Bovine Pancreatic RNAase Research Articles

Thermolabile ribonucleases from antarctic psychrotrophic bacteria: detection of the enzyme in various bacteria and purification fromPseudomonas fluorescens

Thirteen terrestrial psychrotrophic bacteria from Antarctica were screened for the presence of a thermolabile ribonuclease (RNAase-HL). The enzyme was detected in three isolates of Pseudomonas fluorescens and one isolate of Pseudomonas syringae. It was purified from one P. fluorescens isolate and the molecular mass of the enzyme as determined by SDS-PAGE was 16 kDa. RNAase-HL exhibited optimum activity around 40°C at pH 7.4. It could hydrolyse Escherichia coli RNA and the synthetic substrates poly(A), poly(C), poly(U) and poly(A-U). Unlike the crude RNAase from mesophilic P. fluorescens and pure bovine pancreatic RNAase A which were active even at 65°C, RNAase-HL was totally and irreversibly inactivated at 65°C.

Conformational stability of proteins and peptide-peptide interactions in the presence of carbohydrates

The denaturation temperatures and enthalpies of bovine pancreatic RNAase A in the presence of different amounts of D-glucose or its oligomers have been determined from DSC measurements and compared with literature results for other globular proteins in the presence of oligosaccharides or polyhydroxylated compounds. Both parameters increase almost proportionally for RNAase A at increasing sugar concentration (the denaturation appearing as a reversible, one-step process) and the evaluated Gibbs energy-temperature plots show an expansion of the stability range and an increase in relative stability. Isothermal measurements were also obtained by dilution-flow calorimetry to determine the virial coefficients of the excess enthalpies for aqueous solutions of some model peptides (N-acetylamides of simple amino acids) in the presence of l M D-glucose. These results provide an insight into the role of sugars in preventing peptide-peptide interactions.

Sensitivity of monomeric and dimeric forms of bovine seminal ribonuclease to human placental ribonuclease inhibitor

We have studied the inhibition of bovine pancreatic RNAase (RNAase A) and bovine seminal RNAase in its native dimeric form (RNAase BS-1) and in monomeric carboxymethylated form (MCM RNAase BS-1) by human placental RNAase inhibitor (RNAase inhibitor) in order to understand the effect of enzyme structure on its response to the inhibitor. Study of the inhibition as a function of inhibitor concentration revealed that RNAase A and MCM RNAase BS-1 were inhibited fully and the inhibitor-sensitivities of the two were comparable. But under identical inhibitor concentrations RNAase BS-1 was found to be virtually insensitive to the inhibitor; at higher (3-10-fold) inhibitor concentrations marginal inhibition of the native enzyme could be observed. When RNAase BS-1 was pretreated with 5 mM-dithiothreitol (DTT) and assayed, it exhibited greater inhibitor-sensitivity, presumably as a result of its partial monomerization on exposure to DTT. This DTT-mediated change in the response of RNAase BS-1 to the inhibitor did not, however, seem to occur either in the assay conditions (which included DTT) or even when the enzyme was pretreated with DTT in the presence of the substrate, suggesting an effect of the substrate on the enzyme behaviour towards the inhibitor. Independently, gel-filtration runs revealed that, although DTT treatment caused monomerization of RNase BS-1, this change did not take place when DTT treatment was carried out in the presence of the substrate. From our observations, we infer that differential inhibitor-sensitivity of the dimeric and monomeric forms of RNAase BS-1, the relative contents of the two forms and the influence of the substrate on them may be important determinants of the net enzyme activity in the presence of the inhibitor.

Chemical modification by pyridoxal 5′-phosphate and cyclohexane-1,2-dione indicates that Lys-7 and Arg-10 are involved in the p2 phosphate-binding subsite of bovine pancreatic ribonuclease A

