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We have developed a computational method of protein design to detect amino acid sequences that are adaptable to given main-chain coordinates of a protein. In this method, the selection of amino acid types employs a Metropolis Monte Carlo method with a scoring function in conjunction with the approximation of free energies computed from 3D structures. To compute the scoring function, a side-chain prediction using another Metropolis Monte Carlo method was performed to select structurally suitable side-chain conformations from a side-chain library. In total, two layers of Monte Carlo procedures were performed, first to select amino acid types (1st layer Monte Carlo) and then to predict side-chain conformations (2nd layers Monte Carlo). We applied this method to sequence design for the entire sequence on the SH3 domain, Protein G, and BPTI. The predicted sequences were similar to those of the wild-type proteins. We compared the results of the predictions with and without the 2nd layer Monte Carlo method. The results revealed that the two-layer Monte Carlo method produced better sequence similarity to the wild-type proteins than the one-layer method. Finally, we applied this method to neuraminidase of influenza virus. The results were consistent with the sequences identified from the isolated viruses.

(1) Escherichia coli 70S ribosomes showed intrinsic ATPase and GTPase activities, although they were much lower than those of rat liver ribosomes. The latter activity was higher than the former one. (2) The ATPase activity was inhibited by GTP and GMP-P(NH)P, and the GTPase activity was inhibited by ATP and AMP-P(NH)P, indicating a close relationship between the two enzymes. (3) Elongation components alone or in combination enhanced the ATPase activity, indicating the possible correlation of ribosomal ATPase with elongational components. (4) Vanadate at the concentrations that did not inhibit the GTPase activities of EF-Tu and EF-G, depressed the poly(U)-dependent polyphe synthesis, suggesting that ribosomal ATPase (GTPase) participates in peptide elongation by inducing positive conformational changes of ribosomes required for the attachment of elongational components.

Treatment of 30S-5SRNP with 1 M Cs(2)SO(4) at 2 degrees C overnight followed by sucrose density-gradient centrifugation yielded particles smaller than 30S-5SRNP, designated as CsS-particles. CsCl density-gradient centrifugation of CsS-particles showed the homogeneity of the particles containing about half the amount of proteins in 30S-5SRNP particles. The particles contained 18SrRNA, 5SRNP and about half the number of proteins in 30S-5SRNP. The ATPase activity of freshly prepared CsS-particles was about half the original 30S-5SRNP level although it was unstable even at 2 degrees C. Poly(U) slightly enhanced the activity, and phe-tRNA(phe) stimulated it concentration-dependently. EF-1a alone enhanced it, and in combination with poly(U) and phe-tRNA(phe) stimulated it markedly. EF-2 alone markedly increased it. The activity with the full components for elongation described above became very high, being comparable to that of the original 30S-5SRNP and twice that of 40S subunits. A two-dimensional electrophoretogram of the protein in CsS-particles revealed 9 small subunit protein species, in addition to L5, which included proteins interacting with mRNA and two elongation factors. Taken together with the results of our preceding study indicating the participation of ATPase of 80S ribosomes in peptide elongation, the present results indicate CsS-particles may be a part of the ATPase centre of 80S ribosomes.

The DNA-binding domain of c-Myb consists of three imperfect tandem repeats, R1, R2, and R3. Each repeat contains three helices. The minimal DNA-binding domain is an R2R3 fragment. Here, we have examined the backbone dynamics of R2R3 in its DNA-bound form by NMR. Upon binding to DNA, the N- and C-termini, and the linker between R2 and R3 become less flexible. In the free form the third helix of R2 exhibits slow conformational exchange fluctuations owing to a cavity in the hydrophobic core of R2. Upon binding to DNA, the conformational exchange contributions in R2 are reduced but remain significant in NMR relaxation measurements. Upon binding to DNA, the third helix of R3 comes to exhibit significant chemical exchange contributions. These findings suggest that the orientations of the third helices of both R2 and R3 as to DNA are being chemically exchanged. In the DNA-bound form both R2 and R3 exhibit similar dynamical characters, except for amino acids Trp 95, Thr 96, and Val 103 of R2, which are located around the cavity of the unbound form. Upon binding to DNA, since Trp 95 moves into the cavity to fill it up, the local conformational exchange contributions seem to be still observable around the filled cavity.

