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PubMed1977

Studies on salivary gland ribonucleases. II. Purification of ribonucleases from bovine submaxillary gland and the effects of polyamines on their activities.

Four alkaline ribonucleases [EC 3.1.4.22] were purified 2,050- to 3,460-fold from bovine submaxillary gland by repeated CM-Sephadex C-25 chromatography and Sephadex G-50 gel filtration, with a total recovery of about 13%. These were designated as RNase BS1, BS2, BS3, and BS4, based on their order of elution from a CM-Sephadex C-25 column. The molecular weights of these enzymes were estimated by gel filtration to be 19,000, 17,500, 17,000, and 12,000, respectively. These enzymes are very similar to RNase A in that they are inhibited by heparin, show preferential hydrolysis of C5'-O-P linkages adjacent to a cytosine nucleotide rather than a uracil nucleotide, and in their antigenic properties. Spermine was found to stimulate the activities of these enzymes; the degree of stimulation was in the order RNase BS4 greater than BS3 greater than BS2 greater than BS1. The stimulation by spermine is due to the increased cleavage of C5'-O-P linkages adjacent to cytosine nucleotide. The reason for the differences in the degree of spermine stimulation of these enzymes is discussed.

Animals↗

PubMed2002

Multiple ribonuclease H-encoding genes in the Caenorhabditis elegans genome contrasts with the two typical ribonuclease H-encoding genes in the human genome.

Database searches of the Caenorhabditis elegans and human genomic DNA sequences revealed genes encoding ribonuclease H1 (RNase H1) and RNase H2 in each genome. The human genome contains a single copy of each gene, whereas C. elegans has four genes encoding RNase H1-related proteins and one gene for RNase H2. By analyzing the mRNAs produced from the C. elegans genes, examining the amino acid sequence of the predicted protein, and expressing the proteins in Esherichia coli we have identified two active RNase H1-like proteins. One is similar to other eukaryotic RNases H1, whereas the second RNase H (rnh-1.1) is unique. The rnh-1.0 gene is transcribed as a dicistronic message with three dsRNA-binding domains; the mature mRNA is transspliced with SL2 splice leader and contains only one dsRNA-binding domain. Formation of RNase H1 is further regulated by differential cis-splicing events. A single rnh-2 gene, encoding a protein similar to several other eukaryotic RNase H2L's, also has been examined. The diversity and enzymatic properties of RNase H homologues are other examples of expansion of protein families in C. elegans. The presence of two RNases H1 in C. elegans suggests that two enzymes are required in this rather simple organism to perform the functions that are accomplished by a single enzyme in more complex organisms. Phylogenetic analysis indicates that the active C. elegans RNases H1 are distantly related to one another and that the C. elegans RNase H1 is more closely related to the human RNase H1. The database searches also suggest that RNase H domains of LTR-retrotransposons in C. elegans are quite unrelated to cellular RNases H1, but numerous RNase H domains of human endogenous retroviruses are more closely related to cellular RNases H.

Amino Acid Sequence↗

PubMed1989

Antigenic sites on ribonuclease A. Synthetic peptides corresponding to the sites 37-42 and 83-88 on ribonuclease A show immunogenicity.

Two hexapeptides NH2-Lys-Asp-Arg-Cys-Lys-Pro-COOH and NH2-Asp-Cys-Arg-Glu-Thr-Gly-COOH corresponding to the strong hydrophilic regions 37-42 and 83-88, respectively, on ribonuclease A were synthesized by solid-phase method. These synthetic peptides showed antigenic characteristics and provided an experimental validity to the prediction made earlier, supporting the view that highly hydrophilic regions on the protein have a good correlation with their being potentially antigenic.

Animals↗

PubMed1985

Two-dimensional 1H NMR investigation of ribonuclease A and ribonuclease-A--pyrimidine-nucleotide complexes.

