Control of the formation of extracellular ribonuclease in Neurospora crassa.

A finding was made that a species of ribonuclease is released into mycelial culture media when a wild-type strain of Neurospora crassa was grown on limiting amounts of phosphate. The ribonuclease activity in the fully derepressed state extends to about 60 to 100 fold of that in the repressed state. The synthesis of the ribonuclease was inhibited by the addition of rifampicin, cycloheximide or orthophosphate. Three molecular species of the ribonuclease were found. Two enzyme fractions showing larger molecular weights were suspected to be aggregates containing the enzyme showing the smallest molecular weight (molecular weight of 10 300). All three fractions showed pH optima of around 7, preferential hydrolysis of polyguanylic acid and poor hydrolysis of guanosine 2',3',-cyclic monophosphate. These characteristics were the same as those of ribonuclease N1, and it was suggested that ribonuclease N1 is a repressible extracellular enzyme. Mutations in the genes nuc-1 and nuc-2 caused loss of ability to derepress this enzyme, but heterokaryon between them partially restored the ability. The nuc-1 mutation was epistatic to the nuc-2 alleles which are partly constitutive in the ribonuclease production.     

Induction of an extracellular cyclic nucleotide phosphodiesterase as an accessory ribonucleolytic activity during phosphate starvation of cultured tomato cells

During growth under conditions of phosphate limitation, suspension-cultured cells of tomato (Lycopersicon esculentum Mill.) secrete phosphodiesterase activity in a similar fashion to phosphate starvation-inducible ribonuclease (RNase LE), a cyclizing endoribonuclease that generates 2':3'-cyclic nucleoside monophosphates (NMP) as its major monomeric products (T. Nürnberger, S. Abel, W. Jost, K. Glund [1990] Plant Physiol 92: 970-976). Tomato extracellular phosphodiesterase was purified to homogeneity from the spent culture medium of phosphate-starved cells and was characterized as a cyclic nucleotide phosphodiesterase. The purified enzyme has a molecular mass of 70 kD, a pH optimum of 6.2, and an isoelectric point of 8.1. The phosphodiesterase preparation is free of any detectable deoxyribonuclease, ribonuclease, and nucleotidase activity. Tomato extracellular phosphodiesterase is insensitive to EDTA and hydrolyzes with no apparent base specificity 2':3'-cyclic NMP to 3'-NMP and the 3':5'-cyclic isomers to a mixture of 3'-NMP and 5'-NMP. Specific activities of the enzyme are 2-fold higher for 2':3'-cyclic NMP than for 3':5'-cyclic isomers. Analysis of monomeric products of sequential RNA hydrolysis with purified RNase LE, purified extracellular phosphodiesterase, and cleared -Pi culture medium as a source of 3'-nucleotidase activity indicates that cyclic nucleotide phosphodiesterase functions as an accessory ribonucleolytic activity that effectively hydrolyzes primary products of RNase LE to substrates for phosphate-starvation-inducible phosphomonoesterases. Biosynthetical labeling of cyclic nucleotide phopshodiesterase upon phosphate starvation suggests de novo synthesis and secretion of a set of nucleolytic enzymes for scavenging phosphate from extracellular RNA substrates.     

The kinetics and specificity of the reaction of 2'(3')-O-bromoacetyluridine with bovine pancreatic ribonuclease A.

2'(3')-O-Bromoacetyluridine reacts rapidly and selectively with bovine pancreatic ribonuclease A at pH 5.5 and 25 degrees. Under conditions of high molar ratios of nucleoside derivative to enzyme, the only derivative is N-3-carboxymethylhistidine-12 ribonuclease A. The reaction occurs almost exclusively with the histidine-12 residue at the active site inactivation of the enzyme is accompanied by the stoichiometric disappearance of unmodified ribonuclease A and appearance of the product, N-3-carboxymethylhistidine-12 ribonuclease A. Kinetic studies indicate a mechanism involving saturation of the enzyme by the nucleoside derivative. The inhibitor constant, Kb, is 0.087 M and k3 is 35.1 times 10(-4) sec minus 1. The reaction of 2'(3')-O-bromoacetyluridine with the enzyme occurs at a rate approximately 3100 times greater than that corresponding to the reaction with L-histidine. The alkylation reaction is inhibited competitively by uridine with a Ki of 0.013 M. 2'(3')-O-Bromoacetyluridine inactivates ribonuclease A 4.5 times faster than bromoacetic acid and the specificity for alkylation of active-site histidine residues is different. 2'(3')-O-Bromoacetyluridine reacts 1000 times more rapidly with ribonuclease A than iodoacetamide. The contribution of nucleoside binding to the overall rate of alkylation is discussed.     

