Starch usually is composed of a mixture of two polysaccharides: amylose and amylopectin. Amylose is a α-1, 4-linked D-glucopyranose polymer and amylopectin has a number of α-1,4-linked D-glucopyranose chains, joined to other α-1,4-linked D-glucopyranose chains by 5-6 % α-1,6 branch glycosidic linkages [1]. Most starches contain 75-80 % amylopectin with 25-20 % amylose, respectively. There are, however, some starches that contain 100% amylopectin (waxy varieties) and some that are high amylose starches, containing 50-80% amylose and 50-20% amylopectin, respectively. Amylopectins are branched 5-6 %, giving average branch chain lengths of 20-16 D-glucopyranose units, [1] respectively. Using pulse and chase ;;k;kpreactions of hjk;;k'''lighly purified Starch-Synthase (SS) with ADP-[¹⁴C] Glc and nonlabeled ADPGlc, respectively, we recently have shown that starch chains are biosynthesized de novo from the reducing-end, without a primer requirement [2]. The synthesis starts by forming two D-glucopyranosyl covalent intermediates at the active-site of SS; the C-4-OH group of one of the glucopyranosyl intermediates then makes an attack on the C-1-carbon of the other glucopyranosyl intermediate to transfer the glucose and make a α-1,4-glycosidic linkage. The free catalytic group then attacks another ADPGlc unit and forms a D-glucopyranosylintermediate. The synthesis then proceeds processively, going backand- forth between the covalent D-glucopyranosyl-C-4-OH groups and the growing D-glucopyranose-chains. The D-glucopyranosyl units are transferred to the reducing-ends of the growing chains. The synthesis is called a “two catalytic-site insertion mechanism” and results in the processive addition of D-glucopyranose from ADPGlc to the reducingends of growing starch chains [2] (Figure 1) During the pulse reaction, the reducing-ends become labeled with ¹⁴C-D-glucose and that when the pulsed-polysaccharides are isolated, reduced with NaBH4, and acid hydrolyzed, ¹⁴C-D-glucitol is obtained; and in the chase reaction, the ¹⁴C-D-glucose is chased from the reducing-ends into the chain, and in reduction and acid hydrolysis, the ¹⁴C-D-glucitol is decreased, because nonlabeled D-glucose units are being added to the reducingends in the chase. If the synthesis had been to the nonreducing-ends of primers, no D-glucitol would ever have been formed in the pulsed reactions and therefore decreased in the chase reactions. In the present study, the In vitro biosynthesis of starch was performed by using only two highly purified potato starch enzymes: Starch-Synthase (SS) and Starch-Branching-Enzyme (SBE). The amounts of synthesized amylose was approximately the same for each reaction and ten different kinds of starches were obtained by the conversion of different amounts of amylose into amylopectin by the reaction of SBE with the amyloses for different lengths of time or by different amounts of SBE.

Figure 1: Distributions of amylopectin and amylose in the ten in vitro synthesized starches by highly purified potato Starch-Synthase and Starch-Branching-Enzyme.

Materials

Adenosine 5’-diphospho-α-D-glucose (ADPGlc);dithiothreitol (DTT); polyvinyl alcohol 50K; PPO and PoPoP were obtained from Sigma-Aldrich Chem. Co. (St. Louis, MO. USA). ADP-[¹⁴C]Glc (333 mCi/mmol) was obtained from American Radiolabeled Chemicals, Inc. 101 Arc Drive, St. Louis, MO 63147, USA. Amylose was obtained by fractionation of potato starch as given in section, Separation of Amylose from Amylopectin, in the Methods Section [3], or obtained from Sigma-Aldrich Chem. Co. Starch-Synthase (SS) used was Fraction 23 [3] a highly purified enzyme, with a specific activity of 944 mIU/mg (1.0 IU is an International Unit that incorporates 1.0 μmole of D-glucose into starch/min) and was shown to be primer-free [4]. Starch-Branching-Enzyme (SBE) was Fraction 4 [3]. It had a specific activity of 198 mIU/mg, where 1.0 IU equal 1.0 μM of α-1,6 branch linkages formed/min and also was shown to be primer-free, along with the Standard Buffer [2]. The Standard Buffer was pH 8.4 and contained 10 mM glycine, 2 mM EDTA, 0.04% (w/v) polyvinyl alcohol 50K, and 1 mM DTT. Liquid Scintillation Cocktail was prepared, containing 5.0 g PPO and 0.1 g PoPoP in 1 L of toluene (Table 1).

