Nucleases in chloroplast of barley

Two barley chloroplast nuclease fractions were separated by the affinity chromatography and gel electrophoresis. Both were about 2 times more active to RNA than to native DNA and about half as active to denaturated DNA as to native DNA. Both fractions were as active to UV-irradiated (270 J m^-2) native DNA as to intact DNA but their action was inhibited by apurinic sites. The enzyme activities were inhibited by high concentrations of EDTA, NaCl, Mn²⁺, Ca²⁺, Zn²⁺ ions and by N-ethylmaleimide. They do not require Mg²⁺ ions but are stimulated or at higher concentration inhibited by their presence. Both RNase and DNase were active over a wide pH range (5.5–9), the optimum for DNase action in the presence of Mg²⁺ being 6.5, for RNA decomposing activity at pH 8.0. As no mononucleotides were detected in acid soluble form, it seems likely that DNase acts in the endonucleolytic way.     

Formation of Hydrogen Peroxide by a Polyvinyl Alcohol Degrading Enzyme

A method for purification and crystallization, and some properties of ribonuclease from Aspergillus sp. [EC 2.7.7.17] (RNase L) are reported. The purification procedure consisted of six steps, including acetone precipitation, column chromatographies on Duolite A–2 and DEAE-cellulose, repeated chromatography on DEAE-Sephadex A–50 column and affinity chromatography on 5’-AMP-Sepharose 4B column. Crystallization was performed by the dialysis against ammonium sulfate solution at 60% saturation.

The crystalline enzyme was shown to be homogeneous by polyacrylamide disc electrophoresis and ultracentrifugation. Svedberg value of the crystalline enzyme was 4.2. The enzyme was the most active at pH 3.5 and 60~65°C, and it was inhibited markedly with Fe.³⁺

RNase L has no absolute base specificity, and produces four kinds of 3’-mononucleotides from yeast RNA. However, the susceptibility of four nucleotide residues to RNase L increases in the order; G < A < C < U and is quite different from those of RNase T₂, RNase M and RNase R.     

Purification, Characterization, and Fractionation of the delta-Aminolevulinic Acid Synthesizing Enzymes from Light-Grown Chlamydomonas reinhardtii Cells

The synthesis of δ-aminolevulinate from glutamate by Chlamydomonas reinhardtii membrane-free cell homogenates requires Mg²⁺, ATP, and NADPH as cofactors. The pH optimum is about 8.3. When analyzed by a Fractogel TSK gel filtration column the δ-aminolevulinate synthesizing enzymes, including glutamate-1-semialdehyde aminotransferase, elute with an apparent molecular weight of about 45,000. The enzymes obtained from the gel filtration column were separated into three fractions by affinity column chromatography. One fraction binds to heme-Sepharose, one to Blue Sepharose, while the enzyme converting the putative glutamate-1-semialdehyde to δ-aminolevulinic acid is retained by neither column. All three fractions are necessary for the conversion of glutamate to δ-aminolevulinate. The δ-aminolevulinate synthesizing enzymes from Chlamydomonas are sensitive to inhibition by heme but not sensitive to inhibition by protoporphyrin.     

Ferrous Iron and Sulfur Oxidation and Ferric Iron Reduction Activities of Thiobacillus ferrooxidans Are Affected by Growth on Ferrous Iron, Sulfur, or a Sulfide Ore

