Affinity Chromatography of Glyceraldehyde-3-phosphate Dehydrogenase. A Comparative Study of the Enzymes from Yeast and Sturgeon Muscle

Sturgeon glyceraldehyde-3-phosphate dehydrogenase has been strongly adsorbed on an 8-azo-linked NAD+-Sepharose derivative. Specific elution of the matrix-bound enzyme was obtained with relatively low concentrations of NAD+ (20–100 μM) in elution buffer. Recovery of adsorbed enzyme amounted to 90–95% when an hydrophilic 1,3-diaminopropan-2-ol spacer arm was used instead of the more conventional diaminohexyl derivative. A capacity of 0.8 mg of sturgeon enzyme per g of derivative was estimated either under batchwise conditions or by frontal chromatography. Under similar conditions, purified apoglyceraldehyde-3-phosphate dehydrogenase from yeast did not bind significantly to the same NAD+ derivative. Crude extracts of sturgeon muscle can be purified more than 15-fold upon chromatography on this derivative. Unlike the sturgeon enzyme, yeast glyceraldehyde-3-phosphate dehydrogenase was significantly adsorbed to an N6-(6-aminohexyl)-AMP derivative under chromatographic or batchwise conditions. At least 40 units (i.e. 0.2–0.3 mg) of yeast glyceraldehyde-3-phosphate dehydrogenase can be bound per g of matrix. An 8-fold purification of the enzyme can be obtained upon specific elution with NAD+. Relatively large amounts of yeast enzyme (i.e. up to 100 mg) can be partially purified by this one-step procedure. The distinct adsorption properties of yeast and sturgeon glyceraldehyde-3-phosphate dehydrogenases are discussed in the light of the known cooperative coenzyme binding properties of these enzymes. The relevance of affinity chromatography to the preparation of sizeable amounts of glyceral-dehyde-3-phosphate dehydrogenase is discussed and compared with conventional procedures. Possible interference of glyceraldehyde-3-phosphate dehydrogenase with the ‘general ligand’ binding of other dehydrogenase and kinases in crude extracts is discussed.