John H. Frenster, V. G. Allfrey and A.E. Mirsky
Rockefeller Institute
New York, NY 10021

Abstract:
Introduction:

Materials and Methods:
Isolation of Nuclear Ribosome Fractions:
Incubation Procedure:
Measurement of Protein, RNA, DNA and Radioactivity:

Results:
Requirements for Amino Acid Incorporation by Isolated Nuclear Ribosomes:
Loss of Function in Hypertonic Media:
Sensitivity to Incubation with Nucleases:
Effects of Added Polyanions and Polycations:

Discussion:
Acknowledgements:
References:
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Abstract:

Ribonucleoprotein particles of diverse composition and metabolic activity can be extracted from the isolated thymus cell nucleus. If properly supplemented these ribosomes remain capable of active amino acid incorporation into their constituent proteins. This process requires adenosine triphosphate, amino acid activating enzymes, and guanosine triphosphate. Thymus nuclear ribosomes are irreversibly inactivated by brief exposure to hypertonic sucrose solutions, either before or after extraction from the nucleus. While in the nucleus they are resistant to ribonuclease digestion, but after their isolation, ribonuclease attacks the particles and destroys their activity. The addition of deoxyribonucleic acid of diverse sources stimulates amino acid incorporation by isolated nuclear ribosomes, but other polyanions may be inhibitory. This role of deoxyribonucleic acid and the influence of position within the cell nucleus upon the metabolism of nuclear ribosomes is discussed.

Introduction:

Ribonucleoprotein particles of diverse composition and metabolic activity can be extracted from the isolated cell nucleus of calf thymus lymphocytes (1). That such ribosomes are of nuclear origin has been shown by electron microscopy, and by the demonstration that their metabolic activity within the nucleus satisfies three requirements for nuclear localization:

(a). The uptake of amino acids into the proteins of the ribosomal fractions is blocked if the nuclei are first treated with deoxyribonuclease (1, 2). (This test for DNA dependence , apart from indicating nuclear localization, also suggests the need for the nuclear phosphorylating system as a source of ATP (3).

(b). As long as the ribonucleoprotein particles remain within the nucleus their metabolism is insensitive to treatment with ribonuclease.

(c). Amino acid incorporation by RNP particles within the nucleus is stimulated by the addition of sodium ions to the medium. The sodium ion dependence of amino acid uptake has been described previously for intact nuclei (2) as well as for nuclear ribosomes (1). It reflects the need for sodium ions in the process of amino acid transport into the cell nucleus (4). This need for sodium in nuclear protein metabolism contrasts with the usual observation in cytoplasmic systems that amino acid incorporation specifically requires the presence of potassium ions (5).

When isolated nuclear ribosomes are properly supplemented and incubated in a nucleus-free system they remain capable of a progressive incorporation of amino acids into their constituent proteins (1). The experiments to be described in this report are concerned with the chemical requirements of this in vitro synthetic system, with its sensitivity to changes in the osmolarity of the incubation medium, with the effects of added nucleases, and with the results produced by adding natural or synthetic polyanions and polycations.

These studies in isolated nuclear ribosomes are compared and contrasted with the earlier results obtained in intact isolated nuclei, and in nuclear ribosomes in situ.

Materials and Methods:

Isolation of Nuclear Ribosome Fractions:

All isolation procedures were carried out at 2^o. Nuclei were isolated from calf thymus tissue in 0.25 M sucrose-0.003 M MgCl₂ as previously described (1) and freed of contaminating cytoplasmic fragments by two washings in an incubation medium of the following composition: 0.1875 M sucrose, 0.02 M glucose, 0.0285 M NaCl, 0.008 M MgCl₂, buffered with 0.025 M Tris-hydrochloric acid at pH 7.4.

