A re-examination of the kinetic properties of UDP-glucose: (1-.3)-i% glucan (callose) synthases from mung bean seedlings ( Vigna radiata) and cotton fibers (Gossypium hirsutum) shows that these enzymes have a complex interaction with UDP-glucose and various effectors. Stimulation of activity by micromolar concentrations of Ca2" and millimolar concentrations of j-glucosides or other polyols is highest at low (<100 micromolar) UDP-glucose concentrations. These effectors act both by raising the V. of the enzyme, and by lowering the apparent K,. for UDPglucose from >1 millimolar to 0.2 to 0.3 millimolar. Mg2" markedly enhances the affinity of the mung bean enzyme for Ca2" but not for fglucoside; with saturating Ca2', Mg2' only slightly stimulates further production of glucan. However, the presence of Mg2' during synthesis, or NaBH. treatment after synthesis, changes the nature of the product from dispersed, alkali-soluble fibrils to highly aggregated, alkali-insoluble fibrils. Callose synthesized in vitro by the Ca24, #-glucoside-activated cotton fiber enzyme, with or without Mg2', is very similar in size to callose isolated from cotton fibers, but is a linear (1-.3)-#-glucan lacking the small amount of branches at C0-6 found in vivo. We conclude that the high degree of aggregation of the fibrils synthesized with Mg2' in vitro is caused either by an alteration of the glucan at the reducing end or, indirectly, by an effect of Mg2' on the conformation of the enzyme. Rate-zonal centrifuption of the solubilized mung bean callose synthase confirms that divalent cations can affect the size or conformation of this enzyme.Essentially all higher plants contain a UDP-glucose: (1-+3)-f3-glucan (callose) synthase. This enzyme is largely found on the plasma membrane, and in most cases is latent and only becomes activated by perturbed conditions which lead to some loss of membrane permeability (5, 16). Some years ago, Ray (26) named this enzyme Glucan Synthetase II, and assayed it at high concentrations of UDP-Glc in the absence of divalent cations, although others have reported that activity can be enhanced by Mg2' (6),

In pollen tubes of Nicotiana alata, a membrane-bound, Ca2+-independent callose synthase (CalS) is responsible for the biosynthesis of the (1,3)-β-glucan backbone of callose, the main cell wall component. Digitonin increases CalS activity 3- to 4-fold over a wide range of concentrations, increasing the maximum initial velocity without altering the Michaelis constant for UDP-glucose. The CalS activity that requires digitonin for assay (the latent CalS activity) is not inhibited bythe membrane-impermeant, active-site-directed reagent UDP-pyridoxal when the reaction is conducted in the absence of digitonin. This is consistent with digitonin increasing CalS activity bythe permeabilization of membrane vesicles. A second group of detergents, including 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS), Zwittergent 3–16, and 1-α--lysolecithin, activate pollen tube CalS 10- to 15-fold, but only over a narrow range of concentrations just below their respective critical micellar concentrations. This activation could not be attributed to any particular chemical feature of these detergents. CHAPS increases maximum initial velocity and decreases the Michaelis constant for UDP-glucose and activates CalS even in the presence of permeabilizing concentrations of digitonin. Inhibition studies with UDP-pyridoxal indicate that activation by CHAPS occurs by recruitment of previously inactive CalS molecules to the pool of active enzyme. The activation of pollen tube CalS by these detergents therefore resembles activation of the enzyme by trypsin.