The properties and role in peptide elongation of ATPase intrinsic to rat liver ribosomes were investigated. (i) Rat liver 80S ribosomes showed high ATPase and GTPase activities, whereas the GTPase activity of EF-1alpha and EF-2 was very low. mRNA, aminoacyl-tRNA, and elongation factors alone enhanced ribosomal ATPase activity and in combination stimulated it additively or synergistically. The results suggest that these translational components induce positive conformational changes of 80S ribosomes by binding to different regions of ribosomes. Translation inhibitors, tetracyclin and fusidic acid, inhibited ribosomal ATPase with or without elongational components. (ii) Two ATPase inhibitors, AMP-P(NH)P and vanadate, did not inhibit GTPase activities of EF-1alpha and EF-2 assayed as uncoupled GTPase, but they did inhibit poly(U)-dependent polyphe synthesis of 80S ribosomes. (iii) Effects of AMP-P(NH)P and ATP on poly(U)-dependent polyphe synthesis at various concentrations of GTP were examined. ATP enhanced the activity of polyphe synthesis even at high concentrations of GTP, suggesting a specific role of ATP. At low concentrations of GTP, the extent of inhibition by AMP-P(NH)P was very low, probably owing to the prevention of the reduction of the GTP concentration. (iv) Vanadate inhibited the translocation reaction by high KCl-washed polysomes. These findings together indicate that ribosomal ATPase participates in peptide translation by inducing positive conformational changes of mammalian ribosomes, in addition to its role of chasing tRNA from the E site.

To elucidate the roles of conserved Asp residues of Bacillus cereus sphingomyelinase (SMase) in the kinetic and binding properties of the enzyme toward various substrates and Mg2+, the kinetic data on mutant SMases (D126G and D156G) were compared with those of wild type (WT) enzyme. The stereoselectivity of the enzyme in the hydrolysis of monodispersed short-chain sphingomyelin (SM) analogs and the binding of Mg2+ to the enzyme were not affected by the replacement of Asp126 or Asp156. The pH-dependence curves of kinetic parameters (1/Km and kcat) for D156G-catalyzed hydrolysis of micellar SM mixed with Triton X-100 (1:10) and of micellar 2-hexadecanoylamino-4-nitrophenylphosphocholine (HNP) were similar in shape to those for WT enzyme-catalyzed hydrolysis. On the other hand, the curves for D126G lacked the transition observed for D156G and WT enzymes. Comparison of the values and the shape of pH-dependence curves of kinetic parameters indicated that Asp126 of WT SMase enhances the enzyme's catalytic activity toward both substrates and its binding of HNP but not SM. The deprotonation of Asp126 enhances the substrate binding and slightly suppresses the catalytic activity toward both substrates. Asp156 of WT SMase acts to decrease the binding of both substrates and the catalytic activity to HNP but not SM. From the present study and the predicted three-dimensional structure of B. cereus SMase, Asp126 was thought to be located close to the active site, and its ionization was shown to affect the catalytic activity and substrate binding.

The modes of Mg2+ binding to SMase from Bacillus cereus were studied on the basis of the changes in the tryptophyl fluorescence intensity. This enzyme was shown to possess at least two binding sites for Mg2+ with low and high affinities. The effects of Mg2+ binding on the enzymatic activity and structural stability of the enzyme molecule were also studied. The results indicated that the binding of Mg2+ to the low-affinity site was essential for the catalysis, but was independent of the substrate binding to the enzyme. It was also indicated that the alkaline denaturation of the enzyme was partly prevented by the Mg2+ binding, whereas no significant protective effect was observed against the denaturation by urea. The pH dependence of the kinetic parameters for the hydrolysis of micellar HNP and mixed micellar SM with Triton X-100 (1:10), catalyzed by SMase from B. cereus, was studied in the presence of a large amount of Mg2+ to saturate both the low- and high-affinity sites. The pH dependence curves of the logarithm of 1/Km for these two kinds of substrates were similar in shape to each other, and showed a single transition. On the other hand, the shapes of the pH dependence curves of the logarithm of kcat for these two kinds of substrates were different from each other. The pH dependence curve for micellar HNP showed three transitions and, counting from the acidic end of the pH region, the first and third transitions having tangent lines with slopes of +1 and -1, respectively. On the other hand, the curve for mixed micellar SM with Triton X-100 showed one large transition with a slope of +1 (the first transition) and a very small transition (the third transition). On the basis of the present results and the three-dimensional structure of bovine pancreatic DNase I, which has a primary structure similar to that of B. cereus SMase, we proposed a catalytic mechanism for B. cereus SMase based on general-base catalysis.

