To determine non specific substrate degradation, the assays were also performed by preincubating total protein samples for 15 min at 37 °C with or without the specific caspase inhibitor (200 nM Ac-DEVD-CHO) before substrate addition.
(B ) Images of Coomassie-stained Phos-Tag SDS-PAGE in which the non specific substrate and product (GSTP and GST, upper panel) and the specific substrate and product (eIF2aP and eIF2a, lower panel) have been resolved.
All of the recombinant enzymes (wild type and engineered) were also assayed with non-specific substrates, such as xylan, avicel and filter paper (FP).
Moreover, a certain degree of intermingling between the mistargeted proteins and the endosymbiont enzymes catalyzing similar, preceding or subsequent (e.g., fortuitous substrate channeling; use of common or non-specific substrates) reactions possibly sustained a steady state of novel catalyzed reactions inside the photosynthetic organelle.
The activity of all of the mutated enzymes with non-specific substrates indicates that the substrate binding pocket is altered and has become flexible toward other substrates after mutation.
These studies have been on either vermicast generated from non-specific substrates in nature or from blends of soil and phytomass.
Then, the specific activities of the four variants were assayed by using the specific substrate GD4K-βNA and the non-specific substrates Boc-E(OBzl -AR-MCA and Z-FR-MCA.
In the absence of conflicting or non-specific substrates, the TransportDB reaction attributes are included in the standard.
A large number of genes encoding proteins with functions in the transport of water, ions, sugars, and non-specific substrates (ABC transporters) showed differential expression within berry tissues.
DOI: http://dx.doi.org/10.7554/eLife.04871.014 10.7554/eLiFigure71.015 Figure 4 figure supplement 3. Incorporation of G-actin inhibits PPP1R15B-PP1G phosphactivitytivity directed against non-specific substrates.
A more formal quantitative analysis of enzyme kinetics of the different enzyme complexes with regard to eIF2α and non-specific substrates was also deemed important.
