A lubricious formulation exhibiting reduced thrombogenicity, cell proliferation, and protein adsorption.

The adhesion of human platelets, erythrocytes, and leukocytes, the adsorption of protein, and the proliferation of human umbilical vein endothelial cells (HUVEC) on the surface of electropolished stainless steel and the lumen of polyurethane tubing coated with Hydromer's lubricious Duality T8B formulation was evaluated. Following exposure to a platelet-enriched suspension from citrated human whole blood, stainless steel coated with this formulation exhibited significantly reduced adhesion of platelets, erythrocytes, and granulocytes. This reduction in adhesion was confirmed using an immunohistochemical method utilizing antibodies to CD41, CD235, and CD15, respectively. The proliferation of HUVEC cells were significantly reduced when cultured on coated stainless steel. This formulation was also able to significantly reduce the adsorption of plasma proteins and the major protein in tear fluid (lysozyme) to the surface of stainless steel. The nonthrombogenic properties of Duality T8B after application to the lumen of polyurethane tubing were also examined. Following a short-term (3 h) static exposure to citrated human whole blood, microscopic examination revealed that the adhesion of platelets and erythrocytes was reduced significantly, a finding confirmed using anti-CD41 and anti-CD235 antibodies in the immunohistochemical method. A long-term (12 day) study yielded essentially identical results indicating a significant reduction in the adhesion of blood components on the luminal surface of coated polyurethane tubing. In summary, these data indicate that the application of Duality T8B onto surfaces of medical devices, such as catheters, extracorporeal circuitry, and coronary stents, could aid in reducing or preventing not only thrombus formation but also the process of restenosis.

Metal ion activation of S-adenosylmethionine decarboxylase reflects cation charge density.

S-Adenosylmethionine decarboxylase from Escherichia coli is a pyruvoyl cofactor-containing enzyme that requires a metal cation for activity. We have found that the enzyme is activated by cations of varying charge and ionic radius, such as Li+, A13+, Tb3+, and Eu3+, as well as the divalent cations Mg2+, Mn2+, and Ca2+. All of the activating cations provide kcat values within 30-fold of one another, showing that the charge of the cation does not greatly influence the rate-limiting step for decarboxylase turnover. Cation concentrations for half-maximal activation decrease by >100-fold with each increment of increase in the cation charge, ranging from approximately 300 mM with Li+ to approximately 2 microM with trivalent lanthanide ions. The cation affinity is related to the charge/radius ratio of the ion for those ions with exchangeable first coordination sphere ligands. The exchange-inert cation Co(NH3)63+ activates in the presence of excess EDTA (and NH4+ does not activate), indicating that direct metal coordination to the protein or substrate is not required for activation. The binding of metal ions (monitored by changes in the protein tryptophan fluorescence) and enzyme activation are both cooperative with Hill coefficients as large as 4, the active site stoichiometry of this (alphabeta)4 enzyme. The Hill coefficients for Mg2+ binding and activation increase from 1 to approximately 4 as the KCl concentration increases, which is also observed with NaCl or KNO3; neither Na+ nor K+ activates the enzyme. The single tryptophan in the protein is located 16 residues from the carboxyl terminus of the pyruvoyl-containing alpha chain, in a 70-residue segment that is not present in metal ion independent AdoMet decarboxylases from other organisms. The results are consistent with allosteric metal ion activation of the enzyme, congruent with the role of the putrescine activator of the mammalian AdoMet decarboxylase.

Catalytic properties of the archaeal S-adenosylmethionine decarboxylase from Methanococcus jannaschii.

