Starch Phosphorylase Inhibitor from Sweet Potato - NCBI

Plant Physiol. (1986) 80, 534-538

0032-0889/86/80/0534/05/$0 1.00/0

Starch Phosphorylase Inhibitor from Sweet Potato' Received for publication July 19, 1985 and in revised form October 21, 1985

TSUNG-CHAIN CHANG2 AND JONG-CHING SU*

Department ofArgicultural Chemistry, National Taiwan University, and Institute of Biological Chemistry, Academia Sinica, I Roosevelt Road Section 4, Taipei 107, Republic of China ABSTRACT A protein, starch phosphorylase inhibitor, was purified from the root of sweet potato (Ipomoea batatas [L.] Lam. cv Tainon 65). It had a molecular weight of 250,000 and could be composed of five identical subunits. The isoelectric point of the inhibitor was 4.63. It was a noncompetitive inhibitor toward the sweet potato enzyme with a K; value of 1.3 x 10-' molar when glucose-l-P was the variable substrate. Because cross-reacting materials of rabbit antiphosphorylase inhibitor of sweet potato were found in three arbitrarily selected plant materials, viz. potato tuber, spinach leaf, and rice grain, the occurrence of this protein seemed universal in higher plants. By an immunofluorescence technique, the inhibitor was located in the amyloplast and cell wall where phosphorylase was also found. This implies that they may interact in vivo, and the inhibitor may play an unknown regulatory role apinst the plant enzyme.

Since its discovery from the extracts of potato and pea in 1940 (8), starch phosphorylase (a-1,4-glucan:orthophosphate a-D-glucosyltransferase, EC 2.4.1.1) has been found in a variety of plants including spinach, banana, sweet potato, algae, etc. (for review, see ref. 5). It is generally regarded as a starch degrading enzyme although the possibility of its playing a synthetic role in some plants is also suggested (21-23). Contrary to the plant enzyme, however, muscle glycogen phosphorylase is assigned a definite role of a-1,4-glucan breakdown in animals and is well known for being under allosteric control. Upon activation through a cascade of successive enzymic reactions, phosphorylase a is formed and catalyzes the conversion of glycogen and phosphate to glucoseI-P. However, for decades, plant biochemists and physiologists have failed to discover a similar regulatory mechanism with respect to plant phosphorylase (5). As a part of our effort to elucidate the biochemical mechanism of rapwid starch accumulation in sweet potato root, starch phosphorylase was purified from it. The total enzyme activity increased about 3-fold after ion exchange chromatography, suggesting the presence of a phosphorylase inhibitor. Indeed, we were able to purify the inhibitor to homogeneity and studied its properties, subcellular localization and presence in plants other than sweet potato. MATERIALS AND METHODS Purification of Starch Phosphorylase Inhibitor. All purification steps were carried out at 0 to 4°C. Approximately 300 g of

