Kikuo Ogata

Proposed Uniform Nomenclature for Mammalian Ribosomal Proteins

SummaryThe numbering systems for mammalian ribosomal proteins used in several laboratories have been correlated and a proposal for a standard system is presented.

Functional differences in protein synthesis between free and bound polysomes of rat liver

1. 1. Free polysomes and bound polysomes, prepared from rat liver were incubated with [3H]- and [14C]leucine, respectively, in a cell-free system. The labelled supernatant fractions were then combined and fractionated by DEAE-cellulose column chromatography1, followed by acrylamide gel electrophoresis. The serum albumin fraction was found to be preferentially synthesized by bound polysomes. 2. 2. From the supernatant fraction, doubly labelled with [14C]- and [3H]leucine in the cell-free system as described above, the serum albumin fraction and the catalase fraction were prepared by immunological methods i.e. by employing specific antisera against them, after the removal of unspecific proteins. It was found that bound polysomes synthesized serum albumin exclusively. It was further suggested that catalase was synthesized on both kinds of polysomes although the results were not conclusive, owing to the low incorporation of the amino acid into this enzyme fraction. 3. 3. The results of time-course experiments of the incorporation of [14C]leucine in vivo into the serum albumin fraction, which was prepared from the “nascent protein” fractions of both kinds of polysomes by the immunological methods described above, showed that the labelled albumin fraction was located only on bound polysomes and supported the indication of exclusive synthesis of this protein by bound polysomes. 4. 4. The DEAE-cellulose chromatography1 of the supernatant fraction, doubly-labelled by both kinds of polysomes in a cell-free system, showed a fraction (Fraction I) eluted by 15 mM NaH2PO4, which was more effectively synthesized by free polysomes.

Release of ribonucleoprotein particles containing rapidly labeled ribonucleic acid from rat liver nuclei: Effect of adenosine 5′-triphosphate and some properties of the particles

Abstract Nuclei labeled with [6-14C]orotic acid for 40 min in vivo were isolated from rat liver by the modified method of Chauveau et al.13. Release of rapidly labeled RNA from nuclei was studied by incubation with cytoplasmic ribonuclease inhibitor in vitro. 1. 1. Release of rapidly labeled RNA from isolated nuclei is dependent upon the following factors. (a) Temperature and duration of the incubation: The release is directly proportional to temperature in the range between 10 and 37° in the presence of 5 mM Mg2+ and 1 mM ATP. (b) The concentration of ATP in the reaction mixture: ATP specifically stimulates the release. GTP, CTP and UTP are less stimulative, and ADP and AMP have almost no effect on the release. (c) The concentrations of bivalent cations: All of the bivalent cations tested prevent the release. Alkaline earth metals, such as Mg2+, Ca2+ and Ba2+, react antagonistically to ATP. EDTA inhibits the release somewhat. (d) The pH of the incubation: The release of rapidly labeled RNA is directly proportional to pH in the range between pH 6.0 and 8.0. 2. 2. On incubation with 5 mM Mg2+ and 3 mM ATP at 20°, rapidly labeled RNA is released in the form of ribonucleoprotein particles having a predominant s value of 45 S. 3. 3. The particles have a low RNA/protein ratio (1:5.6) and a low buoyant density in CsCl (1.40). They are very sensitive to ribonuclease, but quite insensitive to deoxyribonuclease, deoxycholate treatment and EDTA treatment. 4. 4. The specific activity of the released RNA (incubation at 20° for 5 min) labeled with [14C]orotic acid for 40 min in vivo is 3.3 times higher than that of RNA from cytoplasmic 45-S particles. Total nuclear RNA has the highest specific activity, which is 5 times higher than that of released RNA. 5. 5. Analyses of base composition of RNA released from nuclei by ATP show a high A+U G+C ratio, both by the ultraviolet absorption method (1.05) and by the radioactivity of 32P-short labeling (1.17). Sedimentation profiles of rapidly labeled RNA in the released fraction show the main peak at 14 S with a wide distribution from 6 to 28 S. 6. 6. The mechanism of the release of rapidly labeled RNA from nuclei by ATP is discussed. On the basis of a comparison of the 45-S ribonucleoprotein particles released from nuclei by ATP with other nuclear ribonucleoprotein particles and cytoplasmic 45-S particles, it is concluded that 45-S ribonucleoprotein particles released by ATP constitute the main form in which messenger RNA appears during its transportation from the nucleus to the cytoplasm.

