Plant Physiol. 1974 Apr;53(4):533-42. doi: 10.1104/pp.53.4.533.

The chloroplast and cytoplasmic ribosomes of euglena: I. Stability of chloroplast ribosomes prepared by an improved procedure.

Plant physiology

S D Schwartzbach, G Freyssinet, J A Schiff

PMID: 16658740 PMCID: PMC541394 DOI: 10.1104/pp.53.4.533

Abstract

A new isolation procedure has resulted in an improved yield of stable 68S chloroplast ribosomes from Euglena gracilis var. bacillaris. Chloroplasts are isolated by suspending the cells in buffer I (sorbitol, 250 mm; sucrose, 250 mm; Ficoll, 2.5% [w/v]; magnesium acetate, 1 mm; bovine serum albumin, 0.01% [w/v]; mercaptoethanol, 14 mm; N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid, pH 7.6, 5 mm) and passing through a French press at less than 1500 pounds per square inch. The crude chloroplasts are purified by three washings with buffer II (sorbitol, 150 mm; sucrose, 150 mm; Ficoll, 2.5% [w/v]; magnesium acetate, 1 mm; bovine serum albumin, 0.01% [w/v]; mercaptoethanol, 14 mm; N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid, pH 7.6, 5 mm). Stable 68S chloroplast ribosomes are obtained when the isolated chloroplasts are resuspended in ribosome buffer (tris-HCI, pH 7.6, 10 mm; magnesium acetate, 12 mm; KCI, 60 mm) containing spermidine, 0.5 mm; mercaptoethanol, 14 mm; sucrose, 8% (w/w), passed through a French press at 4000 pounds per square inch and extracted with either 0.1% (w/v) sodium deoxycholate or 1.0% (v/v) Triton X-100. At 0 to 4 C in ribosome buffer, the purified 68S chloroplast monosome forms a 53S particle while the 35S particle, an expected product of monosome dissociation, cannot be detected. Spermidine and mercaptoethanol prevent the formation of 53S particles from 68S monosomes. The purified 53S particles derived from 68S monosomes contain 23S RNA as well as a significant amount of 16S RNA, suggesting that this particle may not be a true ribosomal subunit.

References

1. Plant Physiol. 1974 Apr;53(4):543-54 - PubMed
2. Dev Biol. 1972 May;28(1):253-73 - PubMed
3. J Mol Biol. 1971 Aug 14;59(3):491-503 - PubMed
4. Biochemistry. 1968 Feb;7(2):668-74 - PubMed
5. Biochim Biophys Acta. 1971 Aug 12;246(1):81-99 - PubMed
6. Biochim Biophys Acta. 1969 Oct 22;190(2):368-80 - PubMed
7. J Mol Biol. 1969 Apr 14;41(1):139-47 - PubMed
8. Science. 1970 Oct 9;170(3954):171-3 - PubMed
9. J Cell Biol. 1971 Nov;51(21):499-513 - PubMed
10. J Cell Sci. 1972 Mar;10(2):267-305 - PubMed
11. Biochemistry. 1967 Jun;6(6):1818-27 - PubMed
12. J Biol Chem. 1972 Dec 25;247(24):8043-50 - PubMed
13. J Cell Biol. 1969 May;41(2):431-40 - PubMed
14. Anal Biochem. 1969 Jan;27(1):130-49 - PubMed
15. J Biol Chem. 1960 Jul;235:2112-6 - PubMed
16. FEBS Lett. 1972 Aug 1;24(2):219-224 - PubMed
17. J Bacteriol. 1969 Oct;100(1):260-8 - PubMed
18. Science. 1971 Jul 16;173(3993):241-2 - PubMed
19. FEBS Lett. 1970 Oct 5;10(3):149-152 - PubMed
20. Biochim Biophys Acta. 1971 Sep 24;246(3):507-16 - PubMed
21. Biochem J. 1969 Oct;114(4):847-54 - PubMed
22. Plant Physiol. 1967 Jul;42(7):922-32 - PubMed
23. Plant Physiol. 1971 Feb;47(2):217-21 - PubMed
24. Biochem J. 1972 Jun;128(2):353-65 - PubMed
25. Biochem Biophys Res Commun. 1973 Jan 23;50(2):443-51 - PubMed

Publication Types

• Journal Article