Mutagenesis of residues flanking Lys-40 enhances the enzymatic activity and reduces the angiogenic potency of angiogenin.

The primary structure of the blood vessel inducing protein angiogenin is 35% identical with that of pancreatic ribonuclease (RNase) and contains counterparts for the critical RNase active-site residues His-12, Lys-41, and His-119. Although angiogenin is a ribonucleolytic enzyme, its activity toward conventional substrates is lower than that of pancreatic RNase by several orders of magnitude. Comparison of the amino acid sequences of RNase and angiogenin reveals several striking differences in the region flanking the active-site lysine, including a deletion and a transposition of aspartic acid and proline residues. In order to examine how these sequence changes alter the functional properties of angiogenin, an angiogenin/RNase hybrid protein (ARH-II), in which residues 38-41 of angiogenin (Pro-Cys-Lys-Asp) have been replaced by the corresponding segment of bovine pancreatic RNase (Asp-Arg-Cys-Lys-Pro), was prepared by regional mutagenesis. Compared to angiogenin, ARH-II has markedly diminished angiogenic activity on the chick embryo chorioallantoic membrane but 5-75-fold greater enzymatic activity toward a variety of polynucleotide and dinucleotide substrates. In addition, the specificity of ARH-II toward dinucleotide substrates differs from that of angiogenin and is qualitatively similar to that of pancreatic RNase. Thus, non-active-site residues near Lys-40 in angiogenin appear to play a significant role in determining enzymatic specificity and reactivity as well as angiogenic potency. An additional angiogenin/RNase hybrid protein (ARH-IV), in which residues 59-71 of ARH-II have been replaced by the corresponding segment of pancreatic RNase, was also prepared.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enzymatically active angiogenin/ribonuclease A hybrids formed by peptide interchange

The primary structures of the blood vessel inducing protein human angiogenin and human pancreatic ribonuclease (RNase) are 35% identical. Angiogenin catalyzes the limited cleavage of ribosomal RNA (18 and 28 S), yielding a characteristic pattern of polynucleotide products, but shows no significant activity toward conventional pancreatic RNase substrates [Shapiro, R., Riordan, J. F., & Vallee, B. L. (1986) Biochemistry 25, 3527-3532]. Angiogenin/RNase hybrid enzymes--wherein particular regions of primary structure in RNase are replaced by the corresponding segments of angiogenin--serve to explore the structural features underlying angiogenin's characteristic activities. Herein we show that synthetic angiogenin peptides, Ang(1-21) and Ang(108-123), form noncovalent complexes with inactive fragments of bovine RNase A--RNase(21-124) (i.e., S-protein) and RNase(1-118), respectively--with regeneration of activity toward conventional RNase substrates. Maximal activities for the Ang(1-21)/S-protein complex (Kd = 1.0 microM) are 52%, 45%, and 15% toward cytidine cyclic 2',3'-phosphate, cytidylyl(3'----5')adenosine, and yeast RNA, respectively. In contrast, activities of the RNase(1-118)/Ang(108-123) hybrid (Kd = 25 microM) are 1-2 orders of magnitude lower toward cyclic nucleotides and dinucleoside phosphates. However, substitution of phenylalanine for Leu-115 in Ang(108-123) increases activity up to 100-fold. Both His-13 and His-114 in the angiogenin peptides are required for activity since their substitution by alanine yields inactive complexes. Importantly, the pattern of polynucleotide products formed during cleavage of ribosomal RNA by the Ang(1-21)/S-protein hybrid shows a striking resemblance to that formed by angiogenin, demonstrating that the hybrid retains features of both angiogenin and RNase A.(ABSTRACT TRUNCATED AT 250 WORDS)     

The complete amino acid sequence of bovine milk angiogenin

The amino acid sequence of angiogenin isolated from bovine milk was deduced by gas-phase sequencing of the protein and its fragments. The protein contains 125 residues and has a calculated molecular mass of 14,577 Da. The sequence is highly homologous (65% identity) to the sequence of human angiogenin, most of the differences being the result of conservative replacements. Like human angiogenin, the bovine protein is also homologous to bovine pancreatic RNase A (34% identity) and the three major active site residues known to be involved in the catalytic process, His-14, Lys-41 and His-115, are conserved. When tested against conventional substrates for RNase A activity, bovine angiogenin displays the same selective ribonucleolytic activity as human angiogenin. The sequence of bovine angiogenin contains the cell recognition tripeptide Arg-Gly-Asp which is not present in the human protein.     

