Pyruvate kinase isozymes from the green alga, Selenastrum minutum. I. Purification and physical and immunological characterization.
Pyruvate kinase from the green alga Selenastrum minutum consists of two isoforms (PK1 and PK2) separable by Q-Sepharose chromatography. The two isoforms have been highly purified to respective final specific activities of 42 and 23 (mumol pyruvate produced/min)/mg protein. Purification steps included salt fractionation, anion-exchange, hydrophobic interaction, and gel filtration chromatography. The final enzyme preparations differ significantly in physical and immunological properties. PK1 is heat labile and is completely inactivated following reaction with N-ethylmaleimide. In contrast, PK2 is heat-stable and is only partially inactivated following N-ethylmaleimide treatment. PK1 appears to be homotetrameric with a native molecular mass of about 240 kDa, whereas PK2 appears to be homodecameric with a native molecular mass of approximately 590 kDa. The antigenic reaction of both final PK preparations to rabbit antiserum prepared against homogeneous germinating castor bean endosperm cytosolic pyruvate kinase was tested by immunoprecipitation and Western blotting. The two algal pyruvate kinases are immunologically unrelated as only PK2 cross-reacts with the cytosolic pyruvate kinase antibodies. These data indicate that the S. minutum pyruvate kinase isoforms, PK1 and PK2, are not interconvertible forms of the same protein, but probably represent chloroplastic and cytosolic isozymes, respectively.
Lin
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Department of Biology, Queen's University, Kingston, Ontario, Canada.
Turpin
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DH
Plaxton
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WC
D000645:Ammonium Sulfate; D000818:Animals; D015153:Blotting, Western; D005591:Chemical Fractionation; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D000460:Chlorophyta / Q000201:enzymology*; D002847:Chromatography, Agarose; D002851:Chromatography, High Pressure Liquid; D005033:Ethylmaleimide; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D011233:Precipitin Tests; D011770:Pyruvate Kinase / Q000302:isolation & purification / Q000378:metabolism*; D011817:Rabbits
D007527:Isoenzymes; D011770:Pyruvate Kinase; D005033:Ethylmaleimide; D000645:Ammonium Sulfate