geoscience.net logo
+ Resolve Article
+ Follow Us
Follow on FacebookFollow on Facebook
Follow on TwitterFollow on Twitter

+ Translate
+ Subscribe to Site Feed
GeoScience Most Shared ContentMost Shared Content

Recombinant glutathione S-transferase (GST) expressing cells purified by flow cytometry on the basis of a GST-catalyzed intracellular conjugation of glutathione to monochlorobimane


, : Recombinant glutathione S-transferase (GST) expressing cells purified by flow cytometry on the basis of a GST-catalyzed intracellular conjugation of glutathione to monochlorobimane. Cytometry 12(7): 651-665

COS cells transiently expressing glutathione S-transferase (GST) pi, Ya, or Yb1 (human Pi, rat Alpha or Mu, cytosolic classes) were purified by flow cytometry and used in colony-forming assays to show that GST confers cellular resistance to the carcinogen benzo[a]pyrene (+/-)-anti-diol epoxide (anti-BPDE). We developed a sorting technique to viably separate recombinant GST+ cells (20%) from the nonexpressing electroporated population (80%) on the basis of a GST-catalyzed intracellular conjugation of glutathione to the fluorescent labeling reagent monochlorobimane (mClB). The concentration of mClB, length of time cells are exposed to mClB, and activity of the expressed GST isozyme determined the degree to which recombinant GST+ cells fluoresced more intensely than controls. On-line reagent addition ensured that all cells were exposed to 25 microM mClB for 30-35 s during transit before being analyzed for fluorescence intensity and sorted. The apparent Km for mClB of the endogenous COS cell GST-catalyzed intracellular reaction was 88 microM. Stained GST Ya+ or Yb1+ cells catalyzed the conjugation 2 or 5 times more effectively than GST pi+ cells. Enzyme activity in cytosolic fractions prepared from sorted recombinant GST+ cells was 1.8 +/- 0.3-fold greater than that of the control (80 +/- 4 nmol/min/mg protein). Upon a 5-fold purification of GST pi+ cells in the electroporated population, resistance to anti-BPDE in colony-forming assays increased 5 times, from 1.1-fold (unsorted) to 1.5-fold (sorted) (P less than 0.001).

US$19.90

PMID: 1664313

DOI: 10.1002/cyto.990120710


Other references

Kraus, C.K.; Marco, C.A., 2016: Shared decision making in the ED: ethical considerations. The process of shared decision making (SDM) is an ethical imperative in the physician-patient relationship, especially in the emergency department (ED), where SDM can present unique challenges because patients and emergency physicians often have n...

Guillén, M.; Corella, D.; Cabello, M.L.; González, J.I.; Sabater, A.; Chaves, J.F.; Hernández-Yago, J., 2005: Identification of novel SLC3A1 gene mutations in Spanish cystinuria families and association with clinical phenotypes. Cystinuria is an inherited metabolic disease characterized by an abnormal urinary excretion of cystine and dibasic amino acids, leading to kidney stone formation. Incidence of cystinuria in the Mediterranean Spanish population is one of the highes...

Li, J.; Ge, Z.; Liang, C.; An, N., 2017: Present status of recycling waste mobile phones in China: a review. A large number of waste mobile phones have already been generated and are being generated. Various countries around the world have all been positively exploring the way of recycling and reuse when facing such a large amount of waste mobile phones....

Degawa, M.; Arai, H.; Kubota, M.; Hashimoto, Y., 1995: Ionic lead, but not other ionic metals (Ni2+, Co2+ and Cd2+), suppresses 2-methoxy-4-aminoazobenzene-mediated cytochrome P450IA2 (CYP1A2) induction in rat liver. Male F344 rats were pretreated with lead nitrate, nickel chloride, cobalt chloride or cadmium chloride, and their effects on the induction of cytochrome P450 (CYP) enzymes, mainly CYP1A2 enzyme, with 2-methoxy-4-aminoazobenzene (2-MeO-AAB) in the...

Buess, C.; Guggenbuehl, W.; Harnoncourt, K., 1995: Technology of ultrasonic spirometry. Ultrasonic spirometry is based on the measurement of 2 absolute transit times of sound waves across a tubular sensor form which the flow velocity and volume of breathing can be determined. The inherent advantage of this method to be described in t...

Verhulst, S.; Best, J.; van Grunsven, L.A.; Dollé, L., 2015: Advances in hepatic stem/progenitor cell biology. The liver is famous for its strong regenerative capacity, employing different modes of regeneration according to type and extent of injury. Mature liver cells are able to proliferate in order to replace the damaged tissue allowing the recovery of...

Kikkawa, Y.; Abe, H.; Iwata, T.; Inoue, Y.; Doi, Y., 2001: In situ observation of crystal growth for poly[(S)-lactide] by temperature-controlled atomic force microscopy. The crystallization behavior and crystalline morphologies of poly[(S)-lactide] (P[(S)-LA]) in thin films crystallized isothermally at over 160 degrees C were characterized by transmission electron microscopy and atomic force microscopy (AFM). The...

Bonanno, L.M.; Delouise, L.A., 2007: Steric crowding effects on target detection in an affinity biosensor. This work quantifies the impact of steric crowding on whole antibody (Ab) receptor immobilization and target Ab detection and also demonstrates how the versatile biotin/streptavidin receptor immobilization system must be tuned to optimize target d...

Fricke, R.F.; Longmore, W.J., 1979: Effects of insulin and diabetes on 2-deoxy-D-glucose uptake by the isolated perfused rat lung. Journal of Biological Chemistry 254(12): 5092-5098

Boj, E.; Kamińska, M.; Wrzolkowa, T.; Przymanowski, Z., 1973: Light- and electron-microscopic pattern of early experimental pneumonia induced by Klebsiella pneumoniae bacilli. Patologia Polska 24(1): 69-83