Differences between the protein moieties of active subunits and EDTA-treated subunits of rat liver ribosomes with specific references to a 5 S rRNA - protein complex.

When active 40 S subunits of rat liver ribosomes were treated with EDTA, the conversion of 40 S subunits to 30 S subunits occurred with partial release of 13 kinds of proteins out of 29 kinds of 40 S proteins. Buy contrast, 60 S subunits completely lost one protein (L3) by EDTA-treatment with concomitant release of the fraction sedimenting at about 7 S (7 S fraction). It was found that the 7 S fraction contained 5 S ribosomal RNA as well as L3 protein having a molecular weight of 38,000. Buoyant density in CsCl of the 7 S fraction was 1.60 g/cm3, suggesting that this fraction consisted of RNA and protein at an approximately equal ratio on a weight basis. These findings, taken together with the molecular weights of 5 S rRNA (40,000) and L3 protein, may indicate that the 5 S ribosomal RNA - protein complex from rat liver 60 S subunits consists of one molecule of 5S ribosomal RNA and one molecule of L3 protein.

Isolation of serum albumin-synthesizing polysomes from rat liver.

Abstract The procedures for the purification of rat liver polysomes synthesizing serum albumin was developed, employing the quantitative precipitin method with rat serum albumin as a carrier and its antibody, and ribonuclease inhibitor from rat liver. The addition of ribonuclease inhibitor to polysomes during the incubation with antibody was found to prevent their degradation. Under these conditions, about 12 % of the membrane-bound polysomes of rat liver was found in the specific precipitate of serum albumin and its antibody, while a negligible amount of free polysomes was precipitated. It is concluded that polysomes synthesizing serum albumin are isolated by this method.

THE INCORPORATION OF [14C]GLYCINE INTO THE PROTEIN OF GUINEA PIG BRAIN CORTEX SLICES

WITHIN recent years circumstantial evidence has accumulated in favour of the view that brain tissue is very inactive in protein metabolism; it was observed by several workers that the in v i m incorporation of [14C]amino acid into brain protein after the intravenous or intraperitoneal injection of labelled amino acid was very low compared with that into other tissue protein (SPRINSON and RITTENBERG, 1949; TARVER and MORSE, 1948). However, GAITONDE and RICHTER (1956) found that after intracisternal administration, labelled amino acids were incorporated rapidly into brain protein and indicated that the low incorporation observed in the previous experiments was due to the low permeability of the blood-brain barrier to amino acids. LAJTHA, FURST, GERSTEIN and WAELSCH (1957) used intravenous administration of [14C]lysine and arrived at a fairly high value for the cerebral protein turnover rate by measuring the specific activities of free as well as protein bound amino acids in the brain. Furthermore, GREENBERG et al. (1948) reported that the incorporation of [14C]glycine into protein by brain homogenate was of the same order as that by liver homogenate. Our recent experiments indicated that [14C]leucine was incorporated into the proteins of the guinea pig cerebral cortex and liver in a cell-free system (SATAKE, MASE, TAKAHASHI and OGATA, 1960). In order to obtain some characteristic of protein synthesis in brain tissues, the [14C]glycine uptake into protein by brain slices was observed under various conditions, and comparison was made between incorporation of radioactivity by brain slices and that by liver slices. A preliminary report of some of these experiments has appeared (OGATA, TAKAHASHI and MASE, 1957).

