Inhibition and labeling of red beet uridine 5'diphospho-glucose: (1,3)-β-glucan (callose) synthase by chemical modification with formaldehyde and uridine 5'diphospho-pyridoxal

The effects of the lysine-reactive chemical modification reagents, uridine 5’ diphospho (UDP)-pyridoxal and formaldehyde (HCHO), on the activity of membrane-bound and solubilized UDP-Glc: (1,3)-β-D-glucan synthase (callose synthase) from red beet (Beta vulgaris L.) storage tissue were compared. Exposure to micromolar levels of UDP-pyridoxal, or millimolar levels of HCHO in the presence of NaCNBH₃, resulted in complete enzyme inactivation. Conditions for inhibition of membrane-bound enzyme activity by the two reagents were markedly similar; divalent cations were required for inactivation, and complete protection of activity was obtained with EDTA or EGTA. The substrate, UDP-Glc, protected membrane-bound callose synthase against inactivation by UDP-pyridoxal or HCHO, but protected the solubilized enzyme only against inhibition by UDP-pyridoxal, suggesting that the lysine residue modified by both these reagents is at the enzyme active site, and that the site is more open or has a certain conformational flexibility in the solubilized enzyme. Potential UDP-Glc-binding polypeptides of callose synthase were identified by a two-step labeling procedure. First, nonessential lysine residues were blocked by irreversible modification reaction with HCHO or UDP-pyridoxal in the presence of UDP-Glc to protect lysines at UDP-Glc-binding sites. In the second step, proteins were recovered, reacted with [¹⁴C]-HCHO in the absence of UDP-Glc, and polypeptide labeling patterns analyzed by SDS-polyacrylamide gel electrophoresis and fluorography. This procedure reduced incorporation of label by 5- to 8-fold compared to a procedure omitting the preblocking step, and with enzyme partially purified by solubilization in CHAPS followed by product entrapment, labeling was limited to a small set of polypeptides. Taken together with the results of other studies, the data suggest that one or more polypeptides migrating in the 54–57 kDa region are good candidates for the UDP-Glc-binding components of callose synthase.     

UDP-Glucose: (1,3)-beta-Glucan Synthase from Daucus carota L. : Characterization, Photoaffinity Labeling, and Solubilization

The membrane-bound UDP-glucose-β-(1,3)-glucan synthase from Daucus carota L. was characterized and a solubilization procedure was developed. The enzyme exhibited maximal activity in the presence of 0.75 millimolar Ca²⁺, 0.5 millimolar EGTA, and 5 millimolar cellobiose at pH 7.5 and 30°C at 1 millimolar UDPG. Reaction products were confirmed to be (1,3)-linked glucan. Polypeptides of 150, 57, and 43 kilodaltons were labeled with the photoactivatible affinity label 5-azido-uridine 5′-β-[³²P] diphosphateglucose. Labeling of the 150 and 57 kilodalton polypeptides was completely protected against by 1 millimolar non-radioactive UDPG suggesting that one or both of these polypeptides may represent the UDPG binding subunit of glucan synthase. Carrot glucan synthase was solubilized with the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) in the absence of divalent cations and chelators; however, the percentage of enzyme which could be solubilized showed variability with membrane source. With microsomal membranes, up to 80% of the enzyme was released with 0.7% CHAPS. Solubilized enzyme was stable for at least 9 hours at 4°C. When more highly purified membrane fractions were isolated from sucrose step gradients a slightly different picture emerged. Activity from the 20/30% interface (Golgi and tonoplast enriched) was readily solubilized and expressed. Activity from the 30/40% interface (plasma membrane enriched) was also solubilized; however, it was necessary to add heat inactivated microsomes to assay mixtures for full activity to be expressed. A requirement for endogenous activators is suggested.     

CHAPS Solubilization and Functional Reconstitution of beta-Glucan Synthase from Red Beet Root (Beta vulgaris L.) Storage Tissue

A method for the solubilization and reconstitution of red beet (1,3)-β-d-glucan synthase with the detergent 3-[(3-cholamidopropyl) dimethylammonio]-1-propane sulfonate (CHAPS) was developed. Glucan synthase was effectively solubilized from microsomal or plasma membranes by 0.6% CHAPS in the presence of EGTA and EDTA. Chelators were found essential for effective solubilization and divalent cations inhibitory. A preextraction of membranes with 0.3% CHAPS and 5 millimolar Mg²⁺ prior to the solubilization step was found to remove protein contaminants and increase the specific activity of the solubilized enzyme. Conditions for recovering activity from Sepharose 4B gel filtration columns were defined. Addition of phospholipids and low levels of CHAPS in column elution buffers resulted in complete functional reconstitution with 100% recovery of added activity. Specific activities were increased 20- to 22-fold over microsomes. Active vesicles were recovered by centrifugation. These results provide independent and direct confirmation of the enzyme's requirement for a phospholipid environment.     

