Design and concise synthesis of fully protected analogues of l-gamma-carboxyglutamic acid.

The design and synthesis of four nonnaturally occurring amino acid analogues of l-gamma-carboxyglutamic acid (Gla), appropriately protected for Fmoc-based solid-phase peptide synthesis (SPPS), is described. These amino acids are Bu-Mal 2, BCAH 3, Pen-Mal 4, and Cm-Gla 5. These Gla analogues have been designed to replace the glutamic acid of position 1 in the cyclic decapeptide G1TE, which is a potent inhibitor of tyrosine kinase, to further enhance binding to the Grb2-SH2 domain of signal transduction receptors. In the new amino acids, the propionic acid side chain of Glu has been replaced by a malonyl or a carboxymethylmalonyl moiety located at different distances from the alpha-carbon to optimize interactions in the phosphotyrosine-binding cavity of the Grb2-SH2 domain. Additionally, a direct and efficient synthetic route for the preparation of Fmoc-protected l-gamma-carboxyglutamic acid, which is amenable to large-scale production, has been developed to provide this important and unique amino acid(1) in 55% overall yield.

γ-Glutamyl Transpeptidase Activity Assay.

Gamma-glutamyl transpeptidase catalyzes the transfer of a gamma-glutamyl bond and is of importance in glutathione metabolism. The most common assay methods for this enzyme, in which gamma-glutamyl derivatives of p-nitroaniline are used as a gamma-glutamyl donor, are described in this unit.

High-affinity calcium-binding site in the gama-carboxyglutamic acid domain of bovine factor VII.

The calcium-mediated interaction of factor VIIa with tissue factor is considered to be the primary trigger of blood coagulation. To determine the role of calcium ions in the action of factor VII, we prepared monoclonal antibodies whose binding to factor VII was calcium-dependent. A monoclonal antibody designated C6 strongly inhibited factor VII-induced clotting at a molar ratio of factor VII to antibody of 1:1. The half-maximal binding of factor VII to the C6 antibody was observed at a concentration of calcium ions of 80 microM. Proteolytic fragments of factor VII were assayed for their ability to inhibit competitively the binding of 125I-factor VII to immobilized C6 antibody. The binding was inhibited by increasing amounts of factor VII, by a fragment that contained the gamma-carboxyglutamic acid (Gla) domain linked to first epidermal growth factor-like domain, and by a Gla domain peptide (residues 1-41), over a range of concentration of 10(-9) to 10(-7) M. The antigenic site recognized by the monoclonal antibody C6, which was generated upon the high-affinity binding of calcium ions, was located in the Gla domain. The C6 antibody inhibited the activation of factor X and the amidolytic activity of factor VIIa in the presence of tissue factor. These results demonstrate that a high-affinity calcium-binding site(s) is located in the Gla domain of factor VII, which is concerned with the initiation of tissue factor-mediated blood coagulation by factor VIIa.

Comparison of kinetic property between human seminal and renal gamma-glutamyltransferase.

The initial-velocity kinetics, optimal pH, acceptor specificity and the influence of metal ions, EDTA and urea were studied on the human seminal gamma-glytamyltransferase (GGT) in comparison with the human renal GGT. The activity was measured with glycylglycine as an acceptor and with gamma-glutamyl-4-nitroanilide or gamma-glutamyl-3-carboxy-4-nitroanilide as a donor. Because the double-reciprocal plots showed paralled lines, the reaction of seminal GGT proceeds in nonsequence (Ping Pong Bi Bi) mechanism. The acceptor Michaelis constants for the seminal GGT were about 2 times higher than those for the renal enzyme with gamma-glutamyl-3-carboxy-4-nitroanilide as well as gamma-glutamyl-4-nitroanilide as donors, which the donor michaelis constants for seminal GGT were very similar to those for renal enzyme. The optimal pH and pK values were 8.2-8.6 and about 7.7, respectively. There was little difference in the specificity for various acceptors between the seminal and renal enzyme. Glycylglycylglycine was an effective acceptor other than glycylglycine, showing 80% of the activity with glycylgycine. Various substrates including metal ions tested were practically neither inhibitory nor stimulatory for seminal and renal GGTs.

A practical synthesis of optically pure and fully protected L-gamma-carboxyglutamic acid derivatives and its application in peptide synthesis.

