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A Density Labelling Method for the Quantitation of Radioactive Label Recycling in Studies on Individual Protein Turnover
RAINER EISING and BENNO SÜSELBECK
Journal of Experimental Botany
Vol. 42, No. 240 (July 1991), pp. 947-955
Published by: Oxford University Press
Stable URL: http://www.jstor.org/stable/23692906
Page Count: 9
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Recycling of radioactive label from degraded protein is a major drawback in pulse—chase experiments for the measurement of protein turnover, because it leads to underestimations of degradation constants. This paper describes a density labelling method for the quantitation of label recycling into individual proteins, whereby underestimated degradation constants can be corrected. The method requires a density difference between native (light) and density-labelled (heavy) protein of less than 10 g dm-3 and, therefore, allows using 2H2O concentrations of only 50% (v/v) or even considerably lower. Plants are pulse-labelled with 14C and grown on 2H2O either during the pulse or following the chase. Distribution of 14C-label between light and heavy protein is determined from a 14C-superimposition curve obtained by isopycnic centrifugation of the purified protein in a CsCl gradient. The superimposition curve mathematically is described by the sum of two single Gaussian curves. It proved impossible to determine all six unknown parameters of this function by an algorithm. Therefore, mean values (i.e. the peak positions in the gradient) of light and heavy protein are determined from internal markers (3H-labelled light or heavy protein). Evaluation of data is performed by a computerized curve fitting program which provides a numerical and a graphical result of label recycling. The detection limit of the method is 10% of the minor component in a heterogenous population of light and heavy protein differing in density by only 8.0 g dm-3.
Journal of Experimental Botany © 1991 Oxford University Press