Enzymatic Activity Determination in Tobacco Leaves

Example 4

To determine enzymatic activities, 500 mg of tobacco leaf tissue collected from leaf 23 of three biological replicates of was ground in 1 ml HEPES extraction buffer (100 mM HEPES, 2 mM dithiothreitol (DTT), 1 mM EDTA, pH 7.5) and 100 mg of polyvinylpyrrolidone was added during grinding. Following centrifugation (13,000 g, 10 min, 4° C.), the enzyme activities were measured using an isotopic method as described by Capell et al. (1998) by measuring the release of ¹⁴CO2. L-[1-¹⁴C]Arg and L-[1-¹⁴C]Orn were used as radioactive substrates.

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US11859192B2. Compositions and methods for producing tobacco plants and products having altered alkaloid levels with desirable leaf quality (2024-01-02). Altria Client Services LLC [US]. Inventors: Marcos Fernando de Godoy Lusso [US], James A. Strickland [US], Jesse Frederick [US], Dongmei Xu [US], Chengalrayan Kudithipudi [US].

Patent 2024

Biological Buffer Centrifugation Dithiothreitol Edta Enzyme activities Hepes Isotopic Leaf Polyvinylpyrrolidone Radioactive Tissue Tobacco

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Variable analysis

independent variables

Tissue source (leaf 23 of three biological replicates)

dependent variables

Enzymatic activities (measured by the release of 14CO2)

control variables

Extraction buffer composition (100 mM HEPES, 2 mM dithiothreitol (DTT), 1 mM EDTA, pH 7.5)
Grinding conditions (500 mg tobacco leaf tissue, 1 ml extraction buffer, 100 mg polyvinylpyrrolidone)
Centrifugation conditions (13,000 g, 10 min, 4° C.)

positive controls

L-[1-14C]Arg and L-[1-14C]Orn used as radioactive substrates

negative controls

None explicitly mentioned

Annotations

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