Reinvestigation on the Amino Acid Composition and C-Terminal Group of Taka-Amylase A. By Kozo NARITA*, HIRONORI MURAKAMI* and TOKUJI IKENAKA**

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1 The Journal of Biochemistry, Vol. 59, No. 2, 1966 Reinvestigation on the Amino Acid Composition and C-Terminal Group of Taka-Amylase A By Kozo NARITA*, HIRONORI MURAKAMI* and TOKUJI IKENAKA** (From *the Institute for Protein Research and **Faculty of Science, Osaka University, Osaka) (Received for publication, October 7, 1965) Amino acid analysis of crystalline Takaamylase A [EC , ƒ -1, 4-glucan 4- glucanohydrolase, Aspergillus oryzae] prepared from "Takadiastase Sankyo " was performed by A k a b o r i et al. (1) by mainly classical starch column chromatographic method. Later, Stein et al. (2) reported amino acid composition of crystalline a-amylase prepared from " Clarase 900" using ion exchange chromato graphy. The latter amylase seems to be identical with the present Taka-amylase, since both are commercial materials produced from Aspergillus oryzae. However, a slight difference between the analytical results by the two groups has been recognized. Physicochemical and amino acid sequence studies, which have been developed in this institute, require precise data of its amino acid composition. Recent development of the automatic recording equipment (3) for the analysis of amino acid mixture afforded us to reinvestigate amino acid composition of the amylase. One of the purposes of the present communication is to describe the results of the amino acid analysis performed by the use of an automatic equipment. Ikenaka (4) has reported based on his experiments using the technique of hydrazino lysis that the C-terminal groups of Takaamylase A were alanine, serine and glycine, and he postulated a branched polypeptide chain structure terminating at an alanine residue at the amino end. However, no direct evidence in support of such a structure has so far been presented. In consequence reinvestigation on the C-terminal group by some other methods is required. In the present studies, carboxypeptidase A [EC ] was used to make clear the terminal residue. In contrast to the inference made by I k e n a k a, our experiments indicated that the amylase has a single open chain structure ending with C-terminal serine. Described herein are also the experiments that had led us to this conclusion. MATERIALS AND METHODS Crystalline Taka-Amylase A-The amylase was extracted and crystallized from " Takadiastase Sankyo " according to the direction of A k a b o r i et al. (5). Three times recrystallized amylase was used in some experiments. However three times recrystallized amylase was demonstrated to be not homogeneous by chromatography on a column of diethylaminoethyl-cellulose (6). Therefore amino acid analysis, hydrazinolysis and a part of the experiments of the carboxypeptidase (Worthington Biochemical Corp., Freehold, New Jersey) digestion were performed using the chromatographically purified amylase. Nitrogen content of the chromato graphically purified amylase was 15.40%, whereas Akabori et al. (1) and Stein et al. (2) reported 14.98% and 14.86%, respectively, on their crystalline samples. Reduced and Carboxymethylated Taka-Amylase A (RCM-amylase)-Samples of the RCM-amylase were prepared as reported previously (7) Amino Acid Analysis-The chromatographically purifed amylase was hydrolyzed with redistilled hydrochloric acid in evacuated sealed tubes at 110 C for 24, 48 and 72 hours. Techniques of the treatment of the hydrolysates and amino acid analysis by an automatic amino acid analyzer, Beckman/Spinco, Model MS, were the same as those described previously (8). Half cystine content was estimated by ampero metric titration on the reduced protein with sodium borohydride and the results were already reported

2 Amino Acid Composition and C-Terminal Group of Taka-Amylase 171 (7). Tryptophan content was determined by the spectrophotometric method of G o o d w i n and M o r ton on a sample dissolved in 0.1 N NaOH (9). Carboxypeptidase A Digestion-The ph of 1%o amylase solution was adjusted to 8.0 with 0.2 M sodium bicarbonate and carboxypeptidase A was added to the substrate solution in the ratio of 10-50: 1 by weight. The digestion was performed at 37 C. An aliquot corresponding to lƒÊmole of the substrate was pipetted out at each scheduled interval and the sample was acidified to ph 2 with 0.1 N hydrochloric acid or with citrate buffer of ph 2 to inactivate the enzyme. The denatured amylase was removed by centrifugation and released amino acids in the supernatant were estimated as their dinitrophenyl (DNP) derivatives as