Osteocalcin binds tightly to the γ-glutamylcarboxylase at a site distinct from that of the other known vitamin K-dependent proteins

Vitamin K-dependent proteins contain a propeptide that is required for recognition by the enzyme gamma-glutamylcarboxylase. Substrates used in vitro for carboxylation studies lacking a prosequence are characterized by Km values in the millimolar range, whereas the Km for peptides containing a prosequence is three or four orders of magnitude smaller. Here we report that descarboxy-osteocalcin is an exception in this respect. With descarboxy-osteocalcin in purified propeptide-free recombinant carboxylase, the Km was 1.8 microM. Furthermore, osteocalcin was an inhibitor of descarboxy-osteocalcin carboxylation with a Ki of 76 microM. In contrast with the other vitamin K-dependent proteins, free propeptides do not inhibit descarboxy-osteocalcin carboxylation. Moreover, propeptide-containing substrates were inhibited neither by osteocalcin nor by its propeptide. From our studies we conclude that descarboxy-osteocalcin must have an internal recognition sequence that binds to gamma-glutamylcarboxylase at a site different from the propeptide-recognition site

Vitamin K-antagonistic effect of plastoquinone and ubiquinone derivatives in vitro

AbstractDecyl-ubiquinone and decyl-plastoquinone were used as model compounds to test the potential effect of quinone derivatives on two enzymes of the vitamin K cycle in vitro. Substantial inhibition of γ-glutamate carboxylase was found, whereas vitamin K-epoxide reductase was inhibited to a much lesser extent. The inhibitory effect of both decylquinones was eliminated in a time-dependent way by solubilized microsomes, but not by purified carboxylase. Since a wide variety of prenylquinones occur as micronutrients, these results are of potential relevance for the effects of natural quinones in the human diet

Isoenzymes of vitamin-K-dependent carboxylase

Vitamin-K-dependent carboxylase was prepared from bovine liver, kidney, lung and testis and it was checked that these systems obeyed the laws of normal enzyme kinetics. Four carboxylatable substrates were obtained from different sources and the apparent Michaelis constants of the various carboxylase for these four substrates were measured. From the results thus obrained we conclude that carboxylase is a group name for a number of isoenzymes which are present in heaptic as well as in various non-hepatic tissues

The quantification of gammacarboxyglutamic acid residues in plasma-osteocalcin

In this paper we describe an assay procedure for determining the amount of gammacarboxyglutamic acid (Gla) residues in serum- or plasma-osteocalcin. The test includes removing by ethanol precipitation the majority of the proteins from the plasma (e.g., the Gla-containing coagulation factors) and the specific extraction of osteocalcin with the aid of immobilized immunopurified antibodies. It is demonstrated that the Gla-content of circulating osteocalcin from normal cows is similar to that of osteocalcin obtained from bone. Hence, the fact that part of the newly synthesized osteocalcin does not bind to the hydroxyapatite matrix in bone cannot be explained by an undercarboxylation of the molecule