Particulate glucan synthetase activity: Generation and inactivation after treatments with indoleacetic acid and cycloheximide

The 2-to 4-fold rise in particle-bound 3-glucan synthetase (uridine diphosphate-glucose:,6-1 ,4-glucan glucosyltransferase) activity that can be induced by indoleacetic acid in pea stem tissue is not prevented by concentrations of actinomycin D or cycloheximide that inhibit growth and macromolecule synthesis. The rise is concluded to be a hormonally induced activation of previously existing, reversibly deactivated enzyme. The activation is not a direct allosteric effect of indoleacetic acid or sugars. It is blocked by inhibitors of energy metabolism, by 2-deoxyglucose, and by high osmolarity, but not by Ca'+ at concentrations that inhibit auxin-induced elongation and prevent promotion of sugar uptake by indoleacetic acid, and not by a, a'-dipyridyl at concentrations that inhibit formation of hydroxyproline. Regulation of the system could be due either to an ATP-dependent activating reaction affecting this enzyme, or to changes in levels of a primer or a lipid cofactor.Under conditions previously defined (37), treatment of pea stem tissue with IAA or other auxins causes a rapid, temperature-dependent rise in the level of particle-bound /8-glucan synthetase activity (UDP-glucose:/8-1 ,4-glucan glucosyltransferase) obtainable from the tissue. This report considers how this hormonal effect depends upon cellular metabolism. A somewhat similar investigation of the effect of IAA, applied in lanolin paste, on GDP-glucose-dependent glucan synthetase activity in stem tissue of whole decapitated pea seedlings was published recently (48). METHODSPlant material and glucan synthetase preparation and assay were as described previously (37). Briefly, pea stem segments 8 mm long cut from the region of maximal elongation rate were kept in a moist atmosphere at 35 C for 2.5 to 3 hr to reduce glucan synthetase activity substantially below the initial level; then a medium containing usually 30 mm sucrose was added and incubation at 35 C was continued for 1 hr. Then the tissue was treated with plus-or minus-IAA media at a specified temperature. In the case of inhibitor experiments the media also contained 16 mm K phosphate buffer, pH 6.1, and 'Supported by a grant from the National Science Foundation. when necessary to ensure establishment of the appropriate inhibition by the time of addition of IAA the sucrose pretreatment media also contained the inhibitor, as specified for the experiments reported. At the end of the treatment period the tissue was chilled on ice, washed with ice water, and used for preparation and assay of glucan synthetase (37).In Vivo Isotope Incorporation Experiments. Samples of 50 segments were incubated in 3.0 ml of labeled substrate in 16 mM K phosphate buffer, pH 6.1, on a reciprocating shaker (120 oscillations/min) at 25 C. At the conclusion of incubation the segments were washed thoroughly with ice water. For assay of incorporation from sugars into particles and cell wall the segments were ground in the same manner (37) as for glucan synthetase assay; the cell wall residue (1,OOOg) and particle pe...

Section: Data Incontrasting

confidence: 99%

“…This report considers how this hormonal effect depends upon cellular metabolism. A somewhat similar investigation of the effect of IAA, applied in lanolin paste, on GDP-glucose-dependent glucan synthetase activity in stem tissue of whole decapitated pea seedlings was published recently (48).…”

mentioning

confidence: 89%

Regulation of β-Glucan Synthetase Activity by Auxin in Pea Stem Tissue

Ray

1973

“…This could be attributableto physical damage or disruption (e.g., dissociation of enzyme from primer) of surface enzyme complexes or to release of intracellular inactivators (22). In either event, present data demonstrates that initial rates of cellulose synthetase activity assayed with UDP-glucose in tissue slices (up to 5.6 ,ug glucose incorporated/hr.…”

Section: Resultsmentioning

confidence: 59%

“…Finally, it should be pointed out that studies of effects of hormones and antibiotics on levels of particulate glucan synthetase activity (14,22) have generally assumed that the changes observed result from altered biosynthesis or metabolic turnover of essential components of the active enzyme comPlant Physiol. Vol.…”

Section: Resultsmentioning

confidence: 99%

The Site of Cellulose Synthesis

Shore¹,

Raymond²,

Maclachlan³

1975

“…The intent was also to examine their properties, bearing in mind that it might be possible to exploit a natural activator and minimize effects of any destructive agent during tissue homogenization in order to prepare a more effective ,8-glucan synthetase complex for studies in vitro.Brief reports have appeared indicating that crude extracts from pea epicotyl (12,13,20) and lupin hypocotyl (21) contain a heatlabile component which suppresses membrane-bound 81-glucan synthetase activity, and a heat-stable component which enhances it. Little is known of the specificities or modes of action of these factors beyond the observations that they have different effects on the products synthesized from UDP-glucose and GDP-glucose (13), implying specificity, and the labile component appears to act with a pH optimum close to 7 (20).…”

mentioning

confidence: 99%

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

Chao

Maclachlan²

1978

(14,17) that at least part of total fl-glucan synthetase activity is subject to rapid turnover in vivo.The aims of the present study were, in part, to assess the potential of the pea extract components to act as synthetasemoderating regulators in vivo. The intent was also to examine their properties, bearing in mind that it might be possible to exploit a natural activator and minimize effects of any destructive agent during tissue homogenization in order to prepare a more effective ,8-glucan synthetase complex for studies in vitro.Brief reports have appeared indicating that crude extracts from pea epicotyl (12,13,20) and lupin hypocotyl (21) contain a heatlabile component which suppresses membrane-bound 81-glucan synthetase activity, and a heat-stable component which enhances it. Little is known of the specificities or modes of action of these factors beyond the observations that they have different effects on the products synthesized from UDP-glucose and GDP-glucose (13), implying specificity, and the labile component appears to act with a pH optimum close to 7 (20). It is possible that these components represent natural regulators of cellulose or callose synthesis in a manner analogous to the role postulated for specific proteases in controlling fungal chitin (3, 9) and f8-1,3-glucan (23) synthetase activities, as well as turnover of numerous other plant enzymes. It is also possible that the net deleterious effect of crude extracts on synthetase activity, which is particularly evident when assays are conducted with tissue slices (13), i.e. cell surface synthetase (15, 18), is responsible in part for the difficulties encountered in achieving rates of f,-glucan synthesis by isolated particulate fractions comparable to those observed in vivo (7,13,15,18 MATERIALS AND METHODSThe source of growing tissue used throughout this study is the apical 10 mm, defined here as a "segment" (about 25 mg fresh wt), cut from the third internode of pea epicotyls. Seedlings were grown as described earlier (15,18). Methods for assaying fB-glucan synthetase activity in this tissue are based on previous results (17,18,20) and basic information on optimal conditions (15