Pancreatic RNAase Research Articles

Binding sites of ribosomal proteins on two specific fragments derived from Escherichia coli 50 S ribosomes.

Escherichia coli 50 S ribosomes were digested by pancreatic RNAase under conditions which lead to the cleavage of the 23 S into two specific fragments. After removal of about half of the proteins (split proteins) by treatment with lithium chloride, the subparticles (core fragments) were separated and their proteins (core proteins) analyzed. Only four core proteins were bound exclusively to one or the other of the core fragments; each of the other core proteins was found on both fragments in constant proportion. In reconstitution experiments, a class of split proteins was found which did not recombine with the individual core fragments but which bound to a mixture of both. Without demonstrating the homogeneity of the 50 S ribosomes population, these results suggest that the ribosomal proteins are not randomly distributed in the complete 50 S subunit although most of them bind to multiple sites.

The incorporation of amino acids into the protein of isolated soya bean mitochondria

A system involving the incorporation of amino acids into the protein of mitochondria isolated aseptically from soya bean hypocotyls has been partially characterized. Incorporation is optimal at pH 7.4, is dependent on magnesium, phosphate and succinate, is resistant to pancreatic RNAase and cycloheximide, but is sensitive to D-threo-chloramphenicol, oligomycin, DNP, and changes in osmotic concentration.

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.

Spaltung von löslicher ribonucleinsäure und serin-spezifischen transfer-ribonucleinsäure-fraktionen mit pankreas-ribonuclease

Several RNA samples were split with pancreatic RNAase (polyribonucleotide 2-oligonucleotidotransferase (cyclizing), EC 2.7.7.16). The fragments were separated by chromatography on DEAE-cellulose columns and in some cases by subsequent paper electrophoresis. The amounts of mono- and dinucleotides from various unfractionated and partially fractionated soluble RNA's were compared (Table I). In hydrolysates of highly purified serine transfer RNA fractions 7 di-, 7 tri-, 7 tetra-, 2 penta- and 2 hexanucleotides - coming from 2 serine transfer-RNAs - were identified and quantitatively determined (Tables II and III), as well as some oligonucleotides, which occur in smaller amounts and probably come from contaminations of the serine transfer-RNA fractions. 3 oligonucleotides could not be identified, and one only partially. Several oligonucleotides contain odd nucleotides.

The inhibition of deoxyribonucleotidyl transferase, DNAase and RNAase by sodium poly ethenesulfonic acid. Effect of the molecular weight of the inhibitor

1. 1. The nature of the inhibition of DNA nucleotidyltransferase (EC 2.7.7.7) from KB cells (DNA polymerase) by poly ethenesulfonic acid (PES) and other polyanionic materials were examined. The inhibition was found to be competitive with the DNA used to prime the enzyme. The K 1 was found to be 1.72 ± 0.36 μg PES/ml for the 12 900 mol. wt. material in the range of zero to 10 μg inhibitor/ml. The inhibitory action of PES was strongly dependent on the molecular weight of the sample used in the range of 5000 to 13 000 and further increases in molecular weight did not seem to affect efficacy. Other polyanionic polymers, such as heparin, carboxymethyl cellulose, alginic acid, chitosan sulfate, RNA and chondroitin sulfate also inhibited this enzyme to varying degrees. 2. 2. Spermine, which by itself inhibits the DNA polymerase, reversed the inhibition caused by PES. This is explained by the competition between DNA and PES for salt formation (and precipitation) with spermine. 3. 3. The inhibition of pancreatic RNAase (EC 2.7.7.16) by PES exhibited a similar dependence on the molecular weight of the polymer as did the inhibition of DNA polymerase. 4. 4. Addition of PES to DNAase I (EC 3.1.4.5) assays resulted in a bimodal, concentration-dependent curve. At intermediate concentrations the compound stimulated DNA hydrolysis, while at higher concentrations it inhibited.

Some nucleotide sequences from partially purified transfer ribonucleic acids

1. 1. Transfer RNA from baker's yeast and from Escherichia coli have been fractionated by partition and ion-exchange chromatography. 2. 2. Partially purified samples of transfer RNA from yeast specific for serine and valine and from E. coli specific for phenylalanine and lysine, as well as unfractionated transfer RNA from both sources have been subjected to digestion with alkali, pancreatic RNAase (EC 2.7.7.16), and takadiastase RNAase, T 1 (EC 3.1.4.8). The products of the digestions were separated by paper ionophoresis and chromatography in a two-dimensional system and identified. 3. 3. It was found that: (a) The nucleotide sequences of unfractionated transfer RNA's are non-random. (b) It appears unlikely that methylated purines occur in the serine transfer RNA reported on, and none have been identified in the partially purified samples from E. coli. (c) In transfer RNA from E. coli no runs of adenylic acid longer than 3 residues were found. (d) The following provision list of nucleotide sequences occurring in pancreatic ribonuclease digest of serine transfer RNA from yeast has been deduced: AC, 1; AU, 2; GC, 4; GU, 2; A 2C, 1; A 2U, 1; G 2C, 2; G 2U, 1; G 2ψ, 1; [AG]C, 1; [AG]U, 2; [AG 2]U, 2; C, 11; U, 10; and ψ, 1.

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.

Nucleotide-sequence differences among ribosomal ribonucleic acid fractions and soluble ribonucleic acid from various bacterial species

Bacterial RNA fractions with identical base compositions have been compared by digesting the RNA with pancreatic RNAase (EC 2.7.7.16) and determining the relative frequency of nucleotide fragments. 1. There were no detectable differences among several strains of Escherichia coli nor for the same strain grown under a variety of conditions. 2. The 70-S ribosomal RNA's from bacteria with widely varying guanine-cytosine contents of their DNA were compared. There were no detectable differences among bacteria closely related to E. coli. Only species with very high or low guanine-cytosine contents showed considerable differences in their RNA from each other and from E. coli. 3. The 50-S and 30-S RNA's of these species differ from each other and from the corresponding fractions of E. coli. 4. Nucleotide mapping of s-RNA's also showed no detectable differences among species closely related to E. coli but considerable differences for bacteria with high or low guanine-cytosine content of their DNA. The implications of these results in terms of the synthesis and function of the various RNA fractions are discussed.