Ribonuclease A was studied by two-dimensional 1H NMR spectroscopy. 10 out of 12 alanine and 9 out of 10 threonine spin systems as well as all valine [9], leucine [2] and isoleucine [3] spin systems were identified from the correlated spectroscopy (COSY) and relayed coherence transfer spectroscopy (RCT). Sequence-specific assignments were obtained from nuclear Overhauser effect spectra for proton resonances of 21 amino acid moieties. 2' and 3'-pyrimidine-nucleotide-RNase-A complexes were also investigated by two-dimensional NMR. We were able to monitor structural changes in the active center, the vicinity of the active center and in regions far from the catalytic region. Chemical shift changes of resonances of protons near Thr-45 reflected the binding of the same moiety. This in turn is also dependent on the position of the nucleotide phosphate group. Binding of 2' nucleotides led to characteristic changes in protein regions not affected by the binding of 3' nucleotides. These results are interpreted in terms of structural differences between the 2' and 3'-nucleotide-RNase-A complexes; the structure of the complex of the native 3' nucleotide inhibitor being more closely related to that of the free protein.

Amino Acid Sequence↗

PubMed1992

Dissection of the ribonuclease T1 subsite. The transesterification kinetics of Asn36Ala and Asn98Ala ribonuclease T1 for minimal dinucleoside phosphates.

Ribonuclease T1 contains a subsite which by interacting with the leaving nucleoside N of GpN dinucleoside phosphate substrates, contributes to catalysis [Steyaert, J., Wyns, L. & Stanssens, P. (1991) Biochemistry 30, 8661-8665]. The Asn36Ala and Asn98Ala mutations reduce the transesterification rates of GpA, GpC and GpU considerably whereas they have virtually no effect on the transesterification kinetics of the synthetic substrate guanosine 3'-(methyl phosphate) (GpMe) (in which the leaving nucleoside is replaced by methanol), indicating that the Asn36 and Asn98 side chains are part of the RNase T1 subsite [Steyaert, J., Haikal, A. F., Wyns, L. & Stanssens, P. (1991) Biochemistry 30, 8666-8670]. The kinetics of the Asn36Ala, Asn98Ala and wild-type catalyzed transesterification of guanosine 3'-(5'-D-ribosyl phosphate) (GpRib), another GpN analog in which the leaving groups is replaced by D-ribose, enables the mapping of the subsite interactions provided by Asn36 and Asn98. We find that the Asn36 amide function contributes 4.6 kJ/mol to catalysis through interactions with the ribose moiety of the leaving nucleoside. Asn98 is at least in part responsible for the subsite preference for cytidine; the Asn98 side chain preferentially binds cytosine as the leaving nucleoside base.

Alanine↗

PubMed1989

Role of ribonuclease and ribonuclease inhibitor activities in Alzheimer's disease.

It has been suggested that defects in the relationship between ribonuclease and its proteinaceous inhibitor could be a contributory factor in Alzheimer's disease. We have investigated this possibility further by analysing free and bound enzyme activities and the activity of the inhibitor in nine regions of diseased and normal brain. These were chosen to include areas known to be affected by the disease, regions not histologically affected but thought to be involved in the disease process, and areas not thought to be involved in the disease. Neither the enzyme nor its inhibitor is defective in its activities in the chosen areas of Alzheimer's disease brain when compared with those of carefully age-matched controls.

Alzheimer Disease↗

PubMed1991

Effect of aging, castration, and testosterone administration on ribonuclease and ribonuclease-inhibitor activities in the prostate of rats.

Free ribonuclease (RNase)-inhibitor activities in both ventral and dorsal prostates had their highest peaks in 4-week-old rats and smallest peaks in around 7-week-old animals. Total RNase activity in the ventral prostate decreased overall with age, while that in the dorsal prostate increased. No significant amount of free RNase activity was found in either prostate. Weight, protein content, and free RNase-inhibitor activity in both prostates decreased after castration and increased after administration of testosterone to castrated rats. Total RNase activity in the ventral prostate was increased by castration and decreased by testosterone administration. In the dorsal prostate, total RNase activity had two peaks, 7 d after castration and 2 d after testosterone administration. A large amount of free RNase activity was found in the ventral prostate 7 d after castration and this activity was decreased by testosterone administration. In the dorsal prostate, free RNase activity was not detected after castration and testosterone administration. These results suggest that changes in the level of RNase-inhibitor in both prostates are involved in the regulation of their RNA content through the control of free RNase activity.