The washed nuclei were then extracted with five volumes of 0.1 M Tris buffer containing 0.003 M MgCl₂ at pH 7.1. This 0.1 M Tris extract I was then centrifuged at 300 x g for 30 min. and the resulting nucleus-free supernate was centrifuged in the Spinco ultracentrifuge at 105,000 x g for 60 min. This procedure sediments the nuclear ribosome particles in the range 120-7000 S.

The resulting supernate was brought to Ph 5.2 with 1 M acetic acid, and the precipitate collected as nuclear pH 5 amino acid activating enzymes (6).

Incubation Procedures:

The nuclear ribosome pellets were resuspended (by syringing) in a final volume of 2.0 ml of the incubation medium described above, to which was added 0.20 mg pyruvate kinase, 20 umoles of sodium phosphenolpyruvate, 2.0 umoles ATP (sodium salt), 1.0 umole guanosine triphosphate (sodium salt), DL-[1-¹⁴C]leucine (2 uC/0.05 mg), and a measured amount of the nuclear pH 5 enzyme fraction. Incubations were carried out aerobically at 37^o with shaking.

Measurement of Protein, RNA, DNA and Radioactivity:

After incubation, cold trichloroacetic acid was added to the nuclear suspensions to bring the final concentration to 5 % TCA. The nucleic acids were determined in the TCA hydrolyzates obtained after heating for 30 min at 90^o. DNA was measured by the diphenylamine reaction (7), and RNA by the p-bromophenylhydrazine reaction (8). The protein content was determined as previously described (2). The radioactivity of the protein in the TCA precipitate was measured after removal of the nucleic acids and lipids (2), using a thin-window, gas-flow counter and correcting the counts for self-absorption (2).

Results:

Requirements for AminoAcid Incorporation by Isolated nuclear Ribosomes:

When isolated nuclear ribosomes are incubated in a nucleus-free systemthey are found to be capable of an active and sustained incorporation of DL-[1-¹⁴C]leucine into the proteins of the isolated particles (Fig. 1):

Fig. 1. Incorporation of [1-¹⁴C]leucine into the proteins of isolated nuclear ribosomes in a nuclear-free system. The nuclear ribosomes (120-7000 S) and nuclear pH 5 enzymes (<120 S) were prepared as described in Materials and Methods. The complete incubation system consisted of nuclear ribosomes (5.5 mg protein, 1.23 mg RNA, < 0.04 mg DNA), pH 5 enzymes (0.96 mg protein, 0.04 mg RNA, <0.001 mg DNA), 20 umoles sodium phosphoenolpyruvate, 2.0 umoles sodium ATP, 1.0 umole sodium GTP, and 0.20 mg pyruvate kinase. 0.05 mg DL-[1-¹⁴C]leucine (2.0 uC) was added at time zero. Final volume of incubation medium = 2.0 ml. The incubation was carried out aerobically at 37^o, with shaking.

Complete system: Isolated RNP particles, 120-7000 S (5.5 mg protein, 1.23 mg RNA), nuclear pH 5 enzymes <120 S (0.96 mg protein, 0.04 mg RNA), 0.20 mg pyruvate kinase, 20 umoles sodium phosphenolpyruvate, 2.0 umoles sodium ATP, 1.0 umole sodium GTP, 1.0 umoleCTP, and DL[1-¹⁴C]leucine (2 uC/0.05 mg) in 2.0 ml incubation medium. Incubated for 60 min at 37^o.

In this isolated sub-nuclear system, replacement of half of the Na⁺ by an equivalent amount of K⁺ produced no change in the rate of leucine incorporation. Such indifference to the type of univalent cation present contrasts to the requirement for sodium ion displayed by the intact isolated nucleus (2) and its contained nuclear ribosomes (1) in incorporating amino acids into their constituent proteins. The difference in behavior is due to the fact that sodium is primarily involved as a cofactor in the process of transporting amino acids into the nucleus (4). In the isolated subnuclear particulates, access of the amino acid to the site of synthesis apparently does not involve a similar enzymic transport mechanism.

