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A topological study of the human gamma-glutamyl carboxylase.

Publication ,  Journal Article
Tie, J; Wu, SM; Jin, D; Nicchitta, CV; Stafford, DW
Published in: Blood
August 1, 2000

gamma-Glutamyl carboxylase (GC), a polytopic membrane protein found in the endoplasmic reticulum (ER), catalyzes vitamin K-dependent posttranslational modification of glutamate to gamma-carboxyl glutamate. In an attempt to delineate the structure of this important enzyme, in vitro translation and in vivo mapping were used to study its membrane topology. Using terminus-tagged full-length carboxylase, expressed in 293 cells, it was demonstrated that the amino-terminus of the GC is on the cytoplasmic side of the ER, while the carboxyl-terminus is on the lumenal side. In addition, a series of fusions were made to encode each predicted transmembrane domain (TMD) followed by a leader peptidase (Lep) reporter tag, as analyzed by the computer algorithm TOPPRED II. Following in vitro translation of each fusion in the presence of canine microsomes, the topological orientation of the Lep tag was determined by proteinase K digestion and endoglycosidase H (Endo H) cleavage. From the topological orientation of the Lep tag in each fusion, the GC spans the ER membrane at least 5 times, with its N-terminus in the cytoplasm and its C-terminus in the lumen.

Duke Scholars

Published In

Blood

ISSN

0006-4971

Publication Date

August 1, 2000

Volume

96

Issue

3

Start / End Page

973 / 978

Location

United States

Related Subject Headings

  • Structure-Activity Relationship
  • Immunology
  • Humans
  • Dogs
  • Cell Membrane
  • Carbon-Carbon Ligases
  • Animals
  • 3213 Paediatrics
  • 3201 Cardiovascular medicine and haematology
  • 3101 Biochemistry and cell biology
 

Citation

APA
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MLA
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Tie, J., Wu, S. M., Jin, D., Nicchitta, C. V., & Stafford, D. W. (2000). A topological study of the human gamma-glutamyl carboxylase. Blood, 96(3), 973–978.
Tie, J., S. M. Wu, D. Jin, C. V. Nicchitta, and D. W. Stafford. “A topological study of the human gamma-glutamyl carboxylase.Blood 96, no. 3 (August 1, 2000): 973–78.
Tie J, Wu SM, Jin D, Nicchitta CV, Stafford DW. A topological study of the human gamma-glutamyl carboxylase. Blood. 2000 Aug 1;96(3):973–8.
Tie, J., et al. “A topological study of the human gamma-glutamyl carboxylase.Blood, vol. 96, no. 3, Aug. 2000, pp. 973–78.
Tie J, Wu SM, Jin D, Nicchitta CV, Stafford DW. A topological study of the human gamma-glutamyl carboxylase. Blood. 2000 Aug 1;96(3):973–978.

Published In

Blood

ISSN

0006-4971

Publication Date

August 1, 2000

Volume

96

Issue

3

Start / End Page

973 / 978

Location

United States

Related Subject Headings

  • Structure-Activity Relationship
  • Immunology
  • Humans
  • Dogs
  • Cell Membrane
  • Carbon-Carbon Ligases
  • Animals
  • 3213 Paediatrics
  • 3201 Cardiovascular medicine and haematology
  • 3101 Biochemistry and cell biology