Ribonuclease in plant vacuoles: purification and molecular properties of the enzyme from cultured tomato cells

Abel, Steffen; Glund, Konrad

doi:10.1007/bf00403030

Cited by 38 publications

(21 citation statements)

References 31 publications

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“…Additionally, vacuolar RNAs of destabilized isolated organelles are optimally degraded by inherent ribonucleolytic activities at pH 5.7 ( Fig. 4B), a value that coincides well with the pH optimum of purified tomato vacuolar RNase 1 (2).…”

supporting

confidence: 60%

“…4B), a value very similar to the pH optimum of the purified tomato vacuolar RNase I [pH 5.6 (21)]. This enzyme has previously been characterized as an acid phosphotransferase that hydrolyzes single-stranded RNA preferentially adjacent to purine residues (2,3).…”

Section: Evidence For a Vacuolar Nucleolytic Compartmentmentioning

confidence: 92%

“…2) is indicative of RNA degradative processes that occur in vacuoles, probably as a result of the action of vacuolar RNase I. This enzyme is the predominant cellular nucleolytic and the only vacuolar ribonucleolytic activity in logarithmically growing cultured tomato cells (1) and has been characterized as a relatively purine-specific cyclizing RNase (2,3). A rough characterization of vacuolar oligoribonucleotides revealed mainly phosphorylated 3'-termini (Fig.…”

mentioning

confidence: 99%

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Evidence for RNA-Oligonucleotides in Plant Vacuoles Isolated from Cultured Tomato Cells

Abel

Blume²,

Glund³

1990

Plant Physiol.

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We have shown that highly purified vacuoles of suspensioncultured tomato (Lycoperskon esculentum) cells contain RNAoligonucleotides, using two different approaches to label and detect RNA: (a) in vivo labeling of cellular RNA with [5-3H]uridine, followed by preparation of vacuoles from protoplasts and by quantification of radioactively labeled material; and (b) in vitro labeling and analysis on sequencing gels of nucleic acids prepared from tomato vacuoles and their identification as RNA. The intravacuolar location of the RNA found in vacuolar preparations was concluded from analyzing for RNA intact organelles after repeated flotation steps as well as ribonuclease A treatment.About 3% of the RNA in protoplasts was localized within vacuoles, exceeding by severalfold the contribution made by contamination with unlysed protoplasts and subcellular organelles. Investigation of the size distribution of vacuolar RNA revealed an oligonucleotide pattern strikingly different from that which would arise from contaminating protoplasts; vacuolar RNA fragments are considerably shorter than 80 nucleotides. Characterization of these oligoribonucleotides (3'-phosphorylated termini; relatively rich in pyrimidines) as possible products of tomato vacuolar ribonuclease I action, and, in addition, enzymatic hydrolysis of vacuolar RNA by inherent enzyme activities in lysed vacuole preparations support the hypothesis that plant vacuoles are involved in cellular nucleolytic processes.Involvement in degradative pathways of the cellular turnover of macromolecules is thought to be one of the most prominent functions of vacuoles in plants (for a review, see refs. 5 and 6). This has mainly been concluded from the occurrence of a broad spectrum of acid hydrolases in plant vacuoles (24), rendering this organelle homologous to the lysosomes in animal cells (6).Until now, proteolysis is the best-characterized lytic process in that plant vacuoles have been found to participate. In most cases, intracellular proteases have been found to be located in the vacuole (see ref. 6). This organelle was shown to be directly involved in the breakdown of storage proteins during germination in seeds (22) and to be able to acquire and degrade intracellular proteins in suspension-cultured sycamore cells (8,9).In contrast to these studies, which support the role of vacuolar proteases in the continuous turnover of cellular proteins, little is as yet known about a possible nucleolytic function of plant vacuoles. Although vacuoles contain a set of potentially nucleolytically acting enzymes [nuclease, ribonuclease, acid phosphatase (24)], which could completely digest nucleic acid substrates down to nucleosides and phosphate, the biological significance of the individual vacuolar enzymes and of the vacuolar compartment per se in intracellular nucleolytic events remains to be proven.In

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supporting

confidence: 60%

Section: Evidence For a Vacuolar Nucleolytic Compartmentmentioning

confidence: 92%

mentioning

confidence: 99%

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Evidence for RNA-Oligonucleotides in Plant Vacuoles Isolated from Cultured Tomato Cells

Abel

Blume²,

Glund³

1990

Plant Physiol.

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“…RNase and DNase activities were assayed as described (2). The enzyme unit was defined according to Wilson (29) acid-NaOH (pH 5.5).…”

Section: Assaysmentioning

confidence: 99%

Induction of an Extracellular Ribonuclease in Cultured Tomato Cells upon Phosphate Starvation

Nürnberger¹,

Abel²,

Jost³

et al. 1990

Plant Physiol.

