Since the main melanocyte-stimulating hormone (MSH)-release-inhibiting factor or hormone (MIF or MRIH) has been isolated from bovine hypothalami and its structure determined to be H-Pro-Leu-Gly-NH2, the corresponding synthetic hormones H-Pro-[14C]Leu-Gly-NH2 and [3H]Pro-Leu-Gly-NH2 were prepared to facilitate studies on the inactivation of MIF. Incubation of [14C]MIF for 1 hr at 37° with human plasma, followed by recovery and bioassay, showed complete inactivation. At 0°, inactivation was negligible. The radioactivity recovered from the inactivated products of MIP incubated at 37°, and from the active, intact hormone incubated at 0°, was similar. Thin-layer Chromatographic and electrophoretic separation of the inactivation products of [14C]MIF yielded four peptide materials which were identified by amino acid analyses, Edman-dansyl degradation, and measurement of radioactivity as: proline, [14C]Leu-Gly-NH2, and traces of Pro-[14C]Leu-Gly-OH and [14C]Leu-Gly-OH. The peptides, Pro-Leu-Gly-OH, Leu-Gly-NH2 and Leu-Gly-OH, were then synthesized and shown to be inactive in the MIF assay. Electrophoretic and Chromatographic comparison of the mobilities of these synthetic peptides and proline with that of the purified inactivation products obtained from labeled or unlabeled MIF confirmed Leu-Gly-NH2 and proline as the main fragments as well as traces of Pro-Leu-Gly-OH. These studies show that incubation of MIF with human plasma caused cleavage of its Pro-Leu bond and destruction of biological activity.
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