Electrophoretic and chromatographic separation of two fructose-1,6-bisphosphatase forms from Synechococcus leopoliensix |
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Authors: | Klaus-Peter Gerbling Martin Steup Erwin Latzko |
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Institution: | (1) Botanisches Institut der Westfälischen Wilhelms-Universität, Schloßgarten 3, D-4400 Münster, Federal Republic of Germany |
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Abstract: | d-Fructose-1,6-bisphosphatase (EC 3.1.3.11) activity in crude extracts of the blue-green alga Synechococcus leopoliensis (Anacystis nidulans) has been investigated using high resolving electrophoretic and chromatographic separation techniques. Two catalytically active enzyme forms which exhibited isoelectric points of 4.7–4.8 (designated from A) and 4.5–4.6 (designated form B) were resolved by isoelectric focusing. Both enzyme forms acted specifically on fluctose-1,6-bisphosphate. No interconversion between the A and B forms of fructose bisphosphatase activity was detected after refocusing. The apparent molecular weight of the two enzyme forms was determined by non denaturing polyacrylamide gradient electrophoresis; the values were 67,000–70,000 and 60,000–65,000 for A and B, respectively. Both enzyme forms were separated by preparative scale chromatofocusing. Kinetic measurements performed with the separated and partially purified fructose bisphosphatase forms indicated that both enzyme forms differ in their AMP sensitivity. The two enzymes were completely inactivated by the addition of cysteamine and reactivated by dithiols but the reactivation kinetics were different.Abbreviations DTT
dl-Dithiothreithol
- MTT
3(4,5-dimethylthiazolyl-2)-2,5-diphenyl tetrazolium bromide
- PMS
phenazine methosulfate
- TCA
trichloroacetic acid
- Tris
tris(hydroxymethyl)-aminomethane |
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Keywords: | Fructose-1 6-bisphosphatase Synechococcus leopoliensis Anacystis nidulans Isoelectric focusing Polyacrylamide gel electrophoresis Chromatofocusing |
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