Tamikazu Kume, Hiroshi Watanabe, Masaaki Takehisa and Tomotaro Sato*

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1 Agric. Biol. Chem., 45 (6), , Radiosensitivity of Glucose Isomerase in vivo and in vitro Tamikazu Kume, Hiroshi Watanabe, Masaaki Takehisa and Tomotaro Sato* Takasaki Radiation Chemistry Research Establishment, Japan Atomic Energy Research Institute, Takasaki, Gunma , Japan Received October 14, 1980 The radiosensitivity of glucose isomerase was investigated under various irradiation conditions. The inactivation of glucose isomerase irradiated in a cell-bound state was exponential, and an increase in inactivation was recognized in an oxygenated condition. The cell-free enzymewas highly radiosensitive and had a small oxygen effect compared to that in a cell-bound state. The oxygen enhancement ratio (OER) decreased with a degree in enzyme purification. Released glucose isomerase was protected by the addition of glutathione, and the inactivation curve in nitrogen almost agreed with that in the cell. The protective effect of glutathione in oxygen decreased at higher doses because glutathione in oxygen was easily decomposed by irradiation. The effect of irradiation on glucose isomerase has been investigated from a practical point of view. Streptomyces glucose isomerase is conveniently used to compare radiosensitivity in vitro and in vivo since the enzymeis an intracellular one and the activity can be measured in an intact cell. Enzymes, in general, are inactivated significantly in vitro by irradiation, while those in vivo are hardly affected. It is also knownthat the inactivation of the enzyme in a cell is enhanced in the presence of oxygen during irradiation similar to the lethal effect on living cells. However, the role of oxygen in the system has not been satisfactorily elucidated. In a previous paper,1] we investigated the inactivation of purified glucose isomerase in a dilute solution by y-irradiation. The results showed that the hydroxyl radical and hydrogen atom were efficient for the inactivation and the hydrated electron contributed a little. This paper describes the inactivation of glucose isomerase irradiated in vivo and in vitro, and the oxygen effect in various states of the enzyme. * Present address: Food Research Institute, Tokai Bussan Co., Ltd., Fukuroi, Shizuoka 437, Japan. MATERIALS AND METHODS Microorganisms. Streptomyces phaeochromogenus cells provided by Nagase Sangyo Co., Ltd., were mainly used. Streptomyces strain No. 41 isolated by Tsumura et al.2) was also used, which was cultivated according to the method of Tsumura and Sato.3) These cells were deepfreeze stored until use. Enzymepreparation. Streptomyces cells were suspended in distilled water, centrifuged at 12,000 rpm for 30min, and washed twice with distilled water. The enzyme in the precipitated cells is referred to as the cell-bound enzyme. The enzymein supernatant is referred to as the released enzyme. The crude enzymewas obtained as follows: the cells were disrupted by Dyno-Mill Type KDL (WAB Machinenfabrik, Switzerland) with 0.1 mmglass beads, and insoluble materials were removedby centrifugation at 3000rpm for 5 min. Irradiation. The cell suspensions and the enzyme solutions were saturated with nitrogen before irradiation or bubbled with oxygen or nitrous oxide during irradiation, except for irradiation in the presence of air. The samples were irradiated at room temperature with the use of 60kCi cobalt-60 slab source. The dose rate used was in the range of 20 toloookrad/hr as determined by Fricke dosimetry. Enzyme assay. Assays of enzymatic activities of glucose isomerase were performed as described in the previous paper.1} Protein concentration was determined by the micro-kjeldahl method.4)

2 1352 RESULTS T. Kume, H. Watanabe, M. Takehisa and T. Sato AND DISCUSSION 1. Inactivation of glucose isomerase in cell Frozen Streptomyces phaeochromogenus cells were suspended in distilled water (ca. 2.4 mg wet weight per milliliter) and the suspensions were irradiated as they were. The inactivation of glucose isomerase is shown in Fig. 1. The inactivation curve in nitrogen has an initially high radiosensitivity and is followed by an exponential portion with low sensitivity, whereas the inactivation curve in oxygen is an exponential one. It is assumed from the curve in nitrogen that there are different type enzymes which have a different radiosensitivity. Whenthe cell suspension was centrifuged at 12,000 rpm for 30min, about 45% of activity was present in the supernatant. Therefore, about 45%of the glucose isomerase is present in a cell-free state in this suspension. This shows the remaining activity belongs to the cell-bound enzyme. The cell-bound enzyme was collected in the conditions described above and the suspension was irradiated in nitrogen, nitrous oxide and oxygen. The exponential inactivation was observed and the significant Fig. 1. Inactivation of Glucose Isomerase in Cell Suspension Irradiated in Nitrogen and in Oxygen. O, in nitrogen; #, in oxygen. oxygen enhancement effect was observed as shown in Fig. 2. Nitrous oxide also increases the inactivation of cell-bound glucose isomerase compared to that in nitrogen. From this result, it is considered that the hydroxyl radical contributes to the inactivation in a cell-bound state the same as in the purified glucose isomerase solution.1} In the case of the Streptomyces strain No. 41, more than 50%of the glucose isomerase is present in a cell-free state. However, the inactivation of cell-bound and released enzyme after separation was the same as that of Streptomyces phaeochromogenus. Since glucose isomerase is easily released from cells during storage, it is desirable to inhibit the enzymerelease from the view point of application. If the release of glucose isomerase is inhibited, such as in heating the cells,5} the inactivation of the enzyme by irradiation can be minimized. The inactivation of glucose isomerase in a dry state was investigated. The cell suspensions were lyophilized and sealed in vacuo. The dry cells were irradiated in vacuo and in air by opening the vials prior to irradiation. Figure 3 shows the inactivation curves of glucose isomerase irradiated in dry cells. The enzymatic activity decreases exponentially in both conditions. The enzyme is more radiosensitive in air than in vacuo. When the cell suspension in nitrogen was irradiated at liquid nitrogen temperature ( C), the inactivation curve was similar to that of a dry cell in vacuo. The oxygen enhancement ratio (OER) is 1.9 from the ratio ofd37 in vacuo (4.2 Mrad) to in air (2.2 Mrad). This value is within the range of Dose(x10 rad) 12 Fig. 2. Inactivation of Glucose Isomerase Irradiated in Cell-bound State. O, in nitrogen; A, in nitrous oxide; #, in oxygen.

