Choice of precursors for the measurement of protein turnover by the double-isotope method. Application to the study of mitochondrial proteins

Abstract

1. The double-isotope concept [Arias, Doyle & Schimke (1969) J. Biol. Chem. 224, 3303--3315] for the measurement of protein turnover was used to estimate the turnover of proteins in subcellular and submitochondrial fractions prepared from rat liver. 2. Double-isotope experiments with [3H]leucine as first precursor and [14C]leucine as second precursor were used to measure the turnover rates of proteins in subcellular and submitochondrial fractions. Solvent extraction procedures designed to remove lipids and nucleic acids from trichloroacetic acid precipitates only changed the isotope ratio of the microsomal fraction. It was not possible to measure turnover of proteins in mitochondrial and submitochondrial fractions with these precursors. 3. Double-isotope experiments were designed to minimize first-precursor reutilization by employing NaH14CO3. [3H]Arginine was used as second precursor. The turnover rates of protein in subcellular and submitochondrial fractions was measured. Solvent extraction procedures designed to remove lipids and nucleic acids showed changes in the isotope ratio for all subcellular fractions, especially in microsomal and detergent-soluble mitochondrial fractions. Isotope ratios of precipitates after solvent extraction indicate that, whereas considerable heterogeneity exists for the average rates of protein turnover in subcellular fractions, little heterogeneity is observed in the average rates of protein turnover in submitochondrial fractions.

Protein degradation in rat liver during post-natal development

“…It is clear that, in the adult-rat microsomal fraction, proteins are degraded more rapidly than are proteins in other subcellular fractions. The low rate of degradation of adult liver mitochondrial proteins has also been found by other workers (Arias et al, 1969;Aschenbrenner et al, 1970;Burgess et al, 1978). A low rate of degradation was observed for the adult-rat particle-free supernatant proteins, which is at variance with the results of Arias et al-(1969) and Burgess et al (1978).…”

“…Other amino acids are labelled by H14CO3-, most notably glutamate/glutamine and in the mitochondrial proteins aspartate/asparagine, but the specific radioactivity of these amino acids is low relative to that of arginine (Swick & Handa, 1956). Despite the use of 12.5 mCi of H14CO3per rat in these experiments the 14C specific radioactivities for subcellular and submitochondrial proteins (Table 3) are similar to the specific radioactivities obtained by Burgess et al (1978) using rats of similar size and only 5 mCi of H14CO3per rat. The large loss of 14C label (approx.…”

“…The low rate of degradation of adult liver mitochondrial proteins has also been found by other workers (Arias et al, 1969;Aschenbrenner et al, 1970;Burgess et al, 1978). A low rate of degradation was observed for the adult-rat particle-free supernatant proteins, which is at variance with the results of Arias et al-(1969) and Burgess et al (1978). No satisfactory explanation was found for this apparently anomalous result.…”

“…Subcellular fractionation of rat liver. The method of subcellular fractionation has been described in Burgess et al (1978).…”

“…Cytochrome oxidase. The method of preparation of anti-(cytochrome oxidase) serum and coupling to Sepharose 4B is given by Burgess et al (1978). The binding capacity of the prepared immunoadsorbent was 67nmol *min"-1 *(ml of Sepharose)-l.…”