The ATPase activity of rat liver 30S-5SRNP particles prepared by EDTA treatment of 80S ribosomes, and that of 40S subunits were investigated in correlation with polypeptide elongation. The ATPase activity of 30S-5SRNP particles was higher than that of 40S subunits. Poly(U) and TMV RNA stimulated the ATPase activity of 30S-5SRNP particles more markedly than that of 40S subunits. These two kinds of particles also showed intrinsic GTPase. Poly(U) enhanced the GTPase activity of 30S-5SRNP particles but not that of 40S subunits. An elongation factor (EF-1alpha, EF-2, or EF-1alphabetagamma) alone or in combination with poly(U) and/or other elongation factors stimulated the ATPase activities of both particles. The extent of stimulation of the ATPase activity by a combination of these components was usually somewhat higher than or similar to the sum of those with the individual components. The extents of stimulation by these components were higher in the case of 30S-5SRNP particles than that of 40S subunits, indicating the importance of the 5SRNP moiety in the former particles. The intactness of 18SrRNA was required for promotion of the ATPase activity of 30S-5SRNP particles by Phe(+), (-)tRNA(Phe). The ATPase activities of the two kinds of particles by themselves or those observed with the combinations of the components mentioned above were inhibited by several kinds of translation inhibitors. The degrees of inhibition were generally higher for 30S-5SRNP particles. The ATPase activity of 40S subunits was enhanced by spermidine, suggesting the importance of the conformational change induced by it. These results imply the participation of the intrinsic ATPase of 30S-5SRNP particles and 40S subunits in polypeptide elongation, and the important role of the 5SRNP moiety of 30S-5SRNP particles in the ATPase activity.

To obtain direct evidence for the attachment of 5SrRNA-ribosomal L5 protein particles (5SRNP) and methionine-tRNA (tRNA(met)) to methionyl-tRNA synthetase (MetRS) in the macromolecular aminoacyl-tRNA synthetase (ARS) complex of rat liver, a MetRS-5SRNP-tRNA(met) complex was dissociated from the macromolecular ARS complex fraction by n-octyl-beta-D-glucoside (Method I) or by omega-aminooctyl agarose (Method II) chromatography. The dissociated MetRS complex fraction was purified by gel filtration followed by tRNA-Sepharose chromatography using partially purified tRNA(met) in Method I, and by hydrophobic interaction chromatography in Method II. In both methods, final Superdex200 chromatography showed that MetRS activity was present in the region corresponding to the molecular weight of the MetRS-5SRNP-tRNA(met) complex (M(r) 200,000). One main protein band corresponding to the molecular weight of MetRS was observed on SDS-PAGE of the final product, which was concentrated by lyophilizing after dialysis against water. Using serum albumin as an inhibitor of adhesion of L5 to the microconcentrators which was used to concentrate the final product, a distinct L5 band was detected on SDS-PAGE, the intensity of which was comparable to that of the MetRS band. Northern blot analysis of RNA prepared from the tRNA-Sepharose fraction showed the presence of 5SrRNA. Dot blot analysis using an antibody against ribosomal protein L5 showed that L5 was present in the Superdex200 fractions prepared by both methods. The MetRS specific activities in MetRS complex fractions incubated without tRNA increased during the purification procedures, indicating that endogeneous tRNA(met) exists stably in the MetRS complex. 5SRNP and 5SrRNA markedly enhanced the MetRS activity in the MetRS complex, indicating that 5SRNP(A) plays a role as a positive effector of MetRS.

