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Plant Physiol. 1994 Apr; 104(4): 1287–1294.
PMCID: PMC159292

Characterization of the kinetic, regulatory, and structural properties of ADP-glucose pyrophosphorylase from Chlamydomonas reinhardtii.

Abstract

ADP-glucose pyrophosphorylase (ADP-Glc PPase) from Chlamydomonas reinhardtii cells was purified over 2000-fold to a specific activity of 81 units/mg protein, and its kinetic and regulatory properties were characterized. Inorganic orthophosphate and 3-phosphoglycerate were the most potent inhibitor and activator, respectively. Rabbit antiserum raised against the spinach leaf ADP-Glc PPase (but not the one raised against the enzyme from Escherichia coli) inhibited the activity of the purified algal enzyme, which migrated as a single protein band in native polyacrylamide gel electrophoresis. Two-dimensional and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicate that the enzyme from C. reinhardtii is composed of two subunits with molecular masses of 50 and 53 kD, respectively. The molecular mass of the native enzyme is estimated to be 210 kD. Antisera raised against the spinach leaf holoenzyme and against the 51-kD spinach subunit cross-reacted with both subunits of the algal ADP-Glc PPase in immunoblot hybridization, but the cross-reaction was stronger for the 50-kD algal subunit than for the 53-kD subunit. No cross-reaction was observed when antiserum raised against the spinach leaf pyrophosphorylase 54-kD subunit was used. These results suggest that the ADP-Glc PPase from C. reinhardtii is a heterotetrameric protein, since the enzyme from higher plants and its two subunits are structurally more related to the small subunit of the spinach leaf enzyme than to its large subunit. This information is discussed in the context of the possible evolutionary changes leading from the bacterial ADP-Glc PPase to the cyanobacterial and higher plant enzymes.

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Selected References

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  • Burnette WN. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. [PubMed]
  • Charng YY, Kakefuda G, Iglesias AA, Buikema WJ, Preiss J. Molecular cloning and expression of the gene encoding ADP-glucose pyrophosphorylase from the cyanobacterium Anabaena sp. strain PCC 7120. Plant Mol Biol. 1992 Oct;20(1):37–47. [PubMed]
  • Copeland L, Preiss J. Purification of Spinach Leaf ADPglucose Pyrophosphorylase. Plant Physiol. 1981 Nov;68(5):996–1001. [PMC free article] [PubMed]
  • Dickinson DB, Preiss J. Presence of ADP-Glucose Pyrophosphorylase in Shrunken-2 and Brittle-2 Mutants of Maize Endosperm. Plant Physiol. 1969 Jul;44(7):1058–1062. [PMC free article] [PubMed]
  • Ghosh HP, Preiss J. Adenosine diphosphate glucose pyrophosphorylase. A regulatory enzyme in the biosynthesis of starch in spinach leaf chloroplasts. J Biol Chem. 1966 Oct 10;241(19):4491–4504. [PubMed]
  • Hannah LC, Nelson OE., Jr Characterization of ADP-glucose pyrophosphorylase from shrunken-2 and brittle-2 mutants of maize. Biochem Genet. 1976 Aug;14(7-8):547–560. [PubMed]
  • Haugen TH, Ishaque A, Preiss J. Biosynthesis of bacterial glycogen. Characterization of the subunit structure of Escherichia coli B glucose-1-phosphate adenylyltransferase (EC 2.7.7.27). J Biol Chem. 1976 Dec 25;251(24):7880–7885. [PubMed]
  • Hunkapiller MW, Lujan E, Ostrander F, Hood LE. Isolation of microgram quantities of proteins from polyacrylamide gels for amino acid sequence analysis. Methods Enzymol. 1983;91:227–236. [PubMed]
  • Iglesias AA, Barry GF, Meyer C, Bloksberg L, Nakata PA, Greene T, Laughlin MJ, Okita TW, Kishore GM, Preiss J. Expression of the potato tuber ADP-glucose pyrophosphorylase in Escherichia coli. J Biol Chem. 1993 Jan 15;268(2):1081–1086. [PubMed]
  • Iglesias AA, Kakefuda G, Preiss J. Regulatory and Structural Properties of the Cyanobacterial ADPglucose Pyrophosphorylases. Plant Physiol. 1991 Nov;97(3):1187–1195. [PMC free article] [PubMed]
  • Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. [PubMed]
  • Lin TP, Caspar T, Somerville C, Preiss J. Isolation and Characterization of a Starchless Mutant of Arabidopsis thaliana (L.) Heynh Lacking ADPglucose Pyrophosphorylase Activity. Plant Physiol. 1988 Apr;86(4):1131–1135. [PMC free article] [PubMed]
  • Miziorko HM, Lorimer GH. Ribulose-1,5-bisphosphate carboxylase-oxygenase. Annu Rev Biochem. 1983;52:507–535. [PubMed]
  • Nakamura Y, Imamura M. Regulation of ADP-Glucose Pyrophosphorylase from Chlorella vulgaris. Plant Physiol. 1985 Jul;78(3):601–605. [PMC free article] [PubMed]
  • Okita TW. Is there an alternative pathway for starch synthesis? Plant Physiol. 1992 Oct;100(2):560–564. [PMC free article] [PubMed]
  • Okita TW, Nakata PA, Anderson JM, Sowokinos J, Morell M, Preiss J. The Subunit Structure of Potato Tuber ADPglucose Pyrophosphorylase. Plant Physiol. 1990 Jun;93(2):785–790. [PMC free article] [PubMed]
  • Pettersson G, Ryde-Pettersson U. Metabolites controlling the rate of starch synthesis in the chloroplast of C3 plants. Eur J Biochem. 1989 Jan 15;179(1):169–172. [PubMed]
  • Plaxton WC, Preiss J. Purification and Properties of Nonproteolytic Degraded ADPglucose Pyrophosphorylase from Maize Endosperm. Plant Physiol. 1987 Jan;83(1):105–112. [PMC free article] [PubMed]
  • Preiss J. Bacterial glycogen synthesis and its regulation. Annu Rev Microbiol. 1984;38:419–458. [PubMed]
  • Preiss J, Ball K, Smith-White B, Iglesias A, Kakefuda G, Li L. Starch biosynthesis and its regulation. Biochem Soc Trans. 1991 Aug;19(3):539–547. [PubMed]
  • Preiss J, Danner S, Summers PS, Morell M, Barton CR, Yang L, Nieder M. Molecular Characterization of the Brittle-2 Gene Effect on Maize Endosperm ADPglucose Pyrophosphorylase Subunits. Plant Physiol. 1990 Apr;92(4):881–885. [PMC free article] [PubMed]
  • Preiss J, Romeo T. Physiology, biochemistry and genetics of bacterial glycogen synthesis. Adv Microb Physiol. 1989;30:183–238. [PubMed]
  • Sanwal GG, Preiss J. Biosynthesis of starch in Chlorella pyrenoidosa. II. Regulation of ATP: alpha-D-glucose 1-phosphate adenyl transferase (ADP-glucose pyrophosphorylase) by inorganic phosphate and 3-phosphoglycerate. Arch Biochem Biophys. 1967 Mar;119(1):454–469. [PubMed]
  • Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC. Measurement of protein using bicinchoninic acid. Anal Biochem. 1985 Oct;150(1):76–85. [PubMed]
  • Tsai CY, Nelson OE. Starch-deficient maize mutant lacking adenosine dephosphate glucose pyrophosphorylase activity. Science. 1966 Jan 21;151(3708):341–343. [PubMed]

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