Linearization of the Bradford Protein Assay Increases Its Sensitivity: Theoretical and Experimental Studies

Zor, Tsaffrir; Selinger, Zvi

doi:10.1006/abio.1996.0171

Cited by 922 publications

(571 citation statements)

References 3 publications

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“…The protein was then exchanged into 6 M guanidinium HCl using a PD-10 column, and the concentration of protein was determined by absorbance at 280 nm using the method of Pace et al (20). Protein concentration of the same sample was determined using a Bradford assay where the BSA standard curve was linearized by dividing the 590 nm absorbance by the 450 nm absorbance, according to the method of Zor and Selinger (21). The comparison of these two values provides a conversion factor for PqqE of 0.855 (i.e.…”

Section: Methodsmentioning

confidence: 99%

Demonstration That the Radical S-Adenosylmethionine (SAM) Enzyme PqqE Catalyzes de Novo Carbon-Carbon Cross-linking within a Peptide Substrate PqqA in the Presence of the Peptide Chaperone PqqD

Barr

Latham

Iavarone

et al. 2016

Journal of Biological Chemistry

108

184

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The radical S-adenosylmethionine (SAM) protein PqqE is predicted to function in the pyrroloquinoline quinone (PQQ) biosynthetic pathway via catalysis of carbon-carbon bond formation between a glutamate and tyrosine side chain within the small peptide substrate PqqA. We report here that PqqE activity is dependent on the accessory protein PqqD, which was recently shown to bind PqqA tightly. In addition, PqqE activity in vitro requires the presence of a flavodoxin-and flavodoxin reductasebased reduction system, with other reductants leading to an uncoupled cleavage of the co-substrate SAM. These results indicate that PqqE, in conjunction with PqqD, carries out the first step in PQQ biosynthesis: a radical-mediated formation of a new carbon-carbon bond between two amino acid side chains on PqqA.Pyrroloquinoline quinone (PQQ) 2 is employed by a wide variety of bacteria, where it functions as a redox cofactor in substrate oxidation via an alternate (non-glycolytic) pathway for production of cellular ATP (1). Hundreds of bacterial species are thought to produce PQQ, based on the presence in their genomes of the strongly conserved pqq operon (2) (Fig. 1A). Although the existence of this important redox cofactor has been known for decades, knowledge of the biosynthetic pathway for PQQ has remained scant (3, 4). The PQQ molecule derives from the evolutionarily conserved glutamate and tyrosine side chains within a ribosomally produced peptide substrate PqqA (Fig. 1, B and C). To produce PQQ, a large number of chemical modifications are required: (i) the formation of a new carbon-carbon bond between the ␥-carbon of glutamate and the 3-position of tyrosine (labeled in green in Fig. 1C); (ii) the addition of two additional hydroxyl groups to the tyrosine ring; (iii) a condensation between the 4-OH of tyrosine and the backbone amine of glutamate to produce a heterocyclic ring; and (iv) an 8-electron oxidation catalyzed by PqqC, a cofactorless enzyme that converts 3a-(2-amino-2-carboxyethyl)-4,5-dioxo-4,5,6,7,8,9-hexahydroquinoline-7,9-dicarboxylic acid (AHQQ) to PQQ in the final step of the pathway (5-7).The radical SAM enzyme PqqE has been the primary candidate as the catalyst for the formation of the new carbon-carbon bond between glutamate and tyrosine. Radical SAM proteins use the reductive cleavage of S-adenosyl methionine to initiate free radical chemistry, and can accomplish a wide variety of reactions, including carbon-carbon bond formation (8, 9). PqqE is a founding member of the SPASM domain-containing radical SAM proteins, which contain one or more auxiliary clusters in their C-terminal regions, and of which several are known to modify small peptides or proteins (10, 11). Recently, a small (ϳ10 kDa) protein, PqqD, has been shown to form a strong, sub-micromolar K D complex with the peptide substrate PqqA. This complex was further demonstrated to associate with PqqE (12). These findings suggested that PqqD would serve as a chaperone to deliver PqqA to PqqE, functioning as a necessary and heretofore missing componen...

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Section: Methodsmentioning

confidence: 99%

Demonstration That the Radical S-Adenosylmethionine (SAM) Enzyme PqqE Catalyzes de Novo Carbon-Carbon Cross-linking within a Peptide Substrate PqqA in the Presence of the Peptide Chaperone PqqD

Barr

Latham

Iavarone

et al. 2016

Journal of Biological Chemistry

108

184

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“…Total protein was quantified by the Bradford assay method using Bio-Rad Protein Assay Dye Reagent (Bio-Rad, Cat #500-0006) and a bovine serum album standard 70 . A Bio-Rad 10% Mini-PROTEAN TGX gel (Bio-Rad, Cat #456-1034) was run using the Mini-PROTEAN Tetra Cell electrophoresis set up.…”

