Activity of Particulate Starch Synthetase'

An investigation was made to determine the univalent cation requirements of s.tarch synthetase from a variety of plant species of economic importance. The particulate enzyme from sweet corn was shown to have an absolute requirement for potassium, with the optimum activation occurring at 0.05 M KCI. Rubidium, cesium, and ammonium were 80 as effective as potassium while sodium and lithium were respectively 21 % and 8 % as effective as potassium. The KA for potassium was determined to be 6 mm. In the case of the particulate staroh synthetase from wheat, bush beans, field corn, soybeans, peas, or potatoes, oonsiderable stimulation of enzyme activity was obtained by the addition of potassium *to -the reaction mixture. In these studies, low enzyme activity was observed in th.e absence of added potassium, but the content of endogenous univalent cations in the reactions may be sufficient to account for the activities observed. Anions of various types had no effect on starch synthetase activity. Divalent cations produced slight activation in the presence or absence of potassium. All efforts to show a potassium requirement for glycogen synthetase from rat liver have been nega-tive. A consistent consequence of potas.sium deficiency in plants is the inhibition of starch synthesis and the accuimulation of solu.ble carbohydrates and reducing sugars (4). In higher plants, carbohydrate and reducing sugar utilization might be impaired as a result of the loss of activity of one or more of several enzymes k'nown to require potassium or other univalent cations (4, 12). In the case of starch synthesis, a dual role of potassium has only recently been suggested; . In 1966 Akatsuka and Nelson (1) demonstrated that potassium stimulated the activities of both the embryonic and endospermic derived particulate starch synthetase systems from immature maize seeds. They reported that potassium stimulatedboth systems when adenosine diphosphate glucose (ADPG) was the substrate, but inhibited at the same level when uridine diphosphate glucose (UDPG) was the substrate. EDTA in the presence of potassium stimulated enzyme activity but was inhibitory when 'Technical paper 2683 of the Oregon Agricultural Experiment Station. This research was supported in part by a research grant (AM-08123) from the United States Public Health Service. This research will be included in a thesis to be submitted as partial fulfillment of the requirements for a Ph.D. degree at Oregon State University. 2 Present address: Departmenit of Biology, Southern Oregoln College, Ashland, Oregon 97520. potassium was omitted from the reaction mixture containing the embryonic starch synthetase. Saturation curves for potassium activation were presented although no attempt was made to eliminate potassium from the enzyme components or the reagents. In fact. dipotassium salts of the substrates were utilized in their investigation. Potassium also was shown to have a protective effect against thermal inactivation of the enzymes at 600. In an investigation of the starch synthesizing svstem from Chlorella pyrenoidosa, Preiss and Greenberg (16) showed that the partially purified soluble starch synthetase was stimulated 15 to 30 % by the addition of either KCI, GSH, or bovine plasma albumin. If all 3 of these components were omitted from the reaction mixture, activity was reduced 60 %. Similar results also were obtained in an investigation of the synthesis of bacterial glycogen '(6). These authors did not indicate whether or not potassium was removed from the components of the assay mixture. In a preliminary report in 1968 (15), Nitsos and Evans presented evidence that partially purified particulate starch synthetase from sweet corn was completely dependent upon univalent cations for activity. Potassium was the most effective activator, but rubidium, cesium, and ammonium also were effective. Sodium, lithium,. and tris were ineffective in the assay system. More recently, Murata and Akazawa (13) examined the role of potassium in the starch synthetase 1260 www.plantphysiol.org on January 22, 2018 Published by Downloaded from Copyright © 1969 American Society of Plant Biologists. All rights reserved. NITSOS AND EVANS-UNIVALENT CATIONS AND STARCH SYNTHESIS from sweet potato roots and certain other sources. In their report, a 7-fold stimulation of the activity of the enzyme was observed by the addition of 0.1 M KCl. From kinetic studies, the Michaelis-Menten constant for activation, KA, for KCI was determined to be 13 mm. The enzyme was saturated at 0.05 M KCI, and no effect of KCI on the Km for ADPG could be demonstrated. A protective effect of potassium against thermal inactivation of the enzyme also was shown. Since a univalent cation requirement for the starch synthesizing systems of various sources has been indicated, it seemed that a study of the detailed alkali metal requirements of the starch synthetase systems would lead to a better understanding of the factors controlling