usual (10) or by an amino acid analyzer (Beckman/Spinco, Model MS or Shibata Chemical Apparatus Manu facturing Co., Model AA 400). In some cases the released amino acids were separated from the remaining protein fragment by the aid of molecular sieve action of ion exchange resin (11). Hydrazinolysis-For this experiment the chromato graphically purified amylase was used. About 1 lemole of the protein was placed in a small test tube and 0.5 ml. of anhydrous hydrazine was added. The tube was carefully sealed and heated at 100 C for various periods of time. The hydrazinolyzate was dried in an evacuated desiccator over concentrat ed sulfuric acid for hours. The residue was dissolved in 2 ml. of water, and 0.5 ml. of benzalde hyde was added. The mixture was immediately shaken vigorously for 1 hour at room temperature to allow the amino acid hydrazides to react with benzaldehyde. The mixture was centrifuged to remove the insoluble matter and the supernatant was quantitatively transferred into a flask for lyophilization. The separated oily material in the centrifuge tube was washed with 1 ml. of water and centrifugation was repeated. The supernatant and the washings were quickly lyophilized. Just before it was subjected to amino acid analysis, the lyophilized material was dissolved in 1.2 ml. of citrate buffer of ph 2.2 and 1 ml. of the solution was placed on a 50 cm. column of the automatic amino acid analyzer (12). Chromatography was performed using the same buffer system as for usual 150 cm. column. In another experiment, C-terminal amino acid was characterized as their DNP-derivatives by the method of Niu and Fraenkel-Conrat (13). RESULTS AND DISCUSSION Amino Acid Composition q f Taka-Amylase A- Amino acid composition of the amylase is listed in Table I. The enzyme sample used contained 11.3% moisture and 2.25% ash and figures in Table I are obtained after their corrections. Number of amino acid residues in Table I was calculated on the basis of molecular weight of 51,000 (14, 15). Half cystine residue was estimated by amperometric titration after reduction of disulfide bonds with sodium borohydride and cysteine residue was estimated on the denatur ed amylase sample as reported previously (7). Tryptophan content was determined spectro photometrically (9). Tyrosine content was also estimated spectrophotometrically and the value of 33.4 moles per mole protein was obtained. This value is in agreement with that estimated by the analyzer on the acid hydrolysates listed in Table I. Totally 462 residues were calculated to be involved in one molecule of the amylase. Basing on the amino acid composition, molecular weight of 50,772 is calculated. It is known that the amylase contained approximately 10 residues of hexose and hexosamine (16). Therefore the molecular weight becomes close to 52,000 and this value is not inconsistent with the physicochemical estimate. Action of Carboxypeptidase A on Taka- Amylase and RCM-Amylase-The amounts of released amino acids from the crystalline amylase by the carboxypeptidase digestion are estimated as their DNP-derivatives and are plotted as a function of time in Fig. 1. The release rates of amino acids in the initial stage were in the order of serine, leucine and the other amino acids under the conditions used. However in some experiments, the initial release rates of serine and leucine were reverse under similar conditions. It has been demonstrated that when the crystalline amylase alone was incubated at ph's 4.9, 7.1 and 8.3, respectively, significant amounts of amino acids were released in every case without appreciable loss of the activity (17 ). In the present experiments, prolonged incubation (14 days) of the amylase with carboxypeptidase A released about 42 moles of amino acids (estimated by the ninhydrin reaction and expressed as leucine

3 172 K. NARITA, H. MURAKAMI and T. IKENAKA TABLE Amino Acid Composition of Taka-Amylase A1) I 1) The value for each amino acid is an average of three determinations. Tryptophan was estimated by spectrophotometric method (9) and cysteine and half cystine were estimated by amperometric titration on the samples before and after sodium borohydride reduction of the disulfide bonds in the amylase, respectively (7 ). Cysteine content is not involved in half cystine value. 