Solubilization of the Starch-Granule-Bound Starch Synthase of ... - NCBI

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May 13, 1983 - FRASER D. MACDONALD AND JACK PREISS. Department ofBiochemistry .... 50.2 rotor in a Beckman L5-50 ultracentrifuge. The supernatant.
Plant Physiol. (1983) 73, 175-178 0032-0889/83/73/01 75/04/$00.50/0

Short Communication

Solubilization of the Starch-Granule-Bound Starch Synthase of Normal Maize Kernels' Received for publication May 13, 1983

FRASER D. MACDONALD AND JACK PREISS Department of Biochemistry and Biophysics, University ofCalifornia, Davis, California 95616 ABSTRACr The starch-granule-bound starch synthase from Zca mays has been solubilized with a recovery of between 50 and 84%. Chromatography of the solubilized enzyme on DEAE-Sepharose resolves two fractions of activity which may be distinguished by their response to citrate. Neither solubilized isoenzyme displays any significant activity with UDPglucose.

pany, Rockford, IL; a-amylase (pig pancreas, phenylmethylsulfonyl fluoride treated, 980 units/mg protein) from Worthington,

Freehold, NJ and amyloglucosidase (Aspergillus niger, 14 units/

mg protein) from Boehringer-Mannheim Biochemicals. Acarbose and BAY e 4609, inhibitors of a-amylase and amyloglucosidase (15), were gifts of Drs. E. Truscheit and D. Schmidt of Bayer AG, D-5600 Wuppertal, West Germany. Other reagents were of the highest purity available. Preparation of Starch Granules. Kernels (100 g) were separated into 50-g lots and each aliquot was ground with a pestle and mortar in 50 ml ice-cold Tris-acetate buffer (50 mm, pH 8.0) containing 2.5 mM dithioerythritol and mm EDTA. The homogStarch synthase is the major starch biosynthetic enzyme in enate was filtered through four layers of cheesecloth and the solid plant tissues. A substantial portion of the starch synthase activity residue ground twice more with 25 ml extraction buffer. The in nonphotosynthetic plants cells is particulate, due to its tight material passing through the cheesecloth was combined and association with starch granules (4). This starch-granule-bound centrifuged at 16,000g for 10 min. The yellow layer of storage synthase is the only form of the enzyme known to utilize UDP protein that forms over the pellet of starch granules was scraped glucose as well as the more active substrate ADPglucose (12). In off and discarded with the supernatant before resuspending the maize kernels, there are two forms of the soluble starch synthase, starch granules in extraction buffer (100 ml). The starch granules one of which shows an activity in the absence of exogenous were then washed in extraction buffer five times by centrifugation primer which is strongly stimulated by 0.5 M citrate (1). The so that all traces of soluble starch synthase activity were removed. relationship between the soluble and particulate enzymes and The granules were then washed three times in acetone (-20°C) their possible interaction in the synthesis of starch is not under- and allowed to dry. The dry starch granules were stored at -20°C stood. To gain information on the properties of the granule- in which state enzyme activity was stable for several months. bound enzyme, we have solubilized and partially purified the Between 15 and 18 g of starch granules were recovered by this activity from maize kernels. Previous attempts to solubilize procedure and 42 to 65% of the total starch synthase activity granule-bound starch synthase while retaining enzyme activity resided in these granules. Solubilization and Partial Purification of Starch Synthase. have been unsuccessful (2, 3, 4, 14). Some solubilization of activity was obtained using 50% dimethyl sulfoxide (8). However, Starch granules (7 g) were ground in 500-mg aliquots in a pestle the starch synthase was still associated with the dissolved starch and mortar at 0 to 4°C. Each aliquot was ground no longer than granule. The solubilization of UDPglucose-specific starch syn- 3 min. The ground material was combined and suspended in thase from sweet potato starch granules has been reported (13). 120 ml of ice-cold Tris-acetate buffer (100 mM, pH 7.5) containNo ADPglucose-specific activity was solubilized. However, no ing 100 mm KCI, 2.5 mm dithioerythritol, 10% (v/v) glycerol, data were presented for the recovery of enzyme activity and the and 0.1% BSA. The suspension was stirred at 4°C for 40 min nature of the product formed by 14C transfer from UDPglucose before being centrifuged for 1 h at 100,000g and 4°C in a type was not investigated. We have sought to improve on these 50.2 rotor in a Beckman L5-50 ultracentrifuge. The supernatant procedures by digesting starch granules with a-amylase and was removed and will be referred to as the untreated supernatant. The pellet was resuspended in 120 ml of the suspension buffer amyloglucosidase. and incubated with a-amylase (10 units/ml starch suspension) and amyloglucosidase (5.6 units/ml starch suspension) for 90 MATERIALS AND METHODS min at room temperature with gentle stirring. The digested starch Materials. Dent maize (Zea mays L.), inbred W64A was field suspension was then centrifuged at 100,000g as described above. grown in 1978. Plants were self- or sib-pollinated and ears were The supernatant was removed and will be referred to as the harvested 22 d after pollination, frozen on dry ice, and stored at treated supernatant; the pellet was retained for assay of enzyme -15°C until use. ADP[U-'4C]glucose was prepared enzymically activity. (11) and UDP[U-'4C]glucose was obtained from Amersham/ The untreated and treated supernatant were applied separately Searle. Amylopectin was purchased from Pierce Chemical Com- to DEAE-Sepharose CL-6B columns (1.5 x 23 cm) equilibrated with 50 mm Tris-acetate (pH 8.0), 2.5 mm dithioerythritol, 10 'Supported in part by the National Science Foundation Grants PCM mm EDTA, 10% (v/v) glycerol. The columns were washed with 78-16127 and PCM 82-05705. 150 ml of the above Tris-acetate/glycerol buffer and then eluted 175

