Abstract
Proteins from Nicotiana tabacum cytoplasmic and chloroplast ribosomes and their subunits have been isolated under a variety of conditions and resolved by two-dimensional polyacrylamide gel electrophoresis. Average absolute mobility maps, constructed from the resultant electropherograms, were used to compare ribosomal proteins from cytoplasmic and chloroplast ribosomes. A novel technique for the estimation of molecular weights from two-dimensional electrophoretic mobilities is described. The cytoplasmic ribosome of N. tabacum possesses 73-80 distinct proteins with pI greater than 5, 26-30 associated with the 40 S subunit, and 47-50 with the 60 S subunit. The 60 S cytoplasmic ribosomal subunit has, in addition, at least three acidic polypeptides (pI less than 5). The chloroplast ribosome has 55-58 unique basic proteins with 22-23 occurring in the 30 S subunit and 33-35 in the 50 S subunit. A few additional acidic polypeptides are associated with the 30 S and 50 S subunits (2-3 and 1, respectively). There is little similarity between the electrophoretic patterns or molecular weight frequency distributions of proteins of analogous cytoplasmic and chloroplast ribosomal subunits of N. tabacum. The electrophoretic patterns and molecular weight frequency distributions of the proteins of N. tabacum chloroplast ribosomal subunits are quite similar to those of the Escherichia coli ribosome and the chloroplast ribosome of the alga Chlamydomonas. N. tabacum cytoplasmic ribosomal protein electrophoretic patterns and molecular weights are very similar to proteins from the Chlamydomonas cytoplasmic ribosome. These data substantiate: 1) the close affiliation between higher plant chloroplast and prokaryotic ribosomes and 2) a general similarity between angiosperm cytoplasmic ribosomal proteins and other classes of eukaryotic cytoplasmic ribosomal proteins.
Highlights
Proteins from Nicotiunutabacum cytoplasmic and chloroplast ribosomes and their subunits have been isolated under a variety of conditions and resolved by two-dimensional polyacrylamide gel electrophoresis
In terms of mass and density of their subunits, number of their component proteins, and sizes and degree of sequence homology between analogous rRNA species, organellar ribosomes bear more resemblance to prokaryotic ribosomes than they do to the cytoplasmic ribosome found in the same cell
The subunit-monosome equilibria of cytoplasmic and chloroplast ribosomes have very different ionic strength and magnesium ion concentration dependencies [7,8,9], a factthat is exploited in the separation of the two ribosomesand their subunits
Summary
Ribosome Isolation-Approximately 300 g of deribbed leaves (3-7 cm) of N . tabacum Chloroplast ribosomal subunits and cytoplasmic monosomes were in tandem on a series of slab gels large enough to accommodate two resolved by zonal centrifugation through an equivolumetric gradient first dimension capillary gels. Chloroplast ribosomal subunit and cy- pensates (normalizes) for systematic sources of variation in absolute toplasmic monosome fractions from sucrose gradients were diluted mobility map coordinates obtained from different gels Native chloroplast and cyto- Molecular Weight Estimation-Proteins extracted from 1 M plasmic monosomes from these gradients were diluted 1:l with dis- NH&l-washed E. coli ribosomes were resolved in tandem with ChRP sociation buffer, repelleted, resuspended in 8 M urea Cells were harvested by sedimentation and disrupted ChRP maps were appropriately averaged to yield an absolute mobility by grinding with alumina in 5 m~ MgCh, 25 mM KCl, 25 r m Tris- map of E. coli ribosomal proteins, possessing the same origin and HC1, pH 8 , 4 "C. Ribosomal Protein Extraction-Purified ribosomal precipitates were dissolved in 8 M urea (Schwarz-Mann,ultrapure), 1%2-mercap-
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