Stabilization in microsomal membranes of the fifth transmembrane segment of the Na+,K+-ATPase α subunit with proline to leucine mutation

1993 ◽  
Vol 71 (7-8) ◽  
pp. 410-415 ◽  
Author(s):  
Haruo Homareda ◽  
Kiyoshi Kawakami ◽  
Kei Nagano ◽  
Hideo Matsui
Keyword(s):  
Insertion Site ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Membrane Insertion ◽  
Transmembrane Segment ◽  
Α Subunit ◽  
Microsomal Membranes

We have reported that the fifth transmembrane segment of the Na+,K+-ATPase α subunit could not be inserted into microsomal membranes when it was synthesized from the truncated α1 cDNA. In this study, three proline residues in the segment were changed into leucine residues by site-directed mutagenesis. Replacement of all three proline residues by leucine residues allowed the insertion of this segment and its translocation. This result confirms our previous finding and demonstrates that three proline residues in the fifth transmembrane segment prevent insertion of the segment into the membrane.Key words: Na+,K+-ATPase, transmembrane segment, membrane insertion, site-directed mutagenesis.

Location of functional regions of acetylcholine receptor α-subunit by site-directed mutagenesis

Nature ◽  
1985 ◽  
Vol 313 (6001) ◽  
pp. 364-369 ◽  
Author(s):  
Masayoshi Mishina ◽  
Takamasa Tobimatsu ◽  
Keiji Imoto ◽  
Ken-ichi Tanaka ◽  
Yoshihiko Fujita ◽  
...  
Keyword(s):  
Acetylcholine Receptor ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Α Subunit ◽  
Functional Regions

scholarly journals Site-directed mutagenesis of two conserved charged amino acids in the N-terminal region of α subunit ofE. coli-F0F1

FEBS Letters ◽  
1996 ◽  
Vol 382 (1-2) ◽  
pp. 171-174 ◽  
Author(s):  
Barbara Hase ◽  
Sabine Werner-Grüne ◽  
Gabriele Deckers-Hebestreit ◽  
Heinrich Strotmann
Keyword(s):  
Amino Acids ◽  
Terminal Region ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Α Subunit ◽  
Charged Amino Acids

Introduction of DPR, an Enterostatin Fragment Peptide, into Soybean β-Conglycinin α′ Subunit by Site-directed Mutagenesis

2004 ◽  
Vol 68 (1) ◽  
pp. 253-256 ◽  
Author(s):  
Yasuyuki TAKENAKA ◽  
Naomi DOYAMA ◽  
Nobuyuki MARUYAMA ◽  
Shigeru UTSUMI ◽  
Masaaki YOSHIKAWA
Keyword(s):  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Α Subunit

Site-directed mutagenesis of the α subunit of tryptophan synthase from salmonella typhimurium

1988 ◽  
Vol 151 (2) ◽  
pp. 672-678 ◽  
Author(s):  
Syed Ashrafuddin Ahmed ◽  
Haruhiko Kawasaki ◽  
Ronald Bauerle ◽  
Hatsué Morita ◽  
Edith Wilson Miles
Keyword(s):  
Salmonella Typhimurium ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Tryptophan Synthase ◽  
Α Subunit

scholarly journals Human Thyrotropin (TSH) Variants Designed by Site-Directed Mutagenesis Block TSH Activity in Vitro and in Vivo

Endocrinology ◽  
2005 ◽  
Vol 146 (6) ◽  
pp. 2845-2850 ◽  
Author(s):  
Naiel Azzam ◽  
Rinat Bar-Shalom ◽  
Zaki Kraiem ◽  
Fuad Fares
Keyword(s):  
Chorionic Gonadotropin ◽  
Biologically Active ◽  
Site Directed Mutagenesis ◽  
Human Thyroid ◽  
Directed Mutagenesis ◽  
Β Subunit ◽  
Single Chain ◽  
Glycoprotein Hormone ◽  
Α Subunit

Abstract TSH is a heterodimeric glycoprotein hormone synthesized in the pituitary and composed of a specific β-subunit and a common α-subunit shared with FSH, LH, and human chorionic gonadotropin. The heterodimer was previously converted into a biologically active single chain protein by genetic fusion of the genes coding to both subunits in the presence of the carboxy-terminal sequence of human (h) chorionic gonadotropin-β subunit as a linker [hTSHβ-carboxyl-terminal peptide (CTP)-α]. N-linked carbohydrate-free single-chain TSH variants were constructed by site-directed mutagenesis and overlapping PCR: one devoid of both N-linked oligosaccharide chains on the α-subunit (hTSHβ-CTP-αdeg) and the other lacking also the oligosaccharides on the β-subunit (hTSHβdeg-CTP-αdeg). These variants were expressed in Chinese hamster ovary cells and secreted into the culture media. We have previously reported that the variants block the activities of hTSH and thyroid-stimulating immunoglobulins in cultured human thyroid follicles. In the present study, binding affinity of hTSH variants to hTSH receptor and the localization of the antagonistic effect were examined. Moreover, the effect of these variants on TSH activity was tested in vivo. The results of the present study indicate that the hTSH variants bind to the hTSH receptor with high affinity. Experiments using forskolin also indicated that the N-linked carbohydrate-free TSH single-chain variants inhibit TSH activity at the receptor-binding site and not at a postreceptor level. Moreover, the variants significantly inhibited (about 50%) TSH activity with respect to thyroid hormone secretion in vivo in mice. These variants may offer a novel therapeutic strategy in treating hyperthyroidism.

scholarly journals Site-directed Mutagenesis of the α Subunit of Human Prolyl 4-Hydroxylase

1995 ◽  
Vol 270 (17) ◽  
pp. 9926-9931 ◽  
Author(s):  
Arja Lamberg ◽  
Taina Pihlajaniemi ◽  
Kari I. Kivirikko
Keyword(s):  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Α Subunit
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