Rubisco mutagenesis provides new insight into limitations on photosynthesis and growth in Synechocystis PCC6803

Orthophosphate (Pi) stimulates the activation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) while paradoxically inhibiting its catalysis. Of three Pi-binding sites, the roles of the 5P- and latch sites have been documented, whereas that of the 1P-site remained unclear. Conserved residues at the 1P-site of Rubisco from the cyanobacterium Synechocystis PCC6803 were substituted and the kinetic properties of the enzyme derivatives and effects on cell photosynthesis and growth were examined. While Pi-stimulated Rubisco activation diminished for enzyme mutants T65A/S and G404A, inhibition of catalysis by Pi remained unchanged. Together with previous studies, the results suggest that all three Pi-binding sites are involved in stimulation of Rubisco activation, whereas only the 5P-site is involved in inhibition of catalysis. While all the mutations reduced the catalytic turnover of Rubisco (Kcat) between 6- and 20-fold, the photosynthesis and growth rates under saturating irradiance and inorganic carbon (Ci) concentrations were only reduced 40–50% (in the T65A/S mutants) or not at all (G404A mutant). Analysis of the mutant cells revealed a 3-fold increase in Rubisco content that partially compensated for the reduced Kcat so that the carboxylation rate per chlorophyll was one-third of that in the wild type. Correlation between the kinetic properties of Rubisco and the photosynthetic rate (Pmax) under saturating irradiance and Ci concentrations indicate that a >60% reduction in Kcat can be tolerated before Pmax in Synechocystsis PCC6803 is affected. These results indicate that the limitation of Rubisco activity on the rate of photosynthesis in Synechocystis is low. Determination of Calvin cycle metabolites revealed that unlike in higher plants, cyanobacterial photosynthesis is constrained by phosphoglycerate reduction probably due to limitation of ATP or NADPH

Activation of mTORC1 by leucine is potentiated by branched-chain amino acids and even more so by essential amino acids following resistance exercise

Protein synthesis is stimulated by resistance exercise and intake of amino acids, in particular leucine. Moreover, activation of mammalian target of rapamycin complex 1 (mTORC1) signaling by leucine is potentiated by the presence of other essential amino acids (EAA). However, the contribution of the branched-chain amino acids (BCAA) to this effect is yet unknown. Here we compare the stimulatory role of leucine, BCAA, and EAA ingestion on anabolic signaling following exercise. Accordingly, eight trained volunteers completed four sessions of resistance exercise during which they ingested either placebo, leucine, BCAA, or EAA (including the BCAA) in random order. Muscle biopsies were taken at rest, immediately after exercise, and following 90 and 180 min of recovery. Following 90 min of recovery the activity of S6 kinase 1 (S6K1) was greater than at rest in all four trials (Placebo&amp;lt;Leucine&amp;lt;BCAA&amp;lt;EAA; P &amp;lt; 0.05 time × supplement), with a ninefold increase in the EAA trial. At this same time point, phosphorylation of eukaryotic translation initiation factor 4E (eIF4E)- binding protein 1 (4E-BP1) at Thr37/46 was unaffected by supplementation, while that of Thr46 alone exhibited a pattern similar to that of S6K1, being 18% higher with EAA than BCAA. However, after 180 min of recovery this difference between EAA and BCAA had disappeared, although with both these supplements the increases were still higher than with leucine (40%, P &amp;lt; 0.05) and placebo (100%, P &amp;lt; 0.05). In summary, EAA ingestion appears to stimulate translation initiation more effectively than the other supplements, although the results also suggest that this effect is primarily attributable to the BCAA.CODEN: AJPCD</p

Resistance exercise-induced S6K1 kinase activity is not inhibited in human skeletal muscle despite prior activation of AMPK by high-intensity interval cycling

Combining endurance and strength training in the same session has been reported to reduce the anabolic response to the latter form of exercise. The underlying mechanism, based primarily on results from rodent muscle, is proposed to involve AMPK-dependent inhibition of mTORC1 signaling. This hypothesis was tested in eight trained male subjects who in randomized order performed either resistance exercise only (R) or interval cycling followed by resistance exercise (ER). Biopsies taken from the vastus lateralis before and after endurance exercise and repeatedly after resistance exercise were assessed for glycogen content, kinase activity, protein phosphorylation, and gene expression. Mixed muscle fractional synthetic rate was measured at rest and during 3 h of recovery using the stable isotope technique. In ER, AMPK activity was elevated immediately after both endurance and resistance exercise (similar to 90%, P &lt; 0.05) but was unchanged in R. Thr(389) phosphorylation of S6K1 was increased severalfold immediately after exercise (P &lt; 0.05) in both trials and increased further throughout recovery. After 90 and 180 min recovery, S6K1 activity was elevated (similar to 55 and similar to 110%, respectively, P &lt; 0.05) and eukaryotic elongation factor 2 phosphorylation was reduced (similar to 55%, P &lt; 0.05) with no difference between trials. In contrast, markers for protein catabolism were differently influenced by the two modes of exercise; ER induced a significant increase in gene and protein expression of MuRF1 (P &lt; 0.05), which was not observed following R exercise only. In conclusion, cycling-induced elevation in AMPK activity does not inhibit mTOR complex 1 signaling after subsequent resistance exercise but may instead interfere with the hypertrophic response by influencing key components in protein breakdown