Integrated electroosmotic perfusion of tissue with online microfluidic analysis to track the metabolism of cystamine, pantethine, and coenzyme A.
We have developed an approach that integrates electroosmotic perfusion of tissue with a substrate-containing solution and online microfluidic analysis of products, in this case thiols. Using this approach we have tracked the metabolism of cystamine, pantethine and CoA in the extracellular space of organotypic hippocampal slice cultures (OHSCs). Currently, little is known about coenzyme A (CoA) biodegradation and even less is known about the regulation and kinetic characteristics for this sequential multienzyme reaction. We found that the steady state percentage yields of cysteamine from cystamine and pantethine during the transit through OHSCs were 91% ± 4% (SEM) and 0.01%-0.03%, respectively. The large difference in the yields of cysteamine can be used to explain the drugs' different toxicities and clinical effectiveness against cystinosis. The kinetic parameters of the enzyme reaction catalyzed by the ectoenzyme pantetheinase are KM,C/α = 4.4 ± 1.1 mM and Vmax,C = 29 ± 3 nM/s, where α is the percentage yield of pantethine to pantetheine through disulfide exchange. We estimate that the percentage yield of pantethine to pantetheine through disulfide exchange is approximately 0.5%. Based on the formation rate of cysteamine in the OHSCs, we obtained the overall apparent Michaelis constant and maximum reaction rate for sequential, extracellular CoA degradation in an in situ environment, which are K'M = 16 ± 4 μM, V'max = 7.1 ± 0.5 nM/s. Kinetic parameters obtained in situ, although difficult to measure, are better representations of the biochemical flux in the living organism than those from isolated enzymes in vitro.
Department of Chemistry, University of Pittsburgh , Pittsburgh, Pennsylvania 15260, United States.
Juanfang
J
Wu
Mats
M
Sandberg
Stephen G
SG
Weber
D002138:Calibration; D003065:Coenzyme A / Q000378:metabolism*; D003538:Cystamine / Q000378:metabolism*; D053841:Electroosmosis / Q000379:methods*; D005110:Extracellular Space / Q000378:metabolism; D006624:Hippocampus / Q000166:cytology; D046210:Microfluidic Analytical Techniques / Q000379:methods*; D010204:Pantetheine / Q000031:analogs & derivatives* / Q000378:metabolism; D010477:Perfusion / Q000379:methods*; D018511:Systems Integration
D010204:Pantetheine; C005425:pantethine; D003538:Cystamine; D003065:Coenzyme A