Modification of a specific tyrosine residue of ribonuclease A with a diazonium inhibitor analog

The diazonium salt of 5'-(4-aminophenyl phosphoryl)-uridine 2'(3')-phosphate reacts stoichiometrically with pancreatic ribonuclease and modifies only one tyrosyl residue, which was identified as Tyr 73 in the amino acid sequence. The modification does not inhibit the biological activity of RNAase on ribonucleic acid, although a large change in the binding constant towards cytidine cyclic phosphate was observed. The modification can be inhibited by addition of the competitive inhibitor cytidine 2'(3')5'-diphosphate and may indicate that Tyr 73 is the most exposed residue or has a unique reactivity towards this reactive substrate analog.     

The degradation of ribonucleic acid in the cotyledons of Pisum arvense

1. A ribonuclease has been partially purified from the cotyledons of germinating seed of Pisum arvense. 2. The enzyme degrades ribopolynucleotides to adenosine 3'-phosphate, guanosine 3'-phosphate and the cyclic nucleotides cytidine 2',3'-phosphate and uridine 2',3'-phosphate; no resistant ;core' remains. 3. The activity of RNA-degrading enzymes in the cotyledons increases to a maximum during the first 5 days of germination, passes through a minimum around the eighth day, and thereafter increases again. 4. Ion-exchange chromatography of methanol-soluble extracts of cotyledons revealed the presence, amongst other components, of the 2'-, 3'- and 5'-phosphates of cytidine and uridine, the 3'- and 5'-phosphates of adenosine, and guanosine 5'-phosphate. 5. Seed soaked in a solution containing [(32)P]orthophosphate gave a methanol-soluble fraction containing labelled nucleoside 5'-phosphates, but nucleoside 2'- and 3'-phosphates were not labelled. 6. It is believed that the nucleoside 2'- and 3'-phosphates arise by the action of ribonuclease on cotyledon RNA.     

Ribonuclease activity of vaccinia DNA topoisomerase IB: kinetic and high-throughput inhibition studies using a robust continuous fluorescence assay

Vaccinia type I DNA topoisomerase exhibits a strong site-specific ribonuclease activity when provided a DNA substrate that contains a single uridine ribonucleotide within a duplex DNA containing the sequence 5' CCCTU 3'. The reaction involves two steps: attack of the active site tyrosine nucleophile of topo I at the 3' phosphodiester of the uridine nucleotide to generate a covalent enzyme-DNA adduct, followed by nucleophilic attack of the uridine 2'-hydroxyl to release the covalently tethered enzyme. Here we report the first continuous spectroscopic assay for topoisomerase that allows monitoring of the ribonuclease reaction under multiple-turnover conditions. The assay is especially robust for high-throughput screening applications because sensitive molecular beacon technology is utilized, and the topoisomerase is released during the reaction to allow turnover of multiple substrate molecules by a single molecule of enzyme. Direct computer simulation of the fluorescence time courses was used to obtain the rate constants for substrate binding and release, covalent complex formation, and formation of the 2',3'-cyclic phosphodiester product of the ribonuclease reaction. The assay allowed rapid screening of a 500 member chemical library from which several new inhibitors of topo I were identified with IC(50) values in the range of 2-100 microM. Three of the most potent hits from the high-throughput screening were also found to inhibit plasmid supercoil relaxation by the enzyme, establishing the utility of the assay in identifying inhibitors of the biologically relevant DNA relaxation reaction. One of the most potent inhibitors of the vaccinia enzyme, 3-benzo[1,3]dioxol-5-yl-2-oxoproprionic acid, did not inhibit the closely related human enzyme. The inhibitory mechanism of this compound is unique and involves a step required for recycling the enzyme for steady-state turnover.     

