JDP2 suppresses adipocyte differentiation by regulating histone acetylation.

Among the events that control cellular differentiation, the acetylation of histones plays a critical role in the regulation of transcription and the modification of chromatin. Jun dimerization protein 2 (JDP2), a member of the AP-1 family, is an inhibitor of such acetylation and contributes to the maintenance of chromatin structure. In an examination of Jdp2 'knock-out' (KO) mice, we observed elevated numbers of white adipocytes and significant accumulation of lipid in the adipose tissue in sections of scapulae. In addition, mouse embryo fibroblasts (MEFs) from Jdp2 KO mice were more susceptible to adipocyte differentiation in response to hormonal induction and members of the CCAAT/enhancer-binding proteins (C/EBP) gene family were expressed at levels higher than MEFs from wild-type mice. Furthermore, JDP2 inhibited both the acetylation of histone H3 in the promoter of the gene for C/EBPdelta and transcription from this promoter. Our data indicate that JDP2 plays a key role as a repressor of adipocyte differentiation by regulating the expression of the gene for C/EBPdelta via inhibition of histone acetylation.

cDNA-derived amino acid sequence of acetoacetyl-CoA synthetase from rat liver.

In order to examine the primary structure of acetoacetyl-CoA synthetase (acetoacetate-CoA ligase, EC 6.2.1.16; AA-CoA synthetase), the cDNA clone encoding this enzyme has been isolated from the cDNA library which was prepared from the liver of rat fed a diet supplemented with 4% cholestyramine and 0.4% pravastatin for 4 days. Nucleotide sequence analysis of cloned cDNA revealed that AA-CoA synthetase of rat liver contains an open reading frame of 2019 nucleotides, and the deduced amino acid sequence (672 amino acid residues) bears 25.0 and 38.9% homologies with acetyl-CoA synthetases of Saccharomyces cerevisiae and Archaeoglobus fulgidus, respectively.

Amino acids and peptides. LVII. Synthetic peptide with a sequence of ribonuclease from Sulfolobus solfataricus, SSR(1-62), does not function as an RNase.

The 62 residue peptide, SSR(1-62), whose sequence corresponds to that of ribonuclease (RNase) from Sulfolobus solfataricus, and its related peptides, SSR(1-22) and SSR(10-62), were chemically synthesized and their RNase activity and DNA-binding activity were examined. The RNase activity assay using yeast RNA or tRNA(fMet) as substrate showed that the synthetic peptide SSR(1-62) did not hydrolyze yeast RNA or tRNA(fMet). These data were not consistent with previous reports that both the native peptide isolated from S. solfataricus [Fusi et al. (1993) Eur. J. Biochem. 211, 305-311] and the recombinant peptide expressed in Escherichia coli [Fusi et al. (1995) Gene 154, 99-103] were able to hydrolyze tRNA(fMet). However, the synthetic SSR(1-62) exhibited DNA-binding activity. In the presence of synthetic SSR(1-62), the cleavage of DNA (plasmid pUCRh2-4) by restriction endonuclease (EcoRI) was not observed, suggesting that synthetic SSR(1-62) bound to DNA protected DNA from its enzymatic digestion. Neither SSR(1-22) nor SSR(10-62) prevented DNA from being cleaved by a restriction enzyme. These findings strongly suggest the importance of not only the N-terminal region of SSR(1-62) but also the C-terminal region for DNA-binding. Circular dichroism spectroscopy of synthetic SSR(1-62) indicated a beta-sheet conformation, in contrast with synthetic SSR(1-22), which exhibited an unordered conformation.

Alterations in glycosaminoglycans of the aorta of vitamin E-deficient rats.

To elucidate whether or not a vitamin E-deficient diet affects the rat aorta extracellular matrix, we examined the alterations in glycosaminoglycans (GAGs), as one of the components of the extracellular matrix of the aorta. The total amount of uronic acid, as an index of GAG, decreased significantly in the aorta of vitamin E-deficient rats. The components of GAG were identified as hyaluronic acid (HA), heparan sulfate (HS), dermatan sulfate (DS) and chondroitin sulfate (CS) by electrophoresis together with enzymic digestion. The amount of sulfated GAGs, especially the amount of DS and CS, decreased in the aorta of vitamin E-deficient rats. The biosynthetic activity of GAG was determined by using [3H]glucosamine and [35S]sulfate. The total biosynthetic activity of GAG and the incorporation of [3H]glucosamine into HA, HS, DS and CS decreased markedly in the aorta of vitamin E-deficient rats. The decrease in the production of sulfated GAGs, especially DS, which is involved in the potent antithrombogenic activity, could be related to the lower anticoagulant activity in the aorta of vitamin E-deficient rats.