carbohydrate metabolism. This report represents an elaboration of a preliminary report (15) of the univalent cation requirements of starch synthetase from a variety of plant species of economic importance. Materials and Methods Preparation of Reagents. Glassware utilized for the preparation and storage of reagents was acid washed and thoroughly rinsed with douibly distilled, deionized water. Reagents were prepared with deionized water. Reagent grade sucrose, EDTA, cysteine, and glycine were recrystallized before use. All other chemicals also were reagent grade and utilized without further purification. The buffers used throughout this investigation were either tris (hydroxymethyl) -aminomethane (tris) or N-tris (hydroxymethyl)methyl glycine (tricine). The pH of the tris solutions were adjusted with HC1, and that of the tricine solutions with tetramethyl-ammonium hydroxide. Potassium concentrations of the various reagents and solutions were determined by flame photometry '(7). Maximum concentration of endogenous potassium in all assays was less than 0.01 mM. The dipotassium and disodium salts of both ADPG and UDPG, and the barium salt of glucose 6-P were purchased from Sigma Che-mical Company (St. Louis, Missouri). The nucleoside diphosphate sugars were converted to their respective tris salts by passing them through a cation exchange column (Amberlite CG-50) which had been equilibrated with 0.01 M tris, pH 8.0. Concentrations of the substrates were determined on the basis of their molar extinction coefficients at pH 8.0 '(E259mu = 15.4 X 103 for ADPG, and iE260mu = 1.0 X 104 for UDPG). The barium salt of glucose 6-P was converted to the tris salt by precipitation of the barium with a slight excess of tris-sulfate. These reagents were stored at -15° until used. Adenosine diphosphate glucose-14C (glucose-14C U.L.) was purchased from New England Nuclear Corporation (Boston, Massachusetts). .The ethanolic solution of ADPQ-'4C was evaporated in the cold under reduced pressure, and the substrate dissolved in 0.5 ml of 0.05 M tricine, pH 8.0. Specific radioactivity of this compound was determined to be 225 mC/mmole. Preparation of Enzymes. Starch granules were prepared from freshly harvested immature seeds of sweet corn (Zea mays L.), peas (Pisum sativum L.), bush beans (Phaseolus vulgaris L.), field corn (Zea mays L.), wheat (Triticiumii aestivum Linn.), soybeans (Glycine max. L.), and from potato tubers (Solanum tuberosum L.) utilizing a modified method of Leloir, de Fekete, and Cardini (10). T'he seeds or potato tubers were macerated in an Omnimixer for 2 min in 4 volumes of cold (40) deionized water. The homogenate was filtered through cheesecloth and centrifuged for 5 min at 10OOg in a refrigerated centrifuge (0-4o). The white pellet was washed 4 times with cold, deionized water and suspended in 4 volumes of redistilled acetone at 150. The starch preparation was collected by filtration on a Buchner funnel at 150, and was washed 4 additional times with 4 volumes of cold acetone. The final enzyme preparation (starch granules) was dried under reduced pressure at 0° and stored at -200 until utilized. Prior to assaying for starch synthetase activity, the granules (240 mg) were washed at least 3 times with 6 ml portions of 0.1 M tricine, pH 8.0. The final pellet was resuspended in 3 ml of 0.05 M tricine, pH 8.0. Glycogen synthetase was obtained from freshly excised rat livers by a modified method of Leloir and Goldemberg (8). Rats were fed 30 % sucrose ad libitum for 12 hr. killed by CO., suffocation, the livers immediately removed and placed on ice. The livers were macerated in a Ten Broeck homogenizer in 3 volumes of cold 0.25 M sucrose containing 0.001 M EDTA. The resulting homogenate was centrifuged at 2000g for 10 min, and the supernatant (crude enzyme) stored at -150 until assayed. In this form, the glvcogen synthetase is reported to be stable for at least 1 month (8). Further purification of the glycogen synthetase was accomplished by centrifugation of 30 ml of the crude preparation at 25,Q00g for 10 min at 0 to 40 and the transparent, yellow pellet (particulate glycogen) was resuspended in 3 ml of 0.05 M tricine, pH 8.5, containing 0.05 M glucose 6-P and 50 mg/ml soluble starch. The suspension was centrifuged at 25,000g and the washing procedure repeated 3 times. The final pellet was resuspended in 3 ml of 0.05 M tricine, pH 8.5, containing 0.05 M glucose 6-P, and assayed immediately. A 300-fold purification of the enzyme resulted from the above procedure. Enzyme Assays.. The reaction mixture for the assay of starch synthetase contained the folloving reagents in a total volume of 0.2 ml: tricine buffer pH 8.0, 10 ,urmoles; ADPG, 1.0 ,umole; 0.05 ml of the enzyme preparation (4 mg granules containing 20 ug protein); and the appropriate cation concen-tration.The reaction was initiated bv the addition 1261 www.plantphysiol.org on January 22, 2018 Published by Downloaded from Copyright © 1969 American Society of Plant Biologists. All rights reserved.