Jules A.T.P. Meuwissen

Critique of the Assay and Significance of Bilirubin Conjugation

Publisher Summary This chapter describes the methodology and properties of uridine diphosphate (UDP)-glucuronyltransferase and of the related UDP-glycosyltransferase activities. It also delineates the applications to human disease. Bilirubin derives largely from senescent erythrocyte hemoglobin. In most normal adult animals, conjugation of bilirubin with various sugars represents the major mechanism for its transformation. The reaction products are eliminated from the organism by excretion into the bile. Glucuronyl transfer is of predominant importance in the biotransformation of bilirubin in man and rat. Glucuronyl transference to bilirubin and to many other acceptor substrates (G5, L14, M14, P4, W4), with the exception of phenolphthalein (G5), is higher in the rough endoplasmic reticulum than it is in the smooth endoplasmic reticulum. The main field of application of the in vitro assays of bilirubin UDP glycosyltransferase activities is the evaluation of stimulation or inhibition—by drugs or endogenous substances—of the enzyme activities or of their maturation. Development of unconjugated hyper-bilirubinemia in some neonates is related to breast feeding.

Modelling of chemical reactions catalysed by membrane-bound enzymes. Determination and significance of the kinetic constants

A model of multiphasic systems, based on the assumption of zero-order partition of substrates and products into the membranes, is applied to reversible mono-substrate and bi-substrate reactions catalysed by membrane-bound enzymes. Apart from replacement of single-phase kinetic constants by apparent kinetic constants, the derived kinetic expressions are formally identical with those for corresponding single-phase systems. The model confers to the apparent kinetic constants an experimentally verifiable meaning. For full characterization of membrane-kinetic systems, experiments at various concentrations of enzyme-embedding phospholipid are required. Extrapolation to zero phospholipid concentration of each Km app then yields the corresponding true kinetic constant characteristic of the membrane-bound enzyme and also provides a technique for determination of the membrane-partition constants. The procedure implies that the phospholipid content should be assayed for full characterization of membrane-bound enzymes. If, for practical reasons, the assays have to be limited to a single enzyme concentration, correction of the apparent kinetic constants is still possible provided the phospholipid concentration and the partition constants of the reactants are known. The model has permitted prediction of a number of previous observations reflecting the multiphasic nature of the systems. The assumptions, underlying the model, and their implications are examined as well as some commonly used experimental designs for determination of the type of enzymic site.

Selective absorption and scattering of light by solutions of macromolecules and by particulate suspensions

For particulate suspensions and for solutions that scatter light measurably the total absorbance A generally contains contributions due to specific absorption (Aa) and scattering of light (As). The quantity As is closely related to the turbidity tau. In general, spectrophotometry of such systems requires proper modification of the spectrophotometer used in order to permit accurate determination of the absorbance A and of the derived quantities Aa and As. Apparent deviation from Beers law in such systems is often due to inappropriate experimental technique. After a discussion of the parameters that determine the intensity of light scattered by solutes, an account is given of the experimental precautions to be taken for determination of the absorbance of light scattering suspensions and solutions and of techniques for correcting absorbance spectra for scattering of light. Measurement of the turbidity is briefly confronted with determination of the scattering ratio i90 degrees/Io and the impact of erroneous turbidity measurements on derived molecular parameters is discussed.