Purification and characterization of two ribonucleases from developing tomato fruit

Two neutral ribonucleases have been purified from developing tomato fruit. Their activity is maximal 5 days after anthesis, declines during maturation, and then increases slightly in the mature green through breaker stages. The ribonucleases Tf1 and Tf2 have molecular weights of 59 and 29 K, respectively, based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and are glycoproteins. The reduced and denatured Tf1 is composed of two subunits, 30 and 29 K, of which only the 30-K subunit displays ribonuclease activity after renaturation. Reduced and denatured Tf2 is a single 29-K polypeptide that is renaturable to an active ribonuclease. Only the 30-K, active subunit of Tf1 is immunologically cross-reactive with Tf2. Both ribonucleases are cyclyzing endoribonucleases with a strong preference for cleavage at pyrimidine residues, thus generating oligonucleotide products ending with pyrimidine 2',3'-cyclic phosphate. These tomato fruit ribonucleases share a number of properties in common with the S-glycoprotein ribonucleases that are involved in self-incompatibility reactions in some solanaceous plants.     

Purification of a non-histone protein with properties of antizyme to ornithine decarboxylase from germinated barley seeds

The purification of a chromatin-bound antizyme to ornithine decarboxylase from germinated barley seeds is described. This antizyme was extracted from chromatin by 2 M NaCl and purified to homogeneity. Its molecular weight was found to be 9000 with an isoelectric point of 4.1. It reacts with both cytosolic and chromatinbound ornithine decarboxylase from germinated barley seeds and E. coli, but it does not inhibit ornithine decarboxylase of Tetrahymena pyriformis or rat liver.     

Purification and properties of bovine kidney ribonucleases

Two RNases (RNases K1 and K2) were purified from bovine kidney by means of column chromatography on phospho-cellulose, Sephadex G-50, CM-cellulose, heparin-Sepharose, nd agarose-APUP. They were named RNase K1 and RNase K2 in order of elution from the heparin-Sepharose column. The purity of RNase K1 thus obtained was about 90% by SDS-disc electrophoresis. RNase K2 was purified to homogeneity by SDS- and pH 4.3 disc electrophoresis. The yield of RNase K2 was 3.4 mg from 11 kg of kidneys. The antigenic properties of the two bovine renal RNases were studied by Ouchterlony's double diffusion analysis. RNase K1 and RNase A were serologically indistinguishable. RNase K2 did not cross-react immunologically with RNase K1 or RNase A. The molecular weights of these RNases determined by gel-filtration on Sephadex G-50 were 13,400 and 14,600 for RNase K1 and RNase K2, respectively. The pH optima for RNase K1 and RNase K2 were 8.5 and 6.5, respectively. Both RNase K1 and RNase K2 were as acid stable as RNase A. RNase K2 was less heat-stable than RNase K1 and RNase A. Although both renal RNases were pyrimidine nucleotide-specific enzymes, RNase K1 and RNase A were more preferential or cytidylic acid than RNase K2. The chemical composition of RNase K2 was determined. RNase K2, like human urinary RNase Us, contained one tryptophan residue. The N-terminal sequences of RNase K2 and RNase Us were determined by Edman degradation. Rnase K2 had a homologous sequence of about 10 amino acid residues with the sequence of RNase Us, a typical non-secretory RNase, within the N-terminal 30 residues.     

Partial purification and properties of nuclease 1 from barley malt

A sugar-unspecific nuclease has been purified 260-fold from barley malt diastase. The enzyme, a glycoprotein of 37 000 MW, is highly active on single-stranded polynucleotides at pH 5–6. The nuclease is inhibited by several adenine nucleotides, and it binds weakly to NADP-agarose and ATP-agarose.     

Purification and some properties of a nuclease from rye germ nuclei

1. An endonuclease has been isolated from the nuclei of rye (Secale cereale L) germ and partially purified. The enzyme shows optimum activity over the pH range 5.4-7.4 towards both DNA and RNA, and has no phosphomonoesterase or phosphodiesterase activity. 2. DNA is degraded by the rye germ nuclease to oligonucleotides of similar size, and RNA to oligonucleotides and mononucleotides containing a C-terminal 5'-phosphate group. 3. The rate of hydrolysis of nuclear acids by the enzyme decreases in the following order: native DNA greater than denatured DNA greater than RNA. Synthetic polynucleotides are hydrolysed at a rate decreasing in the order: poly(A) greater than poly(U) greater than poly(C) greater than poly(G).     