Transportation of messenger RNA from nuclei to polysomes in rat liver cells

Abstract For the purpose of studying the transportation of messenger RNA (mRNA) in eucaryotic cells, rat liver nuclei containing rapidly labeled RNA and rapidly labeled proteins, and the unlabeled postmitochondrial fraction (S12 fraction) were incubated with ATP, GTP and an energy-generating system. The transfer of rapidly labeled nRNA and rapidly labeled nuclear proteins from nuclei to polysomes in this reaction mixture was investigated and the following results were obtained: 1. 1. The incorporation of rapidly labeled nRNA and rapidly labeled nuclear proteins into the polysomal fraction increased in parallel for 30 min. This incorporation was confirmed by sucrose density gradient centrifugation of the deoxycholate-treated S12 fraction after incubation. 2. 2. Their incorporation into the polysomal fraction was dependent upon ATP, GTP and an energy-generating system. 3. 3. CsCl buoyant density gradient centrifugation of polysomes prepared from the reconstituted system after incubation, suggested that rapidly labeled nRNA and rapidly labeled nuclear proteins were incorporated into polysomes in the form of a ribonucleoprotein complex, about half of which could be separated from ribosomes by EDTA treatment to the lighter region with a peak at α = 1.41. 4. 4. Sedimentation analysis and the base composition of rapidly labeled nRNA incorporated into polysomes during incubation suggested that a greater part of the radioactivity incorporated into polysomes was messenger-type RNA distributed heterogeneously in the region from 4 to 30 S. 5. 5. From the results on the time course of rapidly labeled nRNA/rapidly labeled nuclear proteins ratios in the polysomal fraction, it was suggested that rapidly labeled nuclear proteins may stay in the polysomes during the translation of newly supplied mRNA. These experimental results may lend support to the hypothesis concerning the transportation of mRNA in eucaryotic cells which proposes that mRNA will be transported from nucleus to cytoplasm in the form of a mRNA-informofer complex and will be incorporated into polysomes to manifest its genetic information.

Preparation and some properties of active subunits from rat liver ribosomes

Abstract Rat liver ribosomes were dissociated to 60S and 40S fractions by incubation with 1 M KCl at 37° for 2 h, and separated by subsequent density-gradient centrifugation. While the 40S fraction contained 40S subunits only, the 60S fraction consisted of 60S subunits as a major fraction and dimers of 40S subunits, which could be removed by a 2nd sucrose gradient centrifugation in Medium IV containing 3 mM Mg ++ . The pure 60S and 40S subunits thus prepared could be reassociated to 80S particles which were active in poly U-dependent polyphenylalanine synthesis. 3 H-poly U interacted only with the smaller subunits.

The role of ATP in the transport of rapidly-labeled RNA from isolated nuclei of rat liver in vitro.

To understand the mechanism of the action of ATP on the in vitro transport of the rapidly-labeled RNA from isolated nuclei, the fate of ATP during the incubation as well as the effect of ATP, its analogues and other ribonucleoside triphosphates on the transport was examined and the following results were obtained. (1) More than 97% of added ATP remained acid soluble. No polyadenylation of the rapidly-labeled RNA in the released fraction by added ATP occurred although new polyadenylate segments smaller than 10 S were synthesized. (2) The addition of an ATP-generating system to the reaction mixture restored the initial rate of the release of the rapidly-labeled RNA from isolated nuclei. (3) Among the ribonucleoside triphosphates tested, ATP was most effective in stimulating the release. GTP was about 2/3 as effective as ATP. UTP showed some effect, but CTP showed no effect. EDTA was also non-effective. (4) When no ATP-generating system was added to the reaction mixture, AMP failed to mimic the effect of ATP. However, the combination of AMP and pyrophosphate could take the place of ATP. (5) Both AMP-CPP and AMP-PCP, the ATP analogues, showed the equal degree of their effect on the release, regardless of the position of the methylene bond. From these results, the principal role of ATP in the in vitro transport systems seemed to be its interaction with isolated nuclei to dissociate a structure which retains the rapidly-labeled RNA in the nucleus.

Messenger ribonucleoprotein complexes released from rat liver nuclei by ATP: I. Characterization of the RNA moiety of messenger ribonucleoprotein complexes

Abstract The chemical and metabolic properties of the RNA moiety of the 45-S component which can be released from rat liver nuclei by ATP were investigated. 1. 1. Disc electrophoresis of RNA from the 45-S component labeled with [ 14 C]-orotic acid in vivo for 40 min revealed that it contained rapidly labeled RNA ranging heterogeneously from 18 S to 6-4 S as the main component. 2. 2. RNA from the 45-S component showed a high stimulatory activity for polypeptide synthesis in an Escherichia coli cell-free system. 3. 3. Base composition analysis of RNA from the 45-S component showed a high A+U G+C ratio, as obtained both by the ultraviolet absorption method (1.17) and by the measurement of radioactivity after 32 P short-labeling (1.18). From these results it is concluded the 45-S component contains mRNA as a major component.