Kinetic studies on the interaction of phosphatidylcholine liposomes with Triton X-100.

Sonicated unilamellar and large multilamellar liposome suspensions have been treated with the non-ionic detergent Triton X-100, and the subsequent changes in turbidity have been studied as a function of time. Sonicated liposome suspensions exhibit an increase in turbidity that takes place in two stages, a fast, low-amplitude one is completed in less than 100 ms, and a slow large-amplitude one occurs in 20-40 s. The first increase in turbidity is associated to detergent incorporation into the bilayer, and the second one, to vesicle fusion. The fast stage may be detected at all detergent concentrations, while the slow one is only seen above the critical micellar concentration of Triton X-100. Both processes may be interpreted in terms of first-order kinetics. Studies of the variation of kexp with lipid and detergent concentration suggest a complex multi-step mechanism. In the case of multilamellar liposomes, a fast increase in turbidity is also seen after detergent addition, which is followed by a slow (20-60 s) decrease in turbidity and a very slow (up to 12 h) large scale decrease in turbidity. These processes do not conform to single-exponential patterns. The fast stage is also thought to reflect surfactant incorporation, while the decrease in turbidity is interpreted as bilayer solubilization starting with the outer bilayer (slow stage) and proceeding through the remaining ones (very slow stage).

A kinetic study of the interaction between mitochondrial F1 adenosine triphosphatase and adenylyl imidodiphosphate and guanylyl imidodiphosphate.

1. The presence of 5'-adenylyl imidodiphosphate, a non-hydrolysable analogue of ATP, in the solution used to assay the soluble bovine heart mitochondrial F1-ATPase produced slow competitive inhibition. If the enzyme was preincubated with the inhibitor before the substrate, MgATP, was added, a partial re-activation was obtained. 2. The slow inhibitory process showed first-order rate kinetics, and therefore it seems likely that a conformational change of the enzyme occurs following a faster binding process. A reaction scheme is suggested. At pH 7.8 the rate constant for the inhibition reaction was calculated to be 6.7 X 10(-2)s-1 and that for the re-activation 3.8 X 10(-3)s-1, with Keq. 17.6, indicating that the inhibited enzyme-inhibitor complex will be favoured over the non-inhibited enzyme-inhibitor complex. 3. The presence of 5'-guanylyl imidodiphosphate in the solution used to assay F1-ATPase produced rapid competitive inhibition, which was then slowly reversed until a steady state was reached. This might be explained by a rapid but reversible shift of the inhibition pathway induced by this non-hydrolysable analogue of ATP. A complex rate constant for the displacement of the inhibitor by the substrate of 7.6 X 10(-3)s-1 was calculated. 4. The results are discussed in the light of other recent observations about binding of 5'-adenylyl imidodiphosphate to F1-ATPase and with reference to the binding-site-change mechanism of hydrolysis of ATP by F1-ATPase.

Enzymatic assays: optimization of systems by using pyruvate kinase and lactate dehydrogenase as auxiliary enzymes.

A method to optimize enzymatic assays by using pyruvate kinase and lactate dehydrogenase enzymes is presented and applied to mitochondrial ATPase as an example. Optimum amounts of auxiliary enzymes, to obtain either a 99% of the initial rate in a given time (t99) or a given lag period (L), are calculated from their apparent Michaelis constants (Kapp) in the medium used and their prices per enzymatic international unit.

The role of pH in the melanin biosynthesis pathway.

Having oxidized 3,4-dihydroxyphenylalanine (dopa) with sodium periodate or mushroom tyrosinase in a pH range from 3.5 to 6.0, it has been possible to detect spectrophotometrically 4-(2-carboxy-2-aminoethyl)-1,2-benzoquinone with the amino group protonated (o-dopaquinone-H+), a postulated intermediate in the melanogenesis pathway. When the pH value was greater than 4, the final product obtained was 2-carboxy-2,3-dihydroindole-5,6-quinone (dopachrome); however, for pH values lower than 4, two different products were identified by means of cyclic voltammetry: 5-(2-carboxy-2-aminoethyl)-2-hydroxy-1,4-benzoquinone and dopachrome. These products appeared when oxidation was achieved with the enzyme as well as with periodate. This suggests that two chemical pathways can arise from alpha-dopaquinone-H+, whose relative importance is determined by the pH. The steps of these pathways would be dopa leads to o-dopaquinone-H+ leads to o-dopaquinone leads to leukodopachrome leads to dopachrome, for the first one, and dopa leads to o-dopaquinone-H+ leads to 2,4,5-trihydroxyphenylalanine leads to 5-(2-carboxy-2-aminoethyl)-2-hydroxy-1,4-benzoquinone very slowly leads to intermediate compound leads to dopachrome, for the second one. The stoichiometry for the conversion of dopaquinone-H+ into dopachrome for pH values greater than 4 followed equation of 2 o-dopaquinone-H+ leads to dopa + dopachrome. No participation of oxygen was detected in the conversion of leukodopachrome (2,3-dihydro-5,6-dihydroxyindole-2-carboxylate) into dopachrome.