Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis for Mr Estimations of High-Molecular-Weight Polypeptides

Quandt, N.; Stindl, A.; Keller, Urs

doi:10.1006/abio.1993.1527

Cited by 9 publications

(6 citation statements)

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“…Therefore, it cannot be excluded that a contaminating activity has been purified. On the other hand, the size of SnbD protein was not determined in the best conditions now used for such large peptide synthetases (26,32), and it is probably underestimated. In fact, it could be close to 400 or 500 kDa, the expected size for a four-module peptide synthetase.…”

Section: Discussionmentioning

confidence: 99%

Purification of peptide synthetases involved in pristinamycin I biosynthesis

et al. 1997

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Several assays of pristinamycin I synthetases based on adenylate or thioester formation were developed. Purification to near homogeneity of these enzymatic activities from cell extracts of Streptomyces pristinaespiralis showed that three enzymes could activate all pristinamycin I precursors. SnbA, a 3-hydroxypicolinic acid: AMP ligase activating the first pristinamycin I residue, was purified 200-fold, using an ATP-pyrophosphate exchange assay. This enzyme was shown to be a monomer with an M r of 67,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Then a multifunctional enzyme, consisting of two identical subunits (SnbC) with M r s of 240,000 and able to bind covalently L-threonine as a thioester, was purified 100-fold. This protein also activated L-aminobutyric acid, which is further epimerized to generate the third residue of the pristinamycin I macrocycle. A third protein, consisting of two identical subunits (SnbD) with M r s estimated to be between 250,000 and 350,000, was purified 200-fold. This large enzyme catalyzed thioesterification and subsequent N-methylation of 4-dimethylamino-L-phenylalanine, the fifth pristinamycin I residue. SnbD could also activate L-proline, the fourth pristinamycin I residue, and some preparations retained a low but significant activity for the last two pristinamycin I precursors. Finally, a single polypeptide chain (SnbE) with an M r of 170,000, catalyzing L-phenylglycine-dependent ATP-pyrophosphate exchange, was purified 3,000-fold and characterized. Stepwise Edman degradation of the entire polypeptides or some of their internal fragments provided amino acid sequences for the four isolated proteins. The purified SnbE protein was further shown to be a proteolytic fragment of SnbD.

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Section: Discussionmentioning

confidence: 99%

Purification of peptide synthetases involved in pristinamycin I biosynthesis

et al. 1997

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“…When membrane samples were heated for 5-20 min at 95°C to denature the protein in preparation for SDS-PAGE, as is the conventional method [22][23][24], no protein was detected likely due to HsPCFT aggregation and immobilization in the stacking gel. To establish con-ditions that obviate aggregation, plasma membrane protein fractions were prepared from HeLa cells 48 h after transfection with HsPCFT cDNA, then incubated in the loading buffer containing SDS with and without DTT at RT, 50°C, 75°C or 95°C for 10 min before PAGE.…”

Section: The Effect Of Temperature On Hspcft Protein Stabilitymentioning

confidence: 99%

N-linked glycosylation and its impact on the electrophoretic mobility and function of the human proton-coupled folate transporter (HsPCFT)

Unal

Zhao

Qiu

et al. 2008

Biochimica et Biophysica Acta (BBA) - Biomembranes

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The human proton-coupled folate transporter (HsPCFT, SLC46A1) mediates intestinal absorption of folates and transport of folates into the liver, brain and other tissues. On Western blot, HsPCFT migrates as a broad band (~55 kDa), higher than predicted (~50 kDa) in cell lines. Western blot analysis required that membrane preparations not be incubated in the loading buffer above 50 degrees C to avoid aggregation of the protein. Treatment of membrane fractions from HsPCFT-transfected HeLa cells with peptidyl N-glycanase F, or cells with tunicamycin, resulted in conversion to a ~35 kDa species. Substitution of asparagine residues of two canonical glycosylation sites to glutamine, individually, yielded a ~47 kDa protein; substitution of both sites gave a smaller (~35 kDa) protein. Single mutants retained full transport activity; the double mutant retained a majority of activity. Transport function and molecular size were unchanged when the double mutant was hemagglutinin (HA) tagged at either the NH(2) or COOH terminus and probed with an anti-HA antibody excluding degradation of the deglycosylated protein. Wild-type or deglycosylated HsPCFT HA, tagged at amino or carboxyl termini, could only be visualized on the plasma membrane when HeLa cells were first permeabilized, consistent with the intracellular location of these domains.

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“…A measurement macro (Scion Image) was used to determine T1 and T2 relative migration distances. Relative mobility (R f ) was calculated from the migration distance of the protein band divided by the migration distance of the dye front (Quandt et al 1993;Shapiro and Maizel 1969;Granzier and Wang 1993). Samples were run separately and then in combination to compare subtle differences in the mobility of T1 and T2 (Spierts et al 1997).…”

Section: Gel Electrophoresismentioning

confidence: 99%

Characteristics of titin in strength and power athletes

McBride¹,

Triplett-McBride²,

Davie

et al. 2003

Eur J Appl Physiol

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The purpose of this investigation was to identify characteristics of the muscle protein titin in different athletic populations with increased levels of strength and power relative to non-athletes. Subjects fell into one of four groups: (1) non-athletes (NA) ( n=5), (2) weightlifters (WL) (n=5), (3) powerlifters (PL) (n=5), (4) sprinters (S) (n=5). A one repetition maximum in the squat exercise was performed to assess strength. In addition, countermovement vertical jump trials were performed to assess power capabilities. Peak power (W(peak)) was calculated for the vertical jumps from force plate measurements. From gel electrophoresis analyses of muscle samples, titin-1 (T1) and titin-2 (T2) protein bands were identified, quantified and expressed relative to each other. In addition the relative mobility (R(f)) of T1 and T2 was determined as an estimate of molecular weight. The NA group [%T1=47.8 (5.1), %T2=52.2 (5.1), mean (SE)] had lower T1 and higher T2 percentages than WL [%T1=62.3 (6.6), %T2=37.7 (6.6)], PL [%T1=66.8 (5.0), %T2=33.2 (5.0)] and S [%T1=65.9 (4.9), %T2=34.1 (4.9)] groups (P< or =0.10, preliminary investigation into titin and exercise justifies more liberal alpha level). No significant differences were found in R(f) of T1 or T2 between the groups. This investigation has shown that there is a differential expression of titin protein bands in competitive athletes with increased levels of strength and power in comparison to untrained non-athletic individuals. Some relationships between titin characteristics and athletic performance were observed; however, no conclusions can be made based on these data as to the contribution of titin to strength or power capabilities.

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Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis for Mr Estimations of High-Molecular-Weight Polypeptides

Cited by 9 publications

References 0 publications

Purification of peptide synthetases involved in pristinamycin I biosynthesis

Purification of peptide synthetases involved in pristinamycin I biosynthesis

N-linked glycosylation and its impact on the electrophoretic mobility and function of the human proton-coupled folate transporter (HsPCFT)

Characteristics of titin in strength and power athletes

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