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.     

Purification and characterization of a novel UpN-specific endoribonuclease VI from Artemia larvae

Artemia larval ribonuclease (Sebastián, J., and Heredia, C. F., (1978) Eur. J. Bichem. 90, 405-411) has been purified near homogeneity and its properties were studied. It consists of a single polypeptide chain of 38,000 daltons. It requires a divalent cation for activity. Ca2+ is the most effective among the metals tested. The metal dependence of the activity is biphasic. Maximal activity is obtained at 5-10 mM. In the absence of metals and chelating agents in the assay, 30-40% of the activity is observed. However, if chelating agents are added, the activity is abolished. At low concentrations of free metal (1-20 microM), 30-40% of maximal activity is obtained with Ca2+ or Mn2+, but not with Mg2+, Ca2+, but not Mn2+ or Mg2+, protects the enzyme from thermal inactivation. The best substrates for Artemia ribonuclease are poly(U) and poly(A), although with the latter it has only 10% the activity shown with the former. Using poly(U) as substrate, the products of a terminal digestion are P-2':3'-Urd and 3'-UMP. Using dinucleoside monophosphates as substrates, the enzyme is highly specific for a U residue at the 3' side of the phosphodiester bond (UpN), especially UpA, being inactive if the U residue is at the 5' side (NpU). Although some of its properties are similar to other eukaryotic or prokaryotic ribonucleases, its high specificity for UpN bonds suggest that this is a new type of ribonuclease. Moreover, it is a potentially useful enzyme for RNA analysis and/or sequencing.     

Chemical modification of ribonuclease A with 4-arsono-2-nitrofluorobenzene

4-Arsono-2-nitrofluorobenzene reacts selectively at the anion binding site of bovine pancreatic ribonuclease A. The major derivative is the inactive 41-(4-arsono-2-nitrophenyl) ribonuclease A (45% yield). Additional products are 1-alpha-(4-arsono-2-nitrophenyl) ribonuclease A (11% yield) which is enzymatically active and the disubstituted, inactive 1,41-bis-(4-arsono-2-nitrophenyl) ribonuclease A (25% yield). 2' (3')-O-Bromoacetyluridine reacts with 41-(4-arsono-2-nitrophenyl) ribonuclease A exclusively at the histidine-12 residue at a rate which is approximately one-fourth the rate observed with the unmodified enzyme. Saturation kinetics are observed and the dissociation constant for the protein-inhibitor complex is 0.096 +/- 0.023 M. The first-order unimolecular decomposition constant for complex breakdown is 8.9 +/- 2.9 X 10(-4) s-1. 2'-Bromoacetamido-2'-deoxyuridine reacts with 41-(4-arsono-2-nitrophenyl) ribonuclease A 25 times more slowly than 2'(3')-O-bromoacetyluridine. Bromoacetate reacts with 41-(4-arsono-2-nitrophenyl) ribonuclease A predominantly at the histidine-119 residue at a rate 45 times less than that found for the unmodified enzyme. The results of the alkylation studies imply that the dianionic arsonate does not occupy the phosphate binding site in the enzyme but is sufficiently proximate to account for a decrease in bromoacetate binding as well as a reduction in the nucleophilic reactivity of histidine-12 and -119. All these effects may be accounted for in terms of a local electrostatic perturbation of the active site region by the arsononitrophenyl group.     

Properties of a cyclic 3'5'-nucleotide phosphodiesterase from Vigna mungo

Cyclic AMP phosphodiesterase (PDE) partially purified from roots of Vigna mungo exhibited optimum activity at pH 5.5 to 6.0 and maximum enzyme activity at 50 degrees C. Levels of PDE activity in roots remained relatively constant from the first to the eleventh day after germination; on the twelfth day there was a 400% increase in PDE activity. The enzyme was stable for at least 48 hours at 28 degrees C, retaining 92% of its original activity. Plant growth hormones including gibberellic acid, indoleacetic acid and kinetin at 1.0 and 10.0 microM concentrations did not have any significant effect on enzyme activity. Nucleotides tested including cyclic 2'3' AMP, cyclic 2'3' GMP completely abolished enzyme activity at 1.0mM while cyclic 3'5' GMP, cyclic 3'5' GMP, 2'deoxy 5' ATP, 2'deoxy 5'GTP and 5'ADP were also inhibitory to the enzyme. The enzyme was stimulated by Mg2+, Fe2+ and NH4+ while Cu2+ and Fe3+ were inhibitory. Theophylline, caffeine, phosphate, pyrophosphate and EDTA were inhibitory to the enzyme.     