Steric and chemical evidence had previously shown that residues Lys-7 and/or Arg-10 of bovine pancreatic RNAase A could belong to the p2 phosphate-binding subsite, adjacent to the 3' side of the main site p1. In the present work chemical modification of the enzyme with pyridoxal 5'-phosphate and cyclohexane-1,2-dione was carried out in order to identify these residues positively as part of the p2 site. The reaction with pyridoxal 5'-phosphate yields three monosubstituted derivatives, at Lys-1, Lys-7 and Lys-41. A strong decrease in the yield of derivatives at Lys-7 and Lys-41 was observed when either p1 or p2 was specifically blocked by 5'-AMP or 3'-AMP respectively. These experiments indicate that both sites are needed for the reaction of pyridoxal 5'-phosphate with RNAase A to take place. The positive charge in one of the sites interacts with the phosphate group of pyridoxal 5'-phosphate, giving the proper orientation to the carbonyl group, which then reacts with the lysine residue present in the other site. The absence of reaction between pyridoxal 5'-phosphate and an RNAase derivative that has the p2 site blocked supports this hypothesis. Labelling of Lys-7 with pyridoxal 5'-phosphate has a more pronounced effect on the kinetics with RNA than with the smaller substrate 2',3'-cyclic CMP. In addition, when the phosphate moiety of the 5'-phosphopyridoxyl group was removed with alkaline phosphatase the kinetic constants with 2',3'-cyclic CMP returned to values very similar to those of the native enzyme, whereas a higher Km and lower Vmax. were still observed for RNA. This indicates that this new derivative has recovered a free p1 site and, hence, the capability to act on 2',3'-cyclic CMP, but the presence of the pyridoxyl group bound to Lys-7 is still blocking a secondary phosphate-binding site, namely p2. Finally, reaction of cyclohexane-1,2-dione at Arg-10 is suppressed in the presence of 3'-AMP but only a 19% decrease is observed with 5'-AMP, suggesting that Arg-10 is also close to the p2 phosphate-binding subsite.

Reversible inactivation of ribonucleases by ADPribosylation

The activity of purified bovine seminal RNAase and pancreatic RNAase A (EC 3.1.27.5) has been investigated following in vitro ADPribosylation in the presence of nuclear ADPribosyltransferase (EC 2.4.2.30) and NAD +. ADPribosylation of these enzymes was correlated with a significant decrease in their activities. Approximately three residues of ADPribose were present per mol of enzyme. Removal of the bound ADPribose restored enzyme activity to near normal levels. Similar results were obtained with nuclei isolated from bull seminal vesicles as an endogenous source of seminal RNAase and nuclear ADPribosyltransferase. The findings suggest that in vitro ADPribosylation has a reversible inactivating effect on ribonucleases.

1H-NMR studies on the binding subsites of bovine pancreatic ribonuclease A

The titration curves of the C-2 histidine protons of an RNAase derivative (a covalent derivative obtained by reaction of bovine pancreatic RNAase A (EC 3.1.27.5) with 6-chloropurine 9-β- d-ribofuranosyl 5′-monophosphate) were studied by means of 1H-NMR spectroscopy at 270 MHz. The interaction of natural (5′AMP, 5′GMP, 5′IMP) and halogenated purine mononucleotides (cl 6RMP, br 8AMP) with RNAase A was also monitored by using the same technique. The slight change observed in thep K values of the active centre histidine residues of the RNAase derivative, with respect to those in the native enzyme, can be considered as evidence that the phosphate of the label does not interact directly either with His-12 or 119 in the p 1 site, but in the p 2 site as proposed previously (Parés, X., Llorens, R., Arús, C. and Cuchillo, C.M. (1980) Eur. J. Biochem. 105, 571–579). Lys-7 and/or Arg-10 are proposed as part of the p 2 phosphate-binding subsite. The p K values of His-12 and 119 and the shift of an aromatic resonance of the native enzyme found on interaction with some purine nucleotides, can be interpreted by postulating that the interaction of 5′AMP, 5′GMP and 5′IMP takes place not only in the so-called purine-binding site B 2R 2p 1 but also in the primary pyrimidine-binding site B 1R 1 and p 0 of RNAase A.

Effects of macrocyclic polyamines and polymethylenediamines on reactions influenced by polyamines

The effects of macrocyclic polyamines and polymethylenediamines on various reactions influenced by polyamines have been studied. Among the amines tested, 2,3,4,3- and 3,3,3,4-cyclic polyamines, NH 2(CH 2) 6NH 2 and NH 2(CH 2) 8NH 2 had some ability to stimulate polyphenylalanine synthesis, globin synthesis and rat liver isoleucyl-tRNA formation. The degree of stimulation was at most 40% of that obtained by polyamines. In the degradation of poly(C) by bovine pancreatic RNAase A, all tested amines stimulated the degradation. In the NADPH-dependent lipid peroxidation of rat liver microsomes, the degree of inhibition by 2,3,2,3- or 2,3,3,3-cyclic polyamine was greater than that by spermine. The hydrolysis of ATP by an oligomycin-sensitive ATPase was inhibited by 2,3,4,3- and 3,3,3,4-cyclic polyamines, NH 2(CH 2) 10NH 2 and spermine at somewhat comparable levels. None of the macrocyclic polyamines or polymethylenediamines stimulated the growth of a polyamine-requiring mutant of Escherichia coli. Possible explanations for the differences in the effects of amines on the various reactions are discussed.

