A ribonuclease from a callus cell culture of Panax ginseng C.A. Mey strain R1 was isolated. A pure protein with an apparent molecular mass of 18 kDa was obtained. The N-terminal sequences of the protein and of the C-terminal CNBr peptide were determined. No homology with other ribonucleases was found, but there was 60-70% sequence identity with two intracellular pathogenesis-related (IPR) proteins from parsley, indicating that not only these two proteins, but also homologous IPR proteins identified in other plant species are ribonucleases.

With the use of a high yield prokaryotic expression system, large amounts of human eosinophil cationic protein (ECP) have been obtained. This has allowed a thorough kinetic study of the ribonuclease activity of this protein. The catalytic efficiencies for oligouridylic acids of the type (Up)nU>p, mononucleotides U>p and C>p, and dinucleoside monophosphates CpA, UpA, and UpG have been interpreted by the specific subsites distribution in ECP. The distribution of products derived from digestion of high molecular mass substrates, such as poly(U) and poly(C), by ECP was compared with that of RNase A. The characteristic cleavage pattern of polynucleotides by ECP suggests that an exonuclease-like mechanism is predominantly favored in comparison to the endonuclease catalytic mechanism of RNase A. Comparative molecular modeling with bovine pancreatic RNase A-substrate analog crystal complexes revealed important differences in the subsite structure, whereas the secondary phosphate-binding site (p2) is lacking, the secondary base subsite (B2) is severely impaired, and there are new interactions at the po, Bo, and p-1 sites, located upstream of the P-O-5' cleavable phosphodiester bond, that are not found in RNase A. The differences in the multisubsites structure could explain the reduced catalytic efficiency of ECP and the shift from an endonuclease to an exonuclease-type mechanism.

The preparation of oligodeoxynucleotide derivatives with regiospecifically incorporated dialdehyde reactive groups and their successful use as affinity reagents for different enzymes is described.

The aim of this work is to determine the conformation of the nucleobase adjacent to the cleavable phosphodiester bond in the productive enzyme-substrate complex of RNA-depolymerizing enzymes. To this end the kinetic parameters of hydrolysis of UpA, 2'-C-Me- and 3'-C-Me-UpA were determined for RNase A, RNase Pb2, nuclease S1 and snake venom phosphodiesterase. In these derivatives the ranges of the allowed orientation of uridine residues are restricted due to the substitution of methyl groups for the ribose hydrogen atoms. The results described demonstrate that the proposed method is of general value for the estimation of the nucleotide glycoside angles in the productive enzyme-substrate complexes.

The amino acid sequences of two ribonucleases from a callus cell culture of Panax ginseng were determined. The two sequences differ at 26% of the amino acid positions. Homology was found with a large family of intracellular pathogenesis-related proteins, food allergens and tree pollen allergens from both dicotyledonous and monocotyledonous plant species. There is about 30% sequence difference with proteins from species belonging to the same plant order (Apiales: parsley and celery), 60% with those from four other dicotyledonous plant orders and about 70% from that of the monocotyledonous asparagus. More thorough evolutionary analyses of sequences lead to the conclusion that the general biological function of members of this protein family may be closely related to the ability to cleave intracellular RNA and that they have an important role in cell metabolism. As the three-dimensional structure of one of the members of this protein family has been determined recently [Gajhede et al., Nature Struct Biol 3 (1996) 1040–1045], it may be possible to assign active-site residues in the enzyme molecule and make hypotheses about its mode of action. Structural features in addition to the cellular site of biosynthesis indicate that this family of ribonucleases is very different from previously investigated ones.

To establish the mechanism of dsRNA degradation by mammalian single-stranded-preferring ribonucleases, and, in particular, the influence of their positively charged non-catalytic amino acid residues, we have studied the kinetic parameters of the depolimerization of single- and double-stranded polyribonucleotides such as poly(U), poly(U).poly(A), poly(C) and poly(C).poly(I) by the action of human seminal RNase, bovine seminal RNase and ox pancreas RNase A. While the activities of these RNases on poly(I).poly(C) were definitely lower than those on poly(C), the activities of human seminal and bovine seminal RNases on poly(U).poly(A) and poly(U) were of the same order of magnitude under physiological salt conditions. The ratio of the RNase A degrading activities towards poly(U) and poly(U).poly(A) at I = 0.16 M is ten times higher than the corresponding ratios determined with bovine seminal and human seminal ribonucleases. The high activities of these two RNases towards poly(U).poly(A) are discussed on the basis of their efficient estabilishing action on this double-helical nucleic acid due to their high affinity for poly(A). The destabilizing action of human seminal RNase and bovine seminal RNase on the poly (U).poly(A) duplex is higher than that measurable with bovine RNase A because of the higher number of positive charges present on those enzyme molecules. This may therefore explain why human seminal and bovine seminal ribonucleases are more efficient than RNase A in the depolymerization of poly(U).poly(A) at physiological ionic strength.