Amino acid sequence of bovine angiogenin

The amino acid sequence and disulfide bridges of bovine plasma derived angiogenin were determined by sequencer analysis of the intact protein and fragments derived by enzymatic and chemical digestion. Bovine angiogenin is a single-chain protein of 125 amino acids; it contains six cysteines and has a calculated molecular weight of 14,595. In contrast to the human protein its amino terminus is unblocked. It has the following sequence: H2N-Ala1-Gln-Asp-Asp-Tyr-Arg-Tyr-Ile-His-Phe10-Leu-Thr-Gln-His-Tyr -Asp-Ala-Lys- Pro-Lys20-Gly-Arg-Asn-Asp-Glu-Tyr-Cys-Phe-Asn-Met30-Met-Lys- Asn-Arg-Arg-Leu-Thr - Arg-Pro-Cys40-Lys-Asp-Arg-Asn-Thr-Phe-Ile-His-Gly-Asn50-Lys- Asn-Asp-Ile-Lys-Ala - Ile-Cys-Glu-Asp60-Arg-Asn-Gly-Gln-Pro-Tyr-Arg-Gly-Asp-Leu70- Arg-Ile-Ser-Lys-Ser - Glu-Phe-Gln-Ile-Thr80-Ile-Cys-Lys-His-Lys-Gly-Ser-Ser-Arg90- Pro-Pro-Cys-Arg-Tyr - Gly-Ala-Thr-Glu-Asp100-Ser-Arg-Val-Ile-Val-Val-Gly-Cys-Glu-Asn1 10-Gly-Leu-Pro- Val-His-Phe-Asp-Glu-Ser-Phe120-Ile-Thr-Pro-Arg-His-OH. Disulfide bonds link Cys(27)-Cys(82), Cys(40)-Cys(93), and Cys(58)-Cys(108). Bovine angiogenin is 64% identical with human angiogenin; like the human protein, it is homologous to the pancreatic ribonucleases, with conservation of active site residues. Two regions, 6-22 and 65-75, are highly conserved between the angiogenins but are significantly different from those of the ribonucleases, suggesting a possible role in the molecules' biological activity.     

A covalent angiogenin/ribonuclease hybrid with a fourth disulfide bond generated by regional mutagenesis

Human angiogenin is a blood vessel inducing protein whose primary structure displays 33% identity to that of bovine pancreatic ribonuclease A (RNase A). Angiogenin catalyzes limited cleavage of 18S and 28S ribosomal RNA and is several orders of magnitude less potent than RNase A toward conventional substrates. A striking structural difference between angiogenin and RNase is the virtual absence of sequence similarity within the region of RNase that contains the Cys-65--Cys-72 disulfide bond. Indeed, angiogenin lacks this disulfide linkage. The present report describes the use of regional mutagenesis to generate a covalent angiogenin/RNase hybrid protein, ARH-I, where residues 58-70 of angiogenin have been replaced by the corresponding segment of RNase A (residues 59-73). The protein expressed in Escherichia coli readily folds at pH 8.5 to form the four expected disulfide bonds. The in vivo angiogenic potency of ARH-I is markedly diminished compared with that of angiogenin when examined using the chick chorioallantoic membrane assay. In contrast, its enzymatic activity is dramatically increased. With high molecular weight wheat germ RNA and tRNA, ARH-I is 660- and 300-fold more active than angiogenin, respectively, while with poly(uridylic acid), poly(cytidylic acid), cytidylyl(3'----5')adenosine (CpA), and uridylyl(3'----5')adenosine (UpA) activity is enhanced by about 200-fold. In addition, the specificity of ARH-I toward dinucleoside 3',5'-phosphates is qualitatively similar to RNase A; while angiogenin prefers cytidylyl(3'----5')guanosine (CpG) to UpA, both RNase and the hybrid prefer UpA to CpG. ARH-I also displays greater than 10-fold enhanced activity toward rRNA in intact ribosomes, while abolishing the capacity of the ribosome to support cell-free protein synthesis. The enhanced enzymatic properties of ARH-I parallel a 2-fold increase in chemical reactivity of active-site lysine and histidine residues based on rates of chemical modification. The data indicate that introduction of a region of RNase A containing the Cys-65--Cys-72 disulfide bond into angiogenin dramatically increases RNase-like enzymatic activity while reducing its angiogenicity.     