108

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Suspension-cultured cells of tomato (Lycopersicon esculentum) start to secrete an RNA-degrading enzyme activity during transition from logarithmic to stationary growth phase. Using affinity chromatography on agarose-5-(4-aminophenyl-phosphoryl) uridine 3'(2') monophosphate as a powerful and final enrichment step, the enzyme was purified to homogeneity and characterized as ribonuclease I (RNase I) according to the following data: (a) it has an Mr of 22,000 (sodium dodecyl sulfatepolyacrylamide gel electrophoresis), a pH-optimum of pH 5.5, a pi of 3.9, and its activity was found to be insensitive to EDTA; (b) the enzyme splits single-stranded RNA endonucleolytically by a phosphotransferase reaction yielding 2',3'-cNMPs as primary monomeric products; (c) as studied with diribonucleoside monophosphates as substrates, the enzyme exhibits a pronounced preference for 5' purine residues adjacent to the cleavage site.

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“…Therefore, the structure of the B2 site of RNase Os is very similar to that of RNase LE and other S-like enzymes. These results could explain the similar base specificity of RNase LE 40) and RNase Os. The notation is the same as in Fig.…”

Section: Discussionmentioning

confidence: 54%

Amino Acid Sequence and Characterization of a Rice Bran Ribonuclease

Iwama

Ogawa

Yamagishi

et al. 2001

Biological & Pharmaceutical Bulletin

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RNase T2 family enzymes which are base non-specific and have a molecular mass of about 24 kDa, distributed widely from viruses to microbes, plants and animals. 1)They share two well conserved active site sequences, CAS I and CAS II. Therefore, they are a good model with which to study the evolution of RNases. In the plant kingdom, members of the RNase T2 family are classified into two classes, S-RNases which are the products of S-genes and function as self-incompatibility factors, 2) and S-like RNases.3) The latter enzymes are known to be induced by phosphate deprivation [Jost et al.,4) Bariola et al. 5)], in the case of senescence [Lers et al. 6) ], and exist in the seeds probably for defense [Ide et al.,7) Rojo et al. 8,9) ]. In terms of the evolutional change of plant RNases, knowledge is limited to those plants belonging to Dicotyledoneae, except for barley. 10) These include several plants of Solanaceae [tomato,4,11) and tobacco, 3,12) petunia 13) and a kind of potato 14) ], Cucurbitaceae [bitter gourd, 7) cucumber, 18) and others. In this report, we will describe the amino acid sequence of an RNase from rice bran (Oryza sativa) which is a member of Monocotyledoneae, and its enzymatic properties. MATERIALS AND METHODSEnzymes Lysylendopeptidase and g-pyroglutamine aminopeptidase were obtained from Wako Pure Chemical Ind., Ltd. (Osaka, Japan) and Calbiochem-Novabiochem. Co., Ltd., (LaJolla, LA, U.S.A.), respectively.Other Reagents Yeast RNA was a product of Marin Bio (Tokyo, Japan). Sixteen dinucleoside phosphates were purchased from Sigma (St. Louis, MO, U.S.A.). Phospho-cellulose and DE-52 were obtained from Whatman (Clifton, NJ, U.S.A.). A Mono Q column was obtained from Amersham Pharmcia Biotech (Buckinghamshire, England).Enzyme Assay (i) RNA as substrate: RNase assay was carried out as described 19) with RNA as substrate at pH 5.5 and 37°C. (ii) Dinucleosidephosphates as substrate: The rates of hydrolysis of 16 dinucleoside phosphates (XpYs), where X and Y were one of A, G, C and U, were measured spectrophotometrically as described by Imazawa et al. 20) in 0.1 M sodium acetate buffer (pH 5.5) at 22°C.Protein Concentration The protein concentration was measured spectrophotometrically assuming the absorbancy at 280 nm of a 0.1% protein solution to be 1.0.Purification of an RNase from Rice Bran Purification of an RNase from rice bran was performed by modification of the procedure reported by Yokoyama et al. 21)Step 1. Four kilograms of rice bran (product from a strain of Oryza sativa, Koshihikari) was suspended in 12 l of 20 mM sodium phosphate buffer (pH 7.0) and extracted by vigorous stirring at 4°C. The extract was filtered through a layer of cotton sheet, and the filtrate was centrifuged at 10000 rpm for 10 min. The pH of the supernatant was adjusted to 5.0 by addition of dil. HCl. The supernatant was kept at 4°C overnight, and the precipitate formed was eliminated by centrifugation at 10000 rpm for 10 min (crude extract).Step 2. The crude extract was fractionated by ammonium sulfate, and the pr...

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Ribonuclease in plant vacuoles: purification and molecular properties of the enzyme from cultured tomato cells

Cited by 38 publications

References 31 publications

Evidence for RNA-Oligonucleotides in Plant Vacuoles Isolated from Cultured Tomato Cells

Evidence for RNA-Oligonucleotides in Plant Vacuoles Isolated from Cultured Tomato Cells

Induction of an Extracellular Ribonuclease in Cultured Tomato Cells upon Phosphate Starvation

Amino Acid Sequence and Characterization of a Rice Bran Ribonuclease

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