Adrenomedullin Binding Protein in the Plasma of Multiple Species: Characterization by Radioligand Blotting

Abstract

Frequently, peptide hormones circulate in plasma associated with specific binding proteins that modify the clearance and biochemical activities of the peptide. Our experimental approach was to use ¹²⁵I-ligand blotting procedures to probe for the presence of specific adrenomedullin (AM) binding proteins (AMBPs). Plasma proteins from chick, calf, dog, goat, guinea pig, human, mouse, pig, rabbit and sheep blood were separated electrophoretically in 10% nonreducing SDS-polyacrylamide gels and transferred to nitrocellulose. Nonspecific binding of tracer was blocked on the nitrocellulose with a hydrolyzed casein matrix. Blots were probed with synthetic human ¹²⁵I-AM. Autoradiogram scanning of blots revealed a mixture of 140- and/or 120- kD protein complexes that bound ¹²⁵I-AM in all species tested. Binding of the ligand was specific as judged by a linear competitive displacement of the tracer binding from human, bovine and pig plasma AMBP bands with increasing concentrations of nonlabelled AM in the binding buffer. A series of peptide fragments of AM representing amino- and carboxyterminal regions of the hormone, or amylin, calcitonin gene-related peptide (CGRP), or insulin failed to displace intact ¹²⁵I-AM from ligand blot bands. Bovine plasma proteins from healthy and parasitized calves with an infection-related stunting syndrome were separated electrophoretically, transferred to nitrocellulose and probed with ¹²⁵I-AM; phosphoimage densitometry analysis revealed a 67% decrease in AMBP band intensity in the 120 and 140 kD proteins from infected calves. We conclude that a specific binding protein(s) for AM exists in mammalian and avian blood that might impact on the bioactivity and function of AM in health and disease.