Functional study of the vitamin K cycle in mammalian cells.

We describe a cell-based assay for studying vitamin K-cycle enzymes. A reporter protein consisting of the gla domain of factor IX (amino acids 1-46) and residues 47-420 of protein C was stably expressed in HEK293 and AV12 cells. Both cell lines secrete carboxylated reporter when fed vitamin K or vitamin K epoxide (KO).

Effect of vitamin K-dependent protein precursor propeptide, vitamin K hydroquinone, and glutamate substrate binding on the structure and function of {gamma}-glutamyl carboxylase.

The γ-glutamyl carboxylase utilizes four substrates to catalyze carboxylation of certain glutamic acid residues in vitamin K-dependent proteins. How the enzyme brings the substrates together to promote catalysis is an important question in understanding the structure and function of this enzyme. The propeptide is the primary binding site of the vitamin K-dependent proteins to carboxylase.

Transmembrane domain interactions and residue proline 378 are essential for proper structure, especially disulfide bond formation, in the human vitamin K-dependent gamma-glutamyl carboxylase.

We used recombinant techniques to create a two-chain form (residues 1-345 and residues 346-758) of the vitamin K-dependent gamma-glutamyl carboxylase, a glycoprotein located in the endoplasmic reticulum containing five transmembrane domains. The two-chain carboxylase had carboxylase and epoxidase activities similar to those of one-chain carboxylase. In addition, it had normal affinity for the propeptide of factor IX.

Chemical modification of cysteine residues is a misleading indicator of their status as active site residues in the vitamin K-dependent gamma-glutamyl carboxylation reaction.

The enzymatic activity of the vitamin K-dependent proteins requires the post-translational conversion of specific glutamic acids to gamma-carboxy-glutamic acid by the integral membrane enzyme, gamma-glutamyl carboxylase. Whether or not cysteine residues are important for carboxylase activity has been the subject of a number of studies. In the present study we used carboxylase with point mutations at cysteines, chemical modification, and mass spectrometry to examine this question.

Binding of the factor IX gamma-carboxyglutamic acid domain to the vitamin K-dependent gamma-glutamyl carboxylase active site induces an allosteric effect that may ensure processive carboxylation and regulate the release of carboxylated product.

Propeptides of the vitamin K-dependent proteins bind to an exosite on gamma-glutamyl carboxylase; while they are bound, multiple glutamic acids in the gamma-carboxyglutamic acid (Gla) domain are carboxylated. The role of the propeptides has been studied extensively; however, the role of the Gla domain in substrate binding is less well understood. We used kinetic and fluorescence techniques to investigate the interactions of the carboxylase with a substrate containing the propeptide and Gla domain of factor IX (FIXproGla41).

Determination of disulfide bond assignment of human vitamin K-dependent gamma-glutamyl carboxylase by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Vitamin K-dependent gamma-glutamyl carboxylase is a 758 amino acid integral membrane glycoprotein that catalyzes the post-translational conversion of certain protein glutamate residues to gamma-carboxyglutamate. Carboxylase has ten cysteine residues, but their form (sulfhydryl or disulfide) is largely unknown. Pudota et al.

Circulating and binding characteristics of wild-type factor IX and certain Gla domain mutants in vivo.

Residue K5 in factor IX gamma-carboxyglutamic acid (Gla) domain participates in binding endothelial cells/collagen IV. We injected recombinant factor IX containing mutations at residue 5 (K5A, K5R) into factor IX-deficient mice and compared their behavior with that of wild-type factor IX. The plasma concentration of factor IX that binds to endothelial cells/collagen IV (recombinant wild type and K5R) was consistently lower than that of the one that does not bind (K5A).

The putative vitamin K-dependent gamma-glutamyl carboxylase internal propeptide appears to be the propeptide binding site.

The vitamin K-dependent gamma-glutamyl carboxylase binds an 18-amino acid sequence usually attached as a propeptide to its substrates. Price and Williamson (Protein Sci. (1993) 2, 1997-1998) noticed that residues 495-513 of the carboxylase shares similarity with the propeptide.

