Purification and characterization of two cellulases from auxin-treated pea epicotyls.

Byrne, Henry; Christou, N. V.; Verma, Desh Pal S.; Maclachlan, Gordon

doi:10.1016/s0021-9258(19)41885-1

Cited by 72 publications

(8 citation statements)

References 29 publications

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“…To test for the association of leghemoglobin with the membrane envelope or its possible occurrence inside the envelope, bacteroids enclosed in the membrane envelope were prepared (Fig. 4, Fraction D) and sonicated, or the membrane was solubilized with 0.2% NP-40 and then reacted with antibodies in Ouchterlony plates (8).…”

Section: Localization O F Leghemoglobinmentioning

confidence: 99%

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Isolation and characterization of the membrane envelope enclosing the bacteroids in soybean root nodules.

Dp¹,

Kazazian²,

Zogbi³

et al. 1978

The Journal of Cell Biology

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107

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The membrane envelope enclosing the bacteroids in soybean root nodules is shown by ultrastructural and biochemical studies to be derived from, and to retain the characteristics of, the host cell plasma membrane. During the early stages of the infection process, which occurs through an invagination, Rhizobium becomes surrounded by the host cell wall and plasma membrane, forming the infection thread. The cell wall of the infection thread is degraded by cellulolytic enzyme(s), leaving behind the enclosed plasma membrane, the membrane envelope. Cellulase activity in young nodules increases two-to threefold as compared to uninfected roots, and this activity is localized in the cell wall matrix of the infection threads.Membrane envelopes were isolated by first preparing bacteroids enclosed in the envelopes on a discontinuous sucrose gradient followed by passage through a hypodermic needle, which released the bacteroids from the membranes. This membrane then sedimented at the interface of 34-45% sucrose (mean density of 1.14 g/cma). Membranes were characterized by phosphotungstic acid (PTA)-chromic acid staining, ATPase activity, and localization, sensitivity to nonionic detergent Nonidet P-40 (NP-40) and sodium dodecyl sulfate (SDS) gel electrophoresis. These analyses revealed a close similarity between plasma membrane and the membrane envelope. Incorporation of radioactive amino acids into the membrane envelope proteins was sensitive to cycloheximide, suggesting that the biosynthesis of these proteins is primarily under host-cell control. No immunoreactive material to leghemoglobin antibodies was found inside or associated with the isolated bacteroids enclosed in the membrane envelope, and its location is confined to the host cell cytoplasmic matrix. KEY WORDS Rhizobium leghemoglobin 9 membrane envelope immunohistochemistryIn symbiotic nitrogen-fixing root nodules of leguminous plants, the bacteroids are enclosed in a membrane envelope which was first observed by Bergersen and Briggs (5). This membrane envelope compartmentalizes the bacteroids from the host cell cytoplasm, and thus may play an important role in segregating eu-and procaryote metab-J. C~LL BIOLOGy 9 The Rockefeller University Press 9

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Section: Localization O F Leghemoglobinmentioning

confidence: 99%

“…Uninfected root tissue was used as a control. Cellulase was assayed viscometrically at 35 ~ by using carboxymethylcellulose as a substrate (8).…”

Section: Measurement Of Cellulose Activitymentioning

confidence: 99%

Isolation and characterization of the membrane envelope enclosing the bacteroids in soybean root nodules.

Dp¹,

Kazazian²,

Zogbi³

et al. 1978

The Journal of Cell Biology

Self Cite

107

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“…Cellulolytic enzymes (~-l,4-glucan, 4-glucanohydrolase, EC 3.2.1.4) occur in specific locations in tissues of higher plants at particular stages of development. Their possible regulatory roles in cell growth and differentiation (1, 7-10, 12, 14-18), and the hormonal control of their induction (5)(6)(7)(8)21), have been emphasized in earlier work. Although cellulases have been purified and partly characterized (5,6), their actual localization at the cellular level has not been demonstrated.…”

mentioning

confidence: 99%

“…Their possible regulatory roles in cell growth and differentiation (1, 7-10, 12, 14-18), and the hormonal control of their induction (5)(6)(7)(8)21), have been emphasized in earlier work. Although cellulases have been purified and partly characterized (5,6), their actual localization at the cellular level has not been demonstrated. In elucidating the physiological role of these enzymes, it is imperative that their exact subcellular loci be defined.…”

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confidence: 99%

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Subcellular localization of cellulases in auxin-treated pea.

Bal¹,

Verma²,

Byrne³

et al. 1976

The Journal of Cell Biology

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Two forms of cellulase, buffer soluble (BS) and buffer insoluble (BI), are induced as a result of auxin treatment of dark-grown pea epicotyls. These two cellulases have been purified to homogeneity. Antibodies raised against the purified cellulases were conjugated with ferritin and were used to localize the two cellulases. Tissue sections were fixed in cold paraformaldehyde-glutaraldehyde and incubated for 1 h in the ferritin conjugates. The sections were washed with continuous shaking for 18 h and subsequently postfixed in osmium tetroxide. Tissue incubated in unconjugated ferritin was used as a control. A major part of BI cellulase is localized at the inner surface of the cell wall in close association with microfibrils. BS cellulase is localized mainly within the distended endoplasmic reticulum. Golgi complex and plasma membrane appear to be completely devoid of any cellulase activity. These observations are consistent with cytochemical localization and biochemical data on the distribution of these two cellulases among various cell and membrane fractions.Cellulolytic enzymes (~-l,4-glucan, 4-glucanohydrolase, EC 3.2.1.4) occur in specific locations in tissues of higher plants at particular stages of development. Their possible regulatory roles in cell growth and differentiation (1, 7-10, 12, 14-18), and the hormonal control of their induction (5-8, 21), have been emphasized in earlier work. Although cellulases have been purified and partly characterized (5, 6), their actual localization at the cellular level has not been demonstrated. In elucidating the physiological role of these enzymes, it is imperative that their exact subcellular loci be defined. Attempts made to localize cellulase in higher plants (3, 4) and snail (20) have not met the refinements necessary for precise ultrastructural localization.Auxin-induced growth in etiolated pea epicotyls offers a unique system for localization of cellulases. Two different species of cellulases, one buffer insoluble (Bi) and the other buffer soluble (BS), have been purified and characterized from this system (5, 6). These two proteins have proved to be immunologically different. With the use of ferritin-conjugated antibodies to these two cellulases, an immunocytochemical method was developed to localize the enzymes at subcellular levels. The results were supplemented by cytochemical (4) and cell fractionation procedures. MATERIALS AND METHODS Plant Material and Hormone TreatmentSeeds of Pisum sativum L. var. Alaska were soaked for 20 min in 0.5% sodium hypochlorite. After 8 h of imbibition in tap water, seeds were planted in moistened vermiculite and grown in darkness at room temperature.

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β-Glucanases in Higher Plants: Localization, Potential Functions, and Regulation

Verma

Kumar

Maclachlan

1982

Cellulose and Other Natural Polymer Systems

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Purification and characterization of two cellulases from auxin-treated pea epicotyls.

Cited by 72 publications

References 29 publications

Isolation and characterization of the membrane envelope enclosing the bacteroids in soybean root nodules.

Isolation and characterization of the membrane envelope enclosing the bacteroids in soybean root nodules.

Subcellular localization of cellulases in auxin-treated pea.

β-Glucanases in Higher Plants: Localization, Potential Functions, and Regulation

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