Isolation and Characterization of a High Energy Iron-Sulfur Protein From Mammalian Cells

Author

Beth Feland, University of Tennessee, Knoxville

Date of Award

8-1973

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Nutrition

Major Professor

John T. Smith

Committee Members

Ada Marie Campbell, Jane R. Savage, Mary Rose Gram

Abstract

A sulfur-containing protein (S-protein) was proposed to be an intermediate in the sulfation of cellular constituents containing ester sulfate after it was observed that S-protein isolated from the particulate fraction of the cell was unaffected by avitaminosis E, the stress of malathion intoxication or the level of inorganic sulfate in the diet. The present investigation was undertaken in an attempt to identify the cellular role of the S-protein in sulfate utilization.

The role of the S-protein as a possible intermediate in the sulfation of ester sulfates was determined using both a natural sulfate acceptor, mucopolysaccharide, and an artificial sulfate acceptor, p-nitrophenol. The S-protein appeared to possess the energy required to raise inorganic sulfate to the level of ester sulfate when muco-polysaccharide was the acceptor, but not when the artificial acceptor, p-nitrophenol, was used.

Since the S-protein appeared to possess a high energy configuration, and in view of its particulate origin, further experiments were designed to determine the relationship of the high energy configuration to the oxidative reactions of the cell. Since other investigators had previously suggested that an Fe-S protein is involved in sulfhydryl-disulfide oxidoreductions in the energy conservation at Site I, the modified sulfhydryl-disulfide exchange reaction between the S-protein and 2-mercaptoethanol led to the assumption that the S-protein might be involved in energy conservation at Site I.

The evidence that the S-protein (Fe-S protein) was involved in energy conservation was strengthened by feeding rats 2,4 dinitrophenol, an uncoupler of oxidative phosphorylation, prior to isolation of Fe-S protein. Results of this experiment and an experiment determining the uptake of a test dose of ⁵⁹Fe⁺⁺ and ³⁵SO⁼₄ suggested that rats increased the synthesis of Fe-S protein to compensate for the decreased energy conservation that resulted from the injection of the uncoupler of oxidative phosphorylation.

The-relationship between the Fe-S protein and the formation of ATP was then investigated. When the Fe-S protein was incubated with inorganic phosphate and ADP there was an increase in the disappearance of phosphate which paralleled the amount of Fe-S protein in the incubation mixture. The actual esterification of inorganic phosphate with ADP was demonstrated using ³²PO≡₄ in the incubation medium and isolating pure ATP containing ³²P. Therefore, it is concluded that the Fe-S protein functioned to trap a portion of the energy of the respiratory chain by oxidatively binding inorganic sulfate, which it can then exchange for inorganic phosphate. The high energy phosphate could then be transferred to ADP to generate ATP.

Recommended Citation

Feland, Beth, "Isolation and Characterization of a High Energy Iron-Sulfur Protein From Mammalian Cells. " PhD diss., University of Tennessee, 1973.
https://trace.tennessee.edu/utk_graddiss/3802