Ribonucleoprotein preparation

Figure 1 represents a schematic illustration of the separation of ribosomes, sRNA, and the enzymes involved in the transfer reaction, described in detail below. At the end of the incubation period, the ribonucleoprotein and supernatant fractions were separated by ultracentrifugation, the perchloric acid-insoluble fraction was prepared from each, and the nucleic acids and proteins were isolated from the acid-insoluble residue (17). 

HeLa cells were made permeable in order to allow the passage of the [ P]-labeled ADP-ribose precursor NAD into the cell. After a short labeHng period, the nuclei were isolated and the nuclear matrices were prepared. For complete removal of chromatin constituents and ribonucleoprotein complexes during the subsequent extraction with high salt buffer, nuclei were incubated with hi concentrations of DNase 1 and RNase A. 

Fia. 7. Time curve of the incorporation of leucine-C into the RNA and protein of the ribonucleoprotein particles and the soluble fraction in intact ascites cells. The tumor cells (about 10 gm of packed cells) were incubated at 25° in 50 ml of their own ascitic fluid fortifled with glucose and Tris buffer, and contiuning 3 jimoles with 1.06 X 10 cpm of L-leucine. The NaCl-insoluble and -soluble fractions were prepared from the supernatant of a 16,000 g centrifugation. [Taken from Hoagland et al. (186).]... 

In the electron microscope the particles appear as rather uniform, nearly spherical particles, with diameters clustering around 150 and 250 A. (It is not known whether the larger ones correspond to 80 S particles.) A typical picture, taken of a purified preparation of ribonucleoprotein particles from pea seedlings, is reproduced in Fig, 9. 

Fia. 11. Time curves for the net increase in protein (solid line) and the incorporation of phenylslanine-C (broken line) by isolated ribonucleoprotein particles from pea seedlings. The incubation mixture contained, in a volume of 1 ml fiOMmoles phosphate buffer, pH 7.5 10 Mmoles MgCh 1 Mmole ATP 1 Mmole nL-phenylalanine with 30,000 counts/min and unwashed particles containing 1-1.5 mg protein, a. Freshly prepared particles b. particles stored for 1 day. Each curve represents the average of at least four experiments. [Taken from Raacke ( 57).]... 

The membrane systems from the other organisms are prepared from protoplasts, and hence do not contain cell wall components. Nevertheless, they are very complex, as is perhaps illustrated best by the great differences in the level of incorporation reported for different systems—and for the same system, under different conditions. The data available from the literature are reproduced in Table XII. Despite the great variations in the reported data, it is seen that this system has a vastly greater capacity for amino acid incorporation than isolated microsomes or ribonucleoprotein particles. The only system of the latter kind which approaches the membranes in activity, is the one from pea seedlings (see Table X, Section III, B, 4, d). These two systems have other points of similarity both remain active for 2-3 hr of incubation, and the kinetics are often similarly complex. 

The role of the ribonucleoprotein particles which (presumably) are attached to the cytoplasmic membranes, and vdiich, according to the elegant experiments of McQuillen and co-workers (67) are the seat of nascent protein in vivo, has not been determined in the in vitro system. When, however the membrane preparations are subjected to sonic treatment and then centrifuged, it is uniformly found that the supernatant is devoid of activity, and the sediment (which would contain any existing nucleoprotein particles) retains some, albeit much reduced, activity. 

A number of early observations pointed the way towards our current concepts of protein synthesis. Cells that are particularly active in protein synthesis are rich in cytoplasmic ribonucleic acid (RNA) and ribonucleoprotein (RNA-protein). Further, in a number of studies with intact cells it was found that their rate of protein synthesis varied directly as their cytoplasmic nucleic acid content and that their synthesis of protein was strongly inhibited by ribonuclease (which degrades RNA) and also by chloramphenicol. More recently, limited protein synthesis has been achieved in cell-free preparations obtained from bacterial and higher plant cells and reinforced by the addition of appropriate mixtures of the 20 protein amino acids and a suitable energy source such as ATP. Such in-vitro protein synthesis, most readily followed by studying the incorporation of C-labelled amino acids into protein, is prevented by adding ribonuclease or chloramphenicol. 

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