Protein A:ACP-I fusion

SOME CHARACTERISTICS OF A PROTEIN A ACP-I FUSION IN PLANT FATTY ACID SYNTHESIS... 

Recombinant ACP-I Protein A ACP-I Fusion fmol product/ Xl enzyme/min... 

Table 1 shows that the fusion protein was active in the standard assay for ACP, but that reactivity was not comparable to E. coli ACP at lower concentrations (up to 4 lg added to 50 X reaction 8.5 XM). We have calculated that the Km app for the fusion ACP was 0.5 mM and the equivalent figure for E. coli ACP was 50 jiM. This 10-fold increase in Km value suggests that the ACP portion of the fusion requires higher concentration to be recognized by the acyl ACP synthetase. This effect is probably caused by the unrecognized portion (Ca 75% by mass) taken up by protein A. During the linear phase of reactivity (i.e., between 1.6 to 4.0 lg 50 it appears that the fusion... 

Table 2 shows the result of using either recombinant ACP-I alone or the fusion protein in a spinach chloroplast extract for de novo FAS (7). In this instance the amount of protein added was based on the acyl-ACP synthetase assay for ACP-equivalent reactivity. Therefore, the amount of protein added was not equivalent (i.e., 4 x more fusion protein at each ACP-equivalent concentration). Our results confirmed previous efforts from our lab (8) that increasing levels of ACP relative to available carbon source caused a decrease in net de novo FAS when recombinant ACP-I was used as co-factor/co-substrate. Surprisingly, the fusion protein did not follow this kinetic... 

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