Carboxymethylation of a minor ribonuclease from Aspergillus saitoi.

(1) RNase Ms was inactivated by iodoacetate. The inactivation was most rapid at pH 6.0, and was inhibited in the presence of a denaturant such as 8 m urea or 6 m guanidine-HCL. (2) Competitive inhibitors protected RNase Ms from inactivation by iodoacetate; the effect was in the order 2',(3')-GTP greater than 2',(3')-AMP, 2',(3')-UMP greater than or equal to 2',(3')-CMP. The order is not consistent with that of the binding constants of the 4 nucleotides towards RNase Ms (A is greater than C greater than G greater than U). (3) RNase Ms was inactivated with the concomitant incorporation of one molar equivalent of carboxymethly group. The following evidence indicated that the carboxymethyl group was incorporated into the carboxyl group of an aspartic acid or glutamic acid residue. (i) The carboxymethyl group incorporated into RNase Ms was liberated by treatment with 0.1 n NaOH or 1 m hydroxylamine. (ii) The amino acid composition of carboxymethylated RNase Ms (CM RNase Ms) after acid hydrolysis is similar to that of RNase Ms. (4) 14C-Labeled CM RNase Ms was digested successively with alkaline protease and amino-peptidase M. The radioactive amino acid released was eluted just before aspartate on an amino acid analyzer. After hydrolysis with 6 n HCL, glutamic acid was produced exclusively from the radioactive amino acid. The specific radioactivity of this amino acid calculated from the radioactivity and glutamic acid formed was practctically the same as that of CM RNase Ms. Thus, it was concluded that a carboxymethyl group was incorporated at the carboxyl group of a glutamic acid residue of RNnase Ms. (5) CM RNase Ms bound with 2'-AMP to the same extent as native RNase Ms, but bound to a lesser extent with 2',(3')-GMP. (6) Although the conformation of CM RNase Ms as judged from the CD spectrum was practically the same as that of native RNase Ms, the reactivity of CM RNase Ms towards dinitrofluorobenzene was different from that of native RNase Ms, indicating some difference in the conformation. (7) These results indicate that one glutamic acid residue is involved in the active of RNase Ms.     

Primary structure of an alkaline ribonuclease from bovine liver

A pyrimidine base specific and most basic alkaline RNase named RNase BL4 was isolated from bovine liver as a protein showing a single band on slab gel-electrophoresis. The enzyme is most active at pH 7.5. The enzyme was immunologically distinguishable from the known bovine RNases such as pancreatic RNase (RNase A), seminal RNase, kidney non-secretory RNase (RNase K2), and brain RNase (RNase BRb). The primary structure of this pyrimidine base-specific RNase was determined to be less than EDRMYQRFLRQHVDPDETG- GNDSYCNLMMQRRKMTSHQCKRFNTFIHEDLWNIRSICSTTNIQCKNGQMNCHEGVVRV- TDCRETGSSRAPNCRYRAKASTRRVVIACEGNPEVPVHFDK. It consists of 119 amino acid residues, and is 5 amino acid residues shorter than RNase A. The sequence homology of RNase BL4 with RNase A is 46.2%, and optimal alignment of RNase A and RNase BL4 requires five deletions, one at the 24th position, two at the 75th and 76th positions, and two at the C-terminus in RNase BL4. The RNase BL4 was highly homologous with a porcine liver RNase (RNase PL3, 94.1% homology) studied by Hofsteenge et al. (personal communication from Hofsteenge, J., Matthies, R., and Stones, S.R.).     

Expression of RNase Rh from Rhizopus niveus in yeast and characterization of the secreted proteins.

