Multiple sampling in single-cell enzyme assays using CE-laser-induced fluorescence to monitor reaction progress.
A novel method for assaying enzymes from a single cell or small cell populations is described. The key advantage of this method is the ability to repeatedly sample a single cell enzyme reaction. Whereas multiple sampling has been achieved for larger cell types with a diameter of 1 mm, we report a technique by which single cell enzyme assays of small cells (15 microm in diameter) can be repeatedly carried out. Individual cells were isolated using an in-house-built micromanipulator and placed in nanoliter-scale reaction vessels. The cells were lysed with solution containing substrate, and enzyme activity was assayed by removing 5-nL aliquots with a recently developed nanopipettor. The reaction aliquot was then analyzed using capillary electrophoresis with laser-induced fluorescence detection to quantitate enzyme activity. Sf9 cells were assayed at the single cell level and found to be highly heterogeneous with respect to alpha-glucosidase II activity. Since only 5 nL of the single cell reaction was removed, multiple sampling was possible, allowing triplicate analysis of enzyme activity for each individual cell. Multiple sampling also permitted a single cell reaction to be monitored over time. The sensitivity of this method was demonstrated in the analysis of a low-abundance enzyme, alpha1,3-N-acetylgalactosaminyltransferase, from single HT29 cells. Detecting the product of this enzyme reaction required minimizing the dilution of cellular contents. To demonstrate the potential applications of this methodology in small scale biochemical analyses, single Arabidopsis knf embryos lacking the alpha-glucosidase I encoding KNOPF gene were assayed. Mutant embryos demonstrated insignificant conversion of a triglucose substrate, as compared to wild type, confirming the deletion of alpha-glucosidase I. Embryos were simultaneously assayed for a second enzyme, beta-galactosidase, illustrating that the mutants were viable except for their lack of alpha-glucosidase I activity.
Shoemaker
Glen K
GK
Chemistry Department, University of Alberta, Edmonton, Alberta, Canada T6G 2G2.
Lorieau
Justin
J
Lau
Leon H
LH
Gillmor
C Stewart
CS
Palcic
Monica M
MM
D000818:Animals; D017360:Arabidopsis / Q000166:cytology / Q000196:embryology / Q000201:enzymology / Q000235:genetics; D001681:Biological Assay / Q000379:methods*; D019075:Electrophoresis, Capillary / Q000379:methods*; D004789:Enzyme Activation; D005453:Fluorescence; D005947:Glucose / Q000378:metabolism; D019073:HT29 Cells / Q000166:cytology / Q000201:enzymology; D006801:Humans; D007834:Lasers; D009154:Mutation; D017350:N-Acetylgalactosaminyltransferases / Q000032:analysis / Q000378:metabolism; D036103:Nanotechnology / Q000379:methods*; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence / Q000379:methods; D018411:Spodoptera / Q000166:cytology / Q000196:embryology / Q000201:enzymology / Q000235:genetics; D013997:Time Factors; D000520:alpha-Glucosidases / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D001616:beta-Galactosidase / Q000032:analysis / Q000235:genetics / Q000378:metabolism
D017350:N-Acetylgalactosaminyltransferases; D000520:alpha-Glucosidases; D001616:beta-Galactosidase; D005947:Glucose