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

1978 ◽  
Vol 176 (3) ◽  
pp. 919-926 ◽  
Author(s):  
R J Burgess ◽  
J H Walker ◽  
R J Mayer
Keyword(s):  
Solvent Extraction ◽  
Nucleic Acids ◽  
Protein Turnover ◽  
Isotope Ratio ◽  
Subcellular Fractions ◽  
Turnover Rates ◽  
Extraction Procedures ◽  
Mitochondrial Fractions ◽  
Double Isotope ◽  
Submitochondrial Fractions

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.

scholarly journals Influence of Subcellular Localization and Functional State on Protein Turnover

Cells ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1747
Author(s):  
Roya Yousefi ◽  
Kristina Jevdokimenko ◽  
Verena Kluever ◽  
David Pacheu-Grau ◽  
Eugenio F. Fornasiero
Keyword(s):  
Subcellular Localization ◽  
Functional State ◽  
Protein Turnover ◽  
Protein Localization ◽  
Small Gtpase ◽  
Turnover Rates ◽  
Cellular Behavior ◽  
Two Factors ◽  
Brain Creatine Kinase ◽  
Selection Of

Protein homeostasis is an equilibrium of paramount importance that maintains cellular performance by preserving an efficient proteome. This equilibrium avoids the accumulation of potentially toxic proteins, which could lead to cellular stress and death. While the regulators of proteostasis are the machineries controlling protein production, folding and degradation, several other factors can influence this process. Here, we have considered two factors influencing protein turnover: the subcellular localization of a protein and its functional state. For this purpose, we used an imaging approach based on the pulse-labeling of 17 representative SNAP-tag constructs for measuring protein lifetimes. With this approach, we obtained precise measurements of protein turnover rates in several subcellular compartments. We also tested a selection of mutants modulating the function of three extensively studied proteins, the Ca2+ sensor calmodulin, the small GTPase Rab5a and the brain creatine kinase (CKB). Finally, we followed up on the increased lifetime observed for the constitutively active Rab5a (Q79L), and we found that its stabilization correlates with enlarged endosomes and increased interaction with membranes. Overall, our data reveal that both changes in protein localization and functional state are key modulators of protein turnover, and protein lifetime fluctuations can be considered to infer changes in cellular behavior.

scholarly journals Radiocarbon Dating of Alkenones from Marine Sediments: III. Influence of Solvent Extraction Procedures on 14C Measurements of Foraminifera

Radiocarbon ◽  
2005 ◽  
Vol 47 (3) ◽  
pp. 425-432 ◽  
Author(s):  
Naohiko Ohkouchi ◽  
Timothy I Eglinton ◽  
Konrad A Hughen ◽  
Ellen Roosen ◽  
Lloyd D Keigwin
Keyword(s):  
Solvent Extraction ◽  
Climate Changes ◽  
Extraction Procedures ◽  
Surface Ocean ◽  
The Past ◽  
Proxy Records ◽  
Temporal Relationships ◽  
Ocean Properties ◽  
Bermuda Rise ◽  
Influence Of Solvent

As a result of the growing use of multiple geochemical proxies to reconstruct ocean and climate changes in the past, there is an increasing need to establish temporal relationships between proxies derived from the same marine sediment record and ideally from the same core sections. Coupled proxy records of surface ocean properties, such as those based on lipid biomarkers (e.g. alkenone-derived sea surface temperature) and planktonic foraminiferal carbonate (oxygen isotopes), are a key example. Here, we assess whether 2 different solvent extraction procedures used for isolation of molecular biomarkers influence the radiocarbon contents of planktonic foraminiferal carbonate recovered from the corresponding residues of Bermuda Rise and Cariaco Basin sediments. Although minor Δ14C differences were observed between solvent-extracted and unextracted samples, no substantial or systematic offsets were evident. Overall, these data suggest that, in a practical sense, foraminiferal shells from a solvent-extracted residue can be reliably used for 14C dating to determine the age of sediment deposition and to examine age relationships with other sedimentary constituents (e.g. alkenones).

