Human granulocyte ribonuclease.

Human granulocytes contain an RNase which 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 the secondary phosphate esters of uridine 3′-phosphates. It has no action on uridine 2′: 3′-cyclic phosphates. Poly (A) and poly (G) are inert to its action. Its rate of hydrolysis of poly (C) is about 1% of that of poly (U). It differs from bovine pancreatic RNase and human serum RNase. Because of its unique specificity, this enzyme might serve as a biochemical marker in certain granulocyte disorders.     

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 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.     

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.     

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.     

The mode of action of divalent cations on the RNase catalyzed hydrolysis of RNA and uridylyl (3'-5') uridine

Pseudo first order rate constants (k′) have been measured for the RNase A catalyzed hydrolysis of uridylyl (3′–5′) uridine at several ionic strengths and compositions. The k′ values are independent of Mg²⁺ concentration between 0 and 10 mM. This shows that for hydrolysis of RNA, in which Mg²⁺ concentration does change k′, the perturbation must be through binding of Mg²⁺ to the substrate RNA rather than to the enzyme RNase.     

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.     

New sequence-specific human ribonuclease: purification and properties.

A new sequence-specific RNase was isolated from human colon carcinoma T84 cells. The enzyme was purified to electrophoretical homogeneity by pH precipitation, HiTrapSP and Superdex 200 FPLC. The molecular weight of the new enzyme, which we have named RNase T84, is 19 kDa. RNase T84 is an endonuclease which generates 5'-phosphate-terminated products. The new RNase selectively cleaved the phosphodiester bonds at AU or GU steps at the 3' side of A or G and the 5' side of U. 5'AU3' or 5'GU3' is the minimal sequence required for T84 RNase activity, but the rate of cleavage depends on the sequence and/or structure context. Synthetic ribohomopolymers such as poly(A), poly(G), poly(U) and poly(C) were very poorly hydrolysed by T84 enzyme. In contrast, poly(I) and heteroribopolymers poly(A,U) and poly(A,G,U) were good substrates for the new RNase. The activity towards poly(I) was stronger in two colon carcinoma cell lines than in three other epithelial cell lines. Our results show that RNase T84 is a new sequence-specific enzyme whose gene is abundantly expressed in human colon carcinoma cell lines.     

Quantitation of the loss of the bacteriophage lambda receptor protein from the outer membrane of lipopolysaccharide-deficient strains of Escherichia coli.

Incubation of Neurospora crassa conidia with ribonuclease (RNase) A reduces transport of L-phenylalanine by those cells. Under similar conditions, oxidized RNase A, RNase T1, and RNase T2 do not have this effect. Incubation of conidia with active RNase covalently attached to polyacrylamide beads reduces L-phenylalanine transport. This indicates that the site of enzymatic action is at the cell surface. At the lower concentration of enzyme used in this study, incubation with RNase A reduces transport of L-phenylalanine by the general (G) amino acid permease. Increasing the enzyme concentration results in reduction of transport by the neutral aromatic (N)-specific permease. The increased transport activity that accompanies onset of conidial germination is also sensitive to incubation with RNase A. Application of the enzyme to actively transporting cells does not release amino acid transported prior to enzyme addition. Cells cultured on media supplemented with [2-14C] uridine release isotopic activity after RNase A incubation. Analogous treatments with Pronase, RNase T1, RNase T2, or deoxyribonuclease I do not release isotope activity. Pronase treatment does reduce L-phenylalanine transport. Incubation of conidia with RNase A also inhibits germination of those conidia.     

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.     