(1-->3)-beta-d-Glucan Synthase from Sugar Beet : I. Isolation and Solubilization

A (1→3)-β-glucan synthase has been isolated from petiole tissue of sugar beet (Beta vulgaris L.). Enzyme activity is associated with a membrane fraction with a density of 1.03 grams per cubic centimeter when subjected to isopycnic density gradient centrifugation in Percoll. The reaction product was determined to be a linear (1→3)-β-glucan by methylation analysis and by glucanase digestion. (1→3)-β-Glucan synthase activity is markedly stimulated by Ca²⁺; activation is half-maximal at about 50 micromolar Ca²⁺ and is nearly saturated at 100 micromolar. Other divalent cations tested, Mg²⁺, Mn²⁺, and Sr²⁺, also stimulate enzyme activity but are less effective. Enzyme activity was also stimulated up to 12-fold by β-glucosides. Sirofluor, the fluorochrome from aniline blue, inhibited enzyme activity 95% when included at 1 millimolar. The enzyme was solubilized in Zwittergent 3-14; 85% of total enzyme activity was solubilized in 0.03% detergent and the optimal detergent-to-protein ratio was 0.3 at 3 milligrams per milliliter protein.     

UDP-Glucose: (1-->3)-beta-Glucan Synthases from Mung Bean and Cotton: Differential Effects of Ca and Mg on Enzyme Properties and on Macromolecular Structure of the Glucan Product

A re-examination of the kinetic properties of UDP-glucose: (1→3)-β-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 β-glucosides or other polyols is highest at low (<100 micromolar) UDP-glucose concentrations. These effectors act both by raising the V_max of the enzyme, and by lowering the apparent K_m for UDP-glucose 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 β-glucoside; with saturating Ca²⁺, Mg²⁺ only slightly stimulates further production of glucan. However, the presence of Mg²⁺ during synthesis, or NaBH₄ 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 Ca²⁺, β-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)-β-glucan lacking the small amount of branches at C-0-6 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 centrifugation of the solubilized mung bean callose synthase confirms that divalent cations can affect the size or conformation of this enzyme.     

Relationship between Cottonseed Malate Synthase Aggregation Behavior and Suborganellar Location in Glyoxysomes and Endoplasmic Reticulum

Malate synthase (EC 4.1.3.2) (MS), an enzyme unique to the glyoxylate cycle, was studied in cotyledons of dark-grown cotton (Gossypium hirsutum, L.) seedlings. MS has generally been regarded as a peripheral membrane protein in glyoxysomes and believed by some to be synthesized on rough ER. Immunocyto-chemical localization of MS in both in situ and isolated cottonseed glyoxysomes, however, showed that MS was located throughout the matrix of glyoxysomes, not specifically associated with their membranes. Biochemical data also supported matrix localization. Isolated glyoxysomes were diluted in variously-buffered salt solutions (200 millimolar KCl or 100 millimolar K-phosphate) or detergents (0.1% Triton X-100, 10 millimolar deoxycholate, or 1.0% Triton X-114) and centrifuged to pellet membranes. Greater than 70% of the MS was recovered in supernatants after treatment with salt solutions, whereas generally less than 30% was released following detergent treatments. MS in pellets derived from glyoxysomes burst in low ionic strength buffer solutions was aggregated (observed on rate-zonal gradients). MS released following salt treatments was the 20S nonaggregated form indicating that salt solutions either disaggregated (or prevented aggregation of) glyoxysomal MS rather than releasing it from membranes. We confirmed reports by others that MS comigrated with ER (NADH: cytochrome c reductase) in sucrose (20-40% w/w) gradients buffered with 100 millimolar Tricine (pH 7.5) after 3 hours centrifugation. However, cottonseed MS did not comigrate with ER in gradients buffered with 10 millimolar Hepes (pH 7.0) or 20 millimolar K-phosphate (pH 7.2) after 3 hours centrifugation, or after 22 hours centrifugation in Tricine or Hepes. Collectively, our data with cotton seeds indicate that MS is not a peripheral membrane protein, and that the aggregation behavior of MS (in various buffers) very likely has led to misinterpretations of its putative associations with ER and glyoxysomal membranes.     