Human milk ribonuclease

Two components having ribonuclease (EC 3.1.27.5) activity were isolated from human milk. Each component of human milk ribonuclease (RNAase) moved at a slightly different rate when electrophoresed on polyacrylamide gel but at the same rate when ultracentrifuged. The major component had a molecular weight of approx. 14 000, an isoelectric point of pH 7.9, and exhibited a broad absorbance maximum between 277 and 281 nm. Human milk RNAase hydrolyzed yeast RNA, poly(cytidylic acid) and poly(uridylic acid) but not DNA, poly(adenylic acid) or poly(guanylic acid). Maximum activity occurred at pH 7.7 and 60°C. Amino acid analysis of the major component revealed a large number of alanine, valine, glycine and aspartic acids but no truptophan or free sulfhydryl groups. Lysine was the N-terminal amino acid. Tryptic hydrolysis yielded 18 peptides, some of which are similar to those from bovine pancreatic RNAase. Human milk RNAase activity was increased in the presence of NaCl, KCl and sodium citrate and decreased by CaCl 2, MgCl 2, FeSO 4, ZnSO 4 and CuSO 4.

A new pyrimidine-specific ribonuclease from bovine seminal plasma that is active on both single and double-stranded polyribonucleotides and that can distinguish between Mg 2+-containing and Mg 2+-depleted naturally occurring RNAs

The purification to homogeneity of a new ribonuclease, named RNAase SPL, from bovine seminal plasma is described. This nuclease, like the bovine pancreatic RNAase A, is pyrimidine specific. Its activity on single-stranded synthetic polyribonucleotides such as poly(rU) is significantly higher than that of RNAase A. However, unlike RNAase A, RNAase SPL is highly active on a double-stranded RNA such as poly[r(A · U)], and shows extremely limited activity on naturally occurring RNAs, such as Escherichia coli RNA, prepared with Mg 2+ present throughout the isolation procedure. Under conditions of limiting hydrolysis in which RNAase A degrades 60 to 90% of total E. coli RNA to acid-soluble material and the remaining to material having a molecular weight lower than that of transfer RNA, RNAase SPL does not yield any acid-soluble products: it does not appear to degrade tRNA or 5 S RNA, and causes only a small number of nicks in the remaining RNAs to yield a limiting digest containing products with molecular weights ranging between 10,000 and 150,000. Absence of Mg 2+ during the isolation procedure, or heat denaturation of the RNA makes it as susceptible to RNAase SPL as it is to RNAase A. The above and other related observations reported here support the view that there are Mg 2+-dependent structural features, besides single and doublestrandedness, in naturally occurring RNAs, that can be distinguished by using the two nucleases RNAase SPL and RNAase A.

Liver ribonucleases from the bullfrog, Rana catesbeiana Purification, properties and changes in activity during metamorphosis

A major and three minor RNAases exhibiting activity at neutral pH were found in bullfrog liver homogenates. These RNAases were purified to electrophoretic homogeneity. Total purification was 1100 to 1600-fold, and the average recovery was 35%. The molecular weight estimated by gel filtration and SDS-urea-polyacrylamide gel electrophoresis was 12 000 for all the components. An antibody against the major RNAase (component B) reacted with the major RNAase to form a single precipitin line on double immunodiffusion and inhibited the activity of the major RNAase but did not cross-react with the other three components and bovine pancreatic RNAase A. Amino acid analyses revealed that the major RNAase differed markedly from the other three RNAase components in the contents of ten amino acid residues, whereas the minor RNAases were similar to each other. The major RNAase was contained in both frog and tadpole liver and its activity in the liver markedly increased at the metamorphic climax.

How much is secondary structure responsible for resistance of double-stranded RNA to pancreatic ribonuclease A?

1. 1. Double-stranded f 2 sus11 or Qβ RNAs, resistant to bovine pancreatic RNAase A in 0.15 M NaCl 0.015 M sodium citrate (SSC), are quickly and completely degraded at 10-fold lower ionic strength (0.1 × SSC) under otherwise similar conditions. At this ionic strength the secondary structure of double-stranded RNA is maintained, as judged by the following: (a) the unchanged resistance of double-stranded RNA and DNA, under similar low ionic strength conditions, to nuclease S 1 from Aspergillus oryzae, in contrast with the sensitivity of the corresponding denatured nucleic acids to this enzyme, specific for single-stranded RNA and DNA; (b) the co-operative pattern of the thermal-transition profile of double-stranded RNA (with a T m of 89°C) in 0.1 × SSC. 2. 2. Whereas in SSC bovine seminal RNAase (RNAase BS-1) and whale pancreatic RNAase show an activity on double-stranded RNA significantly higher than that of RNAase A, in 0.1 × SSC the activity of the latter enzyme on this substrate becomes distinctly higher than that of RNAase BS-1, and similar to that of whale RNAase. 3. 3. From these results it is deduced that the secondary structure is probably not the only nor the most important variable in determining the susceptibility of double-stranded RNA to ribonuclease. Other factors, such as the effect of ionic strength on the enzyme and/or the binding of enzyme to nucleic acids, may play an important role in the process of double-strnaded RNA degradation by ribonucleases specific for single-stranded RNA.

