A physiological role of the adenosine A3 receptor: Sustained cardioprotection

Adenosine released during cardiac ischemia exerts a potent, protective effect in the heart. A newly recognized adenosine receptor, the A3 subtype, is expressed on the cardiac ventricular cell, and its activation protects the ventricular heart cell against injury during a subsequent exposure to ischemia. A cultured chicken ventricular myocyte model was used to investigate the cardioprotective role of a novel adenosine A3 receptor. The protection mediated by prior activation of A3 receptors exhibits a significantly longer duration than that produced by activation of the adenosine A1 receptor. Prior exposure of the myocytes to brief ischemia also protected them against injury sustained during a subsequent exposure to prolonged ischemia. The adenosine A3 receptor-selective antagonist 3-ethyl 5-benzyl-2-methyl-6-phenyl-4-phenylethynyl-1,4-(±)-dihydropyridine-3,5-dicarboxylate (MRS1191) caused a biphasic inhibition of the protective effect of the brief ischemia. The concomitant presence of the A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) converted the MRS1191-induced dose inhibition curve to a monophasic one. The combined presence of both antagonists abolished the protective effect induced by the brief ischemia. Thus, activation of both A1 and A3 receptors is required to mediate the cardioprotective effect of the brief ischemia. Cardiac atrial cells lack native A3 receptors and exhibit a shorter duration of cardioprotection than do ventricular cells. Transfection of atrial cells with cDNA encoding the human adenosine A3 receptor causes a sustained A3 agonist-mediated cardioprotection. The study indicates that cardiac adenosine A3 receptor mediates a sustained cardioprotective function and represents a new cardiac therapeutic target.

A link between protein structure and enzyme catalyzed hydrogen tunneling

We present evidence that the size of an active site side chain may modulate the degree of hydrogen tunneling in an enzyme-catalyzed reaction. Primary and secondary kH/kT and kD/kT kinetic isotope effects have been measured for the oxidation of benzyl alcohol catalyzed by horse liver alcohol dehydrogenase at 25°C. As reported in earlier studies, the relationship between secondary kH/kT and kD/kT isotope effects provides a sensitive probe for deviations from classical behavior. In the present work, catalytic efficiency and the extent of hydrogen tunneling have been correlated for the alcohol dehydrogenase-catalyzed hydride transfer among a group of site-directed mutants at position 203. Val-203 interacts with the opposite face of the cofactor NAD+ from the alcohol substrate. The reduction in size of this residue is correlated with diminished tunneling and a two orders of magnitude decrease in catalytic efficiency. Comparison of the x-ray crystal structures of a ternary complex of a high-tunneling (Phe-93 → Trp) and a low-tunneling (Val-203 → Ala) mutant provides a structural basis for the observed effects, demonstrating an increase in the hydrogen transfer distance for the low-tunneling mutant. The Val-203 → Ala ternary complex crystal structure also shows a hyperclosed interdomain geometry relative to the wild-type and the Phe-93 → Trp mutant ternary complex structures. This demonstrates a flexibility in interdomain movement that could potentially narrow the distance between the donor and acceptor carbons in the native enzyme and may enhance the role of tunneling in the hydride transfer reaction.

Human recombinant soluble guanylyl cyclase: Expression, purification, and regulation

The α1- and β1-subunits of human soluble guanylate cyclase (sGC) were coexpressed in the Sf9 cells/baculovirus system. In addition to the native enzyme, constructs with hexahistidine tag at the amino and carboxyl termini of each subunit were coexpressed. This permitted the rapid and efficient purification of active recombinant enzyme on a nickel-affinity column. The enzyme has one heme per heterodimer and was readily activated with the NO donor sodium nitroprusside or 3-(5′-hydroxymethyl-2′furyl)-1-benzyl-indazole (YC-1). Sodium nitroprusside and YC-1 treatment potentiated each other in combination and demonstrated a remarkable 2,200-fold stimulation of the human recombinant sGC. The effects were inhibited with 1H-(1,2,4)oxadiazole(4,3-a)quinoxalin-1one (ODQ). The kinetics of the recombinant enzyme with respect to GTP was examined. The products of the reaction, cGMP and pyrophosphate, inhibited the enzyme. The extent of inhibition by cGMP depended on the activation state of the enzyme, whereas inhibition by pyrophosphate was not affected by the enzyme state. Both reaction products displayed independent binding and cooperativity with respect to enzyme inhibition. The expression of large quantities of active enzyme will facilitate structural characterization of the protein.

Purification and Characterization of a NADPH-Dependent Aldehyde Reductase from Mung Bean That Detoxifies Eutypine, a Toxin from Eutypa lata1

Eutypine (4-hydroxy-3-[3-methyl-3-butene-1-ynyl] benzaldehyde) is a toxin produced by Eutypa lata, the causal agent of eutypa dieback in the grapevine (Vitis vinifera). Eutypine is enzymatically converted by numerous plant tissues into eutypinol (4-hydroxy-3-[3-methyl-3-butene-1-ynyl] benzyl alcohol), a metabolite that is nontoxic to grapevine. We report a four-step procedure for the purification to apparent electrophoretic homogeneity of a eutypine-reducing enzyme (ERE) from etiolated mung bean (Vigna radiata) hypocotyls. The purified protein is a monomer of 36 kD, uses NADPH as a cofactor, and exhibits a Km value of 6.3 μm for eutypine and a high affinity for 3- and 4-nitro-benzaldehyde. The enzyme failed to catalyze the reverse reaction using eutypinol as a substrate. ERE detoxifies eutypine efficiently over a pH range from 6.2 to 7.5. These data strongly suggest that ERE is an aldehyde reductase that could probably be classified into the aldo-keto reductase superfamily. We discuss the possible role of this enzyme in eutypine detoxification.

Intermediates of Salicylic Acid Biosynthesis in Tobacco1

Salicylic acid (SA) is an important component of systemic-acquired resistance in plants. It is synthesized from benzoic acid (BA) as part of the phenylpropanoid pathway. Benzaldehyde (BD), a potential intermediate of this pathway, was found in healthy and tobacco mosaic virus (TMV)-inoculated tobacco (Nicotiana tabacum L. cv Xanthi-nc) leaf tissue at 100 ng/g fresh weight concentrations as measured by gas chromatography-mass spectrometry. BD was also emitted as a volatile organic compound from tobacco tissues. Application of gaseous BD to plants enclosed in jars caused a 13-fold increase in SA concentration, induced the accumulation of the pathogenesis-related transcript PR-1, and increased the resistance of tobacco to TMV inoculation. [13C6]BD and [2H5]benzyl alcohol were converted to BA and SA. Labeling experiments using [13C1]Phe in temperature-shifted plants inoculated with the TMV showed high enrichment of cinnamic acids (72%), BA (34%), and SA (55%). The endogenous BD, however, contained nondetectable enrichment, suggesting that BD was not the intermediate between cinnamic acid and BA. These results show that BD and benzyl alcohol promote SA accumulation and expression of defense responses in tobacco, and provide insight into the early steps of SA biosynthesis.