Eight strains of Thiobacillus ferrooxidans (laboratory strains Tf-1 [= ATCC 13661] and Tf-2 [= ATCC 19859] and mine isolates SM-1, SM-2, SM-3, SM-4, SM-5, and SM-8) and three strains of Thiobacillus thiooxidans (laboratory strain Tt [= ATCC 8085] and mine isolates SM-6 and SM-7) were grown on ferrous iron (Fe²⁺), elemental sulfur (S⁰), or sulfide ore (Fe, Cu, and Zn). The cells were studied for their aerobic Fe²⁺ - and S⁰-oxidizing activities (O₂ consumption) and anaerobic S⁰-oxidizing activity with ferric iron (Fe³⁺) (Fe²⁺ formation). Fe²⁺-grown T. ferrooxidans cells oxidized S⁰ aerobically at a rate of 2 to 4% of the Fe²⁺ oxidation rate. The rate of anaerobic S⁰ oxidation with Fe³⁺ was equal to the aerobic oxidation rate in SM-1, SM-3, SM-4, and SM-5, but was only one-half or less that in Tf-1, Tf-2, SM-2, and SM-8. Transition from growth on Fe²⁺ to that on S⁰ produced cells with relatively undiminished Fe²⁺ oxidation activities and increased S⁰ oxidation (both aerobic and anaerobic) activities in Tf-2, SM-4, and SM-5, whereas it produced cells with dramatically reduced Fe²⁺ oxidation and anaerobic S⁰ oxidation activities in Tf-1, SM-1, SM-2, SM-3, and SM-8. Growth on ore 1 of metal-leaching Fe²⁺-grown strains and on ore 2 of all Fe²⁺-grown strains resulted in very high yields of cells with high Fe²⁺ and S⁰ oxidation (both aerobic and anaerobic) activities with similar ratios of various activities. Sulfur-grown Tf-2, SM-1, SM-4, SM-6, SM-7, and SM-8 cultures leached metals from ore 3, and Tf-2 and SM-4 cells recovered showed activity ratios similar to those of other ore-grown cells. It is concluded that all the T. ferrooxidans strains studied have the ability to produce cells with Fe²⁺ and S⁰ oxidation and Fe³⁺ reduction activities, but their levels are influenced by growth substrates and strain differences.     

Binding of adriamycin to sulphated mucopolysaccharides

Two distinct, Mn²⁺- and Mg²⁺-dependent enzymes with ribonuclease H (RNase H) activities, which degrade RNA-DNA hybrid have been purified from rat liver nuclei. These two enzymes were eluted at 0.16 M and 0.37 M of potassium chloride in phosphocellulose chromatography, respectively, and further purified by blue Sepharose. They are distinguished from one another by their ionic requirement, molecular weight, sedimentation coefficient, optimal pH, sensitivity to the -SH reagent and mode of cleavage.     

Two ribonucleases H from cultured plant cells.

Two forms of enzyme with ribonuclease H (RNase H) [EC 3.1.4.34] activities, have been partially purified from cultured plant cells, strain GD-2, derived from carrot root. One is an Mn2+-dependent RNase H, and the second is an Mg2+-dependent RNase H. These enzymes degrade RNA specifically in RNA-DNA hybrid structures. They were eluted at around 0.2 M and 0.4 M potassium chloride in phosphocellulose chromatography, and were further purified using blue Sepharose. Mg2+-dependent RNase H exhibits maximal activity at pH 9.0, and requires 10 to 15 mM Mg2+ for maximal activity, whereas the Mn2+-dependent enzyme is most active at pH 8.0, is maximally active at an Mn2+ concentration of 0.4 mM, and has some activity with Mg2+. Both enzymes require a sulfhydryl reagent for maximal activity. The enzymes liberate a mixture of oligonucleotides with 5'-phosphate and 3'-hydroxyl termini. The apparent molecular weight of the Mg2+-dependent RNase H was estimated to 18--20 X 10(4) and that of the Mn 2+- dependent RNase H was estimated to be 14 x 10(4) by gel filtration.     

3-Phenoxy-α-cyano-benzyl Esters,the Most Potent Synthetic Pyrethroids

Three fractions with nucleolytic activities were isolated from rice bran by DEAE-cellulose column chromatography and designated as RB-1, RB-2 and RB-3. RB-1, RB-2 and RB-3 had molecular weights of approximately 6,200, 35,000 and 14,500, respectively, by gel filtration. The main fraction (RB-3) was purified by Sephadex G-75 and CM-cellulose column chromatography. The pH optimum was 5.0. The nucleolytic activity of RB-3 was strongly inhibited by Cu^{2 +} , while EDTA had no effect on the activity. Seventy-five percent of the original activity of RB-3 still remained after 16 minutes of heating at 100°C. It appeared to be an endonuclease which hydrolyzes yeast RNA to yield purine nucleotides.     

