Pancreatic RNAase Research Articles

A Novel Ribonuclease from Rana Chensinensis and Its Potential for the Treatment of Human Breast Cancer.

Onconase, a member of the pancreatic RNAase A superfamily of ribonucleases, is a chemotherapeutic agent, which has demonstrated selective antitumor activity in a variety of human malignancies. However, little is known about the mechanisms of it's action on human breast cancer cells. To investigate a novel Onconase from the frog of Rana chensinensis changbaishanensis on human breast cancer cells and the underlying mechanisms, a novel Onconase named Rdchonc from Rana chensinensis changbaishanensis was cloned by polymerase chain reaction. SDS-PAGE revealed that the Rdchonc had a high heterologous expression in Escherichia coli BL21(DE3). The MTT assay indicated that purified Rdchonc was cytotoxic to human breast cancer MCF-7 and MD-MB-231 cells. Treatment with 20 μg/mL Rdchonc protein significantly reduced the invasive capacities of MCF-7 and MD-MB-231 cells. Interestingly, the authors found that such inhibitory effort on tumor cell growth induced by Rdchonc treatment may be explained by the regulation of proapoptotic Bcl-2 family proteins and inhibition of MEK/ERK phosphorylation.

A protein disulfide-thiol interchange activity of HeLa plasma membranes inhibited by the antitumor sulfonylurea N-(4-methylphenylsulfonyl)- N′-(4-chlorophenyl)urea (LY181984)

Plasma membrane vesicles isolated from HeLa cells grown in suspension culture contain a protein disulfide-thiol interchange (protein disulfide-like) activity. The activity was estimated from the restoration of activity to inactive (scrambled) pancreatic RNAase. RNAase activity was measured either by hydrolysis of cCMP or by a decrease in acid precipitable yeast RNA. The ability of plasma membrane vesicles to restore activity to inactive (scrambled) pancreatic ribonuclease was inhibited by the antitumor sulfonylurea N-(4-methylphenylsulfonyl)- N′-(4-chlorophenyl)urea (LY181984). The activity correlated with that of a cyanide-resistant NADH oxidase also associated with the plasma membrane vesicles that exhibited a similar pattern of drug response. The activity was stimulated by reduced glutathione and inhibited by oxidized glutathione but did not depend on either for activity. The antitumor sulfonylurea-inhibited activity was greatest in the presence of reduced glutathione and least in the presence of oxidized glutathione. The antitumor sulfonylurea-inhibited activity was unaffected by a monoclonal antibody to protein disulfide isomerase. Also the antitumor sulfonylurea-inhibited activity was unaffected by peptide antisera to the consensus active site sequence of protein disulfide isomerase. Thus the antitumor sulfonylurea-inhibited activity appeared to reside with a novel cell surface protein capable of oxidation of both NADH and protein thiols and of carrying out a protein disulfide isomerase-like protein disulfide-thiol interchange activity in the absence of NADH or other external reductants.

Inactivation during denaturation of ribonuclease A by guanidinium chloride is accompanied by unfolding at the active site

Inactivation of pancreatic RNAase A occurs in guanidinium chloride (GdmCl) at low concentrations before the unfolding of the molecule as a whole can be detected [Liu and Tsou (1987) Biochim. Biophys. Acta 916, 455-464]. We have now shown that the rate of digestion of the RNAase molecule by either trypsin or proteinase K increases significantly at low concentrations of GdmCl where the enzyme is largely inactivated, but fluorescence and absorption measurements reveal no conformational changes. N-Terminal sequence analysis of the peptide fragments generated shows that proteolysis occurs primarily at or near the active site. The decrease in activity of RNAase at low concentrations of GdmCl is therefore due to partial unfolding of the molecule, particularly at the active site and not to an inhibition by the denaturant.

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.

