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(Rnase T,) Contains Surpriz Ingly Poor Amount of Basic Amino Acids-Only

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(Rnase T,) Contains Surpriz Ingly Poor Amount of Basic Amino Acids-Only The Journal of Biochemistry, Vol. 52, No. 4, 1962 The Structure and Function of Ribonuclease T V. Deamination of Ribonuclease T, By YUICHI SHIOBARA, KENJI TAKAHASHI and Fujio EGAMI (From the Department of Biophysics and Biochemistry, Faculty of Science, University of Tokyo, Tokyo) (Received for publication, May 16, 1962) As has been demonstrated before (1), ribonuclease T, (RNase T,) contains surpriz ingly poor amount of basic amino acids-only three histidine, one lysine and one arginine Determination of protein concentration was based residues per molecule, while pancreatic on the dry weight or the absorption at 278 mƒÊ of the enzyme solution as described before (1). All the ribonuclease (RNase I) contains four histidine, spectrophotometric measurements were carried out in ten lysine and four arginine residues per a Beckman model DU spectrophotometer. molecule (2, 3). Thus RNase T, possesses Deamination-Experiment 1 : 0.2 ml. of RNase T, only two free amino groups-the a-amino solution (about 0.3 mg. enzyme) was added to the :group of the N-terminal alanine (4) and the mixture of 0.8 ml. of M sodium acetate buffer of pH s-amino group of the lysine residue in the 3.5 and 1.0 ml. of M sodium nitrite solution (freshly molecule, while 11 free amino groups are pre prepared before use) preincubated at 2°C or 30°C. sent in RNase I. As to the histidine residues The reaction was allowed to proceed for hours at 2°C in RNase T,, photooxidation experiments or 30°C and at intervals aliquots of 0.1 ml. were re- moved and diluted with 10 ml. of 0.01M sodium have shown that one or two moles of the three histidine residues are of essential importance phosphate buffer of pH 7.5 to stop the reaction. These aliquots were further diluted five-fold with the for the catalytic function of this enzyme (5), same buffer and the enzyme activity was determined. and similar results have also been reported Control samples without sodium nitrite were incubat with RNase I (6-8). With RNase I, certain ed in the same way as above to correct the changes ,of the amino groups in the molecule have in the activity. Examinations for the effect of pH also been reported to be essential for the on the activity were made with the native enzyme enzyme activity, while some others not (9, 10). and two samples deaminated at 2°C for 30 minutes ,On deamination even some increase in the and for over 50 hours respectively. activity has been observed (10). Experiment 2: The reaction was carried out at In this connection, the two amino groups 0°C in the same manner as above in the reaction mixture containing 10 ml. of RNase T1 solution (about present in RNase T, have been investigated 15 mg. enzyme), 10 ml. of M sodium acetate buffer, for their relation to the enzyme activity by pH 3.5, and 10ml. of M sodium nitrite solution at deamination experiment with nitrous acid 0°C for 30 minutes and for 65 hours respectively, and and the results are reported in this paper. was stopped by addition of solid sodium bicarbonate to bring the pH up to ca. 7.0. The neutralized solu EXPERIMENTALS tion was dialyzed against several changes of cold RNase T,-RNase T, was prepared according to distilled water entirely and lyophilized. The two the procedure reported before (1, 11) samples were analyzed for the N-terminal amino Determination of RNase Activity and Protein Concent acids and for the amino acid compositions. The ration-Determination of RNase activity was made enzyme activity was also determined with aliquots of according to the spectrophotometric procedure em 0.1 ml. from each reaction mixture. ployed before with yeast RNA as the substrate (11). Experiment 3: The reaction was carried out at Effects of pH on the enzyme activity of native as well 30°C in the same reaction mixture as in Experiment as deaminated RNase T, were investigated in the pHrange2, exceptfrom that3.9 to10 8.5.ml. usingof 2 MO.2M sodium Tris (pHnitrite 7.2-8."/),solutioncitrate(pH 5.7-6.8)sodium andsodium acetate(pH buffers. 3.9-5.6) 267 268 Y. SHIOBARA, K. TAKAHASHI and F. EGAMI was used instead of M solution of the same volume. 