A rapid method for isolation of purified physiologically active chloroplasts used to study the intra cellular distribution of amino acids in pea pisum sativum cultivar little marvel leaves

Mills, W.R.; Joy, K.W.

Planta (Heidelberg) 148(1): 75-83


ISSN/ISBN: 0032-0935
Accession: 004616406

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A procedure is described for the rapid (< 5 min) isolation of purified, physiologically active chloroplasts from P. sativum L. Mitochondrial and microbody contamination is substantially reduced and broken chloroplasts are excluded by washing through a layer containing a treated silica sol. On average the preparations contain 93% intact chloroplasts and show high rates of 14CO2 fixation and CO2-dependent O2 evolution (over 100 .mu.mol/mg chlorophyll(chl)/h); they are also able to carry out light-driven incorporation of leucine into protein (4 nmol/mg chl/h). The amino-acid contents of chloroplasts prepared from leaves and from leaf protoplasts were determined. Asparagine is the most abundant amino acid in the pea chloroplast (> 240 nmol/mg chl), even though it is proportionately lower in the chloroplast relative to the rest of the cell. The chloroplasts contain about 20% of many of the amino acids of the cell, but for individual amino acids the percentage in the chloroplast ranges from 8-40% of the cell total. Glutamic acid, glutamine and aspartic acid are enriched in the chloroplasts, while asparagine, homoserine and .beta.-(isoxazolin-5-one-2-yl)-alanine are relatively lower. Leakage of amino acids from the chloroplast during preparation or repeated washing was approximately 20%. Some differences exist between the amino-acid composition of chloroplasts isolated from intact leaves and from protoplasts. In particular, .gamma.-aminobutyric acid accumulates to high levels, while homoserine and glutamic acid decrease, during protoplast formation and breakage.