We have identified a 52 kilodalton polypeptide as being a likely candidate for the catalytic subunit of the UDP-glucose: (1-..3)-,Bglucan (callose) synthase of developing fibers of Gossypium hirsutum (cotton). Such a polypeptide migrates coincident with callose synthase during glycerol gradient centrifugation in the presence of EDTA, and can be directly photolabeled with the radioactive substrate, a-[32PJUDP-glucose. Interaction with the labeled probe requires Ca2 , a specific activator of callose synthase which is known to lower the Km of higher plant callose synthases for the substrate UDP-glucose. Using this probe and several other related ones, several other proteins which interact with UDP-glucose were also identified, but none satisfied all of the above criteria for being components of the callose synthase.The glycosyltransferase UDP-glucose: (l-3)-3-glucan synthase (callose synthase) is an intriguing enzyme in higher plants for a variety of reasons. Located in the plasma membrane, it is normally latent and only becomes activated under conditions of stress such as mechanical damage or pathogen invasion; however, synthesis of callose can also occur without apparent stress, for example, at the cell plate and surrounding plasmodesmata, in pollen tubes, and in cotton fibers at one stage of development (for reviews, see refs. 3, 5, 14), and the question of whether this enzyme may share subunits with the elusive cellulose synthase of plants has been raised several times (3,12 (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.Several other techniques of labeling polypeptides may also provide information relevant to 13-glucan synthesis in plants. Our group (23) reported that a 73 kD mung bean membrane polypeptide could bind with high affinity, but apparently not react with, UDP-['4C]glucose, after renaturation of polypeptides from SDS gels; we also found a 44 kD polypeptide which was apparently self-glycosylated with one residue of glucose following incubation of mung bean membranes with Mg2+ and UDP-['4C]glucose. Since the glucose moeity showed turnover in pulse-chase experiments, this polypeptide could be a candidate for a primer protein in glucan synthesis. This polypeptide may relate to a 40 kD self-glycosylating polypeptide found by Ingold and Seitz (11) in both soluble and membrane fractions of Daucus carota L, the soluble form of which has now been purified (21), but whose function remains unclear.This paper reports our fu...

The herbicide 2,6-dichlorobenzonitrile (DCB) is an effective and apparently specific inhibitor of cellulose synthesis in higher plants. We have synthesized a photoreactive analog of DCB (2, for use as an affinity-labeling probe to identify the DCB receptor in plants. This analog retains herbicide activity and inhibits cellulose synthesis in cotton fibers and tobacco cells in a manner similar to DCB.When cotton fiber extracts are incubated with IHIDCPA and exposed to ultraviolet light, an 18 kilodalton polypeptide is specifically labeled. About 90% of this polypeptide is found in the 100,000g supernatant, the remainder being membrane-associated. Gel filtration and nondenaturing polyacrylamide gel electrophoresis of this polypeptide indicate that it is an acidic protein which has a similar size in its native or denatured state.The amount of 18 kilodalton polypeptide detectable by IHiDCPA-labeling increases substantially at the onset of secondary wall cellulose synthesis in the fibers. A similar polypeptide, but of lower molecular weight (12,000), has been detected upon labeling of extracts from tomato or from the cellulosic alga Chara corallina. The specificity of labeling of the 18 kilodalton cotton fiber polypeptide, coupled with its pattern of developmental regulation, implicate a role for this protein in cellulose biosynthesis. Being, at most, only loosely associated with membranes, it is unlikely to be the catalytic polypeptide of the cellulose synthase, and we suggest instead that the DCB receptor may function as a regulatory protein for f-glucan synthesis in plants. synthesis of a cell wall component, that for the hydroxyprolinerich protein extensin, has been identified and cloned (4, 5). No genes involved in synthesis of the carbohydrate polymers of the wall have been identified, due primarily to the limited information available concerning the enzymes involved in plant cell wall synthesis (10). In the case of the most abundant polymer, cellulose, it has not yet been possible even to demonstrate convincing synthesis of this glucan in vitro (9). This report describes a strategy, alternative to enzyme characterization, for the identification of a polypeptide which may be involved in cellulose synthesis in higher plants.The herbicide DCB3 is now recognized as an effective and apparently specific inhibitor of cellulose synthesis in algae and higher plants (10 Effects of DCB and DCPA in Vivo. Cotton ovules, with their associated fibers, were cultured for 17 d post-anthesis. The ovules and fibers were carefully removed from flasks and drained on paper towels, rinsed in culture medium lacking glucose, and 4 ovules per incubation were placed in 2 ml of the same medium lacking glucose with or without DCB or DCPA. DCB and DCPA were dissolved in dimethylsulfoxide and diluted into the medium to give the appropriate concentration. All incubations, including controls, were then adjusted to give a final concentration of dimethylsulfoxide of 0.4% (v/v). Following preincubation in the dark for 15min at 30°C,[14C]gl...

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...