The full-length cDNA encoding RNase Rh, which is secreted extracellularly by Rhizopus niveus, was isolated and its nucleotide sequence was determined. It was placed under control of the promoter of the glyceraldehyde 3-phosphate dehydrogenase gene of Saccharomyces cerevisiae in a high expression vector in yeast. Since yeast cells transformed by this plasmid poorly secreted RNase into the medium, the plasmid pYE RNAP-Rh was constructed, in which the signal sequence of RNase Rh was replaced by the prepro-sequence of aspartic proteinase-I, one of the extracellular enzymes secreted by R. niveus. Yeast cells harboring pYE RNAP-Rh produced RNase efficiently (ca. 40 micrograms/ml) into the medium. The product was a mixture of six enzymes (RNase RNAP-Rhs) having 3, 5, 9, 13, 14, and 16 additional amino acid residues attached to the amino terminus of the mature RNase Rh. The major product was the RNase with three additional amino acids at the amino terminus. Limited digestion of RNase RNAP-Rhs with staphylococcal V8 protease succeeded in shortening the various lengths of extra amino acid residues attached to the amino terminus of RNase Rh, yielding an RNase that has 3 additional amino acids at the amino terminus. It has been named RNase RNAP-Rh. The RNase RNAP-Rh showed the same specific activity and CD spectra as those of RNase Rh, suggesting that the two have similar conformations to each other around aromatic amino acid residues and the peptide backbone.     

Residues 36-42 of liver RNase PL3 contribute to its uridine-preferring substrate specificity. Cloning of the cDNA and site-directed mutagenesis studies.

Within the superfamily of homologous mammalian ribonucleases (RNases) 4 distinct families can be recognized. Previously, representative members of three of these have been cloned and studied in detail. Here we report on the cloning of a cDNA encoding a member of the fourth family, RNase PL3 from porcine liver. The deduced amino acid sequence showed the presence of a signal peptide, confirming the notion that RNase PL3 is a secreted RNase. Expression of the cDNA in Escherichia coli yielded 1.5 mg of purified protein/liter of culture. The recombinant enzyme was indistinguishable from the enzyme isolated from porcine liver based on the following criteria: amino acid analysis, N-terminal amino acid sequence, molecular weight, specific activity toward yeast RNA, and kinetic parameters for the hydrolysis of uridylyl(3',5')adenosine and cytidylyl(3',5')adenosine. Interestingly, the kinetic data showed that RNase PL3 has a very low activity toward yeast RNA, i.e., 2.5% compared to pancreatic RNase A. Moreover, using the dinucleotide substrates and homopolymers it was found that RNase PL3, in contrast to most members of the RNase superfamily, strongly prefers uridine over cytidine on the 5' side of the scissile bond. Replacement, by site-directed mutagenesis, of residues 36-42 of RNase PL3 by the corresponding ones from bovine pancreatic RNase A resulted in a large preferential increase in the catalytic efficiency for cytidine-containing substrates. This suggests that this region of the molecule contains some of the elements that determine substrate specificity.     

Primary structure of a base non-specific and adenylic acid preferential ribonuclease from Aspergillus saitoi

The complete primary structure of a base non-specific and adenylic acid preferential RNase (RNase M) from Aspergillus saitoi was determined. The sequence was determined by analysis of the peptides generated by digestion of heat-denatured RNase M with lysylendopeptidase, and the peptides generated from RCM RNase M by digestion with staphylococcal V8 protease or chemical cleavage with BrCN. It consisted of 238 amino acid residues and carbohydrate moiety attached to the 74th asparagine residue. The molecular weight of the protein moiety deduced from the sequence was 26,596. The locations of 10 half cystine residues are almost superimposable on those of RNase Rh from Rhizopus niveus and RNase T2 from Aspergillus oryzae which have similar base specificity. The homology between RNase M and RNase Rh and RNase T2 amounted to 97 and 160 amino acid residues, respectively. The amino acid sequences conserved in the three RNases are concentrated around the three histidine residues, which are supposed to form part of the active sites of these RNases.     