Binding of placental ribonuclease inhibitor to the active site of angiogenin

The importance of specific residues in angiogenin for binding to placental ribonuclease inhibitor (PRI) has been assessed by examining the interaction of angiogenin derivatives with PRI. PRI binds native angiogenin with a Ki value of 7.1 X 10(-16) M [Lee, F. S., Shapiro, R., & Vallee, B. L. (1989) Biochemistry 28, 225-230]. Substitution of a Gln for Lys-40 in angiogenin by site-specific mutagenesis decreases the association rate constant 3-fold and increases the dissociation rate constant 440-fold, resulting in a 1300-fold weaker Ki value. The half-life of the mutant.PRI complex is 3.4 h compared to approximately 60 days for the native angiogenin.PRI complex. The magnitude of the change in Ki value suggests that in the complex, Lys-40 forms a salt bridge or hydrogen bond with an anionic moiety in PRI. Carboxymethylation of His-13 or His-114 with bromoacetate increases the Ki value 15-fold, and oxidation of Trp-89 by means of dimethyl sulfoxide and hydrochloric acid increases it 2.4-fold, suggesting that these residues also form part of the contact region with PRI. The changes in Ki value reflect an increase in the dissociation rate constant. On the other hand, dinitrophenylation of either Lys-50 or Lys-60 with 1-fluoro-2,4-dinitrobenzene does not significantly alter the Ki value, suggesting that these residues are not part of the contact region. These results indicate that PRI inhibition minimally involves the three residues critical for the activity of angiogenin--Lys-40, His-13, and His-114--and to a lesser extent its single tryptophan, Trp-89.     

HnRNP from HeLa cells contain a ribonuclease active on double-stranded RNA.

A ribonuclease D, i-e acting against double-stranded RNA structures like poly r(AU), was identified in ribonucleoprotein structures containing the heterogenous nuclear RNA (hnRNP) from HeLa cells. This activity could not however be detected in intact hnRNP but only after passage through a DEAE-cellulose column or digestion by a combination of ribonucleases A+T₁. This enzyme does not degrade poly r(A)-poly d(T) nor poly r(A), nor does it yield mononucleotides, excluding the possibility of a non-specific exonuclease type of activity like phosphodiesterase. It is inhibited by ethidium bromide and double-stranded RNA and resembles in all respects so far investigated the ribonuclease D previously isolated from Krebs cells by Rech et al (Nucl. Acids Res. 1976, 3, 2055–2065).     

Replacement of residues 8-22 of angiogenin with 7-21 of RNase A selectively affects protein synthesis inhibition and angiogenesis.

