Affinity chromatography ofd-lactate dehydrogenases fromLimulus polyphemus (horseshoe crab) andHaliotus sp. (abalone) voluntary muscle on 8-(6-aminohexyl)-amino-adenine nucleotide-sepharose
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An improved method of purification employing sequential isoenzyme elution on DEAE-cellulose at pH 6.5 and biologically specific elution with the reduced NAD-pyruvate adduct from 8-(6-aminohexyl)-amino-NAD⊕-Sepharose is described forLimulus (horseshoe crab) muscle D- lactate dehydrogenase. The protein is judged as being at least 98%pure by its constant specific activity in the terminal purification steps, a molar extinction coefficient (280nm) identical with that previously reported for the purified enzyme, and its protein and enzyme electrophoretic patterns on starch and polyacrylamide gel electrophoresis at three pHs. The binding of ammonium sulfate fractionated D- lactate dehydrogenase from crude cell- free Limulus andHaliotus (abalone) muscle homogenates to 8-(6-aminohexyl)-amino-AMP-and 8-(6-aminohexyl)-amino-NAD®-Sepharose columns is demonstrated. A comparison of the binding properties of these two enzymes with those of vertebrate L-lactate dehydrogenases suggests that they may be significantly different in terms of their binding sites for the adenine portion of the coenzyme.
KeywordsLactate Dehydrogenase Ammonium Sulfate Horseshoe Crab Sepharose Column Disodium EDTA
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- 1.Pesce, A., McKay, R. H., Stolzenbach, F., Cahn, R. D., andKaplan, N. O. (1964) J. Biol. Chem. 239: 1753.Google Scholar
- 3.Pesce, A., Fondy, T. P., Stolzenbach, F., Castillo, F., andKaplan, N. O. (1967) J. Biol. Chem. 242: 2151.Google Scholar
- 4.Fondy, T. P., Everse, J., Driscoll, G. A., Castillo, F., Stolzenbach, F. E., andKaplan, N. O. (1965) J. Biol. Chem. 240: 4219.Google Scholar
- 5.Kaloustian, H. D., Stolzenbach, F. E., Everse, J., andKaplan, N. O. (1969) J. Biol. Chem. 244: 2891.Google Scholar
- 6.Kaloustian, H. D., andKaplan, N. O. (1969) J. Biol. Chem. 244: 2902.Google Scholar
- 7.Eichner, R. D., andKaplan, N. O. (1977)Arch. Biochem. Biophys., in press.Google Scholar
- 11.Long, G. L. (1975) “D-Lactate Dehydrogenase from the Horseshoe Crab,”In Methods in Enzymology, Vol. 41,Wood, W. A. (ed.), Academic Press, New York, pp. 313–318.Google Scholar
- 13.Rossmann, M. G. (1975) “Evolutionary and Structural Relationships among Dehydrogenases,”In The Enzymes, Vol. 11,Boyer, P. D. (ed.), Academic Press, New York, pp. 62–102.Google Scholar
- 17.Gabriel, O. (1971) “Analytical Disc Gel Electrophoresis,”In Methods in Enzymology, Vol. 22,Jakoby, W. B. (ed.), Academic Press, New York, pp. 565–578.Google Scholar
- 20.Dixon, J. E. Personal communication (manuscript in preparation).Google Scholar