A one-step, low background Coomassie staining procedure for polyacrylamide gels

Zehr, Bradley D.; Savin, Thomas J.; Hall, Robert E.

doi:10.1016/0003-2697(89)90734-3

Cited by 127 publications

(83 citation statements)

References 5 publications

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“…Protein concentration was determined according to Bradford (20), using BSA as standard. SDS gels (10%) were prepared according to Laemmli (21), and protein bands were visualized by Coomassie staining (22).…”

Section: Methodsmentioning

confidence: 99%

Mechanism of ATP-driven electron transfer catalyzed by the benzene ring-reducing enzyme benzoyl- CoA reductase

Unciuleac

Boll

2001

Proc. Natl. Acad. Sci. U.S.A.

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Benzoyl-CoA reductase (BCR) from the bacterium Thauera aromatica catalyzes the two-electron reduction of benzoyl-CoA (BCoA) to a nonaromatic cyclic diene. In a process analogous to enzymatic nitrogen reduction, BCR couples the electron transfer to the aromatic ring to a stoichiometric hydrolysis of 2 ATP͞2e ؊ . Reduced but not oxidized BCR hydrolyzes ATP to ADP. In this work, purified BCR was shown to catalyze an isotope exchange from [ 14 C]ADP to [ 14 C]ATP, which was Ϸ15% of the ATPase activity in the presence of equimolar amounts of ADP and ATP. In accordance, BCR (␣␤␥␦-composition) autophosphorylated its ␥-subunit when incubated with [␥-32 P]ATP. Formation of the enzyme-phosphate was independent of the redox state, whereas only dithionitereduced BCR catalyzed a dephosphorylation associated with the ATPase activity. This finding suggests that the ATPase-and autophosphatase-partial activities of BCR exhibit identical redox dependencies. BCoA or the nonphysiological electron-accepting substrate hydroxylamine stimulated the redox-dependent effects; the rates of both the overall ATPase and the autophosphatase activities of reduced BCR were increased 6-fold. In contrast, BCoA and hydroxylamine had no effect on oxidized and phosphorylated BCR. The reactivity of the phosphoamino acid fits best with a phosphoamidate or acylphosphate linkage. The results obtained suggest a mechanism of ATP hydrolysis-driven electron transfer, which differs from that of nitrogenase by the transient formation of a phosphorylated enzyme.B enzoyl-CoA (BCoA) is a central intermediate in the metabolism of many aromatic compounds in anaerobic bacteria. This compound becomes reduced by benzoyl-CoA reductase (BCR), a key enzyme in anaerobic aromatic metabolism (1, 2). It catalyzes the reductive dearomatization of BCoA to cyclohexa-1,5-diene-1-carbonyl-CoA ( Fig. 1; ref. 3). In this reaction, the hydrolysis of two ATP to ADP and Pi is stoichiometrically coupled to the transfer of two single electrons from reduced ferredoxin to the aromatic substrate. Notably, BCR also catalyzes the ATP-dependent reduction of the nonphysiological substrate hydroxylamine (2). The stoichiometric coupling of ATP hydrolysis to an electron transfer process has long been considered as a unique feature of nitrogenases (4, 5). However, there are no amino acid sequence similarities between BCR and nitrogenases.The reduction of the aromatic ring is a mechanistically and energetically difficult process that in organic synthesis requires solvated electrons, which are generated by dissolving alkali metals in liquid ammonia. In a so-called Birch mechanism, the reduction of the aromatic ring proceeds in single electron and proton transfer steps by means of radical intermediates (6). A similar mechanism has been proposed for enzymatic benzene ring reduction (7). The crucial step is the first electron transfer to the aromatic ring yielding a nonaromatic radical anion, which requires a redox potential of Ϫ1.9 V (3). BCR overcomes this high redox barrier by coupling a stoichiomet...

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Section: Methodsmentioning

confidence: 99%

Mechanism of ATP-driven electron transfer catalyzed by the benzene ring-reducing enzyme benzoyl- CoA reductase

Unciuleac

Boll

2001

Proc. Natl. Acad. Sci. U.S.A.

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“…Gels were then incubated in 2.5% Triton X-100 for 30 minutes, further incubated for 18 hours at 37°C in 50 mmol/L Tris/10 mmol/L CaCl 2 , pH 8.2, and finally stained with 0.008% Coomassie blue R (Sigma). 32 Various proteinase inhibitors (30 mmol/L EDTA [Sigma]), recombinant TIMP-1 (a gift from Dr H. Nagase), or 1 mmol/L PMSF (Sigma) was added to the incubating buffer to assess the class of proteinase. Activation with 1 mmol/L APMA was used to identify MMP activities.…”

Section: Gelatin and Casein Zymography Of Mmpsmentioning

confidence: 99%

Prevention of Aneurysm Development and Rupture by Local Overexpression of Plasminogen Activator Inhibitor-1