Residues Y179 and H101 of a hydrophobic patch of factor VII are involved in activation by factor Xa.

We studied factor Xa activation of human factor VII in hopes of identifying factor VII residues, not adjacent to the cleavage site, involved in this interaction. We made eight factor VIIs with single mutations (N100A, H101A, D102Q, L144A, R147A, Y179A, D186A, and F256A) and two factor VIIs with multiple mutations [MM3 (L144A/R147A/D186A) and MM4 (N100A/H101A/Y179A/F256A)]. Residues in MM3 have previously been identified as affecting factor X activation, and the residues of MM4 are located at a hydrophobic patch of factor VII on the opposite side of the catalytic domain from those in MM3.

Factor VIIa's first epidermal growth factor-like domain's role in catalytic activity.

Factor VIIa-tissue factor complex formation initiates the extrinsic blood coagulation pathway. We investigated factor VIIa's first epidermal growth factor-like (egf1) domain's role in the catalytic activity increase caused when factor VIIa binds tissue factor. Starting with a factor VIIa with factor IX's egf1 domain (factor VII(IXegf1)a), we made 4 proteins with egf1 residues changed to those in factor VIIa, including E51A, D64Q, FG74-75PA, and K79R.

Changing residue 338 in human factor IX from arginine to alanine causes an increase in catalytic activity.

This study was designed to identify functionally important factor IX (FIX) residues. Using recombinant techniques and cell culture, we produced a mutant FIX with arginine at 338 changed to alanine (R338A-FIX). This molecule had approximately 3 times greater clotting activity than that of wild type FIX (wt-FIX) in the activated partial thromboplastin assay.

A coagulation factor IX-deficient mouse model for human hemophilia B.

Coagulation factor IX deficiency causes hemophilia B in humans. We have used gene targeting to develop a coagulation factor IX-deficient (factor IX-knockout) mouse strain. Mouse embryonic stem (ES) cells were targeted by a socket-containing vector that replaces the promoter through exon 3 of the factor IX gene by neoDeltaHPRT, which is a functional neo gene plus a partially deleted hypoxanthine phosphoribosyl transferase minigene.

The roles of factor VII's structural domains in tissue factor binding.

Factor VIIa binds to tissue factor in one of the initial steps of blood clotting. In order to determine the role of the various domains of the factor VII molecule in this interaction, we made several chimeric factor VII proteins using recombinant DNA techniques. The molecules have factor IX domains substituted into factor VII and vice versa.

The role of the epidermal growth factor-1 and hydrophobic stack domains of human factor IX in binding to endothelial cells.

To determine the function and specificity in factor IX of the first epidermal growth factor (EGF)-like domain and the eight-amino acid hydrophobic stack encoded by exon C (residues 39-46), these domains were replaced by the corresponding polypeptide regions of factor X and chimeric proteins were produced in human embryo kidney cells. Both chimeras were activated by factor XIa at a rate similar to plasma factor IX and exhibited calcium-dependent fluorescence quenching similar to plasma factor IX. Both chimeras competed equally for binding to the endothelial cell receptor.

Binding of alpha 2-macroglobulin-thrombin complexes and methylamine-treated alpha 2-macroglobulin to human blood monocytes.

The binding of alpha 2-macroglobulin (alpha 2M) to human peripheral blood monocytes was investigated. Monocytes, the precursors of tissue macrophages, were isolated from fresh blood by centrifugal elutriation or density gradient centrifugation. Binding studies were performed using 125I-labeled alpha 2M.

Homo- and heterodimer formation with prothrombin and prothrombin fragment 1 in the presence of calcium ions.

The purpose of the current study is to present further evidence for prothrombin self-association as assessed by chemical crosslinking. When the self-association (evaluated by covalent crosslinking with dithiobis(succinimidylpropionate) of prothrombin or fragment 1 was evaluated at the same molar concentration of protein, similar rates of dimer formation were observed for either protein. When prothrombin and fragment 1 were incubated together with the crosslinking reagent and calcium ions, a heterodimer consisting of prothrombin and fragment 1 was observed in addition to prothrombin dimer and fragment 1 dimer.