In connection with the requirements for amino acid incorporation by isolated nuclear ribosomes it should be mentioned that it is not necessary to add a soluble-RNA as amino acid carrier; the transfer-RNA of the isolated thymus nucleus (6) does not occur in a soluble form but instead remains associated with the ribosome fraction.

Loss of Function in hypertonic media:

If nuclear ribosomes are exposed briefly to hypertonic solutions of sucrose (e.g. 1.9 M sucrose - 0.003 M MgCl₂for 15 min) and then returned to the usual isotonic incubation medium, they are unable to display the usual incorporation of amino acids into their constituent proteins (Fig. 2):

Fig. 2. Effect of brief exposure of isolated nuclear ribosomes to hypertonic sucrose solution. An aliquot of nuclear ribosomes (120 - 7000 S) prepared in isotonic sucrose solution was suspended in 10 volumes of 1.9 M sucrose - 0.003 M MgCl₂ solution for 15 min before being returned to the usual isotonic incubation medium described in the legend for Fig. 1.

Sensitivity to Incubation with Nucleases:

Isolated thymus nuclear ribosomes contain a high concentration of RNA and only trace amounts of DNA (1). If they are incubated in the presence of pancreatic ribonuclease there is a progressive decline in their RNA content and one observes a corresponding inhibition of amino acid incorporation into their constituent proteins (Fig. 3):

Fig. 3. Effect of RNase on RNA content and [1-¹⁴C]leucine uptake by isolated nuclear ribosomes in a nucleus-free system. The incubation system is as described in the legend for Fig. 1. In this experiment the nuclear ribosomes comprised 3.4 mg protein, 0.93 mg RNA and <0.04 mg DNA, and the nuclear pH 5 enzymes comprised 6.8 mg protein, 0.23 mg RNA, <0.001 mg DNA. The RNase concentration was 0.5 mg/ml. 

By contrast, the incubation of isolated nuclear ribosomes with pancreatic DNase produces no effect on the rate of amino acid uptake. Yet DNase incubation of whole nuclei results in a marked inhibition of amino acid incorporation into the proteins of the nuclear ribosome fraction (1). This apparent contradiction is readily explained in terms of the energy requirements of the two systems. In both the intact nucleus and in the isolated ribosomes, the process of amino acid incorporation involves the participation of ATP. But in the whole nucleus, ATP synthesis requires the presence of DNA, and treatment with DNase impairs the operation of the nuclear aerobic phosphorylating system (3). As a result, the ATP concentration falls off and amino acid activation comes to a halt. However, in the isolated ribosome system, the energy source is externally supplied in the form of ATP plus an ATP-generating system. Under these conditions, the amino acid uptake need not display an obvious DNase sensitivity.

Effects of Added Polyanions and Polycations:

Addition of calf thymus DNA (prepared as sodium deoxyribonucleate (13)) to the isolated ribosomes of the thymus nucleus, consistently stimulates the incorporation of DL-[1-¹⁴C]leucine into their constituent proteins. As shown in Fig. 4, a DNA supplement may give incorporations up to 50% above "control" values.

Fig. 4. Effects of added DNA, DNA + DNase, and polyethylene sulfonate on [1-¹⁴C]leucine incorporation into the proteins of isolated nuclear ribosomes in a nucleus-free system. The incubation system is that described in the legend for Fig. 1. The DNA was added as the sodium salt to give a final concentration of 1.0 mg/ml.

It has been shown previously that intact isolated nuclei of calf thymus lymphocytes are capable of a rapid incorporation of radioactive amino acids into the proteins of the nucleus and its contained ribosomes (1,2). Preincubation of such intact nuclei with deoxyribonuclease markedly reduces such incorporation, but amino acid uptake can be restored by the subsequent adition of DNA or other polyanions such as RNA, polyethylene sulfonate, chondroitan sulfate, or polyacrylic acid (14). The importance of negative charge in the substituting molecule is shown by a comparison of the effects of polyacrylic acid and its neutral derivative, polyacrylamide. Only the acid restores amino acid uptake to DNA-depleted nuclei; the uncharged amide has no effect. On the other hand, amino acid incorporation is antagonized by the addition of polycationic molecules such as histone or polylysine (14, 15).