Section: For Strain Details)mentioning

confidence: 99%

Retro-biosynthetic screening of a modular pathway design achieves selective route for microbial synthesis of 4-methyl-pentanol

et al. 2014

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Increasingly complex metabolic pathways have been engineered by modifying natural pathways and establishing de novo pathways with enzymes from a variety of organisms. Here we apply retro-biosynthetic screening to a modular pathway design to identify a redox neutral, theoretically high yielding route to a branched C6 alcohol. Enzymes capable of converting natural E. coli metabolites into 4-methyl-pentanol (4MP) via coenzyme A (CoA)-dependent chemistry were taken from nine different organisms to form a ten-step de novo pathway. Selectivity for 4MP is enhanced through the use of key enzymes acting on acyl-CoA intermediates, a carboxylic acid reductase from Nocardia iowensis and an alcohol dehydrogenase from Leifsonia sp. strain S749. One implementation of the full pathway from glucose demonstrates selective carbon chain extension and acid reduction with 4MP constituting 81% (90±7 mg l À 1 ) of the observed alcohol products. The highest observed 4MP titre is 192±23 mg l À 1 . These results demonstrate the ability of modular pathway screening to facilitate de novo pathway engineering.

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“…Subcellular fractions were prepared as described (24). Cell wall and cell membrane fractions were resuspended in lysis buffer and protein content was measured by a modified Bradford procedure (25).…”

Section: ϩmentioning

confidence: 99%

A Mycobacterial Cyclic AMP Phosphodiesterase That Moonlights as a Modifier of Cell Wall Permeability

Podobnik

Tyagi

Matange

et al. 2009

Journal of Biological Chemistry

110

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Mycobacterium tuberculosis utilizes many mechanisms to establish itself within the macrophage, and bacterially derived cAMP is important in modulating the host cellular response. Although the genome of M. tuberculosis is endowed with a number of mammalian-like adenylyl cyclases, only a single cAMP phosphodiesterase has been identified that can decrease levels of cAMP produced by the bacterium. We present the crystal structure of the full-length and sole cAMP phosphodiesterase, Rv0805, found in M. tuberculosis, whose orthologs are present only in the genomes of slow growing and pathogenic mycobacteria. The dimeric core catalytic domain of Rv0805 adopts a metallophosphoesterase-fold, and the C-terminal region builds the active site and contributes to multiple substrate utilization. Localization of Rv0805 to the cell wall is dependent on its C terminus, and expression of either wild type or mutationally inactivated Rv0805 in M. smegmatis alters cell permeability to hydrophobic cytotoxic compounds. Rv0805 may therefore play a key role in the pathogenicity of mycobacteria, not only by hydrolyzing bacterial cAMP, but also by moonlighting as a protein that can alter cell wall functioning.Mycobacterium tuberculosis is probably one of the most successful human pathogens known so far, being singly responsible for the largest number of deaths worldwide due to an infectious disease. M. tuberculosis is phagocytosed by the macrophage and is able to subvert the defenses of the host by a number of mechanisms. These include the presence of a complex cell wall that prevents free passage of potentially toxic material, the ability of the bacteria to withstand the acidic environment of the phagolysosome, and to neutralize reactive oxygen and nitrogen species produced by the activated macrophage (1). An increased understanding of mechanisms utilized by this pathogen to evade the host immune system and continue to reside in the hostile environment of the macrophage, would no doubt provide avenues for the development of drugs to novel targets in the bacterium.Cross-communication between the pathogen and host could involve the utilization of signaling molecules that are conserved evolutionarily. Cyclic AMP is found in all kingdoms of life, and proteins that synthesize and degrade the cyclic nucleotide have been well characterized. The genome of M. tuberculosis H37Rv encodes 16 mammalian-like nucleotide cyclase-like genes (2), and intracellular and extracellular levels of cAMP are very high in both pathogenic and non-pathogenic (e.g. Mycobacterium smegmatis) mycobacteria (2, 3). Recent evidence has highlighted the importance of cAMP in modulating the host macrophage response to M. tuberculosis infection (4). A single adenylyl cyclase was shown to be responsible for the burst of cAMP that is seen in the macrophage following phagocytosis of M. tuberculosis, and this bacterially derived increase in cAMP was essential to attenuate the response of the macrophage to the pathogen.The regulated degradation of cAMP is as important as its synthesi...

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Linearization of the Bradford Protein Assay Increases Its Sensitivity: Theoretical and Experimental Studies

Cited by 922 publications

References 3 publications

Demonstration That the Radical S-Adenosylmethionine (SAM) Enzyme PqqE Catalyzes de Novo Carbon-Carbon Cross-linking within a Peptide Substrate PqqA in the Presence of the Peptide Chaperone PqqD

Demonstration That the Radical S-Adenosylmethionine (SAM) Enzyme PqqE Catalyzes de Novo Carbon-Carbon Cross-linking within a Peptide Substrate PqqA in the Presence of the Peptide Chaperone PqqD

Retro-biosynthetic screening of a modular pathway design achieves selective route for microbial synthesis of 4-methyl-pentanol

A Mycobacterial Cyclic AMP Phosphodiesterase That Moonlights as a Modifier of Cell Wall Permeability

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