2°C, the activity was lost only slowly; less At intervals aliquots of 3 ml. were withdrawn, neutra than 10% inactivation was obtained after 1.5 lyzed with addition of solid sodium bicarbonate, dia hours and more than 50 per cent of the origi lyzed against distilled water and adjusted to a final nal activity was still retained after 25 hours. volume of 5 ml. to measure the ultraviolet spectra. At 30°C, on the other hand, the rate of inacti The enzyme activity was determined as above. vation was N-Terminal Amino Acid Analysis-The N-terminal ,far greater than that at 2°C; about amino acid residue of deaminated RNase T, (Ex- 50% inactivation occurred in 1.5 hours and periment 2) was examined by the dinitrofluorobenzene only 10 per cent of the original activity re method of S a n g e r (12) with the two-dimensional mained after prolonged incubation (50 to 100 paper chromatography of Levy (13) with slight hours). modifications as described before (4). Dinitropheny Examinations of the effect of pH on the lated derivative of the deaminated RNase T, was enzyme activity of both native and deaminat hydrolyzed with 1.0 ml. of 6 N glass-distilled HC1 at ed RNase T1 (Experiment 2) showed that the 105°C for 6 hours in a sealed evacuated test tube. enzyme activity was hardly affected on de Ether-soluble DNP-amino acids were examined by two amination at 2°C for 30 minutes (Fig. 2). dimensional paper chromatography in the solvent system of n-butanol-2 N aq. NH3 and 1.5 M sodium The pH - activity curve of RNase T1 de aminated at 2°C for over 50 hours was some- phosphate buffer of pH 6.0. Amino Acid Analysis-The lyophilized material of what changed as compared with that of the the deaminated RNase T, (Experiment 2) was hydro native enzyme (Fig. 2) ; the activity was about lyzed with 1.0 ml. of 6 N glass-distilled HC1 at 105°C 50 per cent of the original one as measured for 24 hours and the hydrolysate was analyzed for in the pH range of about 7 to 8, while below the amino acid composition with the use of a Beck pH 7, more than 50 per cent of the original man-Spinco model MS automatic amino acid analyzer activity was retained, the activity being almost according to the procedure of S p a c k m a n et al. (16). identical or even somewhat higher than that The results are expressed in molar ratios of the amino of the native enzyme as measured below pH acid residues assuming the number of glycine residue 5. These results are somewhat similar to to be 12.0 per molecule. those reported for RNase I (10), although not RESULTS AND DISCUSSION Changes in the enzyme activity of RNase T1 on deamination is shown in Fig. 1. At pH FIG. 2. Effect of pH on the enzyme activity REACTION TIME (hours) of native and deaminated RNase Ti. FIG. 1. Changes in the enzyme activity of The enzyme activity is expressed as increase RNase T, on deamination. in the optical density at 260mƒÊ of acid-soluble The reaction mixture contained 0.2 ml. of RNase T, solution (0.3 mg. of the enzyme), 1.0 ml. digestion products from yeast RNA. The condi of M sodium nitrite solution and 0.8m1. of M tion of deamination is given in Fig. 1. Native sodium acetate buffer, pH 3.5. The reaction was enzyme: -•-; deaminated enzyme: ---0--- (at allowed to proceed at 2°C (-•-) and at 30°C 2°C for 30 minutes), -.-•~-.- (at 2°C for over 50 (-0-). hours). Ribonuclease T1. V 269 so significant rise in the activity was observed about 55 per cent of the original activity. at around pH 4.5 on the deamination of The N-terminal amino acid residue, alanine, RNase T1. was completely absent from either of the two Analyses of the N-terminal amino acid deaminated preparations, indicating that the residue and the amino acid composition were N-terminal a-amino group was deaminated carried out with the control enzyme, and the very rapidly and is not essential for the cata two deaminated enzyme preparations (Experi lytic activity. The results of amino acid ment 2)-one was deaminated at 0°C for 30 analyses are summarized in Table I. As to minutes, retaining full activity and the other the 30 minutes-deaminated RNase T1, signifi deaminated at 0°C for 65 hours, retaining cant changes occurred only with alanine and TABLE I Amino Acid Composition of Deaminated RNase T, 1) The numbers of residues are calculated by assuming the number of glycine residue per molecule to he 12.0. 2) The values for the amino acid residues which suffered a significant change are given in bold face type. 3) Corrections for the hydrolytic losses of 1.3, 5.b, 8.5 and 4.3 per cent are made for aspartic acid, threonine, serine and proline respectively and the ammonia values are corrected by 15.7 per cent for the increase during the hydrolysis according to the previous results (1). 4) Unknown components. 5) Not determined. 270 Y. SHIOBARA, K.
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