The use of bentonite in the isolation of plant polyribosomes

Watts, R. L.; Mathias, A. P.

doi:10.1016/0005-2787(67)90142-6

Cited by 38 publications

(11 citation statements)

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“…SA). The proportion is similar to that obtained by Stutz and Noll (16) and not much lower than has been found in preparations made with RNase inhibitors, e.g., as shown by Watts and Matthias (20), suggesting that there was no extensive breakdown during preparation. When the leaves undergo rapid expansion the proportion of polysomes decreases markedly (Table II).…”

Section: Eilam Butler and Simonsupporting

confidence: 89%

“…However, it was found that polyvinyl sulfate causes a decrease in the sedimentation constant of the purified monosomes, from 80S to 70S and a dissociation to 35S and 50S subunits (18). A high proportion of polysomes was detected when bentonite was used to remove the ribonuclease from the extract (20). Nevertheless, bentonite has been shown to reduce the yield of ribosomal RNA, perhaps by a selective removal of monosomes (17).…”

Section: Eilam Butler and Simonmentioning

confidence: 99%

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Ribosomes and Polysomes in Cucumber Leaves during Growth and Senescence

Eilam¹,

Butler²,

Simon³

1971

Plant Physiol.

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The quantity of RNA in the ribosomal fraction of the first leaf of cucumber (Cucumis sativus) increases during growth, reaches a maximum before the final fresh weight is attained, and then decreases. The main changes are in the free ribosome fraction, the quantity of membrane-bound ribosomes remaining about constant. Few 65.5S chloroplast ribosomes are present in small leaves; however, they increase in quantity rapidly during growth and form about half of the ribosomes present in the mature fully green leaf. The cytoplasmic ribosomes have a sedimentation coefficient of 77.6S. Ribonuclease-sensitive polysomes were present in leaves of all ages except possibly the very oldest. The proportion of ribosomes in polysome form decreases during growth and then remains roughly constant during senescence. Following maturation of the leaf, the rate of incorporation of 'P into ribosomal-fraction RNA begins to decline. This decline could account for the loss of ribosomes during the early stages of senescence. The possibility that leaf ribonuclease might be responsible for the final, more rapid loss of RNA, is discussed.The quantity of protein in a leaf increases during growth but then, at the time of senescence, it declines, although the evidence suggests that the leaf has not completely lost the power to synthesize protein (14). In view of their role in protein synthesis it was thought that a study of leaf ribosomes might throw some light on the mechanism controlling protein metabolism in leaves.Two classes of ribosome are found in leaf tissues, 70S in the chloroplasts and 80S in the cytoplasm (8). Most studies have been made with young tissues, and little is known about the changes that occur with aging. A decrease in the proportion of polysomes during growth has been indicated in Chinese cabbage leaves (6) and maize shoots (10), and an electron microscope study revealed the presence of polysome-like clusters in aging cucumber cotyledons (5). An over-all loss of ribosomes during aging has been observed in several plant tissues both by electron microscopy (1, 4,5,13) and from biochemical studies (6,15) Preparation of Ribosomes and Polysomes. The leaves (10 to 40 g) were rapidly washed, blotted dry, and weighed; then they were cooled and ground by pestle and mortar with enough acid-washed sand to facilitate grinding and with one to two times their volume of tris-magnesium buffer containing 0.05 M tris, pH 7.8, and 0.01 M magnesium acetate. The low molarity of the grinding medium allowed chloroplast lysis. The homogenate was squeezed through four layers of muslin, and the material in the muslin was extracted three times with fresh buffer and the extracts combined. This preparation and all subsequent handling of extract or ribosomes were made at about 0 C unless otherwise stated. Two types of preparations were made from the extract. Differential Centrifugation. To determine the quantity of free and membrane-bound ribosomes, the extract was divided into halves, and Triton X-100 in tris-magnesium buffer was added to one o...

