Artifacts in sodium dodecyl sulfate-polyacrylamide gel electrophoresis due to 2-mercaptoethanol

Tasheva, Bistra; Dessev, George

doi:10.1016/0003-2697(83)90057-x

Cited by 162 publications

(43 citation statements)

References 13 publications

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“…3 A, the subunit M , of a-L-iduronidase was estimated to be 65000. After staining with silver however, two minor contaminants were revealed of M , 60000 and 54000 which are attributable to dithioerythritol as described by Tasheva and Dessev [19]. They were present in dithioerythritol-only controls and in all other lanes with dithioerythritol added.…”

Section: Subunit Molecular Mass Of Purified A-l-iduronidusesupporting

confidence: 52%

Human alpha-L-iduronidase. 1. Purification, monoclonal antibody production, native and subunit molecular mass

et al. 1985

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Human α‐L iduronidase from liver was purified about 20 000‐fold with a new rapid three‐step, five‐column procedure which consisted of a Concanavalin‐A–Sepharose/Blue‐A–Agarose coupled step, a CM‐Sepharose/Bio‐Gel HT coupled step followedby a cupric‐ion‐chelating Sepharose 6B step. The behaviour of α‐L‐iduronidase on gel permeation chromatography was dependent upon both pH and ionic strength of the eluting buffer. The formation of species with enzyme activity which behaved as large‐molecular‐mass aggregates was favoured under conditions of low ionic strength and neutral pH. The amount of high‐Mr species diminished as the pH decreased or the ionic strength increased to favour a single active species of Mr 65000. A specific monoclonal antibody was generated against liver α‐L‐iduronidase. The antibody specifically immunoprecipitated enzyme activity from both crude and purified sources. The subunit Mr of liver α‐L‐iduronidase was estimated to be 65000 using SDS‐PAGE. Monoclonal antibody immunoprecipitation of radiolabelled enzyme was used to provide definitive confirmation of this subunit size.

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Section: Subunit Molecular Mass Of Purified A-l-iduronidusesupporting

confidence: 52%

Human alpha-L-iduronidase. 1. Purification, monoclonal antibody production, native and subunit molecular mass

et al. 1985

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“…11, lane 6, the 38-kDa band is found only in the fractions containing the kinase activity. The two bands of 68 and 54 kDa that appear in all lanes of the gel are an artefact due to sulfhydryl reagents, as recently shown by Tasheva and Dessev [20]. Fractions were collected and they were assayed for kinase activity and analyzed by NaDodS0,-polyacrylamide gel electrophoresis.…”

Section: Molecular Mass and Structure Of The Enzymementioning

confidence: 86%

Purification and properties of a cAMP‐independent nuclear protein kinase from Dictyostelium discoideum

Renart

Sastre

Sebastián

1984

European Journal of Biochemistry

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A cyclic-AMP-independent nuclear protein kinase has been purified from Dictyostelium discoideum amoebae. The purification procedure involves chromatography of DEAE-Sephadex, phosphocellulose and heparinSepharose. The purified enzyme phosphorylates threonine and serine of acidic proteins as casein and phosvitin. Phosphorylation of casein is stimulated by spermine. The kinase requires Mg2 + and can utilize both ATP and GTP as phosphoryl donors. Heparin is a potent inhibitor of the enzyme, being the protein kinase activity fully inhibited at concentrations of 0.5 pg/ml. One polypeptide of molecular mass 38 kDa was the major protein band present in the purified kinase preparation as estimated by NaDodSO, denaturing polyacrylamide gel electrophoresis. This band belongs to the protein kinase because it is the only one that is observed associated with the protein kinase activity when the enzyme preparation is centrifuged in glycerol gradients. The 38-kDa polypeptide is also the major product of autophosphorylation of the enzyme preparation. The enzymatic properties allow to classify the enzyme as a type-I1 casein kinase. However, its structural properties are different from the mammalian type-I1 casein kinases and make the D. discoideum enzyme more similar to the plants type-I1 casein kinases.Phosphorylation/dephosphorylation of proteins represents an important mechanism in the control of cellular processes, including the regulation of gene expression in eukaryotic cells I1,21.Among the enzymes that catalyze the postranslational phosphorylation of proteins are cyclic-nucleotide-dependent an independent protein kinases. Extensive investigations of CAMP-dependent protein kinases have shown that these enzymes mediate all of the biological effects of cyclic AMP. In contrast, little is known about the physiological role of the CAMP-independent protein kinases, although several types of these kind of enzyme have been characterized in different biological systems, mainly higher eukaryotes [3].The nuclear phosphorylation of chromatin-associated proteins, both histones and non histones, have been implicated in the regulation of gene transcription [4, 51. A cyclic-nucleotideindependent, polyamine-sensitive protein kinase has been involved in the control of transcription of ribosomal RNA genes by means of phosphorylation of RNA polymerase I [6] and some transcriptional factors [7].In the slime mold Dictyostelium discoideum a lot of effort has been dedicated to the study of the involvement of CAMPdependent protein kinases in the expression of its differentiation program. CAMP is the chemical agent that mediates aggregation of the amoebae [8] and it is also implicated in the regulation of latter stages of differentiation [9]. CAMPdependent protein kinases have been identified in D. discoideum both in vegetative cells [lo] and during differentiation [I 1, 121. Besides, the characterization of the regulatory subunit of a CAMP-dependent protein kinase has also been carried out using a photo-affinity probe [13]. However, the character...