Binding modes of inhibitors to ribonuclease T1 as elucidated by the analysis of two-dimensional NMR

Aromatic proton and high field shifted methyl proton resonances of RNase T1 complexed with Guo, 2'GMP, 3'GMP or 5'GMP were assigned to specific amino acid residues by 2D-NMR spectra in comparison with the crystal structure of RNase T1-2'GMP complex. The spatial proximities of amino acid residues as elucidated by NOESY spectra were found to be quite similar among free RNase T1 and the inhibitor complexes, showing that large conformational changes did not occur upon complex formation. However, small but appreciable conformational changes were induced which were reflected by the systematic chemical shift changes of some amino acid residues in the active site. Furthermore, we confirmed that RNase T1 contains two specific binding sites, one for the guanine base and the other for the phosphate moiety. The inhibitors are forced to adapt their conformations to fit the guanine base and the phosphate moiety to each binding site on the enzyme. This is consistent with our previous studies that 2'GMP and 3'GMP take syn form as a bound conformation, while 5'GMP takes anti conformation around glycosidic bonds.     

Amino-acid sequence of ribonuclease T2 from Aspergillus oryzae

The amino acid sequence of ribonuclease T2 (RNase T2) from Aspergillus oryzae has been determined. This has been achieved by analyzing peptides obtained by digestions with Achromobacter lyticus protease I, Staphylococcus aureus V8 protease, and alpha-chymotrypsin of two large cyanogen bromide peptides derived from the reduced and S-carboxymethylated or S-aminoethylated protein. Digestion with A. lyticus protease I was successfully used to degrade the N-terminal half of the S-aminoethylated protein at cysteine residues. RNase T2 is a glycoprotein consisting of 239 amino acid residues with a relative molecular mass of 29,155. The sugar content is 7.9% (by mass). Three glycosylation sites were determined at Asns 15, 76 and 239. Apparently RNase T2 has a very low degree of sequence similarity with RNase T1, but a considerable similarity is observed around the amino acid residues involved in substrate recognition and binding in RNase T1. These similar residues may be important for the catalytic activity of RNase T2.     

Primary structure of a base non-specific and adenylic acid preferential ribonuclease from the fruit bodies of Lentinus edodes.

The complete primary structure of a base non-specific and adenylic acid preferential RNase (RNase Le2) from the fruit bodies of Lentinus edodes was analyzed. The sequence was mostly determined by analysis of the peptides generated by V8 protease digestion and BrCN cleavage (including alpha-chymotryptic, and V8 protease digest of BrCN fragments). It consists of 239 amino acid residues. The molecular weight is 25831. The location of 10 half cystine residues were almost superimposable on those of known fungal RNases of the RNase T2 family. The sequence homologies between RNase Le2 and four known fungal RNases of the RNase T2 family, RNase T2, RNase M, RNase Trv, and RNase Rh, are 102, 103, 109, and 74, respectively. The homologous sequences are concentrated around the three histidines, which are supposed to form the active site of RNase T2 family RNases.     

The amino acid sequence of ribonuclease N1, a guanine-specific ribonuclease from the fungus Neurospora crassa

The complete amino acid sequence of ribonuclease N1 (RNase N1), a guanine-specific ribonuclease from a fungus, Neurospora crassa, was determined by conventional protein sequencing, using peptide fragments obtained by tryptic digestion of cyanogen bromide-treated RNase N1 and by Staphylococcus aureus V8 protease digestion of heat-denatured RNase N1. The results showed that the protein is composed of a single polypeptide chain of 104 amino acid residues cross-linked by two disulfide bonds and has a molecular weight of 11,174: (sequence; see text) (Disulfide bonds: C2-C10, C6-C103) The amino acid sequence was homologous with those of RNase T1 (65% identity) and related microbial RNases.     

Methylation of ribonuclease and albumin by methyl cobalamin

The capacity of methyl cobalamine (14CH3-B12) to methylate RNase and albumin in in vitro systems was studied. Under the experimental conditions, 14CH3-B12 methylated proteins about 100--1000 times more actively than S-adenosyl-methionine, a universal donor of methyl groups. The nature of buffer and pH 2 to 8 had no noticeable effect on the methylating capacity of 14CH3-B12. However, at higher pH rate of incorporation of methyl groups slightly increased. The 14CH3-groups incorporated into RNase amino acids remained stable upon illumination, in the presence of KCN in the incubation medium, and when heated in 20% HCl for 24--72 hr at 105 degrees. Methylation did not influence the enzymic properties of RNase. The automatic amino acid analyzer showed three peaks of the label of modified amino acids in the hydrolzates of methylated RNase, one of which corresponded to methylated methionine.     

