Comparative proton NMR studies of bovine semen and pancreas ribonucleases

The fine structure of bovine semen RNAase was studied with proton NMR spectroscopy making use of the four-protein system constituted by dimeric bovine semen RNAase, its catalytically active monomeric bis-(S-carboxymethyl-31,32) derivative, the naturally monomeric RNAase A from the pancrease of the same species, and dimerized RNAase A. Only four histidine C-2 H resonances were observed in the aromatic spectrum of bovine semen RNAase, which belong to the four histidine residues present in the sequence of bovine semen RNAase subunits at positions identical with those of the histidines of RNAase A. This is indicative of identical environments for the individual histidine residues in both subunits. These resonances were assigned (i) by comparing their titration curves with the corresponding curves obtained with RNAase A and with monomeric bovine semen RNAase and (ii) by evaluating the effects on their titration curves of nucleotide binding. Very similar NMR parameters were measured for His-105 and also for His-119 of seminal and pancreatic RNAase, while His-12 was found to have different environments in the two proteins. The distinctive NMR features of His-48 in bovine semen RNAase confirmed the role of the hinge regions of the subunits in maintaining the dimeric structure of the protein. While monomerization of the seminal enzyme reduced the differences between the histidine C-2 H resonances of RNAase A and bovine semen RNAase, dimerization of RNAase A did not affect the NMR spectrum of this protein, thus indicating as unlikely the possibility that the quaternary structure of bovine semen RNAase resembles that of dimerized RNAase A.     

Bull semen RNAase revisited

A new RNAase, RNAase SPL, was discovered (Reddy et al., 1979), which constituted most of bull semen RNAase activity; it was reminiscent in many of its properties of the bovine seminal RNAase we have studied for many years (see References), but different from it in other respects. When the procedure devised by those authors for its isolation was repeated, we found that an RNAase SPL such as that described in the above-mentioned paper is not to be found in bovine seminal plasma.     

A new pyrimidine-specific ribonuclease from bovine seminal plasma that is active on both single and double-stranded polyribonucleotides and that can distinguish between Mg2+-containing and Mg2+-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 Mg²⁺ 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 Mg²⁺ 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 Mg²⁺-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.     

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+ X 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.     

Bovine seminal ribonuclease precursor synthesized in vitro

Native bovine seminal ribonucelase is a dimeric protein, whose identical subunits (M_r 14 500), linked through two disulfide bridges, can be dissociated by a selective reduction procedure. Evidence is presented that the synthesis in vitro, under reducing conditions, of bovine seminal RNAase, directed by polyadenylated RNA isolated from bull seminal vesicles (where the enzyme is synthesized in vivo), occurs in the form of a precursor, 18 000-Da polypeptide. The precursor nature of this translation product was deduced by two criteria: (1) its specific immunoprecipitation with anti-bovine seminal RNAase antibodies; (2) its processing by dog pancreas microsomal membranes to produce a protein with a molecular weight similar to that of the subunit(s) of bovine seminal RNAase. Moreover, evidence is offered that the precursor polypeptide is able to form in vitro a dimeric molecule under conditions where no exogenous reducing agents were added.     

Identity of the ribonuclease from bovine seminal plasma with ribonuclease BS-1, and its sensitivity to polyvinyl sulphate

A ribonuclease (RNAase BSD) isolated by us earlier from bovine seminal plasma by DNA-affinity chromatography is shown to be homogeneous on polyacrylamide gel electrophoresis, analytical ultracentrifuge and high performance liquid chromatography. From amino acid analysis, high performance liquid chromatography and enzymatic and physicochemical studies, this enzyme is shown to be identical to RNAase BS-1 reported by D'Alessio et al. ((1972) Eur. J. Biochem. 26, 153-161). Immunological studies support this observation. It has also been shown that, as compared to RNAase A, this enzyme is more sensitive to polyvinyl sulphate inhibition.     

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

1. Double-stranded f2 sus11 or Qbeta 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 X 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 S1 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 Tm of 89 degrees C) in 0.1 X SSC. 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 X 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. From these results it is deduced that the secondary structure is probably not the only nor the most important variable in determining the susceptibility 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-stranded RNA degradation by ribonucleases specific 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.     

A ribonuclease from human seminal plasma active on double-stranded RNA

A ribonuclease, active on single- and double-stranded RNAs, has been isolated from human seminal plasma 3-5 micrograms of enzyme were recovered per ml of seminal plasma, equivalent to 71% of total activity and a 2500-fold purification (measured with poly(A) X poly(U) as substrate) from the initial dialyzed material. Similar amounts of RNAase were found per g (wet weight) of human prostate, where the enzyme appears to be produced. Human seminal RNAase degrades poly(U) 3-times faster than poly(A) X poly(U), and poly(C) or viral single-stranded RNA about 10-times faster than poly(U). Degradation of poly(A) X poly(U), viral double-stranded RNA, and poly(A) by human seminal RNAase is 500-, 380- and 140-times more efficient, respectively, than by bovine RNAase A. The enzyme, a basic protein with maximum absorbance at 276 nm, occurs in two almost equivalent forms, one of which is glycosylated. Mr values of the glycosylated and non-glycosylated form are 21000 and 16000, respectively. The amino-acid composition of the RNAase is very similar to that of human pancreatic RNAase. The same is true for the carbohydrate content of its glycosylated form.     

