Regulation of Plasma Membrane β-Glucan Synthase from Red Beet Root by Phospholipids

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...

Section: Resultsmentioning

confidence: 99%

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

Read¹,

Delmer²

1987

“…Because activity is restored when the CHAPS levels are diluted to 0.12% or less following solubilization in the presence of chelators, the ability of the enzyme to catalyze callose formation may be directly dependent on reversible changes in the physical state of the enzyme protein complex and the phospholipid that surrounds it. A phospholipid requirement for CS is well established (23,24,26). Divalent cations at millimolar levels not only activate CS and change the solubility in alkali of the glucan product derived from mung bean (12), but also strongly influence the aggregation state of the enzyme.…”

Section: Discussionmentioning

confidence: 99%

Rapid Enrichment of CHAPS-Solubilized UDP-Glucose: (1,3)-β-Glucan (Callose) Synthase from Beta vulgaris L. by Product Entrapment

Harriman²,

Frost³

et al. 1991

Self Cite

Rapid enrichment of CHAPS-solubilized UDP-glucose:(1,3)-#-glucan (callose) synthase from storage tissue of red beet (Beta vulgaris L.) is obtained when the preparation is incubated with an enzyme assay mixture, then centrifuged and the enzyme released from the callose pellet with a buffer containing EDTA and CHAPS (20-fold purification relative to microsomes). When centrifuged at high speed (80,000g), the enzyme can also be pelleted in the absence of substrate (UDP-Glc) or synthesis of callose, due to nonspecific aggregation of proteins caused by excess cations and insufficient detergent in the assay buffer. True time-dependent and substrate-dependent product-entrapment of callose synthase is obtained by low-speed centrifugation (7,000-11,000g) of enzyme incubated in reaction mixtures containing low levels of cations (0.5 millimolar Mg2+, 1 millimolar Ca2 ) and sufficient detergent (0.02% digitonin, 0.12% CHAPS), together with cellobiose, buffer, and UDP-Glc. Entrapment conditions, therefore, are a compromise between preventing nonspecific precipitation of proteins and permitting sufficient enzyme activity for callose synthesis. Further enrichment of the enzyme released from the callose pellet was not obtained by rate-zonal glycerol gradient centrifugation, although its sedimentation rate was greatly enhanced by inclusion of divalent cations in the gradient. Preparations were markedly cleaner when product-entrapment was conducted on enzyme solubilized from plasma membranes isolated by aqueous two-phase partitioning rather than by gradient centrifugation. Product-entrapped preparations consistently contained polypeptides or groups of closely-migrating polypeptides at molecular masses of 92, 83, 70, 57, 43, 35, 31/29, and 27 kilodaltons. This polypeptide profile is in accordance with the findings of other callose synthase enrichment studies using a variety of tissue sources, and is consistent with the existence of a multi-subunit enzyme complex. The procedure known as product entrapment has recently gained widespread application for the rapid and straightforward enrichment of solubilized polysaccharide synthases. Its use has been reported for purification of chitin (13) and cellulose synthase (15), and for partial purification of red beet (11) and mung bean (12) CSs.3 The procedure consists of incubating solubilized enzyme with substrate (UDP-GlcNAc for chitin synthase or UDP-Glc for cellulose and callose synthase) under conditions that allow synthesis of insoluble polymeric product. Enzyme is thought to become trapped within or bound to the resulting meshwork of microfibrils, and is recovered in concentrated form by centrifugation. The polysaccharide synthase can then be released and the procedure repeated.In the course of our work on CS (1 1), however, it became clear that sedimentation of activity and concomitant purification need not actually require formation of any glucan product, but could also occur simply from precipitation of the enzyme in the assay mixture (which differs in composition from th...

“…Membranes were centrifuged at 80,000g for 45 min, resuspended in 50 mM Hepes-NaOH (pH 7.5), and recentrifuged. Pellets were resuspended in 50 mm Hepes-NaOH (pH 7.5), containing 15% glycerol to their initial volume and were assayed for activity, protein, extracted with chloroform/methanol, and assayed for phospholipid phosphorous as described (21 (Fig. IA) and time-dependent (Fig.…”

Section: Recovery Of Phospholipase and Triton X-100 Extracted Membranesmentioning

confidence: 99%

“…Wash solutions were devoid of enzyme activity. Washed pellets were assayed in phosphatidylethanolamine and phosphatidylcholine since positively-charged phospholipids restored activity to glucan synthase that had been depleted ofphospholipids by detergent extraction (20,21). The simple addition of phosphatidylethanolamine (Fig.…”

Section: Recovery Of Phospholipase and Triton X-100 Extracted Membranesmentioning

confidence: 99%

See 1 more Smart Citation

Susceptibility of UDP-Glucose:(1,3)-β-Glucan Synthase to Inactivation by Phospholipases and Trypsin

Sloan

Wasserman²

1989

Self Cite

UDP-glucose:(1,3)-j%-glucan synthase from Beta vulgaris L. was rapidly inactivated by treatment with phospholipases C, D, and A2. Enzyme activity could not be restored to the phospholipasetreated enzyme by the addition of phosphatidylethanolamine or other phospholipids. Membrane-bound and solubilized glucan synthase were also trypsin-labile with inactivation rates equal in the presence or absence of divalent cations or chelators. Gradual activity declines were observed in membranes incubated with divalent cations, but not with chelators.