Purification and properties of a light-inducible nuclease from Euglena gracilis.

The nuclease described by Carell, E.F., Egan, J.M. and Pratt, E.A. [Arch. Biochem. Biophys. (1970) 138, 26-31] has been purified 1000-fold from Euglena gracilis strain Z. The enzyme catalyzes the hydrolysis of both polyribonucleotides and polydeoxyribonucleotides. The relative rates of hydrolysis of synthetic and natural polynucleotides was found to be: poly (U) 100, poly (dT) 33, denatured calf-thymus DNA 33, yeast tRNA 9, E. coli total RNA 6, poly (dA dT) 5, poly (A) less than 1, poly (C) less than .05, and poly (G) less than .05. The enzyme attacks polynucleotides in an endonucleolytic fashion, yielding products terminated with a 3'-phosphate. Poly (U) appears to be hydrolyzed completely to 3'-UMP; both RNA and DNA appear to have some phosphodiester bonds resistant to enzyme catalyzed hydrolysis. Because of its mode of action and its inducibility by light, we propose the name endonuclease L for this enzyme.     

Purification and properties of extracellular nuclease from tobacco pollen

Proteins diffusing from tobacco pollen grains exhibit different phosphohydrolytic activities. Molecular sieving produces nuclease fractionation into forms I, II and III with apparent molecular masses ≥ 60 × 10³, 32.9 × 10³ and 24.6 × 10³, respectively, and separation of principal forms II and III from phosphatase and major part of 5′- and 3′-nucleotidase activities. These forms did not differ in the mode of substrate attack and were combined for further enzyme characterization. The preparation had 3′-nueleotidase activity even after further purification by DEAE-cellulose chromatography. The enzyme is an endonuclease with preference for single stranded molecules. The endolytical cleavage of native DNA occurs simultaneously in both strands and generates limit products of about 58 pairs of nucleotides. DNA duplex polymers are also cleaved by a terminally-directed, exonuclease-like process. The products of DNA degradation are oligonucleotides and 5′-mononucleotides. In the presence of NaCl, both endolytical and exonucleaselike activities on bihelical DNA are inhibited and the proportion of mono-to oligonucleotides produced increases. The enzyme can rapidly convert superhelical plasmid DNA to a nicked open circular form, and then to a unit-length linear molecule. On the basis of these properties and of those found earlier (sugar-unspecificity, acidic pH optimum, activation by Zn²⁺ ions), the extracellular nuclease of tobacco pollen can be classified as plant nuclease I (EC 3.1.30.x).     

Plant nucleases. I. Separation and purification of two ribonucleases and one nuclease from corn

Three enzymes with ribonuclease activity, one of which also had deoxyribonuclease activity, have been isolated and partially purified from corn seeds and seedlings. The purification of Ribonuclease I from mature seed was previously reported. This enzyme has a pH optimum near 5.0, is loosely adsorbed to carboxymethyl-cellulose, and has a molecular weight of 23,000, determined by gel filtration.Ribonuclease II was isolated from the microsomes of corn roots, and was partially purified by gel filtration. It has a pH optimum plateau from 5.4 to 7.0, and molecular weight of 17,000.Nuclease I hydrolyzes both RNA and DNA. It was isolated from the large particles of a corn root homogenate and was partially purified on a carboxymethyl-cellulose column. It has a pH optimum at 6.2 and a molecular weight of 31,000.The relative activities of the 3 enzymes for deoxyribonuclease and at pH 5 and pH 6.2 for ribonuclease may be used to characterize them during purification operations. Assays on homogenates of corn roots, and especially of the root tips, suggested that a fourth enzyme, which possesses deoxyribonuclease activity, is also present.     

