Characterization of enzymic specificity of a ribonuclease from Ustilago sphaerogena

Rushizky, George W.; Mozejko, Jan H.; Rogerson, David L.; Sober, Herbert A.

doi:10.1021/bi00827a021

Cited by 22 publications

(14 citation statements)

References 22 publications

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“…It displays a strong preference for 3'-linked purine nucleotide phosphodiester linkages [4,5] and a rather restricted pH range for its ribonucleolytic activity, with an optimum centered at pH 4.5 [1]. RNase U2 contains about 50% polar amino acid residues; it has 3 Arg but lacks Lys residues and has a high content of acidic residues (Asp 1 Glu = 16) which results in a very low isoelectric point (pI) value of 2.8-3.3 [6,7].…”

Section: Introductionmentioning

confidence: 99%

Anomalous electrophoretic behavior of a very acidic protein: Ribonuclease U2

et al. 2005

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Ribonuclease U2 is a low-molecular-weight acidic protein with three disulfide bridges. This protein displays an anomalous electrophoretic behavior on standard SDS-PAGE. The electrophoretic mobility of the nonreduced protein roughly corresponds to its molecular mass while the migration of the reduced protein would be in accordance with the expected molecular mass of the protein dimer. This study reveals that the protein does not bind SDS under the SDS-PAGE conditions, its electrophoretic mobility being only determined by its electrostatic charge and hydrodynamic properties. In addition, the nonreduced protein cannot be blotted to a membrane. Unfolding of the protein upon reduction of its disulfide bridges enables electrotransference to membranes due to a restricted diffusion along the electrophoresis gel.

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Section: Introductionmentioning

confidence: 99%

Anomalous electrophoretic behavior of a very acidic protein: Ribonuclease U2

et al. 2005

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“…28). RNase U2 displays a low specificity beyond a strong preference for 3Ј-linked purine nucleotide phosphodiester bonds (A Ͼ G Ͼ Ͼ C Ͼ U) (29,30). Both proteins, ␣-sarcin and RNase U2, are cyclizing RNases because they produce a 2Ј,3Ј-cyclic intermediate as a result of the cleavage reaction (26,30).…”

mentioning

confidence: 99%

Deletion of the NH2-terminal β-Hairpin of the Ribotoxin α-Sarcin Produces a Nontoxic but Active Ribonuclease

Garcı́a-Ortega¹,

Masip²,

Mancheño³

et al. 2002

Journal of Biological Chemistry

105

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Ribotoxins are a family of highly specific fungal ribonucleases that inactivate the ribosomes by hydrolysis of a single phosphodiester bond of the 28 S rRNA. ␣-Sarcin, the best characterized member of this family, is a potent cytotoxin that promotes apoptosis of human tumor cells after internalization via endocytosis. This latter ability is related to its interaction with phospholipid bilayers. These proteins share a common structural core with nontoxic ribonucleases of the RNase T1 family. However, significant structural differences between these two groups of proteins are related to the presence of a long amino-terminal ␤-hairpin in ribotoxins and to the different length of their unstructured loops. The aminoterminal deletion mutant ⌬(7-22) of ␣-sarcin has been produced in Escherichia coli and purified to homogeneity. It retains the same conformation as the wild-type protein as ascertained by complete spectroscopic characterization based on circular dichroism, fluorescence, and NMR techniques. This mutant exhibits ribonuclease activity against naked rRNA and synthetic substrates but lacks the specific ability of the wild-type protein to degrade rRNA in intact ribosomes. The results indicate that ␣-sarcin interacts with the ribosome at two regions, i.e. the well known sarcin-ricin loop of the rRNA and a different region recognized by the ␤-hairpin of the protein. In addition, this latter protein portion is involved in interaction with cell membranes. The mutant displays decreased interaction with lipid vesicles and shows behavior compatible with the absence of one vesicle-interacting region. In agreement with this conclusion, the deletion mutant exhibits a very low cytotoxicity on human rhabdomyosarcoma cells.

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“…Its three‐dimensional structure closely resembles that of other microbial extracellular RNases such as RNase T1 from Aspergillus oryzae [2]. However, RNase U2 displays unique enzymatic properties such as an optimum pH for enzymatic activity at pH 3.5–5.5 [1] and a specificity for hydrolyzing phosphodiester bonds of 3′‐linked purine nucleotides, A/GpN [3,4]. Both characteristics are shared by α‐sarcin, a specific RNase secreted by Aspergillus giganteus [5–7].…”

Section: Introductionmentioning

confidence: 92%

Ribonuclease U2: cloning, production inPichia pastorisand affinity chromatography purification of the active recombinant protein

Martı́nez-Ruiz

Garcı́a-Ortega

Kao

et al. 2000

FEMS Microbiology Letters

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RNase U2 is an endoribonuclease secreted by the fungus Ustilago sphaerogena. Its genomic DNA (rnu2), containing an intron of 116 bp, has been isolated and cloned. The corresponding cDNA has also been synthesized. The recombinant RNase U2 was successfully produced in Pichia pastoris, fused to the yeast alkaline phosphatase signal peptide. The recombinant RNase U2, purified by affinity chromatography, contains three extra amino acids at its amino-terminal end and retains the enzymatic and spectroscopic properties of the natural fungal protein.

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Characterization of enzymic specificity of a ribonuclease from Ustilago sphaerogena

Abstract: A ribonuclease from Ustilago sphaerogena (ATCC 12421) was purified 1100-fold with a yield of 32%. The isolated enzyme was free of contaminating phosphatases, ribonucleases, and deoxyribonucleases. The rate and specific-

Cited by 22 publications

References 22 publications

Anomalous electrophoretic behavior of a very acidic protein: Ribonuclease U2

Anomalous electrophoretic behavior of a very acidic protein: Ribonuclease U2

Deletion of the NH2-terminal β-Hairpin of the Ribotoxin α-Sarcin Produces a Nontoxic but Active Ribonuclease

Ribonuclease U2: cloning, production inPichia pastorisand affinity chromatography purification of the active recombinant protein

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