Useful notes on Protein Turnover in Cells

The rate of breakdown and replacement of protein in cells was badly misunderstood prior to 1939.

In grow­ing animals generally and in secretory tissues in par­ticular (liver, pancreas, endocrine glands, etc.), active synthesis of protein was known.

However, the amount of protein synthesis taking place in other tissues of the adult was believed to be very low and confined to that necessary to replace protein lost in damaged or dying cells.

These small protein losses, together with the catabolism of dietary amino acids, were believed to be responsible for the urea and ammonia measurable in the urine excreted from the body. Proteins were thus regarded as highly stable constituents lasting virtu­ally the entire lifetime of the cell.

The first serious challenge to the “wear and tear” view of protein turnover came as a result of the work of R. Schoenheimer in 1938. Schoenheimer synthe­sized a number of amino acids in which the ¹⁵N content of the alpha-amino nitrogen was considerably in­creased over the naturally occurring amount of this isotope. Schoenheimer then injected ¹⁵N-containing glycine and leucine into rats and noted that these la­beled amino acids were quickly incorporated into the proteins of many tissues.

Although ¹⁵N is not a radio­active isotope of nitrogen, it may nonetheless be dis­tinguished chemically from the more common ¹⁴N form and is called a “heavy” isotope of nitrogen. The results clearly indicated that protein synthesis in adult animals is not restricted to growing and secre­tory tissues but occurs in nearly all cells and that tis­sue proteins are in a continuous state of metabolic flux, being broken down and replaced by newly syn­thesized molecules.

Although the radioactive isotope of carbon, ¹⁴C, was produced in the Berkeley cyclotron in 1940, it was not until 1947 that ¹⁴C-labeled amino acids became avail­able for use as biological tracers. The availability of ra­dioactive amino acids was followed by a series of clas­sic tracer experiments by H. Borsook, T. Hultin, P. Zamecnik, and P. Siekevitz, which verified the find­ings of Schoenheimer that most tissues readily incor­porate amino acids into protein and also added crucial details to the newly emerging view of protein synthe­sis and metabolic turnover.

The first attempts to determine the subcellular site of amino acid incorporation into protein were carried out in 1950 by Borsook. Minutes after injecting ¹⁴C- labeled amino acids into the bloodstreams of guinea pigs, Borsook removed the animals’ livers and, using the technique of differential centrifugation (see Chap­ter 12), prepared subcellular fractions of the tissue. Borsook showed that it was the microsomal fraction that contained the greatest amount of radioactivity and suggested that the microsomes were the reposi­tory of the cell’s protein-synthesizing apparatus. In the same year, Hultin demonstrated that it was the microsomal fraction of chick liver tissue homogenates that incorporate intravenously injected ¹⁵N-glycine into protein.

By 1952, Siekevitz and Zamecnik had been able to demonstrate the in vitro incorporation of ¹⁴C-labeled amino acids into liver cell proteins by both tissue slices and. tissue homogenates. By measuring and compart­ing protein synthetic activity in cell-free whole ho­mogenates, individual cell subfractions, and various combinations of subfractions, Siekevitz showed that the incorporation of amino acids into proteins by mi­crosomes was dependent on an energy source pro­vided by the mitochondrial subtraction and required enzymes and other factors present in the cytosol.

The demonstration that amino acid incorporation into pro­tein required metabolic energy laid to rest a view pop­ular in the 1940s that polypeptide synthesis might be brought about by the reversal of the enzymatic reac­tions in which proteins are degraded. It is especially interesting to note that Siekevitz demonstrated the existence in the cytosol of a MgCl₂-precipitable factor required for protein synthesis. As MgCl₂ was known to precipitate RNA, Siekevitz suggested that RNA might somehow be involved in protein synthesis, a fact not fully recognized until many years later.

The studies described above established the general cytological and chemical basis of protein biosynthesis. Exhaustive research since the 1950s by dozens of groups of investigators has revealed the step-by-step, reaction-by-reaction details of the process and has given us an astounding insight into the molecular or­ganization of the cell’s protein-synthesizing appa­ratus.