Aging↗

PubMed2004

Acid ribonuclease and alkaline ribonuclease isoenzymes in plasma of patients with decreased glomerular filtration rate.

Removal of low molecular weight proteins from plasma by kidneys depends on glomerular filtration rate (GFR), protein-glomerular membrane electric charge, steric interactions and a number of functionally active nephrons present in the kidneys. There is a well documented relationship between the concentration of low molecular weight proteins in plasma and GFR value in patients with impaired renal function. Accumulation of low molecular weight proteins in plasma along with a decrease in GFR value may in the long run enhance formation of protein tissue deposits known as various forms of amyloidosis. In this paper we present studies on plasma concentrations of acid leukocyte-type ribonuclease (RNase) and alkaline pancreatic-type RNase and GFR value in 54 patients with renal failure. RNase isoenzymes' activities were assayed by measuring their enzyme activities manifested as ability to decompose yeast RNA and assay of digestion products' concentration by spectrophotometry. The studies show that decreasing filtration rate produces an increase in serum activities of both acid and alkaline RNases, which is proportional to the logarithm of GFR value. However, the increase rate vs. GFR value is by four times higher for acid RNase than for alkaline RNase. Acid RNase in human plasma is mostly of leukocytic origin and differs from pancreatic-type alkaline RNase, which is of pancreatic origin. The obtained results may suggest that leukocyte originating proteins essentially contribute to low molecular weight protein accumulation in plasma of patients with chronic renal insufficiency.

Adult↗

PubMed1978

Possible sites of origin of human plasma ribonucleases as evidenced by isolation and partial characterization of ribonucleases from several human tissues.

The ribonucleases (RNases) present in a number of human tissues, including heart, brain, lung, and kidney, were purified, partially characterized, and compared in their properties to the previously described RNases from human liver, spleen, pancreas, and serum. The enzymes appeared to fall into two major classes: liver-spleen type RNase and plasma-type RNase. These two types of enzymes were present in varying proportions in all tissues examined. The extent to which the tissues studied possibly contribute to serum RNase levels is discussed.

Brain↗

PubMedDate not supplied

[Modified method for producing ribonuclease A from amorphous and crystalline ribonucleases].

The paper describes a method for producing gram-scale quantities of beef pancreatic ribonuclease A (RNase A) (EC 3.1.4.22) from commercial, amorphous and crystalline RNases. The method involves enzyme purification by chromatography on SP-Sephadex G-25, rechromatography on Bio-Rax 70 followed by concentration, storage and desalting in optimal conditions. In their properties the resultant preparations of RNase A are not inferior to the best commercial samples of the enzyme.

Animals↗

PubMed1998

Extracellular ribonuclease from Rhizopus stolonifer: characteristics of an atypical--guanylic acid preferential--enzyme from ribonuclease T2 family.

An extracellular ribonuclease from Rhizopus stolonifer (designated as RNase Rs) was purified to homogeneity by chromatography on DEAE-cellulose followed by CM-cellulose. The Mr of the purified enzyme determined by gel filtration and SDS-PAGE is 25,000 and 28,200, respectively. RNase Rs is a glycoprotein and contains 10.5% neutral sugar. It is an acidic protein with a pI of 5.0 and has a blocked N-terminus. The optimum pH and temperature are 5.5 and 45 degrees C, respectively. RNase Rs shows high stability between pH 6.0-10.0. Divalent cations like Zn2+, Hg2+ and Cu2+ inhibit the enzyme activity whereas, mononucleotides does not have any significant effect. The enzyme cleaves RNA to 3'-mononucleotides via 2',3'-cyclic nucleotides, with preferential liberation of 2',3'-cyclic GMP, suggesting that RNase Rs is a guanylic acid preferential cyclizing RNase. Moreover, cyclic nucleotides generated are highly resistant to further hydrolysis.