The Effects of Sucrose and Light on the Level of Soluble and Particle-bound Ribonuclease Activities in Excised Avena Leaves

Incubation of excised Avena leaves in a wet chamber in darkness resulted in an increase in both soluble and particle-bound Rnase activities. Illumination promoted the increase in the total RNase which occurred upon leaf excision. The light-induced increase in total RNase was due to an increase in soluble RNase. The increase in RNase activity in the particulate fraction was inhibited by illumination. Feeding 2 per cent sucrose to the tissues in the dark increased the level of soluble RNase and decreased the activity found in the particulate fraction. Treatment of the illuminated tissues with 10^?4M dichlorophenyldimethylurea (DCMU) inhibited the effects of light on the RNase level. It is concluded that the light-effect is explained at least in part by the photosynthetic production of sugars. In excised leaves kept in darkness the RNA content rapidly decreased. Feeding sugars to or illumination of the tissues lowered the rate of RNA breakdown due to leaf excision. DCMU counteracted the light effect. In general, the decrease of RNA was repressed by all treatments leading to an inhibition of the increase of particulate RNase. On the other hand, the observed changes of the soluble RNase were not related with the variations of RNA. Treatment with 3 M urea increased the RNase activity both in the particulate and the soluble fractions. The RNase activity of soluble preparations, partially purified on a Sephadex G-50 column or by (NH₄)₂SO₄ fractionation, was also stimulated by 3 M urea. Treatment with 10^?5M kinetin repressed the increase in RNase activity due to leaf excision both in the soluble and the particulate fractions.     

Separation and characterization of some ribonucleic acids from imaginal discs of Drosophila melanogaster

Summary The RNA obtained from mass isolated imaginal discs of Drosophila melanogaster cultured in vitro in ³H-5-uridine has been studied. A separation procedure employing benzoylated, naphthoylated DEAE cellulose column chromatography was used to obtain several fractions of RNA, including one consisting primarily of ribosomal RNA, and three others relatively free of rRNA and transfer RNA. Other than its behavior in BND-cellulose chromatography, the RNA fractions have been examined with the following procedures: (1) sucrose gradient centrifugation, (2) heat labilities with apparent shifts in S-values, (3) changes in apparent secondary structure due to monovalent cation concentration, (4) Cs₂SO₄–CsCl equilibrium gradient centrifugation, (5) dimethyl sulfoxide gradient centrifugation, (6) melting out studies in different salt (Na⁺) concentrations, (7) limited digestion with different kinds of RNase, (8) kinetics of labeling, and (9) DNA-RNA hybridization. Marked differences were found among the RNA fractions using these techniques. The addition of -ecdysone to the cultured imaginal discs induces the production of quantitatively and perhaps qualitatively different RNA when compared with RNA obtained from discs cultured without the hormone. The possible secondary structures of the RNA fractions are also discussed.     

Purification and Properties of a Nuclease Preferential for Thymidine Residue from Neurospora crassa Mycelia

From the mycelia of Neurospora crassa (wild type No. 6068) multiple forms of a nuclease which had very close isoelectric points (pI = 9.6 (peak I), 9.4 (peak II)) were isolated by ampholine electrofocusing column chromatography (pH 8.5 ~ 10). The nuclease was about 300-fold purified from the crude extract. The two fractions of Peak I, II were indistinguishable in their enzymatic properties and were considered as manifestation of the same enzyme with minor physicochemical differences. The molecular weight was around 41,000 as estimated by the gel filtration method. The enzyme could hydrolyze both DNA and RNA in the order of heat-denatured DNA > native DNA DNA ≧ RNA. RNA competitively inhibited DNA degradation with this enzyme. The enzyme was therefore regarded as a nuclease. The pH optimum was around pH 6.5 toward native DNA, pH 6.7 toward heat-denatured DNA and pH 7.9 toward RNA. The temperature optimum was around 40°C toward these substrates and most of the activities were lost by heating at 55°C for 15 min. The enzyme required Mg²⁺ for action toward heat-denatured DNA and Mg²⁺, Mn²⁺ or Co²⁺ toward native DNA. In the presence of EDTA, the activities toward both types of DNA were lost and recovered by addition of the respective activating metallic ions. p-CMB inhibited this nuclease, but β-mercapto-ethanol and glutathione had no effect. Polyamìnes showed no activation of the nuclease for DNA degradation.     

