Physiological Study of Cysteine Synthase Isozymes inSpinacia oleraceaSeedlings

A spinach O-acetylserine(thiol)lyase homologue, SoCSaseLP, suppresses cysteine biosynthesis catalysed by other enzyme isoforms

“…5 ) is that SoCSaseLP inactivates the tobacco cytosolic OASTL isoform after formation of a complex between them. We tried to detect the recombinant SoCSaseLP and SoCSaseA proteins in the tobacco protein extracts prepared from the corresponding transgenic plants, but we could not detect them with an anti-spinach OASTL antibody [26] . The failure to detect the recombinant proteins may be explained by their low level of accumulation in tobacco plants.…”

“…In lane 4, the crude protein fraction was subjected to immobilized metal affinity chromatography. The recombinant His-CSA was detected by western blotting with an anti-spinach OASTL antibody [26] . The fusion proteins with GST and GST by itself were detected by an anti-GST antibody.…”

“…The blots were washed three times with washing buffer (0.15 M PBS, pH 7.3, 0.1% Tween 20). The recombinant His-CSA proteins on the PVDF membranes were cross-reacted with a rabbit anti-spinach OASTL antibody [26] for 1 h. After washing three times for 10 min each, the blots were incubated for 60 min with a goat anti-rabbit IgG-HRP (Tanpaku Seisei Kogyo, Isezaki, Japan) at a 1:2000 dilution. The blots were washed as before and the colour was developed by adding EzWestBlue (ATTO Corp.).…”

“…The mean enzyme activity in buffer G was 121 ± 5% (n = 3) of the corresponding activity in buffer H. The OASTL activity of the GST-CSLP and His-CSA complex that had been eluted in buffer G was normalized using the coefficient shown above. The His-CSA protein level was determined by quantification of immunologically detected bands marked with the anti-spinach OASTL antibody [26] . The OASTL activities of serial dilutions of purified His-CSA protein were determined.…”