennessee 37232.
L W
LW
Cunningham
B C
BC
Crews
P
P
Gettins
D002152:Calorimetry, Differential Scanning; D055598:Chemical Phenomena; D002621:Chemistry; D004136:Dinitrobenzenes / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007455:Iodine / Q000494:pharmacology; D007700:Kinetics; D008623:Mercaptoethanol / Q000494:pharmacology; D008744:Methylamines / Q000494:pharmacology*; D011487:Protein Conformation; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D014508:Urea / Q000494:pharmacology; D000511:alpha-Macroglobulins / Q000378:metabolism
D004136:Dinitrobenzenes; D008744:Methylamines; D013438:Sulfhydryl Compounds; D000511:alpha-Macroglobulins; D008623:Mercaptoethanol; D014508:Urea; D007455:Iodine; C010936:2,4-dinitrothiocyanatobenzene