These experiments show that polyanions, by virtue of their negative electrical charge, have a general capacity to restore amino acid uptake in isolated cell nuclei after incubation with DNase. It is likely that this is largely a secondary effect following the restoration of nuclear ATP synthesis by polyanions (3).

In isolated nuclear ribosomes, however, a DNA supplement appears to play a more specific role, because not all polyanions resemble DNA in its stimulatory effect on isolated nuclear ribosome metabolism. Thus, polyethylene sulfonate is inhibitory rather than stimulatory when added to suspensions of isolated nuclear ribosomes (Fig. 4) and in this respect resembles the effect of a natural polycation such as the arginine-rich histone of the thymus nucleus, which is also inhibitory to amino acid incorporation into ribosome proteins (Fig. 5).

Fig. 5. Effects of added DNA, histone, and DNA + histone on [1-¹⁴C]leucine incorporation into the proteins of isolated nuclear ribosomes in a nucleus-free system. The incubation system is that described in the legend for Fig. 1. 

The effect of various preparations of RNA and of other synthetic polyanions is under study.

Discussion:

Nuclear ribosomes are a site of intense protein and RNA metabolism while within the cell nucleus (1), and they remain capable of incorporating amino acids into their proteins following isolation from the nucleus. A comparison of the characteristics of such protein metabolism in nuclear ribosomes before and after extraction from the cell nucleus may eventually permit an analysis of the special influences exerted upon the nuclear ribosomes by virtue of their location within various regions of the cell nucleus. Thus, it has been suggested that those nuclear ribosomes derived from the nucleolus display a far more active RNA metabolism than do those derived from non-nucleolar regions of the cell nucleus (1). It is not yet known whether such differences in metabolism persist after removal of the ribosomes from the cell nucleus, that is, whether such differences are intrinsic to the particular particle or are primarily a function of the micro-environment of the particle.

A number of synthetic processes in intact isolated nuclei and their contained ribosomes are irreversibly inactivated by brief exposure to hypertonic sucrose solutions, and such irreversible inactivation is similarly displayed by isolated nuclear ribosomes exposed briefly to hypertonic sucrose. By contrast, isolated nuclear ribosomes do not display the requirement for a sodium environment, nor are they resistant to ribonuclease digestion, as is typical of ribosomes within the nucleus. It may be inferred that the requirement for sodium and the resistance to the action of ribonuclease are functions of the intranuclear position of the ribosomes rather than an intrinsic property of the nuclear ribosomes themselves. It has already been pointed out that the sodium requirement reflects the operation of a transport system bringing amino acids into the nucleus.

The role of DNA and synthetic polyanions in restoring amino acid uptake by DNase-treated nuclei can be viewed as a secondary aspect of restored ATP synthesis. This restoration of nuclear phosphorylation is a non-specific effect; it requires only the presence of a molecule with many repeating negative charges (3). This is not the case with respect to the stimulation of amino acid uptake by isolated nuclear ribosomes. Here DNA plays a more specific role, and polyanions such as polyethylene sulfonate are actually inhibitory to the process. This stimulation of protein synthesis in the ribosomes by DNA and the corresponding inhibition by histones suggests that the synthetic activity of ribosomes within the nucleus will vary depending on their proximity to DNA or histone at different loci of the chromosome. This may prove to be a physiological mechanism for the control of the rates of synthesis of different nuclear proteins.

Acknowledgements:

The first author is a Post Doctoral Fellow of the American Cancer Society. This investigation was supported in part by a Grant (RG 4919) from the U.S. Public Health Service.

References:

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