Removal of proteases from DNase I by chromatography over agarose with covalently attached lima bean protease inhibitor

Otsuka, Allen S.; Price, Paul A.

doi:10.1016/0003-2697(74)90379-0

Cited by 32 publications

(8 citation statements)

References 16 publications

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“…This result was in agreement with previous studies showing that chymotrypsin and trypsin were the major proteases contaminating commercial DNase I [1,4]. Because chymotrypsinogen (precursor of chymotrypsin) is similar to DNase I in molecular weight and isoionic point (pH 5.0), during the preparation commercial DNase I products tend to be contaminated with chymotrypsinogen even at a larger level than active forms (in this order: chymotrypsinogen > chymotrypsin > trypsin) [4,5]. Thus, it seemed very likely that the DNase I products (especially Bio Basic DNase) were also contaminated with a large amount of chymotrypsinogen, which also has some activity toward target proteins [6].…”

supporting

confidence: 93%

“…It looked like PMSF had more effect on the contaminating proteases than the inhibitor cocktail. It could be elucidated by the faster rate of PMSF than AEBSF (the major ingredient in S8830 inhibitor cocktail) in terms of inhibiting chymotrypsin [7], which contaminates DNase I at a level dozens of times higher than trypsin [4].…”

mentioning

confidence: 99%

“…Based on this knowledge, 5 mg/ml Bio Basic DNase stock solution (named solution I) prepared in 100 mM Tris-HCl (pH 8.0) containing 5 mM CaCl 2 was first incubated at 50°C for 1 h for the autodigestion of proteolytic enzymes. It was noted that DNase I can be stable at up to 60°C for several hours [21], and the decline of DNase I activity resulted only from proteolytic enzymes [4,5]. In addition, although DNase I was highly susceptible to proteases, it could be fully stabilized against proteolytic digestion in the presence of 5 mM CaCl 2 [21].…”

mentioning

confidence: 99%

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An efficient method to eliminate the protease activity contaminating commercial bovine pancreatic DNase I

Lee

Jin

2015

Analytical Biochemistry

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supporting

confidence: 93%

mentioning

confidence: 99%

mentioning

confidence: 99%

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An efficient method to eliminate the protease activity contaminating commercial bovine pancreatic DNase I

Lee

Jin

2015

Analytical Biochemistry

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“…The nuclei were resuspended in lysis buffer with use of a Dounce homogenizer and centrifuged through a cushion of 30% sucrose in 10 mM Tris-HCl (pH 7.2), 10 mM NaCl, and 3 mM MgClj. The nuclei were then suspended in the same buffer without sucrose and containing 0.1 mM CaClj at an A260 oi 20 (determined on an aliquot digested with micrococcal nuclease and dissolved in 1 M NaOH), and digested for 10 min at 37°C with varying concentrations of protease-free DNase I (Otsuka and Price 1974). Digestion was stopped by the addition of EDTA to 10 mM and SDS and proteinase K to final concentra tions of 1% and 200 ixg/ml, respectively.…”

Section: Dnase Digestion Of Endo B Genesmentioning

confidence: 99%

Identification of the gene coding for the Endo B murine cytokeratin and its methylated, stable inactive state in mouse nonepithelial cells.

Oshima¹,

Trevor²,

Shevinsky³

et al. 1988

Genes Dev.

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The Endo B type-I keratin intermediate filament protein is first expressed at the 4-to 8-cell stage of mouse development. In the adult, its expression is restricted to a variety of simple epithelial cell types. To investigate the mechanisms responsible for the restricted expression of Endo B, the gene coding for Endo B has been identified from among the five different Endo B genes found in the mouse genome by Southern hybridization analysis and cloning all or part of four of the genes. Nuclear run-on experiments demonstrate that Endo B expression is regulated at the level of transcription. The 5' end of the active gene, designated Endo pi, was found to be highly methylated and in a relatively nuclease-resistant chromatin conformation in fibroblasts and myoblasts that do not express Endo B, but undermethylated and relatively sensitive to nuclease digestion in endodermal cells or F9 embryonal carcinoma cells. The inactive state of the Endo B pi gene in fibroblast appears to be very stable, because somatic cell hybrids formed by the fusion of HeLa cells, which express the homologous human protein, keratin 18, and mouse fibroblasts, continue to express keratin 18 but do not activate Endo B expression. Similarly, the fusion of mouse endodermal cells and fibroblasts results in hybrids that do not extinguish Endo B expression. These results suggest that Endo B transcription is limited by two different mechanisms. In somatic cells such as fibroblasts or myoblasts, expression may be restricted by methylation and a stable, nonpermissive transcriptional state. However, in embryonal carcinoma cells, the Endo B pi gene is undermethylated and in a relatively nuclease-sensitive conformation, but it is restricted by an additional, negative regulatory mechanism.

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“…Beynon, unpublished work). Samples from the digestions were removed and treated with two successive quantities of lima-bean trypsin inhibitor, covalently attached to agarose and prepared as described by Otsuka & Price (1974). The capacity of the adsorbent was determined to be 1.7mg of chymotrypsin/ml of settled gel.…”

Section: Preparation Ofsamplesfor Gel Electrophoresismentioning

confidence: 99%

The chymotrypsin-catalysed activation of bovine liver glutamate dehydrogenase

Place¹,

Beynon²

1982

Biochemical Journal

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1. Ox liver glutamate dehydrogenase is activated by bovine pancreatic alpha-chymotrypsin, but the extent of activation is dependent on the age of the dehydrogenase preparation. 2. The degree of activation is constant and the pseudo-first-order rate constant of activation is directly proportional to the concentration of proteinase used. 3. Commercial preparations of alpha-chymotrypsin differ in their ability to produce a secondary inactivation phase, and this was shown to be due to low tryptic contamination. The 'superactive' form of glutamate dehydrogenase has an increased sensitivity to tryptic inactivation as compared with the native enzyme. 4. Analysis of the activation by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed that the subunit molecular weight of 'superactive' glutamate dehydrogenase differs by less than 5% from that of the native subunit.

show abstract

Removal of proteases from DNase I by chromatography over agarose with covalently attached lima bean protease inhibitor

Cited by 32 publications

References 16 publications

An efficient method to eliminate the protease activity contaminating commercial bovine pancreatic DNase I

An efficient method to eliminate the protease activity contaminating commercial bovine pancreatic DNase I

Identification of the gene coding for the Endo B murine cytokeratin and its methylated, stable inactive state in mouse nonepithelial cells.

The chymotrypsin-catalysed activation of bovine liver glutamate dehydrogenase

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