2) The values for tryptophan, cysteine and half cystine are not involved. equivalents) from one molecule of the amylase, and a control experiment without addition of the carboxypeptidase also showed the release of about 37 moles of amino acids. This phenomenon might be attributed to the combined action of contaminated proteolytic enzyme in the crystalline amylase preparation and of the carboxypeptidase on the denatured amylase, which seems to be present in trace amount. Therefore it is hard to conclude from the result that which amino acid is the C-terminal one of the amylase. It has been shown by diethylaminoethyl cellulose chromatography that the three times, recrystallized amylase contained a small amount of rather low molecular peptidic materials (6) which were probably derived from the contaminated denatured amylase by the action of proteolytic enzyme present in. the crystalline amylase as contaminants. When the solution of the chromatographically purifiedd amylase was incubated at 37 C for 72 days, the release of about 7 moles of amino acids. was observed. Therefore the purified enzyme

4 Amino Acid Composition and C-Terminel Group of Taka-Amylase 173 FIG. 1. The released amino acids from the crystalline Taka-amylase A by the action of carboxypeptidase A. One per cent of the amylase solution was incubated with the enzyme in the substrate to the enzyme ratio of 33: 1. The released amino acids were estimated as their DNP-derivatives. seemed still to contain a trace amount of the proteolytic enzyme. The incubated amylase was then precipitated by adding cold acetone, and crystallized from aqueous acetone by the procedures of A k a b o r i et al. (5). This material was used for carboxypeptidase diges tion. The released amino acids as a function of time are shown in Fig. 2. In contrast to the pattern in Fig. 1, serine was predominant throughout the digestion. No decrease of the amylase activity was observed up to 24 hours' incubation. These results suggest that the C-terminal group is serine and that the C-terminal part is not essential for the activity. When RCM-amylase was incubated with carboxypeptidase A, similar pattern to that in Fig. 2 was obtained with respect to the order -of released amino acids as is shown in Fig. 3. Namely the release of serine was predominant for entire period of the experiment, but the rate of the release was faster than that in the case shown in Fig. 2, in which the substrate was a native protein contrary to the denatured one in Fig. 3. In the present case amino acids released were estimated by an amino acid analyzer and leucine and isoleucine could be determined separately. The amount of leucine released in Fig. 2 apparently exceeded that of the any other amino acids except FIG. 2. The released amino acids from the chromatographically purified Taka-amylase A by carboxypeptidase A digestion. The ratio of the substrate to the enzyme was 13: 1. The released amino acids were estimated as their DNPderivatives. serine, but its amount did not mean for only leucine, since leucine and isoleucine were estimated together as their DNP-derivatives. Thus we concluded that the C-terminal structure of Taka-amylase A was...[thr, Val, Ala, Leu].Ser.OH. FIG. 3. The released amino acids from RCM-amylase by the action of carboxypeptidase A. The ratio of the substrate to the enzyme was 50: 1. The released amino acids were estimated by an amino acid analyzer. Hydraziaolysis-It is clear from the carbo xypeptidase experiments that the C-terminal group of the amylase is serine as was described, but I k e n a k a (4) previously reported three C-terminal amino acid residues, serine, glycine and alanine, by the hydrazinolysis method. Therefore the hydrazinolysis experiments for

5 174 K. NARITA, H. MURAKAM! and T. IKENAKA the amylase were repeated in a similar manner to that performed by Ikenaka, but the DNP-derivatives of the C-terminal amino acids were characterized and estimated by two-dimensional paper chromatography. The results without correction are shown in Fig. 4. Appearence of serine and glycine as a func tion of hydrazinolysis time was similar to that found by Ikenaka but the amount of alanine was slight. When aqueous solution of the hydrazino lyzate was allowed to stand for hours at room temperature, glycine, alanine and serine increased suggesting hydrolysis of their hydrazides to free amino acids. Therefore the treatment of hydrazinolyzate has to be made quickly according to the direction described in the Methods, otherwise non-c-terminal amino acids, especially glycine, alanine and serine, will be mischaracterized as the C- terminal groups. Since Taka-amylase containss a large amount of glycine and alanine, their hydrazides remained after benzaldehyde treatment of the protein hydrazinolyzate would be hydrolyzed during dinitrophenyla tion in a basic medium and I k e n a k a hadd mischaracterized these two amino acids as the C-terminal residues in addition to the real C-terminal serine. FIG. 4. The liberated amino acids from the