Table 1: Percent distribution of amylopectin and amylose in ten in vitro synthesized starches, obtained by the reaction of potato Starch-Synthase and Starch- Branching-Enzyme in different amounts or for different lengths of time.

Methods

Assay for starch-synthase (SS): The SS assay reagent contained (75 μL) Standard Buffer (pH 8.4) with 20 mM (0.05 μCi) ADP-[¹⁴C] Glc; 25 μL of SS samples were added to the assay reagent and incubated at 37°C for 30 min; 25 μL aliquots were then taken in triplicate and each added to 1.5 cm² Whatman 3MM paper, which is immediately added to 100 mL of MeOH, with stirring for 10 min, to stop the reaction and precipitate the synthesized ¹⁴C-amylose onto the paper, and wash away ADP-[¹⁴C] Glc, and buffer material. The papers are washed 2-more times with 100 mL of MeOH and then dried and counted in 10 mL of toluene scintillation cocktail. A control blank is prepared by adding the Standard Buffer and the ADP-[¹⁴C] Glc substrate in the same volume as the reaction digest, with Standard Buffer substituted for the SS enzyme sample. 1.0 mIU=1.0 nmole of D-glucose incorporated into amylose/ min.

Assay for starch-branching-enzyme (SBE): Starch-Branching- Enzyme (SBE) (360 μL) in Standard Buffer (pH 8.4) is added to 200 μL (3 mg/mL) amylose and incubated at 37°C for 60 min; the reaction was stopp'nl'mk'.ll'l'fed by placing the digest in a boiling water bath for 5 min and then cooled and centrifuged to remove denatured protein. The supernatant was removed and 2-10 μL of potassium triiodide reagent (2 mg iodine and 20 mg potassium iodide) was added to the digest supernatant to the point of just giving a slight blue triiodide color, and oxidize the DTT in the buffer. This was followed by a micro-titration (Hamilton Syringe) with 0.15 mM of silver nitrate, until the blue color just disappeared; the resulting silver iodide was removed by centrifugation. The pH was then made 5.2 by the addition of 10 μL of 40 mM pyridine-acetate buffer, containing 200 units of isoamylase (where 1 unit=1.0 nmole of α-(1→6) bonds hydrolyzed/min), and the isoamylase reaction was allowed to proceed for 30 min at 37°C. Three 100-μL aliquots were added to the wells of a microplate, containing 100 μL of copper bicinchoninate reagent, and the reducing value was determined, using maltose (20-200 μg/mL), as a standard [4]. The number of nmoles of α-(1→6) branched linkages formed/min by SBE was calculated from the reducing value obtained from the isoamylase reaction. A control blank was prepared by adding the Standard Buffer with the substrate, substituting water for the enzyme sample, and then carrying it through the isoamylase reaction, giving the exact reaction conditions used for the samples. One unit of SBE = 1.0 nmol of α-(1→6) branch linkages formed/min.

Separation of Amylose from Amylopectin in the synthesized starches [5]: Potato starch (1.0 g in 100 mL) was added to each of the enzyme digests as a carrier when the reaction was over. The pH was adjusted to 7.0 and stirred for 30 min at 21-22°C; 22 mL of 1-butanol was added and stirred for 24 hr at 22°C to precipitate the amylose/1- butanol complexes and were then centrifuged at 20K rpm for 30 min, washed with 20 mL of water; and the amylose was then suspended in 40 mL of water, and the 1-butanol was removed by heating to boiling with the addition of water to maintain the volume. The amylose was then precipitated by the addition of 3 volumes of ethanol and placed at 4°C for 24 hr. The amylopectin that remained in the supernatant from the precipitation of the amylose by butanol-1 was precipitated by the addition of 3 volumes of ethanol and placed at 4°C for 24 hr. The amylose and amylopectin were centrifuged and their pellets were washed with 40 mL of acetone/water, 80/20 (v/v), and then treated with 100 mL of acetone, and dried under vacuo for 24 hr.

