Vitamin K-dependent carboxylation of peptide-bound glutamate. The active species of “CO2” utilized by the membrane-bound preprothrombin carboxylase.

J.P Jones,E.J Gardner,T.G Cooper,R.E Olson

doi:10.1016/s0021-9258(17)41030-1

Abstract

Vitamin K participates in the post-translational carboxylation of peptide-bound glutamate to form the gamma-carboxy-glutamate residues of prothrombin. The reaction requires reduced vitamin K, bicarbonate, oxygen, and a membrane-bound carboxylase. The active species of "CO2," i.e. CO2 or HCO3-, utilized in this carboxylation was determined by the low temperature method of Filmer and Cooper ((1970) J. Theor. Biol. 29, 131-145), taking advantage of the fact that menaquinone-2, in contrast to phylloquinone, is very active at 10 degrees. Microsomes from livers of vitamin K-deficient rats, were incubated in the presence of cycloheximide, avidin, NADH, menaquinone-2, 1 mM acetazolamide (to inhibit carbonic anhydrase), and either 14CO2 or H14CO3-. At 1-min intervals aliquots were removed from the reaction mixture. gamma-Carboxyglutamate was isolated from these samples by ion exchange chromatography after alkaline hydrolysis. After 1 min the incorporation of 14CO2 into gamma-carboxyglutamate was 8 to 10 times as great as that found with H14CO3-. When the carbonic anhydrase inhibitor was omited (with or without addition of exogenous carbonic anhydrase) the two incorporation curves approximated each other at a rate near that exhibited by bicarbonate alone. Similar results were obtained in a microsomal carboxylase system solubilized with Triton X-100. It is concluded that CO2 is the active species of "CO2" initially participating in the vitamin K-dependent carboxylation of preprothrombin and that neither ATP nor biotin is required for the reaction.

Highlights

It is concluded that CO, is the active species of “C02” initially participating in the vitamin K-dependent carboxylation of preprothrombin and that neither Adenosine triphosphate (ATP) nor biotin is required for the reaction
The results presented here demonstrate that CO, is the initially bound species of “CO,” in the vitamin K-dependent carboxylation of glutamate residues in preprothrombin. This finding is consistent with the observation t.hat ATP is not required for this reaction. Together these findings argue against a role for biotin in this carboxylation
We have studied the effect of avidin in three systems without observing any inhibition of peptide-bound glutamate carboxylation

Summary

AND METHODS

Chemicals -Adenosine triphosphate (ATP), phosphocreatine, creatine phosphokinase, reduced nicotinamide adenine dinucleotide (NADH), and adenyl-5’-yl imidodiphosphate (AMP-P(NH)P). Microsomes were prepared from this postmitochondrial supernatant by centrifuging at 105,000 x g for 60 min in Beckman model L-2 ultracentrifuge. System - Microsomes, prepared as indicated above, were homogenized in an amount of buffer identical with that used for the original post-mitochondrial supernatant. The composition of this buffer was the same as described above except here it contained 1.5% Triton X-100. Selected samples were tested for acid lability of the radioactive material by heating with 6 N HCl for 3 h at 90” followed by a second radioactivity measurement Under these conditions malonate derivatives decarboxylate rapidly

RESULTS

Y Q3 2 u2

DISCUSSION