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 mg of enzyme were recovered per ml of seminal plasma, equivalent to 71% of total activity and a 2500-fold purification (measured with poly(A) · poly (U) as substrate) from the inital 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) · poly(U), and poly(C) or viral single-stranded RNA about 10-times faster than poly(U). Degradation of poly(A) · 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. M r 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.

Purification and characterization of ribonucleases from human seminal plasma.

Four ribonucleases (RNAases I-IV) have been purified to homogeneity from human seminal plasma by precipitation with 40-75%-satd. (NH4)2SO4, followed by chromatographies on concanavalin A-Sepharose 4B, DEAE-cellulose phosphocellulose, agarose-5'-(4-aminophenylphospho)uridine 2'(3')-phosphate (RNAase affinity column) and Sephadex G-75 or G-100. The homogeneity of these RNAases was confirmed by polyacrylamide-gel electrophoresis. Mr values for these purified RNAases were 78 000, 16 000, 13 300 and 5000 as estimated by gel filtration. Enzyme activities of RNAases I, III and IV were inhibited by Mn2+, Zn2+ and Cu2+ and activated by Na+, K+, Ba2+, Mg2+, Fe2+ and EDTA, whereas that of RNAase II was inhibited by Ba2+, Mg2+, Fe2+, Mn2+, Zn2+ and Cu2+ and activated by Na+, K+ and EDTA. RNAases I, II and IV demonstrated a higher affinity for poly(C) and poly(U) or yeast RNA, whereas RNAase III preferentially hydrolysed poly(U) over poly(C) and yeast RNA. In the presence of 5 mM-spermine, RNAase I was dissociated to a low-Mr (5000) enzyme with an increase in total RNAase enzymic activity. Xenoantiserum to each RNAase was raised and evaluated by immunoprecipitation and immunohistochemical methods. Anti-(seminal RNAase III) antiserum showed no immunological cross-reaction with RNAases of other human origin, whereas anti-(seminal RNAase I), -(RNAase II) and -(RNAase IV) antisera exhibited indistinguishable immunological reactions with serum RNAase and other human RNAases, except that anti-(seminal RNAase I) and -(RNAase antisera IV) did not react with pancreatic RNAases. Seminal RNAases I and IV were identical immunologically as shown by anti-(RNAase I) and anti-(RNAase IV) in immunodiffusion. Immunohistochemical study revealed that, among human tissues examined, only prostate expressed seminal RNAase III. These results suggested that human seminal RNAase I may be an aggregated molecule of RNAase IV and that seminal RNAases II and IV are similar to serum RNAases, whereas seminal RNAase III is a prostate-specific enzyme.

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.

Cloning of genes for bacteriophage T5 stable RNAs

One EcoRI-generated fragment (440 basepairs) and two EcoRI/ HindIII fragments (220 and 960 basepairs) from the deletion region of T5 phage have been inserted into the phage λ XIII and the plasmid pBR322 as vectors. Recombinant DNA molecules were studied by hybridization with in vivo 32P-labeled T5 4–5 S RNAs on nitrocellulose filters. Two-dimensional polyacrylamide gel electrophoretic fractionation and fingerprint analysis of the RNAs eluted from the filters were carried out to identify RNAs coded by cloned fragments. For the accurate localization of the genes for these RNAs, RNA-DNA hybrids were treated with T1 and pancreatic RNAases, and the eluted RNA fragments stable against RNAase action were electrophoresed. It was shown that the EcoRI ∗440 fragment contains the gene for tRNA 10 (tRNA Asp), the EcoRI/ HindIII ∗220 fragment contains the gene for RNA III (107 bases) and parts of the genes for RNA I (107 bases) and tRNA 12 (tRNA His), and the EcoRI/ HindIII ∗960 fragment contains only a part of the gene for tRNA 9 (tRNA Gln). The arrangement of these genes on the physical map of T5 phage was as follows: -tRNA Gln-tRNA His-RNA III-RNA I-…-tRNA Asp.

