Synthesis of Fibrils in Vitro by a Solubilized Cellulose Synthase from
            <i>Acetobacter xylinum</i>

Lin, Fong Chyr; Brown, R. Malcolm; Cooper, J. B.; Delmer, Deborah P.

doi:10.1126/science.230.4727.822

Cited by 60 publications

(15 citation statements)

References 12 publications

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“…There are three possibilities for the decreased number of cellulose molecules in the brittle cell walls: (a) involvement of a brittle gene in the number of cellulose-forming complexes. Itoh et al (13) reported that the estimated number ofterminal complexes is nearly similar to the estimated number ofglucan chains within the microfibrils; (b) impediment in the assembly process of cellulose-forming complexes; and (c) decrease in UDP-Glc precursor in cellulose synthesis (8,16).…”

Section: Discussionmentioning

confidence: 99%

Culm Brittleness of Barley (Hordeum vulgare L.) Mutants Is Caused by Smaller Number of Cellulose Molecules in Cell Wall

Kokubo

Sakurai²,

Kuraishi³

et al. 1991

Plant Physiol.

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The physicochemical nature of the cell wall was determined in the fourth internode of three isogenic brittle mutants of barley (Hordeum vulgare L.) and corresponding nonbrittle strains. Cellulose contents of the brittle culms were 17.5 to 20.3% of those of corresponding nonbrittle strains. No major difference was found in lignin and noncellulose components (except glucose) between brittle and nonbnttle strains. Maximum bending stresses of brittle culms were 38.0 to 54.2% of those of corresponding nonbrittle strains. The degree of polymerization of cellulose, measured by viscometry, was similar between the brittle and the nonbrittle strains. Mole number of cellulose molecules in a unit length of brittle culms, calculated by dividing cellulose mass by molecular weight, was 7.7 to 17.3% of those of the nonbrittle strains. These results indicate that brittleness of mutant culms is due to fewer numbers of cellulose molecules in the cell walls.Our previous study (15) of brittle culms of three barley mutants generated by a mutagen and Kamairazu mutant whose genetic background was not known revealed that the mutants contain less cellulose in the cell wall than the normal strains. Comparison between normal and isogenic brittle strains is necessary for genetic and biochemical analyses.The low cellulose content in the mutants could be due to a lower level of UDP-Glc, breakdown of cellulose-forming enzymes, disordered cellulose-forming complex (terminal complex), and low production of cellulose-forming enzymes. Determination of mol wt of cellulose produced in the mutants affords a clue to the mechanism of brittleness and decreased cellulose content. If the mol wt of cellulose in the mutants is less than that in normal strains, less cellulose content results from the lower mol wt. Alternatively, if the mol wt ofcellulose of the mutant is similar to that in the normal strains, then fewer cellulose-forming complexes is the cause. Furthermore, determination of mol wt of cellulose can afford a clue to the complex mechanism of cellulose synthesis including assembly ofcellulose-forming enzymes, interaction ofthe enzymes with plasma membrane, and movement of the enzyme assembly, '

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Section: Discussionmentioning

confidence: 99%

Culm Brittleness of Barley (Hordeum vulgare L.) Mutants Is Caused by Smaller Number of Cellulose Molecules in Cell Wall

Kokubo

Sakurai²,

Kuraishi³

et al. 1991

Plant Physiol.

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“…The most reliable methods to prove the presence of crystalline cellulose are x-ray and electron diffraction analyses (Lin et al, 1985;Bureau and Brown, 1987). Highly purified cellulase conjugated to colloidal gold has been used as a probe to identify cellulose by TEM (Lin et al, 1985).Cotton (Gossypium hirsutum) fiber cells have been used by many investigators for the study of cellulose biosynthesis (Delmer et al, 1984;Buchala and Meier, 1985). Experiments in which intact fibers from cotton fruit capsules (Pillonel and Meier, 1985), intact fibers from ovule cultures (Francey et al, 1989), and anucleate protoplasts isolated from fiber cells (Gould et al, 1986) were used have demonstrated cellulose synthesis.…”

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confidence: 99%

“…The AN reagent (Updegraff, 1969) has been used for the selection of cellulose because only crystalline cellulose can remain after treatment with this reagent. The most reliable methods to prove the presence of crystalline cellulose are x-ray and electron diffraction analyses (Lin et al, 1985;Bureau and Brown, 1987). Highly purified cellulase conjugated to colloidal gold has been used as a probe to identify cellulose by TEM (Lin et al, 1985).…”

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confidence: 99%

[beta]-Glucan Synthesis in the Cotton Fiber (I. Identification of [beta]-1,4- and [beta]-1,3-Glucans Synthesized in Vitro)

Okuda

Kudlicka

et al. 1993

Plant Physiol.

