Biosci. Biotechnol. Biochem., 67 (4), 698–703, 2003 Ribonuclease Inhibitors in Malus x domestica (Common Apple): Isolation and Partial Characterization Takao KOSUGE,1 Mamoru ISEMURA,2 Yoshiaki TAKAHASHI,3 Sumiko ODANI,4 and Shoji ODANI1,† 1Department of Biology, Faculty of Science, Niigata University, Ikarashi, Niigata 950-2181, Japan 2Department of Cellular Biochemistry, School of Food and Nutritional Sciences, University of Shizuoka, Tanida, Shizuoka 422-8526, Japan 3Department of Medical Technology, School of Health Science, Faculty of Medicine, Niigata University, Asahimachi, Niigata 951-8518, Japan 4Department of Home Economics, Faculty of Education and Human Science, Niigata University, Ikarashi, Niigata 950-2181, Japan Received August 22, 2002; Accepted January 6, 2003 A ribonuclease inhibitory activity was detected in the sion, and carbohydrate- and ATP-binding.1) Self- fruits of common apple, Malus x domestica,cv.Fuji, incompatibility in the ‰owering plants is also and puriˆed by a‹nity chromatography on ribonuclease mediated by prevention of self-pollination by S- A-Sepharose. It inhibited hydrolysis of cyclic-2?:3?- ribonuclease, which is a homologue of ribonuclease CMP by bovine pancreatic ribonuclease A with an T2.2) Therefore, a collective name of RISBASES apparent inhibition constant of about 5×10－8 M. (RIbonucleases with Special Biological Actions) was 1) Matrix-assisted laser desorptionWionization time-of- proposed for these RNase homologues. These ‰ight mass spectrometry of the puriˆed protein gave two diverse activities must be under precise control peaks corresponding to the mass numbers of 55,658 and responding to individual physiological conditions, 62,839, while three bands of 43-, 34-, and 21-kDa were but their mechanisms seem not fully understood. It detected by SDS-PAGE. These results suggested that may be possible that protein inhibitors of RNase are the inhibitor preparation was a mixture of two proteins regulatory factors. RNase inhibitors, either synthetic comprised of 43- and 21-kDa subunits or of 34- and or natural, have been intensively sought after for 21-kDa subunits. Attempts to separate these two therapeutic purposes for cancer, because angiogene- proteins were unsuccessful. Amino acid composition sis is frequently promoted by a pancreatic RNase A and N-terminal amino acid sequence of these subunits homologue, angiogenin, and inhibition of angioge- were also identiˆed and N-terminal sequences showed nin is expected to result in suppression of growth and some similarity to that of cottonseed storage globulin. metastasis of solid tumors.3–5) RNase inhibitors are of The signiˆcance of the presence of ribonuclease inhibi- another practical use to protect RNA molecules from tors in apple fruits is not clear, but it might allow some unwanted degradation by RNases during prepara- speculation about their possible involvement in the tion, and a recombinant human placental RNase control of the self-incompatibility ribonuclease of inhibitor is commercially available. In contrast to a Rosaceae plants. vast number of proteinase- and amylase-inhibitors, reports on protein inhibitors of RNase are quite Key words: Malus x domestica; ribonuclease inhibi- rare.6–8) We searched for inhibitors of RNase in some tor; amino acid sequence; seed storage plants and fungi and found a substantial inhibitory protein; protein puriˆcation activity in the fruits of apple (Malus x domestica). In this paper, we describe puriˆcation and partial Riboncleases (RNases), which have long been characterization of apple RNase inhibitors. regarded as mere digestive enzymes of animal and microorganisms, have a wide range of hitherto unexpected biological activities such as angiogenesis, neurotoxicity, aspermatogenicity, immuno-suppres- † To whom correspondence should be addressed. Fax: ＋81-25-262-6174; E-mail: sodani＠bio.sc.niigata-u.ac.jp Abbreviations: MALDI, matrix-assisted laser desorptionWionization; PBS, phosphate buŠered saline; PVDF, poly(vinylidene di‰uoride); RNase, ribonuclease; TOF-MS, time-of-‰ight mass spectrometry Apple Fruit Ribonuclease Inhibitors 699 Materials and Methods was placed and incubated for 30 min at 379C. The solution was removed and the tube was washed four Materials. Fruits of M. x domestica cv. Fuji, other times with 200 ml of PBS. Reproducibility of ˆxation plant materials, and mushrooms were obtained from of RNase to the tubes was conˆrmed by the activity a local market. Bovine pancreatic RNase A (EC measurement. To this was added 100 mlofasample 3.1.27.5) and cytidine 2?:3?-cyclic monophosphate or PBS and the tube was incubated for 30 min at were obtained from Sigma-Aldrich Co. (St. Louis) . 