Soluble Factors in Pisum Extracts Which Moderate Pisum β-Glucan Synthetase Activity

Abstract

Homogenates of growing regions of the pea (Pisum sativum L.) epicotyl contain soluble factors (130,000g supernatant) which alter pea β-glucan synthetase activity, as assayed using the substrate UDP-glucose and either particulate fractions or tissue slices as source of enzyme. A heat-stable dialyzable component is present which enhances as much as 3-fold the synthesis of alkali-soluble and -insoluble products from millimolar levels of substrate. A heat-labile nondialyzable component is also present which suppresses synthesis. This component dominates (the net effect of total crude extract) when low (μm) levels of substrate are employed. Methylation analysis shows that both components primarily affect the proportion of β-1,4 rather than β-1,3 linkages which are synthesized. The enhancing factor increases V_max of the synthetase system and only activates in the presence of high levels of substrate. The suppressing factor appears to inactivate the synthetase, since losses of product or substrate are not significant during brief incubation with extract, the factor acts progressively with time with a pH optimum, and it destroys activity during preincubation with particles or slices. It co-precipitates with a protease (gelatinase) at between 20% and 40%-saturated (NH₄)₂SO₄, and it co-fractionates with a major component of total protease on Sephadex gel columns (G-200) with an elution volume corresponding to molecular weight 65,000. The concentrations of these factors are such that they could be natural moderators of synthetase activity in vivo if the two were ever brought in contact, and the inactivator could account for the lability of β1,4-glucan synthetase which occurs upon tissue homogenization.