Proton nuclear magnetic resonance studies of histidine residues in rat and other rodent pancreatic ribonucleases. Effects of pH and inhibitors

Proton nuclear magnetic resonance spectra of the histidine residues in bovine and rat ribonuclease have been compared. The changes in chemical shift on titration and on binding of cytidine-3′-monophosphate and cytidine-2′-monophosphate have been followed. In the presence of the cytidine derivatives the spectra of both enzymes resemble each other more than those of the free enzymes. With these inhibitors, two histidines in rat ribonuclease exhibit the same pK values and shifts as the active site residues histidine 12 and 119 in the bovine enzyme. Their pK values in the inhibitor-free rat enzyme are about 0.4 higher than in the beef enzyme, which can be explained by the substitution at the entrance of the active site cleft of arginine 39 in the beef enzyme by serine in the rat enzyme. Rat ribonuclease contains one histidine with a rather high pK value of 7.6. The cytidine derivatives affect its chemical shift in exactly the same way as the shift of histidine 48 in bovine ribonuclease. The high pK value of this residue in rat ribonuclease can be explained by assuming a strong hydrogen bridge with glutamic acid 16. The other two histidines in rat ribonuclease have rather low pK values of 6.1 and 6.3. The histidine with a pK value of 6.3 has been assigned to position 105 and that with a pK value of 6.1 to position 73.The closer resemblance of the active sites of bovine and rat ribonuclease in the presence of inhibitors than in the inhibitor-free enzymes makes the concept of induced fit interesting from an evolutionary point of view.The characteristic downfield shift of the protonated form of histidine 119 in the complexes of bovine and rat ribonuclease with cytidine-3′-monophosphate is not observed with uridine-3′-monophosphate, suggesting non-identical binding of these pyrimidine nucleotides.Some preliminary results on the nuclear magnetic resonance properties of the histidine residues in coypu and chinchilla pancreatic ribonuclease have been obtained.     

Purification of rat liver particulate neutral ribonuclease and comparison of properties with pancreas and serum ribonucleases.

Rat liver particulate neutral ribonuclease (EC 3.1.4.22) was extensively purified (up to 40000-fold). It is shown to be an endonuclease, specific for pyrimidine bases, hydrolysing 5'-phosphate ester bonds. The enzyme specificity, Km, pH optimum, stability in acid medium and thermal stability at high temperature are the same as those of rat pancreatic and serum ribonucleases. Like pancreatic and serum neutral ribonucleases, the hepatic enzyme is sensitive to the liver natural inhibitor. This inhibitor was purified 8000-fold; its association with ribonuclease follows zero-order kinetics. These identical properties for ribonuclease of rat liver, pancreas and serum support the hypothesis [Bartholeyns, Peeters-Joris & Baudhuin (1975) Eur. J. Biochem. 60, 385-393] of an extrahepatic origin for the liver enzyme, the plasma ribonuclease of pancreatic origin being taken up by endocytosis in the liver. Neutral ribonuclease activity was detected in all rat organs investigated; its distribution among tissues is different from the distribution of the natural ribonuclear inhibitor.     

An acid ribonuclease from rye germ cytosol

Acid ribonuclease from rye germ cytosol was purified 1200-fold. The enzyme is homogeneous on polyacrylamide gel. The optimum pH for ribonuclease activity is 5.8, its MW is 28 500. The enzyme is an endonuclease yielding in the first step of its activity oligonucleotides with a free —OH group in the 3′ position. The end products of RNA hydrolysis are cyclic purine and pyrimidine nucleoside phosphates and the corresponding nucleoside 3′-phosphates. This ribonuclease preferentially attacks sites close to the adenine base and shows a lag in the release of the cytosine base. Specificity tests on natural and synthetic substrates are in good agreement.     

The crystal structure of ribonuclease A in complex with thymidine‐3′‐monophosphate provides further insight into ligand binding

Thymidine‐3′‐monophosphate (3′‐TMP) is a competitive inhibitor analogue of the 3′‐CMP and 3′‐UMP natural product inhibitors of bovine pancreatic ribonuclease A (RNase A). Isothermal titration calorimetry experiments show that 3′‐TMP binds the enzyme with a dissociation constant (K_d) of 15 μM making it one of the strongest binding members of the five natural bases found in nucleic acids (A, C, G, T, and U). To further investigate the molecular properties of this potent natural affinity, we have determined the crystal structure of bovine pancreatic RNase A in complex with 3′‐TMP at 1.55 Å resolution and we have performed NMR binding experiments with 3′‐CMP and 3′‐TMP. Our results show that binding of 3′‐TMP is very similar to other natural and non‐natural pyrimidine ligands, demonstrating that single nucleotide affinity is independent of the presence or absence of a 2′‐hydroxyl on the ribose moiety of pyrimidines and suggesting that the pyrimidine binding subsite of RNase A is not a significant contributor of inhibitor discrimination. Accumulating evidence suggests that very subtle structural, chemical, and potentially motional variations contribute to ligand discrimination in this enzyme. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

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 ribonucleases of bovine skeletal muscle.

