The adenosine 5'-triphosphatase (ATPase) isolated in the microsomal fraction from gastric mucosas is necessary and sufficient for acid secretion into the stomach. Hypersecretion is the immediate cause of ulcers. A new generation of drugs for the pharmacological management of the disease reduces acid secretion by inhibiting the gastric ATPase. Detailed knowledge of the enzyme's structure and mechanism were needed to understand how the first drugs in this class inactivated the enzyme and even more detailed information will be required to develop more effective agents with fewer side effects. The long term goal of this research is to explain how the gastric ATPase catalyzes ATP hydrolysis and couples the energy released to proton transport across the gastric epithelium. That means learning the structure of the enzyme and relating it to function. This proposal focuses on the role of protons in the catalytic mechanism, a peptide that may be a functional subunit of the enzyme, identification of functional amino acids, and conformational changes in the protein that may be the key to energy coupling and the physical transport of ions. A change in the distribution of oxygen isotopes in inorganic phosphate when it reacts with the enzyme in solutions of different acidity indicates that a weakly basic group on the enzyme functions in catalysis. Which step in the catalytic mechanism is affected by protonating this group will be assessed by measuring the rates of the individual steps in the reaction. Chemical modification of a peptide that has been isolated from gastric microsomes inactivates the ATPase. The peptide will be sequenced and the hypothesis that it forms a functional complex with the enzyme will be tested. Amino acids in the enzyme's active site will be identified by chemical modification with a substrate analogue, isolation of the labeled peptide, and microsequence analysis of its structure. Three conformational changes in the gastric enzyme have been observed with fluorescent reporter groups. The rates of these changes will be measured and compared with the rates of steps in the catalytic cycle to learn which changes in structure are functional.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK036873-06
Application #
3235431
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1986-09-29
Project End
1994-08-31
Budget Start
1991-09-01
Budget End
1992-08-31
Support Year
6
Fiscal Year
1991
Total Cost
Indirect Cost
Name
University of California Los Angeles
Department
Type
Schools of Medicine
DUNS #
119132785
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
Smirnova, I N; Lin, S H; Faller, L D (1995) An equivalent site mechanism for Na+ and K+ binding to sodium pump and control of the conformational change reported by fluorescein 5'-isothiocyanate modification. Biochemistry 34:8657-67
Smirnova, I N; Faller, L D (1995) Mechanism of the conformational change in sodium pump reported by eosin. Biochemistry 34:13159-69
Smirnova, I N; Faller, L D (1993) Mechanism of K+ interaction with fluorescein 5'-isothiocyanate-modified Na+,K(+)-ATPase. J Biol Chem 268:16120-3
Phan, B C; Faller, L D; Reisler, E (1993) Kinetic and equilibrium analysis of the interactions of actomyosin subfragment-1.ADP with beryllium fluoride. Biochemistry 32:7712-9
Lin, S H; Faller, L D (1993) Time resolution of fluorescence changes observed in titrations of fluorescein 5'-isothiocyanate-modified Na,K-ATPase with monovalent cations. Biochemistry 32:13917-24
Smirnova, I N; Faller, L D (1993) Role of Mg2+ ions in the conformational change reported by fluorescein 5'-isothiocyanate modification of Na+,K(+)-ATPase. Biochemistry 32:5967-77
Faller, L D; Diaz, R A; Scheiner-Bobis, G et al. (1991) Temperature dependence of the rates of conformational changes reported by fluorescein 5'-isothiocyanate modification of H+,K(+)- and Na+,K(+)-ATPases. Biochemistry 30:3503-10
Faller, L D (1990) Binding of the fluorescent substrate analogue 2',3'-O-(2,4,6-trinitrophenylcyclohexadienylidene)adenosine 5'-triphosphate to the gastric H+,K(+)-ATPase: evidence for cofactor-induced conformational changes in the enzyme. Biochemistry 29:3179-86
Faller, L D; Diaz, R A (1989) Evidence from 18O exchange measurements for steps involving a weak acid and a slow chemical transformation in the mechanism of phosphorylation of the gastric H+, K+-ATPase by inorganic phosphate. Biochemistry 28:6908-14
Faller, L D (1989) Competitive binding of ATP and the fluorescent substrate analogue 2',3'-O-(2,4,6-trinitrophenylcyclohexadienylidine) adenosine 5'-triphosphate to the gastric H+,K+-ATPase: evidence for two classes of nucleotide sites. Biochemistry 28:6771-8