sweet potato roots (Ipomoea batatas (L.) Lam. cv Tainon 65) were homogenized in an equal volume (w/v) of 50 mM imidazoleHCI buffer (pH 6.4), containing 2 mM DTT, 0.1 mM EDTA, and 10% (w/v) sucrose (buffer A). The homogenate was centrifuged at 23,000g for 15 min to remove cell debris. Solid (NH4)2SO4 was added to the supematant to 25% saturation (based on 697 g of (NH4)2S04 saturates 1 L of water at 0C). The precipitate which formed was removed by centrifugation. The supernatant was made to 45% (NH4)2S04 and the precipitate collected by centrifugation. The pellet was dissolved in a minimal amount of buffer A, dialyzed against the same buffer, and applied to a column of DEAE-Sephacel (Pharmacia) (2.6 x 30 cm) equilibrated with buffer A. After washing with 400 ml of the same buffer, the column was eluted with a linear gradient of 0 to 0.4 M NaCl in buffer A (total volume, 1 L), with a flow rate of 24 ml/h (8 ml/fraction) and finally eluted by 1 M NaCl. The fractions with the inhibitor activity were pooled and concentrated by ultrafiltration (Amicon, YM1O membrane). The concentrate was filtered through a column of Sephadex G-100 (1.6 x 97 cm) with buffer A as the eluant at a flow rate of 6 ml/h (4 ml/ fraction). The inhibitor emerged at the void volume and was found to be homogeneous at this stage. Protein was determined by the method of Lowry et al. (16) using BSA as the standard. Activity Assay. Phosphorylase inhibitor was assayed in the direction of glucan synthesis in a 0.25 ml reaction mixture containing 1 Mg purified sweet potato phosphorylase, 40 mM Mes-NaOH buffer (pH 5.9), 10 mm glucose-I-P (Sigma, No. G6875), 0.3% (w/v) soluble starch, and 50 to 10 Mul of column effluent. After incubation at 37°C for 3 min, the Pi released was determined (3). One IU3 was defined as the amount of inhibitor which caused a decrease of 1 unit (1 ,umol Pi/min) of sweet potato starch phosphorylase. Polyacrylamide Gel Electrophoresis. All gels used were 0.8 mm thick. For mol wt determination, gradient polyacrylamide (pore-limit) gel was used (17, 24). SDS-PAGE was carried out according to the method of Laemmli (13). After electrophoresis, gels were stained with Coomassie brilliant blue and dried by the sandwich method (12). Preparation and Testing of Antibody. The purified inhibitor (0.3 mg) was emulsified with a complete Freund's adjuvant and injected subcutaneously into several sites on the back of an adult New Zealand white rabbit. The animal was boosted three times at 10 to 14 d intervals in the same manner, except that an incomplete Freund's adjuvant was used and the protein dosage was reduced to 0.15 mg each. Ten d after the final injection, 25 ml of blood were drawn from the ear vein. The y-globulin fraction was isolated by DEAE chromatography as described by Linn et al. (15). Ouchterlony (19) double diffusion test and immunoelectrophoresis were performed on microscope slides coated with 2% agarose in a barbital buffer (40 mm, pH 8.2).

'Supported by the National Science Council Grant NSC-73-0409B080-01.

2 Present address: Food Industry Research and Development Institute, P.O. Box 246, Hsinchu 300, Taiwan, Republic of China.

3Abbreviations: PBS, phosphate buffered saline, 10 mm phosphate buffer, pH 7.2, 140 mm NaCl; FITC, fluorescein isothiocyanate; IU, inhibitor unit; IEF, isoelectric focusing. 534

