Human platelet ribonuclease

Human platelets contain an RNase which has a pH optimum at 5.0. It hydrolyzes the secondary phosphate esters of uridine 3′-phosphates. It slowly converts uridine 2′:3′-and cytidine 2′:3′-cyclic phosphates to their corresponding nucleoside 3′-phosphates. Poly (A), poly (G) and poly (C) are not only refractory to the action of this enzyme, but also inhibit its action on poly (U). It differs from human granulocyte RNase, human serum RNase and bovine pancreatic RNase. Because of its unique property, this enzyme could serve as a biochemical marker in disorders involving the platelet destruction.     

Purification and Properties of a Ribonuclease in Human Urine that Hydrolyses Polycytidylic Acid

Human urine RNase was purified about 2000-fold. The preparation is free from phosphatase, phosphodiesterase and DNase activities. On electrophoresis through polyacrylaraide gel at pH 8.3, it migrates toward the anode and stains with periodic acid-Schiff reagent, suggesting that it is aci c and glycoprotein in nature. Its isoelectric point is at pH 4.1. It has a molecular weight of about 21, 500.

It is thermostable at pH 4.2 and thermolabile at pH 8.5. It has a pH optimum at 6.5. It exhibits highest preference for cytidine 3′-phosphate linkages. Its activity on poly (C) is endonucleolytic. It cleaves poly (C) via intramolecular transphosphorylation. It has no action on cytidine 2′: 3′-cyclic phosphate or uridine 2′:3′-cyclic phosphate.

Its rate of hydrolysis of poly (U) is less than 2% of that of poly (C). Poly (A) and poly (G) are totally inert to its action. Its action on poly (C) is inhibited by poly (G), poly (A) and poly (U).

It differs from bovine pancreatic RNase A in its physical, chemical and catalytic properties. It is, however, similar to human serum and pancreatic RNase in all its properties, suggesting that pancreas is its likely source.     

Nature and possible origin of Human Serum ribonuclease

Human Serum contains an acidic RNase which is glycoprotein in nature. It is thermostable at pH 4.2 and thermolabile at pH 8.5. It has a pH optimum at 6.5. Its activity either on poly (C) or RNA is endonucleolytic and is absolutely dependent on citrate or phosphate. It exhibits highest preference for the secondary phosphate esters of cytidine 3′-phosphates. It has no action on cytidine 2′:3′-cyclic phosphate. Poly (A) and poly (G) are not only refractory to its action, but also inhibit its action on poly (C). Its rate of hydrolysis of Poly (U) is about 2% of that of poly (C). It differs from bovine pancreatic RNase. It is, however, similar to human pancreatic RNase suggesting that its primary source is pancreas.     

Purification and properties of a ribonuclease in human urine that hydrolyses polycytidylic acid.

Human urine RNase was purified about 2000-fold. The preparation is free from phosphatase, phosphodiesterase and DNase activities. On electrophoresis through polyacrylamide gel at pH 8.3, it migrates toward the anode and stains with periodic acid-Schiff reagent, suggesting that it is acidic and glycoprotein in nature. Its isoelectric point is at pH 4.1. It has a molecular weight of about 21,500. It is thermostable at pH 4.2 and thermolabile at pH 8.5. It has a pH optimum at 6.5. It exhibits highest preference for cytidine 3'-phosphate linkages. Its activity on poly (C) is endonucleolytic. It cleaves poly (C) via intramolecular transphosphorylation. It has no action on cytidine 2': 3'-cyclic phosphate or uridine 2':3'-cyclic phosphate. Its rate of hydrolysis of poly (U) is less than 2% of that of poly C). Poly (A) and poly (G) are totally inert to its action. Its action on poly (C) is inhibited by poly (G), poly (A) and poly (U). It differs from bovine pancreatic Rnase A in its physical, chemical and catalytic properties. It is, however, similar to human serum and pancreatic RNase in all its properties, suggesting that pancreas is its likely source.     

Purification and characterization of a ribonuclease from barley roots

A ribonuclease isolated from barley malt roots exhibited characteristics that conformed to those of RNase I (EC 3.1.27.1). It differed from RNase I from barley leaves and barley seeds in its action on polynucleotides and on 3′,5′-dinucleoside monophosphates, and from barley seed RNase I in its optimum pH. Gel electrophoresis indicated that the enzyme was present in the embryo, roots, shoot and endosperm of germinating barley. The enzyme showed pH optimum at 5.0, isoclectric pH at 4.5, a thermal optimum of 50°, and an apparent molocular weight of 19 000.     