Membrane fractionation and enrichment of callose synthase from pollen tubes of Nicotiana alata Link et Otto

The callose synthase (UDP-glucose: 1,3-β-d-glucan 3-β-d-glucosyl transferase; EC 2.4.1.34) enzyme (CalS) from pollen tubes of Nicotiana alata Link et Otto is responsible for developmentally regulated deposition of the cell wall polysaccharide callose. Membrane preparations from N. alata pollen tubes grown in liquid culture were fractionated by density-gradient centrifugation. The CalS activity sedimented to the denser regions of the gradient, approximately 1.18 g · ml⁻¹, away from markers for Golgi, endoplasmic reticulum and mitochondria, and into fractions enriched in ATPase activity and in membranes staining with phosphotungstic acid at low pH. This suggests that pollen-tube CalS is localised in the plasma membrane. Callose synthase activity from membranes enriched by downward centrifugation was solubilised with digitonin, which gave a 3- to 4-fold increase in enzyme activity, and the solubilised activity was then enriched a further 10-fold by product entrapment. The complete procedure gave final CalS specific activities up to 1000-fold higher than those of pollen-tube homogenates. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that several polypeptides co-fractionated with CalS activity through purification, with a polypeptide of 190 kDa being enriched in product-entrapment pellets. Received: 24 September 1997 / Accepted: 12 November 1997     

Proteins in Intercellular Washing Fluids from Leaves of Barley (Hordeum vulgare L.)

Primary leaves of barley were detached, infiltrated with variousbuffers, and centrifuged to yield ‘intercellular washingfluid’ (IWF). Effective pH control of the IWF was obtainedonly with Tris, among all buffers tried. In these liquids, upto 30 proteins were detected by gradient gel electrophoresis.Intracellular protein from injured cells at the cut ends ofleaves was present in IWF but did not contribute significantlyto the total protein recovered in this liquid. The yield ofprotein in the IWF depended on the buffer used for infiltrationand on the concentration of the buffer. Higher concentrationsof buffer yielded more protein. In other experiments leaves were infiltrated with Tris, centrifuged,and then infiltrated a second time with this buffer containingvarious concentrations of the zwitterionic detergent CHAPS,a sulphobetaine derivative of cholate. Gel electrophoresis ofthe IWF obtained after the second centrifugation revealed protein‘bands’ not detected when the detergent had beenomitted from the infiltration buffer. The electrophoretic patternsof protein ‘bands’ in the gels differed dependingon the CHAPS concentration used for infiltration. The effect of CHAPS on plasmalemma integrity was studied byobserving infiltrated tissue with the electron microscope andby treating isolated protoplasts with the detergent. After infiltrationwith CHAPS at 0.6 mM or 2.0 mM no plasmalemma breaks were detectedin leaves, and isolated protoplasts survived exposure to CHAPSat these concentrations for 2 h without bursting. Evidently,CHAPS at these low concentrations did not destroy the integrityof the plasmalemma; the additional protein recovered in theIWF under these conditions probably originated in the cell wall.Infiltration of leaves with 6.0 mM CHAPS resulted in breaksof the plasmalemma, in tissue collapse and leaf tip necrosis.Isolated protoplasts burst within minutes after being exposedto CHAPS at this concentration. Key words: Cell wall permeability, Intercellular space, Detergent, CHAPS, Protoplasts     

Identification of the UDP-glucose-binding polypeptide of callose synthase from Beta vulgaris L. by photoaffinity labeling with 5-azido-UDP-glucose