The immunological characterization of several human ribonucleases by using primary binding tests

RNAases (ribonucleases), purified from four human tissues, as well as bovine pancreatic RNAase (RNAase A), were studied by immunodiffusion methods and by two different primary binding tests. The enzymes fell into two groups immunologically, those purified from plasma and pancreas in one and those from spleen and liver in the other. No antigenic cross-reaction between the two groups was detected by any of the immunoassays used. There was a slight antigenic cross-reaction between the human and bovine pancreatic RNAases. The liver and spleen RNAases were immunologically identical by all criteria used, whereas a small but consistent antigenic difference between the human plasma and human pancreas enzymes was detected. The significance of this difference between the human plasma and pancreas RNAases is discussed in relation to similarities and differences in their properties.

Human pancreatic-type ribonucleases with activity against double-stranded ribonucleic acids

Purified acid-thermostable ribonuclease (Ribonucleate 3′-pyrimidino-oligonucleotidohydrolase, EC 3.1.4.22) from human pancreas degrades double-stranded RNA at 2% the rate for single-stranded RNA. The activities against single-stranded RNA and double-stranded RNA were shown to be due to a single enzyme with properties similar to bovine pancreatic RNAase A. For purposes of comparison the activities against double-stranded RNA of crystalline ribonucleases of the whale, rat and cow were assayed and found to be 0.4%, 0.03% and 0.003%, respectively, of their activities against single-stranded RNA. Both human serum and urine contain RNAase components of pancreatic origin which hydrolyze double-stranded RNA at 2% and 0.4%, respectively, of the rates against single-stranded RNA. By contrast, purified acid-thermostable RNAases from human spleen and liver hydrolyze double-stranded RNA at least 20-fold more slowly than human pancreatic RNAase, relative to the corresponding rates against single-stranded RNA. The human pancreatic and serum enzymes exhibit appreciable activity against the poly(C) component of the double-stranded poly(I) · poly(C); they also attack poly(C) itself at approximately 25 times the rate for poly(U) and at more than 50 times the rate for single-stranded RNA.

An allosteric model for ribonuclease.

Data from two assay systems show that the kinetics of the hydrolysis of cytidine 2':3'-cyclic monophosphate by bovine pancreatic RNAase (ribonuclease) is not consistent with conventional models. An allosteric model involving a substrate-dependent change in the equilibrium between two enzyme conformations is proposed. Such a model gives rise to a calculated curve of velocity versus substrate concentration which fits the experimental data. The model is also consistent with the results of an examination of the tryptic digestion of RNAase. Substrate analogues are able to protect RNAase against hydrolysis by trypsin and the percentage of RNAase activity which remains after digestion increases sigmoidally as the analogue concentration is increased. The model also explains the pattern seen in the Km values quoted in the literature and is consistent with strong physical evidence for a ligand-induced conformational change for RNAase reported in the literature.

Interchain disulfide bridges in ribonuclease BS-1

RNAase BS-1, a dimeric ribonuclease isolated from bovine seminal plasma, is made up of two identical subunits whose amino acid sequence is homologous to the sequence of bovine pancreatic RNAase A. The dimeric structure, resistant to denaturating agents, is sensitive to thiol reagents even in the absence of denaturants. The isolation and characterization of a cystine peptide containing two adjacent 1 2 cystine residues is reported. As the peptide molecular weight is halved after reductive cleavage with dithiothreitol, a structure based on two interchain disulfide bonds between the two adjacent 1 2 cystine of each subunit is proposed. The singularity of such a structure for a small enzymatic protein is discussed.

The Phylogeny of the Ribonuclease-Ribonuclease Inhibitor System: Its Distribution in Tissues and its Response During Leukaemogenesis and Aging

Acid RNAase, alkaline RNAase, and alkaline RNAase inhibitor appear to be widespread in higher animals. The livers of all mammalian species tested including that ofa monotreme contained inhibitor active against bovine pancreatic RNAase. Chick liver contained inhibitor active against "activated" chick liver supernatant RNAase, but not against bovine pancreatic RNAase. The rat liver inhibitor showed partial activity against chick liver supernatant RNAase.

