GENERAL AND COMPARATIVE ENDOCRINOLOGY 80, 59-67 (1990) Molecular lsoforms of Chicken Growth Hormone (cGH): Different Bioactivities of cGH Charge Variants CARLOS ARAMBURO,’ Jo& LUIS MONTIEL, GERARDO PERERA, SANDRA NAVARRETE, AND Rocfo SANCHEZ Departamento de Fisiologt’a, Institute de Investigaciones BiomCdicas, Universidad National Autdnoma de Mbxico, Mkxico, D.F. 04510, Mbxico Accepted September 20, 1989 It has been suggested that the functional diversity of growth hormone (GH) is related to its molecular complexity. Here we report a characterization of charge and mass variants of chicken growth hormone (cGH) through a variety of electrophoretic systems [nondenaturing (ND-PAGE), denaturing (SDS-PAGE), under reducing and nonreducing conditions, iso- electrofocusing (IEF), and bidimensional electrophoresis] followed by Western blot and immunostaining with a specific antibody directed against pure cGH. We also report the biological properties of two charge variants on two homologous assays. The studies were carried out with purified cGH and with fresh chicken pituitary extracts. Three charge vari- ants were obtained by ND-PAGE (Rr = 0.23, 0.30, and 0.35), which showed the same molecular weight (26 kDa), while in IEF eight isoforms were observed, the most conspic- uous being those with pI = 6.86, 7.5, 7.9, 8.05, and 8.18. In SDS-PAGE under reducing conditions four immunoreactive bands were observed: the monomer (26 kDa), a dimer (52 kDa), a fragment (16 kDa), and a minor band at 22 kDa. Higher MW variants were found under nonreducing conditions. Bidimensional analysis also showed several charge variants for the monomer and the dimer. Bioactivity of two charge variants (0.23 and 0.3) was evaluated with a lipolytic and an antilipolytic assay on chicken adipose tissue explants. It was shown that variant 0.23 was mainly lipolytic, in a dose-dependent response, but lacked antilipolytic effect. On the other hand, variant 0.30 did not show lipolytic effect but pre- sented a clear antilipolytic activity. 0 1990 Academic press, IX. It has been shown that pituitary growth hormone is a family of proteins which when taken as a whole manifests the complex functional diversity of the hormone (Lewis et al., 1980). It has also been suggested that each member of the family could be en- gaged in the expression of a particular ac- tivity, thus relating the molecular heteroge- neity of the hormone with its functional complexity (Lewis, 1984). This information has come mainly from studies with mam- mals, where several mass and charge vari- ants of growth hormone have been de- scribed (Lewis et al., 1980; Chawla et al., 1983). ’ To whom correspondence should be addressed. In birds, little is known about GH heter- ogeneity. Houston and Goddard (1988) re- cently described that chicken pituitary con- tains different forms of GH, identifying up to 10 bands after isoelectrofocusing and im- munoblot analysis, and suggested that some of them may be developmentally reg- ulated. Berghman et al. (1987) also de- scribed the existence of a glycosylated variant of chicken growth hormone (cGH). We recently described the presence of cGH charge variants in chicken pituitary ex- tracts (Aramburo et al., 1989b) and have developed a method to purify them (At-&n- buro et al., 1989a). Furthermore, in that work we reported that a significant fraction of cGH is phosphorylated. Much less is 59 001~6480/90 $1.50 Copyright 0 W!Xl by Academic Press, Inc. All rights of reproduction in any form reserved.