Lipoxygenases 1 and 2 were localized in etiolated germinating soybean seeds (Glycine max [L.]. Meff. var. Williams) by an indirect immunofluorescence staining technique. Sections of paraffin-embedded seedlings were stained with affinity-purified antibodies directed against lipoxygenase 1 or 2. The specificity of the immunofluorescence technique was examined by use of nonimmune serum or immunoglobulin G preparations after total adsorption with the appropriate lipoxygenase coupled to Sepharose 4B.After immunofluorescence staining with antilipoxygenase 1 or 2 IgG storage tissues of cotyledons fluoresce strongly the first days of germination. After 3 days, the abaxial hypodermis, the epidermis, and the vascular bundle sheaths show fluorescence, especially after incubation with antilipoxygenase 2 IgG. Fluorescence in cortex and pith of the hypocotyl migrates to the vascular cylinder during germination. In primary leaves, all tissues show fluorescence after I day of germination. In storage tissues of cotyledons, cytoplasm around the protein bodies fluoresces, whereas in other tissues protein bodies or other large cell organelles fluoresce.It is reasonable to suggest that lipoxygenase exerts its function in cells at the time that rigorous changes in metabolism take place, namely at the start of mobilization of reserves in storage tissues and start of biosynthesis of chloroplastids in several tissues.The enzyme lipoxygenase (linoleate:oxygen reductase, EC 1.13.11.12) catalyzes the dioxygenation of fatty acids containing a methylene-interrupted cis,cis-pentadiene structure. In 1947, Theorell et al. (23) fatty acids (17).As yet the physiological role of lipoxygenase is far from clear. It appears that lipoxygenase activity is maximal during the early stages of seed germination. Although soybeans are epigeous, seedlings remain essentially heterotrophic during the first 10 d (18). Sugars, primarily sucrose and stachyose in the cotyledons and embryonic axis, are the principal carbon sources during the first 3 d ofgermination (1). Subsequently, fat utilization increases and continues for about 10 d. Protein utilization which starts almost immediately, accelerates during the first 10 d and continues up to cotyledonary senescence (12). Holman (1 1) studied the relationship between lipoxygenase activity and changes in fat composition during germination ofsoybean seeds. He found that lipoxygenase activity at pH 9.0 declines sharply after the 2nd d while from the 3rd d linoleic acid and linolenic acid contents begin to decrease. Both variations in lipoxygenase activity of different plant tissues during germination and data dealing with subcellular localization of lipoxygenases have been reviewed by Douillard (6, 7). Lipoxygenase usually is soluble and localized in storage tissues of the seeds of most plants. Knowledge on the exact cellular and subcellular localization in germinating seeds can throw new light on the physiological function of lipoxygenase. Hitherto, localization in subcellular fractions has been examined by...
Soybean lipoxygenases-I and -2 were localized intracellularly in seeds at various stages of germination by indirect labeling of cryosections with protein A-colloidal gold complexes. Two sizes of gold particles (Au' and Au") were used in single-and double-labeling experiments. In distinct metabolic function, a physiological function of the enzymes can be postulated. Therefore, we examined the intracellular localization of lipoxygenases in tissues of germinating soybean seeds. We used an indirect labeling procedure for immunoelectron microscopy with protein A-collodial gold complexes. Preservation of antigenicity and attainability of lipoxygenases in sections was obtained by cryosectioning.Lipoxygenase (EC 1.13.11.12) oxidizes unsaturated fatty acids containing a 1,4-cis,cis-pentadiene system to conjugated hydroperoxy acids. Seeds of soybean (Glycine max [L.] Merr.) contain significant amounts of the isoenzymes lipoxygenase-1 and -2 (respectively, 1.4 and 2.8 mg/g dry weight) (16). Linoleic acid and structurally related fatty acids are substrates for both lipoxygenase-l and lipoxygenase-2. Besides, lipoxygenase-2 is able to oxidize methyl linoleate, and mono-and trilinolein (1, 9, 13). With 1.8 mm linoleic acid as substrate in a standard polarographic assay, lipoxygenase-I shows maximal activity at pH 9.0 and lipoxygenase-2 at pH 6.6. The activity of lipoxygenase-2 at pH 9.0 increases ifthe substrate concentration is lowered or Ca2" is added (17, 25). Product-specificity of the reaction with lipoxygenase-2 also is higher at pH 9.0 (21) from which we suggest a pH 9.0 optimum for both isoenzymes in vivo.
Duckweed colonies were grown on 1 l of nutrient solution supplied with 10 μM L-[(14)C]leucine or with 25 μM L-[(14)C]valine. Under these conditions the exogenously supplied amino acid did not inhibit growth, but caused in the plants a moderately increased pool of that amino acid, which remained essentially constant during the culture period. The effect of the increased pool of valine or leucine on the biosynthesis of these amino acids was determined from isotope dilution in the protein-bound valine and-or leucine. An increase in the leucine pool from 1.1 to 5.0 nmol mg(-1) dry weight resulted in a 21% reduction of metabolite flow through the common part of the valine-leucine biosynthetic pathway; leucine synthesis was reduced by 35%, but valine synthesis by only 5% and isoleucine synthesis was apparently unaffected. An increase in the valine pool from 3.2 to 6.6 nmol mg(-1) dry weight reduced the metabolite flow through the valine-leucine pathway by 48%, valine synthesis by 70%, and leucine synthesis from pyruvate by 29%, which was compensated by leucine synthesis from exogenous valine, whereas the synthesis of isoleucine was not changed. It is concluded that the biosynthesis of valine and leucine is mainly controlled by feedback inhibition of acetohydroxyacid synthetase. In vivo, the feedback inhibition can be exerted in such a way that synthesis of acetolactate (the precursor of valine and leucine) is appreciably reduced, whereas synthesis of acetohydroxybutyrate (the isoleucine precursor) is not inhibited.
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