Reduction by cycloheximide of lysosomal proteolytic enzyme activity and rate of protein degradation in organ-cultured hearts

Wildenthal, K.; Griffin, Edmond E.

doi:10.1016/0304-4165(76)90395-0

Cited by 39 publications

(10 citation statements)

References 19 publications

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“…2 ' 20> 21> 24 A disadvantage of this method should be noted, namely that cycloheximide, itself, causes some alteration in cardiac proteolytic rates, 21 ' 22 as may the use of an amino aciddeficient medium. 25 Thus, as discussed in detail previously, 22 primary inhibition of protein synthesis can secondarily produce a less severe but progressive inhibition of protein breakdown (possibly via alterations in lysosomal lability, depletion of proteins with short half-lives that may be important in regulating proteolysis, or accumulation of metabolites that may influence proteolysis). Nevertheless, if both control and experimental hearts are exposed to cycloheximide, its influence on protein degradation can be assumed to be similar in both, and qualitative differences reasonably can be attributed to the experimental intervention.…”

Section: " 17mentioning

confidence: 97%

Synthesis and degradation of myocardial protein during the development and regression of thyroxine-induced cardiac hypertrophy in rats.

Sanford¹,

Griffin²,

Wildenthal³

1978

Circulation Research

111

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SUMMARY Cardiac hypertrophy was induced in rats by daily injections of L-thyroxine (1.0 mg/kg).Regression from hypertrophy was studied 4 days after discontinuing thyroxine. Isolated, Langendorffperfused hearts were perfused with Krebs-Henseleit buffer, glucose, insulin, and amino acids. To measure protein synthesis, left ventricular tissue was assayed for incorporation of tritiated phenylalanine into protein. Indices of rates of protein degradation were obtained by measuring the release of cold phenylalanine after blocking protein synthesis with cycloheximide. After 3 days of thyroxine (when cardiac growth was maximally increased), the rate of protein synthesis increased by 22% (P < 0.001). After 1 week, synthesis was 8% greater than control (P < 0.05), and by 2 weeks (when hypertrophy was stable and the rate of cardiac growth was similar to controls), synthesis had returned to control levels. In hearts regressing from hypertrophy, synthesis was reduced to 68% of control (P < 0.001). The rate of protein degradation was decreased by 12% ( P < 0.05) after 3 days of thyroxine, but was not different from control at 1 or 2 weeks. During regression, degradation was 12% below control (P < 0.05). Changes in the release of several amino acids that are synthesized or metabolized in heart (e.g., alanine, glycine, serine) were different from changes in phenylalanine release. In conclusion thyroxine-induced cardiac hypertrophy and regression are accompanied by changes in protein synthesis and degradation, and amino acid metabolism. The predominant change in hypertrophy is increased protein synthesis with a minor contribution from reduced degradation. Regression of hypertrophy is accompanied by decreased synthesis, not increased degradation.THEORETICALLY, cardiac hypertrophy may occur as a consequence of increased protein synthesis, reduced protein degradation, or both. Similarly, a decrease in heart size, as in regression of hypertrophy, might be brought about by accelerated protein degradation, a decreased rate of synthesis, or both.The relative importance of these factors is defined imprecisely. There is a general agreement that protein synthesis is accelerated during the development of hypertrophy, 1 " 5 but reports differ regarding whether rates of degradation are retarded 5 or not. 23 The type and magnitude of changes that occur may depend on the nature, intensity, and duration of the stress imposed on the heart, and on whether or not the hypertrophic process is accompanied by cellular damage and/or repair.To clarify the roles of altered synthesis and degradation of cardiac protein in one experimental model of hypertrophy and regression, we have measured their rates in vitro following induction and Received January 13, 1978; accepted for publication July 12, 1978. cessation of a well-defined stimulus to hypertrophy-daily injections of L-thyroxine in rats. The regimen employed produces rapid growth of the heart (over and above that observed in age-matched controls), predominantly involving myocardial cells and without...

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Section: " 17mentioning

confidence: 97%

Synthesis and degradation of myocardial protein during the development and regression of thyroxine-induced cardiac hypertrophy in rats.