In vitro synthesis of starch by SS reacting with ADP-[¹⁴C] Glc and SBE reacting with the synthesized amylose to give amylopectin

Synthesis type I: Two reactions were performed. Both reactions had 1.05 mL containing 400 mIU of SS. The reactions were initiated by the addition of 30 μL of (25.4 mg, 0.1 μCi) ADP-[¹⁴C]Glc and allowed to go for 135 min to give 1.2 Conversion Periods (CP), where 1.0 CP is the theoretical length of time for the complete reaction of ADP- [¹⁴C] Glc. The two reactions were initiated by the addition of 30 μL of SBE (1.0 mIU) to each of the two amylose reactions. The first reaction was allowed to go for 75 sec and the second reaction for 130 sec. The reactions were stopped by the addition of 0.1 M trifluoroacetic acid (TFA) to give a pH of 2.0, and 1.0 g of carrier potato starch was added to each reaction, and the amyloses and amylopectins were separated as described under separation of Amylose from Amylopectin in the synthesized starches [5]. The amounts (20-100 mg) of synthesized amyloses and amylopectins were obtained by Liquid Scintillation Spectrometry. All of the pipetting for 100 μL and less for Synthesis Type I was done, using Hamilton Syringes to give the highest accuracy.

Synthesis type II: Three samples of amylose were synthesized identical to the amyloses in Type I. The reactions were allowed to go for 135 min, 1.2 Conversion Periods, where 1 CP is the theoretical length of time to give complete reaction of ADP-[¹⁴C] Glc. The digests were then precipitated with 3 volumes of ethanol, which was washed 5-times with 80/20 acetone water, and dissolved in 0.6 mL of water by autoclaving at 121° C, followed by making the volume 1.05 mL by adding Standard pH 8.4 Buffer. The branching reactions were then started by the addition of 30 μL (1.0 mIU) of SBE to the three amylose samples and the reactions were allowed to go 15, 45, and 75 sec, respectively. The reactions were stopped by adding 0.1 M TFA to give pH 2, and 1.0 g of carrier potato starch was added to each reaction, and the amyloses and amylopectins were separated as described under Methods. The amounts (20-100 mg) of synthesized amyloses and amylopectins were obtained by Liquid Scintillation Spectrometry. All of the pipetting for 100 μL and less for Synthesis Type II was done, using Hamilton Syringes to give the highest accuracy.

Synthesis type III: Five amyloses were synthesized as in Type I. Varying amounts (0.5 mIU, 0.3 mIU, 0.2 mIU, 0.1 mIU, and 0.01 mIU) of SBE were added and the reactions were allowed to go for 130 sec. They were stopped by the addition of 0.1 M of TFA to give pH 2, followed by the addition of 1.0 g of carrier potato starch to each reaction, and the amyloses and amylopectins were separated as described under Separation of Amylose from Amylopectin in the synthesized starches [5]. The amounts (20-100 mg) of synthesized amyloses and amylopectins were determined by Liquid Scintillation Spectrometry. All of the pipetting for 100 μL or less for Synthesis Type III was done, using Hamilton Syringes to give the highest accuracy.

