Abstract

Our major aim is to elucidate the structure and function of the Escherichia coli coupling factor, F1-ATPase. We will complete purification of a large collection of monoclonal antibodies, directed against the Alpha, Beta, Gamma, Delta, and Epsilon subunits. They will be used to define peptide domains of interest, and to locate exposed parts of F1-ATPase, both for the soluble and the membrane-bound enzyme. We plan to study the assembly of F1-ATPase and to look for evidence of regulation. In other studies, external ATP was seen to cause entry of Na+, exit of K+ and hydrolysis of 1/3 of intracellular ATP in transformed mouse cells. Following this, they became permeable to nucleotides and phosphate esters. We have evidence that Ca++ is involved and plan to use ion selective electrodes and Ca++ indicators such as """"""""quin-2"""""""". Effects of calmodulin on ATP permeabilization will be further examined and mutant studies are underway. Similarities and differences between ATP and ionophore permeabilization will be explored and we aim to get membrane vesicles sensitive to external ATP.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK011789-22
Application #
3224821
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1979-01-01
Project End
1990-09-30
Budget Start
1989-07-01
Budget End
1990-09-30
Support Year
22
Fiscal Year
1989
Total Cost
Indirect Cost

  Institution

Name
Cornell University
Department
Type
Schools of Arts and Sciences
DUNS #
City
Ithaca
State
NY
Country
United States
Zip Code
14850

  Publications

Huang, N N; Wang, D J; Heppel, L A (1993) Stimulation of aged human lung fibroblasts by extracellular ATP via suppression of arachidonate metabolism. J Biol Chem 268:10789-95
Wang, D J; Huang, N N; Gonzalez, F A et al. (1991) Multiple signal transduction pathways lead to extracellular ATP-stimulated mitogenesis in mammalian cells: I. Involvement of protein kinase C-dependent and -independent pathways. J Cell Physiol 146:473-82
Huang, N N; Wang, D J; Gonzalez, F et al. (1991) Multiple signal transduction pathways lead to extracellular ATP-stimulated mitogenesis in mammalian cells: II. A pathway involving arachidonic acid release, prostaglandin synthesis, and cyclic AMP accumulation. J Cell Physiol 146:483-94
Wang, D J; Huang, N N; Heppel, L A (1990) Extracellular ATP shows synergistic enhancement of DNA synthesis when combined with agents that are active in wound healing or as neurotransmitters. Biochem Biophys Res Commun 166:251-8
Gonzalez, F A; Wang, D J; Huang, N N et al. (1990) Activation of early events of the mitogenic response by a P2Y purinoceptor with covalently bound 3'-O-(4-benzoyl)-benzoyladenosine 5'-triphosphate. Proc Natl Acad Sci U S A 87:9717-21
Gonzalez, F A; Alfonzo, R G; Toro, J R et al. (1989) Receptor specific for certain nucleotides stimulates inositol phosphate metabolism and Ca2+ fluxes in A431 cells. J Cell Physiol 141:606-17
Gonzalez, F A; Bonapace, E; Belzer, I et al. (1989) Two distinct receptors for ATP can be distinguished in Swiss 3T6 mouse fibroblasts by their desensitization. Biochem Biophys Res Commun 164:706-13
Huang, N; Wang, D J; Heppel, L A (1989) Extracellular ATP is a mitogen for 3T3, 3T6, and A431 cells and acts synergistically with other growth factors. Proc Natl Acad Sci U S A 86:7904-8
Gonzalez, F A; Rozengurt, E; Heppel, L A (1989) Extracellular ATP induces the release of calcium from intracellular stores without the activation of protein kinase C in Swiss 3T6 mouse fibroblasts. Proc Natl Acad Sci U S A 86:4530-4
Gonzalez, F A; Gross, D J; Heppel, L A et al. (1988) Studies on the increase in cytosolic free calcium induced by epidermal growth factor, serum, and nucleotides in individual A431 cells. J Cell Physiol 135:269-76

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