Biochemical properties and hormonal regulation of barley nuclease

The amino acid composition and NH2-terminal amino acid sequence of barley nuclease (EC 3.1.30.2) were determined. The amino acid composition is similar to that of mung bean nuclease, and therefore the biochemical properties of barley nuclease were characterized and compared with those of mung bean and other plant nucleases. The 3'-nucleotidase activity of barley nuclease is greater for purine than for pyrimidine ribonucleotides. The enzyme has little activity towards ribonucleoside 2' and 5'-monophosphates, and deoxyribonucleoside 3' and 5'-monophosphates, and is also inactive towards the 3'-phosphoester linkage of nucleoside cyclic 2',3' and 3',5'-monophosphates. The enzyme hydrolyzes dinucleoside monophosphates, showing strong preference for purine nucleosides as the 5' residues. Barley nuclease shows significant base preference for homoribonucleic acids, catalyzing the hydrolysis of polycytidylic acid greater than polyuridylic acid greater than polyadenylic acid much greater than polyguanylic acid. The enzyme also has preference for single-stranded nucleic acids. Hydrolysis of nucleic acids is primarily endonucleolytic, whereas the products of digestion possess 5'-phosphomonoester groups. Nuclease activity is inhibited by ethylenediaminetetraacetic acid and zinc is required for reactivation. Secretion of nuclease from barley aleurone layers is dependent on the hormone gibberellic acid [Brown, P.H. and Ho, T.-h. D. (1986) Plant Physiol. 82, 801-806]. Consistent with these results, gibberellic acid induces up to an eight-fold increase in the de novo synthesis of nuclease in aleurone layers. The secreted enzyme is a glycoprotein having an apparent molecular mass of 35 kDa. It consists of a single polypeptide having an asparagine-linked, high-mannose oligosaccharide. The protein portion of the molecule has a molecular mass of 33 kDa.     

Purification and properties of magnesium- and manganese-dependent ribonucleases H from chick embryo

Two RNases H, Mg2+- and Mn2+-dependent RNases H, are present in extracts of chick embryo. These RNases H can be separated by phosphocellulose column chromatography. Mg2+-dependent RNase H was purified over 900-fold and Mn2+-dependent RNase H over 1,700-fold from chick embryo extracts. The molecular weight of the purified Mg2+-dependent RNase H was about 40,000 and of the Mn2+-dependent RNase H about 120,000, when estimated by gel filtration. Mg2+-dependent RNase H exhibits maximal activity at pH 9.5, and requires 15 to 20 mM Mg2+ for maximal activity, whereas Mn2+-dependent RNase H is most active at pH 8.5, and is maximally active at the concentration of 0.4 mM Mn2+, and has some activity with Mg2+. Both enzymes require a sulfhydryl reagent for maximal activity. Mn2+-dependent RNase H was inhibited by o-phenanthroline, pyrophosphate, and those polyamines tested, whereas Mg2+-dependent enzyme was not, although it was inhibited by NaF. Both RNases H liberate a mixture of oligonucleotides with 5'-phosphate and 3'-hydroxyl termini endonucleolytically.     

Purification and chemical properties of two 1,3;1,4-beta-glucan endohydrolases from germinating barley

Two 1,3;1,4-beta-glucan endohydrolases have been purified from extracts of germinating barley by ammonium sulphate precipitation, ion-exchange and gel filtration chromatography. Both enzymes are monomeric, basic proteins. Enzyme I has a molecular weight of 28000 and an isoelectric point of 8.5, while enzyme II has a molecular weight of 33000 and an isoelectric point greater than 10. Enzyme II is a glycoprotein containing 3.6% carbohydrate, of which three residues are probable N-acetylglucosamine, but enzyme I contains only traces of associated carbohydrate. The amino acid compositions of the two 1,3;1,4-beta-glucan endohydrolases are similar and the cross-reactivity of antibodies raised against the purified enzymes suggests that they share common antigenic determinants.     

Purification and properties of nuclease SP.

Single-strand-specific nucleases are a diverse and important group of enzymes that are able to cleave a variety of DNA structures present in duplex molecules. Nuclease SP, an enzyme from spinach, has been purified to apparent homogeneity, allowing for the unambiguous characterization of a number of its physical properties as well as its DNA strand cleavage specificities. The effects of ionic strength, pH, divalent metal cations, and temperature on nuclease SP activity have been examined in detail. Nuclease SP was found to be quite thermostable and could be stimulated by Co2+. In addition, the cleavage of UV-damaged and undamaged supercoiled plasmid substrates under a variety of conditions suggests that at least two types of structures are recognized and processed by nuclease SP: UV photoproduct-induced distortions and unwound "nuclease hypersensitive sites". These studies indicate that nuclease SP is functionally related to other single-strand-specific nucleases and is a potential enzymatic tool for probing and manipulating various types of DNA structures.