Three types of syntheses were performed. All three start with the same amounts of amylose, synthesized by highly purified potato SS to give ¹⁴C-labeled amylose. The results of the different percentages of amylopectin and amylose for the ten syntheses are given in (Table 1). It should be noted that the 75 sec reaction of SBE in the Type I synthesis gave different amounts of amylopectin than did the 75 sec reaction of SBE in Type II synthesis. Type I synthesis gave 53% amylopectin, whereas Type II synthesis only gave 16% amylopectin. In the Type II synthesis, the SS had been completely denatured by precipitating and autoclaving before its addition. The SS in the Type I synthesis was not autoclaved and SS activity remained after SBE had been added and amylose continued to be synthesized during the branching reaction by SBE to give much larger amounts of amylopectin by some synergistic mechanism. In (Table 1), the percent distribution for each synthesized amylopectin and amylose are given. The synthesized starches had (a) “very high amylopectin” (99.9% amylopectin and 0.1% amylose); (b) “normal” amounts of amylopectin (80% amylopectin and 20% amylose) and (70% amylopectin and 30% amylose); (c) intermediate amounts of amylopectin and amylose (57% amylopectin and 43% amylose) and (53% amylopectin and 47% amylose); (d) “high amylose starch” (39% amylopectin and 61% amylose); and (e) “very high amylose starch” (16% amylopectin and 84% amylose) and (10% amylopectin and 90% amylose). These results show that various kinds of starches, containing different amounts of amylopectin and amylose, have been synthesized, In vitro, by only two highly purified enzymes, potato SS and SBE, in which the SBE was allowed to react with the same amounts of amylose for different lengths of time or with different amounts of activity for a constant length of time. Previous studies of starch biosynthesis [6-10] involved various amounts of primers, phosphorylase, and glycogen. During the course of these studies, Hanes found that if he started with α-Glc-1-P and starch, without any phosphate, some glucose units were added to the nonreducing-ends of the starch chains, giving synthesis. This, in essence, was the origin of the requirement for a primer in polysaccharide biosynthesis. It was found shortly thereafter [7], however, that only a few (2-3) glucose units were added to the nonreducing-ends of the starch chains. In plants and animals, it also was found that the concentration ratio of Pi to α-Glc-1-P was 20-40 folds higher in vivo, and phosphorylase was exclusively a degradative enzyme rather than a synthetic enzyme [7-10]. Hanes’ observation [6], however, set the stage for the primer concept for polysaccharide biosynthesis and many investigators made futile searches for the putative primers for polysaccharide biosynthesis. One such study was by Koepsell et al. [11], who added isomaltose and maltose to digests of sucrose and Leuconostoc mesenteroides B-512F dextransucrase, and found that these reactions formed isomaltodextrins and maltosylisomaltodextrins, with maltose at the reducing-ends, respectively. Both also inhibited the biosynthesis of dextran and were therefore not primers. Likewise, Mukerjea and Robyt [12] found that maltose, maltotriose, and maltododecaose (G12) also inhibited starch biosynthesis by maize, wheat, and rice starch granules, very similar to the findings of Koepsell et al. [11] for the biosynthesis of dextran by dextransucrase.In the early 1960s, Leloir et al. [13-15] also found that active Starch-Synthase, was entrapped inside starch granules, and when reacted with adenosine diphospho-¹⁴C-glucose (ADP-[¹⁴C]Glc), ¹⁴C-starch chains were synthesized in the granule. The ¹⁴C-labeled starch was dissolved in buffer and reacted with the exo-acting ß-amylase that hydrolyzes starch from the nonreducing-ends, to give ¹⁴C-labeled maltose. From this result, it has been widely assumed that the starch was being biosynthesized by the addition of ¹⁴C-labeled glucose to the nonreducing-ends of primers. Later, Mukerjea and Robyt, [12] suggested that the Leloir et al. [15] conclusion that the formation of ¹⁴C-maltose by ß-amylase action on their synthesized ¹⁴C-starch, which suggested that the addition of glucose was to the nonreducing-ends of primers, was not necessarily correct: if the starch chains had been synthesized de novo by the addition of D-glucose to the reducing-ends, without the involvement of a primer, the entire starch chains would have been labeled with ¹⁴C-D-glucose and ß-amylase would also have produced ¹⁴C-maltose.In 1948, Wolf et al. [16] assumed that sweet corn starch apparently originated from globules of polysaccharide on the surface of sweet corn. These globules were assumed to be glycogen and, therefore, the starch arose from glycogen. The globules, however, were never shown to be glycogen