Allaire¹,

Hasenstab²,

Kenagy³

et al. 1998

Circulation

130

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Background-Arterial aneurysms exhibit a loss of elastin and an increase in the plasminogen activators urokinase plasminogen activator (u-PA) and tissue plasminogen activator (t-PA). Because u-PA, t-PA, and plasmin have a limited proteolytic activity against elastin, the role of plasminogen activators in the aneurysmal disease is unclear. To investigate this question, we overexpressed plasminogen activator inhibitor-1 (PAI-1), an inhibitor of t-PA and u-PA, in a rat model of aortic aneurysm. Methods and Results-Guinea pig-to-rat aortic xenografts were seeded with syngeneic Fischer 344 rat smooth muscle cells retrovirally transduced with the rat PAI-1 gene (LPSN group) or the vector alone (LXSN group). Some grafts were not seeded with cells (NO group). Western blots showed increased PAI-1 in grafts from the LPSN group compared with LXSN and NO groups. All grafts in the NO group (nϭ8) and 40% in the LXSN group ruptured between days 4 and 14. At 4 weeks in the LXSN group, the remaining unruptured grafts (nϭ6) were aneurysmal (diameter increase Ն100%), whereas in the LPSN group (nϭ6) none of the grafts had ruptured or were aneurysmal. Elastin was preserved in the LPSN group. t-PA, the major PA expressed in the model, was decreased in the LPSN group compared with the other groups, as determined by zymography. Quantitative zymography showed decreased levels of two matrix metalloproteinases (MMPs), a 28-kD caseinase, and activated MMP-9 in the LPSN group. Conclusions-The blockade of plasminogen activators prevents formation of aneurysms and arterial rupture by inhibiting MMP activation. (Circulation. 1998;98:249-255.)

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“…Proteins were stained by silver staining [31] and with Coomassie blue [32]. Gels stained by either method were scanned with a videodensitometric system.…”

Section: Protein Determinationmentioning

confidence: 99%

Enzymes of anaerobic metabolism of phenolic compounds

Brackmann

Fuchs

1993

European Journal of Biochemistry

110

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The reductive removal of aromatic hydroxyl functions plays an important role in the anaerobic metabolism of many phenolic compounds. We describe a new enzyme from a denitrifying Pseudornonas sp., 4-hydroxybenzoyl-CoA reductase (dehydroxylating), which reductively dehydroxylates 4-hydroxybenzoyl-CoA to benzoyl-CoA. The enzyme plays a role in the anaerobic degradation of phenol, 4-hydroxybenzoate, p-cresol, 4-hydroxyphenylacetateate, and other aromatic compounds of which 4-hydroxybenzoyl-CoA is an intermediate. The enzyme is therefore induced only under anoxic conditions with these aromatic substrates, but not with benzoate or under aerobic conditions. A similar enzyme which reductively dehydroxylates 3-hydroxybenzoyl-CoA is induced during anaerobic growth with 3-hydroxybenzoate. The soluble enzyme 4-hydroxybenzoyl-CoA reductase was purified. It has a molecular mass of 260 kDa and consists of three subunits of 75, 35, and 17 kDa. The subunit composition is likely to be a2b2c2. The enzyme contains 12 mol irodmol and 12 mol acid-labile sulfur/mol and exhibits a typical ultraviolethisible spectrum of an iron-sulfur protein.The reaction requires a reduced electron donor such as reduced viologen dyes; no other co-catalysts are required, the product is benzoyl-CoA and oxidized dye. The reductase is rapidly inactivated by oxygen. The inactivation by low concentrations of cyanide or azide in a pseudo-first-order time course suggests that it may contain a transition metal in an oxidation state which reacts with these ligands. 4-Hydroxybenzoyl-CoA reductase represents a type of enzyme which is common in anaerobic aromatic metabolism of phenolic compounds. A similar enzyme is demonstrated in Rhodopseudornonas palustris anaerobically grown with 4-hydroxybenzoate. The biological significance of reductive dehydroxylation of aromatics and a possible reaction mechanism similar to the Birch reduction are discussed.Aromatic compounds comprise the second largest group of natural products. Most of the naturally occurring aromatics carry one or several hydroxyl functions which are often protected against undesired oxidation and other side reactions by methyl ether formation. They are metabolized by microorganisms following two fundamentally different strategies. Under aerobic conditions aromatic compounds are transformed by monooxygenases and dioxygenases into dihydroxylated intermediates such as catechol, protocatechuate, and gentisate. These dihydroxylated compounds are suitable for an oxidative cleavage of the aromatic ring (reviewed recently by [l]).Hydroxylation and oxidative ring cleavage by oxygenases cannot exist under anoxic conditions and therefore a different strategy is required for the anaerobic aromatic metabolism (for recent reviews see 22-61). Notably, in contrast to aerobic metabolism, hydroxylated aromatics are

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A one-step, low background Coomassie staining procedure for polyacrylamide gels

Cited by 127 publications

References 5 publications

Mechanism of ATP-driven electron transfer catalyzed by the benzene ring-reducing enzyme benzoyl- CoA reductase

Mechanism of ATP-driven electron transfer catalyzed by the benzene ring-reducing enzyme benzoyl- CoA reductase

Prevention of Aneurysm Development and Rupture by Local Overexpression of Plasminogen Activator Inhibitor-1

Enzymes of anaerobic metabolism of phenolic compounds

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