title: Increased thermo-stability of rat prolactin after replacing glutamic acid at position 118 by lysine.
abstract: We examined the structural stabilities after heat treatment of 22 mutants of rat prolactin (rPRL) with amino acid replacements at 15 different positions and recombinant wild-type rPRL (WT-PRL) as part of our series of studies on site-directed mutagenesis of rPRL. When WT-PRL at low concentrations (0.1-10 ng/ml) was heated at 100 degrees C for 20 min, it lost its Nb2 proliferation activity, whereas at high concentrations (above 1 microgram/ml), its activity remained. Temperature-dependent loss of the proliferation activity of 10 ng/ml WT-PRL after heat treatment for 5 min was observed. Next, we examined the proliferation activities of the 22 mutants heated at 60 and 70 degrees C for 5 min. After treatment at 60 degrees C, all the mutants retained their initial proliferation activities, whereas treatment at 70 degrees C reduced their activities to about 63%, except for one in which glutamic acid at position 118 was replaced by lysine (E118K), suggesting that the mutations did not induce structural instability. The mutant E118K retained 84% of its initial activity after treatment at 70 degrees C, significantly (P < 0.01) higher than the WT-PRL value. The temperature-dependency profile of the Nb2 proliferation activity of E118K also showed it had significantly increased thermo-stability. Meanwhile another mutant (E118Q) at the same residue showed no increased thermo-stability, suggesting that changing a negative charge (E) to a positive one (K) at position 118 induces ionic bond formation with a neighboring negative charge, resulting in thermostabilization of the structure of PRL.
authors.affiliation: Biosignal Research Center, Gunma University, Japan.
authors.forename: H O
authors.initials: HO
authors.lastname: Chung
authors.forename: K
authors.initials: K
authors.lastname: Tomizawa
authors.forename: T
authors.initials: T
authors.lastname: Kato
authors.forename: K
authors.initials: K
authors.lastname: Wakabayashi
authors.forename: Y
authors.initials: Y
authors.lastname: Kato
mesh_terms: D000818:Animals; D002455:Cell Division; D018698:Glutamic Acid / Q000235:genetics*; D006358:Hot Temperature; D008223:Lymphoma / Q000473:pathology; D008239:Lysine / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D011388:Prolactin / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D014407:Tumor Cells, Cultured
chemical_list: D018698:Glutamic Acid; D011388:Prolactin; D008239:Lysine