Acridine orange as an indicator of the cytoplasmic ribosome state.
In hippocampal neurons of ground squirrels and neuroblastoma culture the ribosome state was analyzed by staining with acridine orange (AO), labeling with radioactive amino acids, and electron microscopy. Electron microscopy indicated that the extent to which ribosomes associated in polysomes varied from 25% in brain cells of torpid ground squirrels up to 93% in growing neuroblastoma cells. In control rat neurons, it was 75%. The fluorescence of the AO-rRNA complex in ribosomes changed with the polysome/monosome ratio. The red fluorescence of AO-single-stranded rRNA complex as well as ribosome association in polysomes decreased greatly as a function of the shift from polysomes to monosomes. The green fluorescence of AO intercalated in the double-stranded rRNA changed insignificantly. As a result, the ratio of red to green fluorescence intensity, Kalpha, changed more than 3-fold with the changing polysome/monosome ratio. Protein labeling also showed strong positive correlation with Kalpha. Thus, rRNA showed different accessibility for AO binding in active and inactive ribosomes. A possible mechanism of partial rRNA shielding with proteins is proposed. AO fluorescence in the cytoplasm, i.e., AO binding to rRNA in ribosomes, is presumed to reflect adequately the profound changes in the state of cell protein synthesizing system that can be regulated by both functional activity and stress factors.
Gordon
R Y
RY
Institute of Cell Biophysics, Russian Academy of Sciences, Pushchino, Moscow Region. khutzian@mars.ibioc.serpukhov.su
Bocharova
L S
LS
Kruman
I I
II
Popov
V I
VI
Kazantsev
A P
AP
Khutzian
S S
SS
Karnaukhov
V N
VN
D000165:Acridine Orange; D000818:Animals; D003513:Cycloheximide / Q000494:pharmacology; D003593:Cytoplasm / Q000737:chemistry*; D005456:Fluorescent Dyes; D006624:Hippocampus / Q000166:cytology; D007202:Indicators and Reagents; D007700:Kinetics; D008297:Male; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012330:RNA, Double-Stranded / Q000032:analysis; D012335:RNA, Ribosomal / Q000032:analysis; D051381:Rats; D017208:Rats, Wistar; D012270:Ribosomes / Q000737:chemistry*; D012589:Sciuridae; D014407:Tumor Cells, Cultured
D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D011500:Protein Synthesis Inhibitors; D012330:RNA, Double-Stranded; D012335:RNA, Ribosomal; D003513:Cycloheximide; D000165:Acridine Orange