The optically active and fully protected gamma,gamma-di-t-butyl N-Fmoc-L-gamma-carboxyglutamate was synthesized from the relatively inexpensive D-serine. The overall yield of the synthesis was about 30%. Our studies review that, under TFA and various acidic conditions, L-Gla and its derivatives were stable with no decarboxylation. Finally, gamma,gamma-di-t-butyl N-Fmoc-L-gamma-carboxyglutamate was successfully used in peptide synthesis by Fmoc strategy on solid phase.

Gamma-glutamyl transferase ectoactivity in the intact rat liver: effect of chronic alcohol consumption.

The localization of gamma-glutamyl transferase (GGT) in the intact rat liver was studied by a new approach in which the chromogenic gamma-glutamyl donor substrate of GGT gamma-glutamyl-p-nitroanilide is perfused through the portal vein to yield p-nitroaniline, which is monitored spectrophotometrically. GGT activity was markedly increased by the gamma-glutamyl acceptors glycyl-glycine, cystine and methionine, following Michaelis-Menten kinetics. Infusion of glutathione (GSH), the natural substrate of GGT, was shown to markedly reduce or to abolish the formation of p-nitroaniline without entering the liver cells, indicating the existence of a GGT ectoactivity accessible to the sinusoidal circulation. This ectoenzyme was shown to remove significant amounts of GSH from the circulation, amounting, in the naive rat, to 20-25% of the net rate at which GSH is contributed by the liver into the circulation. Chronic alcohol consumption is known to increase hepatic GGT activity, although the biological significance of such an effect remains unknown. Present studies show that chronic administration of alcohol to rats leads to a significant (40-75%) increase in hepatic GGT ectoactivity. GGT ectoactivity significantly correlates with total liver GGT, both in control and alcohol-treated animals (r = .76 and r = .90, respectively). Livers of alcohol-fed rats showed an increased (80-110%) capacity to remove circulating GSH which strongly correlated with total liver GGT (r = .96; p less than 0.0001).(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of gamma-carboxyglutamic acid by reverse phase HPLC of its phenylthiocarbamyl derivative.

A method is presented for analysis of gamma-carboxyglutamic acid based on its derivatization with phenylisothiocyanate and reverse phase HPLC analysis of the resulting phenylthiocarbamyl derivative. Proteins were hydrolyzed with sodium hydroxide and the hydrolysates were desalted on Dowex 50 eluted with ammonium hydroxide. The resulting amino acid mixtures were derivatized with phenylisothiocyanate and the phenylthiocarbamyl derivatives were separated under isocratic conditions on either C18 or C8 reverse phase columns using 0.14 M Tris, 0.05% triethylamine, titrated to pH 7.5 with glacial acetic acid, plus 2% acetonitrile, and detected by absorbance at 254 nm. The method is linear over the range from 10 to 1000 pmol of gamma-carboxyglutamic acid and the limit of detection is near 2 pmol. The utility of the method was verified for analysis of purified prothrombin yielding a value of 10.3 mol of gamma-carboxyglutamic acid per mole in agreement with sequence data. No gamma-carboxyglutamate was detectable for acid-hydrolyzed samples of prothrombin, nor in acid- or base-hydrolyzed samples of bovine serum albumin. Application of this method failed to corroborate the reported presence of gamma-carboxyglutamate in a putative mitochondrial gamma-carboxyglutamate-containing calcium-binding protein. The method was also tested for determination of beta-carboxyaspartate, beta-hydroxyaspartate, phosphoserine, phosphothreonine, and phosphotyrosine in an attempt to identify an unknown material which appeared in preparations of the mitochondrial protein.

Behaviour of L-gamma-glutamyl-4-nitroanilide and L-gamma-glutamyl-3-carboxy-4-nitroanilide with respect to gamma-glutamyltransferases of different origin.

In this paper we compare the measurement of catalytic activity concentrations of gamma-glutamyltransferase with the non-carboxylated and the carboxylated substrate in preparations of different origin. Fresh human sera, commercial test sera and preparations of gamma-glutamyltransferase purified from human liver, porcine kidney and bovine kidney were used as sample materials. When assayed with both substrates preparations of gamma-glutamyltransferase from bovine kidney behaved in a different manner as did the enzyme in preparations from human liver or porcine kidney and the enzyme in fresh human sera. On account of the results obtained with both substrates we classified the commercial test sera for their enrichment using multi-inductive component analysis. The differences observed for the various methods of determination seem to have significance in quality control.

