Missing page 192. Page 194 was repeated, therefore one was omitted. / <p> The synthesis and degradation of proteins were studied during aging of cultured human fibroblasts. Equations were derived to yield expressions for the rates of protein degradation, export, and synthesis during exponential growth and steady state from the approach to equilibrium method of radioactively labeling protein. Old cells (cells from normal donors at late passage, cells from old donors, or cells from subjects which the accelerated aging phenotypes of Hutchinson-Gilford (progeria) and Werner syndromes) have a reduced growth rate (0.3-1.3%/hour) when cultured at low density compared to young cells (early-passage cells from normal donors) (2.0-2.5%/hour). Prior to the terminal passage in old cultures, this reduction in growth rate is related primarily to an increased rate of protein degradation (0.96-1.3%/hour in old cells compared to less than 0.55%/hour in young cells). Early-passage cells achieve rapid growth in low density cultures by increasing the protein synthetic rate and decreasing the degradation rate. In high density cultures where the net growth rate was close to zero, the rates of degradation and synthesis were similar in young and old cells prior to their terminal passage (1.9-2.5%/hour). In all cases the rate of protein export was small (less than 0.5%/hour) compared to the rate of protein synthesis. </p> Proteins synthesized by young and old cells were analyzed by two-dimensional gel electrophoresis and were found to be essentially identical in molecular weight and isoelectric points. Induction of synthesis of aberrant proteins by histidine starvation in the presence of histidinol did not reveal differences between early- and late-passage cells from young or old normal donors or from subjects with progeria or Wener Syndrome. Furthermore, there was no correlation between in vitro lifespan and the synthesis of aberrant protein. </p> <p> It is concluded that the increased degradation of proteins and the slow net growth of old cells and the reduced lifespan of cells from old normal donors and subjects with progeria or Werner Syndrome are not due to abnormal protein synthesis. This is contrary to the predictions of the error catastrophe theory of aging. </p> <p> The aberrant proteins synthesized during amino acid starvation are believed to result from amino acid substitution. Several observations reported here are consistent with this hypothesis: (i) No turnover of either native or substituted actins synthesized during histidine starvation of cultured human cells was; (ii) Changes in the isoelectric points of native and substituted actins are predicted by analyses based on the presumed changes in their amino acid composition; (iii) Estimates of the protein synthetic error rates during normal protein synthesis can be derived from a computer model of mRNA translation based on the proposed mechanism of mistranslation; these estimates are consistent under a variety of starvation conditions and are close to other estimates obtained independently for the error frequency in mammalian cells. </p> <p> In both young and old cultured human fibroblasts the error frquency at the histidine codon was calculated to be 1.1 ± 0.1 x 10⁻⁴ (mean+S.E.). Three lines of Sv40-transformed human fibroblasts had error frequencies 2-5 fold greater than their untransformed counterparts. Studies with a variety of other human and non-human cell types did not support the conclusion that transformation in general increased in rate of mistranslation. The observation of increased error frequencies in SV40-transformed human cells may be restricted to this viral transformation. </p> <p> The computer simulations of mRNA translation have provided a means of extrapolating error frequencies determined during amino acid starvation to the error frequency during normal protein synthesis. This model is of great interest for its potential use as a method of rapidly quantifying protein synthetic error frequencies in cultured cells. </p> / Thesis / Doctor of Philosophy (PhD)
| Identifer | oai:union.ndltd.org:mcmaster.ca/oai:macsphere.mcmaster.ca:11375/18560 |
| Date | 12 1900 |
| Creators | Harley, Calvin Bruce |
| Contributors | Goldstein, Samuel, Biochemistry |
| Source Sets | McMaster University |
| Language | English |
| Detected Language | English |
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