The photoaffinity probe 5-azidouridine 5'-[beta-32P]diphosphate glucose (5N3[32P]UDP-Glc) was used to identify a 57-kDa polypeptide as a strong candidate for the UDP-Glc-binding polypeptide of UDP-glucose: (1,3)-beta-glucan (callose) synthase from red beet (Beta vulgaris L.) storage tissue. Unlabeled 5N3UDP-Glc was a competitive inhibitor of callose synthase with a Ki of 310 microM. Callose synthase was purified from plasma membranes by a two-step solubilization with 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate, followed by product entrapment, and photoincorporation of radioactivity from 5N3[32P]UDP-Glc was used to identify UDP-Glc-binding polypeptides that copurified with callose synthase activity. Photoinsertion into the 57-kDa band was closely correlated with all catalytic properties examined. Photolabeling of the 57-kDa polypeptide was enriched upon purification of callose synthase by product entrapment, was abolished with increasing levels of unlabeled UDP-Glc, was dependent upon the presence of divalent cations, and the pH dependence of photolabeling correlated with the pH activity profile of callose synthase. In addition, photolabeling of the 57-kDa band did not occur after phospholipase treatment, which destroys enzyme activity. The extent of labeling of this polypeptide thus correlates closely with the activity of callose synthase under a wide variety of conditions. These results imply that the polypeptide at 57 kDa represents the substrate-binding and cation-regulated component of the callose synthase complex of higher plants.     

(1,3)-β-Glucan synthase fromSaccharomyces cerevisiae: In vitro activation byβ-lactoglobulin or Brij-35, and photoaffinity labeling of enriched microsomal fractions with 5-azido-UDP-Glc and 8-azido-GTP

Two new activators of (1,3)--glucan synthase fromSaccharomyces cerevisiae were identified, and a procedure for preparing enriched enzyme fractions by removal of peripheral membrane proteins and entrapped soluble proteins was developed. Microsomal enzyme activity, known to be enhanced by bovine serum albumin (BSA), was stimulated threefold by both -lactoglobulin and Brij-35. Both apparently substituted for BSA, since no synergistic effects were observed with activators added in combination. Successive washings of microsomal fractions with the detergents Brij-35 and Tergitol NP-40 to remove peripheral and vesicle-entrapped proteins yielded particulate fractions five-fold enriched in glucan synthase activity. GTP, an important effector of glucan synthase, improved purification of the enzyme during detergent extractions. Various membrane fractions were photolabeled with 5[³²P]N₃UDP-Glc or 8N₃[³²P]GTP, and potential UDP-Glc and GTP-binding polypeptides were identified. However, further enrichment will be required to determine which of these might represent subunits of the yeast glucan synthase complex.     

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

We have identified a 52 kilodalton polypeptide as being a likely candidate for the catalytic subunit of the UDP-glucose: (1→3)-β-glucan (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, α-[³²P]UDP-glucose. Interaction with the labeled probe requires Ca²⁺, a specific activator of callose synthase which is known to lower the K_m 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.     

A comparison of glutamate synthase obtained from maize endosperms and roots

Glutamate synthase (EC 2.6.1.53) has been examined in developing endosperms and roots of maize. KCl is required for maximum activity in each tissue. The effect with KCl is seen with buffer strength of 25 to 100 millimolar in the assay. The optimum concentration for the enzyme from endosperm is 20 millimolar and for the enzyme from root tissue the saturating concentration is about 20 millimolar. In root material the enzyme is labile but activity can be restored if KCl is added to the assay. Divalent cations such as Mg²⁺ or Mn²⁺ also activate the enzyme to some extent.     

Enzymic and protein character of tonoplast from hippeastrum vacuoles

The membrane of anthocyanin containing Hippeatrum petal vacuoles was examined for protein and enzyme content after purification by equilibrium density centrifugation. Light scattering, protein, and a Mg²⁺-dependent nucleotide specific ATPase were associated with membrane having a density of 1.08 to 1.12 grams per cubic centimeter. A small amount of acid phosphatase was also present in this region of the gradient, but this activity peaked at about 1.12 grams per cubic centimeter. A component of yeast tonoplast, α-mannosidase, was not significantly present. UDP-glucose, anthocyanidin-3-O-glucosyltransferase, thought to be a cytosol enzyme in Hippeastrum, was absent from tonoplast of vacuoles isolated by osmotic shock in 0.2 molar K₂HPO₄ or 0.35 molar mannitol. Vacuolar acid phosphatase was insensitive to ethylenediaminetetraacetate but was 80% inhibited by 10 millimolar KF, while ATPase was inactivated by 2 millimolar ethylenediaminetetraacetate and only 50% inhibited by 10 millimolar KF. Five major and about 9 minor polypeptides were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membrane protein on 5 to 30 and 6 to 16% gradient gels.     

