PLANT PHYSIOLOGY , Vol 101, Issue 1 179-186, Copyright © 1993 by American Society of Plant Biologists

METABOLISM AND ENZYMOLOGY

Insensitivity of Barley Endosperm ADP-Glucose Pyrophosphorylase to 3-Phosphoglycerate and Orthophosphate Regulation

L. A. Kleczkowski, P. Villand, E. Luthi, O. A. Olsen and J. Preiss
Plant Molecular Biology Laboratory, The Agricultural Research Council of Norway (NLVF), P.O. Box 51, 1432 As, Norway (L.A.K., P.V., E.L., O.-A.O.)

Crude extracts of starchy endosperm from barley (Hordeum vulgare cv Bomi) contained high pyrophosphorolytic activity (up to 0.5 [mu]mol of glucose-1-P formed min-1 mg-1 of protein) of ADP-glucose pyrophosphorylase (AGP) when assayed in the absence of 3-phosphoglycerate (3-PGA). This high activity was observed regardless of whether AGP had been extracted in the presence or absence of various protease inhibitors or other protectants. Western blot analysis using antibodies specific for either the small or large subunit of the enzyme demonstrated that the large, 60-kD subunit was prone to proteolysis in crude extracts, with a half-time of degradation at 4[deg]C (from 60 to 53 to 51 kD) on the order of minutes. The presence of high concentrations of protease inhibitors decreased, but did not prevent this proteolysis. The small, 51-kD subunit of barley endosperm AGP was relatively resistant to proteolysis, both in the presence or absence of protease inhibitors. For the crude, nonproteolyzed enzyme, 3-PGA acted as a weak activator of the ADP-glucose synthetic reaction (about 25% activation), whereas in the reverse reaction (pyrophosphorolysis) it served as an inhibitor rather than an activator. For both the synthetic and pyrophosphorolytic reactions, inorganic phosphate (Pi) acted as a weak competitive or mixed inhibitor of AGP. The relative insensitivity to 3-PGA/Pi regulation has been observed with both the nonproteolyzed crude enzyme and partially purified (over 60-fold) AGP, the latter characterized by two bands for the large subunit (molecular masses of 53 and 51 kD) and one band for the small subunit (51 kD). Addition of 3-PGA to assays of the partially purified, proteolyzed enzyme had little or no effect on the Km values of all substrates of AGP, but it reduced the Hill coefficient for ATP (from 2.1 to 1.0). These findings are discussed with respect to previous reports on the structure and regulation of higher plant AGP.

This article has been cited by other articles:

				T. Ventriglia, M. A. Ballicora, P. Crevillen, J. Preiss, and J. M. Romero Regulatory Properties of Potato-Arabidopsis Hybrid ADP-Glucose Pyrophosphorylase Plant Cell Physiol., June 1, 2007; 48(6): 875 - 880. [Abstract] [Full Text] [PDF]

				S. K. Boehlein, A. K. Sewell, J. Cross, J. D. Stewart, and L. C. Hannah Purification and Characterization of Adenosine Diphosphate Glucose Pyrophosphorylase from Maize/Potato Mosaics Plant Physiology, July 1, 2005; 138(3): 1552 - 1562. [Abstract] [Full Text] [PDF]

				P. Crevillen, T. Ventriglia, F. Pinto, A. Orea, A. Merida, and J. M. Romero Differential Pattern of Expression and Sugar Regulation of Arabidopsis thaliana ADP-glucose Pyrophosphorylase-encoding Genes J. Biol. Chem., March 4, 2005; 280(9): 8143 - 8149. [Abstract] [Full Text] [PDF]

				I. J. Tetlow, M. K. Morell, and M. J. Emes Recent developments in understanding the regulation of starch metabolism in higher plants J. Exp. Bot., October 1, 2004; 55(406): 2131 - 2145. [Abstract] [Full Text] [PDF]

				J. M. Cross, M. Clancy, J. R. Shaw, T. W. Greene, R. R. Schmidt, T. W. Okita, and L. C. Hannah Both Subunits of ADP-Glucose Pyrophosphorylase Are Regulatory Plant Physiology, May 1, 2004; 135(1): 137 - 144. [Abstract] [Full Text] [PDF]

				P. Crevillen, M. A. Ballicora, A. Merida, J. Preiss, and J. M. Romero The Different Large Subunit Isoforms of Arabidopsis thaliana ADP-glucose Pyrophosphorylase Confer Distinct Kinetic and Regulatory Properties to the Heterotetrameric Enzyme J. Biol. Chem., August 1, 2003; 278(31): 28508 - 28515. [Abstract] [Full Text] [PDF]