3 Radiosensitivity of Glucose Isomerase in vivo and in vitro ~2.4 which is obtained on most enzymes irradiated in a dry state.6) It is considered that the direct action of radiation may contribute up to one-third or one-half of the radiation effect in a cell, with the remainder contributed by indirect action.7] In the case of glucose isomerase irradiated in Streptomyces cell, it was calculated from doses in dry (4.2 Mrad) and in wet cells (2.2 Mrad) that the contribution of direct and indirect action are 52% and 48%. 2. Inactivation of glucose isomerase in various states The released enzyme solution was irradiated after separation from cells and the inactivation curves in nitrogen and in oxygen are shown in Fig. 4. Exponential curves are obtained, and a higher radiosensitivity and a smaller oxygen effect are observed compared to that in cell-bound enzyme (Fig. 2). The inactivation of glucose isomerase in crude extract which was obtained by disruption of the cell by Dyno-Mill is shown in Fig. 5. The exponential inactivation and the oxygen effect Fig. 4. Inactivation of Released Glucose Isome'rase. O, in nitrogen; #, in oxygen. are observed in this case. Fromthese results, the radiosensitivity and degree of oxygen effect are differed with the enzyme state. The D37 doses and OER in various states are summarized in Table I. The enzymatic activity per mgprotein is also shownas a relative valufe to crude enzyme. It was recognized that the inactivation of glucose isomerase was remarkably protected in a cell-bound state or in crude extract. The crude extract has many impurity bands on disc gel electrophoresis, while the released enzyme has only a slight impurity band. The cellular components would contribute to the protection and the following mechanism has been proposed8'9) for the protection in vivo; 1) radical scavenging, 2) protection of active site, 3) reactivation of inactivated enzyme by hydrogen transfer, 4) energy transfer in the complex of 'protective substance and enzyme. Radical scavenging would be a large factor in protection because the contribution of indirect action was about 50%for the inactivation of glucose isomerase in wet cells. The other three reactions would also contribute to the protection of glucose isomerase in cells. It has been reported10) that the radiosensitivity of microorganisms is increased with oxygen by a factor of 3, and the same factor is obtained for the inactivation of enzymes and nucleic acids when irradiated in cells. As shown in Table I, the significant oxygen effect was observed for the inactivation of glucose isomerase in a cell-bound state and the value Table I. Changes in Oxygen Enhancement Ratio on Inactivation of Glucose Isomerase D.. D37dose(xl05rad) Relative 3 / Fig. 5. Inactivation of Crude Glucose Isomerase Extracted from Cell. O» in nitrogen; #, in oxygen. Extract from cell disrupted by Dyno-Mill. Released enzyme from cell. Gave a single band on disc gel electrophoresis.