Epitope structures recognised by antibodies against the major coat protein (g8p) of filamentous bacteriophage fd (Inoviridae).

To map the accessible surface of filamentous bacteriophage fd particles, the epitope structures of polyclonal rabbit serum and three mouse monoclonal antibodies raised against complete phage were analysed. Western blot analysis confirmed the major coat protein, gene VIII product (g8p or pVIII), to be the antigen. Overlapping peptides were synthesised by spot synthesis on cellulose membranes, covering the whole sequence of g8p. Each of the three tested monoclonal antibodies, B62-FE2, B62-GF3/G12 and B62-EA11, reacted with a core epitope covering ten amino acid residues at or near the amino terminus of g8p. The epitope recognised by B62-FE2 consists of the ten N-terminal amino acid residues of g8p. Extension of the amino terminus by various sequences did not inhibit binding, indicating that a terminal amino group is not essential for the interaction. Both B62-GF3/G12 and B62-EA11 recognise internal epitopes covering amino acid residues 3 to 12 of g8p. The epitopes of the polyclonal rabbit serum were also confined to the 12 N-terminal amino acid residues. The contribution of individual amino acid residues to the binding was analysed by a set of peptides containing individual amino acids exchanged by glycine. Accessible residues were Glu2, Asp4, Asp5, Pro6, Lys8, Phe11 and Asp12. The positions of the essential amino acid residues within the epitope are in accordance with a helical conformation of the amino-terminal region of g8p. Further, the results suggest new designs of phage display screening vectors to improve their performance in analysing non-linear epitopes.     

Purification and properties of a new ribonuclease from Aspergillus saitoi.

From a commercial digestive produced from Aspergillus saitoi, a ribonuclease [EC 3.1.4.23] having a molecular weight of 12,500 has been isolated in addition to the RNase reported previously, which had a molecular weight of 38,000. The enzyme was found to be homogeneous by chromatography on DEAE-cellulose, disc electrophoresis on polyacrylamide gel, and ultracentrifugation. The NH2-terminal amino acid was identified as glutamic acid. The amino acid composition indicated the presence of about 13 tyrosyl residues, 3 histidyl residues, and 2 half-cystine residues. The pH optimum of the RNase was 4.5, using RNA as a substrate. The enzyme was stable on heating at 70 degrees for 5 min from pH 2 to 10. It hydrolysed RNA completely to mononucleotides via 2', 3'-cyclic nucleotides. The rates of release of nucleotides and 2', 3'-cyclic nucleotides were in the order: guanylic acid is greater than adenylic acid is greater than cytidylic acid is greater than uridylic acid.     

Purification of acid ribonucleases from bovine spleen

Two acid RNases were purified from bovine spleen by means of ammonium sulfate fractionation, chromatographies on-phospho-cellulose, heparin-Sepharose CL-6B, poly G-Sepharose, and 2', 5'-ADP-Sepharose, and gel filtration on Toyopearl HW 55F. Both purified preparations were homogeneous as judged by disc electrophoresis at pH 4.3. They were designated as RNase BSP1 and RNase BSP2 in the order of elution from a phospho-cellulose column. RNase BSP2 was immunologically indistinguishable from RNase K2 from bovine kidney. RNase BSP1 was a typical pyrimidine base-specific, uridylic acid-preferential RNase and had very sharp pH optimum at 6.5. RNase BSP1 thus obtained was a glycoprotein giving two major bands on SDS-slap electrophoresis. Although the apparent molecular weight of RNase BSP1 was distributed in the range of 27,000-20,000, it decreased to about 17,000-18,000 after endoglycosidase F digestion. The N-terminal amino acid sequence up to the 20th amino acid had no homology to those of RNase K2 and RNase A.     

