title: Photometric method for routine determination of kcat and carbamylation of rubisco.
abstract: Ribulose bisphosphate carboxylase (rubisco) is the first enzyme in photosynthetic CO2 assimilation. It is also the single largest sink for nitrogen in plants. Several parameters of rubisco activity are often measured including initial activity upon extraction, degree of carbamylation, catalytic constant of the enzyme (kcat), and the total amount of enzyme present in a leaf. We report here improvements of the photometric assay of rubisco in which rubisco activity is coupled to NADH oxidation which is continuously monitored in a photometer. The initial lag usually found in this assay was eliminated by assaying rubisco activity at pH 8.0 instead of 8.2, using a large amount of phosphoglycerate kinase, and adding monovalent cations to the assay buffer. We found that when using the photometric assay, the ratio of activity found initially upon extraction divided by the activity after incubating with CO2 and Mg(2+) reflects the degree of carbamylation as determined by (14)carboxyarabinitol bisphosphate/(12)carboxyarabinitol bisphosphate competition. We developed methods for measuring the catalytic constant of rubisco as well as the total amount of enzyme present using the photometric assay and carboxyarabinitol 1,5-bisphosphate. We believe that the photometric assay for activity will prove more useful than the (14)CO2 assay in many studies.
authors.affiliation: Department of Botany, University of Wisconsin, 53706, Madison, WI, USA.
authors.forename: T D
authors.initials: TD
authors.lastname: Sharkey
authors.forename: L V
authors.initials: LV
authors.lastname: Savitch
authors.forename: N D
authors.initials: ND
authors.lastname: Butz