Functional analysis of 2-5A-dependent RNase and 2-5a using 2',5'-oligoadenylate-cellulose

2-5A is an intracellular effector that has been implicated in interferon action, hormonal regulation, and cell growth control. 2-5A action is mediated through its activation of 2-5A-dependent RNase (RNase L, RNase F). Affinity resins [2-5A-cellulose and core (2-5A)-cellulose] were chemically synthesized for purification and immobilization of 2-5A-dependent RNase from mouse L cells and rabbit reticulocyte lysates. The breakdown of poly(U)-[3'-32P]Cp to acid-soluble fragments was demonstrated using the 2-5A-dependent RNase:2-5A -cellulose complex; this activity was enhanced by adding (free) 2-5A. In contrast, RNase activity was measured from the 2-5A-dependent RNase:core (2-5A)-cellulose complex only after the addition of free 2-5A. The rabbit reticulocyte 2-5A-dependent RNase is activated only by tetramer or higher oligomers of 2-5A; therefore there was breakdown of poly(U)-[3'-32P]Cp using core (2-5A)-cellulose-bound reticulocyte 2-5A-dependent RNase after addition of tetramer 2-5A but there was no poly(U) degradation in the presence of trimer 2-5A. The absence of significant general nuclease in the assays was demonstrated by the resistance to breakdown of poly(C)-[3'-32P]Cp (not susceptible to 2-5A-dependent RNase). Moreover, core (2-5A)-cellulose was used to develop a sensitive (subnanomolar) assay for the detection of authentic 2-5A. 2-5A, or the material to be tested, was added to mouse L-cell 2-5A-dependent RNase:core (2-5A)-cellulose complex in the presence of poly(U)-[3'-32P]Cp. The concentration of 2-5A in the sample could be measured from the amount of poly(U) degradation. Several closely related analogs of 2-5A were tested and found to be completely inactive. The technology described herein may be applied to the study of the regulation of 2-5A-dependent RNase, the detection of 2-5A from cells and tissues, and other aspects of the 2-5A system.     

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.

     

Human endothelial cells selectively express large amounts of pancreatic-type ribonuclease (RNase 1)

Pyrimidine-specific ribonucleases are a superfamily of structurally related enzymes with distinct catalytic and biological properties. We used a combination of enzymatic and non-enzymatic assays to investigate the release of such enzymes by isolated cells in serum-free and serum-containing media. We found that human endothelial cells typically expressed large amounts of a pancreatic-type RNase that is related to, if not identical to, human pancreatic RNase. This enzyme exhibits pyrimidine-specific catalytic activity, with a marked preference for poly(C) substrate over poly(U) substrate. It was potently inhibited by placental RNase inhibitor, the selective pancreatic-type RNase inhibitor Inhibit-Ace, and a polyclonal antibody against human pancreatic RNase. The enzyme isolated from medium conditioned by immortalized umbilical vein endothelial cells (EA.hy926) possesses an amino-terminal sequence identical to that of pancreatic RNase, and shows molecular heterogeneity (molecular weights 18,000-26,000) due to different degrees of N-glycosylation. Endothelial cells from arteries, veins, and capillaries secreted up to 100 ng of this RNase daily per million cells, whereas levels were low or undetectable in media conditioned by other cell types examined. The corresponding messenger RNA was detected by RT-PCR in most cell types tested so far, and level of its expression was in keeping with the amounts of protein. The selective strong release of pancreatic-type RNase by endothelial cells suggests that it is endowed with non-digestive functions and involved in vascular homeostasis.     

Isolation and characterization of a human colon carcinoma-secreted enzyme with pancreatic ribonuclease-like activity

A ribonuclease was isolated from serum-free supernatants of the human colon adenocarcinoma cell line HT-29. It was purified by cation-exchange and C18 reversed-phase high-performance liquid chromatography. The protein is basic, has a molecular weight of approximately 16,000, and has an amino acid composition that is significantly different from that of human pancreatic ribonuclease. The amino terminus is blocked, and the carboxyl-terminal residue is glycine. The catalytic properties of this ribonuclease resemble those of the pancreatic ribonucleases in numerous respects. Thus, it exhibits a pH optimum of approximately 6 for dinucleotide cleavage and employs a two-step mechanism in which transphosphorylation to a cyclic 2',3'-phosphate is followed by slower hydrolysis to produce a 3'-phosphate. It does not cleave NpN' substrates in which adenosine or guanosine is at the N position and prefers purines at the N' position. Like bovine ribonuclease A, the HT-29-derived ribonuclease is inactivated by reductive methylation or by treatment with iodoacetate at pH 5.5 and is strongly inhibited by the human placental ribonuclease inhibitor. However, in contrast, the tumor enzyme does not cleave CpN bonds at an appreciable rate and prefers poly(uridylic acid) as substrate 1000-fold over poly(cytidylic acid). It also hydrolyzes cytidine cyclic 2',3'-phosphate at least 100 times more slowly than uridine cyclic 2',3'-phosphate and is inhibited much less strongly by cytidine 2'-monophosphate than by uridine 2'-monophosphate. Other ribonucleases known to prefer poly(uridylic acid) were isolated both from human serum and from liver and were compared with the tumor enzyme. The physical, functional, and chromatographic properties of the serum ribonuclease are essentially identical with those of the tumor enzyme. The liver enzymes, however, differ markedly from the HT-29 ribonuclease. The potential utility of the tumor ribonuclease in the diagnosis of cancer is considered.     