The region of human angiogenin containing residues 8-21 is highly conserved in angiogenins from four mammalian species but differs substantially from the corresponding region of the homologous protein ribonuclease A (RNase A). Regional mutagenesis has been employed to replace this segment of angiogenin with the corresponding RNase A sequence, and the activities of the resulting covalent angiogenin/RNase hybrid, designated ARH-III, have been examined. The ribonucleolytic activity of ARH-III is unchanged toward most substrates, including tRNA, naked 18S and 28S rRNA, CpA, CpG, UpA, and UpG. In contrast, the capacity of ARH-III to inhibit cell-free protein synthesis is decreased 20-30-fold compared to that of angiogenin. The angiogenic activity of ARH-III is also different; it is actually more potent. It induces a maximal response in the chick chorioallantoic membrane assay at 0.1 ng per egg, a 10-fold lower dose than required for angiogenin. In addition, binding of ARH-III to the placental ribonuclease inhibitor is increased by at least 1 order of magnitude (Ki less than or equal to 7 x 10(-17) M) compared to angiogenin. Thus, mutation of a highly conserved region of angiogenin markedly affects those properties likely involved in its biological function(s); it does not, however, alter ribonucleolytic activity toward most substrates.     

Amino acid sequence of the nonsecretory ribonuclease of human urine

The amino acid sequence of a nonsecretory ribonuclease isolated from human urine was determined except for the identity of the residue at position 7. Sequence information indicates that the ribonucleases of human liver and spleen and an eosinophil-derived neurotoxin are identical or very closely related gene products. The sequence is identical at about 30% of the amino acid positions with those of all of the secreted mammalian ribonucleases for which information is available. Identical residues include active-site residues histidine-12, histidine-119, and lysine-41, other residues known to be important for substrate binding and catalytic activity, and all eight half-cystine residues common to these enzymes. Major differences include a deletion of six residues in the (so-called) S-peptide loop, insertions of two, and nine residues, respectively, in three other external loops of the molecule, and an addition of three residues at the amino terminus. The sequence shows the human nonsecretory ribonuclease to belong to the same ribonuclease superfamily as the mammalian secretory ribonucleases, turtle pancreatic ribonuclease, and human angiogenin. Sequence data suggest that a gene duplication occurred in an ancient vertebrate ancestor; one branch led to the nonsecretory ribonuclease, while the other branch led to a second duplication, with one line leading to the secretory ribonucleases (in mammals) and the second line leading to pancreatic ribonuclease in turtle and an angiogenic factor in mammals (human angiogenin). The nonsecretory ribonuclease has five short carbohydrate chains attached via asparagine residues at the surface of the molecule; these chains may have been shortened by exoglycosidase action.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation and characterization of a human colon carcinoma-secreted enzyme with pancreatic ribonuclease-like activity

A ribonuclease was isolated from serum-free supernatants of the human colon adenocarcinoma cell line HT-29. It was purified by cation-exchange and C18 reversed-phase high-performance liquid chromatography. The protein is basic, has a molecular weight of approximately 16,000, and has an amino acid composition that is significantly different from that of human pancreatic ribonuclease. The amino terminus is blocked, and the carboxyl-terminal residue is glycine. The catalytic properties of this ribonuclease resemble those of the pancreatic ribonucleases in numerous respects. Thus, it exhibits a pH optimum of approximately 6 for dinucleotide cleavage and employs a two-step mechanism in which transphosphorylation to a cyclic 2',3'-phosphate is followed by slower hydrolysis to produce a 3'-phosphate. It does not cleave NpN' substrates in which adenosine or guanosine is at the N position and prefers purines at the N' position. Like bovine ribonuclease A, the HT-29-derived ribonuclease is inactivated by reductive methylation or by treatment with iodoacetate at pH 5.5 and is strongly inhibited by the human placental ribonuclease inhibitor. However, in contrast, the tumor enzyme does not cleave CpN bonds at an appreciable rate and prefers poly(uridylic acid) as substrate 1000-fold over poly(cytidylic acid). It also hydrolyzes cytidine cyclic 2',3'-phosphate at least 100 times more slowly than uridine cyclic 2',3'-phosphate and is inhibited much less strongly by cytidine 2'-monophosphate than by uridine 2'-monophosphate. Other ribonucleases known to prefer poly(uridylic acid) were isolated both from human serum and from liver and were compared with the tumor enzyme. The physical, functional, and chromatographic properties of the serum ribonuclease are essentially identical with those of the tumor enzyme. The liver enzymes, however, differ markedly from the HT-29 ribonuclease. The potential utility of the tumor ribonuclease in the diagnosis of cancer is considered.     

