Inhibition of Mung Bean UDP-Glucose: (1→3)-β-Glucan Synthase by UDP-Pyridoxal

Abstract

UDP-pyridoxal competitively inhibits the Ca2`-, cellobiose-activated (1-.3)-#-glucan synthase activity of unfractionated mung bean (Vigna radiata) membranes, with a K, of 3.8 1 0.7 micromolar, when added simultaneously with the substrate UDP-glucose in brief (3 minute) assays. Preincubation of membranes with UDP-pyridoxal and no UDPglucose, however, causes progressive reduction of the V,., of subsequently assayed enzyme and, after equilibrium is reached, 50% inhibition occurs with 0.84 ± 0.05 micromolar UDP-pyridoxal. This progressive inhibition is reversible provided that the UDP-pyridoxylated membranes are not treated with borohydride, indicating formation of a Schiff's base between the inhibitor and an enzyme amino group. Consistent with this, UDP-pyridoxine is not an inhibitor. The reaction of (I-.3)-t,-glucan synthase with UDP-pyridoxal is stimulated strongly by Ca2 and, less effectively, by cellobiose or sucrose, and the enzyme is protected against UDP-pyridoxal by UDP-glucose or by other competitive inhibitors, implying that modification is occurring at the active site. Pyridoxal phosphate is a less potent and less specific inhibitor. Latent (1-.3)-#-glucan synthase activity inside membrane vesicles can be unmasked and rendered sensitive to UDP-pyridoxal by the addition of digitonin. Treatment of membrane proteins with UDP4[3Hlpyridoxal and borohydride labels a number of polypeptides but labeling of none of these specifically requires Ca2' and sucrose; however, a polypeptide of molecular weight 42,000 is labeled by UDP4-3Hjpyridoxal in the presence of Mg2" and copurifies with (1-_3)-O-glucan synthase activity.All living plant cells appear to make the (l1--3)-fl-glucan callose at their plasma membrane primarily as a wound-response, whether the wound is mechanical, due to assault by a pathogen, or due to various other kinds of stress (1, 10, 12, and references therein). In addition, callose is found at particular times in the differentiation of certain unwounded cells, such as cotton fibers, pollen tubes, sieve plates, and the cell plate (5). Successful in vitro assay of (1--3)-fl-glucan synthase at rates approaching physiological polymerization rates requires that the enzyme be activated by micromolar levels of Ca2l and by a ,B-glucoside or sucrose (8,12,19,29 (23), and is of large size but to date has only been partially purified (6,8).At the molecular level, little is known about any ofthe enzymes that catalyze the synthesis of polysaccharides in higher plants. We therefore decided to attempt to identify the catalytic polypeptide of the (1--3)-f3-glucan synthase enzyme protein in unfractionated membranes by a chemical modification approach. Pyridoxal-P has frequently been used as a modifying reagent for lysine residues at the active sites of purified enzymes (1 1, 15, and references therein), and reduction of the resultant Schiffrs bases with borohydride gives stable e-N-pyridoxyl-lysine residues. Addition of a nucleoside moiety can direct pyridoxal -P to nucleotide-binding sites (27, 28); th...

Separation and Partial Purification of 1,3-β-Glucan and 1,4-β-Glucan Synthases from Saprolegnia

“…A multitude of factors (Ca2", Mg2", proteases, nucleotides, phosphatases) have been identified as potential regulators of these enzymes (7,10,11,13,18,19). Purified enzymes would allow a new appraisal of the role of these factors.…”

“…By using various techniques (gradient density centrifugation, column chromatography, electrophoresis) it has been shown that glucan synthases are large protein complexes (>450 kD), and several protein subunits ranging from 18 to 83 kD might be involved in glucan synthesis (5,16,19).…”

Callose synthesis during reproductive development in monocotyledonous and dicotyledonous plants

“…[15][16][17][18] The molecular mass of the catalytic complex in plants has been further defined as having a molecular mass of 32-57 kDa by affinity labeling. [19][20][21][22][23] CalS combines to form a heteromeric complex with phragmoplastin, Ugt1, Rho1-like protein, and, possibly, annexin. [24][25][26] it appears that plant Rop1 controls CalS via UGT.…”

Direct Photolabeling with [32P]UDP-Glucose for Identification of a Subunit of Cotton Fiber Callose Synthase

“…Read and Delmer (22) showed that the substrate analog UDP-pyridoxal, first used to identify the active site of glycogen synthase (25), is an effective inhibitor of callose synthase, apparently through formation of a Schiffs base with an active-site lysine; because inhibition was rendered irreversible after reduction of the Schiffs base with 'This work was supported by contract DE-ACO2- 76ERO-1338 from The U.S. Department of Energy via a subcontract from Michigan State University, East Lansing, MI, and by a grant from the German-Israeli Foundation for Scientific Research and Development (GIF). 2 Present address: Plant Cell Biology Research Centre, School of Botany, The University of Melbourne, Parkville, Vic.…”

“…sodium borohydride, this analog offered the hope that the catalytic subunit might be identified by covalent coupling to the active site. There have been several reports of use of UDP-[3H]pyridoxal as labeled probe; in the first (22), a polypeptide of 42 kD from mung bean membranes was labeled, but labeling did not meet all the criteria expected for the catalytic subunit of callose synthase; a second report (19) indicated that a number of membrane proteins from red beet could be labeled; when the specificity of labeling was improved by a substrate protection technique, these were reduced to labeling ofpolypeptides of 200, 76, 60, and 57 kD. Wasserman's group also demonstrated the feasibility of using 5-azido-[32P]UDPglucose as an affinity labeling probe (16) and, in subsequent work using this probe (9), concluded that the 57 kD polypeptide of red beet is the most likely candidate for catalytic subunit of the enzyme based upon its enrichment upon product entrapment (see 13), its pH optimum for labeling, and its effector requirements.…”