LOCALIZATION OF ENZYMES WITHIN MICROBODIES

Microbodies from rat liver and a variety of plant tissues were osmotically shocked and subsequently centrifuged at 40,000 g for 30 min to yield supernatant and pellet fractions. From rat liver microbodies, all of the uricase activity but little glycolate oxidase or catalase activity were recovered in the pellet, which probably contained the crystalline cores as many other reports had shown. All the measured enzymes in spinach leaf microbodies were solubilized. With microbodies from potato tuber, further sucrose gradient centrifugation of the pellet yielded a fraction at density 1.28 g/cm³ which, presumably representing the crystalline cores, contained 7% of the total catalase activity but no uricase or glycolate oxidase activity. Using microbodies from castor bean endosperm (glyoxysomes), 50–60% of the malate dehydrogenase, fatty acyl CoA dehydrogenase, and crotonase and 90% of the malate synthetase and citrate synthetase were recovered in the pellet, which also contained 96% of the radioactivity when lecithin in the glyoxysomal membrane had been labeled by previous treatment of the tissue with [¹⁴C]choline. When the labeled pellet was centrifuged to equilibrium on a sucrose gradient, all the radioactivity, protein, and enzyme activities were recovered together at peak density 1.21–1.22 g/cm³, whereas the original glyoxysomes appeared at density 1.24 g/cm³. Electron microscopy showed that the fraction at 1.21–1.22 g/cm³ was comprised of intact glyoxysomal membranes. All of the membrane-bound enzymes were stripped off with 0.15 M KCl, leaving the "ghosts" still intact as revealed by electron microscopy and sucrose gradient centrifugation. It is concluded that the crystalline cores of plant microbodies contain no uricase and are not particularly enriched with catalase. Some of the enzymes in glyoxysomes are associated with the membranes and this probably has functional significance.     

Properties of a Partially Purified Nucleoside Triphosphatase (NTPase) from the Chloroplast Envelope of Pea

The Mg-nucleoside triphosphatase activity associated with the inner envelope membrane of the pea chloroplast is comprised of at least two components, a major activity that is sensitive to vanadate and sodium fluoride and a minor insensitive activity. The vanadate/fluoride sensitive activity has been partially purified (about 35-fold) from Triton X-100 solubilized membranes by DEAE-Sephadex chromatography and sucrose density gradient centrifugation. The partially purified enzyme resembles the membrane-bound activity in requiring either Mg²⁺ or Mn²⁺, having a broad specificity for nucleoside triphosphates, having a K_m for ATP of 0.18 millimolar, and being inhibited by N-ethylmaleimide, but insensitive to sodium azide and dicyclohexylcarbodiimide. The partially purified enzyme obtained after sucrose gradient centrifugation has a markedly increased sensitivity to inhibition by inorganic pyrophosphate compared with the less pure enzyme. Pyrophosphate is not a substrate of either the membrane-bound or partially purified enzyme.     

Purification and Characterization of a Chloroplast Outer-Envelope-Bound, ATP-Dependent Protein Kinase

An ATP-dependent protein kinase was partially purified from isolated outer envelope membranes of pea (Pisum sativum L., Progress No. 9) chloroplasts. The purified kinase had a molecular weight of 70 kilodaltons, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was of the cyclic nucleotide and Ca²⁺, calmodulin-independent type. The purification involved the detergent solubilization of purified outer envelopes by 0.5% cholate and 1% octylglycoside, followed by centrifugation on a linear 6 to 25% sucrose gradient. Active enzyme fractions were further purified by affinity chromatography on histone III-S Sepharose 4B and ion exchange chromatography on diethylaminoethyl cellulose. The protein kinase eluted at 100 millimolar and 50 millimolar NaCl, respectively. The protein kinase was essentially pure as judged by Western blot analysis. The enzyme has a K_M of 450 micromolar for ATP and a V_max of 25 picomoles of ³²P incorporated into histone III-S per minute per microgram. Inhibition by ADP is competitive (K_i 150 micromolar).     

Fructokinase (Fraction III) of Pea Seeds

A second fructokinase (EC 2.7.1.4) was obtained from pea seed (Pisum sativum L. var. Progress No. 9) extracts. The enzyme, termed fructokinase (fraction III), was specific for fructose and had little activity with glucose. With fructose concentrations above 0.25 millimolar, there was strong substrate inhibition at the optimum pH (8.0) and also at pH 6.6. The apparent K_m values at pH 8.0 for fructose and glucose were 0.06 millimolar and 0.14 millimolar, respectively. The apparent K_m for Mg adenosine 5′-triphosphate (MgATP) was 0.06 millimolar and excess MgATP was inhibitory. Mg²⁺ was essential for activity but the enzyme was inhibited by excess Mg²⁺ or ATP. Mg adenosine 5′-pyrophosphate was also inhibitory. Activity was stimulated by the addition of monovalent cations: of those tested K⁺, Rb⁺, and NH₄⁺ were the most effective. The possible role of fructokinase (fraction III) is discussed.     

