Evidence for post-translational kinetic compartmentation of protein turnover pools in isolated adult cardiac myocytes.
The kinetics of protein metabolism were evaluated in isolated adult feline cardiomyocytes maintained in long term cell culture. The results of these studies suggested that, rather than individual proteins entering a kinetically homogeneous pool, most newly synthesized proteins were segregated into different kinetic compartments with different probabilities 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 when labeling was conducted over very short or long periods (4 h to 32 days). The observed rates of isotope incorporation over different periods did not fit the expected pattern for a homogeneous pool. A close approximation of observed rates of label incorporation over both short and long labeling intervals could, however, be fit to results predicted using a two-compartment model. When the results of long term equilibration labeling studies were also evaluated relative to short and long term labeling ratios, then it was also possible to identify a unique set of parameters for a two-compartment model which could account for label incorporation in both types of experiment. From this analysis it was estimated that the fast kinetic compartment represented a steady-state level of 10% of total cellular protein with a mean half-life of 21.9 h (ks = 75.9% d-1). The slow kinetic compartment comprised the remaining 90% of protein with a mean t1/2 equal to 15.6 days (ks = 4.4% d-1). Both the observed and predicted equilibration rate of this two compartment mixture was best fit by a single exponential function with an equilibration rate of 5.48% d-1. Based on this outcome, it was also predicted that mixing of nascent and long lived proteins could be followed using a protocol of long and short term labeling in different isotopes followed by a chase period without label. By tracking the isotope ratios in total proteins and SDS-polyacrylamide gel electrophoresis 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 compartment as well as individual proteins, such as myosin heavy chain, were preferentially degraded prior to achieving a state of homogeneous mixing with long lived proteins.(ABSTRACT TRUNCATED AT 400 WORDS)
Department of Medicine, Northwestern University Medical School, Chicago, Illinois 60611.
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Clark
D000185:Actinin / Q000096:biosynthesis / Q000378:metabolism; D000818:Animals; D002250:Carbon Radioisotopes; D002415:Cats; D002478:Cells, Cultured; D007700:Kinetics; D007930:Leucine / Q000378:metabolism; D008433:Mathematics; D008954:Models, Biological; D009206:Myocardium / Q000378:metabolism*; D009218:Myosins / Q000096:biosynthesis / Q000378:metabolism; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000378:metabolism*; D014316:Tritium
D002250:Carbon Radioisotopes; D011506:Proteins; D014316:Tritium; D000185:Actinin; D009218:Myosins; D007930:Leucine