The general goal of this research is to elucidate mechanisms of inorganic cation transport across mitochondrial membranes. Specifically experiments will be aimed at identifying and characterizing mitochondrial K+ and MG++ transport mechanisms. Although the proposed investigations would constitute fundamental research, elucidation of mechanisms by which mitochondrial membranes control and maintain the distribution of inorganic cations between cytoplasmic and mitochondrial matrix compartments should aid in understanding deviations from steady state ion distributions which may occur in disease states. Physiological measurements have indicated that the mechanisms which mediate trans[port of K+ and Mg++ into liver mitochondria are similar in their substrate dependence, pH dependence, and sensitivity to inhibitory and stimulatory reagents. Cross competition is also observed. A protein of approximately 53 kDa has been isolated from rat liver mitochondrial membranes by affinity chromatography using immobilized quinine, an inhibitor of mitochondrial k+ and mg++ transport. Experiments will be aimed at determining whether this protein has a role in mediating K+ transport, and whether it also transports Mg++. Attempts will be made to improve the purity and yield of the 53 kDa protein. It will be reconstituted into phospholipid vesicles and planar lipid membranes, and its transport capabilities will be examined. Preliminary results indicate that the reconstituted protein or reagents known to affect K+ uniporter. Effects on transport by the reconstituted protein of reagents known to affect K+ uniporter. Effects on transport by the reconstituted protein or reagents known to affect K+ and Mg++ flux into intact mitochondria will be studied. Polyclonal antibodies prepared against the 53 kDa protein will be used to identify this protein and any related polypeptides on SDS polyacrylamide gels of mitochondrial membrane extracts. Effects of the antibodies on 42K and 28Mg flux into mitoplasts and on activities of the reconstituted 53 kDa protein will be tested.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM020726-12
Application #
3270136
Study Section
Physical Biochemistry Study Section (PB)
Project Start
1977-01-01
Project End
1991-11-30
Budget Start
1989-12-01
Budget End
1990-11-30
Support Year
12
Fiscal Year
1990
Total Cost
Indirect Cost
Name
Rensselaer Polytechnic Institute
Department
Type
Schools of Arts and Sciences
DUNS #
002430742
City
Troy
State
NY
Country
United States
Zip Code
12180
Paliwal, R; Costa, G; Diwan, J J (1992) Purification and patch clamp analysis of a 40-pS channel from rat liver mitochondria. Biochemistry 31:2223-9
Costa, G; Kinnally, K W; Diwan, J J (1991) Patch clamp analysis of a partially purified ion channel from rat liver mitochondria. Biochem Biophys Res Commun 175:305-10
Diwan, J J; Paliwal, R; Kaftan, E et al. (1990) A mitochondrial protein fraction catalyzing transport of the K+ analog T1+. FEBS Lett 273:215-8
Diwan, J J; Moore, C (1990) Effects of some protein-reactive compounds on K+ flux into mitochondria. Biochem Int 22:843-8
Diwan, J J; Haley, T; Sanadi, D R (1988) Reconstitution of transmembrane K+ transport with a 53 kilodalton mitochondrial protein. Biochem Biophys Res Commun 153:224-30
Diwan, J J; Yune, H H; Bawa, R et al. (1988) Enhanced uptake of spermidine and methylglyoxal-bis(guanylhydrazone) by rat liver mitochondria following outer membrane lysis. Biochem Pharmacol 37:957-61
Diwan, J J; Haley, T; Moore, C (1988) Sensitivity of mitochondrial Mg++ flux to reagents which affect K+ flux. J Bioenerg Biomembr 20:261-71
Diwan, J J (1986) Effect of quinine on mitochondrial K+ and Mg++ flux. Biochem Biophys Res Commun 135:830-6
Diwan, J J; Srivastava, J; Moore, C et al. (1986) Stimulation of K+ flux into mitochondria by phenylarsine oxide. J Bioenerg Biomembr 18:123-34
Diwan, J J (1985) Ba2+ uptake and the inhibition by Ba2+ of K+ flux into rat liver mitochondria. J Membr Biol 84:165-71