An X-ray structure of the F1 portion of the mitochondrial ATP synthase shows asymmetry and differences in nucleotide binding of the catalytic β subunits that support the binding change mechanism with an internal rotation of the γ subunit. Other structural and mutational probes of the F1 and F0 portions of the ATP synthase are reviewed, together with kinetic and other evaluations of catalytic site occupancy and behavior during hydrolysis or synthesis of ATP. Subunit function as related to proton translocation and rotational catalysis is considered. Physical demonstrations of the γ subunit rotation have been achieved. The findings have implications for other enzymatic catalyses.

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The ATP Synthase—A Splendid Molecular Machine

Flavonoids: antioxidants or signalling molecules?

Electrochemical biosensors: recommended definitions and classification.

Rotating ring-disk enzyme electrode for biocatalysis kinetic studies and characterization of the immobilized enzyme layer

The binding change mechanism for ATP synthase — Some probabilities and possibilities

Lipid composition and protein profiles of outer and inner membranes from pig heart mitochondria. Comparison with microsomes.

A new trace glucose analyzer has been designed using electrochemical sensors. The differential device includes (a) a glucose sensor consisting of a modified gas electrode in which ihe pH detector was replaced by a platinum disk and the porous film by a collagen membrane on which beta-D-glucose oxidase has been covalently bound after an acyl-azide activation process; (b) a compensating electrode mounted with a nonenzymatic collagen membrane. After injection of a glucose containing sample into the reaction vessel, where the probes are immersed, an anodic current is detected at the enzyme working electrode. Current outputs of both electrodes are subtracted and twice differentiated; a steady state is reached and the stationary and dynamic responses are recorded. Both responses are proportional to glucose concentration in the 0.1 µM-2 mM range, and the reproducibility was found to be better than 2% using these conditions. The extreme sensitivity exhibited by our system, i.e., 10 nM, is better than previously reported data by 3 orders of magnitude, and is very favorable for trace glucose assays in food and biological samples.

/pdf/enzyme-collagen-membrane-for-electrochemical-determination-g4qh0rwm9j.pdf

Enzyme collagen membrane for electrochemical determination of glucose

Films of highly polymerized collagen, prepared in industrial conditions, were chosen for surface covalent binding of enzymes because of their insolubility, mechanical resistance, proteic nature, hydrophilic properties and for their abundance in chemically activable COOH. Untanned films, previously acid- methylated, were activated by acyl azide formation. After removal of reagents by repeated washing, the coupling of enzyme was performed by immersion of the activated film in the enzyme solutions (2 to 3 hr, 0°C). The procedure is particularly mild since the enzymes never come into contact with chemical reagents, and thus avoid all denaturing processes. All the enzymes tested were successfully bound: glutamate dehydrogenase, lactate dehydrogenase, malate dehydrogenase, aspartate aminotransferase, glutamate pyruvate transaminase, creatine kinase, hexokinase, trypsin, and urease. As tested with aspartate amino transferase, enzymatic activity remained constant for months (100% after 5 months) in spite of repeated use of the film at 30°C, washing and storage in buffer at 4°C between assays.

Danièle C. Gautheron

Papers

Lipid composition and protein profiles of outer and inner membranes from pig heart mitochondria. Comparison with microsomes.

Enzyme collagen membrane for electrochemical determination of glucose

A mild method of general use for covalent coupling of enzymes to chemically activated collagen films

Multipurpose electrode with different enzyme systems bound to collagen films

"Hysteretic" behavior and nucleotide binding sites of pig heart mitochondrial F1 adenosine 5'-triphosphatase