Photooxidation and carbethoxylation of a minor ribonuclease from Aspergillus saitoi.

In order to investigate the nature of amino acid residues involved in the active in the active site of a ribonuclease from Aspergillus saitoi, the pH dependence of the rates of inactivation of RNase Ms by photooxidation and modification with diethylpyrocarbonate were studied. (1) RNase Ms was inactivated by illumination in the presence of methylene blue at various pH's. The pH dependence of the rate of photooxidative inactivation of RNase Ms indicated that at least one functional group having pKa 7.2 was involved in the active site. (2) Amino acid analyses of photooxidized RNase Ms at various stages of photooxidative inactivation at pH's 4.0 and 6.0 indicated that one histidine residue was related to the activity of RNase Ms, but that no tryptophan residue was involved in the active site. (3) 2',(3')-AMP prevented the photooxidative inactivation of RNase Ms. The results also indicated the presence of a histidine residue in the active site. (4) Modification of RNase Ms with diethylpyrocarbonate was studied at various pH's. The results indicated that a functional group having pKa 7.1 was involved in the active site of RNase Ms.

Modification of glucoamylases from Rhizopus sp. with 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide.

To investigate the role of carboxyl groups of glucoamylases [EC 3.2.1.3] from a Rhizopus sp. (Gluc1 and Gluc2), the modification of Gluc1 and Gluc2 with a water-soluble carbodiimide, 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide metho-p-toluenesulfonate (CMC), was studied. The inactivation of Gluc1 proceeded with the incorporation of about 3 CMC moieties. In the presence of maltose, the modification of about 2.2 carboxyl groups of Gluc1 proceeded with a slight loss of enzymatic activity. In the re-modification of Gluc1 modified in the presence of maltose, Gluc1 was inactivated by further modification of about 1.3 carboxyl groups. Therefore, one carboxyl group, which was protected by maltose, was thought to be a crucial one. The inactivation of Gluc2 proceeded similarly to that of Gluc1, but the number of CMC moieties incorporated was about one less than in the case of Gluc1. Thus, it was suggested that one of the reactive carboxyl groups of Gluc1 was located in the N-terminal part of Gluc1, which is deficient in Gluc2. From the results of kinetic studies on CMC-modified Gluc1, it was suggested that the hydrolysis mechanism of malto-oligomers differs somewhat from that of PNPG.

Modification of a minor glucoamylase from Aspergillus saitoi with 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide metho p-toluenesulfonate.

In order to elucidate the structure-function relationship of glucoamylases [EC 3.2.1.3, alpha-D-(1-4)-glucan glucohydrolase] from Aspergillus saitoi, the reaction of a minor component, Gluc M2 with 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide metho p-toluenesulfonate (CMC) was studied at pH 4.5. Inactivation of Gluc M2 with [14C]CMC proceeded with the incorporation of about 5 CMC moieties. From the results of analyses of amino acid and sulfhydryl contents of CMC-modified Gluc M2 and the hydroxylamine treatment of the CMC-modified Gluc M2 at pH 7.0, it was concluded that the sites of CMC-modification were carboxylic acids of Gluc M2. In the presence of maltose, when Gluc M2 was treated with [14C]CMC, ca. 4 CMC moieties were incorporated with a simultaneous decrease in activity (30%). The Gluc M2 modified in the presence of maltose was re-modified with CMC after elimination of maltose. The CMC-modified Gluc M2 (70% activity) was inactivated completely with the further incorporation of ca. 2 CMC moieties. The logarithm of the half-life of the inactivation of Gluc M2 by CMC was a linear function of log[CMC] indicating that one carboxyl group among the modified ones was crucial for the inactivation of Gluc M2. From the results of these modification reactions, it was concluded that one or two carboxylic acids in Gluc M2 were crucial for the catalysis of glucoamylase from A. saitoi. Based on the analysis of the pH-profile of CMC inactivation of Gluc M2, the participation of a carboxylic acid having pKa 5.7 in the active site is proposed.

Modification of a ribonuclease from Rhizopus sp. with 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide p-toluenesulfonate.