The effect of RNA upon the removal of RNase by phenol treatment

A small point is made in this article which nevertheless needs to be known to properly interpret certain experiments; namely, that RNA itself protects pancreatic ribonuclease from reagents employed to achieve its removal. Although it has been suggested that phenol treatment alone does not quantitatively remove ribonuclease activity from preparations of RNA derived from crude homogenates (1,2), it has not been previously recognized that one of the factors responsible for the incomplete removal of nuclease is the presence of RNA. Owing to this circumstance (i.e., the differential removal of nuclease in the presence and absence of RNA), control tubes, set up for the purpose of judging how well nuclease had been removed from the system, may give totally misleading results. This phenomenon is not common to all ribonucleases, since under the conditions employed, RNA does not prevent the complete removal of T₁ nuclease by phenol treatment.     

Amino acid sequence of human tumor derived angiogenin

The amino acid sequence and disulfide bond pairing of human tumor derived angiogenin, the first tumor angiogenesis factor to be isolated in pure form from human sources, have been determined by conventional sequencing techniques adapted and applied to nanomole and subnanomole levels of material. Angiogenin, obtained from conditioned media of a human colonic adenocarcinoma cell line, is a single-chain protein consisting of 123 amino acids with the following sequences: less than Glu1-Asp-Asn-Ser-Arg-Tyr-Thr-His- Phe-Leu-Thr-Gln-His-Tyr-Asp15-Ala-Lys-Pro-Gln-Gly-Arg-Asp-Asp- Arg-Tyr-Cys-Glu-Ser-Ile-Met30- Arg-Arg-Arg-Gly-Leu-Thr-Ser-Pro-Cys-Lys-Asp-Ile-Asn-Thr- Phe45-Ile-His-Gly-Asn-Lys-Arg-Ser -Ile-Lys-Ala-Ile-Cys-Glu-Asn-Lys60-Asn-Gly-Asn-Pro-His-Arg-Glu-Asn -Leu-Arg-Ile -Ser-Lys-Ser-Ser75 -Phe-Gln-Val-Thr-Thr-Cys-Lys-Leu-His-Gly-Gly-Ser-Pro-Trp-Pro90-Pro -Cys-Gln-Tyr -Arg-Ala-Thr-Ala -Gly-Phe-Arg-Asn-Val-Val-Val105-Ala-Cys-Glu-Asn-Gly-Leu-Pro-Val- His-Leu-Asp-Gln-Ser-Ile-Phe120-Arg-Arg-Pro123-OH. Three disulfide bonds link the half-cystinyl residues 26-81, 39-92, and 57-107. The sequence is homologous to that of the pancreatic ribonucleases with 35% identity and many of the remaining residues conservatively replaced. Similarities are especially apparent around the major active-site residues His-12, Lys-41, and His-119 of ribonuclease which are conserved as are three of the four disulfide bonds.(ABSTRACT TRUNCATED AT 250 WORDS)     

The synthesis of polyadenylic acid-containing ribonucleic acid by isolated mitochondria from Ehrlich ascites cells.