Amino acid acceptor activity of enzymically altered soluble RNA from Escherichia coli

1. 1. Some amino acid acceptor functions of Escherichia coli s-RNA were shown to be quite resistant to Bacillus subtilis RNAase or RNAase T 1 in the presence of Mg 2+, whereas these functions were susceptible to bovine pancreatic RNAase under the same incubation conditions (RNAase, polyribonucleotide 2-oligonucleotidotransferase (cyclizing), EC 2.7.7.16). 2. 2. The resistance of individual amino acid s-RNA's for B. subtilis RNAase varied with different amino acids. The s-RNA's interacting with alanine, glycine, histidine, lysine, methionine, phenylalanine, tyrosine, valine and leucine were quite resistant to B. subtilis RNAase whereas the amino acid acceptor function of s-RNA specific for other amino acids such as arginine, aspartic acid, glutamic acid, proline, serine, threonine and tryptophan was rather sensitive to this RNAase. RNAase T 1 gave results similar to those obtained with B. subtilis RNAase except that phenylalanine acceptor activity was inactivated. 3. 3. Specific activities for certain amino acyl RNA's were increased 2–3 times by treatment with B. subtilis RNAase. The base composition of a partially digested s-RNA differed from the original s-RNA in that it contained less guanine and adenine but more cytosine. The slope of the thermal denaturation curve, measured in the presence of Mg 2+, was lower for resistant s-RNA than that of the undigested s-RNA. 4. 4. Partially digested s-RNA's were eluted from a column of methylated albumin of different salt concentration than untreated s-RNA's suggesting that relatively small alterations could be made in resistant s-RNA's without complete loss of acceptor activity for the two amino acids, phenylalanine and leucine.

Extracellular ribonuclease from Bacillus subtilis: II. Its amino acid composition and structural comparison with bovine pancreatic ribonuclease

Analysis of amino acid composition of extracellular RNAase from B. subtilis demonstrated that it differed strikingly from bovine pancreatic RNAase in that it contained trytophan instead of cysteine. Fingerprinting of peptides obtained by proteolytic digestion showed that the spots given by B. subtilis RNAase were all different from those given by bovine pancreatic RNAase. Photooxidation of B. subtilis RNAase in the presence of methylene blue resulted in a complete inactivation of this RNAase accompanied by a specific decrease of histidine residue in the molecule, a result which suggested that histidine played an important role in the catalytic activity of this RNAase. RNAase activity was completely lost by titration with N-bromosuccinimide, but alkylating agents, such as iodoacetic or bromoacetic acid, did not react with the RNAase under any of the conditions tested.

Extracellular ribonuclease from Bacillus subtilis

Analysis of amino acid composition of extracellular RNAase from B. subtilis demonstrated that it differed strikingly from bovine pancreatic RNAase in that it contained trytophan instead of cysteine. Fingerprinting of peptides obtained by proteolytic digestion showed that the spots given by B. subtilis RNAase were all different from those given by bovine pancreatic RNAase. Photooxidation of B. subtilis RNAase in the presence of methylene blue resulted in a complete inactivation of this RNAase accompanied by a specific decrease of histidine residue in the molecule, a result which suggested that histidine played an important role in the catalytic activity of this RNAase. RNAase activity was completely lost by titration with N-bromosuccinimide, but alkylating agents, such as iodoacetic or bromoacetic acid, did not react with the RNAase under any of the conditions tested.

Studies on cellular inhibitors of ribonuclease II. Some properties of the inhibitor from rat liver

Both the rat-liver RNAase inhibitor and heparin inhibited bovine pancreatic RNAase (at pH 7.8 and 5.8), but did not affect the activity of RNAase T 1, an alkaline enzyme of fungal origin, or of several acid RNAases derived from plant tissue. These inhibitors differed in their activity towards the enzymes from rat liver. The acid RNAase was inhibited by heparin, but not by the rat-liver inhibitor. The two alkaline RNAase preparations were inhibited by the rat-liver inhibitor, but only slightly affected by heparin. The purified rat-liver inhibitor was inactivated by low concentrations of papain (of several enzymes tested), periodate, sulfhydryl reactants and protamine, and by high salt concentrations. Inactivation by PCMB and Pb 2+ was partially reversible. Inhibition of RNAase activity by heparin was more sensitive to protamine and to salt concentration than was inhibition by the rat-liver inhibitor. These results indicate the protein nature of the rat-liver inhibitor, but suggest carbohydrate may also be an essential component. The rat-liver inhibitor may be classed as a polyanionic inhibitor, but one which forms a comparatively strong complex with RNAase.