Amino Acid Biosynthesis by Isolated Chloroplasts during Photosynthesis

Abstract

The pool sizes of the common amino acids in purified intact chloroplasts from Vicia faba L. were measured (nanomoles per milligram chlorophyll). The three amino acids present in the highest concentrations were glutamate, aspartate, and threonine. Alanine, serine, and glycine were each present at levels between 15 and 20 nanomoles per milligram chlorophyll and 13 other amino acids were detectable at levels below 10.Only aspartate, alanine, glycine, serine, threonine, and lysine became labeled during photosynthetic "CO2 fixation by isolated chloroplasts: the label in aspartate represented over 60 % of the total '4C found in the amino acids. Glutamate dehydrogenase, glutamate-oxaloacetate, and glutamate-pyruvate aminotransferases were present in the chloroplasts, but no other trans. ferase activities from glutamate could be detected. The chloroplasts were able to synthesize a total of 17 other protein amino acids from either alanine or aspartate, but no synthesis of leucine by aminotransferase reactions could be detected. The synthesis of aspartate was studied in more detail. The enzyme systems required for the generation of oxaloacetate from triose phosphate were virtually absent from the chloroplasts but present in the leaf cytoplasmic fraction. Addition of either a leaf "cytoplasmic" fraction or an oxaloacetate generating system resulted in an increased proportion of the total "C fixed being found in the amino acid fraction during photosynthetic "CO2 fixation.It is suggested that the supply of oxaloacetate from the cytoplasm is one of the important factors controlling the synthesis of amino acids by the chloroplast.During "CO2 fixation in photosynthesizing algal cells (2,6,32) 2-ketoglutarate and that both the synthesis of glutamate and of alanine and aspartate probably take place within the chloroplast. An L-glutamate dehydrogenase would be able to catalyze the reductive amination of 2-ketoglutarate. Isolated chloroplasts have been shown to possess such an enzyme dependent on reduced pyridine nucleotides (23, 30) and are capable of catalyzing the photoreduction of 2-ketoglutarate to glutamate (30, 11). The recorded rates of photoreduction of 2-ketoglutarate by isolated chloroplasts are 0.4 ,umole/mg chl-hr (spinach [35]) and 0.6 ,umole/mg chl-hr (broad bean [11]). These low rates may partly account for the finding that, although chloroplasts can now be prepared which are capable of photosynthetic CO2 fixation at rates comparable with those observed in vivo (7,17,18), in general only 5 to 10% of the total radioactivity is recovered in the amino acid fraction (9,37). In isolated chloroplast suspensions aspartate (37), aspartate and alanine (9, 25), and glycine and serine (8) have been shown to be among the products of photosynthetic "CO2 fixation.Assuming the initial incorporation of ammonia in the chloroplast is into glutamate, a further question arises concerning the mode of transfer of the amino group from glutamate to the other amino acids. Amino transferase systems would provide a possible mechanism...

Changes in Pool Sizes of Free Amino Acids and Amides in Leaves and Plastids of Zea mays during Leaf Development

“…Determinations which use nonaqueous techniques (1) to isolate the chloroplasts must be regarded with some caution because of the problems of cytoplasmic contamination in this type of procedure (4,9,27). Previous experiments in our laboratory (12) showed that chloroplasts isolated in aqueous media retain free amino acids but no attempt was made to estimate the amino acid concentrations.…”

“…Isolated plastids were broken by resuspension in ice-cold water, followed by 10 rapid strokes of a TenBroeck homogenizer and then stirred by a mechanical rotamixer until more than 95% of the plastids had lost their envelopes. The suspension ofbroken plastids was centrifuged at 3,000g for 20 min at 1 C. The supernatant was added to ethanol to a final concentration of 80%o (v/v) ethanol and recentrifuged at 3,000g for 45 min at -5 C. After reducing the volume of the supernatant to 10 ml by rotary evaporation below 40 C, it was mixed with 4 g of Dowex 50W-X8 cation exchange resin (200-400 mesh; hydrogen form) (12). The resin was washed three times with 20 ml glass-distilled H20 and the amino acid fraction removed by three washes with 4 mm NH40H.…”

Short Term Effect of He-Ne Laser on Jatropha curcas Leaf Development and Amino Acid Biosynthesis: More Insights in Developmental Genetics of Oil Producing Plants

“…Interestingly, chloroplast is able to synthesize all protein forming amino acids except leucine (Kirk & Leech, 1972). Soluble amino acids that are rapidly transported to growing root and shoots are those with a high N: C ratio such as asparagine, glutamine, and arginine .…”

“…The main pathway of glutamate is shown in Fig. 6.Glutamate is the most abundant amino acid formed by chloroplast followed by aspartate and threonine (Kirk & Leech, 1972). Glutamate is the precursor for chlorophyll synthesis in developing leaves (Yaronskaya et al, 2006), is crucial signaling molecule (Forde, 2002), is tightly regulated by light (Coschigano et al, 1998) via a phytochrome-mediated pathway (Suzuki et al, 2001) and is directly involved in the assimilation and dissimilation of ammonia (Weber & Flügge, 2002).…”

Primary production of particulate protein amino acids : algal protein metabolism and its relationship to the composition of particulate organic matter

“…;Lve since demonstrated the ability of chloroplasts to synthesize a variety of amino acids (e.g . Kirk and Leech, 1972;Leech and Murphy, 1976;Mills et al, 1980;Schulze-Seibert et al, 1984).…”