Rat liver cytosol was incubated with a trace amount of rat liver 5SrRNA which was highly labeled at the 3'-end with cytidine 3',5'-[5'-32P]biphosphate, and with [35S]methionine in the presence of ATP mixture, and then with an antibody against ribosomal protein L5. The mixture was analyzed by protein A-Sepharose chromatography. The following results were obtained. (i) The eluate with glycine-HCl buffer (pH 3.0) from the protein A-Sepharose column contained an overlapping peak of 32P- and 35S-radioactivities. In a control experiment using the same amount of 32P-labeled Escherichia coli 5SrRNA with the same specific activity, no fraction of the eluate contained 32P-radioactivity. (ii) The fractions containing both 32P- and 35S-radioactivities from the protein A-Sepharose column were crosslinked by UV irradiation. The products was subjected to PAGE, and RNA in each gel slice was eluted and purified. The fraction containing both 32P- and 35S-radioactivities was present in a region of somewhat higher molecular weight than that of 5SRNP, whereas very low 32P- and 35S-radioactivities were present in this region in the control experiment without UV irradiation. This finding suggested that [35S]methionyl-tRNA interacted with 32P-labeled 5SRNP. (iii) The fraction containing overlapping 32P- and 35S-radioactivities described above was subjected to Sephadex G-150 chromatography. The component containing both radioactivities was distributed in the region corresponding to molecular weights of 10,000 to 250,000 with a peak at about 200,000, suggesting the presence of a complex containing Met-RS (Mr 108,000), 5SRNP (Mr 74,000), and methionyl-tRNA (Mr 25,000). Furthermore, this fraction showed definite Met-RS activity.(ABSTRACT TRUNCATED AT 250 WORDS)

1) Rat liver 5SrRNA enhanced the activity of methionyl-tRNA synthetase in the macromolecular aminoacyl-tRNA synthetase complex (Fraction B) purified from a rat liver supernatant. 5SrRNA-L5 protein complexes (5SrRNP) had similar effects, whereas other ribosomal RNAs and E. coli 5SrRNA had no effect. 2) 5SrRNA increased the activity of the complex for methionine-dependent ATP-PPi exchange. 3) 5SrRNA increased the activities of methionyl-, arginyl-, and isoleucyl-tRNA synthetases in the complex, but scarcely affected its leucyl-, lysyl-, and glutamyl-tRNA synthetase activities. 4) 5SrRNA increased the activities of the rat liver supernatant for the attachment of [35S]methionine, [3H]isoleucine, [3H]lysine, [3H]proline, [3H]threonine, [3H]tyrosine, and [3H]phenylalanine to endogenous tRNA markedly, and those for [3H]leucine, [3H]arginine, [3H]aspartic acid, and [3H]histidine slightly, but did not affect those for [3H]glutamic acid, [3H]glycine, [3H]valine, [3H]alanine, and [3H]tryptophan. 5) Preincubation of the rat liver supernatant with an antibody against Artemia salina ribosomal protein L5, that cross-reacted with the rat liver ribosomal protein L5, decreased the attachment of [35S]methionine and [3H]isoleucine to endogenous tRNA, and 5SrRNA and 5SRNP enhanced these activities of the supernatant preincubated with antibody. On the other hand, the antibody did not affect that for [3H]alanine. Immune dot blot analysis using the antibody against L5 showed the presence of immunologically the same protein as L5 in the liver supernatant. Northern blot analysis of RNA in the immunoprecipitate prepared from the liver supernatant incubated with the antibody against L5 indicated that 5SrRNA was complexed with L5.(ABSTRACT TRUNCATED AT 250 WORDS)

The 5SrRNA in the rat liver postmicrosomal supernatant was investigated. Acrylamide gel electrophoresis and Northern blot analysis showed that most of the 5SrRNA was present in the fractions obtained on high molecular weight regions separated by Sephadex G-200 column chromatography of the supernatant, which contained the bulk of the methionyl-tRNA synthetase (Fraction I) and tyrosyl-tRNA synthetase (Fraction II). A high molecular weight complex containing nine aminoacyl-tRNA synthetases [Mirande, M., LeCorre, D., & Waller, J.-P. (1985) Eur. J. Biochem. 147, 281-289] was purified by fractional precipitation with polyethylene glycol 6000, gel filtration on Bio-Gel A-1.5m, and finally tRNA-Sepharose column chromatography, which gave two fractions. Fraction B showed the activities of nine aminoacyl-tRNA synthetases and gave protein bands corresponding to eight previously identified enzymes on SDS-PAGE. Fraction A, eluted with a lower KCl concentration than Fraction B, showed lower activities than fraction B of eight of the aminoacyl-tRNA synthetases, the exception being prolyl-tRNA synthetase. The staining patterns with ethidium bromide of the RNAs after PAGE showed 5SrRNA bands for Fraction A but not for Fraction B. However, Northern blot analysis indicated that 5SrRNA was present in both Fractions A and B. The staining pattern after SDS-PAGE of Fraction A with Coomassie Brilliant Blue showed several protein bands in addition to those observed for Fraction B, one of which, with a staining intensity comparable with those of other bands, was located at the same position as ribosomal protein L5, which is the protein moiety of the 5SrRNA-L5 protein complex of ribosomal 60S subunits.(ABSTRACT TRUNCATED AT 250 WORDS)