Bovine skeletal muscle contains small amounts of at least six heat- and acid-stable RNA-degrading enzymes. Our results are the first evidence for multiple ribonucleases in skeletal muscle. Three of these have been highly purified, and each has been shown to be a pyrimidine-specific endoribonuclease by use of a rapid sequencing technique employing gel electrophoresis. However, synthetic co-polymers containing adenylate or guanylate residues in addition to pyrimidine residues are hydrolysed at higher rates than are the pyrimidine homopolymers. With 0.63 mM yeast RNA as substrate, all three enzymes (ribonucleases I, II and III) are optimally active in alkaline solution (pH 7.5-8.5) containing 0.05-0.15 M univalent salts, do not require bivalent cations, and have molecular weights of 13 000-20 000. The properties of muscle ribonuclease I are very similar to those of bovine pancreatic ribonuclease A. Muscle ribonucleases II and III have characteristics similar to those of ribonucleases found in various other bovine tissues. In common with all previously studied pyrimidine-specific endoribonucleases, the bovine muscle ribonucleases are inhibited by such purine homopolynucleotides as polyadenylate. Furthermore, polyamines, present in low concentrations, can reverse or regulate the amount of inhibition of enzyme activity.     

The primary structure of langur (Presbytis entellus) pancreatic ribonuclease: adaptive features in digestive enzymes in mammals

The primary structure of pancreatic ribonuclease from langur (Presbytis entellus) has been determined. This sequence differs from that of human pancreatic ribonuclease at 14 (11%) of the amino acid positions. Eight of these 14 differences involve changes of charge, with the langur enzyme having five fewer positive charges than the human enzyme. The difference in charge between human and langur ribonuclease may be an adaptation to the different requirements for a nondigestive and a digestive role, respectively. A number of similarities in expression, gene duplications, and properties between mammalian ribonucleases and lysozymes have been observed, indicating similar adaptations in both enzyme systems.     

Covalent attachment of 4-thiouridylic acid to ribonuclease A by near-ultraviolet radiation.

Bovine pancreatic ribonuclease A (EC.2.7.7.16) was irradiated with near-ultraviolet light (334 and 365 nm) in the presence of equimolar amount of a substrate analog 4-thio[¹⁴C]uridine 3′-phosphate. Gel-filtration studies revieled that one to two moles of the nucleotide entered into covalent attachment to the enzyme under either aerobic or anaerobic irradiation. Reduction with dithiothreitol of the irradiated protein released about one-third of the attached materials. A model experiment with oxidized glutathione and radioactive 4-thiouridine suggested the formation of aducts between cystinyl residue and the pyrimidine base. The covalent attachment of nucleotide to ribonuclease was independent of inactivation of the enzyme.     

Cell wall-bound trehalase in cultured cells of Japanese morning-glory

Occurrence and distribution of trehalase were examined in cytoplasmic and cell wall fractions of cultured cells of morning-glory, soybean and persimmon. Also, some enzymatic properties and solubilization of the enzyme from cell walls were examined. Trehalase was present in both fractions of morning-glory and persimmon cells while trehalase was present only in the cytoplasmic fraction of soybean cells. Morning-glory trehalases in both fractions showed the same optimum pH at 5.5, while persimmon trehalases in both fractions showed the same optimum pH at 6.0. Soybean enzyme in the cytoplasmic fraction showed two optimum activities at 4.0 and 6.5. Morning-glory cell wall bound trehalase was solubilized with various IM salts at about 70 to 75%. Also, the enzyme was solubilized with various buffers and the solubilization ratio increased with increasing in pH of a same series buffer. After multiple extractions with IM NaCl, about 15% of the original trehalase activity still remained in cell walls. On the other hand, Triton X-100 and the substrate, trehalose, at the various concentrations did not release trehalase from cell walls. Invertase and cellobiase solubilized from morning-glory cell walls were re-adsorbed to the cell walls. However, readsorption of trehalase to cell walls has not yet been attained. Based on these results, physiological roles of plant cell wall-bound trehalase were discussed.     