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In vitro B-glucan products were synthesized by digitonin-solubilized enzyme preparations from plasma membrane-enriched fractions of cotton (Cossypium hirsutum) fiber cells. The reaction mixture favoring &1,4-glucan synthesis included the following effedors: MgZ+, Caz+, cellobiose, cyclic-3':5'-CMP, and digitonin. The ethanol insoluble fraction from this reaction contained &1,4-glucan and 8-1,3-glucan in an approximate ratio of 25:69. Approximately 16% of the &1,4-glucan was resistant to the acetic/nitric acid reagent. The x-ray diffraction pattern of the treated produd favoring &l,4-glucan synthesis strongly resembled that of cellulose II. O n the basis of methylation analysis, the acetic/nitric acid reagent-insoluble glucan produd was found to be exclusively 8-1,4-linked. Enzymic hydrolysis confirmed that the produd was hydrolyzed only by cellobiohydrolase 1. Autoradiography proved that the produd was synthesized in vitro. l h e degree of polymerization (DP) of the in vitro product was estimated by nitration and size exclusion chromatography; there were two average DPs of 59 (70%) and 396 (30%) for the @-1,3-glucanase-treated sample, and an average DP of 141 for the acetic/nitric acid reagent-insoluble produd. O n the basis of product analysis, the positive identification of in vitro-synthesized cellulose was established.Cellulose is the most abundant macromolecule on earth (Brown, 1985). It serves a major structural role in the cell wall of plants, some algae, and certain fungi and is the primary component of economical products such as wood, cotton, and paper. Because of its tremendous abundance and its physiological and economical importance, many attempts at in vitro cellulose synthesis have been made with cell-free systems from various sources during the past three decades Brown, 1989b;Read and Delmer, 1991). The greatest progress has been made using Acetobacter xylinum as an experimental model (Ross et al., 1987;Brown, 1989a;Lin and Brown, 1989;Lin et al., 1990;Saxena et al., 1990;Wong et al., 1990;Mayer et al., 1991;Saxena et al., 1991). In higher plants, however, numerous failures to detect activity for in vitro cellulose synthase have continued because it has remained impossible to make preparations from higher plant cells capable of synthesizing true microfibrillar cellulose or even appreciable quantities of (3-1,4-glucan (Read and Delmer, 1991).Severa1 methods for the characterization of products synthesized in vitro, such as solubility properties, acid hydrolysis, acetolysis, specific enzymic digestion, and linkage analysis after periodate oxidation or methylation, have been used either alone or in various combinations (Fry, 1988). Insolubility in strong alkali (eg. 24% KOH) has been used for the detection of p-1,4-glucan, but (3-1,3-glucan synthesized in vitro may also be present in the alkali-insoluble fraction (Hayashi et al., 1987). The AN reagent (Updegraff, 1969) has been used for the selection of cellulose because only crystalline cellulose can remain after treatment with this reagent. The mo...

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“…The in vitro product derived from digitoninsolubilized membranes has been suggested to occur as fibrils 17 ± 2 A in diameter having an electron diffraction pattern similar to cellulose I (13).…”

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In vitro synthesis of cellulose II from a cytoplasmic membrane fraction of Acetobacter xylinum

Bureau

Brown

1987

Proc. Natl. Acad. Sci. U.S.A.

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The cytoplasmic and outer membranes of Acetobacter xylinum (ATCC 53582) were isolated by discontinuous sucrose density ultracentrifugation. Both lysozyme (EC 3.2.1.17) and trypsin (EC 3.4.21.4) were required for efficient crude membrane separation. Primary dehydrogenases and NADH oxidase were used as cytoplasmic membrane markers, and 2-keto-3-deoxyoctulosonic acid was used to identify the outer membranes. Cellulose synthetase (UDP-glucose:1,4-(3-Dglucan 4-(3-D-glucosyltransferase; EC 2.4.1.12) activity was assayed as the conversion of radioactivity from UDP-['4C]glucose into an alkali-insoluble P-1,4-D-[14C]glucan. This activity was predominantly found in the cytoplasmic membrane. The cellulose nature of the product was demonstrated by (i) enzymatic hydrolysis followed by TLC, (ii) methylation analysis followed by TLC, and (iii) GC/MS. Further, the weightaverage and number-average degree of polymerization of the in vitro product, determined by high-performance gel permeation chromatography, were 4820 and 5270, respectively. In addition, x-ray diffraction analysis indicated that the in vitro product is cellulose II, which is in contrast to the in vivo product-namely, cellulose I.

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Synthesis of Fibrils in Vitro by a Solubilized Cellulose Synthase from Acetobacter xylinum

Cited by 60 publications

References 12 publications

Culm Brittleness of Barley (Hordeum vulgare L.) Mutants Is Caused by Smaller Number of Cellulose Molecules in Cell Wall

Culm Brittleness of Barley (Hordeum vulgare L.) Mutants Is Caused by Smaller Number of Cellulose Molecules in Cell Wall

[beta]-Glucan Synthesis in the Cotton Fiber (I. Identification of [beta]-1,4- and [beta]-1,3-Glucans Synthesized in Vitro)

In vitro synthesis of cellulose II from a cytoplasmic membrane fraction of Acetobacter xylinum

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