Preparation of transforming deoxyribonucleic acid by phenol treatment

Several procedures employing chloroform and phenol for extraction and deproteinization have been explored for the preparation of highly purified transforming DNA from Bacillus subtilis . The recovery, purity, transforming activity and the sedimentation coefficients of the preparations have been compared. The following procedure has been found to give the best results: lysis of cells with lysozyme (EC 3.2.1.17), followed by freezing and thawing, extraction of nucleic acids with a mixture of phenol and pH-9 buffer in the presence of sodium dodecyl sulfate, digestion of RNA with a mixture of pancreatic RNAase (EC 2.7.7.16) and RNAase T1 from Taka-diastase (EC 3.1.4.8), followed by treatment with phenol. When the digestion is carried out with pancreatic RNAase alone, it is followed by precipitation with isopropapol in order to eliminate RNA. The final DNA preparation has high transforming activity, especially for the joint transformation of linked markers.

Preparation of transforming deoxyribonucleic acid by phenol treatment

Abstract Several procedures employing chloroform and phenol for extraction and deproteinization have been explored for the preparation of highly purified transforming DNA from Bacillus subtilis . The recovery, purity, transforming activity and the sedimentation coefficients of the preparations have been compared. The following procedure has been found to give the best results: lysis of cells with lysozyme (EC 3.2.1.17), followed by freezing and thawing, extraction of nucleic acids with a mixture of phenol and pH-9 buffer in the presence of sodium dodecyl sulfate, digestion of RNA with a mixture of pancreatic RNAase (EC 2.7.7.16) and RNAase T1 from Taka-diastase (EC 3.1.4.8), followed by treatment with phenol. When the digestion is carried out with pancreatic RNAase alone, it is followed by precipitation with isopropapol in order to eliminate RNA. The final DNA preparation has high transforming activity, especially for the joint transformation of linked markers.

Preparation of transforming deoxyribonucleic acid by phenol treatment

Several procedures employing chloroform and phenol for extraction and deproteinization have been explored for the preparation of highly purified transforming DNA from Bacillus subtilis . The recovery, purity, transforming activity and the sedimentation coefficients of the preparations have been compared. The following procedure has been found to give the best results: lysis of cells with lysozyme (EC 3.2.1.17), followed by freezing and thawing, extraction of nucleic acids with a mixture of phenol and pH-9 buffer in the presence of sodium dodecyl sulfate, digestion of RNA with a mixture of pancreatic RNAase (EC 2.7.7.16) and RNAase T1 from Taka-diastase (EC 3.1.4.8), followed by treatment with phenol. When the digestion is carried out with pancreatic RNAase alone, it is followed by precipitation with isopropapol in order to eliminate RNA. The final DNA preparation has high transforming activity, especially for the joint transformation of linked markers.

Nucleotide-sequence differences among ribosomal ribonucleic acid fractions and soluble ribonucleic acid from various bacterial species

Bacterial RNA fractions with identical base compositions have been compared by digesting the RNA with pancreatic RNAase (EC 2.7.7.16) and determining the relative frequency of nucleotide fragments. 1.1. There were no detectable differences among several strains of Escherichia coli nor for the same strain grown under a variety of conditions.2.2. The 70-S ribosomal RNA's from bacteria with widely varying guanine-cytosine contents of their DNA were compared. There were no detectable differences among bacteria closely related to E. coli. Only species with very high or low guanine-cytosine contents showed considerable differences in their RNA from each other and from E. coli.3.3. The 50-S and 30-S RNA's of these species differ from each other and from the corresponding fractions of E. coli.4.4. Nucleotide mapping of s-RNA's also showed no detectable differences among species closely related to E. coli but considerable differences for bacteria with high or low guanine-cytosine content of their DNA.

Effect of oxygen and N-ethylmaleimide on the inactivation of ribonuclease by gamma-radiation.

A 0.01% aqueous solution of pancreatic RNAase was irradiated at room temperature with gamma rays from a Co/sup 60/ source (dose rate about 2.6 x 10/ sup 5/ rad/hr). Air, oxygenfree nitrogen, or various mixtures of the gases were bubbled rapidly into the solution through an open-ended tube for 8 min before and throughout irradiation. In some runs N-ethylmaleimide was present at various concentrations. It was found that N-ethylmaleimide (5 x 10/sup -4/ M) showed a considerable protective effect under anoxic conditions (Nethylamaleimide in the absence of radiation did not affect the activity of the enzyme). Oxygen (when air was bubbled through) also exerted a marked protective affect but this was not observed with unbubbled aerobic solutions. The protection observed with N- ethylmaleimide may be due to its acting as a radical scavenger. (P.C.H.)

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.

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.

Cerebrospinal fluid ribonuclease activity.

Cerebrospinal fluid RNAase was determined by a new turbidimetric method. The normal activity of enzyme for this fluid was found to be equivalent to the activity of .084 ± .02 μg of crystalline pancreatic RNAase. Patients with cerebrovascular accidents, brain tumors and acute multiple sclerosis had significantly elevated enzyme levels in their spinal fluid. The percentage of cases with elevated RNAase was considerably greater than those with elevated transaminase. The concentrations of both enzymes in the various areas of the human brain were measured. RNAase was uniquely higher in the white than in the grey matter. Submitted on April 15, 1958