Removal of gossypol from cottonseed by solvent extraction procedures

1952 ◽  
Vol 29 (8) ◽  
pp. 339-341 ◽  
Author(s):  
J. M. Dechary ◽  
R. P. Kupperman ◽  
F. H. Thurber ◽  
A. M. Altschul
Keyword(s):  
Solvent Extraction ◽  
Extraction Procedures

A reassessment of the double isotope ratio (C-13)O/C(O-18) in molecular clouds

1989 ◽  
Vol 341 ◽  
pp. 293 ◽  
Author(s):  
David K. Taylor ◽  
R. L. Dickman
Keyword(s):  
Isotope Ratio ◽  
Molecular Clouds ◽  
O 18 ◽  
Double Isotope

II. Biochemical Investigations

1972 ◽  
Vol 18 (5) ◽  
pp. 462-470 ◽  
Author(s):  
C Rimington ◽  
W G Sears ◽  
L Eales
Keyword(s):  
Solvent Extraction ◽  
Thin Layer ◽  
Soluble Fraction ◽  
Characteristic Pattern ◽  
Chromatographic Technique ◽  
Extraction Procedures ◽  
Felty's Syndrome ◽  
Chromatographic Procedure ◽  
Felty’S Syndrome

Abstract The urinary porphyrin excretion of the patient (Part I) has been examined. Methods for determination of the various porphyrin fractions have also been compared and critically assessed. A thin-layer chromatographic technique was found to be rapid, discriminating, and precise. Of solvent-extraction procedures, that of Rimington and Benson for the determination of urinary "X" porphyrins gives results that compare well with those obtained by the quantitative chromatographic procedure. The patient excreted relatively large amounts of uroporphyrin, heptacarboxylic porphyrin, and "X" porphyrins. In addition, the hydroxycoproporphyrins 1 and 2 of Elder were present in the ether-soluble fraction of her urine in relatively large quantities during the period when the patient was severely ill. Her fecal porphyrins were not markedly increased. The porphyria exhibited by this case of Felty’s syndrome resembled the characteristic pattern of symptomatic porphyria, usually associated with alcoholism, but in this case other factors must be considered because this patient was a life-long abstainer from alcohol.

Protein turnover during metabolic arrest in turtle hepatocytes: role and energy dependence of proteolysis

1994 ◽  
Vol 266 (4) ◽  
pp. C1028-C1036 ◽  
Author(s):  
S. C. Land ◽  
P. W. Hochachka
Keyword(s):  
Energy Dependence ◽  
Protein Turnover ◽  
Lactate Production ◽  
Significant Inhibition ◽  
Turnover Rates ◽  
Atp Turnover ◽  
Protein Pool ◽  
Chrysemys Picta Bellii ◽  
Metabolic Arrest ◽  
Energy Dependent

Hepatocytes from the western painted turtle (Chrysemys picta bellii) are capable of a coordinated metabolic suppression of 88% during 10 h of anoxia at 25 degrees C. The energy dependence and role of proteolysis in this suppression were assessed in labile ([3H]Phe-labeled) and stable ([14C]Phe-labeled) protein pools. During anoxia, labile protein half-lives increased from 24.7 +/- 3.3 to 34.4 +/- 3.7 h, with stable protein half-lives increasing from 55.6 +/- 3.4 to 109.6 +/- 7.4 h. The total anoxic mean proteolytic suppression for both pools was 36%. On the basis of inhibition of O2 consumption and lactate production rates by cycloheximide and emetine, normoxic ATP-dependent proteolysis required 11.1 +/- 1.7 mumol ATP.g-1.h-1 accounting for 21.8 +/- 1.4% of total cellular metabolism. Under anoxia this was suppressed by 93% to 0.73 +/- 0.43 mumol ATP.g-1.h-1. Summation of this with protein synthesis ATP turnover rates indicated that under anoxia 45% of total ATP turnover rate was directed toward protein turnover. Studies with inhibitors of energy metabolism indicated that the majority of energy dependence was found in the stable protein pool, with no significant inhibition occurring among the more labile proteins. We conclude that proteolysis is largely energy dependent under normoxia, whereas under anoxia there is a shift to a slower overall proteolytic rate that is largely energy independent and represents loss mostly from the labile protein pool.

On using cis-Pt (II)-uracil as a stain for nucleic acids in brain slices and subcellular fractions

1976 ◽  
Vol 49 (3) ◽  
pp. 253-261 ◽  
Author(s):  
Joseph A. Babitch ◽  
Donald L. Helseth ◽  
Tien-Cheng Chiu
Keyword(s):  
Nucleic Acids ◽  
Brain Slices ◽  
Subcellular Fractions
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