Measurement of steady state protein degradation in cultured human muscle cells

Abstract

Double label techniques for measurement of protein turnover in cultured cells are described. In the isotope withdrawal method protein in cultured muscle is labeled with two isotopes of the same amino acid for 24 to 100 h, followed by exposure to fresh medium containing one isotope only at the same specific activity for an additional 24 to 48 h. In the isotope addition method the order of addition of single and double-labeled media is reversed. After incubation the ratio of the two isotopes in the cell protein is a function of the incubation time and the degradation rate constant K d ; K d can readily be calculated using a graphical or iterative method. In mixed cultures of human muscle with initial incubation ranging 24 to 159 h, the K d 's obtained from various incubation times were similar. Both the isotope withdrawal and the isotope addition methods gave a K d value of 0.018 h −1 similar to values obtained by two different single isotope methods which monitor the appearance of free isotope in the medium of previously labeled cells. There were no differences of K d values obtained in cultures of muscle from normal patients and those with denervation, inflammatory myopathies, or nonspecific myopathic biopsy changes. When proteins were separated by gel electrophoresis, those of molecular weight greater than 60,000 had higher average K d values as compared to lower molecular weight proteins. The double isotope labeling method has the advantage of being easily applied to cultures with small numbers of cells and is potentially useful in obtaining the degradation rates of individual cellular proteins. The major disadvantages are (1) in their present form the methods can be used only in steady state cultures and (2) they require rather long (24 h) labeling times.