Vitamin K-dependent carboxylase: affinity purification from bovine liver by using a synthetic propeptide containing the gamma-carboxylation recognition site.

Hubbard, Brian; Ulrich, Magda M. W.; Jacobs, Margaret; Vermeer, Cees; Walsh, Christopher T.; Furie, Bruce; Furie, Barbara C.

doi:10.1073/pnas.86.18.6893

Cited by 53 publications

(36 citation statements)

References 42 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The entire purification can be accomplished in 4 days. It is difficult to compare different purification schemes but, by comparing final specific activities, it appears that our puri- fication results in a carboxylase preparation that 185-fold higher specific activity than the best previously published method (11). From 14CO2 incorporation into the pentapeptide FLEEL, we estimate the specific activity of our carboxylase prepared by Affi-FIXQ/S chromatography eluted by method II (Fig.…”

Section: Discussionmentioning

confidence: 99%

“…Harbeck et al (10) extended this method by eluting the carboxylase from a prothrombin antibody column with a synthetic propeptide achieving a 500-fold purification and a final specific activity of6.6 x 106 cpm per mg per hr. Hubbard et al (11) reported the purification of the carboxylase to homogeneity using a synthetic propeptide sequence as an affinity ligand. However, the reported final specific activity of 1.3 x 107 cpm per mg per hr was still not significantly different than that reported by Girardot (8).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Identification and purification to near homogeneity of the vitamin K-dependent carboxylase.

Morris

Stafford

1991

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Vitamin K-dependent carboxylase catalyzes the modification of specific glutamic acids to y-carboxyglutamic acid in several blood-coagulation proteins. This modification is required for the blood-clotting activity of these proteins and has thus been the subject of intense investigation. We have now identified the bovine vitamin K-dependent carboxylase and purified it to near homogeneity by an affinity procedure that uses the 59-amino acid peptide FIXQ/S (residues -18 to 41 of factor IX with mutations Arg --Gin at residue -4 and Arg -+ Ser at residue -1). The carboxylase as purified has a molecular weight of 94,000. It is also the major protein that can be cross-linked to iodinated FIXQ/S and is the only protein whose cross-linking is prevented by a synthetic factor IX propeptide. The degree of purification is about 7000-fold with reference to ammonium sulfate-fractionated microsomal protein from liver.A number of blood coagulation proteins require a posttranslational vitamin K-dependent modification for biological activity. Stenflo et al. (3) have reported that prothrombin, the prototype of these vitamin K-dependent proteins, contained the modified amino acid y-carboxyglutamic acid (Gla). Prothrombin from animals treated with the vitamin K antagonist warfarin lacked this Gla modification. It was inferred from these observations that the blood-clotting activity of the vitamin K-dependent proteins required y-carboxylation of specific glutamic acid residues. Shortly thereafter, Esmon et al. (4) demonstrated an enzyme activity, vitamin K-dependent carboxylase (hereafter called carboxylase), capable of making this Gla modification.After cDNA sequences were obtained for several of the vitamin K-dependent proteins, Pan and Price (5) compared the deduced amino acid sequences and suggested that the propeptide consensus sequence preceding the amino terminus of the vitamin K-dependent protein was a recognition site for the carboxylase. This suggestion was confirmed by Knobloch and Suttie (6), who demonstrated the importance ofthe propeptide in carboxylation by showing that the synthetic propeptide sequence of human factor X stimulated the activity of the carboxylase for a small substrate (Boc-Glu-GluLeu-OMe) in vitro. Jorgensen et al. (7) extended this observation by showing that factor IX with its propeptide deleted was not carboxylated.In spite of its importance, the carboxylase has not been previously purified. Purification of 400-fold was reported by Girardot (8 that an immobilized factor X antibody would bind the carboxylase, presumably through a factor X precursorcarboxylase complex, and that the bound carboxylase retained its activity for the synthetic peptide substrate FLEEL. Harbeck et al. (10) extended this method by eluting the carboxylase from a prothrombin antibody column with a synthetic propeptide achieving a 500-fold purification and a final specific activity of6.6 x 106 cpm per mg per hr. Hubbard et al. (11) reported the purification of the carboxylase to homogeneity using a synthetic propeptide ...

show abstract

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Identification and purification to near homogeneity of the vitamin K-dependent carboxylase.

