Biochemistry without oxygen

Sw, Englander; Db, Calhoun; Jj, Englander

doi:10.1016/0003-2697(87)90454-4

Cited by 161 publications

(125 citation statements)

References 13 publications

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“…l), equilibrium microdialysis and ultrafilt- higher than when measured anaerobically by the glucose oxidase system [37] (Fig. 2B).…”

Section: Equilibrium Binding Of L-noradrenaline To Native Bovine and mentioning

confidence: 74%

“…In order to perform the binding studies under anaerobic conditions, without directly affecting the redox state of the enzyme-bound iron, all solutions were prepared free of dioxygen by the glucose oxidase method [37]; 2 p1 of a stock solution of glucose oxidase (20 pg/ml) and catalase (2 mg/ ml) were added/ml dialysis buffer (pH 7.25), containing 0.3% (mass/vol.) glucose and incubated for 30 rnin at 20 "C, before phenylalanine hydroxylase was added.…”

Section: Equilibrium Binding Studiesmentioning

confidence: 99%

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Cooperative homotropic interaction of l‐noradrenaline with the catalytic site of phenylalanine 4‐monooxygenase

Martı́nez

Haavik

Flatmark

1990

European Journal of Biochemistry

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Catecholamines (adrenaline, noradrenaline and dopamine) are potent inhibitors of phenylalanine 4-monooxygenase (phenylalanine hydroxylase, EC 1.14.16.1). The amines bind to the enzyme by a direct coordination to the high-spin ( S = 5/2) Fe(II1) at the active site (charge transfer interaction), as seen by resonance Raman and EPR spectroscopy. Experimental evidence is presented that a group with an apparent pK, value of about 5.1 (20°C) is involved in the interaction between the catecholamine and the enzyme. The high-affinity binding of L-noradrenaline to phenylalanine hydroxylase, as studied by equilibrium microdialysis (anaerobically) and ultrafiltration (aerobically), shows positive cooperativity (h = 1.9); at pH 7.2 and 20°C the rat enzyme binds about 0.5 mol Lnoradrenaline/mol subunit with a half-maximal binding (Sso) at 0.25 pM L-noradrenaline. No binding to the ferrous form of the enzyme was observed. The affinity decreases with decreasing pH, by phosphorylation and by preincubation of the enzyme with the substrate L-phenylalanine, while it increases after alkylation of the enzyme with the activator N-ethylmaleimide. Preincubation of the enzyme with L-phenylalanine also leads to a complete loss of the cooperativity of L-noradrenaline binding (h = 1 .O). The many similarities in binding properties of the inhibitor L-noradrenaline and the activator/substrate L-phenylalanine makes it likely that the cooperative interactions of these effectors are due to their binding to the same site. The high-affinity of catecholamines to phenylalanine hydroxylase is a valuable probe to study the active site of this enzyme and is also relevant for the homologous enzyme tyrosine hydroxylase, which is purified as a stable catecholamine-Fe(II1) complex.

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“…l), equilibrium microdialysis and ultrafilt- higher than when measured anaerobically by the glucose oxidase system [37] (Fig. 2B).…”

Section: Equilibrium Binding Of L-noradrenaline To Native Bovine and mentioning

confidence: 74%

Section: Equilibrium Binding Studiesmentioning

confidence: 99%

Cooperative homotropic interaction of l‐noradrenaline with the catalytic site of phenylalanine 4‐monooxygenase

Martı́nez

Haavik

Flatmark

1990

European Journal of Biochemistry

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“…Human Hb was prepared from fresh red cells by standard methods and was stored frozen at Ϫ80°C. Deoxygenation used argon bubbling, dithionite, a glucose oxidase-catalase mixture, and ferrous pyrophosphate (23,24), which also acts as a T state-selective bisphosphoglycerate analog.…”

Section: Methodsmentioning

confidence: 99%

Protein structure change studied by hydrogen-deuterium exchange, functional labeling, and mass spectrometry

