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Purification of chloroplast α-1,4-glucan phosphorylase from spinach leaves by chromatography on sepharose-bound starch
- Source :
- Biochimica et Biophysica Acta (BBA) - Enzymology. 659:123-131
- Publication Year :
- 1981
- Publisher :
- Elsevier BV, 1981.
-
Abstract
- Chloroplast alpha-1,4-glucan phosphorylase (EC 2.4.1.1) has been purified to homogeneity from spinach leaves as revealed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purification procedure is composed of (NH4)2SO4 precipitation, ion-exchange chromatography, and chromatography on Sepharose-bound starch. In order to achieve binding of the chloroplast phosphorylase, a previously described Sepharose-glucan gel (Steup, M. Schächtele, C. and Latzko, E. (1980) Planta 148, 168-173) was modified by introducing hydrophobic groups in addition to the covalently bound starch. The chloroplast phosphorylase exhibited complete binding to this type of gel and could be eluted by a mixture of soluble glucan and NaCl. For the purified chloroplast phosphorylase, sodium dodecyl-sulfate polyacrylamide gel electrophoresis and pyridoxal phosphate determination resulted in a molecular weight estimation of about 110,000 per monomer. The apparent molecular weight of the native enzyme, as determined by polyacrylamide density gradient electrophoresis and gel filtration on Sephadex G-200, was 200,000 and 220,000, respectively. The data indicate that the chloroplast phosphorylase is a dimer with a molecular weight higher than that of the non-chloroplast phosphorylase.
- Subjects :
- Chromatography
Phosphorylases
Chemistry
Starch
Sepharose
Size-exclusion chromatography
Polyacrylamide
food and beverages
General Medicine
Plants
Chromatography, Affinity
Molecular Weight
chemistry.chemical_compound
Glycogen phosphorylase
Biochemistry
Sephadex
Electrophoresis, Polyacrylamide Gel
Sodium dodecyl sulfate
Polyacrylamide gel electrophoresis
Subjects
Details
- ISSN :
- 00052744
- Volume :
- 659
- Database :
- OpenAIRE
- Journal :
- Biochimica et Biophysica Acta (BBA) - Enzymology
- Accession number :
- edsair.doi.dedup.....cfa8b340b409c68cd011898e9a6f29fa