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|Title:||Studies on the Degradation of Dihydrofolate Reductase and Other Intracellular Proteins by Mammalian Cells|
|Authors:||Fox, Elizabeth Bothwell Joan|
|Abstract:||<p>The mechanisms by which mammalian cells degrade intracellular proteins have been investigated. Radioactively labelled proteins of rat hepatoma tissue culture cells were incubated in cell-free systems in an attempt to identify the degrading processes. Although proteins in intact cells are degraded to their constituent amino acids, little acid-solube material was produced from the labelled proteins at neutral pH, even in cell homogenates which contained all the cellular components.</p> <p>Dihydrofolate reductase from a subline of the L1210 lymphoma was radioactively labelled and used as a model protein so that changes in a single protein could be investigated. The enzyme was inactivated during incubation with homegenates of the same cell line. Little inactivation was produced by the 100,000 x g supernatant while inactivation was rapid during incubation with the 27,000 x g pellet which contains mitochondria lysosomes and microsomes, or with the 100,000 x g pellet which contained microsomes.</p> <p>Column chromatography on Sepharose 2B showed that inactivation of dihydrofolate reductase was accompanied by its incorporation into material of high molecular weight. Since inactive enzyme was recovered in several fractions of the column eluate corresponding to the elution volume of the proteins of the subcellular membranes, this material probably represented enzyme which was inactive and bound to these membranes. Greatest binding occurred with membranes of the microsomal fraction.</p> <p>The formation of inactive enzyme of high molecular weight did not depend on prior denaturation of the enzyme nor on prior proteolytic cleavage since no significant production of acid-soluble material, change in molecular weight or change in the charge could be demonstrated. Treatment of the inactive enzyme with urea resulted in reactivation, again suggesting that extensive modification of the enzyme by proteolysis did not occur.</p> <p>Binding of the enzyme to microsomal membranes was not reversed by dilution or by the addition of cofactor and methotrexate. Methotrexate, an inhibitor of dihydrofolate reductase, increases the levels of the enzyme in the cells of patients with cancer treated with the drug, possibly by stabilization against degradation. When methotrexate was present during incubation of dihydrofolate reductase with the subcellular membrane fraction inactivation and binding of the enzyme to microsomal membranes was decreased.</p> <p>The suggestion is made that binding to microsomal membranes could represent the initial step in the degradation of proteins in intact cells. Lysosomes contain proteases and are a likely site of degradation of intracellular proteins. The failure to demonstrate degradation in broken cell preparations could be due to proteins not being able to enter lysosomes. These organelIes are formed from vesicles of the Golgi apparatus. It is suggested that by binding to membranes of the microsomal fraction in intact cells, intracellular proteins could be incorporated into lysosomes at the time of their formation.</p>|
|Appears in Collections:||Open Access Dissertations and Theses|
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