Englander

Mar

et al. 2003

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

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202

191

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An automated high-throughput, high-resolution deuterium exchange HPLC-MS method (DXMS) was used to extend previous hydrogen exchange studies on the position and energetic role of regulatory structure changes in hemoglobin. The results match earlier highly accurate but much more limited tritium exchange results, extend the analysis to the entire sequence of both hemoglobin subunits, and identify some energetically important changes. Allosterically sensitive amide hydrogens located at near amino acid resolution help to confirm the reality of local unfolding reactions and their use to evaluate resolved structure changes in terms of allosteric free energy.H ydrogen exchange (HX) measurements can, in principle, locate protein-binding sites and structure changes and can quantify otherwise unavailable dynamic and energetic parameters (1-4). For relatively small proteins, HX can be measured at an amino acid resolved level by NMR methods. For larger, functionally more interesting proteins, other strategies are required. Earlier work (5, 6) developed a ''functional-labeling'' approach that can selectively label, by hydrogen-tritium (H-T) or hydrogen-deuterium (H-D) exchange, just those sites that change in any functional process. In favorable cases, the label can then be located at medium resolution by a proteolytic fragmentation method in which the fragments are quickly produced and then separated by HPLC under conditions where the loss of isotopic label is slow (6-9).To move toward higher resolution and more comprehensive coverage of target proteins, recent work in many laboratories has coupled the HPLC separation to a second dimension of fragment resolution by online MS (10, 11). These methods tend to be labor intensive and time consuming, with limitations in throughput and comprehensiveness and in the structural resolution of functionally important changes. This article merges previous HX functional labeling and fragment separation methods with an automated MS approach termed deuterium exchange MS (DXMS) (12-18).We are using Hb as a model system to study how protein molecules manage intramolecular signal transduction processes. Hb functions by transducing a part of the binding energy of its initially bound O 2 ligands into structure-change energy. The energy is carried through the protein to distant heme sites in the form of energetic structure changes, and there transduced back into binding energy. The initial reduced binding energy and the later enhanced binding produces the physiologically important sigmoid binding curve. In short, the currency of allosteric interactions is free energy. Trying to understand allostery without measuring free energy is like trying to understand an economic system without measuring money. A great deal of information on regulatory structure change in many proteins is now available, but mainly in a qualitative pictorial sense from ''before and after'' crystallographic or NMR views. How these changes participate in energy transduction and translocation has been little explored (19)(20)(21...

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“…Fluorescence Imaging-After washing, the cells on coverslips were covered with I-MEM containing glucose, glucose-oxidase, and catalase to deplete oxygen from the medium (28). Oxygen depletion virtually eliminates photobleaching of the pyrene chromophore but has no detectable effect on fluorophore distribution or cell morphology (25).…”

Section: Methodsmentioning

confidence: 99%

γ-Cyclodextrins Greatly Enhance Translocation of Hydrophobic Fluorescent Phospholipids from Vesicles to Cells in Culture

Tanhuanpää

Somerharju

1999

Journal of Biological Chemistry

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Short-chain, fluorescent derivatives are commonly used to investigate intracellular phospholipid trafficking. However, their use can yield misleading results because they, unlike the native species, can rapidly distribute between organelles due to their low hydrophobicity. On the other hand, hydrophobic derivatives are very difficult to introduce to cells and thus have hardly been used. Here we show that carboxyethylated ␥-cyclodextrin (CE-␥-CD) greatly enhances transfer of a variety of hydrophobic fluorescent phospholipid derivatives from vesicles to cultured cells. Several lines of evidence indicate that CE-␥-CD enhances transfer of lipid molecules by increasing their effective concentration in the aqueous phase, rather than by inducing membrane fusion or hemifusion. Incubation with CE-␥-CD and donor lipid vesicles does not extract cholesterol or phospholipids from the cells or compromise plasma membrane intactness or long term cell viability. Using CE-␥-CD-mediated transfer, we introduced hydrophobic pyrene-labeled phosphatidylserine to the plasma membrane of fibroblast cells and followed their distribution with time. In contrast to what has been previously observed for other, less hydrophobic species, transport of this lipid to the Golgi apparatus or mitochondria was not detected. Rather, much of this fluorescent PS remained in the plasma membrane or was incorporated to various endocytotic compartments. These findings indicate that the native, typically hydrophobic phosphatidylserine molecules efflux only very slowly via the cytoplasm to intracellular organelles. This helps to explain how cells can maintain a very high concentration of phosphatidylserine in the inner leaflet of their plasma membrane. Furthermore, the present results underline the importance of using hydrophobic analogues when studying intracellular trafficking of many phospholipid classes.

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Biochemistry without oxygen

Cited by 161 publications

References 13 publications

Cooperative homotropic interaction of l‐noradrenaline with the catalytic site of phenylalanine 4‐monooxygenase

Cooperative homotropic interaction of l‐noradrenaline with the catalytic site of phenylalanine 4‐monooxygenase

Protein structure change studied by hydrogen-deuterium exchange, functional labeling, and mass spectrometry

γ-Cyclodextrins Greatly Enhance Translocation of Hydrophobic Fluorescent Phospholipids from Vesicles to Cells in Culture

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