The amino acid sequences of two ribonucleases from a callus cell culture of Panax ginseng were determined. The two sequences differ at 26% of the amino acid positions. Homology was found with a large family of intracellular pathogenesis-related proteins, food allergens and tree pollen allergens from both dicotyledonous and monocotyledonous plant species. There is about 30% sequence difference with proteins from species belonging to the same plant order (Apiales: parsley and celery), 60% with those from four other dicotyledonous plant orders and about 70% from that of the monocotyledonous asparagus. More thorough evolutionary analyses of sequences lead to the conclusion that the general biological function of members of this protein family may be closely related to the ability to cleave intracellular RNA and that they have an important role in cell metabolism. As the three-dimensional structure of one of the members of this protein family has been determined recently [Gajhede et al., Nature Struct Biol 3 (1996) 1040-1045], it may be possible to assign active-site residues in the enzyme molecule and make hypotheses about its mode of action. Structural features in addition to the cellular site of biosynthesis indicate that this family of ribonucleases is very different from previously investigated ones.

Three forms of the extracellular alkaline ribonuclease of B. polymyxa (RNases Bpo I, Bpo II and Bpo III) were obtained in the homogeneous states. Relative molecular weights, N‐ and C‐terminal amino acid sequences of the proteins were determined. The two higher‐molecular weight forms of the enzyme (RNases Bpo I and Bpo III) were shown to be precursor molecules which differed from the mature B. polymyxa RNase (RNase Bpo II) by the presence of a extra twelve and six amino acid residues at the amino terminus, respectively. The study of catalytic properties of the enzyme elucidated that the extent of specificity of B. polymyxa RNase in respect to high‐polymeric substrates is different from that of other natural Bacillus RNases. Amino acid composition of B. polymyxa RNase was established and structural similarity within family of Bacillus RNases is discussed. B. polymyxa RNase was shown to be inhibited by intracellular protein inhibitor of B. amyloliquefaciens RNase with the dissociation constant of 2.7×10‐12 M.

Binase, the extracellular ribonuclease of Bacillus intermedius, is inhibited by barstar, the natural protein inhibitor of the homologous RNase, barnase, of B. intermedius. The dissociation constants of the binase complexes with barstar and its double Cys40.82 Ala mutant are about 10−12 M, only 5 to 43 times higher than those of the barnase‐barstar complex. As with barnase, the denaturation temperature of binase is raised dramatically in the complex. Calorimetric studies of the formation and stability of the binase‐barstar complex show that the binase reaction with barstar is qualitatively similar to that of barnase but some significant quantitative differences are reported.

The kinetics of single-stranded (SS) and double-stranded (ds) polyribonucleotides cleavage by three mammalian pancreatic type ribonucleases have been studied under low and high salt conditions. The values kcat, Km, and kcat/Km for depolymerization of poly(U), poly(A).poly(U), poly(I) and poly(I).poly(C) by bovine RNase A, bovine seminal RNase, and human seminal RNase have been determined and compared to each other. The Km values of bovine RNase A for (ss) or (ds) substrates were of the same order of magnitude under low and high ionic strength conditions, while their kcat values were found to differ considerably. Qualitatively similar results were obtained with bovine and human seminal RNases, i.e., the activity ratios (ssRNA/dsRNA) were mostly determined by the ratio of kcat values. It was shown that the modest levels of activity toward dsRNAs shown by single-strand-preferring RNases may occur by a mechanism consisting in the binding of the RNase to single nucleotides which are wound off the double helix because of thermal fluctuations. A higher activity and its enhancement as a function of number and location of the positive charges present on the RNase surface (human seminal RNase > bovine seminal RNase > bovine RNase A), as well as its increase under low ionic strength conditions, could instead be explained by the increased occurence of the splitting mechanism based on the binding of the RNase to single-stranded RNA sequences transiently exposed from the RNA double-helix.