Optimization of fermentation conditions for production of anti-TMV extracellular ribonuclease by Bacillus cereus using response surface methodology

Bacillus cereus ZH14 was previously found to produce a new type of antiviral ribonuclease, which was secreted into medium and active against tobacco mosaic virus. In order to enhance the ribonuclease production, in this study the optimization of culture conditions using response surface methodology was done. The fermentation variables including culture temperature, initial pH, inoculum size, sucrose, yeast extract, MgSO₄·7H₂O, and KNO₃ were considered for selection of significant ones by using the Plackett–Burman design, and four significant variables (sucrose, yeast extract, MgSO₄·7H₂O, and KNO₃) were further optimized by a 2⁴ factorial central composite design. The optimal combination of the medium constituents for maximum ribonuclease production was determined as 8.50 g/l sucrose, 9.30 g/l yeast extract, 2.00 g/l MgSO₄·7H₂O, and 0.62 g/l KNO₃. The enzyme activity was increased by 60%. This study will be helpful to the future commercial development of the new bacteria-based antiviral ribonuclease fermentation process.     

Studies on the enzymatic and physicochemical behaviour of the trichloroacetic acid-treated and untreated bovine pancreatic ribonuclease

Exposure of ribonuclease A to 5% trichloroacetic acid inactivates the enzyme partially. One of the possible reasons for such inactivation might be the exposure of one of the buried tyrosyl groups to the outside surface of the molecule (Sagar and Pandit (1983) Biochim. Biophys. Acta 743, 303-309). The trichloroacetic acid-treated enzyme hydrolysed 2':3'-cCMP with an efficiency of about 60%; while with rRNA as substrate, it is about 45%. Results indicate that apart from the reduction in the activity on trichloroacetic acid treatment, the enzyme possesses a reduced ability to break down the secondary structures of substrates such as rRNA in the first phase of the reaction. Thermal unfolding of ribonuclease A was followed by various physicochemical techniques such as UV absorbance, CD-spectroscopy and differential scanning microcalorimetry. The results indicate that the enzyme, after trichloroacetic acid-treatment, has a less ordered structure when compared to that of untreated enzyme. Thermal unfolding profiles reveal that trichloroacetic acid-treated ribonuclease A, like the untreated enzyme, follows a one-step transition with relatively lower transition temperature (Tm). NMR-spectral data suggests perturbations in the histidyl environment at the active site.     

A ribonuclease from human skeletal muscle

1. A ribonuclease has been prepared from human muscle by ammonium sulphate fractionation, heat treatment and ion-exchange chromatography. 2. The enzyme degrades polycytidylic acid and polyuridylic acid to the nucleoside 3'-phosphates, with nucleoside 2':3'-cyclic phosphates as intermediates. Polyadenylic acid and polyguanylic acid are not attacked. 3. The enzyme has maximal activity at pH8.5. The molecular weight (by gel filtration) is between 11000 and 12000. It is relatively heat-stable. It exhibits optimum activity in a medium of high ionic strength, and is inhibited by several bivalent cations, particularly Zn(2+).     

2',5'-Oligoadenylate synthetase activity in lymphocytes from normal mouse.

The activity of 2',5'-oligoadenylate synthetase, an enzyme recently discovered in interferon-treated cells, was found in lymphocytes from normal mouse spleen that had received neither exogenous interferon nor its inducers. The oligoadenylate synthesized by lymphocyte cell extracts inhibited protein synthesis in rabbit reticulocyte lysates. The oligomers were composed mainly of trimer and were resistant to digestion by T2 ribonuclease. The level of the enzyme in lymphocytes was about 20 to 30% of that in L929 cells treated with interferon. The activity of the enzyme was further enhanced in lymphocytes in vitro by addition of interferon. The 2',5'-oligoadenylate synthetase was distributed among several lymphoid tissues, but was not detected in cell extracts from brain or liver. The enzyme may play an important role in the regulation of the immune system.     