The carboxyl group in a ribonuclease from Rhizopus sp. (RNase Rh) was modified by a water-soluble carbodiimide, 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide p-toluenesulfonate (CMC). From the relation between the extent of modification and the enzymatic activity, it was concluded that at least the modification of two carboxyl groups seemed to induce the loss in enzymatic activity. In the presence of 1 M cytidine, RNase Rh activity was protected from the CMC-modification. Under conditions in which the enzyme was inactivated to 20% activity, about 70% of the enzymatic activity was retained in the presence of cytidine. The inactivation of the RNase Rh pre-treated with CMC in the presence of cytidine with [14C]CMC indicated that the RNase Rh lost its enzymatic activity with the incorporation of about one [14C]CMC. Therefore, it could be concluded that one carboxyl group is involved in the active site of RNase Rh. The binding of the CMC-modified RNase Rh with 2'-AMP was studied spectrophotometrically. The affinity of the modified RNase Rh towards 2'-AMP decreased markedly upon CMC modification.

Purification and properties of human urine ribonucleases.

1. Two RNases (RNase UL and RNase US) were purified from the urine of human adults by means of column chromatographies on SP-Sephadex C-50, phospho-cellulose and CM-cellulose and gel-filtration on Sephadex G-75 in homogeneous states obtained by SDS-disc electrophoresis. 2. Molecular weights of these RNases determined by gel-filtration were 38,000 and 13,000 for RNase UL and RNase US, respectively. 3. Optimal pH's of urine RNases were 8.0 and 6.75 for RNase UL and RNase US, respectively. 4. Chemical composition of urine RNases was determined. RNase UL contains about 20.7% of neutral sugar and 7.8% of hexosamine. RNase US contains a very small amount of carbohydrate moiety. 5. Base specificity of urine RNases studied with 2',3'-cyclic nucleotides and dinucleoside phosphates as substrates indicated that both RNases were pyrimidine specific and cytosine preferential enzyme, as is bovine pancreatic RNase A. Although base specificity of RNase UL was qualitatively similar to RNase A, that of RNase US was slightly different. That is, RNase US did not hydrolyze UpU and hydrolyzed UpC and 2',3'-cyclic UMP very slowly. 6. Antigenic properties of human urine RNases were studied by Ouchterlony's double diffusion analysis. RNase UL, RNase US, and RNase A were serologically distinguishable.

Modification of a glucoamylase from Aspergillus saitoi with 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide.

1. In order to elucidate the structure-function relation of a glucoamylase [EC 3.2.1.3, alpha-D-(1 leads to 4)-glucan glucohydrolase] from Aspergillus saitoi (Gluc M1), the reaction of Gluc M1 with water-soluble carbodiimides was studied. 2. Gluc M1 was inactivated most effectively by 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide (CMC) at pH 4.5. 3. Inactivation of Gluc M1 with [14C]CMC proceeded with the incorporation of about 12 CMC moieties. From the results of amino acid analysis, titration of SH group with Ellman's reagent and hydroxylamine treatment at pH 7.0, it was concluded that the crucial sites of modification were carboxyl groups of Gluc M1. 4. The CD spectrum of CMC-modified Gluc M1 (residual activity, ca. 9.8%) suggested that the gross conformation of the native enzyme was retained. 5. In the presence of maltose, when Gluc M1 was incubated with [14C]CMC, ca. 10 CMC moieties were incorporated with a simultaneous decrease in enzymatic activity (30%). The Gluc M1 modified in the presence of maltose was remodified with CMC after elimination of maltose. The CMC-modified Gluc M1 was inactivated completely with the incorporation of ca. 4 CMC moieties. 6. The logarithm of the half-life of the inactivation of Gluc M1 by CMC was a linear function of log [CMC] indicating that one carboxyl group among the modified ones was crucial for inactivation of Gluc M1. 7. The protection by maltose of Gluc M1 from inactivation and the increase in K1 values for maltose of CMC-modified Gluc M1's suggested that a crucial carboxyl group(s) was located near or on subsites 2 and 3.

The teratogenic mechanism of 6-aminonicotinamide on limb formation of chick embryos: abnormalities in the biosynthesis of glycosaminoglycans and proteoglycans in micromelia.

