Impact of erythrocyte Duffy antigen genetic polymorphism on the distribution of GroBeta-T, a novel human CXC chemokine.

PURPOSE: Grobeta-T, a human CXC chemokine, has been studied for its potential to mobilize stem cells. Chemokines bind specifically to receptors on target immune cells but also to a homologous erythrocyte blood group antigen, the Duffy Antigen/Receptor for Chemokines (DARC)that is subject to genetic polymorphism in humans.A mutation in the DARC gene is common among African Americans and results in lack of expression of the erythrocyte antigen. We used a combination of in vitro studies of Grobeta-DARC interaction and pharmacokinetic simulation to anticipate the potential impact of this polymorphism on the pharmacokinetics of Grobeta-T. METHODS: [125I]Grobeta-T was incubated in Caucasian blood to characterize the concentration dependence of the blood to plasma concentration ratio (B/P). Affinity and capacity of binding was estimated by Scatchard analysis; specificity was investigated by competitive displacement with a CC chemokine. The B/P value (7 nM) was then determined in blood from 8 African American subjects. Duffy antigen expression was determined by antibody agglutination. A pharmacokinetic model was developed which accounted for blood-cell binding. Simulations were performed to explore effects of dose regimen and DARC expression on the Grobeta-T plasma concentration-time profile. RESULTS: Grobeta-T affinity and capacity for DARC (Caucasian blood)were 23.0 +/- 1.2 and 37.7 +/- 0.6 nM, respectively;excess CC chemokine fully displaced [125I]Grobeta-T. Chemokine binding was highly correlated with the presence or absence of the Duffy antigen (p<0.01) in African American blood; the proportion of subjects for which binding was observed (3/8), was consistent with the reported frequency of DARC expression in this population. Counter to intuition,in the terminal disposition phase at low doses,concentrations of free Grobeta-T in the presence of DARC may be substantially higher than in the absence of DARC. CONCLUSION: Dissociation from the erythrocyte antigen may lead to greater persistence, at low doses, of free Grobetabeta-T in the blood of individuals expressing the chemokine sink.

Isotope and elemental effects indicate a rate-limiting methyl transfer as the initial step in the reaction catalyzed by Escherichia coli cyclopropane fatty acid synthase.

Cyclopropane fatty acid (CFA) synthases catalyze the formation of cyclopropane rings on unsaturated fatty acids (UFAs) that are natural components of membrane phospholipids. The methylene carbon of the cyclopropane ring derives from the activated methyl group of S-adenosyl-L-methionine (AdoMet), affording S-adenosyl-L-homocysteine (AdoHcys) and a proton as the remaining products. This reaction is unique among AdoMet-dependent enzymes, because the olefin of the UFA substrate is isolated and unactivated toward nucleophilic or electrophilic addition, raising the question as to the timing and mechanism of proton loss from the activated methyl group of AdoMet. Two distinct reaction schemes have been proposed for this transformation; however, neither was based on detailed in vitro mechanistic analysis of the enzyme. In the preceding paper [Iwig, D. F. and Booker, S. J. (2004) Biochemistry 43, http://dx.doi.org/10.1021/bi048693+], we described the synthesis of two analogues of AdoMet, Se-adenosyl-L-selenomethionine (SeAdoMet) and Te-adenosyl-L-telluromethionine (TeAdoMet), and their intrinsic reactivity toward polar chemistry in which AdoMet is known to be involved. We found that the electrophilicity of AdoMet and its onium congeners followed the series SeAdoMet > AdoMet > TeAdoMet, while the acidity of the carbons adjacent to the relevant heteroatom followed the series AdoMet > SeAdoMet > TeAdoMet. When each of these compounds was used as the methylene donor in the CFA synthase reaction, the kinetic parameters of the reaction, k(cat) and k(cat) K(M)(-1), followed the series SeAdoMet > AdoMet > TeAdoMet, suggesting that the reaction takes place via methyl transfer followed by proton loss, rather than by processes that are initiated by proton abstraction from AdoMet. Use of S-adenosyl-L-[methyl-d(3)]methionine as the methylene donor resulted in an inverse isotope effect of 0.87 +/- 0.083, supporting this conclusion and also indicating that the methyl transfer takes place via a tight s(N)2 transition state.