Control of Ganglioside Biosynthesis by a Rate Limiting UMP-ASE and Product Ganglioside Inhibition
The removal of UMP by UMP-ase is a rate limiting step in the synthesis of GM1 ganglioside by UDP-Galactose: GM2 Ganglioside Galactosyltransferase (I). I and UMP-ase were found to possess similarities not shared by UDP-ase in homogenates of ten day old rat brain. All three enzymes required Mn++ for activation. Ca++ was equally effective in activating UDP-ase, but did not activate I or UMP-ase. The activity of I and UMP-ase were doubled by raising the incubation temperature from 37° to 45° while UDP-ase activity was unaffected by the temperature increase. Preincubation of the homogenate for 20 min with 0. 25% trypsin resulted in a 50% loss of activity for I and UMP-ase and no loss of activity for UDP-ase. Sulfhydryl group reactive reagents NEM or PCMB totally abolished I and UMP-ase activity, while UDP-ase was only partially inhibited by these reagents. I and UMP-ase had a half life of 2 days in isolated microsomes while UDP-ase does not display this lability. UDP-ase had ten times the activity of UMP-ase. UDP also inhibits UMP-ase. It is concluded that during the course of in vitro GM1 biosynthesis where I is inhibited by UDP and UMP, UDP, a product of I is rapidly cleaved by UDP-ase to UMP, which is more slowly removed by UMP-ase and is therefore a rate limiting enzymatic step in ganglioside biosynthesis. Gangliosides can also regulate the activities of their own biosynthetic metalloenzymes by virtue of their affinity for these metal ions. This affinity by product ganglioside for metal ions would in addition control ganglioside biosynthesis by also inhibiting the metal ion activated UDP-ase and the rate limiting UMP-ase. This would prevent the removal of UDP and UMP which are demonstrated inhibitors of the key ganglioside biosynthetic enzyme I.
KeywordsIncubation Mixture Uridine Nucleotide Monium Acetate Ganglioside Biosynthesis Nucleotide Product
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