J. Biochem., 80, 767-776 (1976)
Ribonucleases from Porcine Brain Partial Purification and Properties1 Kazuya NISHIKAWA,1 Kenji TAKAHASHI,' and Toshio ANDO1 Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Bunkyo-ku, Tokyo 113 Received for publication, April 26, 1976
1. An acid ribonuclease was partially purified from an acetone powder of porcine train. This enzyme was an acidic protein with a molecular weight of around 70,000. It acted on yeast RNA optimally at about pH 5.9, yielding only a mixture of 3'mononucleotides, and therefore appears to be an exonuclease. It did not hydrolyze heat-denatured calf thymus DNA or bis(/>-nitrophenyl) phosphate. It was fairly unstable to heat and acid. 2. An alkaline ribonuclease was partially purified from the same source simultaneously. This enzyme was a basic protein with a molecular weight of 25,000—26,000. It was a pyrimidine-specific endoribonuclease, and acted on yeast RNA optimally at around pH 7.4. It did not hydrolyze heat-denatured calf thymus DNA or bis(/>-nitrophenyl) phosphate. It was fairly stable to heat and acid.
pH optima around pH 6 and 8 was reported in the brain homogenates of some animals, such as the rat ( 3 ) and guinea pig (4). Similar activity with a pH optimum around pH 6 was also reported for bovine brain (5). Until recently, however, there were few reports on the purification and characterization of such enzymes from brain, except for a partial purification of an exonuclease from bovine brain by Kurihara (5). Elucidation of the properties of brain nucleases is of great interest since they are involved in nucleic acid metabolism in the brain, which might be related to the specific function of brain. The present study was initiated in order to obtain data on the properties of such nucleases in porcine brain and also to compare the results with those obtained with bovine
There are various reports on the purification and properties of nucleases from different tissues of vertebrates (2). In brain, the presence of two RNA-depolymerizing activities with 1
This study was supported in part by grants (88516, •958016) from the Ministry of Education, Science and Culture, of Japan. A preliminary account of this study has already appeared ( / ) . 3 Present address: Institute of Molecular Biology, Faculty of Science, Nagoya University, Chikusa-ku, Nagoya 464. 3 Present address: Department of Biochemistry, Primate Research Institute, Inuyama, Aichi 484. Inquiries about this paper should be sent to this address. •* Present address: Department of Chemistry, Faculty of Physical Sciences and Engineering, Meisei University, Hodokubo, Hino, Tokyo 191. Vol. 80, No. 4, 1976
767
K. NISHIKAWA, K. TAKAHASHI, and T. ANDCP
768
brain from the viewpoint of comparative biochemistry. In the course of this study, we partially purified an acid ribonuclease and a pyrimidine-specific alkaline ribonuclease from acetone powder of porcine brain and studied some of their properties. These results were reported previously in a preliminary form ( 1 ) . This paper describes the experimental details of this study. EXPERIMENTAL Materials—Porcine brains were obtained from a slaughterhouse immediately after sacrifice and stored frozen at —20°. Yeast RNA was obtained from Juj5 Paper Co., Akita. pChloromercuribenzoate(PCMB) was purchased from Tokyo Kasei Co., and bis(/>-nitrophenyl) phosphate was from Seikagaku Kogyo Co., Tokyo. DEAE-cellulose (DE-23) and CM-cellulose (CM-52) were Whatman preparations. E. coli alkaline phosphatase [EC 3.1. 3.1] and calf thymus DNA were kindly supplied by Drs. T. Uchida and S. Inoue, respectively. Assay of Enzymatic Activities—Ribonuclease activity was determined with yeast RNA as a substrate. Unless otherwise specified, the assay mixture consisted of 0.25 ml of 0.2 M citrate-phosphate buffer, pH 5.9, or 0.2 M TrisHC1 buffer, pH 8.0, an appropriate amount of enzyme (usually 0.1 ml), 0.1 ml of 0.1 mM aqueous PCMB (£-chloromercuribenzoate)5 and 0.25 ml of 1% RNA in a total volume of 1.0 ml, made up with water. For assay of the samples obtained by column chromatography, 0.1 ml of 0.1% bovine serum albumin was added to the assay mixture, and PCMB was omitted from the assay mixture. The reaction wag allowed to proceed at 37° for 60 min and was then terminated by the addition of 0.25 ml of 0.75% uranyl acetate in 25% perchloric acid. The reaction mixture was centrifuged at 3000 rpm for 7 min, then 0.2 ml of the supernatant was removed and diluted with 5.0 ml of distilled water. The absorbance at 260 5
PCMB was used to inhibit the activity of a nuclease inhibitor present in brain (6"). After chromatography on DE-23, the acid ribonuclease preparation was free from the inhibitor, but became less stable.
