Purification of ribonuclease T1 by diethylaminoethylcellulose chromatography

Fields, Robert; Dixon, H. B. F.; Law, Gillian R.; Yui, Chie

doi:10.1042/bj1210591

Cited by 13 publications

(4 citation statements)

References 15 publications

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“…This activity probably results from a minor contamination by nuclease Si (Ando, 1966) and could be removed by heating concentrated enzyme solutions at pH 6.5 to 100°for 5 min; a slight precipitate formed during this treatment which was removed by filtration. The specific activity of all the ribonuclease T, preparations agreed with that determined previously (Fields et al, 1971) and was not affected by the heat treatment. The concentration of ribonuclease Ti was determined spectrophotometrically using an extinction coefficient of 2.1 X 104 m-1 cm-1 at 278 nm (Egami et al, 1964).…”

Section: Methodssupporting

confidence: 87%

Interaction of guanine ligands with ribonuclease T1

Walz¹,

Hooverman²

1973

Biochemistry

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Section: Methodssupporting

confidence: 87%

Interaction of guanine ligands with ribonuclease T1

Walz¹,

Hooverman²

1973

Biochemistry

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“…Indeed, certain low-molecular-mass proteins are known to behave anomalously on SDS/PAGE [14]. For examples, ribonuclease A (13.7 kDa), ribonuclease T 1 (11.1 kDa) and the heavy chain of aspergillopepsin II (18.3 kDa) were reported to give molecular masses of 18.5 kDa (35 % overestimated) [14], 18.0 kDa (62 % overestimated) [15] and 33.0 kDa (80 % overestimated) [16] respectively on SDS/PAGE. The molecular mass of 23 kDa estimated by gel filtration is somewhat lower than the theoretical value of 33.4 kDa.…”

Section: Discussionmentioning

confidence: 99%

Isolation and characterization of recombinant Drosophila Copia aspartic proteinase

Athauda

Yoshioka

Shiba

et al. 2006

Biochemical Journal

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The wild type Copia Gag precursor protein of Drosophila melanogaster expressed in Escherichia coli was shown to be processed autocatalytically to generate two daughter proteins with molecular masses of 33 and 23 kDa on SDS/PAGE. The active-site motif of aspartic proteinases, Asp-Ser-Gly, was present in the 23 kDa protein corresponding to the C-terminal half of the precursor protein. The coding region of this daughter protein (152 residues) in the copia gag gene was expressed in E. coli to produce the recombinant enzyme protein as inclusion bodies, which was then purified and refolded to create the active enzyme. Using the peptide substrate His-Gly-Ile-Ala-Phe-Met-Val-Lys-Glu-Val-Asn (cleavage site: Phe-Met) designed on the basis of the sequence of the cleavage-site region of the precursor protein, the enzymatic properties of the proteinase were investigated. The optimum pH and temperature of the proteinase toward the synthetic peptide were 4.0 and 70 degrees C respectively. The proteolytic activity was increased with increasing NaCl concentration in the reaction mixture, the optimum concentration being 2 M. Pepstatin A strongly inhibited the enzyme, with a Ki value of 15 nM at pH 4.0. On the other hand, the active-site residue mutant, in which the putative catalytic aspartic acid residue was mutated to an alanine residue, had no activity. These results show that the Copia proteinase belongs to the family of aspartic proteinases including HIV proteinase. The B-chain of oxidized bovine insulin was hydrolysed at the Leu15-Tyr16 bond fairly selectively. Thus the recombinant Copia proteinase partially resembles HIV proteinase, but is significantly different from it in certain aspects.

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“…To surmount this complication, we adapted a column-based method to purify fungal T1 and T2 RNases by binding them to an agarose column coupled with guanosine monophosphate (GMP) (Fields et al, 1971). We found that cell wall digesting enzymes readily passed through GMP columns, while the contaminating RNases remained stuck to the column.…”

Section: Enzyme Purification Is Necessary For Maintaining Rna Qualitymentioning

confidence: 99%

FX-Cell: Quantitative cell release from fixed plant tissues for single-cell genomics

Marchant

Nelms

Walbot

2021

Preprint

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Single-cell RNA-sequencing (scRNA-seq) can provide invaluable insight into cell development, cell type identification, and plant evolution. However, the resilience of the cell wall makes it difficult to dissociate plant tissues and release individual cells. Here, we show that plant tissues can be rapidly and quantitatively dissociated if the tissues are fixed prior to enzymatic digestion. Fixation enables digestion at high temperatures at which enzymatic activity is optimal and stabilizes the plant cell cytoplasm, rendering cells resistant to mechanical shear force. This protocol was applied to maize anthers and provided suitable single-cell expression data for the identification of the tapetum, endothecium, meiocytes, and epidermis, while providing putative marker genes and gene ontology information for the identification of unknown cell types. This approach also preserves morphology of the isolated cells, permitting many cell types to be identified without staining. Our fixation-based protocol can be applied to a range of plant species and tissues with minimal optimization.

show abstract

Purification of ribonuclease T1 by diethylaminoethylcellulose chromatography

Cited by 13 publications

References 15 publications

Interaction of guanine ligands with ribonuclease T1

Interaction of guanine ligands with ribonuclease T1

Isolation and characterization of recombinant Drosophila Copia aspartic proteinase

FX-Cell: Quantitative cell release from fixed plant tissues for single-cell genomics

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