Properties of human prolactin (PRL) and H27A-PRL, a mutant that does not bind Zn++.
The ability of protein hormones to self-associate is likely to play an important role in concentrating hormones into secretory granules; therefore, the aggregation properties of human PRL and H27A mutant were investigated. Human PRL bound (65)Zn++; the Scatchard analysis was convex up, and limited by the solubility of PRL, but at least 0.7 mole Zn++ bound per mole of PRL. Binding of (65)Zn++ to H27A-PRL was greatly reduced. The biological activity in an Nb2 cell assay and the circular dichroism spectrum of wild type and H27A-PRL were similar, indicating the H27A mutant folded correctly, and the binding of Zn++ to the high affinity site is not essential for biological activity. Dynamic light scattering measurements indicated 10 and 20 mu M Zn++ caused some aggregation of both wild type and H27A-PRL. Sedimentation equilibrium analysis indicated that PRL is primarily a monomer in the absence of Zn++ and that there is increasing self-association in the presence of 5 and 10 mu M Zn++. The mutant H27A exhibited a greater tendency to aggregate without changing detectably the mode of association. Although human PRL binds Zn++ as human GH does, it differs in that the ability to bind Zn++ and to self-associate were decoupled in PRL. Human PRL must have two types of interactions with Zn++; one is binding to a site involving histidine 27, and the other is weaker interactions that induce self-association of PRL.
Sun
Z
Z
Department of Pharmacology, Yale School of Medicine, New Haven, Connecticut, USA.
Li
P S
PS
Dannies
P S
PS
Lee
J C
JC
D000818:Animals; D002460:Cell Line; D002942:Circular Dichroism; D006801:Humans; D011388:Prolactin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D015032:Zinc / Q000378:metabolism*
D011388:Prolactin; D015032:Zinc