After a dose of 10 micrograms of 6-aminonicotinamide (6-AN) was administered to day-4- chick embryo in ovo, micromelia was obviously observed in the hind limbs of 7-day chick embryos. We examined the teratogenic mechanism of 6-AN by using the normal or micromelial hind limbs (buds) from day 5 to day 7, with special attention to the biosynthesis of glycosaminoglycan (GAG) and proteoglycan as an index of limb chondrogenesis. The present study provides evidence for abnormalities in the levels of GAG or proteoglycan biosynthesis in the micromelial hind limbs (buds). 1) Both [35S]sulfate and [3H]glucosamine incorporation into GAG per 10 limbs or mg DNA of the micromelia were inhibited, suggesting a decrease of GAG synthesis. 2) The micromelial limbs synthesized low-sulfated chondroitin sulfate (chondroitin) as judged by the 35S/3H ratio, the proportion of unsulfated disaccharide (delta Di-0S), and the result of cellulose acetate electrophoresis, although there were no significant differences in the approximate molecular size of 35S-chondroitin sulfates synthesized between the normal and micromelial limbs. 3) PAPS-synthesizing activity in the micromelial limbs was markedly inhibited, and this may result in the production of low-sulfated proteoglycan. 4) The transition from mesenchymal- to cartilage-specific proteoglycan synthesis did not appear in the micromelial limbs as judged by the sedimentation profiles. 5) 6-AN caused marked reductions in the oxygen consumption and ATP level of the micromelial limbs, thereby causing the defect in PAPS formation. We suggest that these 6-AN-induced sequential molecular defects (the reduction of respiratory activity, ATP and PAPS level, and concomitant interference with GAG and proteoglycan biosynthesis) in the limbs (buds) during the critical period of limb morphogenesis must be major factors resulting in the cartilage growth retardation or disorder, i.e., micromelia.

Enzymatic properties of mutant enzymes at Trp49 and Tyr57 of RNase Rh from Rhizopus niveus.

In order to establish the role of Tyr57 and Trp49 in the enzymatic reaction of RNase Rh, several mutant enzymes at Tyr57 and Trp49 were prepared by protein engineering and their enzymatic properties were investigated. Among the four mutant enzymes at Trp49 (W49F, W49Y, W49A, and W49I), W49F showed 16% of the activity of the native enzyme, but the others (W49Y, W49A, and W49I) showed greatly decreased activity. The data showed that Trp49 is very important for the enzyme activity. Among 8 mutant enzymes at the 57th position, Y57F and Y57W showed similar enzymatic activity toward RNA to that of the wild-type enzyme, but the others (Y57G, Y57A, Y57V, Y57M, and Y57K) are more active toward RNA and less active toward XpGs. The reason for the apparent increase for RNA activity is discussed from the view point of substrate inhibition. It is noteworthy that W49F and Y57W became more pyrimidine base- and purine base-preferential, respectively.

Enzymatic properties of mutant forms of RNase Rh from Rhizopus niveus as to Asp51.

In order to determine the role of Asp51 of RNase Rh from Rhizopus niveus, enzymes with mutations at the 51st position, D51N, D51E, D51Q, D51S, D51T, D51A, and D51K, were prepared, and their enzymatic properties were investigated as to specific activity and base specificity. All the mutant enzymes showed relatively high activity toward poly I and poly C, and markedly reduced activity toward poly A and poly U. In particular, the enzymatic activities toward poly I of D51T and D51S were higher than that of RNase RNAP Rh. Among the mutant enzymes, D51N, D51S, and D51T showed more than ca. 30% of the activity of RNase Rh, when RNA, poly I and poly C were used as substrates, respectively. The substitution of Ala, Glu, or Lys at Asp51 is unfavorable for enzymatic activity. Among XpGs (X = A, G, U, or C), D51N, D51S, and D51T showed higher activity toward GpG then CpG. Therefore, Asp51 in RNase Rh plays a critical role in the adenylic acid preference of RNase T2 family enzymes. Our results obtained with a protein engineering technique provide basic insights into the control of the base specificity of RNase Rh.

Inhibition of glucoamylases from a Rhizopus sp. and Aspergillus saitoi by aminoalcohol derivatives.

