Simple alkanethiol groups for temporary blocking of sulfhydryl groups of enzymes

Smith, Daniel J.; Maggio, E; Kenyon, George L.

doi:10.1021/bi00675a019

Cited by 318 publications

(146 citation statements)

References 36 publications

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“…The inactivation of papain with methylmethanethiol sulphonate (Smith et al, 1975) has been described previously (Roberts et al, 1986;Lindahl et al, 1988). Excess reagents were removed by chromatography on Sephadex G-25 (Pharmacia, Uppsala, Sweden).…”

Section: Methodsmentioning

confidence: 99%

Interaction of chicken cystatin with inactivated papains

Björk

Ylinenjärvi

1989

Biochemical Journal

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Papain which was inactivated by covalent attachment of small substituents to the active-site cysteine, up to the size of a carbamoylmethyl group, bound with high affinity to chicken cystatin (Kd < -15 pM), although less tightly than did active papain (Kd 60 fM). However, as the size of the substituent was increased further, the affinity decreased appreciably, generally in proportion to the size of the inactivating group. For instance the dissociation constants for papain inactivated with N-ethylmaleimide and

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Section: Methodsmentioning

confidence: 99%

Interaction of chicken cystatin with inactivated papains

Björk

Ylinenjärvi

1989

Biochemical Journal

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“…Proteins (50 g of each sample) were denatured by adding 1 l of 2% SDS and reduced by addition of 2 l of 50 mM Tris-(2-carboxyethyl) phosphine. The cysteine residues were blocked using 1 l of 200 mM methyl methanethiosulfonate as described previously (62,68). These samples were digested with trypsin (65), dried completely, and reconstituted in 50 l of the dissolution buffer (500 mM triethylammonium bicarbonate).…”

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confidence: 99%

Transcriptome Analysis of Pseudomonas syringae Identifies New Genes, Noncoding RNAs, and Antisense Activity

et al. 2010

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To fully understand how bacteria respond to their environment, it is essential to assess genome-wide transcriptional activity. New high-throughput sequencing technologies make it possible to query the transcriptome of an organism in an efficient unbiased manner. We applied a strand-specific method to sequence bacterial transcripts using Illumina's high-throughput sequencing technology. The resulting sequences were used to construct genome-wide transcriptional profiles. Novel bioinformatics analyses were developed and used in combination with proteomics data for the qualitative classification of transcriptional activity in defined regions. As expected, most transcriptional activity was consistent with predictions from the genome annotation. Importantly, we identified and confirmed transcriptional activity in areas of the genome inconsistent with the annotation and in unannotated regions. Further analyses revealed potential RpoN-dependent promoter sequences upstream of several noncoding RNAs (ncRNAs), suggesting a role for these ncRNAs in RpoNdependent phenotypes. We were also able to validate a number of transcriptional start sites, many of which were consistent with predicted promoter motifs. Overall, our approach provides an efficient way to survey global transcriptional activity in bacteria and enables rapid discovery of specific areas in the genome that merit further investigation.

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“…3B). The reaction could be completely inhibited with compound 3 or the thiol-labeling reagent methyl methanethiolsulfonate (17). The antibody also utilized compound 6 (R or S methyl group) as substrates with a rate similar to compound 5 without demonstrating appreciable stereospecificity.…”

Section: Resultsmentioning

confidence: 99%

Direct selection for a catalytic mechanism from combinatorial antibody libraries.

Janda

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

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Semisynthetic combinatorial antibody library methodology in the phage-display format was used to select for a cysteine residue in complementari-determinig regions. Libraries (Fig. 1) of each dilution was plated on a LB (Luria-Bertani) plate/ carbenicillin to measure the output. Carbenicillin (20 pg/ml) was added to the cell culture and shaken at 370C for 1 hr. The concentration of antibiotic was increased to 50 gg/ml and shaken at 370C for another hour. The cell culture was diluted into 100 ml of superbroth that contained carbenicillin at 50 Ag/ml, tetracycline at 10 pg/ml, and 1012 plaque-forming units of VCSM13 helper phage (Stratagene). After shaking 2 hr at 370C, kanamycin (70 Ag/ml) was added and shaken at 370C overnight. The cells were removed by centrifugation, and 4% PEG and 3% NaCl were added to the supernatant. After 30 min on ice, the phage particles were centrifuged (9000 x g, 30 min, 40C). The phage were resuspended with TBS/1% BSA and clarified by centrifugation (16,000 x g, 10 min, 4Q.). The phage solution was ready for further panning.For the next four rounds of panning, the washing procedures were modified as follows: second round (twice with washing buffer, once with acid solution); third round (five times with washing buffer, once with acid solution for 5 min); fourth round (10 times with washing buffer/3% BSA, twice with acid solution for 5 min); fifth round (10 times with washing buffer/3% BSA, twice with acid solution for 5 min). For each round, the bound phage were eluted twice with 50 A4 of 20 mM dithiothreitol for 5 min. The eluted phage were measured and amplified as described above. After the fifth round of panning, the phagemid DNA was purified, digested with Spe I and Nhe I, and purified on agarose gel to remove the DNA of gene III. The 4.7-kb fragment was electroeluted, religated, and transformed into E. coli. The colonies were picked up and grown until the OD600 = 0.8, 1 mM isopropyl 13-Dthiogalactopyranoside was added, and the culture was incubated at 300C overnight. The cells were lysed with freezeand-thaw cycles between -700C and 37c in PBS. The supernatant was tested on ELISA plates coated with the Abbreviation: BSA, bovine serum albumin. *To whom reprint requests should be addressed. 2532The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Simple alkanethiol groups for temporary blocking of sulfhydryl groups of enzymes

Cited by 318 publications

References 36 publications

Interaction of chicken cystatin with inactivated papains

Interaction of chicken cystatin with inactivated papains

Transcriptome Analysis of Pseudomonas syringae Identifies New Genes, Noncoding RNAs, and Antisense Activity

Direct selection for a catalytic mechanism from combinatorial antibody libraries.

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