Isolation and polypeptide composition of l,3-ß-glucan synthase from plasma membranes of Brassica oleracea

The l,3-ß-glucan synthase (callose synthase, EC 2.4.1.34) was solubilized from cauliflower ( Brassica oleracea L.) plasma membranes with digitonin, and partially purified by ion exchange chromatography and gel filtration [fast protein liquid chromatography (FPLC)] using 3-[(cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS) in the elution buffers. These initial steps were necessary to obtain specific precipitation of the enzyme during product entrapment, the final purification step. Five polypeptides of 32, 35, 57, 65 and 66 kDa were highly enriched in the final preparation and are thus likely components of the callose synthase complex. The purified enzyme was activated by Ca²⁺, spermine and cellobiose in the same way as the enzyme in situ, indicating that no essential subunits were missing. The polyglucan produced by the purified enzyme contained mainly 1,3-linked glucose.     

Hydroxymethylglutaryl coA reductase (NADPH) in the latex of Hevea brasiliensis

The activity of hydroxymethylglutaryl CoA reductase (NADPH) (EC 1.1.1.34) was studied in the latex of regularly tapped mature trees of Hevea brasiliensis. The reductase activity was found mainly (95% of the total activity) in the pellet fraction (40 000 g) of the centrifuged latex. The enzyme in this fraction had a specific requirement for NADPH as the cofactor and, while not obligatory for activity, was activated by dithiothreitol at the optimum concentration of 2 mM. The pH optimum was found to be 6.6–6.9 in 0.1 M phosphate buffer. Mevalonate and CoA (at 2 mM each) did not affect enzyme activity, while hydroxymethylglutarate (2 mM) was slightly inhibitory. p-Chloromercuribenzoate (1 mM) completely inhibited this enzyme. The reductase activity in the 40 000 g pellet was not easily solubilized either using Triton X-100 or by sonication. The apparent K_m for the washed, membrane-bound enzyme (103 000 g pellet) was 56 μ M (RS-HMG-CoA). Magnesium-ATP (4 mM) inactivated the reductase but this effect was greatly diminished or was absent upon washing the 40 000 g pellet.     

Biosynthesis of (1-->3)-beta-D-glucan (callose) by detergent extracts of a microsomal fraction from Arabidopsis thaliana.

The aim of this work was to develop a biochemical approach to study (1-->3)-beta-D-glucan (callose) biosynthesis using suspension cultures of Arabidopsis thaliana. Optimal conditions for in vitro synthesis of callose corresponded to an assay mixture containing 50 mM Mops buffer, pH 6.8, 1 mM UDP-glucose, 8 mM Ca2+ and 20 mM cellobiose. The enzyme was Ca2+-dependent, and addition of Mg2+ to the reaction mixture did not favour cellulose biosynthesis. Enzyme kinetics suggested the existence of positive homotropic cooperativity of (1-->3)-beta-D-glucan synthase for the substrate UDP-glucose, in agreement with the hypothesis that callose synthase consists of a multimeric complex containing several catalytic subunits. Detergents belonging to different families were tested for their ability to extract and preserve membrane-bound (1-->3)-beta-D-glucan synthase activity. Cryo-transmission electron microscopy experiments showed that n-octyl-beta-D-glucopyranoside allowed the production of micelle-like structures, whereas vesicles were obtained with Chaps and Zwittergent 3-12. The morphology and size of the (1-->3)-beta-D-glucans synthesized in vitro by fractions obtained with different detergents were affected by the nature of the detergent tested. These data suggest that the general organization of the glucan synthase complexes and the properties of the in vitro products are influenced by the detergent used for protein extraction. The reaction products synthesized by different detergent extracts were characterized by infrared spectroscopy, methylation analysis, 13C-NMR spectroscopy, electron microscopy and X-ray diffraction. These products were identified as linear (1-->3)-beta-D-glucans having a degree of polymerization higher than 100, a microfibrillar structure, and a low degree of crystallinity.