The mechanism of inhibition of the two glucoamylases from a Rhizopus sp. and Aspergillus saitoi by aminoalcohol derivatives was investigated. Hydrolysis of maltose by the glucoamylases was inhibited competitively by aminoalcohols at pH 5.0, and tris(hydroxymethyl)aminomethane, 2-amino-2-ethyl-1,3-propanediol and 2-aminocyclohexanol were relatively good inhibitors of the glucoamylases among the aminoalcohol derivatives tested. One hydroxyl group and an amino group in these inhibitors were indispensable for the inhibitory action, and the addition of other hydroxyl, amino or ethyl groups was enhancing. With an increase in pH from 4.0 to 6.0, the Ki values of the aminoalcohols decreased. This result suggested the participation of a carboxyl group, which was related to the glucoamylase activity and had a pKa of 5.7, in the binding of aminoalcohols. The UV difference spectra induced on binding of the aminoalcohol analogues with the glucoamylases may indicate a change of the environment of tryptophan residues to a slightly higher pH on inhibitor binding. The influence of aminoalcohols on the fluorescence intensity due to tryptophan residues and the CD-spectra of the glucoamylases was less than that of maltitol. Thus, the interaction of aminoalcohols with tryptophan residues in the glucoamylases might be less pronounced than that in the case of substrate analogues. The modes of binding of the aminoalcohols with the two glucoamylases were very similar. Therefore, the phenomenon might be a common feature of glucoamylases in general.

Role of Lys108 in the enzymatic activity of RNase Rh from Rhizopus niveus.

In order to elucidate on the mechanism of action of RNase Rh from Rhizopus niveus, we investigated the role of Lys108, which is conserved among the RNase T2 family RNases except for two cases. The RNase activities of Lys108 mutant RNases, RNase RNAP K108R and K108L, are about 33.5 and 3.1% of that of the wild type enzyme, respectively. The relative rates of cleavage of dinucleoside phosphates by these two mutant enzymes were comparable to those with RNA as a substrate. The kinetic parameters of RNases RNAP K108R and K108L towards XpGs (where X is one of A, G, U, and C) were measured. The data indicated that the Km values of the two mutant enzymes are similar to those of the wild-type enzyme. The rates of release of the four nucleotides from RNA by digestion with the mutant enzymes were in the order A > G > U > C, which is qualitatively the same as that of the wild-type enzyme. From these data, we concluded that the Lys108 residue participates in the catalytic process, but not in the binding, and the positive charge of Lys108 is indispensable for the catalytic process, that is, the positive charge of Lys108 may stabilize the pentacoordinated intermediate in the transition state as proposed in the case of Lys41 in RNase A, or may polarize the phosphate moiety of the substrate.

Modification of a major ribonuclease from Aspergillus saitoi with 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide.

In order to investigate the role of carboxyl groups of a base non-specific ribonuclease from Aspergillus saitoi [EC 3.1.27.1] (RNase M, molecular weight 36,000), the modification of RNase M with a water-soluble carbodiimide, 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide(CMC), was studied. The inactivation of RNase M proceeded almost linearly with the incorporation of about 9.5 CMC moieties. The peptide backbone structure of the modified RNase M was practically the same as that of the native RNase M, as assessed from the CD spectra in the region of 200-250 nm. In the presence of competitive inhibitors, adenosine, and cytidine, inactivation of RNase M by CMC was partially inhibited. In the presence of cytidine (1 M), the modification of about 4 carboxyl groups of RNase M proceeded with a slight loss of enzymatic activity (ca. 20%). Further modification inactivated RNase M with the incorporation of ca. 4-5 CMC without any detectable intramolecular peptide bond formation. Therefore, it was concluded that carboxyl groups responsible for enzymatic activity were included among these carboxyl groups protected by cytidine. The logarithm of the half-live of the inactivation of RNase M by CMC was a linear function of log[CMC] with a slope of minus one, indicating that at least one carboxyl group among the modified ones may be essential for catalysis. The digestion of CMC-modified RNase M with carboxypeptidase A eliminated the carboxyl terminal group from the site of CMC modification.

Evidence that three histidine residues of a base non-specific and adenylic acid preferential ribonuclease from Rhizopus niveus are involved in the catalytic function.

