Measurement of local cerebral protein synthesis in vivo: influence of recycling of amino acids derived from protein degradation.

Abstract

A quantitative autoradiographic method for the determination of local rates of protein synthesis in brain in vivo is being developed. The method employs L-[1-'4C]leucine as the radiolabeled tracer. A comprehensive model has been designed that takes into account intracellular and extracellular spaces, intracellular compartmentation of leucine, and the possibility of recycling of unlabeled leucine derived from steadystate degradation of protein into the precursor pool for protein synthesis. We have evaluated the degree of recycling by measuring the ratio of the steady-state precursor pool distribution space for labeled leucine to that of unlabeled leucine. The values obtained were 0.58 in whole brain and 0.47 in liver. These results indicate that there is significant recycling of unlabeled amino acids derived from steady-state protein degradation in both tissues. Any method for the determination of rates of cerebral protein synthesis in vivo with labeled tracers that depends on estimation of precursor pool specific activity in tissue from measurements in plasma must take this recycling into account.The autoradiographic deoxyglucose method (1) for measurement of local cerebral glucose utilization provides a useful model for the design of methods to determine local rates of biochemical processes within tissues in vivo. We have used the same general approach to develop a method for the quantitative determination of local rates of protein synthesis, or, more precisely, amino acid incorporation into protein, in the nervous system (2, 3). The method is an application of the basic principles for the assay of rates of chemical reactions with isotopes adapted for the special conditions encountered in vivo (4). Briefly, the total amount of labeled product specific to the reaction of interest (in this case labeled protein) formed in the selected tissue over an interval of time is divided by the integrated specific activity (SA) of the precursor pool over the same interval and corrected for any isotope effect (4). This relationship can be expressed as follows:

Postadolescent Changes in Regional Cerebral Protein Synthesis: AnIn VivoStudy in theFmr1Null Mouse

“…The pure tRNA fraction was dissolved in 50 mM sodium carbonate, pH 10, and incubated at 37°C for 90 min to deacylate the aminoacyl-tRNA. Deacylated tRNA was precipitated overnight at Ϫ20°C in ethanol and removed by centrifugation (12,000 ϫ g, 20 min) (Smith et al, 1988). The supernatant solution, which contained the previously tRNA-bound but now free amino acids, was dried in a stream of N 2 and redissolved in 40 l of 0.2 M sodium citrate, pH 2.2.…”

“…rCPS was determined with L-[1-14 C]leucine as the radiolabeled tracer used in conjunction with quantitative autoradiography to achieve spatial localization of the labeled protein in the brain (Smith et al, 1988). The operational equation of the method is as follows:…”

“…At the end of the infusions, the mice were decapitated, and their brains were quickly removed and chilled to 0°C in ice-cold 0.25 M sucrose. Each brain was weighed and homogenized in 5 ml of 0.25 M sucrose (0°C) containing 10 mM vanadyl ribonucleoside complex to inhibit ribonuclease, 6 mg of uncharged tRNA as carrier, and L-norleucine (0.02 mM) added as an internal standard and centrifuged at 100,000 ϫ g for 1 h. The tRNA-bound amino acids were purified as described previously (Smith et al, 1988). The pure tRNA fraction was dissolved in 50 mM sodium carbonate, pH 10, and incubated at 37°C for 90 min to deacylate the aminoacyl-tRNA.…”

“…i for whole brain ( WB ) was evaluated in a series of steady-state experiments as described previously (Smith et al, 1988 3 H]leucine in the arterial plasma and tRNA-bound pools in brain were determined as described below. Timed arterial blood samples (ϳ20 l) were collected every 15 or 30 min during the infusion and centrifuged immediately to separate plasma, which was then deproteinized by addition of one-third of a volume of 16% (W/V) sulfosalicylic acid containing L-norleucine (0.04 mM) as an internal standard for amino acid analyses.…”

Transforming Growth Factor-α Attenuates N-Methyl-D-aspartic Acid Toxicity in Cortical Cultures by Preventing Protein Synthesis Inhibition through an Erk1/2-dependent Mechanism

“…precursor pool for the incorporation of amino acids into proteins (32)(33)(34). The immediate precursor pool is not the free amino acid in the intracellular space but the corresponding aminoacyl-tRNA.…”