adenosine-5--o-(3-thiotriphosphate) and thymidine-5--triphosphate

Signal transduction via P2 purinergic receptors was investigated in HSG cells, a continuous cell line originally derived from an irradiated human salivary gland. Ligand specificity for nucleotide receptors in HSG cells was investigated with various nucleotides and their analogues. Inositol 1,4,5-trisphosphate (IP3) production was significantly increased by ATP, UTP and ATP gamma S. The ligand specificity of this effect agreed well with that of the P2U purinergic receptor. On the other hand, 45Ca2+ influx was stimulated by ATP, UTP > ATP gamma S, ADP, UDP > ADP beta S > AMPPNP, GTP, TTP > CTP, GDP, TDP, AMPPCP, AMPCPP. This ligand specificity of 45Ca2+ influx was much broader than IP3 production. Also pertussis and cholera toxin had no effect on both IP3 production and 45Ca2+ influx by ATP or UTP. 3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (Bz-ATP) stimulates 45Ca2+ influx more effectively than IP3 formation. A 53-kDa membrane protein was photolabelled with [alpha-32P]Bz-ATP. This 53-kDa protein is a putative P2 purinergic receptor. In particular, the labelling was inhibited by a ligand profile that corresponded to that for 45Ca2+ influx. These findings suggest that nucleotides stimulate 45Ca2+ influx and IP3 formation by separate pathways via pertussis and cholera toxin-insensitive G proteins. Thus, in HSG cells, IP3 formation is coupled to the P2U subclass, while 45Ca2+ influx is coupled to another subclass, such as P2X, that regulates calcium channels.

Topics: Adenine Nucleotides; Adenosine Diphosphate; Adenosine Triphosphate; Adenylyl Imidodiphosphate; Affinity Labels; Calcium; Calcium Channels; Calcium Radioisotopes; Cell Line; Cholera Toxin; Cytidine Triphosphate; GTP-Binding Proteins; Guanosine Diphosphate; Humans; Inositol 1,4,5-Trisphosphate; Ligands; Membrane Proteins; Pertussis Toxin; Radiopharmaceuticals; Receptors, Purinergic; Salivary Ducts; Signal Transduction; Submandibular Gland; Substrate Specificity; Thionucleotides; Thymine Nucleotides; Uridine Triphosphate; Virulence Factors, Bordetella

The physical basis of ATP binding and activation of DNA polymerase III holoenzyme was studied by an ultraviolet irradiation cross-linking technique. ATP and dATP were photocrosslinked to the alpha, tau, gamma, and delta subunits of holoenzyme; photocrosslinking of dATP was competitively inhibited by ATP. No photocrosslinking was observed with GTP or CTP, nor did GTP, CTP, or UTP inhibit cross-linking of ATP. ADP and adenosine 5'-O-(3-thio)-triphosphate, both potent inhibitors of ATP activation of holoenzyme, inhibited cross-linking of ATP to tau, gamma, and delta subunits, but not to the alpha subunit, suggesting that one or more of these subunits are ATP (or dATP)-binding sites. Photocrosslinking of dTTP to the ATP-activated holoenzyme was exclusively to the epsilon subunit, the dnaQ ( mutD ) gene product; dCTP and dGTP were not photocrosslinked to any subunit. Binding of dTTP was enhanced by ATP, but by no other nucleotide (or deoxynucleotide). This binding of dTTP to epsilon, a subunit likely responsible for regulation of proofreading by the holoenzyme, may function in the control of the fidelity of replication.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Affinity Labels; Binding Sites; Deoxyadenine Nucleotides; DNA Polymerase III; DNA-Directed DNA Polymerase; Escherichia coli; Photochemistry; Thionucleotides; Thymine Nucleotides; Ultraviolet Rays