				M. A. Ballicora, A. A. Iglesias, and J. Preiss ADP-Glucose Pyrophosphorylase, a Regulatory Enzyme for Bacterial Glycogen Synthesis Microbiol. Mol. Biol. Rev., June 1, 2003; 67(2): 213 - 225. [Abstract] [Full Text] [PDF]

				E. Baroja-Fernandez, F. J. Munoz, T. Saikusa, M. Rodriguez-Lopez, T. Akazawa, and J. Pozueta-Romero Sucrose Synthase Catalyzes the de novo Production of ADPglucose Linked to Starch Biosynthesis in Heterotrophic Tissues of Plants Plant Cell Physiol., May 15, 2003; 44(5): 500 - 509. [Abstract] [Full Text] [PDF]

				I. J. Tetlow, E. J. Davies, K. A. Vardy, C. G. Bowsher, M. M. Burrell, and M. J. Emes Subcellular localization of ADPglucose pyrophosphorylase in developing wheat endosperm and analysis of the properties of a plastidial isoform J. Exp. Bot., February 1, 2003; 54(383): 715 - 725. [Abstract] [Full Text] [PDF]

				P. E. Johnson, N. J. Patron, A. R. Bottrill, J. R. Dinges, B. F. Fahy, M. L. Parker, D. N. Waite, and K. Denyer A Low-Starch Barley Mutant, Riso 16, Lacking the Cytosolic Small Subunit of ADP-Glucose Pyrophosphorylase, Reveals the Importance of the Cytosolic Isoform and the Identity of the Plastidial Small Subunit Plant Physiology, February 1, 2003; 131(2): 684 - 696. [Abstract] [Full Text] [PDF]

				P. R. Salamone, I. H. Kavakli, C. J. Slattery, and T. W. Okita Directed molecular evolution of ADP-glucose pyrophosphorylase PNAS, January 1, 2002; (2002) 12603799. [Abstract] [Full Text] [PDF]

				D. M. Beckles, A. M. Smith, and T. ap Rees A Cytosolic ADP-Glucose Pyrophosphorylase Is a Feature of Graminaceous Endosperms, But Not of Other Starch-Storing Organs Plant Physiology, February 1, 2001; 125(2): 818 - 827. [Abstract] [Full Text]

				C. H. Harn, J. M. Bae, S. S. Lee, S. R. Min, and J. R. Liu Presence of Multiple cDNAs Encoding an Isoform of ADP-Glucose Pyrophosphorylase Large Subunit from Sweet Potato and Characterization of Expression Levels Plant Cell Physiol., November 1, 2000; 41(11): 1235 - 1242. [Abstract] [Full Text] [PDF]

				D. N.P. Doan, H. Rudi, and O.-A. Olsen The Allosterically Unregulated Isoform of ADP-Glucose Pyrophosphorylase from Barley Endosperm Is the Most Likely Source of ADP-Glucose Incorporated into Endosperm Starch Plant Physiology, November 1, 1999; 121(3): 965 - 975. [Abstract] [Full Text]

				T. W. Greene and L. C. Hannah Maize Endosperm ADP–Glucose Pyrophosphorylase SHRUNKEN2 and BRITTLE2 Subunit Interactions PLANT CELL, August 1, 1998; 10(8): 1295 - 1306. [Abstract] [Full Text]

				P. Geigenberger, M. Geiger, and M. Stitt High-Temperature Perturbation of Starch Synthesis Is Attributable to Inhibition of ADP-Glucose Pyrophosphorylase by Decreased Levels of Glycerate-3-Phosphate in Growing Potato Tubers Plant Physiology, August 1, 1998; 117(4): 1307 - 1316. [Abstract] [Full Text]

				I. H. Kavakli, J.-S. Park, C. J. Slattery, P. R. Salamone, J. Frohlick, and T. W. Okita Analysis of Allosteric Effector Binding Sites of Potato ADP-glucose Pyrophosphorylase through Reverse Genetics J. Biol. Chem., October 26, 2001; 276(44): 40834 - 40840. [Abstract] [Full Text] [PDF]

				P. R. Salamone, I. H. Kavakli, C. J. Slattery, and T. W. Okita Directed molecular evolution of ADP-glucose pyrophosphorylase PNAS, January 22, 2002; 99(2): 1070 - 1075. [Abstract] [Full Text] [PDF]