Ribonuclease from the hepatopancreas of the red king crab <Emphasis Type="Italic">Paralithodes camtschatica</Emphasis>

Three enzyme preparations, two acid and one alkaline RNases, were isolated from the hepatopancreas of the red king crab Paralithodes camtschatica using DEAE-cellulose chromatography and gel chromatography. The alkaline RNase was activated by Mg²⁺ ions and had a pH optimum of 7.2; the acid RNases, a pH optimum of 5.5. The molecular weight of the alkaline RNase was 19 kDa; two acid RNases, 33 and 70 kDa, respectively. The enzymes exhibited a sufficiently high thermostability (IT₅₀ = 53–55°C) and were strongly inhibited by NaCl (IC₅₀, 0.1–0.25 M). The alkaline RNase exhibited no specificity for heterocyclic bases, whereas the acid RNases hydrolyzed poly(U) and poly(A) at maximum rates.     

Free zinc concentration in bovine milk measured by analytical affinity chromatography with immobilized metallothionein

A new analytical affinity chromatography method was developed for measuring the free [Zn²⁺] concentration in bovine milk. The column was generated by immobilizing avidin and attaching biotinylated metallothionein (MT) on controlled-pore glass beads. Zinc bound to the MT column at physiological free [Zn²⁺] concentration and was dissociated again in an elution buffer of pH 2. The distributions of extrinsically added⁶⁵Zn and native zinc in different fractions of milk were virtually identical, validating the use of extrinsic labeling in studies of the free [Zn²⁺] concentration in milk. Extrinsically labeled whey fractions were mixed with standard solutions whose free [Zn²⁺] concentrations were calculated by computer model.⁶⁵Zn retained by the column provided an indication of free [Zn²⁺] concentration in the mixture, and by interpolation, in the original milk. The free [Zn²⁺] concentration measured by the affinity chromatography method in the milk of a group of six cows was 90.4±29.7 pM. This value is similar to estimates of free [Zn²⁺] concentrations in other biological fluids by entirely different methods. Measurement of free [Zn²⁺] may be helpful in understanding the physiology and biochemistry of zinc metabolism.     

Cowpea ribonuclease: properties and effect of NaCl-salinity on its activation during seed germination and seedling establishment

Pitiúba cowpea [Vigna unguiculata (L.) Walp] seeds were germinated in distilled water (control treatment) or in 100 mM NaCl solution (salt treatment), and RNase was purified from different parts of the seedlings. Seedling growth was reduced by the NaCl treatment. RNase activity was low in cotyledons of quiescent seeds, but the enzyme was activated during germination and seedling establishment. Salinity reduced cotyledon RNase activity, and this effect appeared to be due to a delay in its activation. The RNases from roots, stems, and leaves were immunologically identical to that found in cotyledons. Partially purified RNase fractions from the different parts of the seedling showed some activity with DNA as substrate. However, this DNA hydrolyzing activity was much lower than that of RNA hydrolyzing activity. The DNA hydrolyzing activity was strongly inhibited by Cu²⁺, Hg²⁺, and Zn²⁺ ions, stimulated by MgCl₂, and slowly inhibited by EDTA. This activity from the most purified fraction was inhibited by increasing concentrations of RNA in the reaction medium. It is suggested that the major biological role of this cotyledon RNase would be to hydrolyze seed storage RNA during germination and seedling establishment, and it was discussed that it might have a protective role against abiotic stress during later part of seedling establishment.     

