Increased thermo-stability of rat prolactin after replacing glutamic acid at position 118 by lysine.
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.
Chung
H O
HO
Biosignal Research Center, Gunma University, Japan.
Tomizawa
K
K
Kato
T
T
Wakabayashi
K
K
Kato
Y
Y
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
D018698:Glutamic Acid; D011388:Prolactin; D008239:Lysine