nm of this solution was read against a blank sample treated in the same way except for theabsence of the enzyme. The absorbance at 260 nm thus determined (dAtK) showed a linear relationship to the amount of the enzyme in the range of ^AJM up to about 0.5. The activity corresponding to dAtfi0 = 1.0 wasdefined as 1 enzyme unit and the specific activity as the total dA2K divided by the absorbance at 280 nm of the enzyme solution. T h e protein concentration was determined tentatively assuming /i28oJ-£* = l.O. Deoxyribonuclease activity was determined under the same conditions except that the reaction mixture (1 ml) contained 0.5 ml of a. 0.4% heat-denatured calf thymus DNA solution and was incubated for 8 hr. Phosphodiesterase activity was determined, with bis(£-nitrophenyl) phosphate as a substrate. The reaction mixture contained 0.2 mL (about 1 ribonuclease unit) of enzyme solution, 0.25 ml of 0.2M citrate-phosphate buffer, pHL 5.9, or 0.2 M Tris-HCl buffer, pH 8.0, 0.25 mL of 0.07% aqueous bis(/>-nitrophenyl) phosphate, and 0.3 ml of distilled water. The mixture was kept at 37° for 2.5 hr and the absorbance at 400 nm was read. The mixture without enzyme was treated in the same way and used, as the blank. Determination of Substrate Specificity — Each enzyme sample (0.1 ml, 1—2 units) was. added to a mixture of 0.1 ml of 0.2 M Tris-HCl. buffer, pH 8.0, or 0.2 M citrate-phosphate, buffer, pH 5.9, 0.1 ml of 2% RNA and 0.1 ml. of distilled water. The mixture was kept at 37° for 24 hr. At suitable intervals, aliquots were removed and subjected to paper chromatography on Toyo-Roshi No. 51 or 51A filter paper. The solvent used was isopropyli alcohol-saturated ammonium sulfate solutionwater (2 : 79 : 19, by volume). To determine the base specificity of thealkaline ribonuclease, 10 mg of yeast RNA was. dissolved in 0.25 ml of 0.2 M Tris-HCl buffer, pH 8.0, and to this was added 0.75 ml (3.3. units) of enzyme solution. The reaction mixture was kept at 37° and at suitable intervals. 0.1 ml aliquots were removed. 20 p\ of 2.1 N HC1 was added to each aliquot and the mixture was kept at 5° overnight. Each reaction* / . Biochtm.
ZRIBONUCLEASES FROM PORCINE BRAIN
rmixture was neutralized by the addition of 20 ftl of 2.1 N NaOH, then mixed with 0.25 ml •of 0.2 M Tris-HCl buffer, pH 8.3, and 30 i*\ of -a solution of alkaline phosphatase (22 i*g) and kept at 37° for 18 hr. 80 f\ of 3 N KOH was wadded and the mixture was incubated at 37° for a further 18 hr. After this, 50 A
Ribonucleases from Porcine Brain Partial Purification and Properties1 Kazuya NISHIKAWA,1 Kenji TAKAHASHI,' and Toshio ANDO1 Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Bunkyo-ku, Tokyo 113 Received for publication, April 26, 1976
1. An acid ribonuclease was partially purified from an acetone powder of porcine train. This enzyme was an acidic protein with a molecular weight of around 70,000. It acted on yeast RNA optimally at about pH 5.9, yielding only a mixture of 3'mononucleotides, and therefore appears to be an exonuclease. It did not hydrolyze heat-denatured calf thymus DNA or bis(/>-nitrophenyl) phosphate. It was fairly unstable to heat and acid. 2. An alkaline ribonuclease was partially purified from the same source simultaneously. This enzyme was a basic protein with a molecular weight of 25,000—26,000. It was a pyrimidine-specific endoribonuclease, and acted on yeast RNA optimally at around pH 7.4. It did not hydrolyze heat-denatured calf thymus DNA or bis(/>-nitrophenyl) phosphate. It was fairly stable to heat and acid.