Cytotoxic properties of pancreatic ribonuclease, modified by the surface active substance oxanol KD-6]

Pancreatic RNase modified by the surface active substance oxanole KD-6 (OxRNase) was studied in respect to its cytotoxic action on cells. The studies included in vitro and in vivo tests with intravital staining of the cells by neutral red and the 3H uridine label, as well as the test with the preparation action on fusion of lysosomes and phagosomes. It was shown that in all the tests the hydrophobised RNase had a higher cytotoxic action versus the native enzyme. The analysis of the experimental data suggested that the cytotoxicity of the hydrophobised RNase was due to its action on the cell membrane structures including the lysosome membranes.     

Reassignment of the active site histidines in ribonuclease A by selective deuteration studies.

The C₂H resonance of the active site histidine residue designated AS-2, which has the lower pK_a of the two active site histidines, has been correlated in both RNase A and RNase S by comparing the pH 3 to 5.5 regions of the chemical shift titration curves, the effect of the inhibitor CMP-3′ on the chemical shifts at pH 4.0, and the effect of Cu II on the line widths at pH 3.6. It has been demonstrated that resonance AS-2 is absent in the spectrum of RNase S′ reconstituted using S-peptide deuterated at the C₂ of His 12, and in that of the RNase S′-CMP-3′ complex. We thus demonstrate that histidine AS-2 is in fact His 12 in both enzymes. This finding is in agreement with out previous assignment of the exchangeable NH proton in RNase A to His 12, but reverses the assignments of the active site histidine C₂H resonances made earlier by other authors.     

Ribonuclease in Bovine Milk

The RNase (EC 2.7.7.16) in bovine milk was partially purified from milk whey. The basic properties and the hydrolyzing specificity of this enzyme were studied. This enzyme was heat stable. When RNA was used as the substrate, the pH optimum was 7.5 and 3′- nucleotides were produced, but the extent of the hydrolysis stopped at 31 per cent and core was left. C! and U! were hydrolyzed but purine cyclic nucleotides were not. Poly U was digested to 3′-uridylic acid passing through U! but poly A was not. These properties are quite similar to pancreatic RNase.     

A novel ribonuclease with antiproliferative activity from fresh fruiting bodies of the edible mushroom Hypsizigus marmoreus

An 18-kDa ribonuclease (RNase) with a novel N-terminal sequence was purified from fresh fruiting bodies of the mushroom Hypsizigus marmoreus. The purification protocol comprised ion exchange chromatography on DEAE cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on CM-cellulose and Q-Sepharose and gel filtration by fast protein liquid chromatography on Superdex 75. The starting buffer was 10 mM Tris-HCl buffer (pH 7.2), 10 mM Tris-HCl buffer (pH 7.2), 10 mM NH(4)OAc buffer (pH 5), 10 mM NH(4)HCO(3) buffer (pH 9.4) and 200 mM NH(4)HCO(3) (pH 8.5), respectively. Absorbed proteins were desorbed using NaCl added to the starting buffer. A 42-fold purification of the enzyme was achieved. The RNase was unadsorbed on DEAE cellulose, Affi-gel blue gel and CM-cellulose but adsorbed on Q-Sepharose. It exhibited maximal RNase activity at pH 5 and 70 degrees C. Some RNase activity was detectable at 100 degrees C. It demonstrated the highest ribonucleolytic activity (196 U/mg) toward poly C, the next highest activity (126 U/mg) toward poly A, and much weaker activity toward poly U (48 U/mg) and poly G (41 U/mg). The RNase inhibited [(3)H-methyl]-thymidine uptake by leukemia L1210 cells with an IC(50) of 60 microM.     

Acid ribonuclease from HeLa cell lysosomes.

An acid ribonuclease has been purified from HeLa cell lysosomes. The specific activity of the RNase in lysosomes is 8-fold higher than that in nuclei and 15-fold higher than that in the postlysosomal fraction. The purified enzyme showed no detectable DNase, phosphodiesterase, phosphatase, or alkaline RNase activity. The acid RNase binds to Con A-agarose and is inferred to be a glycoprotein. It has a low isoelectric point at pH 3.0 to 3.5, and the optimal pH for activity is between 5.0 and 5.5. The enzyme requires no divalent cation for optimal activity and is totally inhibited by 1 mM Cu2+ or Hg2+. Monovalent cations including Na+, K+, and NH4+ stimulate the activity in low ionic strength buffer. The enzyme degrades rRNA faster than tRNA, and tRNA faster than poly(U); poly(A) and poly(C) are highly resistant. The products from rRNA are mostly oligonucleotides with 3'-phosphate ends. An acid RNase is also present in the lysosomes of L-cells grown in a medium free of serum; it is probably identical to the one described here.     