Ribonucleoprotein organization of eukaryotic RNA: XV. Different nucleoprotein structures of globin messenger RNA sequences in nuclear and polyribosomal ribonucleoprotein particles

Heterogeneous nuclear RNA and polyribosomal messenger RNA are both complexed with specific sets of proteins in the cell, forming ribonucleoprotein complexes known as hnRNP and mRNP, respectively. In the present investigation, the nucleoprotein structures of globin mRNA sequences in hnRNP and mRNP were probed by digestion with nuclease, under conditions in which RNA-protein rearrangements were shown not to occur. Mild digestion with pancreatic RNAase of a Friend erythroleukemia cell RNP fraction containing both hnRNP and mRNP resulted in a preferential depletion of globin mRNA-homologous sequences, as measured by hybridization of the surviving RNA with globin complementary DNA. Hypersensitivity to nuclease typifies 65% of the globin mRNA-homologous sequences, with the other 35% remaining relatively nuclease-resistant. Removal of polyribosomal mRNP by release with EDTA, followed by re-isolation of hnRNP on a sucrose gradient eliminated the nuclease-hypersensitive class of globin mRNA sequences in favor of the relatively nuclease-resistant class. These results suggest that mRNA sequences are more nuclease-sensitive in polyribosomal mRNP than they are in nuclear hnRNP particles. The implication is that mRNA sequences undergo a significant change in RNP structure at some point during their movement from nucleus to cytoplasm.

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.

Polymeric N-hydroxysuccinimide esters in the synthesis of N-4-aminoacyl- and peptidyl derivatives of cytidine and cytidylic acids

1. The possibility of using poly-N-hydroxysuccinimide esters for the synthesis of N-4-aminoacyl derivatives of cytidine and cytidine 2′,3′-cyclophosphate in a DMFA medium has been shown. 2. A synthesis of an analog of a C-terminal tetrapeptide (121–124) of pancreatic RNA-ase A, BOC-L-Asp-(β-OBu-t)-L-Ala-L-Asp-(OH)-L-Val-OBu-t has been carried out by the method of poly-N-hydroxysuccinimide reagents.

Messenger RNA sequences in nuclear ribonucleoprotein particles are complexed with protein as shown by nuclease protection

Soon after their transcription, eukaryotic messenger RNA precursors (heterogeneous nuclear RNA) are assembled into ribonucleoprotein particles (hnRNP). In the present experiments we have asked whether the mRNA-homologous components of hnRNA are complexed with proteins in hnRNP or, alternatively, exist as stretches of naked RNA in the particles. Mild digestion of HeLa cell hnRNP particles by micrococcal nuclease, pancreatic RNAase or an endogenous particle-bound nuclease, followed by hybridization of the surviving RNA with a complementary DNA probe for poly(A) + mRNA sequences reveals that messengerhomologous sequences are protected from nuclease attack in hnRNP.

Characterization of initiator RNA from replicating simian virus 40 DNA synthesized in isolated nuclei