pH optima around pH 6 and 8 was reported in the brain homogenates of some animals, such as the rat ( 3 ) and guinea pig (4). Similar activity with a pH optimum around pH 6 was also reported for bovine brain (5). Until recently, however, there were few reports on the purification and characterization of such enzymes from brain, except for a partial purification of an exonuclease from bovine brain by Kurihara (5). Elucidation of the properties of brain nucleases is of great interest since they are involved in nucleic acid metabolism in the brain, which might be related to the specific function of brain. The present study was initiated in order to obtain data on the properties of such nucleases in porcine brain and also to compare the results with those obtained with bovine
There are various reports on the purification and properties of nucleases from different tissues of vertebrates (2). In brain, the presence of two RNA-depolymerizing activities with 1
This study was supported in part by grants (88516, •958016) from the Ministry of Education, Science and Culture, of Japan. A preliminary account of this study has already appeared ( / ) . 3 Present address: Institute of Molecular Biology, Faculty of Science, Nagoya University, Chikusa-ku, Nagoya 464. 3 Present address: Department of Biochemistry, Primate Research Institute, Inuyama, Aichi 484. Inquiries about this paper should be sent to this address. •* Present address: Department of Chemistry, Faculty of Physical Sciences and Engineering, Meisei University, Hodokubo, Hino, Tokyo 191. Vol. 80, No. 4, 1976
767
K. NISHIKAWA, K. TAKAHASHI, and T. ANDCP
768
brain from the viewpoint of comparative biochemistry. In the course of this study, we partially purified an acid ribonuclease and a pyrimidine-specific alkaline ribonuclease from acetone powder of porcine brain and studied some of their properties. These results were reported previously in a preliminary form ( 1 ) . This paper describes the experimental details of this study. EXPERIMENTAL Materials—Porcine brains were obtained from a slaughterhouse immediately after sacrifice and stored frozen at —20°. Yeast RNA was obtained from Juj5 Paper Co., Akita. pChloromercuribenzoate(PCMB) was purchased from Tokyo Kasei Co., and bis(/>-nitrophenyl) phosphate was from Seikagaku Kogyo Co., Tokyo. DEAE-cellulose (DE-23) and CM-cellulose (CM-52) were Whatman preparations. E. coli alkaline phosphatase [EC 3.1. 3.1] and calf thymus DNA were kindly supplied by Drs. T. Uchida and S. Inoue, respectively. Assay of Enzymatic Activities—Ribonuclease activity was determined with yeast RNA as a substrate. Unless otherwise specified, the assay mixture consisted of 0.25 ml of 0.2 M citrate-phosphate buffer, pH 5.9, or 0.2 M TrisHC1 buffer, pH 8.0, an appropriate amount of enzyme (usually 0.1 ml), 0.1 ml of 0.1 mM aqueous PCMB (£-chloromercuribenzoate)5 and 0.25 ml of 1% RNA in a total volume of 1.0 ml, made up with water. For assay of the samples obtained by column chromatography, 0.1 ml of 0.1% bovine serum albumin was added to the assay mixture, and PCMB was omitted from the assay mixture. The reaction wag allowed to proceed at 37° for 60 min and was then terminated by the addition of 0.25 ml of 0.75% uranyl acetate in 25% perchloric acid. The reaction mixture was centrifuged at 3000 rpm for 7 min, then 0.2 ml of the supernatant was removed and diluted with 5.0 ml of distilled water. The absorbance at 260 5
PCMB was used to inhibit the activity of a nuclease inhibitor present in brain (6"). After chromatography on DE-23, the acid ribonuclease preparation was free from the inhibitor, but became less stable.
nm of this solution was read against a blank sample treated in the same way except for theabsence of the enzyme. The absorbance at 260 nm thus determined (dAtK) showed a linear relationship to the amount of the enzyme in the range of ^AJM up to about 0.5. The activity corresponding to dAtfi0 = 1.0 wasdefined as 1 enzyme unit and the specific activity as the total dA2K divided by the absorbance at 280 nm of the enzyme solution. T h e protein concentration was determined tentatively assuming /i28oJ-£* = l.O. Deoxyribonuclease activity was determined under the same conditions except that the reaction mixture (1 ml) contained 0.5 ml of a. 0.4% heat-denatured calf thymus DNA solution and was incubated for 8 hr. Phosphodiesterase activity was determined, with bis(£-nitrophenyl) phosphate as a substrate. The reaction mixture contained 0.2 mL (about 1 ribonuclease unit) of enzyme solution, 0.25 ml of 0.2M citrate-phosphate buffer, pHL 5.9, or 0.2 M Tris-HCl buffer, pH 8.0, 0.25 mL of 0.07% aqueous bis(/>-nitrophenyl) phosphate, and 0.3 ml of distilled water. The mixture was kept at 37° for 2.5 hr and the absorbance at 400 nm was read. The mixture without enzyme was treated in the same way and used, as the blank. Determination of Substrate Specificity — Each enzyme sample (0.1 ml, 1—2 units) was. added to a mixture of 0.1 ml of 0.2 M Tris-HCl. buffer, pH 8.0, or 0.2 M citrate-phosphate, buffer, pH 5.9, 0.1 ml of 2% RNA and 0.1 ml. of distilled water. The mixture was kept at 37° for 24 hr. At suitable intervals, aliquots were removed and subjected to paper chromatography on Toyo-Roshi No. 51 or 51A filter paper. The solvent used was isopropyli alcohol-saturated ammonium sulfate solutionwater (2 : 79 : 19, by volume). To determine the base specificity of thealkaline ribonuclease, 10 mg of yeast RNA was. dissolved in 0.25 ml of 0.2 M Tris-HCl buffer, pH 8.0, and to this was added 0.75 ml (3.3. units) of enzyme solution. The reaction mixture was kept at 37° and at suitable intervals. 0.1 ml aliquots were removed. 20 p\ of 2.1 N HC1 was added to each aliquot and the mixture was kept at 5° overnight. Each reaction* / . Biochtm.
ZRIBONUCLEASES FROM PORCINE BRAIN
rmixture was neutralized by the addition of 20 ftl of 2.1 N NaOH, then mixed with 0.25 ml •of 0.2 M Tris-HCl buffer, pH 8.3, and 30 i*\ of -a solution of alkaline phosphatase (22 i*g) and kept at 37° for 18 hr. 80 f\ of 3 N KOH was wadded and the mixture was incubated at 37° for a further 18 hr. After this, 50 A