Cytoplasmic poly(ADP-ribose) polymerase during the HeLa cell cycle.

Poly(ADP-ribose) polymerase, an enzyme that has reportedly been confined to the nucleus of eukaryotic cells, has been found in the cytoplasm of HeLa cells. The enzyme activity is stimulated more than 30-fold by the addition of both DNA and histones. These two macromolecules are absolutely necessary for maximal activity and they act in a synergistic manner. The product of the reaction was characterized as poly(ADP-ribose) by its acid insolubility, its insensitivity to hydrolysis by DNase, RNase, spleen phosphodiesterase or Pronase and by release of 5′-AMP and 2′-(5″-phosphoribosyl)-5′-AMP by incubation with snake venom phosphodiesterase. A covalent attachment between histone F1 and poly(ADP-ribose) has been established by using the cytoplasmic enzyme. The enzyme is primarily associated with ribosomes, both free ribosomes and those found in polysomes. Inhibition of protein synthesis in the intact cell reduced the level of activity in the cytoplasm. The enzyme can be removed from the ribosomes by centrifugation through sucrose gradients containing 0.6 m ammonium chloride. A relationship between this enzyme and DNA replication is suggested by the fact that the specific activity in the cytoplasm parallels the rate of DNA synthesis during the HeLa cell cycle.     

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.     

Antitumor action of seminal ribonuclease, its dimeric structure, and its resistance to the cytosolic ribonuclease inhibitor

Bovine seminal RNase (BS-RNase) is a homodimeric enzyme with a cytotoxic activity selective for tumor cells. In this study, the relationships of its cytotoxic activity to its dimeric structure and its resistance to the cytosolic RNase inhibitor (cRI) are investigated systematically by site-directed mutagenesis. The results show that (1) the dimericity of BS-RNase is essential for its full cytotoxic action; (2) the role of the dimeric structure in the antitumor activity is that of making the enzyme insensitive to the cytosolic RNase inhibitor; (3) a RNase may not be completely insensitive to cRI to exploit a full cytotoxic potential.     

Regeneration of enzyme activity after western blot: activation of RNase L by 2-5A on filter--importance for its detection.

A rapid and convenient new procedure for detecting RNase L activity following Western blot by renaturation of the enzyme on the nitrocellulose sheets is described. This method allows the simultaneous analysis of enzymatic activity (e.g., cleavage of poly(uridylic acid)-3'-[32P]pCp) and RNase L binding to radioactivE probes (e.g., 2-5A-3'-[32P]pCp) in the same sample. Unlike previously published methods, this procedure eliminates interference from proteases or other RNases during the analysis of RNase L activity. The detection of RNase(s) L is also affected by the presence of endogenous 2-5A, 2-5A derivatives, or other possible "inhibitors" in cell extracts; this Western blot assay allows of RNase(s) L to be detected independently of intracellular 2-5A or analogs. Differences between the procedures used so far and this Western blot technique can indeed be demonstrated. It is shown with this Western blot assay that although RNase L has been described as a protein of 185-200 kDa under nondenaturating conditions, its 80-kDa (and 40-kDa) component is able to bind 2-5A and to cleave poly(uridylic acid) in a 2-5A-dependent way, independently of other subunit(s) or cofactor(s).     

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.