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Section: Eilam Butler and Simonsupporting

confidence: 89%

Section: Eilam Butler and Simonmentioning

confidence: 99%

Ribosomes and Polysomes in Cucumber Leaves during Growth and Senescence

Eilam¹,

Butler²,

Simon³

1971

Plant Physiol.

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“…The widespread occurrence of ribonuclease (RNAase), particularly in plant tissues, has hampered efforts to develop such a method. The inclusion of the ribonuclease inhibitors, bentonite (24) or polyvinyl sulfate (2), in the homogenizing medium is not always effective (2, 6,16) and, in addition, may result in considerable loss of polyribosomes (6,20). Recent reports (5,17) Polyribosomge Isolation.…”

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confidence: 99%

Polyribosome Isolation in the Presence of Diethyl Pyrocarbonate

Weeks¹,

Marcus²

1969

Plant Physiol.

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Abstract. Isolation of polyribosomes from wheat embryos and corn root tips in the presence of diethyl pyrocarbonate showed this reagent to have a protective effect on polyribosome structure. In addition, the use of diethyl pyrocarbonate allowed initial homogenization to be performed under less stringent conditions than those normally employed for polyribosome isolation. The use of the reagent is however limited, in that it is deleterious to in vitro ribosomal amino acid incorporation.Numerous studies have been reported in which the size distribution of polyribosomes has been correlated with a particular phvsiological state (1,2,4,9,11,12,18,22,23,25 Polyribosomge Isolation. Imbibed wheat embrvos were blotted dry, frozen on dry ice, and ground in a mortar and pestle packed in dry ice. Corn root tip sections (3 mm) were cut, immediately frozen on dry ice, and ground similarly. The ground wheat embryos (250 mg) or corn root tips (40 tips) were homogenized in a Duall homogenizer in 7 ml of a solution containing 0.25 m sucrose, 20 mM KCl, 5 mM MgAc2, 50 mM tris-CI (pH 7.7), 1 mM Cleland's reagent. If no diethyl pyrocarbonate (DEP) was to be added during homogenization, the final tris-Cl concentration was brought to 0.1 M with 1.0 M tris-Cl (pH 7.7). If DEP was included, the tris-CI con-centration was brought to 0.1 M by adding 1.0 M untitrated tris. Diethyl pyrocarbonate has a short half-life in water (5 hr at 0°), and was therefore added (0.1 ml per 10 ml grinding solution) immediately before homogenization.The remainder of the isolation procedure was similar to the procedure described by Lin, Key, and Bracker (10). The homogenates were cleared by centrifugation for 10 min at 17,000g. They were then layered over 3 ml of 1.66 M sucrose containing 20 mm KCI, 5 mm MgAc2, 50 mM tris-Cl (pH 7.7), 1.0 mM Cleland's reagent and centrifuged for 90 min at 225,000g. Microsomal pellets were resuspended in 0.5 ml of 20 mM KCl, 5 mM MgAc2, 50 mM tris-Cl (pH 7.7), 1.0 mm Cleland's reagent. Approximately 1 mg of ribosomes was layered on a 10 to 34 % sucrose density gradient containing 20 mM KCI, 5 mm MgAc2, 50 mM tris-Cl (pH 7.7), and 1.0 mM Cleland's reagent and centrifuged 2 hr at 90,000g in a SW25 rotor. The gradients were fractionated using a modified ISCO density-gradient fractionator with continual monitoring of absorbance at 254 mM.In Vitro Amino Acid Incorporation. Polyribosomes were obtained from microsomal pellets prepared as described above. Supernatant-(S-23) was obtained from dry wheat embryos (13)

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“…The resulting pellet was washed twice with distilled water and resuspended in 0.1 M-disodium ethylenediaminetetra-acetic acid and stirred overnight. Crude bentonite has a high metal ion content which decreases protein-binding capacity, and EDTA is effective in ion removal (Fraenkel-Conrat et al, 1961;Watts & Mathias, 1967). The EDTA was removed by overnight dialysis against distilled water.…”

Section: Methods Of Isolation and Analysismentioning

confidence: 99%