Brain UDPgalactose: ceramide galactosyltransferase Purification of a catalytically active protein obtained after proteolytic digestion.

A procedure for the purification of UDPgalactose--2-hydroxyacylsphingosine galactosyltransferase (EC 2.4.1.45) including detergent extraction, ion-excharge chromatography and proteolytic digestion was developed. The active fraction obtained by this procedure had about 100 times higher specific activity than microsomes. Enzymic activity resisted destruction by pronase treatment at 4 degrees C. Agarose gel chromatography indicated the presence of an enzyme-phospholipid-detergent complex with a molecular weight between 400 000 and 500 000. Intact phospholipids seemed to be required for full enzymic activity as evidenced by the drastic loss of activity upon treatment with phospholipase A or C.     

Isolation of a novel thermolabile heterodimeric ribonuclease with antifungal and antiproliferative activities from roots of the sanchi ginseng Panax notoginseng.

An isolation procedure, consisting of ion exchange chromatography on CM-Sepharose, affinity chromatography on Affi-gel blue gel, and fast protein liquid chromatography on Mono S, was utilized to purify a base-nonspecific, heterodimeric ribonuclease (RNase) with diverse activities from roots of the sanchi ginseng Panax notoginseng. The RNase is unique in that it consists of two different nonglycoprotein subunits with a molecular weight of 27 and 29 kDa, respectively. The latter subunit is characterized by an N-terminal sequence showing remarkable similarity to that of the bitter gourd RNase. The Panax notoginseng RNase demonstrates potent RNase and translation-inhibitory activities. In addition, it exhibits antiproliferative activity toward leukemia L1210 cells and antifungal activity against Physalospora piricola and Coprinus comatus. Its RNase activity is not heat-resistant, unlike most RNases which are thermostable.     

Expression of soluble bovine pancreatic ribonuclease A in Pichia pastoris and its purification and characterization

A Pichia pastoris expression system for bovine pancreatic RNase A was constructed: the RNase A sequence was fused to the PHO1 signal and the AOX1 promoter was used for efficient secretion. Approximately 5 mg of soluble enzymes were secreted per liter of the culture, but one half of them were glycosylated. After a series of purifications by cation-exchange chromatography, the glycosylated enzyme was removed and the pure recombinant soluble unglycosylated RNase A was obtained in the final yield of 1 mg per liter of the culture. N-Terminal sequence, molecular weight, secondary structure, thermal stability, and activity were completely identical with those of commercial RNase A. Glycosylated RNase A had a decreased kcat, 60-70% of the activity of wildtype RNase A, as in the case of RNase B. Its carbohydrate moiety seemed to destabilize the enzyme differently from RNase B since Tm of the glycosylated RNase A was decreased by 6 degrees C. The carbohydrate moiety of the glycosylated enzyme contained no GlcNAc. The N34A mutant RNase A, in which the only potential N-glycosylation site, Asn34, is mutated to alanine, was also glycosylated, implying that glycosylation is not N-linked but O-linked.     

Isolation and partial characterization of catalytic antibodies with oligonuclease activity from bovine colostrum

Catalytic antibodies (abzymes) which hydrolyze RNA and DNA were isolated from bovine colostrum by sequential chromatography on Protein A Sepharose, denaturated DNA-cellulose, Mono Q, and gel permeation chromatography on Superose 12 at pH 2.3 after acidic shock. Metachromatic agar containing toluidine blue and yeast RNA was used to measure RNase activity. Electrophoresis in agarose showed DNase activity on plasmid DNA from Escherichia coli and DNA from calf thymus in fractions from all 4 purification steps. Gel permeation chromatography showed that the abzymes hydrolysed both a single-stranded polyadenylic acid (Poly A) and single-stranded polycitidylic acid (Poly C), while partially purified RNase from the colostrum hydrolysed Poly (C), but not Poly (A). Electrophoresis of purified abzymes under denaturing conditions showed protein bands of molecular mass corresponding to heavy and light chains of IgG. The abzymes immunoreacted with anti-bovine IgG. The RNase activity of the purified abzymes represented 0.022% of total RNase activity in the colostrum; acid shock and gel filtration at low pH reduced the specific RNase activity of abzymes 3.6-fold. The RNase activity of abzymes at pH 6.6 was reduced by 90% by heat treatment at 75 degrees C for 52 min.     