176

MACDONALD AND PREISS

with a linear gradient (300 ml) of 0 to 0.6 M KCI in the Trisacetate/glycerol buffer. Fractions (8 ml) were collected at 0.9 ml/ min and those containing starch synthase activity were pooled and concentrated with an Amicon ultrafiltrator (model 52, PM10 membrane) at 0.75 kg cm-2. Assay of Starch Synthase. Assay A (Primed Activity). The transfer of glucose into primer from ADPglucose was measured in reaction mixtures containing 200 nmol ADP('4C]glucose (500 cpm/nmol), 20,umol Bicine (pH 8.5), 5 Mmol potassium acetate, 2 ,mol GSH, 1 ,umol EDTA, 1 mg amylopectin and enzyme in a total volume of 0.2 ml. Reactions were incubated at 37°C for 15 min, and the incorporation of radioactivity into methanolinsoluble polysaccharide was measured as described previously

Plant Physiol. Vol. 73, 1983

as a primer was stimulated 2- to 3-fold by 0.5 M citrate and, in the presence of citrate, the total activity recovered was close to 100% of that measured in the ground granules. Between 70 and 84% of the activity was solubilized by this procedure. Starch synthase activity released by grinding alone and that released by subsequent amylolysis were chromatographed separately on DEAE-Sepharose (Table II). Figure 1 shows the elution profile obtained for the amylase-treated enzyme. A virtually identical profile was obtained for the activity released by grinding alone (profile not shown). Two peaks of activity are seen. The first (fraction one) was strongly stimulated by 0.5 M citrate in the presence of exogenous primer (assay B) and also showed a strong unprimed reaction in the presence of citrate (assay C) (Fig. 1; TableII). The second peak (fraction two) contained activity that (5). Assay B (Primed plus Citrate Activity). The transfer of glucose was only 2-fold stimulated by 0.5 M citrate in the presence of in the presence of exogenous primer and 0.5 M citrate was exogenous primer and only showed a small unprimed activity measured as described in assay A except that potassium acetate with citrate. was replaced with 100,g BSA and 100 ,smol sodium citrate. The major part of the a-amylase activity applied to the DEAEAssay C (Unprimed plus Citrate Activity). The transfer of Sepharose column was recovered in the column washings. The glucose in the absence of exogenous primer was measured as amyloglucosidase activity, however, bound to the column and described in assay A except that potassium acetate and amylo- was eluted in a peak between the two fractions of starch synthase. pectin were replaced with 100 Ag BSA and 100,umol sodium Fraction one and fraction two starch synthase activity was not citrate. Reactions were stopped by heating at 100C for1 min. increased by preincubation with the aforementioned hydrolytic Carrier amylopectin (1 mg) was added and radioactivity meas- enzyme inhibitors or by use of the Dowex resin assay suggesting ured as described (5). that the amyloglucosidase contamination was small. The transfer of glucose into primer from UDPglucose was The products formed by incubation of fraction one and fracmeasured as described above except that ADP['4C]glucose was tion two with ADP['4C]glucose were shown to be starch by replaced by 200 nmol UDP[4CJglucose (500 cpm/nmol) or 4 digestion with hydrolytic enzymes. Digestion with a-amylase released more than 80% of the incorporated '4C as glucose, ,umol UDP['4C]glucose (180 cpm/nmol). The assay of starch synthase in the treated supernatant and maltose, and maltotriose. Digestion with amyloglucosidase refinal pellet was strongly interfered with by the a-amylase and leased more than 85% of the incorporated '4C as glucose. amyloglucosidase. Adequate measurements could be obtained The granule-bound starch synthase showed considerable activby preincubating enzyme samples with Acarbose and BAY e ity with high concentrations (20 mM) of UDPglucose (Table III). 4609 (both at 50 Ag/ml) for 15 min at25°C(15). Starch synthase The activity with UDPglucose decreased on grinding of the activity was then determined using the reaction mixtures for granules in contrast to the increase observed for ADPglucose. assays A and B. After incubation at37°C for 15 min, the reaction This UDPglucose-specific activity was further decreased by sowas stopped by heating for1 min at100C and1 ml (assay A) lubilization of the enzyme and the maximum activity with or 4 ml (assay B) of a slurry containing 200 mg/ml of anion- UDPglucose was less than10% of the ADPglucose-specific activexchange resin (Dowex 1-x-8, Cl- form; 200-400 mesh) was ity for the solubilized isoenzymes. added to absorb the unreacted ADP['4C]glucose. After filtration through Whatman No. 1 filter paper, the radioactivity in 0.5 ml DISCUSSION of the filtrate was determined in a liquid scintillation counter. The increase in ADPglucose starch synthase activity brought Product Analysis, Enzymic Digestion. Glucan labeled with14C was produced by incubating enzyme samples in assay systems A, about by grinding of starch granules is consistent with the obserB, and C with ADP['4C]glucose at 2,015 cpm/nmol. The glucan vations of Frydman and Cardini (4) and was due both to a was coprecipitated with1 mg carrier amylopectin, washed as decrease in theKm for ADPglucose and an increase in the Vmax described (5), and dissolved in 0.25 ml water. Aliquots (60jl) of the reaction (results not shown). The stability of the starch synthase during the solubilization were incubated in a total volume of 100 gl with (a) a-amylase procedure is confirmed by the recovery of close to 100% of the (27 units) in 20 mm phosphate buffer (pH 7.0) plus0.1M KCI; (b) amyloglucosidase (0.7 units) in 20 mm acetate buffer (pH initial activity (ground granules) in the final supematants and 4.0). Incubations were for 24 h, and the products were analyzed pellet. The recovery of between 70 and 84% of the activity in a 100,000g supernatant indicates that the bulk of the granuleby paper chromatography as described (10). bound enzyme was successfully solubilized. The activity released Protein Determination. Protein was assayed by the Lowry method using BSA as a standard or by a modification of the by grinding alone and that released by subsequent amylolysis are Lowry method using RNA coprecipitation for dilute protein seen to be similar both in the relative amounts of fraction one and fraction two activity and in the behavior of these fractions samples (9). M in the presence of 0.5 citrate. These similarities suggest that the amylase treatment does not significantly alter the properties RESULTS of the solubilized starch synthase. Preliminary experiments revealed that maize starch granules The differing responses of fraction one and fraction two activM were very resistant to amylolysis unless first mechanically dis- ities to 0.5 citrate and their different elution behavior from rupted. The most consistently effective method of grinding was DEAE-Sepharose indicate that these fractions may represent two by hand using a pestle and mortar. This grinding increased starch different isoenzymes. Fraction one activity displays some simisynthase activity roughly 3-fold and released a small percentage larity to the starch synthase one obtained from the soluble phase ofthe total activity into a (Table I). Further of maize kernel homogenates. Both of these enzymes show IO00,00gsupernatant solubilization was achieved by digestion of the remaining partic- extensive, citrate-stimulated, unprimed activity (10). The enulate material with a-amylase and amyloglucosidase (Table I). hancement of citrate-stimulated activity by the addition of exThe solubilized activity measured with exogenous amylopectin ogenous primer, however (Table II), contrasts with the roughly