Presence of osteocalcin and related higher molecular weight 4-carboxyglutamic acid-containing proteins in developing bone.

Development of a sensitive radioimmunoassay for the vitamin K-dependent bone protein osteocalcin in avian species has provided new information on the biosynthesis of this protein in bone. Chicken osteocalcin shares many structural features, including the sequence positions of its 3 gamma-carboxyglutamic acid (Gla) residues, with osteocalcins of human, monkey, cow, and rat, but is cryptic in the radioimmunoassays for these species. In the chicken assay system, the intact 50-residue (Mr = 5670) protein is required for immunoreactivity. Reduction and alkylation of the disulfide bond (Cys 23-Cys 29) or tryptic removal of the COOH-terminal pentapeptide abolish antibody binding activity. Decarboxylation of the 3 Gla residues enhances the affinity for antibody by 1.5- to 2-fold. Osteocalcin appears coincident with the very earliest detectable perichondral mineralization in developing long bone (tibiotarsus) of the 7- to 8-day-old chick embryo (stages 31-33). However, amino acid analysis demonstrates an excess of Gla in embryonic bone compared to the level of osteocalcin by radioimmunoassay. Two independent experimental approaches have partially resolved this paradox. First, extraction and gel filtration in 4 M guanidine hydrochloride of total bone proteins has revealed high molecular weight species which share antigenic determinants with osteocalcin, namely, 10,000 (+/- 1,000), 15,000 (+/- 2,000), 35,000 (+/- 5,000), and 85,000 (+/- 15,000), in addition to 5,670 osteocalcin. Second, a selective 3H exchange labeling procedure for Gla residues has revealed Gla-containing proteins in bone in the same molecular weight classes. One or more of these may represent precursors in the biosynthetic pathway for osteocalcin.

Measurement of plasma gamma-glutamyltransferase in clinical chemistry: kinetic basis and standardisation propositions.

The conditions for the measurement of gamma-glutamyltransferase (EC 2.3.2.2) activity of human plasma were studied at 30 degrees C using the kinetic technique of Szasz [3]. The optimum pH in Tris (hydroxymethylaminomethane) buffer and 2-amino-2-methyl-1.3-propanediol at a concentration of 100 mmol/1 are 8.0 and 8.1. The kinetic characteristics of human plasma gamma-glutamyltransferase were studied using gamma-L-glutamyl p-nitroanilide and its carboxyl derivative as donor substrates. Glycylglycine was chosen as the best acceptor of the gamma-glutamyl radical. Under these conditions, we have shown that the inhibition by the donor substrate was more important at acidic pH and vanished at alkaline pH. This inhibition was obviously related to the presence of the acceptor, but did not vary with glycylglycine concentration. At pH 8.0, by increasing the acceptor concentration some competition occurs at the donor binding site, as reported by other authors in relation to the known ping-pong bi-bi enzyme mechanism for the gamma-glutamyltransferase. Some displacement of donor substrate by increasing amounts of acceptor substrate could be observed at all pH values we studied. However, the influence of glycylglycine on the enzyme's maximum velocity and affinity for the donor substrate was also pH dependent. Studying the kinetic characteristics of the enzyme as a function of the pH suggests that the enzyme works with more than one active site at pH 7.5-8.0. Based on the results of this study, we propose measurement conditions for gamma-glutamyltransferase at 30 degrees C in routine clinical chemistry without preference in the choice of substrate.

Some kinetic properties of gamma-glutamyltransferase from rabbit liver.

gamma-Glutamyltransferase ((5-glutamyl)-peptide: amino-acid 5-glutamyltransferase, EC 2.3.2.2) of rabbit liver (detergent form) was purified 1100-fold in order to study its kinetic properties. Kinetic studies were conducted from pH 6.0 to 12.0 in the absence and presence of the acceptor substrate glycylglycine using gamma-glutamyl-3-carboxy-4-nitroanilide as the donor. The existence of more than one binding site for both donor and acceptor is postulated on kinetic evidence such as donor substrate activation, donor substrate inhibition and acceptor substrate activation. Homotropic interaction is also observed, in the form of negative cooperativity, in donor substrate binding, in the absence of acceptor at pH less than 9.0 and positive cooperativity (n = 2), in the absence or presence of acceptor at pH greater than 9.0. Hydrolase reaction reaches a maximum of activity at pH 10 (pK 8.6). Transferase activity under conditions of maximal velocity is maximal at pH 9.0 (pK 7.1). The ratio of transferase activity/hydrolase activity is maximal at pH 7.0-7.5. At low donor substrate concentrations, maximal activity is attained at pH 7.5.