1. When the postmitochondrial supernatant (PM-supernatant) from regenerating rat liver was incubated with [3H]methionine, the incorporation of [3H]methinoine into the N-terminal residues of nascent peptides on ribosomes was observed. This incorporation was sensitive to a low cocentration (2X10(-6) M) of pactamycin. The results suggest that PM-supernatant has low but definite activity for the initiation of nascent protein synthesis. Polysomes and cell sap from regenerating rat liver showed negligible pactamycin-sensitive incorporation of [3H]methionine into N-terminal, residues of nascent peptides. 2. PM-supernatant from regenerating rat liver was incubated with [35S]methionine in the complete reaction mixture. After addition of ribosomal proteins labelled with [3H]methionine in vivo, ribosomal structural proteins were prepared from the incubation mixture by acetic acid extraction, CM-cellulose column chromatography, Sephadex G-200 gel filtration and finally by two-dimensional acrylamide gel electrophoresis. Incorporation was observed in the greater part of ribosomal proteins on the two dimensional gel. From the 35S-to-3H ratios of ribsomal protein fractions during the purification procedures, it appeared that the incorporation of labelled methionine into the ribosomal proteins by PM-supernatant was about 3% of that into the total proteins. When [3H]leucine was used, the values were about 4% in the same cell-free system and 5 to 6% in in vivo labelling. The results indicate that ribosomal proteins are synthesized with high efficiency by PM-supernatant from regenerating rat liver.

After rat liver polysomes were irradiated with UV light at 254 nm for 2 h, a cross-linked poly(A)-containing mRNA-protein complex (mRNP) was prepared and a protein moiety was labeled with 125I. After RNase treatment, its protein moiety was analyzed by two-dimensional polyacrylamide gel electrophoresis followed by radioautography. There were radioactive spots which extended from the positions of those of S3/S3a, S6, L5, and L6/L7 towards the origin. In the case of UV irradiation for 30 min, radioactive spots extending similarly from those of S3/S3a, and L5 were observed. Radioactive areas on the two-dimensional gel in the case of irradiation for 2 h were further analyzed by SDS polyacrylamide gel electrophoresis. The peaks of radioactivity were detected at the protein band containing L6, that containing S3a and L5 and that containing S6, L7, and L8. It was proposed that S3a, S6, L5, and L6 proteins, according to the proposed uniform nomenclature (McConkey et al. (1979) Mol. Gen. Genet. 169, 1-6(1)), were cross-linked to mRNA by UV irradiation.

The long-term effects (up to 12 h) of low dose in vivo actinomycin D treatment, which selectively inhibits rRNA synthesis, on the activity of rat liver for the synthesis of ribosomal proteins relative to that for the synthesis of total protein were investigated. The effects of actinomycin D treatment in vivo and in vitro on the template activity of poly(A)-containing mRNA of rat liver for ribosomal proteins were examined by using a wheat germ cell-free system. The following results were obtained. 1. The activity of rat liver for synthesizing total protein observed in vivo and in vitro was inhibited by actinomycin D treatment even at a small dose. 2. A double-labeling technique using [3H] and [14C]leucine in vivo showed that the rate of synthesis of the ribosomal protein fraction relative to that of total protein in actinomycin-treated rat liver (6 + 6 h) was 1.45 times higher than that in the control rat. 3. By using a wheat germ cell-free system, it was shown that the template activity of poly(A)-containing mRNA for the synthesis of total protein was increased slightly by actinomycin D treatment in vivo. Furthermore, the template activity for the ribosomal protein fraction relative to that for total protein was increased. This increase was observed in most of the ribosomal proteins separated on two-dimensional acrylamide gel electrophoresis, although the extents of increase were different among individual ribosomal proteins examined. On the other hand, the selective increase of the template activity for the ribosomal protein fraction was not observed when poly(A)-containing mRNA was incubated with actinomycin D in vitro, although the template activity for total protein was increased slightly.