RNA-Dependent RNA Polymerase in Nuclei of Cells Infected with Influenza Virus

Nuclei purified from chicken embryo fibroblast cells infected with influenza (fowl plague) virus contain an RNA-dependent RNA polymerase. The in vitro activity of this enzyme is insensitive to actinomycin D, and is completely destroyed by preincubation with ribonuclease. Enzyme induction is prevented if cells are treated with actinomycin D or cycloheximide at the time of infection. RNA-dependent RNA polymerase activity increases rapidly in cell nuclei from 1 h postinfection, reaches a maximum at 3 to 4 h, then declines; a similar RNA polymerase activity in the microsomal cell fraction increases from 2 h postinfection and reaches a maximum at 5 to 6 h. The characteristics of the nuclear and microsomal enzymes in vitro are similar with respect to pH and divalent cation requirements. The in vitro products of enzyme activity present in the nuclear and microsomal fractions of cells infected for 3 and 5 h were characterized by sucrose density gradient analysis, and annealing to virion RNA. The microsomal RNA polymerase product contained 67 and 93% RNA complementary to virion RNA at 3 and 5 h, respectively; for the nuclear RNA polymerase product these values were 40% in each case.     

The amino-acid sequence of kangaroo pancreatic ribonuclease

Red kangaroo (Macropus rufus) ribonuclease was isolated from pancreatic tissue by affinity chromatography. The amino acid sequence was determined by automatic sequencing of overlapping large fragments and by analysis of shorter peptides obtained by digestion with a number of proteolytic enzymes. The polypeptide chain consists of 122 amino acid residues. Compared to other ribonucleases, the N-terminal residue and residue 114 are deleted. In other pancreatic ribonucleases position 114 is occupied by a cis proline residue in an external loop at the surface of the molecule. Other remarkable substitutions are the presence of a tyrosine residue at position 123 instead of a serine which forms a hydrogen bond with the pyrimidine ring of a nucleotide substrate, and a number of hydrophobichydrophilic interchanges in the sequence 51-55, which forms part of an alpha-helix in bovine ribonuclease and exhibits few substitutions in the placental mammals. Kangaroo ribonuclease contains no carbohydrate, although the enzyme possesses a recognition site for carbohydrate attachment in the sequence Asn-Val-Thr (62-64). The enzyme differs at about 35-40% of the positions from all other mammalian pancreatic ribonucleases sequenced to date, which is in agreement with the early divergence between the marsupials and the placental mammals. From fragmentary data a tentative sequence of red-necked wallaby (Macropus rufogriseus) pancreatic ribonuclease has been derived. Eight differences with the kangaroo sequence were found.     

Purification of an endonuclease from adenovirus-infected KB cells.

This report describes the purification of an endonuclease from extracts of adenovirus-type-2-infected KB cells. Endonuclease activity can also be detected in extracts of uninfected KB cells and the enzyme activities from extracts of uninfected and adenovirus-infected cells are very similar, if not identical. The enzyme has its maximal activity at pH 4.0. The enzyme found in uninfected and adenovirus-infectedcells is, however, strikingly different from an endonuclease isolated from calf serum. Hence, the endonuclease described is probably not a contaminant derived from the medium in which the KB cells were propagated. The endonuclease in crude extracts from uninfected or adenovirus-infected KB cells can be activated or its activity enhanced by treatment of the extracts with proteolytic enzymes, like pronase or trypsin. Evidence has been presented suggesting that this activation is due to proteolytic cleavage of an inhibitor present in crude extracts of uninfected and adenovirus-type-2-infected KB cells. A second endonuclease has been found in extracts of infected and uninfected cells with optimal activity at pH 7.2 and this endonuclease can be separated from the one with a pH optimum at 4.0.     

Characterization of a ribonuclease from bovine brain.

