Probing the nucleotide binding sites in T7 RNA polymerase using cibacron blue.

T7 RNA polymerase (T7 RNAP) is an enzyme that utilizes ribonucleotides to synthesize the nascent RNA chain in a template-dependent manner. In this work we have studied the interaction of T7 RNAP with cibacron blue, an anthraquinone monochlorotriazine dye, and its effect on the function of the enzyme. T7 RNAP binds to the dye in a bi-phasic manner. The first phase of the binding is characterized by a high affinity (Kd in the nanomolar range) and reversible inactivation of the enzyme. The second binding site is the common substrate binding site. The association of the dye with T7 RNAP is a good model to understand the physiological significance of a high affinity binding of the initiating nucleotide, GTP, earlier reported from our laboratory. The results will be discussed to understand the role of the high affinity GTP binding.

Hemodynamic characteristics of extracellular UTP in the perfused rat liver

Uridine 5'-triphosphate (UTP) is stored in the granules of cells such as platelets and is released into the extracellular space upon cell stimulation. Extracellular UTP is known to influence many biological processes. We investigated the hemodynamic effects of UTP on the perfused rat liver and characterized its receptors. Liver perfusions were performed in a recirculation system under constant pressure (28 cmH2O). The perfusion flow and oxygen consumption rate were measured at 30 second intervals. UTP decreased the perfusion flow and the oxygen consumption rate, dose-dependently. UTP-induced changes were transient and disappeared in about 10 minutes. Suramin (P2-purinergic antagonist, 100 uM) and indomethacin (cyclooxygenase inhibitor, 20 uM) blocked UTP-induced hemodynamic changes significantly. The effects of UTP were also inhibited when Kupffer cells were damaged with treatment of gadolinium chloride (10 mg/kg iv). L-NAME (1 mM), a potent inhibitor of nitric oxide synthase, markedly enhanced and prolonged the contractile response of UTP in the hepatic vessel. These results suggest that UTP acts mainly on suramin-sensitive UTP receptors on the Kupffer cell through prostanoid synthesis. The nitric oxide systems in the endothelium seem to counteract the vasoconstrictile action of UTP in the hepatic circulation.