title: Purification and characterization of 6-phosphogluconate dehydrogenase from the wing-polymorphic cricket, Gryllus firmus, and assessment of causes of morph-differences in enzyme activity.
abstract: Considerable information exists on the physiological correlates of life history adaptation, while molecular data on this topic are rapidly accumulating. However, much less is known about the enzymological basis of life history adaptation in outbred populations. In the present study, we compared developmental profiles of fat body specific activity, kinetic constants of homogeneously purified and unpurified enzyme, and fat body enzyme concentration of the pentose-shunt enzyme, 6-phosphogluconate dehydrogenase (6PGDH, E.C.1.1.1.44) between the dispersing [long-winged, LW(f)] and flightless [short-winged, SW] genotypes of the cricket Gryllus firmus. Neither kcat nor the Michaelis constant for 6-phosphogluconate differed between 6PGDH from LW(f) versus SW morphs for either homogeneously purified or unpurified enzyme. Purified enzyme from the LW(f) morph exhibited reduced KM for NADP(+), but this was not observed for multiple KM(NADP+) estimates for unpurified enzyme. A polyclonal antibody was generated against 6PGDH which was used to develop a chemiluminescence assay to quantify 6PGDH concentration in fat body homogenates. Elevated enzyme concentration accounted for all of the elevated 6PGDH specific activity in the LW(f) morph during the juvenile and adult stages. Finally, activity of another pentose-shunt enzyme, glucose-6-phosphate dehydrogenase, strongly covaried with 6PGDH activity suggesting that variation in 6PGDH activity gives rise to variation in pentose shunt flux. This is one of the first life-history studies and one of the few studies of intraspecific enzyme adaptation to identify the relative importance of evolutionary change in enzyme concentration vs. kinetic constants to adaptive variation in enzyme activity in an outbred population.
authors.affiliation: School of Biological Sciences, University of Nebraska, Lincoln, NE 68588, USA. Electronic address: azera1@unl.edu.
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Zera
authors.affiliation: School of Biological Sciences, University of Nebraska, Lincoln, NE 68588, USA.
authors.forename: Cody
authors.initials: C
authors.lastname: Wehrkamp
authors.affiliation: School of Biological Sciences, University of Nebraska, Lincoln, NE 68588, USA.
authors.forename: Rudolf
authors.initials: R
authors.lastname: Schilder
authors.affiliation: School of Biological Sciences, University of Nebraska, Lincoln, NE 68588, USA.
authors.forename: Christine
authors.initials: C
authors.lastname: Black
authors.affiliation: School of Biological Sciences, University of Nebraska, Lincoln, NE 68588, USA.
authors.forename: Paul
authors.initials: P
authors.lastname: Gribben
mesh_terms: D000222:Adaptation, Physiological; D000818:Animals; D005216:Fat Body / Q000201:enzymology; D006135:Gryllidae / Q000201:enzymology*; D019476:Insect Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D007527:Isoenzymes; D007700:Kinetics; D008163:Luminescent Measurements / Q000379:methods; D009928:Organ Specificity; D010734:Phosphogluconate Dehydrogenase / Q000032:analysis / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D011817:Rabbits; D014921:Wings, Animal / Q000201:enzymology*
chemical_list: D019476:Insect Proteins; D007527:Isoenzymes; D010734:Phosphogluconate Dehydrogenase
keywords: 6-phosphogluconate dehydrogenase; 6PGDH; Biochemical adaptation; Concentration and kinetics; Enzyme purification