EVIDENCE FOR POSTTRANSLATIONAL KINETIC COMPARTMENTATION OF PROTEIN-TURNOVER POOLS IN ISOLATED ADULT CARDIAC MYOCYTES

The kinetics of protein metabolism were evaluated in isolated adult fe line cardiomyocytes maintained in long term cell culture. The results of these studies suggested that, rather than individual proteins enter ing a kinetically homogeneous pool, most newly synthesized proteins we re segregated into different kinetic compartments with different proba bilities of being degraded. Evidence for this conclusion was developed from two types of experiment. The first line of evidence was derived from the kinetics of labeled amino acid incorporation into protein whe n labeling was conducted over very short or long periods (4 h to 32 da ys). The observed rates of isotope incorporation over different period s did not fit the expected pattern for a homogeneous pool. A close app roximation of observed rates of label incorporation over both short an d long labeling intervals could, however, be fit to results predicted using a two-compartment model. When the results of long term equilibra tion labeling studies were also evaluated relative to short and long t erm labeling ratios, then it was also possible to identify a unique se t of parameters for a two-compartment model which could account for la bel incorporation in both types of experiment. From this analysis it w as estimated that the fast kinetic compartment represented a steady-st ate level of 10% of total cellular protein with a mean half-life of 21 .9 h (k(s) = 75.9% d-1). The slow kinetic compartment comprised the re maining 90% of protein with a mean t1/2 equal to 15.6 days (k(s) = 4.4 % d-1). Both the observed and predicted equilibration rate of this two compartment mixture was best fit by a single exponential function wit h an equilibration rate of 5.48% d-1. Based on this outcome, it was al so predicted that mixing of nascent and long lived proteins could be f ollowed using a protocol of long and short term labeling in different isotopes followed by a chase period without label. By tracking the iso tope ratios in total proteins and SDS-polyacrylamide gel electrophores is separated proteins, as well as in counts released into the culture medium during the chase period it was determined that nascent proteins were preferentially degraded for a period of at least 48 h following synthesis. Up to 20% of nascent proteins in both the total protein com partment as well as individual proteins, such as myosin heavy chain, w ere preferentially degraded prior to achieving a state of homogeneous mixing with long lived proteins. These results suggest that in adult c ardiac myocytes the regulation of protein balance functions not only a t the transcriptional level, but also involves post-translational segr egation of nascent peptides into different kinetic compartments in whi ch there are nearly 20-fold differences in half-life.