Rat kidney UDPG: alpha-1,4-glucan alpha-4-glucosyltransferase

Abstract

The enzyme systems responsible for the biosynthesis of glycogen (UDPG: a-1,4- glucan a-4-glucosyltransferase) can be extracted mainly as a D form (dependent on glucose-6-P for activity). Because of the low glycogen content of this tissue, the enzyme becomes soluble after centrifugation at 100,000 X g for 90-120 minutes. The enzyme can be collected as a particulate fraction if high molecular weight glycogen is added to the soluble fraction, and then centrifuged again for 90 minutes at 100,000 X g.

Both D and the very low I activities increase by preincubation of crude extracts in the presence of mercaptoethanol. D activity rises quickly and stabilizes after the first 15 minutes. I activity increases slowly and during longer periods but a complete D to I transformation is not obtained although the ratio of activities + glucose-6-P/-glu- cose-6-P decreases to some extent indicating some D to I form transformation.

Increases of both the D and I activities can be prevented by addition of 50 mM NaF to the extracts, indicating that these reactions are brought about by phosphopro- tein-phosphatases.

Mg ions do not stimulate the D activity or its increase by preincubation. In fact they prevent this increase and even induce an inactivation of this form during time of preincubation. I activity is slightly stimulated by this cation at 0 time of preincubation but the rate of increase of this activity during preincubation does not seem to be stimulated, or perhaps it is very little.

Addition of ATP an Mg inactivates both I and D activities in a time and concen­ tration of ATP dependent reaction and this reaction reverses with time if the phospha­ tase is not inhibited by the presence of NaF. The inactivating reactions induced by the addition of ATP and Mg can be stopped by a further addition of EDTA strong enough to trap the necessary Mg2+. Then the recovery of both D and I activities can be observed in those preparations whose phosphatases were not inhibited by the presence of NaF. In those which were inhibited the addition of EDTA only produces the arrest of the inactivating reaction, but the recovery of the D or I activity can no longer be seen.

AU these experimental data suggest that the D and I forms of glycogen synthetase extracted from rat kidney can be interconverted in part but also they are able to be transformed into inactive forms, more phosphorylated, that can be reconverted into active by dephosphorylations carried out by phosphatases.