Size and charge isomer separation and estimation of molecular weights of proteins by disc gel electrophoresis

Hedrick, Jerry L.; Smith, Alan Jay

doi:10.1016/0003-9861(68)90569-9

Cited by 1,892 publications

(606 citation statements)

References 33 publications

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“…Markers used were bovine serum albumin (M r 68000), alcohol dehydrogenase (M r 150000), catalase (M r 240000) and ferritin (M r 450000). As an alternative, electrophoresis on 7.5 and 10% polyacrylamide gels was used [8] with the markers bovine serum albumin, uricase (M~ 100000), alcohol dehydrogenase and phosphorylase A (Mr 370000). The molecular weight of allantoinase subunits was estimated by polyacrylamide gelelectrophoresis (13% gels) in the presence of SDS as described by Weber and Osborn [9].…”

Section: Materials Arid Methodsmentioning

confidence: 99%

Allantoinase from Pseudomonas aeruginosa Purification, properties and immunochemical characterization of its in vivo inactivation

Janssen

Smits

Drift

1982

Biochimica et Biophysica Acta (BBA) - General Subjects

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The catabolic enzyme allantoinase is rapidly inactivated in cells of Pseudomonas aeruginosa when the stationary phase of growth is reached. This process is irreversible since the protein synthesis inhibitor chloramphenicol completely blocked the reappearance of allantoinase activity that is observed when allantoin is added to stationary cells. Purified allantoinase appeared to be a protein composed of four identical subunits with a molecular weight of 38000. With antibodies raised against purified allantoinase it was found that allantoinase inactivation is accompanied by a parallel decrease in immunologically reactive material. This suggests that ailantoinase inactivation is caused or followed by rapid proteolysis.

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Section: Materials Arid Methodsmentioning

confidence: 99%

Allantoinase from Pseudomonas aeruginosa Purification, properties and immunochemical characterization of its in vivo inactivation

Janssen

Smits

Drift

1982

Biochimica et Biophysica Acta (BBA) - General Subjects

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“…In order to estimate protein subunit molecular weights from electrophoretic runs in gels containing 8 M urea, the procedure of Hedrick and Smith [4] was adapted. Gel concentration was varied as indicated in fig.…”

Section: Methodsmentioning

confidence: 99%

Purification and properties of methionyl‐tRNA synthetase from E. coli K 12 carrying the F32 episome

Cassio¹,

Waller²

1971

FEBS Letters

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“…Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of protein samples was done by the procedure of Laemmli (21), and gels were stained with Coomassie blue. The molecular weight of native IucD was determined by the procedure of Hedrick and Smith (16).…”

mentioning

confidence: 99%

Construction and biochemical characterization of recombinant cytoplasmic forms of the IucD protein (lysine:N6-hydroxylase) encoded by the pColV-K30 aerobactin gene cluster

et al. 1993

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The aerobactin gene cluster in pColV-K30 consists of five genes (iucABCD iutA); four of these (iucABCD) are involved in aerobactin biosynthesis, whereas the fifth one (iutA) encodes the ferriaerobactin outer membrane receptor. iucD encodes lysine:NM-hydroxylase, which catalyzes the first step in aerobactin biosynthesis. Regardless of the method used for cell rupture, we have consistently found that IucD remains membrane bound, and repeated efforts to achieve a purified and active soluble form of the enzyme have been unsuccessful. To circumvent this problem, we have constructed recombinant IucD proteins with modified amino termini by creating three in-frame gene fusions of IucD to the amino-terminal amino acids of the cytoplasmic enzyme 13-galactosidase. Two of these constructs resulted in the addition to the iucD coding region of a hydrophilic leader sequence of 13 and 30 amino acids. The other construct involved the deletion of the first 47 amino acids of the IucD amino terminus and the addition of 19 amino acids of the amino terminus of 1-galactosidase. Cells expressing any of the three recombinant IucD forms were found to produce soluble N6-hydroxylysine. One of these proteins, lucD439, was purified to homogeneity from the soluble fraction of the cell lysates, and it was capable of participating in the biosynthesis of aerobactin, as determined in vitro by a cell-free system and in vivo by a cross-feeding bioassay. A medium ionic strength of 0.25 (250 mM NaCl) or higher was required to maintain the protein in a catalytically functional, tetrameric state. The enzyme was stringently specific with regard to its substrate and cofactor, L-lysine and flavin adenine dinucleotide, respectively. NADPH appeared to be the preferred electron donor used by IucD439 in the N hydroxylation process.

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Size and charge isomer separation and estimation of molecular weights of proteins by disc gel electrophoresis

Cited by 1,892 publications

References 33 publications

Allantoinase from Pseudomonas aeruginosa Purification, properties and immunochemical characterization of its in vivo inactivation

Allantoinase from Pseudomonas aeruginosa Purification, properties and immunochemical characterization of its in vivo inactivation

Purification and properties of methionyl‐tRNA synthetase from E. coli K 12 carrying the F32 episome

Construction and biochemical characterization of recombinant cytoplasmic forms of the IucD protein (lysine:N6-hydroxylase) encoded by the pColV-K30 aerobactin gene cluster

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