49C, washed four times with PBS, and further incu- Sepharose 4B and cyanogen bromide-activated batedwith100mlofyeastRNA(0.43mgWml in PBS) Sepharose were products of Amersham-Pharmacia for 15 min at 379C. Then 400 mlof95z ethanol BioTech (Uppsala). Ribonucleic acid (yeast) and sol- containing 10 mM MgCl2 was added and the mixture vents for HPLC were purchased from Wako Pure was centrifuged to precipitate large RNA molecules. Chemical Industries (Osaka). Reagents for protein The supernatant (400 ml) was mixed with 3.0 ml of sequencing were from Applied Biosystems Japan 66z ethanol and absorbance at 260 nm was meas- (Tokyo). ured. Suitability of the method for measuring RNase inhibitory activity was veriˆed by a commercial Isolation of RNase Inhibitor by A‹nity Chro- placental RNase inhibitor (Rnasin, Promega KK, matography. Whole mature apple fruits (500 g) were Tokyo). RNase activity was also measured by the homogenized in a blender with 500 ml of 50 mM Tris- absorbance change at 286 nm using 2?:3?-cyclic CMP HCl, pH 8.0, containing 0.1 M KCl, 10 mM EDTA, as a substrate according to the procedure of Black- 5z glycerol, 4z soluble poly(vinylpyrrolidone), burn et al.6) A Hitachi 320 recording spectrophotom- 10z dimethylsulfoxide, 5 mM dithiothreitol, 10 mM eter was used. Concentrations of the enzyme and 6-amino-n-caproic acid, and 1 mM phenylmethylsul- substrate were usually 0.073 mM and 1.0 mM, fonyl ‰uoride. The extract was centrifuged at 105,000 respectively, in 0.1 M Tris-acetate (pH 6.5). RNase- ×g for 1 h. The centrifuged supernatant was ˆrst put inhibiting activity was assessed by the diŠerence in on a column of Sepharose (2.5×10 cm) to remove absorbance change in the presence and absence of an proteins having a‹nity for the agarose matrix, and inhibitor sample. Mode of the inhibition was exam- then to an RNase A-Sepharose column (1.5×20 cm), ined by a Lineweaver-Burk plot of the results, and which had been prepared by coupling 25 mg of the inhibition constant was roughly estimated by a RNase to 20 ml of CNBr-activated Sepharose accord- Dixon plot (i.e. plotting 1W[against [I ]atdiŠerent ing to the manufacturer's instruction. Both columns ˆxed substrate concentrations [S]).11) These plots were equilibrated with 50 mM Tris-HCl, pH 7.5, were analyzed by the linear regression method using 0.3 M KCl, and 1 mM EDTA. The RNase A- the PRISM software (Graph Pad Software, Inc., San Sepharose column was extensively washed with the Diego, U.S.A.). same buŠer until absorbance at 280 nm of the eluent became negligible. The bound protein was eluted AminoAcidAnalysis.Samples (1–3 nmol) blotted with 45 mM acetic acid, and then with 4 M urea. onto PVDF membrane were hydrolyzed in sealed The eluted fractions were dialyzed against 100 tubes in vacuo with 5.7 M HCl, 4z (vWv) thioglycolic volumes of 45 mM sodium phosphate buŠer, pH 6.5. acid for 22 h at 1109C. Amino acid compositions All operations were done at 49C. were analyzed on a Hitachi Model 835 amino acid analyzer. Polyacrylamide Gel Electrophoresis. Protein sam- ples were electrophoresed in the presence and absence Amino Acid Sequencing. Amino acid sequences of of the denaturant (sodium dodecyl sulfate, SDS).9,10) proteins (about 200 pmol) were analyzed on an A semilograrithmic plot of molecular mass versus Applied Biosystems 476A gas-phase sequencer after relative mobility was generated with marker proteins blotting onto the PVDF membrane. andusedtoestimatethemolecularmassesofsample proteins. Comparison of Amino Acid Sequence. Amino acid sequences were compared using the data bases at the Measurement of RNase Inhibitory Activity. DNA Information and Stock Center, National Screening of RNase inhibitor activity in the crude Institute of Agrobiological Resources (Tsukuba) by tissue extracts was done by measuring RNase activity the program FASTA.12) using a solid phase method, because the presence of large amounts of UV-absorbing substances in the Mass spectrometry. Protein mass analysis was extract interfered with the spectrophotometric assay. done by matrix-assisted laser-desorptionWionization To a polystyrene round-bottom tube (12×75 mm, time-of-‰ight mass spectrometry using a Shimadzu- Falcon 2008 RIA tube, Becton Dickinson & Co., Kratos instrument (KOMPACT MALDI II) at an Lincoln Park, U.S.A) 100 ml of bovine pancreatic accelerating voltage of 20 keV. Sinapic acid was used RNase (1 mgWml) in phosphate buŠered saline (PBS) as the matrix. 700 T. KOSUGE et al. Fig. 1. A‹nity Chromatography of Apple Extract on RNase A- Sepharose. High-speed supernatant of the extract was put on a column (2.5×7 cm) of RNase A-Sepharose equilibrated with 50 mM Tris-HCl, pH 7.5, 0.3 M KCl, and 1 mM EDTA. The column was extensively washed with the same buŠer. The bound protein was eluted with 45 mM aceticacid(arrowA),andthenwith4M urea Fig. 2. SDS-Polyacrylamide Gel Electrophoresis of Apple RNase (arrow B). Two-ml fractions were collected. Proteins were de- Inhibitor Preparation. tected by their absorbance at 280 nm. Fractions indicated by (1) Fraction eluted with 45 mM acetic acid. (2) Fraction eluted bars were pooled separetely and dialyzed against 50 volumes of with 4 M urea. (3) Marker proteins. Molecular masses are indi- 45 mM sodium phosphate buŠer, pH 6.5. All operations were cated on the right margin.