Expression of Soluble Bovine Pancreatic Ribonuclease A in Pichia pastoris and its Purification and Characterization

A Pichia pastoris expression system for bovine pancreatic RNase A was constructed: the RNase A sequence was fused to the PHO1 signal and the AOX1 promoter was used for efficient secretion. Approximately 5 mg of soluble enzymes were secreted per liter of the culture, but one half of them were glycosylated. After a series of purifications by cation-exchange chromatography, the glycosylated enzyme was removed and the pure recombinant soluble unglycosylated RNase A was obtained in the final yield of 1 mg per liter of the culture. N-Terminal sequence, molecular weight, secondary structure, thermal stability, and activity were completely identical with those of commercial RNase A. Glycosylated RNase A had a decreased k _cat, 60-70% of the activity of wild-type RNase A, as in the case of RNase B. Its carbohydrate moiety seemed to destabilize the enzyme differently from RNase B since T _m of the glycosylated RNase A was decreased by 6°C. The carbohydrate moiety of the glycosylated enzyme contained no GlcNAc. The N34A mutant RNase A, in which the only potential N-glycosylation site, Asn34, is mutated to alanine, was also glycosylated, implying that glycosylation is not N-linked but O-linked.     

Antitumor activity and other biological actions of oligomers of ribonuclease A

Dimers, trimers, and tetramers of bovine ribonuclease A, obtained by lyophilization of the enzyme from 40% acetic acid solutions, were purified and isolated by cation exchange chromatography. The two conformers constituting each aggregated species were assayed for their antitumor, aspermatogenic, or embryotoxic activities in comparison with monomeric RNase A and bovine seminal RNase, which is dimeric in nature. The antitumor action was tested in vitro on ML-2 (human myeloid leukemia) and HL-60 (human myeloid cell line) cells and in vivo on the growth of human non-pigmented melanoma (line UB900518) transplanted subcutaneously in nude mice. RNase A oligomers display a definite antitumor activity that increases as a function of the size of the oligomers. On ML-2 and HL-60 cells, dimers and trimers generally show a lower activity than bovine seminal RNase; the activity of tetramers, instead, is similar to or higher than that of the seminal enzyme. The growth of human melanoma in nude mice is inhibited by RNase A oligomers in the order dimers < trimers < tetramers. The action of the two tetramers is very strong, blocking almost completely the growth of melanoma. RNase A dimers, trimers, and tetramers display aspermatogenic effects similar to those of bovine seminal RNase, but, contrarily, they do not show any embryotoxic activity.     

A 3'-deoxymononucleotide-producing nuclease from the marine sponge Verongia aerophoba. I. Purification.

A four-step procedure for purification of a nuclease from the keratinous sponge Verongia aerophoba is described. The extracted material is lyophilized, acidified, and subjected to chromatography on Sephadex, hydroxyapatite, and phosphocellulose. The nuclease is purified about 1,000-fold from the crude extract and approximately 1,600-fold from concomitant acid RNase. Phosphodiesterase is lost after chromatography on Sephadex. The purified enzyme solution contains one single activity as determined by polyacrylamide gel electrophoresis.     

Large-scale purification, crystallization and some physicochemical properties of extracellular guanyl-RNases C2 and Pch1]

The purification of RNase C2 from 76.5 1 of Asp. clavatus cultural fluid and RNase Pch1 from 160 1 of Pen. chrysogenum 152 A cultural fluid was described. 1150-fold purification of RNase C2 was attained by precipitation with ammonium sulfate, ion-exchange chromatography and rechromatography on DEAE-cellulose, gel chromatography on Sephadex G-75, and crystallization from diluted acidic buffer. During the preparation of RNase Pch1 additional chromatography on CM-cellulose was used before crystallization, the purification being 2220-fold. It was obtained 600 mg RNase C2 and 900 mg RNase Pch1. Some physico-chemical properties of crystalline RNases were studied.     