In order to study the structure-function relationship of an RNase T2 family enzyme, RNase Rh, from Rhizopus niveus, we investigated the roles of three histidine residues by means of site-specific mutagenesis. One of the three histidine residues of RNase RNAP Rh produced in Saccharomyces cerevisiae by recombinant DNA technology was substituted to a phenylalanine or alanine residue. A Phe or Ala mutant enzyme at His46 or His109 showed less than 0.03%, but a mutant enzyme at His104 showed 0.54% of the enzymatic activity of the wild-type enzyme with RNA as a substrate. Similar results were obtained, when ApU was used as a substrate. The binding constant of a Phe mutant enzyme at His46 or His109 towards 2'-AMP decreased twofold, but that at His104 decreased more markedly. Therefore, we assumed that these three histidine residues are components of the active site of RNase Rh, that His104 contributes to some extent to the binding and less to the catalysis, and that the other two histidine residues and one carboxyl group not yet identified are probably involved in the catalysis. We assigned the C-2 proton resonances of His46, His104, and His109 by comparison of the 1H-NMR spectra of the three mutant enzymes containing Phe in place of His with that of the native enzyme, and also determined the individual pKa values for His46 and His104 to be 6.70 and 5.94. His109 was not titrated in a regular way, but the apparent pKa value was estimated to be around 6.3. The fact that addition of 2'-AMP caused a greater effect on the chemical shift of His104 in the 1NMR spectra as compared with those of the other histidine residues, may support the idea described above on the role of His104.

Purification and primary structure of a new guanylic acid specific ribonuclease from Pleurotus ostreatus.

A guanine nucleotide-specific RNase (RNase Po1) was isolated from caps of the fruit bodies of Pleurotus ostreatus. RNase Po1 is most active towards RNA at pH 8.0. The effect of heating on the molar ellipticity at 210 nm of RNase Po1 showed that RNase Po1 is more stable than RNase T1. The primary structure of RNase Po1 was determined to be < ETGVRSCNCAGRSFTGTDVTNAIRSARAGGSGNYPHVYNNFEGFSFSCTPTFFEFPVFRGSVYSGGSPG ADRVIYD- QSGRFCACLTHTGAPSTNGFVECRF. It consisted of 101 amino acid residues, with a molecular weight of 10,760. RNase Po1 has relatively higher sequence homology with RNase T1 family RNase. It contains 6 half cystine residues. The locations of four of them are superimposable on those of RNase U1 and RNase U2. The amino acid residues forming the active site of RNase T1 were well conserved in this RNase. Therefore, RNase Po1 is a unique member of the RNase T1 family in respect of the location of one disulfide bridge, and its stability.

In vitro mRNA synthesis by Sendai virus: isolation and characterization of the transcription initiation complex.

We have developed an in vitro transcription system using purified Sendai virus particles in which viral mRNA synthesis is almost entirely dependent on the addition of cellular proteins (host factors), one of which can be replaced by highly purified cellular tubulin [Mizumoto et al. (1995) J. Biochem. 117, 527-534]. In this study, to elucidate the function of host factors in transcription, we isolated an active initiation complex as a viral ribonucleoprotein, by incubating virus particles with bovine brain extract in the absence of nucleoside triphosphates, followed by ultracentrifugation. RNA products from the isolated initiation complex contained six mRNA species corresponding to all the virus-encoded genes, and most of them had a 5'-cap structure as well as a 3'-poly(A) tail. Immunoblotting showed that tubulin was specifically associated in the active complex. These data suggest that cellular tubulin, one of the host factors essential for Sendai virus transcription, interacts with the viral ribonucleoprotein to form an active complex at the initiation step.

Characterization of poly C preferential ribonuclease from chicken liver.

Poly C preferential RNase previously reported by Levy and Karpetsky [J. Biol. Chem. 255, 2153-2159 (1980)] and Miura et al. [Chem. Pharm. Bull. 32, 4053-4060 (1984)] was extensively purified from chicken liver to homogeneity as determined by SDS-PAGE (RNase CL2). The poly C preference over poly U was slightly higher than that of bovine pancreatic RNase A. However, the kinetic constants for 8 dinucleoside phosphates, CpY and UpY (Y = one of A, G, U, and C) as substrates showed that RNase CL2 was preferential for cytidylic acid, but less so than RNase A, and the influence of Y base on the rate of hydrolysis of CpY or UpY was less marked than in the case of RNase A. The primary structure of RNase CL2 was determined. The molecular weight calculated from the sequence was 13,420. Comparison of the amino acid sequence of RNase CL2 with those of other vertebrate RNases showed that RNase CL2 is a member of the RNase A family, but is not a non-secretory RNase. It retains 3 disulfide bridges of RNase A, but Cys65-Cys72 of RNase A is missing. As for the active site, the amino acid residues of the P0 and P1 sites of RNase A are completely conserved. Among the B1 site components, Thr45 (RNase A numbering) is conserved, but Phe120 and Ser123 are substituted by Leu and Thr, respectively. Among the B2 site residues, Gln69, Asn71, and Glu111 are substituted by other amino acids.