Determination of the kinetic properties of platycodin D for the inhibition of pancreatic lipase using a 1, 2-diglyceride-based colorimetric assay

A new colorimetric assay is described, based on inhibition of protein phosphotases, that enables the rapid, simple and sensitive determination of the concentration of toxins from cyanobacteria.

Download Full-text

The Need for Using An Enzymatic Colorimetric Assay in Creatinine Determination of Peritoneal Dialysis Solutions

Peritoneal Dialysis International ◽

10.1177/089686089001000122 ◽

1990 ◽

Vol 10 (1) ◽

pp. 89-92 ◽

Cited By ~ 17

Author(s):

Liliane Larpent ◽

Christian Verger

Keyword(s):

Peritoneal Dialysis ◽

Reliable Method ◽

Colorimetric Assay ◽

Colorimetric Method ◽

Peritoneal Membrane ◽

Creatinine Kinetics ◽

Dialysis Solutions ◽

Peritoneal Dialysis Solutions ◽

Enzymatic Test

The fate of the peritoneal membrane on continuous ambulatory peritoneal dialysis (CAPD) is usually evaluated through the modification of its permeability to various solutes as glucose, creatinine, and urea. Therefore, the accuracy of the methods used for measurements of creatinine is of great importance. A particular problem does exist for creatinine determination as it may be influenced by the presence of glucose. We studied a new enzymatic colorimetric method for creatinine determination in peritoneal dialysis solutions which contain high dextrose concentrations. Creatinine was measured in plasma, urine, and dialysate from 18 patients on CAPD and in pure dextrose solutions, with an enzymatic test (Boehringer Mannheim) and with Jaffe's reaction on two different analyzers: Astra (Beckman) and Eris (Merck). Creatinine results were similar with both assays (Jaffe's reaction and enzymatic test) when measured in blood and urine. However the Jaffe's reaction gave higher creatinine results than the enzymatic test (p < 0.001), when assays were performed in peritoneal dialysis solutions and in pure glucose solutions. In addition, it appeared that other components of dialysis solutions, mainly calcium chloride, influenced unpredictably the results of creatinine with the Jaffe's reaction. We conclude that specific enzymatic test is a more accurate and reliable method to evaluate creatinine kinetics through the peritoneal membrane when determined in CAPD solutions.

Download Full-text

Gating Competence of Constitutively Open CLC-0 Mutants Revealed by the Interaction with a Small Organic Inhibitor

The Journal of General Physiology ◽

10.1085/jgp.200308784 ◽

2003 ◽

Vol 122 (3) ◽

pp. 295-306 ◽

Cited By ~ 52

Author(s):

Sonia Traverso ◽

Laura Elia ◽

Michael Pusch

Keyword(s):

Chloride Channels ◽

Bound State ◽

Side Chain ◽

Pore Channel ◽

Kinetic Properties ◽

Wild Type ◽

High Affinity ◽

Critical Residue ◽

Fast Gating

Opening of CLC chloride channels is coupled to the translocation of the permeant anion. From the recent structure determination of bacterial CLC proteins in the closed and open configuration, a glutamate residue was hypothesized to form part of the Cl−-sensitive gate. The negatively charged side-chain of the glutamate was suggested to occlude the permeation pathway in the closed state, while opening of a single protopore of the double-pore channel would reflect mainly a movement of this side-chain toward the extracellular pore vestibule, with little rearrangement of the rest of the channel. Here we show that mutating this critical residue (Glu166) in the prototype Torpedo CLC-0 to alanine, serine, or lysine leads to constitutively open channels, whereas a mutation to aspartate strongly slowed down opening. Furthermore, we investigated the interaction of the small organic channel blocker p-chlorophenoxy-acetic acid (CPA) with the mutants E166A and E166S. Both mutants were strongly inhibited by CPA at negative voltages with a >200-fold larger affinity than for wild-type CLC-0 (apparent KD at −140 mV ∼4 μM). A three-state linear model with an open state, a low-affinity and a high-affinity CPA-bound state can quantitatively describe steady-state and kinetic properties of the CPA block. The parameters of the model and additional mutagenesis suggest that the high-affinity CPA-bound state is similar to the closed configuration of the protopore gate of wild-type CLC-0. In the E166A mutant the glutamate side chain that occludes the permeation pathway is absent. Thus, if gating consists only in movement of this side-chain the mutant E166A should not be able to assume a closed conformation. It may thus be that fast gating in CLC-0 is more complex than anticipated from the bacterial structures.

Download Full-text