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SOLUBILIZATION OF STARCH-GRANULE-BOUND STARCH SYNTHASE

Table I. Release of Starch Synthasefrom Starch Granules by Grinding and by Subsequent Treatment with a-Amylase and Amyloglucosidase Starch granules were ground, suspended in buffer, and then centrifuged to give an untreated supernatant and pellet. This pellet was resuspended in buffer and treated with a-amylase and amyloglucosidase before a second centrifugation to give the treated supernatant and pellet. ADPglucose starch synthase activity was measured in each fraction in the presence of amylopectin and with and without 0.5 M citrate. Data are the means and SE from three separate experiments. Ground Granules

Starch Synthase Activity Assay

Ground granules

Intact granules

Untreated

Treated

Treated

supernatant

supematant

pellet

Activity Total Solubilirecovery zation %

nmol/min ml Without citrate (assayA) + 0.5 M citrate (assayB)

12.1 ±0.1

31.8 ± 1.7

3.3 ± 1.3

7.2 ±0.4

7.7 ±0.5

33

57

12.1 ±0.1

35.2±3.6

8.3±2.1

18.7± 1.2

8.2± 1.2

77

100

Table II. Separate Purification of Starch Synthase Activity Released by Grinding and by Subsequent Treatment with a-Amylase and Amyloglycosidase + Unprimed . Primed + . Total Citrate Unprmed Citrate Pnmed Volume Protein Fraction

total units mg ml 3482 3564 36.4 120 Ground granules 546 169 115 142' Untreated supernatant Untreated DEAE eluate 177 47 3.1 2.5 Peak I 28 14 0.3 1.3 Peak II 2262 936 192a 130 Treated supematant Treated DEAE eluate 1005 92 160 7.5 Peak I 64 37 0.7 2.0 PeakII ' The large increase in total protein during solubilization is due to the addition of 120 mg BSA.

0 0

172 5

0 3

675 28

E C

1, @S 0

1.0

C 0 il 0

CM

w C.) z

C

0

0 a 0

-0

0.5