Cations, Divalent↗

PubMed1995

Effects of replacement of Lys25 with Gln on the conformation of ribonuclease T1: sequence-specific 1H NMR resonance assignments of Gln25 ribonuclease T1 by two-dimensional NMR spectroscopy.

Two isozymes of ribonuclease (RNase) T1 exist in nature, i.e. Gln25 RNase T1 and Lys25 RNase T1. Gln25 RNase T1 is less stable than Lys25 RNase T1, although the enzymatic activity is not distinguishable between these two isozymes. To elucidate the effects of the replacement of Lys25 with Gln on the conformation and microenvironments of RNase T1 in detail, two-dimensional NMR spectra were measured, sequence-specific 1H NMR resonance assignments of Gln25 RNase T1 were performed, and then the determined parameters and microenvironments of Gln25 RNase T1 were compared with those of Lys25 isozyme [Hoffmann, E. and Rüterjans, H. (1988) Eur. J. Biochem. 177, 539-560]. The main chain protons were assigned for 101 out of the total of 104 amino acid residues. Secondary structure elements were identified from analysis of characteristic NOE patterns, interstrand NOE connectivities, and hydrogen-deuterium exchange rates of main chain amide protons. The results indicated that Gln25 RNase T1 contains a single alpha-helix and seven beta-strands. The secondary structure of Gln25 RNase T1 is, thus, essentially the same as that of Lys25 RNase T1. On the other hand, comparison of the conformation-dependent shifts of Gln25 RNase T1 with these of Lys25 RNase T1 showed that the replacement of Lys25 with Gln has significant effects on the C-terminal part of the alpha-helix region and the base-binding site. These results may indicate that the base-binding site is relatively flexible in the RNase T1 molecule. Among the residues of the C-terminal part of the alpha-helix region, the protons of Asp29 were most affected in terms of their chemical shifts, which may indicate that the side chain carboxylate anion of Asp29 is the counterpart of the electrostatic interaction of Lys25 in Lys25 RNase T1. The Gln25 of Gln25 RNase T1 may have little or no interaction with Asp29, and this may be the reason why Gln25 RNase T1 is less stable than the Lys25 isozyme.

Amino Acid Sequence↗

PubMed1984

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.

Amino Acids↗

PubMed1990

Purification and characterization of a ribonuclease from human spleen. Immunological and enzymological comparison with nonsecretory ribonuclease from human urine.

A ribonuclease has been isolated from human spleen (RNase HS) by means of acid extraction, ammonium sulphate fractionation, successive column chromatographies on CM-cellulose, heparin-actigel, and poly(G)-agarose, and double gel-filtration on Sephadex G-75. The purified preparation was homogeneous as judged by SDS/PAGE. RNase HS was found to be a glycoprotein, containing three fucose, one mannose and five glucosamine residues/molecule, with a molecular mass of 17 kDa as determined by both SDS/PAGE and gel filtration. The catalytic properties and structural features, including its amino acid composition and the amino acid sequence of the N-terminal 35 residues, indicated that the enzyme was strictly related to nonsecretory RNase isolated from human urine and liver. In particular, the amino acid sequence of the N-terminal was identical with that of urine nonsecretory RNase and eosinophil-derived neurotoxin. Furthermore, analyses using three different antibodies specific to RNase HS, urine nonsecretory RNase and urine secretory RNase, indicated that RNase HS was not immunologically distinguishable from urine nonsecretory RNase, but clearly so from urine secretory RNase. However, the carbohydrate compositions of RNase HS and urine nonsecretory RNase were found to differ. It therefore remains to be resolved whether or not the tissue of origin of nonsecretory RNase in urine is the spleen.

Adult↗

PubMed1964