Examination of Isolated Cell Constituents

Whether the occurrence of an enzyme in a particulate fraction really represents its in vivo location can be substantiated by the following properties (a) close binding to membrane lipids, cf. cyclopenase (D 8.4.2), which is a lipoprotein of the plasma membrane (b) cooperation with other enzymes, cf. the catalytic facilitation in the biosynthesis of cyanogenic glycosides, cinnamic and benzoic acids (A 3.1) and (c) in situ examination by cytochemical methods, cf. the localization of phenol oxidases (C 2.3.1), peroxidases (C 2.4) and thioglucosidases (D 9.4). 

Biochemical tracer experiments on compartmentalization and channeling in living cells take into consideration the cellular microenvironment as well as the interrelations between enzymes and low molecular compounds and other cell constituents. Such experiments are especially useful for studying the dynamic aspects of secondary metabolism. However, they have the drawback of not allowing the spatial localization of existing pools and channels of the metabolites. Thus, they give reliable results only if they are combined with cytochemical experiments or with examination of isolated cell constituents. 

There are two basic types of experiments using tracer molecules. In one, tracers are applied in pulse experiments and the pattern of labeling in the tracer-derived products is determined over time. In the other, the fate of labeled compounds is examined after a chase period following the initial pulse. 

Typical examples are experiments in which COg was administered to intact leaves in competition with mevalonic acid in the location of the site of plastid quinone biosynthesis and experiments with Penicillium cyclopium, where the channeling of the precursor L-phenylalanine and its incorporation into the alkaloids of the cyclopenin-viridicatin group and proteins were studied 

Luckner, M., Diettrich, B., Lerbs, W. Cellular compartmentation and channeling of secondary metabolism in microorganisms and higher plants. Prog. Phytochem. 6, 103-142 (1980) 

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