To elucidate the functional role of some residues in the active site of binase, the extracellular ribonuclease of Bacillus intermedius, we used site-directed mutagenesis. On cleavage of various substrates the catalytic activity of binase mutant His101Glu is 2.0–2.7% of that for native enzyme. The decrease in activity is determined mainly by the decrease in molecular rate constant kcat with almost unchanged affinity of the enzyme for the substrate, characterized by KM. This is the expected result if His101 acts as an general acid, donating a proton to the leaving group on cleavage of a phosphodiester bond. The replacement of Lys26 by Ala causes a reduction in the enzyme activity to 13—33%, depending on the substrate. The activity decreases are due to changes in both kcat and KM for poly(I) and poly(A) but in kcat alone for GpA. In the latter case the effect is far less than that seen in the homologous mutation in the closely related enzyme, barnase.

A complete amino acid sequence of extracellular Bacillus circulans RNase was established and compared with a structure of B. amyloliquefaciens RNase. Gln 15 , Gly 65 and Gln 104 in B. amyloliquefaciens RNase were found to be replaced by Leu, Ala and Lys, respectively, in B circulans RNase. Catalytic properties of B. circulans RNase were studied.

Bacterial ribonucleases from Bacillus amyloliquefaciens and Bacillus intermedius show the specificity towards the nature of a nucleoside at the O3' end of the phosphodiester bond to be split in the preference order G > A >> U > C in the cleavage reactions of polynucleotides. It follows from the X-ray data that the substrate guanosine base is bound at the active site of these RNases in the same manner as for high-specificity guanylic RNases. We supposed that the difference in specificity for the two types of RNases is due to the additional hydrogen bond between the protein and a purine base in the case of bacterial guanyl-preferring RNases in contrast to the high-specificity guanylic RNases. To examine this hypothesis we prepared the Ser57 --> Glu mutant of B. amyloliquefaciens, in which this hydrogen bond is eliminated. Kinetic studies demonstrate that the specificity of this mutant towards guanylic substrates is 35-times greater than that of the wild-type RNases from B. amyloliquefaciens and close to that of RNases T1.

The chemical-shift dependences of the proton signals of the guanosine and uridine moieties were measured as a function of the relative amount of GpcU complexed with RNase Pb1 (EC 3.1.27.3). The equal values of the chemical-shift changes of the guanosine C8-protons on complex formation between GpcU and RNase Pb1 and that of the 3′-GMP and RNase Pb1 allow to conclude that the guanosine base is bound in the same manner in these protein-ligand complexes. The guanosine moiety of GpcU is also most probably bound in the syn-conformation. The absence of changes in both the linewidths and the chemical shifts of the C1′, C5 and C6-proton signals of the uridine on complex formation indicates that the uridine moiety of the dinucleoside phosphonate is not immobilized in the complex. The pH dependences of the chemical shifts of the C2-protons of the histidine-imidazole ring of RNase Pb1 and that of the 31P of GpcU in the RNase complex were studied. The results suggest that there is a direct interaction betweenthe phosphonate group of the ligand and the protonated imidazole ring of His-90. The side groups of His-38 and Glu-56 are hydrogen bonded to each other at neutral pH and they are located in the vicinity of the phosphonate group of GpcU. When the carboxyl group of Glu-56 is protonated the His-38 imidazole ring forms a new hydrogen bond with one of the phosphoryl oxygens of the phosphonate group. On the basis of these results we propose the mechanism of action of RNase Pb1 which is probably also true for RNase T1.

The kinetic parameters of reactions catalyzed by four guanyl-specific RNases T1, Pb1, Th1 and Sa were studied comparatively using three types of substrates; guanosine-2',3'-cyclophosphates, GpN dinucleoside phosphates and synthetic polyribonucleotides. The kinetic parameters were shown to be similar in spite of considerable differences in primary structures of these RNases, including amino acid residues of the active sites. Therefore, primary structures of guanyl RNases allow for a considerable number of substitutions (both in the 'recognising' and catalytical parts of the active site) without changes in the catalytical parameters.

Kinetic parameters kcat and KM were measured for cleavage of poly I, poly A, poly U, poly C and poly I poly C by guanyl-specific RNases Sa, Pb1 and T1 and compared with that of guanyl-preferential RNase Bi. Catalytic efficiencies of the investigated enzymes to polynucleotide substrates vary considerably. The structural basis for specificity of these RNases is discussed. A hypothesis is suggested that Ser-56 plays an important role in recognition of poly A by RNase Bi.

The rates of enzymatic hydrolysis of 2′,5′-oligoadenylates and their synthetic analogs have been measured. These compounds were treated with either NIH 3T3 cell lysates, mouse liver homogenates or snake venom phosphodiesterase. All analogs with 3′-terminal acyclic nucleoside residues demonstrated greater stability compared with the natural compound adenylyl(2′-5′)adenylyl(2′-5′)adenosine.