535

STARCH PHOSPHORYLASE INHIBITOR FROM SWEET POTATO

Immunoelectrophoresis was conducted at 10 V/cm for 2 h with Io the same buffer in the electrode chambers. After electrophoresis, anti-inhibitor y-globulin was put into the central trough. The E slides (either for electrophoresis or double diffusion test) were incubated at4°C for 20 h, then washed overnight in PBS, airdried, and stained with Coomassie brilliant blue. :D Isoelectric Focusing. Analytical IEF was performed according to the method of Hudson and Hay (9). Casting of thin-layer >1 ) 7$ polyacrylamide gel of 0.5 mm thickness containing1% Ampholine (LKB, pH range 3.5-10, or 4-6), and all subsequent steps 0 Dafter IEF, namely fixing, staining, and preservation, were referred pamphlet 1818-P ( 1l). 'LKB the to H-Type Amino Acid Analysis. The purified inhibitor in buffer A was dialyzed against an acetate buffer to remove sucrose, )1 lyophilized, and hydrolyzed in an evacuated tube with 6 N HCi Fraction no. at 108 to 1 10°C for 24 h. The hydrolysate was analyzed by a Shimadzu HPLC-amino acid analyzing system (10). Fluorescence Immunohistochemistry. Antibodies and preimmune serum were diluted with PBS containing 1% BSA. For . .1.2 III indirect immunofluorescence observations, paraffin sections (8 B ) ,gm) on slides were first incubated with rabbit1 antiphosphorylase CIL inhibitor y-globulin (0.3 mg/ml) at 37°C for h. The slides were a rinsed in PBS and then incubated with FITC-conjugated goat 0-*C 16 0 anti-rabbit IgG diluted 1:30 (Cappel Lab., Malvern, PA, No. b 2' 1 1212-0081) at room temperature for 30 min. Control experio ments were performed in the same manner, except that preimA 8' 0~~~~~~~~~~~~~~~ mune rabbit serum (1 mg/ml) was used as the primary antibody. 30 After extensive washing with PBS to remove nonspecific fluores20 soA2 10 40 cu 4' cence, the sections were mounted in glycerol/PBS (9:1) and 0 observed with an Olympus BH-2 fluorescence microscope. Pho- aco 50 40 30 20 10 0 tomicrographs were recorded on Kodak Tri-X film rated at ASA Fraction no. 400. Crude Extracts from Other Plants. Spinach (Spinacia oleracea FIG. 1. Chromatographic elution patterns of sweet potato root phosL.), potato (Solanum tuberosum L.), and rice (Oryza sativa L.) phorylase inhibitor. chromatogram of the 25 to 45% were purchased form the local market (varieties unspecified). (NH4)2SO4 precipitateA, ofDEAE-Sephacel crude extract. The linear NaCl gradient eluted About 25 g of each sample were homogenized with 25 ml of the main activity at about 0.15 M. The arrow indicates where phosphobuffer A and centrifuged (23,000g, 15 min), and the supernatant rylase emerged. B, Sephadex G-100 chromatogram. The first peak which was brought to 65% saturation with solid (NH4)2SO4. The pre- emerged at the void volume contained the inhibitor activity. A at 280 cipitate was collected by centrifugation, dissolved in 1 ml of nm (0); phosphorylase inhibitor activity (0); NaCI gradient (--). buffer A, and dialyzed against the same buffer. The dialyzed solutions were used directly for the detection of cross-reacting Molecular Weight and Subunit Molecular Weight. The mol materials of rabbit antiphosphorylase inhibitor of sweet potato wt of the inhibitor was estimated as 250,000 by gradient PAGE test. diffusion by double of 245,000 was obtained when a gel filtration (Fig. 2A). A value x 82 column (1.6 cm) of Sepharose CL-6B calibrated against RESULTS AND DISCUSSION thyroglobulin (669K), ferritin (440K), catalase (232K), and alPurification of Phosphorylase Inhibitor. Through steps of dolase (158K) was used (Fig. 4). By relating the elution volume (NH4)2SO4 fractionation (25-45% saturation), DEAE-Sephacel to those of standard proteins, the inhibitor was estimated to have ion exchange chromatography and Sephadex G-100 gel filtration a Stoke's radius of 52.5 A. SDS-PAGE (Fig. 2b) showed that the (Fig. 1), phosphorylase inhibitor was purified to homogeneity. In inhibitor was composed of only one kind of subunit with a mol the DEAE-Sephacel step, phosphorylase was eluted at 0.3 M NaCl wt of 51,000. Thus the native inhibitor appeared to be penta(arrow, Fig. la). The enzyme was purified to homogeneity by meric. Isoelectric Point. The value estimated by isoelecric focusing in filtration through Sepharose CL-6B and rechromatography on DEAE-Sephacel. The inhibitor emerged at the void volume of thin-layer polyacrylamide gel was either 4.62 (pH range 3.5-10) the gel filtration step, indicating that a molecular sieve with a or 4.63 (pH range 4-6) (Fig. 5). Inhibition Pattern. The double reciprocal transformation of pore size larger than that of Sephadex G- 100, such as Sepharose CL-6B, would be a better choice. The purification procedure was the initial velocity data (Fig. 6) obtained in the direction of simple and about 5 mg of inhibitor were obtained from 300 g of glucan synthesis with glucose- 1-P as the variable substrate resweet potato roots with a recovery of 66% and a purification of vealed that the proteinaceous effector was a typical noncom140-fold (Table I). Single bands were obtained on gradient and petitive inhibitor against the sweet potato starch phosphorylase; SDS-PAGE (Fig. 2). No spurs or additional arcs were noted by in the presence of the inhibitor, the Vmax decreased while the either immunodiffusion or immunoelectrophoresis (Fig. 3). apparent Km for glucose- 1-P (0.6 mM) remained unchanged. The These observations and the single band obtained on IEF (Fig. 5) dissociation constant of the enzyme-inhibitor complex (Ki) was suggest that the inhibitor has been purified to homogeneity. estimated to be 1.3 x 10- M in the presence of 0.3% soluble The purified inhibitor completely lost its activity when heated starch (pH 5.9, The inhibitor appears to be more potent at 80°C for 30 s, or digested with papain or chymotrypsin; the than several small molecules (such as ATP, UDP-glc and ADPlatter treatment yielded peptide fragments as revealed by SDS- glc) which are competitive inhibitors of starch phosphorylase PAGE. with K, values in the order of 10-' M (3 and TC Chang, JC Su, .-

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37°C).