The synthesis of poly(A)-containing RNA by isolated mitochondria from Ehrlich ascites cells was studied. Isolated mitochondria incorporate [3H]AMP or [3H]UTP into an RNA species that adsorbs on oligo (dT)-cellulose columns or Millipore filters. Hydrolysis of the poly(A)-containing RNA with pancreatic and T1 ribonucleases released a poly(A) sequence that had an electrophoretic mobility slightly faster than 4SE. In comparison, ascites-cell cytosolic poly(A)-containing RNA had a poly(A) tail that had an electrophoretic mobility of about 7SE. Sensitivity of the incorporation of [3H]AMP into poly(A)-containing RNA to ethidium bromide and to atractyloside and lack of sensitivity to immobilized ribonuclease added to the mitochondria after incubation indicated that the site of incorporation was mitochondrial. The poly(A)-containing RNA sedimented with a peak of about 18S, with much material of higher s value. After denaturation at 70 degrees C for 5 min the poly(A)-containing RNA separated into two components of 12S and 16S on a 5-20% (w/v) sucrose density gradient at 4 degrees C, or at 4 degrees and 25 degrees C in the presence of formaldehyde. Poly(A)-containing RNA synthesized in the presence of ethidium bromide sedimented at 5-10S in a 15-33% (w/v) sucrose density gradient at 24 degrees C. The poly(A) tail of this RNA was smaller than that synthesized in the absence of ethidium bromide. The size of the poly(A)-containing RNA (approx. 1300 nucleotides) is about the length necessary for that of mRNA species for the products of mitochondrial protein synthesis observed by ourselves and others.     

Angiogenin cleaves tRNA and promotes stress-induced translational repression

Stress-induced phosphorylation of eIF2α inhibits global protein synthesis to conserve energy for repair of stress-induced damage. Stress-induced translational arrest is observed in cells expressing a nonphosphorylatable eIF2α mutant (S51A), which indicates the existence of an alternative pathway of translational control. In this paper, we show that arsenite, heat shock, or ultraviolet irradiation promotes transfer RNA (tRNA) cleavage and accumulation of tRNA-derived, stress-induced small RNAs (tiRNAs). We show that angiogenin, a secreted ribonuclease, is required for stress-induced production of tiRNAs. Knockdown of angiogenin, but not related ribonucleases, inhibits arsenite-induced tiRNA production and translational arrest. In contrast, knockdown of the angiogenin inhibitor RNH1 enhances tiRNA production and promotes arsenite-induced translational arrest. Moreover, recombinant angiogenin, but not RNase 4 or RNase A, induces tiRNA production and inhibits protein synthesis in the absence of exogenous stress. Finally, transfection of angiogenin-induced tiRNAs promotes phospho-eIF2α–independent translational arrest. Our results introduce angiogenin and tiRNAs as components of a phospho-eIF2α–independent stress response program.     

Base cleavage specificity of angiogenin with Saccharomyces cerevisiae and Escherichia coli 5S RNAs

The base cleavage specificity of angiogenin toward naturally occurring polyribonucleotides has been determined by using rapid RNA sequencing technology. With 5S RNAs from Saccharomyces cerevisiae and Escherichia coli, angiogenin cleaves phosphodiester bonds exclusively at cytidylic or uridylic residues, preferably when the pyrimidines are followed by adenine. However, not all of the existent pyrimidine bonds in the 5S RNAs are cleaved, likely owing to elements of structure in the substrate. Despite the high degree of sequence homology between angiogenin and ribonuclease A (RNase A), which includes all three catalytic as well as substrate binding residues, the cleavage patterns with natural RNAs are unique to each enzyme. Angiogenin significantly hydrolyzes certain bonds that are not appreciably attacked by RNase A and vice versa. The different cleavage specificities of angiogenin and RNase A may account for the fact that the former is angiogenic while the latter is not.     

Isolation and characterization of polyadenylate-containing RNA from Bacillus brevis.

A substantial fraction (30--40%) of pulse-labeled RNA from exponentially growing cells of Bacillus brevis contains polyadenylate sequences, as measured by adsorption to oligo(dT)-cellulose. The weight-average length of poly(A) tracts obtained after digestion with pancreatic and T1 ribonucleases is 60 nucleotide residues. Susceptibility to degradation by snake venom phosphodiesterase after ribonuclease degradation indicates that the poly(A) sequences are located near the 3' ends of the RNA chains, but that in 40% of the material at least one internal pyrimidine nucleotide residue intervenes between the poly(A) tract and the 3'-hydroxyl terminus. These pyrimidine nucleotides consist of 65% cytidylate and 35% uridylate residues. In the remaining RNA chains, the poly(A) sequence is directly at the 3'-terminus, but the possibility cannot be excluded that a small fraction of this material may contain a 3'-hydroxyl terminal guanylate residue. The weight-average sedimentation coefficient of poly(A)-containing RNA is 12.5 S, corresponding to a polynucleotide chain length of 800--900 residues. This is in a size range expected for messenger RNA, a possibility which is also supported by the observation that pulse-labeled RNA has a considerably higher poly(A) content than long-term labeled RNA.     