Kinetic properties of gamma-glutamyltransferase from human liver.

We studied the kinetic properties of purified "heavy" form of human liver gamma-glutamyltransferase (EC 2.3.2.2) in the presence and absence of the acceptor substrate glycylglycine under Vmax conditions and as a function of pH. gamma-Glutamyl carboxynitroanilide was used as the donor substrate. Our data suggest that hydrolysis of donor substrate is the major pathway in the absence of acceptor. Autotransfer, if it occurs, is not important. Hydrolysis and transfer reactions have different pH profiles both for Vmax and Km. The pH dependency of Vmax and Km for both the hydrolase and the transferase reactions most probably reflects a change in the rate-limiting step: deacylation of the enzyme at acidic pH and acylation at alkaline pH. Negative cooperativity, observed for both donor and acceptor substrates near neutral pH, is interpreted in terms of more than one active site per dimer for each substrate.

Synthesis of gamma-carboxyglutamic acid and its derivatives via Mannich-base condensation.

This report describes the synthesis of N alpha-formyl and N alpha-benzyloxycarbonyl-gamma, gamma-di-t-butyl-gamma-carboxyglutamic acid via Mannich-base condensation starting from di-t-butyl malonate and diethylformamido or benzyloxycarbonylamidomalonate.

Structure of a Gla-containing dipeptide. The crystal structure of (+/-)-N-carbobenzoxy-(gamma,gamma'-DI-tertbutyl)-gamma-carboxyglutamylglycine ethyl ester.

The crystal and molecular structure of a dipeptide containing a blocked gamma-carboxyglutamyl (Gla) residue is presented. Two intermolecular hydrogen bonds link the amides with carbonyl groups in the dipeptide backbone, but the protected gamma-carboxy groups on the modified glutamic acid are not hydrogen bonded.

Automated colorimetric method for the determination of serum gamma-glutamyl transpeptidase utilizing the Technicon SMAC alkaline phosphatase flow channel.

An automated procedure for the determination of serum gamma-glutamyltranspeptidase using Technicon SMAC alkaline phosphatase flow channel has been developed. The flow variants are insignificant. We use L-gamma-glutamyl-3-carboxy-4-nitroanilide as a very soluble substrate. With the dyalisis procedure, hemoglobin and bilirubin do not interfere. By employing dialysis, the need of blank corrections is therefore eliminated. The procedure can been run at 130 determinations per hour with insignificant carryover and satisfactory precision.

Gamma-Glutamyl-3-carboxy-14-nitroanilide: the substrate of choice for routine determinations of gamma-glutamyl-transferase activity in serum?

Gamma-Glutamyl-3-carboxy-4-nitroanilide has been tested as donor substrate in the assay of gamma-glutamyltransferase activity in serum, glycylglycine being used as acceptor substrate. This donor substrate is highly solube even in neutral solutions, in contrast to the commonly used gamma-glutamyl-4-nitroanilide. The enzyme which apparently acts accordingly to a ping-pong bi bi kinetic mechanism, shows an absolute KM value for gamma-glutamyl-3-carboxy-4-nitroanilide of about 0.64 mmol/l, and for glycylglycine of about 13.4 mmol/l. The former KM value is significantly lower than that previously found for gamma-glutamyl-4-nitroanilide. The carboxyl derivative exhibits a marked competitive inhibitory effect on the gamma-glutamyltransferase. This effect is more pronounced than that of gamma-glutamyl-4-nitroanilide. The carboxyl derivative has somewhat higher absorbance in the range of wave length (400-420 nm) used to monitor the formation of the product. It is concluded that as donor substrate in the assay of gamma-glutamyltransferase activity of serum, the new derivative is not substantially superior to the gamma-glutamyl-4-nitroanilide conventionally used.