Antitumor action of bovine seminal ribonuclease

Unlike the bovine pancreatic ribonuclease (RNAase A), bovine seminal ribonuclease (BS RNAase) displays various biological activities including antitumor cytotoxicity. To learn more about its antitumor activity, we investigated BS RNAase effect on athymic nude mice bearing various tumors. BS RNAase (250 μg per mouse per day) was administered to the mice with prostate carcinoma for three weeks by three different routes (intraperitoneally—i.p., subcutaneously—s.c., and intratumorally—i.t.). Administration i.p. was ineffective, while s.c. administration reduced significantly size of tumors and i.t. administration abolished half of the tumors in treated mice. The i.t. administration of BS RNase to nude mice bearing melanoma showed even better results. Eighty % of mice were without tumors and in the other mice the tumors were significantly diminished. The best antitumor effect was obtained in case of seminoma. All mice bearing this tumor were cured after ten doses of BS RNAase.     

Fast and high-yielding procedures for the isolation of bovine seminal RNAase

Fast and high yielding procedures for the isolation of bovine seminal RNAase are described. Homogeneous enzyme is prepared from seminal plasma in high yields in a single chromatographic step. Higher amounts (hundreds of mg) are easily prepared from seminal vesicles, a more available source of enzyme. Both procedures can be used also for the direct isolation of the isoenzymes of bovine seminal RNAase. An ultrarapid (1 hour) procedure is described for the preparation of mg amounts of pure enzyme, or of the individual isoenzymes, from seminal plasma.     

DSC study of reversible and irreversible thermal denaturation of concentrated globular protein solutions

A calorimetric study of the thermal denaturation of bovine serum albumin, RNAase and catalase in concentrated solutions (crystals) has been carried out. The results obtained for RNAase studied within the pH range 2.5-8.5 show that for concentrated solutions there is an interval of pH where, on cooling of the solution which had undergone denaturation, its renaturation is observed. In the case of concentrated and dilute solutions of RNAase these intervals coincide. The study of RNAase under such conditions at various heating rates shows that there is a range of rates in which the process of denaturation of concentrated solutions can be considered as reversible. The dependences of Td and Hd on pH and concentration of solutions have been determined. The denaturation enthalpy of concentrated solutions like in dilute ones, has been found to be independent of the pH of solutions, and the experimentally registered change has been proved to be the result of its dependence on temperature. A new method of determination of protein denaturation enthalpy under the conditions of intensive molecule aggregation is suggested. The forms of irreversibility as appearing in the calorimetric experiment were determined by comparing reversible and irreversible denaturation under continuous and step-heating regimes. It is shown that the decrease in Tmax and the narrowing of the heat absorption peak in the case of decreasing heating rates of protein solutions, observed under certain environmental conditions, results from the irreversibility of the denaturation process.     

Differential, structure-dependent susceptibility of poly(A) and RNA to monomeric and dimeric pancreatic ribonuclease A.

Cross-linked dimers of bovine RNAase A are definitely more efficient than monomers at degrading polyadenylic acid under conditions of ionic strength and pH, where the polymer assumes either a double-helical or an ordered single-stranded, base-stacked structure. The opposite occurs, i.e., monomers of RNAase A are definitely more active than dimers,when poly(A) is digested by the two enzyme species under conditions where the conformation of the polymer is essentially that of a random coil. The same pattern of events occurs when total RNA from Escherichia coli or single-stranded RNA of f2 sus11 bacteriophage are used as substrates under opposite ionic-strength conditions. In the presence of high salt concentrations, favouring the formation and the stability of a secondary structure in self-complementary sequences of RNA, the ribonucleic acids are degraded at a higher rate by dimers than by monomers of bovine RNAase A. The opposite occurs in the presence of very low salt concentrations, i.e. when the RNAs are in solution presumably as random coils. These observations are discussed in the light of a hypothesis already advanced to understand the mechanism of enzymic degradation of secondary structures of polyribonucleotides.     

Ionic control of enzymic degradation of double-stranded RNA

The pattern of the degradation of various double-stranded polyribonucleotides by several ribonucleases (bovine RNAase A and its cross-linked dimer, bovine seminal RNAase, and pike-whale pancreatic RNAase) has been studied as a function of ionic strength and pH. It appears that (1) there is no direct correlation between the secondary structure of double-stranded RNA and its resistance against enzymatic breakdown, i.e., the stability of the secondary structure of double-helical RNA is not the main variable in the process. (2) The acstivity responses of the enzymes examined to changes of ionic strength and pH suggest that enzymic degradation of double-stranded RNA is mainly controlled by ion concentration, and that the process may fall within the phenomena interpreted by the theory of the ionic control of biochemical reactions advanced by Douzou and Maurel (Douzou, P. and Maurel, P. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 1013--1015). (3) The activity curves of the enzyme studied show, at a given pH, a shift toward higher ionic strengths as a function of the basicity of the enzyme protein. This finding explains the already observed correlation between number and/or density of positive charges of a ribonuclease molecule and its ability to attack double-stranded RNA in 0.15 M sodium chloride/0.015 M sodium citrate (SSC). (4) A careful analysis of the influence of ionic strength and pH on the reaction appears to be necessary in order to characterize a ribonuclease which shows activity towards double-stranded RNAs, and to allow a meaningful comparison between different enzymes capable of attacking these substrates.     