Sanford¹,

Griffin²,

Wildenthal³

1978

Circulation Research

111

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“…Indeed, we observed two intriguing facts in the present experimental work: (1) although cycloheximide is currently reported as a protein synthesis inhibitor and as effectively blocking PRL biosynthesis (Swearingen, 1971), total protein content of pituitary cell extracts after culture in the presence of cycloheximide was not decreased in comparison with control pituitary cell extracts (Table 1B). A comparable phenomenon associated with reduced lysosomal proteolytic capacity has been observed in organ culture (Wildenthal & Griffin, 1976). However with the particular culture technique we used, the context is different: the bulk of newly synthesized proteins by the pituitary cells consists of PRL and GH (Velkeniers et al, 1988); consequently, total protein content of cellular extracts mainly represents residual cellular hormone content and is not contaminated by tissue protein as is the case for organ culture, where one deals with total tissue protein.…”

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confidence: 56%

Routing, Processing and Export of Rat Pituitary Prolactin: Identification of a 36 kDa Disulphide-bridged Oligomeric Preprolactin

Bollengier

Mahler²,

Vanhaelst³

1999

Archives of Physiology and Biochemistry

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To gain an insight in the routing, processing and export of rat prolactin, rat pituitary cells were cultured in serum-free medium in the presence of cycloheximide, carbonyl cyanide m-chlorophenylhydrazone, Brefeldin A and monensin. The potential influence of these perturbants, whose well documented effects are the altering of protein synthesis and transport, was studied on rat prolactin molecular size isoforms appearing in cellular extracts and in culture medium. The outcome of the culture experiments as recorded in vertical SDS-PAGE, thiol gradient electrophoresis and sequential SDS-PAGE followed by prolactin specific immunoblotting and densitometry, was as follows: (1) at the cellular level we were able to characterize a novel 36 kDa protein as a disulphide-bridged oligomeric precursor prolactin, which is presumably rapidly transformed in the cis/medial Golgi; to designate monomeric rat prolactin as an early Golgi protein and t o advance evidence that the main processing of the glycosylated rat prolactin is a cis/medial Golgi event; (2) in release none of the perturbants disturbed the relative distribution of monomeric and glycosylated rat prolactin, the main molecular size isoforms currently secreted by untreated pituitary cells, or induced the appearance of transformed molecular size isoforms; (3) the secretion mode indicates that rat prolactin is released via the regulated pathway in the presence of the perturbants used.

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“…The first report on a dual inhibitory effect of cycloheximide appeared in 1969 (18). Succeeding studies pointed to an impact of cycloheximide on lysosomal protein degradation because the inhibitory effect on protein degradation correlated with the activity of some lysosomal cathepsins (20,21). More recently, it was found that presence of cycloheximide led to the accumulation of polyubiquitinated murine chains in yeast S. cerevisiae.…”

Section: Discussionmentioning

confidence: 99%

“…The second and third equation describe the time evolution of the DRiPs fraction and the native (folded but nonfluorescent) protein. k 21 , a 1 and k 22 , a 2 denote the elementary rate constants for the synthesis and proteolytic degradation of these two different protein pools. The rate of protein synthesis is set to be proportional to the concentration of the messenger RNA.…”

Section: Kinetic Modelmentioning

confidence: 99%

Quantifying the Contribution of Defective Ribosomal Products to Antigen Production: A Model-Based Computational Analysis

Bulik

Peters

Holzhütter

2005

The Journal of Immunology

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Antigenic peptides (epitopes) presented on the cell surface by MHC class I molecules derive from proteolytic degradation of endogenous proteins. Some recent studies have proposed that the majority of epitopes stem from so-called defective ribosomal products (DRiPs), i.e., freshly synthesized proteins that are unable to adopt the native conformation and thus undergo immediate degradation. However, a reliable computational analysis of the data underlying this hypothesis was lacking so far. Therefore, we have applied kinetic modeling to derive from existing kinetic data (Princiotta et al. 2003, Immunity 18, 343–354) the rates of the major processes involved in the cellular protein turnover and MHC class I-mediated Ag presentation. From our modeling approach, we conclude that in these experiments 1) the relative share of DRiPs in the total protein synthesis amounted to ∼10% thus being much lower than reported so far, 2) DRiPs may become the decisive source of epitopes within an early phase after onset of the synthesis of a long-lived (e.g., virus derived) protein, and 3) inhibition of protein synthesis by the translation inhibitor cycloheximide appears to be paralleled with an instantaneous decrease of protein degradation down to ∼1/3 of the normal value.

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Reduction by cycloheximide of lysosomal proteolytic enzyme activity and rate of protein degradation in organ-cultured hearts

Cited by 39 publications

References 19 publications

Synthesis and degradation of myocardial protein during the development and regression of thyroxine-induced cardiac hypertrophy in rats.

Synthesis and degradation of myocardial protein during the development and regression of thyroxine-induced cardiac hypertrophy in rats.

Routing, Processing and Export of Rat Pituitary Prolactin: Identification of a 36 kDa Disulphide-bridged Oligomeric Preprolactin

Quantifying the Contribution of Defective Ribosomal Products to Antigen Production: A Model-Based Computational Analysis

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