A coupled spectrophotometric assay for routine assessment of carbamylation and k@ of rubisco

Conference · Tue May 01 04:00:00 EDT 1990 · Plant Physiology, Supplement; (USA)

OSTI ID:5725432

Sharkey, T D; Butz, N D ^[1]

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1. Univ. of Wisconsin, Madison (USA)

We have developed methods to use the spectrophotometric assay for rubisco to determine k{sub cat} and carbamylation of rubisco in crude leaf extracts. In this assay, rubisco activity is coupled to NADH oxidation by PGA kinase and NADH dependent GAP dehydrogenase. The difficulty with this method in the past has been an initial lag in the observed signal, presumably because PGA must build up. This problem was solved by including high levels of ATP plus an ATP regenerating system and by using pH 8.0 for the assays. At higher pH the initial lag was observed, at lower pH, rubisco activity declined. The continuous spectrophotometric assay is particularly suited to studies of fallover, the loss of activity of rubisco during assay, and to studies of activation, the increase in activity as rubisco becomes carbamylated. the activity of rubisco measured immediately upon extraction compared to the activity after incubation with CO{sub 2} and Mg{sup 2+} correlated well with the degree of carbamylation as determined by {sup 14}CABP/{sup 12}CABP competition experiments. Rubisco activity was reduced by binding CABP, and the number of active sites were estimated by extrapolation to zero activity. These data allow the calculation of k{sub cat}. These methods allow estimation of many important parameters of rubisco activity in less time than previous methods and without generation of any radioactive waste.

• OSTI ID: 5725432
• Report Number(s): CONF-9007196--
• Conference Information: Journal Name: Plant Physiology, Supplement; (USA) Journal Volume: 93:1
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ATP
CARBON 14 COMPOUNDS
CARBON-CARBON LYASES
CARBOXY-LYASES
ENZYME ACTIVITY
ENZYMES
ISOTOPE APPLICATIONS
LABELLED COMPOUNDS
LEAVES
LYASES
NUCLEOTIDES
ORGANIC COMPOUNDS
PH VALUE
RIBULOSE DIPHOSPHATE CARBOXYLASE
TRACER TECHNIQUES