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Table I. Purification of Phosphorylase Inhibitorfrom Sweet Potato Root The results were based on 300 g of sweet potato. See "Materials and Methods" for details. Purification Protein Total Activity Specific Activity Purification

Recovery

Steps 1. 25-45% (NH4)2SO4 2. DEAE-Sephacel 3. SephadexG-100

AI

2 3

B

mg 952 31.7 4.5

IU/mg 1.6 42.4 225.5

IU 1536 1343.8 1014.9

1 23

-fold (1) 26.3 140.1

% (100) 87.5 66.1

A B

E

Aj|C D

B IM.

B FIG. 3. Ouchterlony double diffusion test (A) and immunoelectrophoresis (B) of phosphorylase inhibitor. A, The central well (A) contained 3 Mg of purified inhibitor and the surrounding wells 0.1 mg (2 td) of preimmune rabbit serum (B), 20 gg (C and D) or 10 jig (E) of y-globulin fraction of anti-sweet potato phosphorylase inhibitor serum. B, Wells A and B contained 4.5 Mg of phosphorylase inhibitor each. After electrophoresis, antiphosphorylase inhibitor y-globulin (2.5 mg/ml) was added to the central trough.

FIG. 2. PAGE of purified phosphorylase inhibitor in the absence (A) or presence (B) of SDS. A, Gradient (5-20%) PAGE. Lane 1, mol wt standards (3 Mg each) from top to bottom are: thyroglobulin (669K), ferritin (440K), catalase (232K), lactate dehydrogenase (140K), and BSA (67K). Lanes 2 and 3, 6, and 3 Mg phosphorylase inhibitor, respectively. B, SDS-PAGE performed in a 7.5% gel. Lane 1, mol wt standards (3.5 Ag each) from top to bottom are: phosphorylase b (94K), BSA (67K), carbonic anhydrase (30K), trypsin inhibitor (20.1 K), and a-lactalbumin (I 4.4K). Lanes 2 and 3, 6, and 3 Mtg phosphorylase inhibitor respectively.

unpublished data). The effect of the inhibitor on the rabbit muscle phosphorylase a (Sigma, No. P-1 261) was also tested, but no inhibition was found. This result could be expected. The sequence of the Nterminal 104 residues of potato phosphorylase was determined by Nakano et al. (18). A comparison of this sequence with the complete sequence of the rabbit muscle enzyme (25) shows that the N-terminal 33-residue regions are completely different from each other. As this region is responsible for both allosteric and covalent controls in the rabbit enzyme (4), the dissimilarity may explain the difference in regulatory properties between the two enzymes (6). Amino Acid Composition. The H-type amino acid composition of the inhibitor is shown in Table II. Fluorescence Immunohistochemistry. The fleshy part of sweet

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0.6 Kave FIG. 4. Mol wt determination of phosphorylase inhibitor by Sepharose CL-6B gel filtration. Standard proteins are: 1, thyroglobulin (669K); 2, ferritin (440K); 3, catalase (232K); 4, aldolase (158K). Standard proteins (0); phosphorylase inhibitor (-). 0.4

STARCH PHOSPHORYLASE INHIBITOR FROM SWEET POTATO

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Table II. H-Type Amino Acid Composition of Phosphorylase Inhibitor Results are given as residues/subunit (±SE), assuming a subunit mol wt of 5 1,000. The last column shows the residue percentage. Residue Amount Percent Asp 59.9 ± 1.4 14.49 Thr 18.0±0.1 4.35 Ser 24.0±0.1 5.81 Glu 42.1 ±0.2 10.18 Pro 26.7 ± 0.3 6.46 39.9 ± 0.3 Gly 9.65 Ala 38.0 ± 0.0 9.19 Val 22.8 ± 0.2 5.52 Met 12.5 ± 0.2 3.02 Ile 16.0 ± 0.1 3.87 Leu 31.9±0.0 7.72 Tyr 17.1 ±0.3 4.14 Phe 18.4 ± 0.2 4.45 His 7.0 ± 0.1 1.69 Lys 22.0 ± 1.3 5.32 17.1 ±0.2 Arg 4.14

FIG. 5. IEF of phosphorylase inhibitor in 0.5 mm thick polyacrylamide gel layers. Lane 1, 6 gg inhibitor, pH range 4 to 6. Lane 2, 3.6 ug inhibitor, pH range 3.5 to 10.

