A fluorescent method to determine vitamin K-dependent gamma-glutamyl carboxylase activity.
The gamma (γ)-glutamyl carboxylase is a key enzyme in vitamin K-dependent carboxylation of proteins involved in hemostasis and inflammation. It is an endoplasmic enzyme posttranslationally converting glutamic acid residues into γ-carboxyglutamic acid residues in proteins. The activity of tissue derived γ-glutamyl carboxylase is commonly assayed by incorporation of H¹⁴CO₃⁻ into synthetic peptides and subsequent quantification using liquid scintillation counting. We present a nonradioactive assay using a fluorescein isothiocyanate-labeled short peptide that can be readily detected in its unmodified and γ-glutamyl carboxylated form by reversed-phase HPLC. This method offers a convenient alternative to the established radioactive labeling techniques.
Division of Nephrology and Clinical Immunology, University Hospital of the RWTH Aachen, Pauwelsstrasse 30, 52074 Aachen, Germany.
Nadine
N
Kaesler
Thomas
T
Schettgen
Vasantha P
VP
Mutucumarana
Vincent
V
Brandenburg
Willi
W
Jahnen-Dechent
Leon J
LJ
Schurgers
Thilo
T
Krüger
D000595:Amino Acid Sequence; D000818:Animals; D019735:Carbon-Carbon Ligases / Q000737:chemistry / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D008297:Male; D013058:Mass Spectrometry; D008970:Molecular Weight; D051381:Rats; D017208:Rats, Wistar; D012588:Scintillation Counting; D013050:Spectrometry, Fluorescence; D014812:Vitamin K / Q000378:metabolism*
D014812:Vitamin K; D019735:Carbon-Carbon Ligases; C020660:glutamyl carboxylase