A rapid and simple method for the purification of rat liver RNase inhibitor

A rapid and simple method for the purification of rat liver alkaline RNase inhibitor from a 105,000 g supernatant is reported. It involves protein precipitations by (NH₄)₂SO₄ and chromatography on carboxymethyl cellulose-RNase column. The purification procedure gives a 1020-fold increase in specific activity with a yield of 32%. This purified inhibitor can be stored for 5 weeks without any loss in activity.     

Primary structure of a ribonuclease from bullfrog (Rana catesbeiana) liver

A pyrimidine base-specific ribonuclease was purified from bullfrog (Rana catesbeiana) liver by means of CM-cellulose column chromatography and affinity chromatography on heparin-Sepharose CL-6B, which gave single band on SDS-slab electrophoresis. The primary structure of the bullfrog liver RNase was determined. It consisted of 111 amino acid residues, including 8 half-cystine residues. From the sequence, it was concluded that three disulfide bridges in RNase A were conserved in the bullfrog RNase, that a disulfide bridge in RNase A [Cys65-Cys126 (RNase A numbering)] was deleted, and that a new disulfide bridge was created in the C-terminal part of the enzyme. In this frog RNase, the amino acid residues thought to be essential for catalysis in bovine pancreatic RNase A were conserved except for Asp121 (RNase A numbering). The sequence homology of the bullfrog liver RNase with bovine pancreatic RNase A was 30.6%. The sequence of bullfrog liver RNase was very similar to those of lectins obtained from bullfrog egg by Titani et al. [Biochemistry (1988) 26, 2189-2194] and R. japonica egg by Kamiya et al. [Seikagaku (in Japanese) (1989) 60, 733; and personal communication from Kamiya, Y., Oyama, F., Oyama, R., Sakakibara, F., Nitta, K., Kawauchi, H., and Titani, K.]. The sequence homology between the bullfrog liver RNase and the two lectins was 70.2 and 64.8%, respectively.     

Polyclonal antibodies against RNase L. Subcellular localization of this enzyme in mouse cells.

RNase L activated by 2-5A (a series of 2'-5'-linked adenylic oligoribonucleotides) is a key enzyme of the interferon system. To study RNase L (endonuclease L) in intact cells independently of intracellular 2-5A and of its activity, we have developed polyclonal antibodies against RNase L. RNase L from mouse spleen was purified on a column of 2-5A-Sepharose and used to immunize rabbits in co-injection with polyadenylic-polyuridylic acid as adjuvant. Antibodies were purified by chromatography on Affi-Gel blue and 2-5A-Sepharose-immobilized RNase L. These polyclonal antibodies immunoprecipitate the 80- and 40-kDa forms of RNase L in mouse spleen. In Western blot, only the 80-kDa form of RNase L is recognized by these antibodies. These purified antibodies were used to localize RNase L in the cytoplasm of intact mouse NIH 3T3 cells by immunofluorescence. The cytoplasmic localization of RNase L was confirmed by its 2-5A binding activity after cellular fractionation.     

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.     

Partial purification and immobilization of ribonuclease T2.

A simple procedure, consisting of water extraction, heat treatment at pH 2.0, negative adsorption on DEAE-cellulose at pH 4.9, and concanavalin A-Sepharose chromatography, was developed for the partial purification of ribonuclease (RNase) T2 from taka-diastase powder with an overall yield of 5.5%. The partially purified enzyme when coupled to aminoethyl Bio-Gel P-60, retained 12-16% of the activity of the soluble enzyme. Temperature stability studies on RNase T2 bound to matrices, activated with increasing concentrations of glutaraldehyde, and the influence of lysine modification on the activity of the soluble enzyme revealed that the low activity observed for the gel-bound enzyme is probably due to the masking of the active site of the enzyme as a result of the involvement of lysine residues, situated near the active site, during coupling. Immobilization did not affect the pH and temperature optima of RNase T2. On repeated use, the bound enzyme retained approximately 55% of its initial activity after six cycles. These results are discussed, taking into consideration the factors affecting immobilized enzymes.     