A cDNA clone specific for rat ribosomal protein S26 was isolated by a positive hybridization translation assay from a cDNA library made for 8-9S poly(A)mRNA from regenerating rat liver. The nucleotide sequence of the cDNA was determined. The sequence contains 23 base pairs in the 5' noncoding region, 345 base pairs in the protein coding region and 67 base pairs in the 3' noncoding region besides the poly(A) tail. The primary structure of the protein S26 was deduced from the nucleotide sequence. It consists of 115 amino acids. Its molecular weight is 13,015 and its pI is about 11.1. The calculated amino acid composition is consistent with the reported composition of S26. From the results of Southern blot analysis, the protein S26 appears to have multiple genes.

Protein-protein neighbors in rat liver 60S ribosomal subunits were investigated by using two bifunctional imidoesters; dimethyl suberimidate (DMS) and dimethyl 3,3'-dithiobispropionimidate (DTP). 1. Complexes cross-linked with DMS were separated by two-dimensional acrylamide/urea gel electrophoresis. Each complex in the gel was labeled with 125I (1) and was cleaved into the original monomeric protein constituents of ammonolysis. The products were analyzed by two-dimensional acrylamide/urea gel electrophoresis, followed by radioautography of the stained gel. Eight protein pairs are proposed according to our numbering system (2): L1-L3(L3-L5), L2-L21 (L4-L26), L5-L13 (L7/L7a-L15), L5-L34 L7/L7a-L36), L6-L34 (L8-L36), L6-L32 (L8-L35), L12-L32 (L13/L13a-L35), and L18-L27 (L18-L27/L27a). The designations according to the proposed uniform nomenclature (3) are given in parentheses. 2. Complexes cross-linked with DTP were analyzed by acrylamide/SDS diagonal gel electrophoresis (4), and the molecular weights of the complexes and their components were determined. The monomer components of cross-linked pairs were labeled with 125I in the gel, and identified by two-dimensional acrylamide/urea gel electrophoresis followed by radioautography. Six pairs are proposed; L1-L5 (L3-L7/L7a), L1-L6 (L3-L8). L5-L19 (L7-/L7a-L21/L23/L23a), L13-L15 (L15-L14), L13-L16 (L15-L19), and L23-L27 (L30-L27/L27a). 3. Two pairs which were formed by protein-protein interaction without the use of cross-linking reagents were identified as L2-L4 (L4-L6) and L4-L30 (L6-L29).

To examine whether serine proteases of rat liver chromatin are also involved in the degradation of newly synthesized and unbound ribosomal proteins and histones, like the nuclear thiol protease which we reported previously (Tsurugi, K. & Ogata, K. (1979) Eur. J. Biochem. 101, 205-213), in vivo experiments were carried out with serine protease inhibitor, PMSF. The following results were obtained. When normal rats received an intraperitoneal injection of PMSF (10 mg per 100 g body weight), nuclear serine proteases were inhibited almost completely for at least 90 min. PMSF did not affect the synthesis of proteins and RNAs of ribosomes and other subcellular fractions. The effects of PMSF treatment in vivo on the degradation of newly synthesized ribosomal proteins and histones in regenerating rat liver pretreated with a low dose of actinomycin D, which preferentially inhibited rRNA synthesis, were examined by using the double-isotope method. It was found that PMSF treatment did not affect their degradation. On the other hand, administration of E-64, a thiol protease inhibitor, to partially hepatectomized rats inhibited the degradation of those proteins markedly. From these results, it is concluded that the nuclear thiol protease, but not serine proteases, is preferentially involved in the degradation of newly synthesized ribosomal proteins and histones which are not associated with rRNA and DNA, respectively.