Molecular cloning of cDNAs encoding ribonuclease-related proteins in Nicotiana glutinosa leaves, as induced in response to wounding or to TMV-infection

We earlier isolated a cDNA clone (NGR1) encoding a wound-inducible ribonuclease (RNase NW) from leaves of Nicotiana glutinosa [Kariu et al. Biosci. Biotechnol. Biochem., 62, 1144-1151 (1998)]. In this study, two distinct cDNA clones, NGR2 and NGR3, encoding proteins with a ribonuclease-related sequence in the N. glutinosa leaves, were amplified and sequenced. The nucleotide sequences of NGR2 and NGR3 consist of 1244 bp and 1069 bp, and have open reading frames encoding 277 (RNase NGR2) and 236 (RNase NGR3) amino acid residues, respectively. The deduced amino acid sequences of the putative RNases NGR2 and NGR3 showed 33% and 58% amino acid sequence identity, respectively, with that of RNase NW and 32% identity with each other. Sequence comparison showed that NGR2 is similar to RNase RNS2 (61%) from Arabidopsis thaliana, while NGR3 is related to RNase LX (84%) from tomato (Lycopersicon esculentum). RNA gel blot analysis showed that the RNase NGR2 gene is constitutively expressed to measurable levels; it is not increased by either wounding or TMV infection. In contrast, the expression of the NGR3 gene is induced after 48 h upon TMV infection.     

Characterization of two forms of base non-specific and adenylic acid preferential ribonuclease from Aspergillus saitoi

Two forms of RNases (RNase ML and RNase MM) from Aspergillus saitoi which are base non-specific and adenylic acid preferential were separated from each other by DEAE-cellulose column chromatography. They are indistinguishable with respect to enzymatic properties such as base preferability, pH optimum, kinetic constants measured with 2',3'-cUMP and 2',3'-cCMP as substrates, and effects of ionic strength, physical properties such as heat stability, isoelectric point and circular dichroism spectra, amino acid composition and immunological property. They only differ in carbohydrate content. The apparent molecular weight determined by SDS-disc electrophoresis was 36,000 for RNase ML and 32,000 for RNase MM. Both RNases were reduced and carboxymethylated, and then digested with trypsin, separately. Glycopeptides were isolated from the both digests by gelfiltration and paper chromatography. The amino acid compositions of glycopeptides obtained from RNase ML (ML TS-IIC) and that obtained from RNase MM (MM TS-IIIC) were the same. The amino acid sequences of both glycopeptides determined by Edman degradation and carboxypeptidase digestion were also the same. The results indicated that RNase ML and RNase MM were the same protein having different sizes of carbohydrate chains at one site on the molecule.     

Binding modes of inhibitors of ribonuclease T1 as elucidated by analysis of two-dimensional NMR

Aromatic proton and high field shifted methyl proton resonances of RNase T1 complexed with Guo, 2'GMP, 3'GMP, or 5'GMP were assigned to specific amino acid residues by analyses of the two-dimensional NMR spectra in comparison with the crystal structure of the RNase T1-2'GMP complex. These assignments were subsequently correlated to those of free RNase T1 [Hoffmann & Rüterjans (1988) Eur. J. Biochem. 177, 539-560]. The spatial proximities of amino acid residues as elucidated by NOESY spectra were found to be quite similar among free RNase T1 and the inhibitor complexes, showing that large conformational changes did not occur upon complex formation. However, small but appreciable conformational changes were induced, which were reflected by the systematic chemical shift changes of some amino acid residues in the active site. Furthermore, we confirmed that RNase T1 contains two specific binding sites, one for the guanine base and the other for the phosphate moiety. The inhibitors are forced to adapt their conformations to fit the guanine base and the phosphate moiety to each binding site on the enzyme. This is consistent with our previous studies that 2'GMP and 3'GMP take the syn form as a bound conformation, while 5'GMP takes the anti conformation around glycosidic bonds [Inagaki et al. (1985) Biochemistry 24, 1013-1020]. The slow-exchange process between free and bound forms involving Tyr42 and Tyr45 was found to be specific to the recognition of the guanine base.     