Acceptor activity of 4-N-acetylcytidine in the synthesis of (3'-5')-internucleotide bond catalyzed by pancreatic nuclease]

Cytidine and 4-N-acetylcytidine were compared as phosphate acceptors in dinucleoside monophosphate synthesis catalyzed by pancreatic ribonuclease with uridine-2',3'-cyclophosphate and cytidine-2',3'-cyclo phosphate as phosphate donors. Because of low solubility of 4-N-acetylcytidine in water, the synthesis was carried out in aqueus-organic media. The results obtained indicate that acetylation of the exoaminogroup of cytidine decreases its acceptor activity. For the first time uridilyl-(3'-5')-4-N-acetylcytidine and cytidilyl-(3'-5')-4-N-acetylcytidine are prepared enzymatically by pancreatic ribonuclease.     

Production, purification, and characterization of lipase from thermophilic and alkaliphilic Bacillus coagulans BTS-3

A thermophilic isolate Bacillus coagulans BTS-3 produced an extracellular alkaline lipase, the production of which was substantially enhanced when the type of carbon source, nitrogen source, and the initial pH of culture medium were consecutively optimized. Lipase activity 1.16 U/ml of culture medium was obtained in 48 h at 55 degrees C and pH 8.5 with refined mustard oil as carbon source and a combination of peptone and yeast extract (1:1) as nitrogen sources. The enzyme was purified 40-fold to homogeneity by ammonium sulfate precipitation and DEAE-Sepharose column chromatography. Its molecular weight was 31 kDa on SDS-PAGE. The enzyme showed maximum activity at 55 degrees C and pH 8.5, and was stable between pH 8.0 and 10.5 and at temperatures up to 70 degrees C. The enzyme was found to be inhibited by Al3+, Co2+, Mn2+, and Zn2+ ions while K+, Fe3+, Hg2+, and Mg2+ ions enhanced the enzyme activity; Na+ ions have no effect on enzyme activity. The purified lipase showed a variable specificity/hydrolytic activity towards various 4-nitrophenyl esters.     

Diguanosine 5',5'-P1,P4-tetraphosphate and other purine nucleotides inhibit endoribonuclease VI from Artemia

The activity of the endoribonuclease VI from Artemia is sensitive to several purine nucleotides. The enzyme is non-competitively inhibited by diguanosine tetraphosphate (Ki = 75 microM), a nucleotide abundant in Artemia encysted gastrulae and located in the same particulate fraction as the gastrular ribonuclease. Diguanosine triphosphate and diadenosine tetraphosphate are less efficient inhibitors (Ki congruent to 200 microM). The ribonuclease is non-competitively inhibited by 5'-AMP (Ki = 10 microM) and 5'-GMP (Ki = 50 microM) but is insensitive to the corresponding 5'-phosphates of cytosine and uridine. Other purine mononucleotides inhibit the enzyme activity less efficiently. The modulation of the enzyme activity by these nucleotides is discussed in relation with the changes in ribonuclease activity during early development of Artemia.     

Characterization of the Extracellular Ribonuclease of Tetrahymena pyriformis W

Several investigations have indicated that Tetrahymena pyriformis secretes ribonuclease activity into culture media. The extracellular ribonuclease from strain W has been purified and partially characterized. The molecular weight was determined by gel filtration to be 26,500. The amino acid composition of the enzyme was compared with those of the three intracellular ribonucleases characterized by Trangas, and substantial differences were demonstrated. The extracellular enzyme hydrolyzed both polyadenylic and polyuridylic acids, indicating lack of absolute base specificity. The hydrolysis of polyadenylic acid followed normal Michaelis-Menten kinetics, but substrate inhibition occurred at high concentrations of polyuridylic acid. The hydrolysis of polyuridylic acid was competitively inhibited by 2'- and 3'-cytidine, guanine, and uridine nucleotides, and by 2'AMP. No inhibition of the hydrolysis of Torula yeast RNA was detected. The kinetic properties of the extracellular ribonuclease are compared with those of the intracellular enzymes.