First demonstration of lactoribonuclease, a ribonuclease from bovine milk with similarity to bovine pancreatic ribonuclease

The isolation of a ribonuclease designated lactoribonuclease, with a molecular weight and an N-terminal amino acid sequence identical to those of bovine pancreatic ribonuclease, was first reported from bovine milk. After removal of globulin from acid whey by precipitation with 1.8 M (NH4)2SO4, (NH4)2SO4 was added to attain a concentration of 3.6 M. Adsorption on the ion exchanger CM-Sepharose and subsequently on Mono S by fast protein liquid chromatography yielded pure lactoribonuclease. The enzyme, like pancreatic ribonuclease, was most active at pH 7.5 with yeast transfer RNA (tRNA) as substrate. Lactoribonuclease and pancreatic ribonuclease showed a strong preference for poly(C) over poly(U). However, pancreatic ribonuclease did so with a higher specific activity, suggesting that the two ribonucleases are not identical. No inhibitory effect was shown by either lactoribonuclease or pancreatic ribonuclease toward poly (A) and poly (G). The effect of lactoribonuclease and pancreatic ribonuclease on tRNA increased with the concentration of tRNA. Lactoribonuclease inhibited cell-free translation in a rabbit reticulocyte lysate system with an IC50 of 3.5 nM while the corresponding IC50 for pancreatic ribonuclease was 0.09 nM.     

Molecular evolution of pancreatic-type ribonucleases

Amino acid sequences of 39 mammalian ribonucleases have been used to construct trees by the maximum parsimony procedure. These trees are in fairly good agreement with the biological classification of the species involved. In the branching order of the six investigated eutherian mammalian orders, the edentates diverge first, followed, probably, by the primates. No definite conclusions can be drawn about the order of divergence of the perissodactyls, the rodents, and the group consisting of artiodactyls plus cetaceans. Nucleic acid sequences of part of the messenger RNAs of rat pancreatic and bovine seminal ribonuclease were compared. Both messengers have a second stop codon at position 129, which is in agreement with the addition of four residues at the C-terminus in several other ribonucleases. Turtle pancreatic ribonuclease and human angiogenin differ from each other and from the mammalian ribonucleases at 55%-70% of the amino acid positions; they share a number of structural features. Mammalian nonsecretory ribonucleases are homologous to the pancreatic ribonucleases in sequence regions where the active-site histidine residues are located.     

Adenylate-rich oligonucleotides of ribosomal and ribosomal precursor RNA from HeLa cells

HeLa cell ribosomal precursor (45S) RNA has been found to contain nucleotide sequences also found in 18S RNA and others found in 28S RNA. Thus, 18S RNA and 28S RNA are readily distinguishable, whereas 45S RNA is similar to 18S and 28S RNA combined. The nucleotide sequences analysed were those resistant to the combined action of pancreatic and T1 ribonucleases.     

Ribonucleases and proteins related to it, as well as ribonuclease inhibitors of proteinaceous nature

The recent discovery of unexpected functional diversity of different RNA molecules and some products of their degradation renewed the interest in ribonucleases. This review mainly presents recent systematized data on animal ribonucleases, including pyrimidine-specific ribonucleases, angiogenin, eosinophil-derived neurotoxin, eosinophil cationic protein, and lectins from frog eggs. The relationship between amino acid sequences and possible pathways of the evolution of this protein superfamily is discussed, as well as properties and primary structures of protein ribonuclease inhibitors.