The chromatin fraction of rat liver exhibited proteolytic activity caused by serine proteases, with maximal activity at pH 8 or 10. They were analyzed by affinity labeling with [3H]diisopropylfluorophosphate followed by SDS-polyacrylamide gel electrophoresis, and partially purified by gel filtration through Sepharose 6B after selective extraction from the chromatin fraction. The following results were obtained. 1. The chromatin fraction contained three DFP-binding proteins with molecular weights of 52,000, 25,000, and 15,000 daltons, and they were tentatively designated proteins A, B, and C, respectively. Unlike proteins A and B, protein C reacted with DFP more strongly at pH 10 than at pH 8. A greater part of protein B was present in the nucleoli, while the others were predominantly distributed in extra-nucleolar chromatin. 2. Proteins A and B were extracted from the chromatin fraction by 5 M urea and 0.7 M NaCl, respectively; while protein C was not extractable by either solution. Proteins A and B were further purified by gel filtration through Sepharose 6B. 3. Protein B was a neutral protease with a maximal activity for 3H-labeled ribosomal proteins at pH 8 and showed high specificity for basic proteins, such as histone and ribosomal proteins. Protein A was an alkaline protease with a maximal activity at pH 10 and showed proteolytic activity not only for basic proteins but also for hydrophobic proteins, such as casein and non-histone proteins of chromatin. 4. Protein A was activated at pH 8 by high concentrations of NaCl, suggesting the presence of some inhibitor(s). Protein A was converted to protein C at pH 10, and also at pH 8 with high concentrations of NaCl. Thus, protein A is suggested to be the complex of protein C and unknown inhibitor(s). 5. When the chromatin fraction was incubated at pH 10, non-histone proteins were degraded much faster than at pH 8, although H1 histone was degraded at similar rates at both pHs. These results indicate that the chromatin fraction of rat liver contains at least two kinds of serine proteases, B and C, and that protease B is probably involved in the metabolism of basic protein, especially H1 histone. Protease C, the greater part of which associates with some inhibitors to form protein A, may play its main role in the metabolism of non-histone proteins.

1. When rats received an intraperitoneal injection of [3H]actinomycin D, it bound to the RNA moiety of free and bound polysomes of rat liver. The labeling increased gradually up to 6 h or 6 + 6 h. The poly(A)-containing mRNA showed definite radioactivity and its specific activity was higher than that of rRNA, although the total radioactivity of rRNA was markedly higher than that of poly(A)-containing mRNAs. 2. An equilibrium dialysis method using rabbit globin mRNA showed that the binding constant of actinomycin D to globin mRNA was 0.056 x 10(6) M-1, and globin mRNA had 2 binding sites per mol for actinomycin D. 3. From the results of the present experiments and those described in the preceding paper, it is suggested that one of the mechanisms by which actinomycin D treatment in vivo and in vitro stimulates the template activity of mRNA may be binding to mRNA, which alters the conformation of mRNA.

Proteins of rat liver nucleolar RNP, especially 110S RNP containing 45S RNA, were investigated and the following results were obtained. 1. Nucleolar extract was prepared from thioacetamide-treated rat liver nucleoli in the presence of 1 mg PVS per ml. Sucrose-density gradient centrifugation of the nucleolar extract showed RNP distributed between 60S and 110S. The 110S particles (110S RNP) contain 45S RNA as judged from the radioactivity profiles of nucleolar extract labelled in vivo with [3H]orotic acid for 10 min and 2 h. The protein components of 110S RNP and total RNP heavier than 60S (total RNP) were analyzed and compared. 2. Since the effects of PVS treatment on the extraction and the electrophoretic pattern of ribosomal proteins were removed by increasing the Mg2+ concentration during acetic acid extraction, as described in our preceding paper (1), proteins of 110S RNP as well as total RNP from PVS-pretreated nucleoli were extracted with 67% acetic acid containing 334 mM Mg2+ and analyzed by two-dimensional acrylamide gel electrophoresis. Ribosomal proteins masked by contaminating histone spots on the gel were identified by SDS-acrylamide gel electrophoresis. 3. 110S RNP contained a large portion of ribosomal proteins from large subunits; 24 protein spots were distinct, 6 protein spots were faint and 5 proteins (L3, L8, L30, L35, and L36) were missing completely. On the other hand, 110S RNP contained a small number of small subunit proteins; only 6 proteins showed distinct spots, 7 proteins showed faint spots and 12 protein spots were missing. 110S particles contained 11 non-ribosomal proteins, although it is difficult to rule out the possibility that some of them were contaminating chromatin proteins. 4. Total RNP showed electrophoretic patterns of 60S ribosomal proteins similar to those of 110S RNP, although L3 protein was present as a faint spot and 26 proteins showed distinct spots. Total RNP contained more small subunit proteins than 110S RNP; 7 proteins spots were distinct, 10 protein spots were faint and 8 protein spots were missing. The results suggest that some kinds of 40S proteins and L3 protein are attached to 110S RNP during the processing of 110S RNP.