Fractionation of RNA from tetrahymena by affinity chromatography on poly-u-sepharose

Summary Exponential growing Tetrahymena pyriformis organisms were labelled with (³H) uridine or (³H) adenosine. The labelled RNA was extracted and isolated by affinity chromatography on poly-uridylic-acid Sepharose and further analysed by means of sucrose gradient centrifugation and RNase digestion.Experimental evidence proved the existence of RNase resistant poly adenylic-acid fragments in the RNA of Tetrahymena cells. This poly adenylic-acid segment has a sedimentation rate of 4-5 S and would be localised in the 10-12S region of the RNA which is probably the m-RNA.Supported by Stiftung Volkswagenwerk Research Grant No.112273.     

The kinetics of pancreatic ribonuclease reaction with alkaline and acidic forms of poly A

Summary The RNase hydrolysis of random-coil (alkaline form) poly A follows biphasic kinetics at low salt concentrations. However, its resistance to RNase increases with the ionic strength. Helical (acidic form) poly A is also susceptible to RNase but its hydrolysis follows first-order kinetics, and its resistance increases as the pH is lowered. These conformation-dependent kinetics of poly A hydrolysis are similar to those obtained in the hydrolysis of cellular RNA and reovirus double-stranded RNA.     

Single-strand-specific nuclease from rat liver endoplasmic reticulum: characterization and mode of action

1. The characteristics and mode of action of a single-strand-specific nuclease isolated from rat liver endoplasmic reticulum are investigated with respect to its DNA and RNA substrates. 2. The RNase activity of the enzyme is slightly influenced by the presence of divalent cations but the DNase activity is enhanced by divalent cations particularly Mn2+. 3. Activity is partially inhibited by the presence of EGTA; this effect is reversed most efficiently by the addition of Mn2+. 4. The enzyme exhibits small pH dependence between pH 6-9 and maximum activity is observed at pH 7-7.5 for both DNase and RNase activities. 5. Sulfhydryl group reagents do not affect its action but histidyl group reagents exert a small but definite effect. 6. The enzyme degrades DNA and RNA endonucleolytically producing fragments which possess 3'-OH and 5'-phosphate termini. 7. Monomers are not produced even after prolonged degradation. 8. The end product of poly(U)degradation ranges between two and four building blocks but the DNA product is longer probably due to considerable percentage of secondary structure.     

Catalytic acitivity of Ntau-carboxymethylhistidine-12 ribonuclease.

Ntau-Carboxymethylhistidine-12 RNase is active with both RNA and uridine cyclic 2':3'-monophosphate as substrates. Experimental evidence is presented to show that the activity cannot be due to contaminating RNase A, or other RNase-type protein, to the presence of a mixed dimer between 12- and 119-substituted RNases, or to the presence of trace amounts of Ntau-carboxymethylhistidine-12 RNase. The carboxymethyl derivative has approximately 1 and 13 per cent the specific activity of native enzyme against cyclic 2',3'-UMP at pH 5.0 and 8.5, respectively.     

Interaction of metal ions with polynucleotides and related compounds. X. Studies on the reaction of silver (I) with the nucleosides and polynucleotides, and the effect of silver(I) on the zinc(II) degradation of polynucleotides