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“…The fractions from step 3 containing 6SS activity (Fig. 2) were pooled and concentrated to 20 ml, dialysed in an ultrafiltration stirred cell against 20 …”

Section: Purification Of 6ssmentioning

confidence: 99%

Human liver N-acetylglucosamine-6-sulphate sulphatase. Purification and characterization

Freeman

Clements

Hopwood

1987

Biochemical Journal

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Human N-acetylglucosamine-6-sulphate sulphatase was purified at least 50000-fold to homogeneity in 780%yield from liver with a simple three-step four-column procedure, which consists of a concanavalin A-Sepharose/Blue A-agarose coupled step, chromatofocusing and Cu2+-chelating Sepharose chromatography. In all, four forms were isolated and partially characterized. Forms A and B, both with a pl greater than 9.5 and representing 30% and 60% respectively of the recovered enzyme activity, were separated by hydroxyapatite chromatography of the enzyme preparation obtained from the Cu2+-chelating Sepharose step. Both forms A and B had native molecular masses of 75 kDa. When analysed by SDS/polyacrylamide-gel electrophoresis, form A consists of a single polypeptide of molecular mass 78 kDa, whereas form B contained 48 kDa and 32 kDa polypeptide subunits. Neither form A nor form B was taken up from the culture medium into cultured human skin fibroblasts. The two other forms (C and D), with pI values of 5.8 and 5.4 respectively, represented approx. 7% and 3% of the total recovered enzyme activity. The native molecular masses of forms C and D were 94 kDa and approx. 75 kDa respectively. Form C contained three polypeptides with molecular masses of 48, 45 and 32 kDa. N-Acetylglucosamine-6-sulphate sulphatase activity was measured with a radiolabelled disaccharide substrate derived from heparin. The development of this substrate enabled the isolation and characterization of N-acetylglucosamine-6-sulphate sulphatase to proceed efficiently. Forms A, B and C had pH optima of 5.0, Km values of 11.7, 14.2 and 11.1 LM respectively and Vmax values of 105, 60 and 53 nmol/min per mg of protein respectively. The molecular basis of the multiple forms of this sulphatase is not known. It is postulated that the differences in structure and properties of the four enzyme forms are due to differences in the state of processing of a large subunit.

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Artifacts in sodium dodecyl sulfate-polyacrylamide gel electrophoresis due to 2-mercaptoethanol

Cited by 162 publications

References 13 publications

Human alpha-L-iduronidase. 1. Purification, monoclonal antibody production, native and subunit molecular mass

Human alpha-L-iduronidase. 1. Purification, monoclonal antibody production, native and subunit molecular mass

Purification and properties of a cAMP‐independent nuclear protein kinase from Dictyostelium discoideum

Human liver N-acetylglucosamine-6-sulphate sulphatase. Purification and characterization

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