Properties and subcellular distribution of ribonuclease activity in Chlorella

RNase activity from Chlorella was partially purified. Two RNase activities were demonstrated, one soluble and the other ribosomal. The effects on ribonuclease activity of variations in pH and temperature, and of Mg²⁺, Na⁺, and mononucleotides were examined. The RNase activities (phosphodiesterases EC 3.1.4.23) were both endonucleolytic, releasing oligonucleotides, and cyclic nucleotide intermediates, but exhibited different specificities in releasing mononucleotides from RNA. The ribosomal activity released 3′-GMP, and after prolonged incubation 3′-UMP, but the soluble activity released 3′-GMP, 3′-AMP and 3′-UMP. Neither ofthe RNase preparations hydrolysed DNA, nor released 5′-nucleotides from RNA. Increased ribosomal RNase activity was related to dissociation of ribosomes, and latency of ribosomal RNase activity was demonstrated. The possible in vivo distribution of RNases is discussed.     

Synthesis of Double-Stranded RNA in a Virus-Enriched Fraction from Agaricus bisporus

Partially purified virus preparations from sporophores of Agaricus bisporus affected with LaFrance disease had up to a 15-fold-higher RNA-dependent RNA polymerase activity than did comparable preparations from healthy sporophores. Enzyme activity was dependent upon the presence of Mg²⁺ and the four nucleoside triphosphates and was insensitive to actinomycin D, α-amanitin, and rifampin. The ³H-labeled enzyme reaction products were double-stranded RNA (dsRNA) as indicated by CF-11 cellulose column chromatography and by their ionic-strength-dependent sensitivity to hydrolysis by RNase A. The principal dsRNA products had estimated molecular weights of 4.3 × 10⁶ and 1.4 × 10⁶; they corresponded in size and hybridized to the major dsRNAs detected in the virus preparation by ethidium bromide staining. Cs₂SO₄ equilibrium centrifugation of the virus preparation resolved a single peak of RNA polymerase activity that banded with a 35-nm spherical virus particle containing dsRNAs with molecular weights of 4.3 × 10⁶ and 1.4 × 10⁶. The data suggest that the RNA-dependent RNA polymerase associated with the 35-nm spherical virus is a replicase which catalyzes the synthesis of the genomic dsRNAs.     

Calcium protects DNase I from proteinase K: a new method for the removal of contaminating RNase from DNase I

DNase I and proteinase K are two enzymes commonly used in the purification of highly polymerized RNA. In the presence of EDTA DNase I is rapidly inactivated by proteinase K while in 10 mm Ca²⁺ DNase is totally immune to proteinase K inactivation even at protease concentrations of up to 1 mg/ml. RNase A, a common contaminant of “RNase-free” DNase was inactivated by proteinase K in the presence or absence of Ca²⁺. Treatment of DNase I with proteinase K in the presence of Ca²⁺ selectively removed RNase A activity as judged by rRNA and poly(A⁺ RNA ribosomal RNA degradation monitored by sucrose gradient centrifugation. These results suggest that (i) DNase A and proteinase K can be used together in the presence of Ca²⁺ to obtain better digestion of nucleoprotein complexes, and (ii) proteinase K treatment of Ca²⁺ DNase can be used to selectively remove contaminating RNase.     

Purification and some properties of the extracellular acid proteases from Mucor renninus

A complex of proteases was fractionated into three enzymes by chromatography of a crude enzyme preparation obtained from culture fluid of the fungus Mucor renninus on biospecific polystyrene adsorbent. Electrophoretically homogeneous proteases I-III were obtained by subsequent rechromatography on biospecific adsorbent and gel filtration on Sephadex G-75. Optimal proteolytic activities occurred at pH 4.25; 3.5 and 2.5 for enzymes I, II and III, respectively. Milk-clotting activity was exhibited only by protease II. All three proteases hydrolysed haemoglobin, Na caseinate and bovine serum albumin. Enzyme I hydrolysed Na caseinate the most effectively, while haemoglobin was the most effective substrate for proteases II and III. Trypsinogen was activated only by protease I. All three enzymes have a molecular weight ～35 000 as determined by gel chromatography on Sephadex G-75 column and by sodium dodecylsulphate disc electrophoresis. Isoelectric points, pH-stability range, amino acid composition, carbohydrate content were determined for each enzyme and the influence of metal ions (Ca²⁺, Mg²⁺, Cu²⁺, Co²⁺) on proteolytic activities of these enzymes studied.     

