But. Mug. Tokyo 81: 411-413 (July 25, 1968)

Short Communication

Hideyuki NAGASHIMA*, Saheye NAKAMURA* and Kazutosi NISIZAWA* : Biosynthesis of Floridean by Preparations from a Marine Red Alga Serraticardia maxima* *

長 島 秀 行*・ 中 村 佐 兵 衛*・ 西 沢 一 俊*:紅 藻 オ オ シ コ ロ の ク ロ ロ プ ラ ス ト標 品 に よ る 紅 藻 デ ン プ ン の 生 合 成

Received June 5, 1968

Floridean starch, which has a branched structure similar to or gly- cogen, is one of the reserve substances in most r ad algae. It has been assumed that a starch synthetase (ADP- : a-1, 4- a-4-glucosyltransferase) in higher is localized in or in amylop' asts of reserve organs, mostly bound to starch granules. In , however, there is no clear differentiation between assimi'atory and reserve organs, and the starch granules are generally found outside the chlorop'asts. Moreover, there is no report on the synthetase of floridean starch except for the most recent paper of J. F. Fredrick1'. He has found the activity of this enzyme in the course of study on phosphorylase in a red alga Rhodymenia ~ertusa, but not described its properties in detail. In the present communication, we describe the presence of floridean starch syn- thetase and its intracellular distribution in a calcareous marine red alga, Serraticardia maxima. Fronds of S. maxima were collected at the rocky shores of Shimoda Bay, Shizu- oka Prefecture. The chloroplasts were iso'atcd from the fronds by two different methods. In one method, 300 g of chi' led fronds were homogenized at 0-4 ° with 250 ml of a solution containing 0.5 M sucrose, 0.2 M Tris-HCl buffer of pH 7.0 and a small amount of cysteine in a Waring Blendor and chilled mortC r, and then sqeezed through two layers of cheese cloth. The filtrate was centrifuged at 200 x g for 5 min. and the supernatant containing chloroplasts was further ceni r a_fuged at 3,000 X g for 15 min. The resu'ti_ng precipitate was washed with the buffer contain~.ng sucrose to remove heavy contaminants. The washed chloroplast preparation was named " SUC-chloro- plasts " and cusp: _nded in 1 m'_ of 0.01 M Tris-HCl buffer of pH 7.0 containing 0.0005 M cysteine a-id 0.001 M EDTA. This preparation contained still starch granu' s, but it was used for enzyme source without further treatment. The " SUC-chlrroplasts " were colored green, since pigments such as phycoerythrin and phycocyanin were leaked from the chloroplasts during the preparation. In the other method, a slightly modified procedure of J. H. McClendon2' was em- ployed; 300 g of chilled fronds were homogenized with 250 ml of a solution containing Botanical Institute, Faculty of Science, Tokyo Kyoiku University, Otsuka, Tokyo, Japan.

** Coetribution from the Shirnoda Marine Biological Station , Tokyo Kyoiku University, No. 176. 412 Bot. Mag. Tokyo Vol. 81

100 g of polyethylenglycol X4000, 0.2 M Tris-HC1 buffer of pH 7.0 and a small amount of cysteine. The subsequent process was almost the same as that for the " SUC- chloroplasts ". These chloroplasts were reddish purple and pigments such as phyco- erythrin were not leaked from chloroplasts. They seemed, therefore, to preserve the original structure. It was named " PEG-chloroplasts ", and suspended in the same buffer as used for another chloroplast preparation. This also contained some starch granules. The activity of starch synthetase was expressed in terms of the extent of incor- poration of 14C-glucose from ADP-14C-glucose or UDP-14C-glucose into floridean starch present in the reaction mixture. The results are summarized in Fig. 1. When " PEG- chloroplasts " were used as enzyme source, it was observed that about 6% of 14C- glucose was incorporated from both ADP-14C-glucose and UDP-14C-glucose. On the other hand, when " SUC-chloroplasts " were used, an appreciable amount of 14C-glucose was incorported from UDP-14C-glucose but almost none from ADP-41C-glucose. The formation of new a-1, 4-glucosidic linkage in the floridean starch was verified by the recovery of radioactive maltose after j3-amylolysis of the starch fractions. The extremely low starch synthetase activity in " SUC-chloroplasts " toward ADP- glucose might be due to the possible leakage of starch synthetase itself from chloro-

Fig. 1. Incorporation of 14C-glucose from ADP-14C-glucose and UDP-14C-glucose into floridean starch by different chloroplast preparations. The reaction mixture, which contained 12 m~c moles of ADP-14C-glucose (6,100 cpm) or 13 mp moles of UDP-14C-glucose (12, 700 cpm), 5;u moles of Tris-HC1 buffer of pH 8.4, O.lp mole of EDTA, 0.05 mole of cysteine, O.75~c mole of NaF, 1.35 mg of floridean starch added as primer and chloroplasts suspension in a total volume of 751 was incubated at 37° for 60 min. or 120 min. The reaction was stopped by boiling the mixture for 2 min. The floridean starch was precipitated by the addition of 0.5 ml of 75% methanol containing 1% KC13> and washed five times with the above solution. The starch was then dissolved in 1 ml of water and insoluble materials were removed. Radioactivity of the starch was counted in a liquid scintillation counter. In the control experiment with boiled chloroplasts, radioactivity corresponding less than 0.10 mp mole of nucleotide glucose was found in the starch fraction and this amount has been subtracted in the figure. PEG and SUC show " PEG-chloroplasts " and SUC-chloroplasts ", respectively. July-August, 1968 NAGASHIMA,H. et al. 413 plasts in the course of the preparation, judging from the release of accessory pigments such as phycoerythrin. However, since the " SUC-chloroplasts " showed the activity toward UDP-glucose, although very slightly, the activities for the two nucleotide would be caused by different enzymatic entities4'. In addition, these results suggest that the chloroplast membranes of the present red alga would be different in the structure from those of the common higher plants. Further study on this enzyme is now in progress. The authors are indebted to Dr. J. Preiss for his generous supply- of ADP-14C- glucose and UDP-14C-glucose.

References 1) Fredrick, J. F., Phytochemistry 6 : 1041 3) Ghosh, H. P., and Preiss, J., Biochemistry (1967). 4 : 1354 (1965). 2) McClendon, J. H., Physiol. 29: 448 4) Frydman, R. B., and Cardini, C. F., J. Biol. (1954). Chem. 242: 312 (1967).