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First published online December 14, 2005
Journal of Experimental Biology 209, 158-170 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.01962

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Fixed metabolic costs for highly variable rates of protein synthesis in sea urchin embryos and larvae

Douglas A. Pace and Donal T. Manahan^*

Department of Biological Sciences, University of Southern California, Los Angeles, CA 90089-0371, USA

View larger version (32K): [in a new window]   Fig. 1. Photomicrographs and primary data sets for protein synthesis and respiration rate measurements of 13-day-old fed larvae of Lytechinus pictus. (A) Photomicrograph of 13-day-old unfed and (B) fed larvae. Scale bars, 200 µm. (C) Linear increase of [14C]alanine incorporation into the protein pool (TCA-insoluble fraction) of 13-day-old fed larvae of L. pictus: y=41.4(±2.71)x-68.4 (r2=0.98; P<0.001). Rates of [14C]alanine incorporation were converted to absolute rates of protein synthesis (ng protein individual-1 h-1) using the change in the specific activity of [14C]alanine in the precursor free amino acid pool (Fig. 1E), the mole-percent of alanine in protein (7.8%; Table 1) and the mole-percent corrected molecular mass for amino acids in protein (129.4 g mole-1; Table 1). (D) Chromatogram of extracted free amino acid pool from 13-day-old larvae of L. pictus separated using high performance liquid chromotagraphy (HPLC). D, aspartate; E, glutamate; N, asparagine; S, serine; H, histidine; G, glycine; R, arginine. Alanine was the [14C]amino acid tracer used to determine rates of protein synthesis. (E) Specific activity of [14C]alanine in the free amino acid pool during protein synthesis experiments with 13-day-old larvae of L. pictus. Specific activity was measured at the specified sampling intervals by quantifying the moles of total alanine with HPLC (as in Fig. 1D). [14C]alanine was measured by liquid scintillation counting of the radioactivity associated with the alanine peak fraction. Increase of specific activity was described by the linear equation: y=39.2(±3.85)x+279.9 (r2=0.96; P<0.001 for ANOVA of regression slope). (F) Respiration rate of 13-day-old fed larvae of L. pictus. Each data point represents a different respiration chamber. Respiration rate was calculated by determining the slope of total oxygen consumption in each respiration chamber for the known number of larvae in that chamber: y=109.5(±8.4)x+1254 (r2=0.97; P<0.0001), where the slope of 109.5 has units of pmol O2 larva-1 h-1.

View larger version (18K): [in a new window]   Fig. 2. Changes in (A) total protein and (B) the amount of alanine in the free amino acid pool during development and growth of the sea urchin Lytechinus pictus. Embryos, open circles; unfed larvae, open squares; fed larvae, filled squares. Values are means ± s.e.m., and where not shown errors fell within the graphical representation of the data point. (A) Changes in total protein content. Each data point is the mean value of 2-3 determinations. The fitted line shows an increase in protein content for fed larvae: y=20.68e0.17x (r2=0.99). (B) Changes in the amount of alanine in the free amino acid pool. Each data point is the mean of 6 measurements. The fitted line shows an increase in alanine in fed larvae: y=2.460e0.19x (r2=0.99).

View larger version (29K): [in a new window]   Fig. 3. Comparison of alanine transport rate in embryos and larvae of L. pictus in the presence and absence of the protein synthesis inhibitor, emetine (see Materials and methods for details). For all alanine transport measurements, the r2 values from the linear regressions of the alanine transport rate with time ranged from 0.93 to 0.99. Open bars, without emetine; solid bars, with emetine. (A) Alanine transport rates for stages of development measured before the start of feeding treatment experiments. BL, blastula; GA, gastrula: PL, prism larva; EP, early pluteus. (B) Alanine transport rates in fed and unfed larvae. Values are means ± s.e. slope of the slope of transport rate. (C) Primary data to show comparison of alanine transport rates in the presence and absence of emetine (fed larvae, 16-day-old). Slopes and intercepts were not significantly different (ANOVA). Open circles + broken line, no emetine (r2=0.95); solid circles + solid line, with emetine (r2=0.93).

View larger version (13K): [in a new window]   Fig. 4. Rates of protein synthesis in embryos (open circles), unfed larvae (open squares) and fed larvae (filled squares) of the sea urchin L. pictus. Each data point is the mean ± 1 s.e.m. of 5-6 determinations; where not shown, errors fell within the graphical representation of the data point. The fitted line shows an increase in rate of protein synthesis for fed larvae: y=0.25e0.23x (r2=0.96).

View larger version (12K): [in a new window]   Fig. 5. Correlative cost of protein synthesis in embryos (open circles), unfed larvae (open squares) and fed larvae (filled squares) of L. pictus. Cost of protein synthesis was calculated from the slope of the relationship between protein synthesis and metabolic rate. The broken line is the regression of metabolic rate and protein synthesis for all stages of development studied (embryos, unfed and fed larvae). Slope of regression=8.41±0.49 J mg-1 protein; r2=0.95; N=17). Solid line is regression for fed larvae alone=7.69±1.36 J mg-1 protein; r2=0.91; N=5).

View larger version (16K): [in a new window]   Fig. 6. Inhibitor analysis to determine the cost of protein synthesis in embryos and larvae of L. pictus. Protein synthesis (PS) and metabolic rates (O2) in the presence and absence of emetine (see Materials and methods for details) are shown for each replicate measurement for each developmental stage studied (blastula, gastrula and larva). Total height of each bar represents metabolic rate or protein synthesis rate when no inhibitor was present. The height of the solid component represents the rate in the presence of emetine. Error bars are calculated as s.e.m. of the slope for each linear regression analysis (error of the estimate of protein synthesis calculated as in Fig. 1C; error for metabolic rate as in Fig. 1F). Costs of protein synthesis (all means ± s.e.m.) were: blastula, 7.76±2.55 J mg-1 protein (N=3); gastrula, 7.75±0.90 J mg-1 protein (N=3); larva, 10.34±0.57 J mg-1 protein (N=2). The costs were not significantly different for all stages of development analyzed (ANOVA; P=0.60, N=8). The average cost of protein synthesis=8.40±0.99 J mg-1 protein.

View larger version (29K): [in a new window]   Fig. 7. (A) Setting metabolic rate. Percent of total metabolic rate (MR; given as µJ individual-1 h-1) for different developmental stages of L. pictus accounted for by protein synthesis and sodium pump activity (data on Na+/K+-ATPase taken from Leong and Manahan, 1997). (B) Protein depositional efficiency. Rate of protein deposition (solid line) calculated from data on total protein content (see equation in legend of Fig. 2A). Rate of protein synthesis (broken line) calculated from protein synthesis rate (see equation in legend of Fig. 4). Rate of protein degradation during development (dotted line) calculated as difference between rates of synthesis and deposition. All rates were calculated on a per-larva basis. For 15-day-old larvae, the depositional efficiency was 21%.