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- W316932 abstract "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 estimationmore » of many important parameters of rubisco activity in less time than previous methods and without generation of any radioactive waste.« less" @default.
- W316932 created "2016-06-24" @default.
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- W316932 date "1990-05-01" @default.
- W316932 modified "2023-09-23" @default.
- W316932 title "A coupled spectrophotometric assay for routine assessment of carbamylation and k@ of rubisco" @default.
- W316932 hasPublicationYear "1990" @default.
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