and could very well have been preamylopectin. Nevertheless, from their observation, it was proposed that glycogen was an intermediate in the biosynthesis of amylopectin and amylose. In 1951 Hobson et al. logically indicated, however, that amylose had to be synthesized first and then part of the amylose converted into amylopectin [17]. However, in 1958 Erlander [18], stated that cumulative evidence indicated that glycogen was the precursor for the formation of the linear amylose chains by the action of isoamylase or debranching-enzyme with the glycogen to give amylose. The released chains, however, that would be released would be quite short, ~10-12 D-glucose units, and did not come close to the number average degree of polymerization (dpn) values obtained for several amyloses: the smallest known was from amylomaize-VII, a high amylose starch, in which the amylose has a dpn of ~400 glucose units; then amylomaize starch, with 20% amylose, has a dpn of ~800 glucose units; potato starch, with 25% amylose, has dpn of ~1,000 glucose units; and wheat starch, with 25% amylose, has a dpn of ~4,000 D-glucose units. In 1990, Baba et al. [19] found that glycogen was the best primer for starch biosynthesis. This observation furthered the hypothesis that starch arose from glycogen. In 1996, Ball et al. [20] proposed that amylopectin was synthesized from glycogen. Glycogen is branched 10- 12%, having an average of 8-10 glucose units per chain. They proposed that glycogen was debranched to give 5-6% branching and the chains were then elongated at the nonreducing-ends by Starch-Synthase to give an average of 20-25 glucose units in amylopectin. In 1998, Ball et al. [21] further hypothesized that the amylose, that was initially released from the glycogen was also elongated by Starch-Synthase addition of several hundred glucose units to the nonreducing-ends to give highmolecular weight amyloses. Ball and Morell [22] proposed that glycogen was a precursor for the formation of starch. There are a number of problems with these hypotheses. One problem is that experimental attempts to elongate linear maltodextrin chains and branched maltodextrin chains from the nonreducing-ends by Starch- Synthase only gives the addition of a few (1-3) glucose units to the nonreducing-ends of linear and branched putative primers [23,24] Mukerjea and Robyt [12] further found, a compounding result for Starch-Synthesis, in which different concentrations of the putative maltodextrin primers (maltose, maltotriose, and maltododecaose) inhibited starch biosynthesis, instead of stimulating it, as would have been expected for primers. Using pulse and chase techniques with ADP-[¹⁴C] Glc, Mukerjea and Robyt [2] recently showed that Starch- Synthase synthesizes linear amylose chains, de novo, by the addition of D-glucose units to the reducing-ends of growing amylose chains and not to the nonreducing-ends of a primer. Further, Robyt et al. [25] showed that the need for a primer in starch biosynthesis had been perpetuated for over 70-years by a primer myth that arose from the use of Tris-type buffers (Tris, Bicine, and Tricine), which they have recently shown to be potent inhibitors for Starch-Synthase [25]. It was then shown that the inhibition can be partially reversed, giving ~10% Starch- Synthase activity for Tris and Bicine, but 0% for Tricine buffers by the addition of the putative primers that actually are activators that release a small percentage of the ADPGlc substrate that is complexed with the Tris-buffers, producing inhibition [25]. Along with the carbohydrate putative primers, a high concentration of a non-carbohydrate, 500 mM Na-citrate, had also been found by Pollock and Preiss to give about the same degree (10%) activation of SS in the presence Tris-type buffers as did 10 mg/mL glycogen [26]. Na-Citrate is not a carbohydrate and its structure could, therefore, not be acting as a primer. Thus, it was shown that these so-called primers (glycogen, starch, maltodextrins, and Na- Citrate) were not primers, but activators that partially reversed the Tris-buffer inhibition [25]. In the present study, the In vitro biosynthesis of ten different kinds of starches, having different percent ratios of amylopectin and amylose, were obtained using only two highly purified potato enzymes, Starch-Synthase and Starch-Branching-Enzyme. The amounts of amylopectin and amylose synthesized are given in Table 1. The ratios range from 99.9% amylopectin and 0.1% amylose to 10% amylopectin and 90% amylose. All ten starches were biosynthesized in this study by first synthesizing identical amounts of amylose by Starch- Synthase and ADP-[¹⁴C] Glc, and then having different amounts or different lengths of time of reaction for Starch-Branching-Enzyme to synthesize the α-1,6-branch linkages with the amylose chains, to give 10 different kinds of ¹⁴C-labeled