Human seminal ribonuclease. A tool to check the role of basic charges and glycosylation of a ribonuclease in the action of the enzyme on double-stranded RNA

Human seminal ribonuclease (a basic protein occurring in a glycosylated and in a non-glycosylated form) is very active against double-stranded RNAs (De Prisco, R., Sorrentino, S., Leone, E. and Libonati, M. (1984) Biochim. Biophys. Acta 788, 356-363). The action of the two enzyme forms on single-stranded and double-stranded substrates was studied as a function of pH and ionic strength. Results indicate (1) that glycosylation of the RNAase molecule does not affect enzyme action on single-stranded RNAs, while (2) degradation of double-stranded RNAs is moderately increased by the presence of carbohydrates in the enzyme molecule. Human seminal RNAase shows a marked helix-destabilizing activity on poly(dA-dT) X poly(dA-dT). Under various conditions, this action (1) is definitely stronger than that of bovine RNAase A, and (2) seems to be less dependent on the glycosylation than on the basicity of the enzyme protein. The remarkable activity of human seminal RNAase on double-stranded RNA may, at least partly, be related to the enzyme properties mentioned above.     

A heat-stable protein inhibitor of cyclic 3′,5′-nucleotide phosphodiesterase

A heat-stable, non-dialyzable inhibitory factor of cyclic nucleotide phosphodiesterase was detected in and partially purified from bovine retina. The factor appears to be a protein, since the inhibitory activity was abolished by trypsin digestion but not by DNAase or RNAase treatment. The protein inhibitor from bovine retina effectively inhibits the Ca²⁺-independent phosphodiesterase from several sources, including bovine retina, bovine rod outer segment, and a human lymphoblastic leukemia cell line, indicating lack of tissue and species specificity.     

A heat-stable protein inhibitor of cyclic 3',5'-nucleotide phosphodiesterase.

A heat-stable, non-dialyzable inhibitory factor of cyclic nucleotide phosphodieterase was detected in and partially purified from bovine retina. The factor appears to be a protein, since the inhibitory activity was abolished by trypsin digestion but not by DNAase or RNAase treatment. The protein inhibitor from bovine retina effectively inhibits the Ca2+-independent phosphodiesterase from several sources, including bovine retina, bovine rod outer segment, and a human lymphoblastic leukemia cell line, indicating lack of tissue and species specificity.     

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.     

1H-NMR study on the tautomerism of the imidazole ring of histidine residues. II. Microenvironments of histidine-12 and histidine-119 of bovine pancreatic ribonuclease A

The NMR titration curves of proton chemical shifts were observed for the C2 protons of histidine residues in intact bovine pancreatic RNAase A (EC 3.1.27.5) and carboxyalkylated RNAase A. By comparing the methyl region of NMR spectra, the 250-340 nm region of circular dichoic spectra, and the NMR titration curves of tyrosine ring protons among intact and modified RNAase A, it was ascertained that the carboxyalkylation of histidine residues at position 12 or 119 did not make any appreciable conformational changes to RNAase A. With the pK values determined for intact and modified RNAase A, the microscopic pK values and molar ratios of tautomers were estimated for His-12 and His-119 by means of the procedure described in the preceding paper. The estimated microscopic pK values of tautomers were 6.2 for the N1-H tautomer of His-12, more than 8 for the N3-H tautomer of His-12, 7.0 for the N1-H tautomer of His-119, and 6.4 for the N3-H tautomer of His-119, respectively. These values were interpreted in terms of the microscopic environments surrounding the histidine residues. The microscopic structure estimated in the present study was discussed, comparing it with those from X-ray crystallography and hydrogen-tritium (or hydrogen-deuterium) exchange technique.     

The antitumor action of seminal ribonuclease tested with the plasmacytoma spleen colonization assay

The antitumor action of bovine seminal ribonuclease was evaluated with a quantitative assay based on the production of tumor foci in the spleens of mice injected with plasmacytoma cells. The antitumor action depended on the integrity of the catalytic site, and on the dimeric structure of the enzyme. A working hypothesis is proposed, based on these results, and on previous results obtained studying the antitumor action of seminal RNAase in vitro on cell cultures. According to this hypothesis, the antitumor action is based on the ability of seminal RNAase to interact at specific receptor sites on the tumor cell membrane, as well as on its RNA degrading ability.