S-Adenosylmethionine decarboxylase (AdoMetDC) is a pyruvoyl cofactor-dependent enzyme that participates in polyamine biosynthesis. AdoMetDC from the Archaea Methanococcus jannaschii is a prototype for a recently discovered class that is not homologous to the eucaryotic enzymes or to a distinct group of microbial enzymes. M. jannaschii AdoMetDC has a Km of 95 microm and the turnover number (kcat) of 0.0075 s(-1) at pH 7.5 and 22 degrees C. The turnover number increased approximately 38-fold at a more physiological temperature of 80 degrees C. AdoMetDC was inactivated by treatment with the imine reductant NaCNBH3 only in the presence of substrate. Mass spectrometry of the inactivated protein showed modification solely of the pyruvoyl-containing subunit, with a mass increase corresponding to reduction of a Schiff base adduct with decarboxylated AdoMet. The presteady state time course of the AdoMetDC reaction revealed a burst of product formation; thus, a step after CO2 formation is rate-limiting in turnover. Comparable D2O kinetic isotope effects of were seen on the first turnover (1.9) and on kcat/Km (1.6); there was not a significant D2O isotope effect on kcat, suggesting that product release is rate-limiting in turnover. The pH dependence of the steady state rate showed participation of acid and basic groups with pK values of 5.3 and 8.2 for kcat and 6.5 and 8.3 for kcat/Km, respectively. The competitive inhibitor methylglyoxal bis(guanylhydrazone) binds at a single site per (alphabeta) heterodimer. UV spectroscopic studies show that methylglyoxal bis(guanylhydrazone) binds as the dication with a 23 microm dissociation constant. Studies with substrate analogs show a high specificity for AdoMet.

Enzymatic properties of S-adenosylmethionine synthetase from the archaeon Methanococcus jannaschii.

S-Adenosylmethionine synthetase (ATP:l-methionine S-adenosyltransferase, MAT) catalyzes a unique enzymatic reaction that leads to formation of the primary biological alkylating agent. MAT from the hyperthermophilic archaeon Methanococcus jannaschii (MjMAT) is a prototype of the newly discovered archaeal class of MAT proteins that are nearly unrecognizable in sequence when compared with the class that encompasses both the eucaryal and bacterial enzymes. In this study the functional properties of purified recombinant MjMAT have been evaluated. The products of the reaction are AdoMet, PP(i), and P(i); >90% of the P(i) originates from the gamma-phosphoryl group of ATP. The circular dichroism spectrum of the dimeric MjMAT indicates that the secondary structure is more helical than the Escherichia coli counterpart (EcMAT), suggesting a different protein topology. The steady state kinetic mechanism is sequential, with random addition of ATP and methionine; AdoMet is the first product released, followed by release of PP(i) and P(i). The substrate specificity differs remarkably from the previously characterized MATs; the nucleotide binding site has a very broad tolerance of alterations in the adenosine moiety. MjMAT has activity at 70 degrees C comparable with that of EcMAT at 37 degrees C, consistent with the higher temperature habitat of M. jannaschii. The activation energy for AdoMet formation is larger than that for the E. coli MAT-catalyzed reaction, in accord with the notion that enzymes from thermophilic organisms are often more rigid than their mesophilic counterparts. The broad substrate tolerance of this enzyme proffers routes to preparation of novel AdoMet analogs.

Methylthioadenosine phosphorylase regulates ornithine decarboxylase by production of downstream metabolites.

The gene encoding methylthioadenosine phosphorylase (MTAP), the initial enzyme in the methionine salvage pathway, is deleted in a variety of human tumors and acts as a tumor suppressor gene in cell culture (Christopher, S. A., Diegelman, P., Porter, C. W., and Kruger, W. D. (2002) Cancer Res. 62, 6639-6644). Overexpression of the polyamine biosynthetic enzyme ornithine decarboxylase (ODC) is frequently observed in tumors and has been shown to be tumorigenic in vitro and in vivo. In this paper, we demonstrate a novel regulatory pathway in which the methionine salvage pathway products inhibit ODC activity. We show that in Saccharomyces cerevisiae the MEU1 gene encodes MTAP and that Meu1delta cells have an 8-fold increase in ODC activity, resulting in large elevations in polyamine pools. Mutations in putative salvage pathway genes downstream of MTAP also cause elevated ODC activity and elevated polyamines. The addition of the penultimate salvage pathway compound 4-methylthio-2-oxobutanoic acid represses ODC levels in both MTAP-deleted yeast and human tumor cell lines, indicating that 4-methylthio-2-oxobutanoic acid acts as a negative regulator of polyamine biosynthesis. Expression of MTAP in MTAP-deleted MCF-7 breast adenocarcinoma cells results in a significant reduction of ODC activity and reduction in polyamine levels. Taken together, our results show that products of the methionine salvage pathway regulate polyamine biosynthesis and suggest that MTAP deletion may lead to ODC activation in human tumors.