l h e intracellular localization of transaldolase, transketolase, ribose-5-phosphate isomerase, and ribulose-5-phosphate epimerase was reexamined in spinach (Spinacia oleracea 1,) leaves. We found a highly predominant if not exclusive localization of these enzyme activities in chloroplasts isolated by isopyknic centrifugation in sucrose gradients. Clucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, glucose phosphate isomerase, and triose phosphate isomerase activity was present in the chloroplast fraction but showed additional activity in the cytosol (supernatant) fraction attributable to the cytosol-specific isoforms known to exist for these enzymes. Anion-exchange chromatography of proteins of crude extracts on diethylaminoethyl-Fractogel revealed only a single enzyme each for transaldolase, transketolase, ribose-5-phosphate isomerase, and ribulose-5-phosphate epimerase. The data indicate that chloroplasts of spinach leaf cells possess the complete complement of enzymes of the oxidative pentose phosphate pathway (OPPP), whereas the cytosol contains only the first two reactions, contrary to the widely held view that plants generally possess a cytosolic OPPP capable of cyclic function. l h e chloroplast enzymes transketolase, ribose-5-phosphate isomerase, and ribulose-5-phosphate epimerase appear to be amphibolic for the Calvin cycle and OPPP.
Class I and class II aldolases are products of two evolutionary non-related gene families. The cytosol and chloroplast enzymes of higher plants are of the class I type, the latter being bifunctional for fructose-1,6-and sedoheptulose-1,7-P P in the Calvin cycle. Recently, class II aldolases were detected for the cytosol and chloroplasts of the lower alga Cyanophora paradoxa. The respective chloroplast enzyme has been shown here to be also bifunctional for fructose-1,6-and sedoheptulose-1,7-P P . Kinetics, also including fructose-1-P, were determined for all these enzymes. Apparently, aldolases are multifunctional enzymes, irrespective of their class I or class II type.z 1999 Federation of European Biochemical Societies.
The 24‐h patterns of tissue thyroid hormone concentrations and type II 5′‐ and type III 5‐iodothyronine deiodinase (5′D‐II and 5D‐III, respectively) activities were determined at 4‐h intervals in different brain regions of male euthyroid rats entrained to a regular 12‐h light/12‐h dark cycle (lights on at 6:00 a.m.). Activity of 5′D‐II, which catalyzes the intracellular conversion of thyroxine (T4) to 3,3′,5‐triiodo‐l‐thyronine (T3) in the CNS, and the tissue concentrations of both T4 and T3 exhibited significant daily variations in all brain regions examined. Periodic regression analysis revealed significant circadian rhythms with amplitudes ranging from 9 to 23% (for T3) and from 15 to 40% (for T4 and 5′D‐II) of the daily mean value. 5′D‐II activity showed a marked nocturnal increase (1.3–2.1‐fold vs. daytime basal value), with a maximum at the end of the dark period and a minimum between noon and 4:00 p.m. 5D‐III did not exhibit circadian patterns of variation in any of the brain tissues investigated. Our results disclose circadian rhythms of 5′D‐II activity and thyroid hormone concentrations in discrete brain regions of rats entrained to a regular 12:12‐h light‐dark cycle and reveal that, in the rat CNS, T3 biosynthesis is activated during the dark phase of the photoperiod. For all parameters under investigation, the patterns of variation observed were in part regionally specific, indicating that different regulatory mechanisms may be involved in generating the observed rhythms.
A cDNA encoding the Calvin cycle enzyme transketolase (TKL; EC 2.2.1.1) was isolated from Sorghum bicolor via subtractive differential hybridization, and used to isolate several full-length cDNA clones for this enzyme from spinach. Functional identity of the encoded mature subunit was shown by an 8.6-fold increase of TKL activity upon induction of Escherichia coli cells that overexpress the spinach TKL subunit under the control of the bacteriophage T7 promoter. Chloroplast localization of the cloned enzyme is shown by processing of the in vitro synthesized precursor upon uptake by isolated chloroplasts. Southern blot-analysis suggests that TKL is encoded by a single gene in the spinach genome. TKL proteins of both higher-plant chloroplasts and the cytosol of non-photosynthetic eukaryotes are found to be unexpectedly similar to eubacterial homologues, suggesting a possible eubacterial origin of these nuclear genes. Chloroplast TKL is the last of the demonstrably chloroplast-localized Calvin cycle enzymes to have been cloned and thus completes the isolation of gene probes for all enzymes of the pathway in higher plants.
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