PROTEIN SYNTHESIS IN VITRO IN RAT BRAIN AFTER SHORT-TERM PROTEIN STARVATION AND REFEEDING

Rats were fed a protein-free diet for 4 or 6 days. They were compared with rats kept on the same diet for 3 or 5 days and on adequate protein for one additional day. The incorporation of ¹⁴C-labelled amino acid into protein was studied in systems containing ATP, GTP, phosphoenolpyruvate, pyruvate kinase and if required, a mixture of unlabelled amino acids and either the 6000 g supernatant fraction of a brain homogenate or microsomes and soluble enzymes. The 6000 g supernatant fraction showed variation in amino acid incorporating activity as well as in RNase activity as measured by breakdown of labelled polyuridylic acid. There was no difference in RNase activity in isolated microsomes, but the amino acid incorporating activity was significantly higher in preparations obtained from rats fed one meal of protein after 5 days of protein-starvation.     

Covalent structure of collagen. Amino acid sequence of an arthritogenic cyanogen bromide peptide from type II collagen of bovine cartilage

Bovine articular type II collagen was prepared by limited pepsin digestion, differential salt fractionation and carboxymethylcellulose chromatography. Cyanogen bromide digestion of purified type II collagen alpha chains yielded twelve distinct peptides designated CB1-12. The peptide alpha 1(II)-CB11 was isolated by carboxymethylcellulose chromatography and Sephadex G-75S gel filtration. Automated Edman degradation together with chymotrypsin, thermolysin and trypsin digestion enabled identification of its complete amino acid sequence. Compared with type I and type III collagen, the data show similarity with alpha 1(I)-CB8 and alpha 1(III)-CB6-1-8-10-2 peptides, respectively. The peptide is located within residues 124-402 of the alpha 1(II) collagen chain and with its identification, now extends the known amino acid sequence of bovine type II cartilage collagen to 660 amino acid residues including alpha 1(II)-CB1-2-6-12-11-8-10 (partial). This corresponds to alpha 1(I)-CB0-1-2-4-5-8-3-7 (partial; 1-660) and alpha 1(III)-CB3A-3B-3C-7-6-1-8-10-2-4-5 (partial; 1-660) of bovine alpha 1(I) and alpha 1(III) collagen chains.     

NMR structure of an archaeal homologue of ribonuclease P protein Rpp29

A protein component of the Archaeoglobus fulgidus RNase P was expressed in Escherichia coli, purified, and structurally characterized using multidimensional NMR methods. The dominant structural feature of this 11 kDa protein is a sheet of six antiparallel beta-strands, wrapped around a core of conserved hydrophobic amino acids. Amide proton exchange and (15)N relaxation rate data provide evidence that the first 16 residues of the protein, located before the start of the first beta-strand, and the last 24 residues, located past the end of the last beta-strand, are relatively flexible; this contrasts with the relatively rigid and well-defined structure of the beta-sheet. Amino acid sequence comparisons among a diverse set of species indicate that the A. fulgidus protein is homologous to the human RNase P protein Rpp29, yeast RNase P protein Pop4, and a known archaeal RNase P protein from Methanobacter thermoautotrophicus; conserved hydrophobic residues indicate that the homologous protein in each of these species contains a similar beta-sheet structure. Conserved surface residues located in the loop connecting strands beta2 and beta3, the loop connecting strands beta4 and beta5, and in the flexible N- and C-terminal tails are most likely to have specific interactions with the RNA and other proteins of RNase P. The structural model of an RNase P protein component provided by the present work provides an essential step toward eventually understanding the overall architecture of this complex enzyme and the mechanism by which it performs its functions.