Nascent DNA chains of papova viruses and animal cells are transiently linked to a 5′-terminal oligoribonucleotide, termed initiator RNA, which is implicated in their priming. To study the biosynthesis and degradation of iRNA † † Abbreviations used: iRNA, initiator RNA; RI DNA, replicative intermediate DNA; HEPES, N-2-hydroxyethyl piperazine- N′-2-ethanesulfonic acid; EGTA, ethyleneglycol-bis(β-aminoethyl ether)- N,N′-tetraacetic acid. , as well as its location within replicating DNA, iRNA was labeled in isolated nuclei of simian virus 40 (SV40)-infected monkey kidney cells with rNTP precursors, [α- 32P]rCTP or [β- 32P]rGTP. Incubation in the presence of up to 200 μg α-amanitin/ml served to reduce non-iRNA transcription, although it did not affect the synthesis of iRNA. This result also confirmed that RNA polymerases II and III are not involved in iRNA synthesis. Replicative intermediate SV40 DNA was then purified by salt/ detergent extraction and gel filtration and treated with pancreatic RNAase to remove residual non-iRNA-bound 32P. Initiator RNA was not affected, however, suggesting that it was entirely base-paired to the parental template. Denaturation of the purified SV40 RI DNA, followed by gel filtration, resolved the nascent DNA into short chains (Okazaki pieces) and long chains, iRNA being associated mainly with the former. Upon DNAase I digestion, both short and long chains yielded intact iRNA, migrating on the gel in the (pA) 9–12 marker range. However, the long chains yielded in addition shorter oligonucleotides, apparently partially degraded iRNA. Further enzymic digestions of iRNA labeled either with [α- 32P]rCTP or [β- 32P]rGTP indicated that a fraction of it contained a 5′-terminal triphosphate group. The retrograde fork polarity of iRNA-containing Okazaki pieces was demonstrated by their highly asymmetric hybridization to single-stranded DNA probes of unidirectional replication fork polarities, as previously seen with Okazaki pieces labeled in their DNA moiety. In contrast, iRNA-tagged long chains hybridized evenly with probes of both fork polarities, indicating that Okazaki pieces become ligated to the continuously growing strand of the opposite fork early in replication.

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.

Purification and properties of ribonucleases from human urine

The two major ribonucleases (EC 3.1.27.5) present in normal human urine have been highly purified and extensively characterized for their enzymatic, physical, chemical and structural properties. One of the enzymes, RNAase C, is a glycoprotein which exhibits a pH optimum of 8.5 with RNA as the substrate and preferentially degrades the synthetic homoribopolymer poly(C). This enzyme is resolved into multiple components by column electrofocusing. However, prior treatment with neuraminidase results in a single form of RNAase C with an isoelectric point of 10.4, indicating that the charge heterogeneity is the result of variability in sialic acid content. Amino acid composition and NH 2- and COOH-terminal sequence analyses of RNAase C show that this enzyme is very similar to mammalian pancreatic RNAases; the data indicate a peptide chain of 126 amino acid residues and a 33% carbohydrate content. The second enzyme isolated from urine, termed RNAase U, is also a glycoprotein which has a pH optimum of 7.0 with RNA as substrate and is virtually inactive against poly(C). RNAase U lacks sialic acid and focuses as a single component with a highly basic isoelectric point of greater than pH 11.0. The NH 2- and COOH-terminal sequences of RNAase U show little homology with the pancreatic RNAases. However, the amino acid composition of this enzyme indicates it is very similar to human spleen RNAase.

Studies on dissociation and reconstitution of nuclear 30-S ribonucleoprotein particles containing pre-mRNA

Treatment of nuclear 30-S ribonucleoprotein (RNP) particles containing pre-mRNA (precursor of mRNA) with 2 M NaCl leads to dissociation of RNA and protein. The protein component is present either as an aggregate with a sedimentation coefficient close to 30 S (a free informofer) or as a slowly sedimenting material (monomers or oligomers of informatin). Most of the informofers and slowly sedimenting material are in the equilibrium state. Iodination or aging of the 30-S particles stabilizes informofers. Lowering of NaCl concentration in the mixture of RNA with informofers or informatin subunits leads to reconstitution of RNP particles. In both cases, the particles formed have a sedimentation coefficient of about 30 S and a buoyant density equal to 1.4–1.41 g/cm 3 but their response to pancreatic RNAase (EC 3.1.27.5) and high salt treatment is very different. Both the particles reconstituted from RNA and informofers and the original particles are very sensitive to pancreatic RNAase and after high salt treatment free informofers are formed. In contrast, the RNA of the particles reconstituted from slowly sedimenting material is much more protected against pancreatic RNAase action. These particles are also rather stable to high salt treatment. Thus, only if a protein in the form of an informofer aggregate is used, faithful reconstitution takes place. The data obtained are discussed in terms of the structure of the nuclear ribonucleoprotein particles containing precursor of messenger RNA.