An alkaline ribonuclease (pH optimum near 8) has been purified from whole beef brains and found to have a base specificity like that of bovine pancreatic ribonuclease, but in most other respects to be distinguishable from the enzymes of bovine pancreas, semen, or brain nuclei. The preparation appears homogeneous in sedimentation equilibrium and probably so in polyacrylamide gel electrophoresis under normal or dissociating conditions. Sedimentation equilibrium and SDS gel electrophoresis both indicate a molecular weight of 2.4-2.6 times 10-4, and tryptic and chymotrypic peptide patterns are consistent with a protein of this size. No dissociation into subunits has been attained. The enzyme is not precipitated by antiserum to pancreatic ribonuclease, although its activity is inhibited by this antiserum with low efficiency. In comparisons of the hydrolysis of RNA the brain enzyme was found to have a similar specificity to pancreatic RNase, but to have a loser Km for RNA and to produce significantly different oligonucleotides upon partial hydrolysis of bacteriophage RNA, suggesting differences in the mechanism of substrate recognition. In contrast, nuclease inactivation by iodoacetate at pH 5.5 is indistinguishable for pancreatic or purified brain RNase.     

The ribonuclease inhibitors from porcine thyroid and liver are slow, tight-binding inhibitors of bovine pancreatic ribonuclease A

Ribonuclease inhibitors were purified from the latent ribonuclease fractions of porcine thyroid and liver and used to test the hypothesis that their inhibition of bovine pancreatic ribonuclease A is correctly described by tight-binding rather than Michaelis-Menton kinetics. Both proteins were found to act as slow, tight-binding inhibitors of the enzyme. These steady-state velocities also showed that both the thyroid and liver inhibitors were competitive inhibitors of bovine pancreatic ribonuclease A with Ki's of 0.1 and 0.4 nM, respectively. In contrast to interpretations based on Michaelis-Menton assumptions that show non-competitive inhibition, these results suggest that an enzyme:inhibitor:substrate complex does not exist.     

The return of pancreatic ribonucleases

A decade after losing favor as an 'uninteresting' digestive enzyme, pancreatic ribonuclease has been found to be homologous to a series of extracellular proteins that may influence tumor cell growth, neurological development and biological differentiation. One surprising outcome of these discoveries has been the confirmation of the hypothesis that extracellular 'communicator RNA' is a messenger important in cell growth and differentiation. The only question is: why wasn't this recognized earlier?     

Ribonucleases expressed by human pancreatic adenocarcinoma cell lines.

Human ribonucleases have been considered as a possible tumor marker for pancreatic cancer, and elevated serum levels of ribonuclease activity in patients with pancreatic cancer have been reported by many authors. The reason for this elevation is unknown. In this study, we demonstrate that human pancreatic adenocarcinoma cell lines synthesize and secrete different ribonucleases. We isolated and characterized human pancreatic, or secretory, ribonuclease (RNase 1) from the conditioned media of the human pancreatic adenocarcinoma cell lines Capan-1, MDAPanc-3, IBF-CP3 and Panc-1, and the ampullary adenocarcinoma cell line MDAAmp-7, which represent a wide range of differentiation stages. Only one of these cell lines, Panc-1, produces significant amounts of nonsecretory ribonuclease. We then established a purification procedure for both secretory and nonsecretory ribonucleases, consisting of concentration of the supernatant by tangential filtration, anion-exchange and cation-exchange liquid chromatography and C4 RP-HPLC. Ribonuclease activity fractions were monitored using both the spectrophotometric and negative-staining zymogram techniques. The results of N-terminal sequence analysis, kinetic analysis and endoglycosidase digestion studies indicate that the main ribonuclease secreted by all the cell lines is the secretory-type ribonuclease and that it is composed of several differently N-glycosylated forms. Northern blot analyses confirm that some of the cell lines express secretory ribonuclease mRNA. The mRNA levels produced by Panc-1 and MDAPanc-28 are too low to be detected. Similar levels of expression of nonsecretory ribonuclease are found by Northern blot analysis in all the cell lines except Panc-1, which expresses higher levels. Here, we describe, for the first time, that several human pancreatic cancer cell lines with different degrees of differentiation express and secrete ribonucleases. This fact indicates that one origin of the elevated serum RNase levels in patients with pancreatic cancer are tumor cells. Analysis of the oligosaccharide moiety of the RNase 1 secreted by Capan-1 shows that it is highly glycosylated and its N-glycan chains are significantly different from that of the RNase 1 produced by normal pancreas. These results renew the possibility of using human serum RNase 1 determination as a tumor marker.     

Ribonuclease H from rat liver. I. Partial purification and characterization of nuclear ribonuclease H1.