Potentiometric titration curves of the silver(I) complexes of cytidine, adenosine, and uridine show little uptake of base below pH 7, unlike the curve for silver(I)-guanosine, which shows extensive base uptake at neutral pH. This observation is correlated with spectrophotometric data showing little difference between the silver complex spectra of adenosine, cytidine, and uridine and the spectra of uncomplexed nucleosides, except at high pH, but showing a great difference between the silver complex spectra of guanosine and inosine and the corresponding uncomplexed nucleosides even at pH 6. Similar comparisons of the silver complexes of poly A, poly C, poly I, and poly U, both by potentiometric titration and by spectrophotometry, show that poly I behaves like guanosine and inosine as expected, differing from poly A and poly C. However, poly U behaves like poly I and thus does not resemble uridine in its complexing behavior. There is thus a dichotomy between poly A and poly C on the one hand in silver complexing phenomena, compared with poly U and poly I on the other. When silver(I) is added to systems containing zinc(II) and various polynucleotides, the same dichotomy is noted. Silver(I) inhibits the degradation by zinc(II) of all four polynucleotides, but the degradation of poly I and poly U is prevented virtually completely. Silver(I) alone has no degradative effect on RNA and inhibits, the zinc(II) degradation of RNA. Polynucleotide complexes in which silver(I) and zinc(II) are simultaneously bound to different positions on the macromolecules are postulated as intermediates in the inhibited degradation reactions.     

Double-stranded RNA specific nuclease from germinating embryos ofPennisetum typhoides

A double-stranded RNA specific nuclease (ds RNase) has been purified from the pearl milletPennisetum typhoides. The purification involved S-30 preparation from the germinating embryos, DEAE-cellulose and DNA-cellulose chromatography. The partially pure enzyme preferentially solubilized the synthetic double-stranded polynucleotide [³H]poly(rA) · poly(rU); the degradation of [³H]poly(rC) was fourteen fold lower under the same assay conditions. Further more, the ds RNase activity was inhibited to an extent of 58% by ethidium bromide, which is known to intercalate with double-stranded RNAs. Active sulfhydryl groups were found to be necessary for the ds RNase activity since the enzyme action was inhibited by N-ethylmaleimide. Ethidium bromide and N-ethyl-maleimide did not significantly inhibit the ss RNase activity. In contrast, diethyl pyrocarbonate inhibited ss RNase activity completely and ds RNase by 58%. Heating the enzyme for 20 min at 50°C resulted in drastic loss of both enzyme activities. The ds RNase showed maximum activity in the pH range of 6.5 to 7.5. The enzyme actsin vitro onE. coli 30S precursor ribosomal RNA and the cleavage products migrated in the region of mature 23S and 16S rRNAs.     

Tracking the elusive 5′ exonuclease activity of <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis> RNase J

Key message

Chlamydomonas RNase J is the first member of this enzyme family that has endo- but no intrinsic 5′ exoribonucleolytic activity. This questions its proposed role in chloroplast mRNA maturation.

Abstract

RNA maturation and stability in the chloroplast are controlled by nuclear-encoded ribonucleases and RNA binding proteins. Notably, mRNA 5′ end maturation is thought to be achieved by the combined action of a 5′ exoribonuclease and specific pentatricopeptide repeat proteins (PPR) that block the progression of the nuclease. In Arabidopsis the 5′ exo- and endoribonuclease RNase J has been implicated in this process. Here, we verified the chloroplast localization of the orthologous Chlamydomonas (Cr) RNase J and studied its activity, both in vitro and in vivo in a heterologous B. subtilis system. Our data show that Cr RNase J has endo- but no significant intrinsic 5′ exonuclease activity that would be compatible with its proposed role in mRNA maturation. This is the first example of an RNase J ortholog that does not possess a 5′ exonuclease activity. A yeast two-hybrid screen revealed a number of potential interaction partners but three of the most promising candidates tested, failed to induce the latent exonuclease activity of Cr RNase J. We still favor the hypothesis that Cr RNase J plays an important role in RNA metabolism, but our findings suggest that it rather acts as an endoribonuclease in the chloroplast.

     

Ribonuclease activity and substrate preference of human eosinophil cationic protein (ECP).

The eosinophil cationic protein (ECP), a potent helminthotoxin with considerable neurotoxic activity, was recently shown to also have ribonucleolytic activity. In this work the substrate preference of ECP ribonuclease action was studied in detail. With single-stranded RNA or synthetic polyribonucleotide substrates ECP showed significant but low activity, 70- to 200-fold less than that of bovine RNase A. ECP hydrolyzed RNA more rapidly than it did any synthetic polynucleotide. Poly(U) was degraded more rapidly than poly(C), and poly(A) and double-stranded substrates were extremely resistant. Defined low molecular weight substrates in the form of the 16 dinucleoside phosphates (NpN') and uridine and cytidine 2',3'-cyclic phosphates were tested, and none showed hydrolysis by ECP at a significant rate. The results link ECP ribonucleolytic activity to the 'non-secretory' liver-type enzymes rather than to the 'secretory' pancreatic-type RNases.