Changes in trypsin-binding properties and conformation of rabbit alpha-2-macroglobulin on reaction with methylamine.
Reactions of rabbit alpha-2-macroglobulin with methylamine and trypsin were studied and the results were compared with those obtained for previously described 2-macroglobulins from other species. Rabbit alpha-2-macroglobulin was cleaved by trypsin at a number of sites, whereas the human homologue was split essentially only in the "bait" region into two fragments of similar sizes. Reaction of native or methylamine-treated rabbit alpha-2-macroglobulin with trypsin resulted in a substantial decrease in the intensity of fluorescence induced by binding of 6-(p-toluidino)-2-naphthalenesulfonate or bis(8-anilino-1-naphthalenesulfonate). Under the same conditions, the fluorescence of the human protein increased. The time course of the reaction of rabbit alpha-2-macroglobulin with methylamine was studied by measuring (i) the generation of thiol groups, (ii) the decrease in trypsin-inhibiting activity with remazol brilliant blue hide powder as the substrate, and (iii) the decrease in trypsin-protein amidase activity. The thiol appearance reaction exhibited a multiphasic time course. The initial phase was found to follow second-order kinetics with an apparent rate constant of 1.2 M-1.s-1. Under the same conditions, the human protein showed monophasic kinetics with a rate constant of 12 M-1.s-1. Both the trypsin-inhibiting activity and the trypsin-protein amidase activity concurrently decreased at a slower rate than the thiol appearance. These results indicate that rabbit alpha-2-macroglobulin is more stable to nucleophilic attack by methylamine but less resistant to proteolysis by trypsin than the human homologue, and that the final conformation induced by methylamine differs considerably from that induced by trypsin.
Tamamizu
S
S
Department of Biochemistry, Faculty of Pharmacy, Kinki University, Higashi-Osaka.
Miyake
Y
Y
Ito
T
T
Sinohara
H
H
D000818:Animals; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008744:Methylamines / Q000494:pharmacology*; D011487:Protein Conformation; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013438:Sulfhydryl Compounds / Q000032:analysis; D014357:Trypsin / Q000378:metabolism*; D014361:Trypsin Inhibitors; D000511:alpha-Macroglobulins / Q000378:metabolism*
D008744:Methylamines; D013438:Sulfhydryl Compounds; D014361:Trypsin Inhibitors; D000511:alpha-Macroglobulins; D014357:Trypsin