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.     

A novel method for isolation and concentration of ribonuclease T1 from Taka-Diastase

A novel method for isolation and concentration of RNase T1 from Taka-Diastase is developed. It is a combination method of bentonite adsorption with dialysis desorption. In the present method, RNase T1 can be concentrated about ten-fold, the recovery of total activity was greater than 95%, and specific activity was raised 8-10 folds. Further purification with ammonium sulfate precipitation and chromatography on DEAE-cellulose and DEAE-Sephadex yields a RNase T1 which contains no pMase. pDase nor RNase T2 activities and a 750 fold increase in specific activity. Our method is more simple, rapid, and efficient than previous methods.     

Degradation of ribonucleic acid in Candida lipolytica: extraction of ribonucleic acid degrading enzymes

We developed an efficient and simple method for RNase extraction from Candida lipolytica cells which consists of predrying the cells with solvents and incubating them for 8 to 15 hr at 37 to 45°C in a slightly acid buffer which contains EDTA or salts. This method is called Solvent Dehydration Buffer Extraction (SDBE) procedure. Predrying with acetone or ethanol, or by lyophilization, followed by washing with acetone or ethylacetate gives the most efficient RNase extraction. The yield and specific activity obtained by this extraction procedure are higher than by any other method examined. An apparent 1.5- to 2.0-fold activation of RNase occurred during the SDBE process. Activation of RNase in homogenates obtained by grinding fresh cells is also observed with EDTA or acetate buffer. The SDBE procedure works efficiently regardless of growth phase for Candida lipolytica, and works also with other Candida yeasts.     

Organosoluble enzyme conjugates with poly(2-oxazoline)s via pyromellitic acid dianhydride

The use of enzymes in organic solvents offers a great opportunity for the synthesis of complex organic compounds and is therefore in focus of current research. In this work we describe the synthesis of poly(2-methyl-1,3-oxazoline) (PMOx) and poly(2-ethyl-1,3-oxazoline) (PEtOx) enzyme conjugates with hen-egg white lysozyme, RNase A and α-chymotrypsin using a new coupling technique. The POXylation was carried out reacting pyromellitic acid dianhydride subsequently with ethylenediamine terminated POx and then with the NH?-groups of the respective enzymes. Upon conjugation with the polymers, RNase A and lysozyme became fully soluble in DMF (1.4 mg/ml). These are the first examples of fully POXylated proteins, which become organosoluble. The synthesized enzyme conjugates were characterized by SDS-PAGE, isoelectric focusing, dynamic light scattering and size exclusion chromatography, which all indicated the full POXylation of the enzymes. The modified enzymes even partly retained their activity in water. With α-chymotrypsin as example we could demonstrate that the molecular weight of the attached polymer significantly influences the activity.     

Partial purification and characterization of a double-stranded RNA-specific nuclease from human placenta

A double-stranded RNA-specific nuclease (ds RNase) has been isolated and partially purified from human placenta by DEAE-cellulose and DNA-cellulose column chromatography. Denatured DNA-cellulose retained most of the single-stranded RNA-specific nuclease (ss RNase) activity, whereas the ds RNase came out in the void volume. N-ethylmaleimide at a concentration of 5 mM, selectively inhibited ds RNase activity by 60% under the conditions in which the ss RNase activity was inhibited to an extent of 7%. The ds RNase was specifically inhibited by Penicillium chrysogenum viral ds RNA and by ethidium bromide. The partially purified ds RNase showed requirements for Mg+ whereas Mn2+ and NH4+ ions were inhibitory. The DEAE-enzyme cleaved 32P-labelled 45S ribosomal precursor RNAs from Yoshida ascites sarcoma cells into species that had similar electrophoretic mobilities as the mature rRNAs.