An SPR-based method for Hill coefficient measurements: the case of insulin-degrading enzyme

Two substrates of insulin-degrading enzyme (IDE), amyloid beta-protein (Abeta) and insulin, are critically important in the pathogenesis of Alzheimer's disease (AD) and type 2 diabetes mellitus (DM2), respectively. We previously identified IDE as a principal regulator of Abeta levels in neuronal and microglial cells. A small chromosomal region containing a mutant IDE allele has been associated with hyperinsulinemia and glucose intolerance in a rat model of DM2. Human genetic studies have implicated the IDE region of chromosome 10 in both AD and DM2. To establish whether IDE hypofunction decreases Abeta and insulin degradation in vivo and chronically increases their levels, we characterized mice with homozygous deletions of the IDE gene (IDE --). IDE deficiency resulted in a >50% decrease in Abeta degradation in both brain membrane fractions and primary neuronal cultures and a similar deficit in insulin degradation in liver. The IDE -- mice showed increased cerebral accumulation of endogenous Abeta, a hallmark of AD, and had hyperinsulinemia and glucose intolerance, hallmarks of DM2. Moreover, the mice had elevated levels of the intracellular signaling domain of the beta-amyloid precursor protein, which was recently found to be degraded by IDE in vitro. Together with emerging genetic evidence, our in vivo findings suggest that IDE hypofunction may underlie or contribute to some forms of AD and DM2 and provide a mechanism for the recently recognized association among hyperinsulinemia, diabetes, and AD. Show more

Enzymes are biological catalysts (also known as biocatalysts) that speed up biochemical reactions in living organisms, and which can be extracted from cells and then used to catalyse a wide range of commercially important processes. This chapter covers the basic principles of enzymology, such as classification, structure, kinetics and inhibition, and also provides an overview of industrial applications. In addition, techniques for the purification of enzymes are discussed. Show more

Hill coefficients (n(H)) derived from four parameter logistic fits to dose-response curves were compared to calculated realistic reaction schemes and related to experimental data: (1) Hill coefficients may give information on the number of interacting sites but cannot distinguish between competitive, non-competitive or ortho-, iso-, or allosteric mechanisms. (2) For enzymatic dose-inhibition curves, Hill coefficients smaller than one do not indicate anticooperative binding but show that at least one ternary complex has enzymatic activity. (3) Hill coefficients different from one are proof for multiple ligand binding. The large variations of reported Hill coefficients corresponds to multiple allosteric binding, where induced conformational changes cause loss of the active conformation. Such a denaturation mechanism is in stark contrast to the desired specificity of drugs. The discussion is open. Show more