Morris

Stafford

1991

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

show abstract

“…OC and mutant OC (the 3 glutamic acid residues, Glu [positions], had been mutated to aspartic acid residues [Asp]) constructs were provided by Caren Gundberg (33). pLIVE expression vector, a vector driven by a liver-specific promoter composed of the minimal mouse albumin promoter and the mouse α-fetoprotein enhancer was purchased from Mirus Bio.…”

Section: Methodsmentioning

confidence: 99%

“…This method of controlling the carboxylation status of osteo calcin has been previously used to inactivate vitamin K-dependent clotting factors (33). Furthermore, for validation purposes (see below), we cloned green fluorescent protein (gfp) into the pLIVE vector.…”

Section: Figurementioning

confidence: 99%

Osteoblasts mediate the adverse effects of glucocorticoids on fuel metabolism

Brennan‐Speranza

Henneicke²,

Gasparini³

et al. 2012

J. Clin. Invest.

168

123

View full text Add to dashboard Cite

Long-term glucocorticoid treatment is associated with numerous adverse outcomes, including weight gain, insulin resistance, and diabetes; however, the pathogenesis of these side effects remains obscure. Glucocorticoids also suppress osteoblast function, including osteocalcin synthesis. Osteocalcin is an osteoblast-specific peptide that is reported to be involved in normal murine fuel metabolism. We now demonstrate that osteoblasts play a pivotal role in the pathogenesis of glucocorticoid-induced dysmetabolism. Osteoblast-targeted disruption of glucocorticoid signaling significantly attenuated the suppression of osteocalcin synthesis and prevented the development of insulin resistance, glucose intolerance, and abnormal weight gain in corticosterone-treated mice. Nearly identical effects were observed in glucocorticoid-treated animals following heterotopic (hepatic) expression of both carboxylated and uncarboxylated osteocalcin through gene therapy, which additionally led to a reduction in hepatic lipid deposition and improved phosphorylation of the insulin receptor. These data suggest that the effects of exogenous high-dose glucocorticoids on insulin target tissues and systemic energy metabolism are mediated, at least in part, through the skeleton.

show abstract

“…Anti-carboxylase-(86-99) antibodies were affinitypurified as described (13 Bovine Liver Carboxylase Purification. Isolation of microsomes from fresh bovine liver was performed as described (10). Carboxylase was solubilized with CHAPS and concentrated by precipitation with ammonium sulfate.…”

mentioning

confidence: 99%

Expression of bovine vitamin K-dependent carboxylase activity in baculovirus-infected insect cells.

Rehemtulla

Kaufman

Walsh

et al. 1993

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

A vitamin K-dependent carboxylase has recently been purified from bovine liver microsomes and candidate cDNA clones have been isolated. Definitive identification of the carboxylase remains circumstantial since expression of candidate carboxylase cDNAs in mammalian cells is confounded by the presence of endogenous carboxylase activity. To overcome this problem, a recombinant strain of baculovirus (Autographa california nuclear polyhedrosis virus, AcMNPV) encoding a putative carboxylase (vbCbx/AcMNPV) was used to infect Sf9 insect cells, which we demonstrate have no endogenous carboxylase activity. Infection with vbCbx/AcMNPV conferred vitamin K-dependent carboxylase activity to Sf9 insect cells. Carboxylase activity was demonstrated to peak 2-3 days after infection with vbCbx/AcMNPV. Metabolic radiolabeling with L-[35S]methionine revealed that the 90-kDa recombinant protein is the major protein synthesized at the time of peak activity after infection. An anti-peptide antibody directed against residues 86-99 reacted with bovine liver carboxylase on Western blot analysis and immunoprecipitated recombinant carboxylase from infected Sf9 microsomal protein preparations. Since Sf9 insect cells lack endogenous vitamin K-dependent carboxylase activity, expression of carboxylase activity in Sf9 insect cells with recombinant baculovirus demonstrates that the protein encoded by this cDNA is a vitamin K-dependent gamma-glutamyl carboxylase.

show abstract

Vitamin K-dependent carboxylase: affinity purification from bovine liver by using a synthetic propeptide containing the gamma-carboxylation recognition site.

Cited by 53 publications

References 42 publications

Identification and purification to near homogeneity of the vitamin K-dependent carboxylase.

Identification and purification to near homogeneity of the vitamin K-dependent carboxylase.

Osteoblasts mediate the adverse effects of glucocorticoids on fuel metabolism

Expression of bovine vitamin K-dependent carboxylase activity in baculovirus-infected insect cells.

Contact Info

Product

Resources

About