Love-Life. An open door to industry.

One method for hospitals to establish new revenue sources and set the stage for future cooperative ventures is to work closely with local businesses. Cooperative efforts, however, not only benefit the hospital, they also help employers reduce health care costs and employees increase health awareness. This article describes the health awareness program developed at Akron General Medical Center in Akron, Ohio.

Structural and functional characterization of the inhibition of urokinase by alpha 2-macroglobulin.

We have investigated the interaction of alpha 2-macroglobulin (alpha 2M) with the serine proteinase urokinase, an activator of plasminogen. Urokinase formed sodium dodecyl sulfate stable complexes with purified alpha 2M and with alpha 2M in plasma. These complexes could be visualized after polyacrylamide gel electrophoresis by protein blots using 125I-labeled anti-urokinase antibody or by fibrin autography, a measure of fibrinolytic activity.

Activation of normal and abnormal human factor IX with trypsin.

Human factor IX is activated to factor IXa beta when factor XIa cleaves two peptide bonds, Arg 145-Ala 146 and Arg 180-Val 181, to release an activation peptide. In factor IX Chapel Hill (IXCH), isolated from a hemophilia B patient with a mild bleeding disorder, the arginine 145 residue has been replaced with a histidine. Thus factor IXCH is activated by factor XIa by cleaving only at the Arg 180-Val 181 bond, leaving the activation peptide attached, and resulting in an activated species, factor IXa alpha CH, that, like normal factor IXa alpha, is only 20% as active as factor IXa beta.

Structural and functional characteristics of activated human factor IX after chemical modification of gamma-carboxyglutamic acid residues.

Activated human factor IX (factor IXa) was treated under mildly acidic conditions with a mixture of formaldehyde and morpholine. This reagent has been shown to react preferentially with gamma-carboxyglutamyl (Gla) residues and to convert these residues to gamma-methyleneglutamyl residues (Wright, S.F.

Inactivation of human blood coagulation factor X by chemical modification of gamma-carboxyglutamic acid residues.

The inactivation of human factor X by incubation with a reagent known to chemically modify gamma-carboxyglutamic acid to gamma-methylene glutamic acid was studied. Incubation of factor X at pH 5.0 with a preincubated formaldehyde/morpholine mixture (0.

Bovine alpha- and beta-thrombin. Reduced fibrinogen-clotting activity of beta-thrombin is not a consequence of reduced affinity for fibrinogen.

beta-Thrombin, a product of the limited proteolysis of alpha-thrombin, is characterized by greatly reduced fibrinogen-clotting activity as compared to alpha-thrombin but with unchanged activity toward ester substrates. The present study was designed to elucidate the basis for the changes in the catalytic activity resulting from the conversion of bovine alpha-thrombin to bovine beta-thrombin. Fibrinogen was utilized as a competitive inhibitor in the hydrolysis of a peptide nitroanilide substrate by bovine alpha- and beta-thrombin.

Characterization of thrombin binding to alpha 2-macroglobulin.

The formation and structural characteristics of the human alpha 2-macroglobulin (alpha 2M)-thrombin complex were studied by intrinsic protein fluorescence, sulfhydryl group titration, electrophoresis in denaturing and nondenaturing polyacrylamide gel systems, and in macromolecular inhibitor assays. The interaction between alpha 2M and thrombin was also assessed by comparison of sodium dodecyl sulfate-gel electrophoretic patterns of peptides produced by Staphylococcus aureus V-8 proteinase digests of denatured alpha 2M-125I-thrombin and alpha 2M-125I-trypsin complexes. In experiments measuring fluorescence changes and sulfhydryl group exposure caused by methylamine, we found that thrombin produced its maximum effects at a mole ratio of approximately 1.