Significant increases in potato Starch-Synthase and Starch-Branching-Enzyme activities by dilution with buffer containing dithiothreitol and polyvinyl alcohol 50 K

We have recently found that the dilution of purified potato Starch-Synthases (SS) and Starch-Branching-Enzymes (SBEs), by a glycine buffer (pH 8.5), containing 1.0 mM dithiothreitol (DTT) and 0.04% (w/v) polyvinyl alcohol (PVA) 50 K, produced a striking and significant increase in activity (mIU/mL and total mIU) when diluted 1→2 to 1→10. As an example, one SS fraction diluted 1→10 went from 259 to 1470 mIU/mL, giving a total of 14,700 mIU. Dilutions of 1→15 usually resulted in a complete loss of activity. Removal of both DTT and PVA also gave the complete loss of activity. Individual removal of just DTT and the removal of just PVA also produced lowered activities on dilution. The addition of the DTT and the PVA back to the diluted fractions did produce an increase in the activity, but never to the extent that occurred when the samples were diluted simultaneously with both DTT and PVA together in the diluting buffer. Dilution of SBE with buffer containing both DTT and PVA, gave moderate increases, with the exception of one fraction that diluted 1→20 gave a significant increase from 18 to 382 mIU/mL and a total of 7640 mIU. It is concluded that there are inactive starch synthesizing enzymes in the purified fractions that are significantly activated by DTT and PVA, giving much greater amounts of enzyme activities.

Dilutions (1 to n) with Standard Buffer, containing10 mM glycine, 2 mM EDTA, 1 mM DTT, and 0.04% (w/v) PVA 50 K.Figure optionsDownload as PowerPoint slideHighlights
► Dilution with pH 8.5 buffer, containing 1.0 mM DTT and 0.04% (w/v) PVA 50 K, gave increases in activities.
► Dilution 1→4 to 1→10 of starch synthesizing enzymes gave significant increases in activities.
► Dilution to 1→15 and beyond gave zero activity.
► Increasing the concentration of DTT from 1.0 to 3.0 mM gave significant increases in activity.
► Removal of both DTT and PVA gave zero activity and removal of 1 of the 2 gave a reduction in activity.