Molecular weight determinations and the influence of gel density, protein charges and protein shape in polyacrylamide gel electrophoresis

Blattler, D.P.; Reithel, F.J.

doi:10.1016/s0021-9673(00)84000-5

Cited by 23 publications

(3 citation statements)

References 10 publications

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“…These findings suggest that T. pallidum 4D consists of large complexes of extensively disulfide-bonded ordered rings. Based on their inability to enter the stacking gel, the molecular masses of these higher oligomers must be at least several million daltons (2).…”

Section: Discussionmentioning

confidence: 99%

Role of disulfide bonds in the oligomeric structure and protease resistance of recombinant and native Treponema pallidum surface antigen 4D

Radolf¹,

Borenstein²,

Kim³

et al. 1987

J Bacteriol

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Recombinant Treponema pallidum surface antigen 4D isolated from Escherichia coli formed a proteaseresistant ordered ring structure composed of 19,000-dalton subunits. On gradient sodium dodecyl sulfatepolyacrylamide gel electrophoresis, the higher oligomers of recombinant 4D migrated with molecular masses that were nearly multiples of the 190,000-dalton basic ordered ring. Reduction at room temperature with 2-mercaptoethanol converted the 190,000-dalton ordered ring and the higher oligomers to a 160,000-dalton form and the dissociated monomer. A 190,000-dalton form of 4D was identified in sodium dodecyl sulfate-solubilized T. pallidum after reduction at room temperature. Disulfide bonds stabilized both native and recombinant 4D oligomers against dissociation by heating in detergent without a reducing agent. Electron microscopy of recombinant 4D revealed that the characteristic ordered ring structure was maintained after reduction. Reduction of 4D under conditions that preserved the ordered ring structure did not affect the resistance of the molecule to digestion with proteinase K. The properties of 4D suggest that it may fulfill an important structural role in the T. pallidum outer membrane.Detailed study of the surface proteins of Treponema pallidum recently has been made possible by the expression of treponemal antigens in Escherichia coli (21,26,31,32). We have recently described a recombinant T. pallidum protein, designated 4D, which is a protease-resistant, 190,000-dalton ordered ring structure composed of 19,000-dalton monomers (6, 7). Several lines of evidence suggest that the native 4D antigen plays a role in the pathogenesis of syphilis. 4D and immunologically related molecules have been detected only in T. pallidum and the other pathogenic treponemes (7, 23; unpublished data). Rabbit antisera to the purified recombinant molecule immobilize T. pallidum cells in vitro (7), and 4D antiserum has been used to demonstrate the surface location of the native 4D antigen by immunoelectron microscopy (22). Rabbits immunized intravenously with the recombinant protein develop significant partial protection against subsequent intradermal challenge (L. Borenstein, J. Radolf, T. Fehniger, J. Miller, and M. Lovett, submitted for publication).Previously we demonstrated that molecules identical to both the 19,000-dalton monomer and the 90,000-dalton proteinase K limit-digestion product of recombinant 4D could be identified in T. pallidum immunoblots reacted with recombinant 4D antiserum (7). The native form of 4D, however, was not identified. In this report we demonstrate that disulfide bonds mediate the higher oligomeric structure of both native and recombinant 4D. After reduction with 2-metcaptoethanol, the 190,000-dalton form of native 4D could be identified. We MATERIALS AND METHODS T. pallidum Nichols was maintained by intratesticular passage in New Zealand White rabbits without the use of cortisone acetate. T. pallidum for immunoblots was obtained from rabbits given daily intramuscular injections of cortisone ac...

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Section: Discussionmentioning

confidence: 99%

Role of disulfide bonds in the oligomeric structure and protease resistance of recombinant and native Treponema pallidum surface antigen 4D

Radolf¹,

Borenstein²,

Kim³

et al. 1987

J Bacteriol

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“…Enzyme solution was treated at 100 "C for 5 min with 2 % dodecyl sulphate/2 % mercaptoethanol before running. Bovine serum albumin, which was used as a standard, was similarly treated but in the absence of mercaptoethanol to obtain dimeric, trimeric and tetrameric forms [5].…”

Section: Gel Electrophoresismentioning

confidence: 99%

Enzymes of Nitrogen Metabolism in Legume Nodules

Boland¹,

Benny

1977

European Journal of Biochemistry

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An NADH-dependent glutamate synthase has been purified 500-fold from the plant cytoplasm fraction of Lupinus angustifolius nodules. It consists of a single polypeptide chain, M , 235000. The optimum pH is 8.5, at which K , values for 2-oxoglutarate, glutamine and NADH are 39 pM, 400 pM and 1.3 pM respectively. The catalytic centre activity is of the order of 70 s-' and is independent of pH between 6.5 and 9.5. Glutamate synthase is inhibited by glutamic acid, oxaloacetic acid, aspartic acid and asparagine, all competitive with 2-oxoglutarate; and by NAD' , which is competitive with NADH.There is evidence of two flavine prosthetic groups per enzyme molecule.Although the nitrogenase enzyme of nitrogen fixation has been extensively studied and is comparatively well understood, the enzymes that metabolise the ammonia produced by the nitrogenase reaction are relatively poorly understood.Robertson et al. [l] first showed the presence of a NADH-dependent glutamate synthase in the plant cytoplasm fraction of the nodule. The level of this enzyme was shown to increase dramatically in developing nodules with the onset of nitrogen fixation, suggesting a role in the overall ammonia assimilation system.Recently Scott et al.[2] suggested a pathway for nitrogen metabolism in the nodule, involving glutamate synthase and three other enzymes : glutamine synthetase, aspartate aminotransferase and asparagine synthetase.This laboratory is currently investigating ammonia assimilation by nitrogen-fixing lupin nodules. We now report the purification and some kinetic properties of the plant cytoplasmic NADH-dependent glutamate synthase. The reaction catalysed is:Abbreviation. Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulphonic acid. EXPERIMENTAL PROCEDURE MaterialsNADH (grade 111), bovine serum albumin (fraction V) and oxaloacetic acid were obtained from Sigma Chemical Co. Amino acids and other keto acids were from BDH Chemicals Ltd. All other reagents were analytical grade purity. PuriJj:cation of EnzymeLupins (Lupinus angustifolius L. Uniwhite) were grown as previously described [I]. Nodules (26.5 g) were harvested from 160 18-day-old plants, extracted and centrifuged to remove bacteroids [l]. The supernatant was then treated with 0.01 vol. of 0.1 M phenylmethylsulphonyl fluoride in isopropanol and centrifuged 20 min at 0 "C, 30000 x g to remove particulate material, giving an extract of the plant cytoplasm fraction. In the subsequent enzyme purification all steps were carried out at 0-4 "C. 0.1 M potassium phosphate buffer, pH 7.6, containing 0.5 % mercaptoethanol was used throughout, with modifications as described.Glutamate synthase was precipitated from the plant cytoplasm extract between 35 % and 50 % saturation with ammonium sulphate (209 g lP1, plus 94 g I-') and was resuspended in 2-3 ml of phosphate buffer to which had been added 1 mg disodium EDTA,

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“…If the distance moved in a gel is plotted against the log of the molecular weight of the monomeric proteins, a straight line is obtained. Blattler and Reithel (1970) have published very useful graphs relating acrylamide concentration to exclusion limits for various proteins. They concluded that shape differences must be rather extreme to affect the results.…”

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confidence: 99%