4 1354 T. Kume, H. Watanabe, M. Takehisa and T. Sato of OERin vitro decreased with the degree of enzyme purification. Since the inactivation of purified glucose isomerase was protected by oxygen, hydroxyl radical and superoxide formed by H+O2 or ea~ +O2 would be ineffective in the inactivation of the enzyme in vivo. It is considered that the inhibition of the repair mechanism by hydrogen transferll) is the most likely mechanism in this case. Thus the competition with "fixation of damage by oxygen" and "repair by hydrogen transfer" would be the main reaction on inactivation of glucose isomerase in vivo under oxygenated conditions. 3. Radioprotection effect ofglutathione A cell contains non-protein sulfhydryl compounds and it is believed that the glutathione, one major cellular sulfhydryl constituent, plays an important role in the radiosensitivity of cells.12) The effect of glutathione on the inactivation of glucose isomerase was investigated using the released enzyme. The inactivation curves of released enzyme containing 10mM and 30mM glutathione (ph 7.0) are shown in Fig. 6. The enzyme is protected remarkably by glutathione in nitrogen and the inactivation curve almost agreed with that of cell-bound enzyme. The following mechanism has been proposed12) to explain the role of glutathione in biological radiosensitivity. This involves; radical scavenging, hydrogen transfer and the formation of mixed disulfides. The last reaction seems inapplicable in the case of glucose isomerase since glucose isomerase is a non-sulfhydryl enzyme.13'14) The other two reactions seem to be acceptable in this case. In oxygen, the protective effect of glutathione disappeares easily as shown in Fig. 6. It has been shown15) that G(-SH) of glutathione is almost 20 (molecules/100ev) at 3 mmconcentration in oxygenated solution at ph 7. Using this value, the dose required to decompose 10mMand 30mMglutathione in oxygenated solution at ph 7 is calculated to be about 4.8 x 105 and 1.4 x 106 rad, respectively. The disappearance of protection with glutathione at each concentration occurs around these doses (Fig. 6). Hutchinson10) has shown that glutathione protects the inactivation of DNAand trypsin in dilute solution, and OER is roughly the same as that in cells. Recently Morse and Dahl16) have also shown the cellular glutathione is a key to the oxygen effect in radiation damage of E. coli using a glutathione deficient mutant. In our experiment, however, the inactivation curve in oxygen with glutathione was different from that of cellbound enzyme irradiated in oxygen. This shows that the oxygen effect in cells cannot be explained as the simple action of glutathione. It could be considered that other nonsulfhydryl components also contribute to the oxygen effect in cells as reported by Hemmen et al}n) Acknowledgments. The authors wish to thank Dr. N. Tsumura of the National Food Research Institute for a gift of Streptomyces strain No. 41. They would also like to show their appreciation to Professor H. Iizuka of the Science University of Tokyo and Dr. H. Ito of this Institute for their encouragement and discussion. REFERENCES Dose (x106fad) Fig. 6. Effect of Glutathione on Inactivation of Released Glucose Isomerase by Irradiation. O, with 10 or 30mM glutathione, in nitrogen; %, with lo nim glutathione, in oxygen; A, with 30 mmglutathione, in oxygen ) T. Kume, H. Watanabe, S. Aoki Biol. Chem., 45, 1311 (1981). and T. Sato, Agric. 2) N. Tsumura, T. Kasumi and M, Ishikawa, Kept. Nat. Food Res. Inst., 3) N. TsumuraandT. No. 31, 71 (1976). Sato, Agric. Biol. Chem., 29, 1129 (1965). 4) W. W. Umbreit, R. H. Burris and J. F. Stauffer, "Manometric Techniques," Burgess Publishing Company, USA, 1957, p ) Y. Takasaki, Y. Kosugi and A. Kanbayashi, "Fermentation Advances," ed. by D. Perlman,

5 Radiosensitivity of Glucose Isomerase in vivo and in vitro 1355 Academic Press Inc., New York, 1969, p ) K. I. Altman, G. B. Gerber and S. Okada, "Radiation Biochemistry," Vol. I, Academic Press, New York, 1970, 7) R. J. Shalek, C. p. 13. E. Smith andj. Hunter, Radiat. Res., 31, 467 (1967). 8) G. Fletcher and S. Okada, Radiat. Res.,, ll, 291 (1959). 9) A. Pihl and T. Sanner, "Radiation Protection and Sensitization," ed. by H. Moroson and M. Quintiliani, p.43. Taylor and Francis Ltd., London, 1970, 10) F. Hutchinson, Radiat. Res., 14, 721 (1961). ll) G. E. Adams, "Advances in Radiation Research," Vol. 3, ed. by M. Burton and J. L. Magee, Wiley- Interscience, New York, 1972, p ) M. Quintiliani, R. Badiello, M. Tamba and G. Gorin, "Modification of Radiosensitivity of Biological Systems" IAEA, Vienna, 1976, p ) M. Suekane, M. Tamura and C. Tomimura, Agric. Biol. Chem., 42, 909 (1978). 14) R. A. Hougi-Angeletti, /. Biol. Chem., 250, 7814 (1975). 15) M. Quintiliani, R. Badiello, M. Tamba, A. Esfandi and G. Gorin, Int. J. Radiat. Biol., 32, 195 (1977). 16) M. L. Morse and R. H. Dahl, Nature, 111, 660 (1978). 17) J. J. Van Hemmen, W. J. A. Meuling and J. F. Bleichrodt, Int. J. Radiat. Biol, 26, 547 (1974).

EFFECT OF IONISING RADIATIONS ON THE HAEMOGLOBIN OF A MARINE BIVALVE SCAPHARCA DEYROLLEI SUB SP. CRISPI PATEL AND PATEL*

J. mar. biol. Ass- India, 1973, 15 (1) : 262 266 EFFECT OF IONISING RADIATIONS ON THE HAEMOGLOBIN OF A MARINE BIVALVE SCAPHARCA DEYROLLEI SUB SP. CRISPI PATEL AND PATEL* S. PATEL AND B. PATEL Health Physics