INVESTIGADORES
IGLESIAS Alberto Alvaro
artículos
Título:
Regulatory and Structural Properties of the Cyanobacterial ADPglucose Pyrophosphorylases
Autor/es:
A.A. IGLESIAS, G. KAKEFUDA, J. PREISS
Revista:
PLANT PHYSIOLOGY.
Referencias:
Año: 1991 vol. 97 p. 1187 - 1195
ISSN:
0032-0889
Resumen:
ADPglucose pyrophosphorylase (EC 2.7.7.27) has been purifiedfrom two cyanobacteria: the filamentous, heterocystic, AnabaenaPCC 7120 and the unicellular Synechocystis PCC 6803.The purification procedure gave highly purified enzymes fromboth cynobacteria with specific activities of 134 (Synechocystis)and 111 (Anabaena) units per milligram protein. The purifiedenzymes migrated as a single protein band in sodium dodecylsulfate-polyacrylamide gel electrophoresis with molecular masscorresponding to 53 (Synechocystis) and 50 (Anabaena) kilodaltons.Tetrameric structures were determined for the native enzymesby analysis of gel filtrations. Kinetic and regulatory propertieswere characterized for the cyanobacterial ADPglucosepyrophosphorylases. Inorganic phosphate and 3-phosphoglyceratewere the most potent inhibitor and activator, respectively. TheSynechocystis enzyme was activated 126-fold by 3-phosphoglycerate,with saturation curves exhibiting sigmoidicity (Ao.s = 0.81millimolar; nH = 2.0). Activation by 3-phosphoglycerate of theenzyme from Anabaena demonstrated hyperbolic kinetics (Ao.5 =0.12 millimolar, n,H = 1.0), having a maximal stimulation of 17-fold.10.5 values of 95 and 44 micromolar were calculated for theinhibition by inorganic phosphate of the Synechocystis and Anabaenaenzyme, respectively. Pyridoxal-phosphate behaved as anactivator of the cyanobacterial enzyme. It activated the enzymefrom Synechocystis nearly 10-fold with high apparent affinity (Ao.5= 10 micromolar; n,H = 1.8). Phenylglyoxal modified the cyanobacterialenzyme by inactivating the activity in the presence of 3-phosphoglycerate. Antibody neutralization experiments showedthat anti-spinach leaf (but not anti-Escherichia co/i) ADPglucosepyrophosphorylase serum inactivated the enzyme from cyanobacteria.When the cyanobacterial enzymes were resolved onsodium dodecyl sulfate- and two-dimensional polyacrylamide gelelectrophoresis and probed with Westem blots, only one proteinband was recognized by the anti-spinach leaf serum. The samepolypeptide strongly reacted with antiserum prepared against thesmaller spinach leaf 51 kilodalton subunit, whereas the anti-54kilodalton antibody raised against the spinach subunit reactedweakly to the cyanobacterial subunit. Regulatory and immunologicalproperties of the cyanobacterial enzyme are more related tothe higher plant than the bacterial enzyme. Despite this, resultssuggest that the ADPglucose pyrophosphorylase from cyanobacteriais homotetrameric in structure, in contrast to the reportedheterotetrameric structures of the higher plant ADPglucose pyrophosphorylase.