chromatographically purified Taka-amylase A by hydrazinolysis. The amino acids were characteriz ed and estimated as their DNP-derivatives by two-dimensional paper chromatography. In another experiment, free amino acids present in the hydrazinolyzates at 4, 6, 8, 10, 12 and 15 hours were estimated by the automatic amino acid analyzer (18) as is shown in Fig. 5, in which quantities of amino acids were not corrected for their losses during hydrazinolysis. About 0.5 ƒêmole of serine at the maximum point was obtained per mole of the protein, and when the values at longer hydrazinolysis periods were extrapolated to zero time, about I mole of serine was obtained. Amounts of glycine and alanine did not exceed 0.2 mole and the other amino acids were lesser than 0.05 mole throughout entire period of hydrazinolysis. Decrease of the amount of serine at longer hydrazinolysis times can be explained by the fact that several amino acids, of which side chain groups are small, converted partly into their hydrazides by the action of anhydrous hydrazine at 100 Ž (19, 20). FIG. 5. The liberated C-terminal amino acids from the chromatographically purified Takaamylase A by hydrazinolysis. The amino acids were estimated by an amino acid analyzer. In a chromatographic elution pattern of the hydrazinolyzate, two unknown peaks were observed, the one eluted at the break-through point and the other small and broad peak eluted at the position of proline. It seems to, be unlikely that these peaks are amino acid hydrazides escaped their removals by the benzaldehyde treatment, since amino acid hydrazides are basic materials and may not elute at such positions. However, the breakthrough peak may be contaminated condensa tion products of amino acid hydrazides with benzaldehyde, which dissociate into the two, components at 100 Ž and may react with the ninhydrin reagent. Control experiment using

6 Amino Acid Composition and C-Terminal Group of Taka-Amylase 175 synthetic aspartic and glutamic monohyd razides gave no appreciable peaks under the conditions used, although in the previous experiments the monohydrazides gave clear peaks using 15 cm. column at room tempera ture (19). Lability of amino acid hydrazides with the contact of sulfonic acid type ion exchange resin was previously described (19). SUMMARY Amino acid analysis of the chromato graphically purified crystalline Taka-amylase A was undertaken with an automatic amino acid analyzer and the following amino acid composition was determined : Asp66, Thr3s, Sera., G1u30, Pro21, Gly40, Ala36, Va128, Met,, Ileu28, Leu31, Tyr33, Phe14, Lys18, His,, Arg9, Try,,, CySH1, CyS-8 and (NH3)44. In total 462 residues are involved in one molecule of the amylase and the molecular weight of 50,772 could be calculated excluding constituent carbohydrates. The C-terminal group was determined by the use of carboxypeptidase A and by the modified hydrazinolysis method, and the following C-terminal structure was tentatively proposed :... [Val, Thr, Ala, Leu].Ser.OH The authors are grateful to Sankyo Co., Ltd., for the supply of " Takadiastase Sankyo ". The present studies were partly aided by a Scientific Grant from the Ministry of Education. REFERENCES (1) Akabori, S., Ikenaka, T., Hanafusa, H., and Okada, Y., J. Biochem., 41, 803 (1954) (2) Stein, E.A., Junge, J.M., and Fischer, E.H., J. Biol. Chem., 235, 371 (1960) (3) Spackman, D.H., Stein, W.H., and Moore, S. Anal. Chem., 30, 1190 (1958) (4) Ikenaka, T., J. Biochem., 43, 255 (1956) (5) Akabori, S., Ikenaka, T., and Hagihara, B., J. Biochem., 41, 577 (1954) (6) Toda, H., and Akabori, H., J. Biochem., 53, 102 (1963) (7) Seon, B.K., Toda, H., and Narita, K., J. Biochem., 58, 348 (1965) (8) Narita, K., Murakami, H., and Titani, K., J. Biochem., 56, 216 (1964) (9) Goodwin, T.W., and Morton, R.A., Biochem. J., 40, 628 (1946) (10) Fraenkel-Conrat, H., Harris, J.I., and Levy, A.L., Methods of Biochem. Anal., 2, 360 (1955) (11) Thompson, A.R., Nature, 169, 495 (1952) (12) Tsugita, A., J. Mol. Biol., 5, 284 (1962) (13) Niu, C.I., and Fraenkel-Conrat, H., J. Am. Chem. Soc., 77, 5882 (1955) (14) Isemura, T., `Symposium on Cytochemistry' 3, 1 (1954) (15) Isemura, T., and Fujita, S., J. Biochem., 44, 443 (1957) (16) Hanafusa, H., Ikenaka, T., and Akabori, S., J. Biochem., 42, 55 (1955) (17) Akabori, S., Ikenaka, T., Oikawa, A., and Tsugita, A., `Symposium on Enzyme Chem.' 9, 13 (1956) (18) Tsugita, A., Gish, D.T., Young, J., Fraenkel- Conrat, H., Knight, C. A., and Stanley, W.M., Proc. Natl. Acad. Sci., 46, 1463 (1960) (19) Narita, K., and Ollta, Y., Bull. Chem. Soc. Japan, 32, 1023 (1959) (20) Narita, K., Biochem. Biophys. Research Communs., 5, 160 (1961)