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A variety of Sepharose derivatives containing DL-O-phosphorylserine or adenosine nucleotides with different points of attachment, has been synthesized and tested for affinity to phosphoglycerate kinase. The most effective gels contained periodate-oxidized ATP or ADP bound via the ribose by hydrazone formation to adipoyl-dihydrazo-Sepharose. The effect of pH, magnesium and buffer ions on the binding capacity of the ATP derivative of Sepharose has been examined. Optimal elution of phosphoglycerate kinase was investigated using different combinations of adenosine nucleotides, 3-phosphogylcerate and magnesium ions. A method is presented giving conditions for the purification of phosphoglycerate kinase from different sources (spinach, human erythrocytes, human, rabbit and trout muscle). It includes extract preparation, affinity chromatography and gel filtration. The method is greatly superior to known isolation procedures by virtue of its technical simplicity, excellent yield (85-100%) and reproducability. The capacity of the ATP-ribosyl-adipoyl-dihydrazo-Sepharose was 5 mg phosphoglycerate kinase per 1 g of matrix. Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate indicated that the final products are homogeneous. The phosphoglycerate kinases from different sources appear to have the same affinity for this ATP derivative of Sepharose, the same molecular weight and the same specific activity.
G W Kuntz, and
S Eber, and
W Kessler, and
H Krietsch, and
W K Krietsch
January 1977,
Archives of biochemistry and biophysics,
G W Kuntz, and
S Eber, and
W Kessler, and
H Krietsch, and
W K Krietsch
July 1971,
Biochemistry,
G W Kuntz, and
S Eber, and
W Kessler, and
H Krietsch, and
W K Krietsch
January 1974,
Methods in enzymology,
G W Kuntz, and
S Eber, and
W Kessler, and
H Krietsch, and
W K Krietsch
September 1981,
Bioscience reports,
G W Kuntz, and
S Eber, and
W Kessler, and
H Krietsch, and
W K Krietsch
March 1983,
Journal of chromatography,
G W Kuntz, and
S Eber, and
W Kessler, and
H Krietsch, and
W K Krietsch
January 1974,
Methods in enzymology,
G W Kuntz, and
S Eber, and
W Kessler, and
H Krietsch, and
W K Krietsch
January 1974,
Biochimie,
G W Kuntz, and
S Eber, and
W Kessler, and
H Krietsch, and
W K Krietsch
June 1973,
Biochemical and biophysical research communications,
G W Kuntz, and
S Eber, and
W Kessler, and
H Krietsch, and
W K Krietsch
January 1975,
Biokhimiia (Moscow, Russia),
G W Kuntz, and
S Eber, and
W Kessler, and
H Krietsch, and
W K Krietsch
January 1977,
Enzyme,