Nuclear location of ribonuclease H and increased level of magnesium-dependent ribonuclease H in rat liver on thioacetamide treatment

Rat liver nuclei were isolated in aqueous solutions of low ionic strength or anhydrous glycerol. The presence of ribonuclease H (RNase H) [EC 3.1.4.34] activity in the cytoplasm is due to extraction of the nuclear enzyme by buffer and inorganic salts. Two forms of RNase H were separated from rat liver nuclei by affinity chromatography using a DNA-cellulose column. When the RNase H in the wash solution of nuclei with 0.3 M sucrose and in nuclear solution extracted with 0.15 M NaCl were fractionated on a single-stranded DNA-cellulose column, two peaks corresponding to Mn2+- and Mg2+-dependent RNases H were eluted at 0.1 M and 0.2 M NaCl, respectively, and a peak having both RNase H activities was recovered in the wash-through fraction from the column. Among the enzymes from these two fractions in the nuclei, the activity of the Mg2+-dependent RNase H which binds to DNA-cellulose increased several-fold within 24 h of a single injection of thioacetamide. The activities of Mg2+-dependent RNase H extracted with higher-salt solution from the nuclei and recovered in the flow-through fraction from the DNA-cellulose column and the Mn2+-dependent RNase H activities were relatively unaffected by an injection of thioacetamide.     

Purification and Characterization of Extracellular Pectinolytic Enzymes Produced by Sclerotinia sclerotiorum

An exopolygalacturonase (exoPG) and an exopolymethylgalacturonase (exoPMG) produced by Sclerotinia sclerotiorum have been purified by ammonium sulfate precipitation, gel filtration, and ion exchange chromatography. The exoPG and the exoPMG were purified 66- and 50-fold, respectively, by using a series of separation procedures that included ammonium sulfate precipitation and gel chromatography. Molecular masses of the native proteins were 68 kDa for exoPG and 140 kDa for exoPMG. The pH optima of the enzymes were about pH 5, and their optimum temperature was 45°C. Activities of both enzymes were inhibited by Hg²⁺, Zn²⁺, Cu²⁺, and p-chloromercuribenzoate. ExoPMG activity, in contrast to exoPG activity, was stimulated by Mn²⁺ and Co²⁺. ExoPMG hydrolyzed only citrus pectin, while exoPG degraded sodium polygalacturonate and, to a lesser extent, citrus pectin. The exo mode of action of the enzymes was revealed by thin-layer chromatography of substrate hydrolysates. Antibodies raised against each purified protein exhibited no cross-reaction, thus confirming the biochemical specificities of the enzymes.     

Properties of peroxidases from tobacco cell suspension culture

An enzyme preparation from suspension cultured tobacco cells oxidized IAA only in the presence of added cofactors, Mn²⁺ and 2,4-dichlorophenol, and showed two pH optima for the oxidation at pH 4·5 and 5·5. Effects of various phenolic compounds and metal ions on IAA oxidase activity were examined. The properties of seven peroxidase fractions separated by column chromatography on DEAE-cellulose and CM-Sephadex, were compared. The peroxidases were different in relative activity toward o-dianisidine and guaiacol. All the peroxidases catalysed IAA oxidation in the presence of added cofactors. The pH optima for guaiacol peroxidation were very similar among the seven isozymes, but the optima for IAA oxidation were different. The anionic and neutral fractions showed pH optima near pH 5·5, but the cationic isozymes showed optima near pH 4·5. With guaiacol as hydrogen donor, an anionic peroxidase (A-1) and a cationic peroxidase (C-4) were very different in H₂O₂ concentration requirements for their activity. Peroxidase A-1 was active at a wide range of H₂O₂ concentrations, while peroxidase C-4 showed a more restricted H₂O₂ requirement. Gel filtration and polyacrylamide gel studies indicated that the three cationic peroxidases have the same molecular weight.