starches, with different percentages of amylopectin and amylose see Table 1. The amounts of amylose and amylopectin are also given in Figure 1 so that a comparison can be readily made between the ten synthesized starches. It should be noted that the relative activity of Starch-Branching-Enzyme must be considerably less than the activity of Starch-Synthase, as amylopectin is only branched 5-6%, although the amount of amylopectin is usually considerably larger than the amount of amylose; however, the amount of branching enzyme activity must then be considerably less than the amount of starch synthesizing activity to obtain a starch with significant amounts of amylose e.g., 20-25%.Starch-Debranching-Enzyme(s) that supposedly would react with glycogen and amylopectin were not present in the reactions, and the putative primers (glycogen, maltodextrins, or pre-formed starch) were also not present. The earlier hypotheses put forward by Ball et al. [20,21], in which it was proposed that glycogen was a precursor for the biosynthesis of both amylose and amylopectin, by the action of a Debranching-Enzyme, followed by the elongation of the chains of the partially debranched glycogen and the debranched amylose chains by Starch-Synthase to give amylopectin and an extensively elongated debranched amylose chains also by Starch-Synthase shows that the Ball et al. [20,21] hypotheses are impossible and do not occur because (i) Debranching-Enzyme(s) are not involved in the synthesis and have not been shown to be involved in any study and certainly was not involved in the present study, and (ii) it has been shown that Starch-Synthase also does not elongate starch chains from the nonreducing-ends [2], as is required by the Ball et al. hypotheses [21,22]. Starch-Synthase synthesizes linear, α-1,4 linked chains, de novo, processively from the reducing-end, as shown by Mukerjea and Robyt [2]. The present study, thus, shows that glycogen is not involved in starch biosynthesis as was proposed by Ball and Morell [22], and linear α-1,4-glucose chains are not synthesized from the nonreducing-end of a primer, as has been hypothesized by Ball et al. [21] and many others.While the In vitro biosynthesis is not identical to the in vivo biosynthesis that occurs in chloroplasts and amyloplasts, this study closely mimics the in vivo biosynthesis, capable of giving different kinds of starches. An important point here is that if this synthesis, to give ten different kinds of starches, with different ratios of amylopectin and amylose, can occur In vitro, using only two enzymes (highly purified Starch-Synthase and Starch-Branching- Enzyme), it certainly should be able to also occur in vivo in chloroplasts and amyloplasts. The study, further, shows that glycogen, Debranching- Enzyme(s), and amylose elongation from the nonreducing-ends, are not involved in starch biosynthesis, and the synthesis only requires two kinds of enzymes, Starch-Synthase(s) that synthesize amylose chains, de novo, from the reducing-end, and Starch-Branching-Enzyme(s) that synthesize α-1,6-linked branches to give amylopectin from the amylose in different proportions to produce different kinds of starches. What the study does not show is how the Starch-Branching-Enzyme is regulated and controlled in vivo to give the different ratios of amylopectin and amylose. In this study, it was controlled by first synthesizing amylose and then adding small amounts of Branching- Enzyme for varying lengths of time to convert a portion of the amylose into amylopectin. The major synthesis was performed by Starch- Synthase synthesis (400 mIU of SS reacting with 400 nmoles of ADP- [¹⁴C] Glc reaction for 135 min) to synthesize ¹⁴C-amylose; and the amylopectin fraction was then synthesized by the addition of 1.0-0.01 mIU of Starch-Branching-Enzyme for 130 sec to 15 sec with the ¹⁴C-amyloses to give the different percentages of the amylopectin fractions. The action of the Branching-Enzyme is, thus, only a fraction of the Starch-Synthase reaction, as amylopectin is only branched to the extent of 5-6% and, therefore, only a relatively small amount of the branching enzyme activity is required to obtain the varying amounts of the amylopectin fractions, to give the different ratios of amylopectin and amylose (Table 1 for the individual number of SBE units and number of seconds that they reacted to obtain the 10 starches). Schwall et al. [27] published a paper in 2000, in which a very high-amylose, potato starch was obtained by simultaneously inhibiting two SBE, A and B isoforms, to give about 1% of the wild-type activities. This project and result further indicates that it is SBE that controls the amount of amylopectin and not a Debranching-Enzyme action on glycogen, followed by extension of the nonreducing-ends of the resulting debranched glycogen and short amylose chains by Starch-Synthase.