A ribonuclease H, an enzyme that specifically degrades the RNA moiety of RNA-DNA hybrid, has been partially purified from rat liver nuclei and characterized. Neither native or denatured DNA, nor single or double-stranded synthetic polyribonucleotides were degraded by the enzyme. The enzyme possesses a molecular weight of about 36,000 and requires alkaline pH, magnesium ions, and ammonium sulphate for maximum activity. The enzyme acts on the hybrid as an endonuclease, resulting in oligonucleotides with 3'-hydroxyl termini. The properties of this enzyme were distinct from those of the rat liver cytosol enzyme reported by Roewekamp and Sekeris in many respects, such as molecular weight, optimal pH and requirements for divalent cations. Preliminary experiments suggest that the nuclear enzyme is localized in the nucleoplasm and nucleoli. These results indicate that multiple forms of ribonuclease H exist in different regions of rat liver cells.     

Identification of a ribonuclease P-like activity from human KB cells.

An endoribonuclease which cleaves tRNA precursor molecules has been partially purified from human KB tissue culture cells. This activity is found in cytoplasmic fractions but is not detectable in the nucleoplasm. tRNA precursor molecules from both E. coli and KB cells are cleaved by this novel activity to produce 5' phosphate-terminated oligonucleotides. E coli RNAase P and the KB cell nuclease both make a single endonucleolytic scission in E. coli tRNATyr precursor, thereby separating the 41 extra nucleotides on the 5' end of the precursor molecule from the 5' terminal sequence of the mature tRNATyr molecule. The cleavage products generated from other E. coli tRNA precursors by the KB cell activity are identical in size to those produced by RNAase P. The KB cell endoribonuclease requires Mg2+ and a monovalent cation (Na+, K+, or NH4+) for function. The enzymatic activity has a broad pH optimum, centered near pH 8.0, and the activity is inhibited by tRNA. Several KB cell RNAs with long half-lives in vivo, including 5S and bulk 4S RNA, are not cleaved by this nuclease. The KB cell endoribonuclease resembles E. coli RNAase P in its substrate specificity, pH optimum, ion requirements, and sensitivity to tRNA. These properties and the cytoplasmic localization of the novel endoribonuclease indicate its involvement in the biosynthesis of KB cell tRNA.     

Human platelet ribonuclease

Human platelets contain an RNase which has a pH optimum at 5.0. It hydrolyzes the secondary phosphate esters of uridine 3′-phosphates. It slowly converts uridine 2′:3′-and cytidine 2′:3′-cyclic phosphates to their corresponding nucleoside 3′-phosphates. Poly (A), poly (G) and poly (C) are not only refractory to the action of this enzyme, but also inhibit its action on poly (U). It differs from human granulocyte RNase, human serum RNase and bovine pancreatic RNase. Because of its unique property, this enzyme could serve as a biochemical marker in disorders involving the platelet destruction.     

Purification of cytosolic,latent endoribonuclease from porcine thyroid

An alkaline endoribonuclease was purified 1800-fold from the cytosolic, latent ribonuclease fraction of porcine thyroids by gentle procedures specifically designed to exclude both heating and acidification steps. Polyacrylamide gel electrophoresis revealed a broad peak of enzyme activity that was coincident with the stained protein band. As estimated by gel filtration chromatography the major form of the enzyme (59%) had a molecular weight of 51,000; the remainder of the activity was distributed among six minor forms. Carboxymethyl-cellulose chromatography showed that the enzyme had at least three interconvertible forms. The latent alkaline ribonuclease had a pH optimum of 8.1 in both Tris and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffers and was stimulated by a number of monovalent chloride and potassium salts at ionic strengths between 10 and 70 mm; above 100 mm the salts were all inhibitory with the exception of ammonium chloride. At 1 mm both MgCl₂ and CaCl₂ were stimulatory, whereas CuCl₂ ZnCl₂ and EDTA were inhibitors. Both native and denatured DNA were slightly stimulatory. The porcine thyroid latent alkaline ribonuclease was specific for pyrimidine homopolymers and yielded a mixture of cyclic mononucleotides and oligonucleotides when incubated with poly(C). It did not hydrolyze 2′(3′)-cyclic CMP, purine homopolymers, native or denatured DNA or poly(A) · poly(U). Its activity toward rRNA was greater than toward tRNA and it cleaved the former to a mixture of mononucleotides and oligonucleotides. The properties of the intracellular, cytosolic, latent, alkaline ribonuclease distinguish it from pancreatic ribonuclease A and other nonsecretory ribonucleases.