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0.4

537

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1.2

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FIG. 6. Double-reciprocal plot of inhibition kinetic data. Each assay contained I gcg of sweet potato phosphorylase. In the inhibited reaction, 0.047,ug of the inhibitor was used per assay. No inhibitor (-) or inhibitor included (-).

potato is derived from the proto- and metaxylem during root development. Parenchymal cells ofthis part contain a lot of large amyloplasts full of starch. Tissue sections of this part specifically bound the antibody at the surface as well as the inside of starch granules, as revealed by fluorescence (Fig. 7A). In addition, the cell wall also highly fluoresced (Fig. 7A). When the sections of a very young root in size no more than 1 cm in diameter were tested, distinct fluorescence was also shown by the same subcellular sites (figures not shown). In control experiments, which were performed with preimmune rabbit serum as the primary antibody, the fluorescence of amyloplast and cell wall could hardly be detected (Fig. 7B). Schneider et al. (21) located starch phosphorylase of potato tuber in the amyloplast, and now in addition to this locus, we found the enzyme in the cell wall of root parenchymal cell of sweet potato (TC Chang et al., unpublished data). These results indicate that both phosphorylase and its inhibitor are present in the same subcellular loci from a very early stage of root development and thus their interaction in vivo seems unavoidable. Presence of the Inhibitor in Other Plants. Figure 8 shows that cross-reacting materials of rabbit anti-sweet potato phosphorylase inhibitor were present in three arbitrarily selected plant materials, viz. potato tuber, spinach leaf, and rice grain. The precipitin lines formed with the three plant extracts completely fused, while spurs toward wells containing the crude extracts (wells B and C) were found; this means that the sweet potato inhibitor has additional unique antigenic determinants detected by the antiserum. In addition, two precipitin lines were observed for each of these samples, indicating that there could be two forms of phosphorylase inhibitors in these plant tissues with one dominant over the other. Providing the diverse occurrence of phosphorylase inhibitor in different species (mono- and di-cotyledons) and organs (root, tuber, leaf and grain), it may be reasonable to conclude that this inhibitor is of universal occurrence in higher plants. Although starch phosphorylase has been purified from a variety of sources (1, 2, 14, 20, 21), no report of its inhibitor of protein nature has appeared. Why it has escaped detection for so long is an intriguing question. Hammond and Preiss (7) reported an ATP-stimulated inactivating factor(s) of starch phosphorylase. However, the inactivation appeared to be due to proteinase action. The discovery of starch phosphorylase inhibitor implies the presence of a new type of regulatory mechanism of the enzyme in plants, which may be completely different from that of the glycogen phosphorylase system.

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FIG. 7. Immunofluorescence micrographs of the tissue sections of sweet potato root. A, Paraffin sections were first treated with rabbit antiphosphorylase inhibitor ay-globulin, followed by FITC-conjugated goat anti-rabbit IgG. Starch granules (G) and cell walls (arrow head) highly fluoresced (x200). B, Control experiment. Sections were processed as in (a) except that preimmune rabbit serum was used as the primary antibody (x200). 9. HUDSON L, FC HAY 1980 Practical Immunology, Ed 2. Blackwell Scientific

B

FIG. 8. Presence of inhibitor in other plants. The central wells contained 20 jsg of y-globulin fraction of antiphosphorylase inhibitor serum (A) or 0.1 mg of preimmune rabbit serum protein (H), and the surrounding wells contained 3 gg phosphorylase inhibitor (B), 2 Ml each of extracts from potato tuber (C and F), rice grain (D and G), and spinach leaf (E). Acknowledgments-We thank Dr. H. J. Su for his assistance in immunofluoresMiss Y. D. Chang for the preparation of paraffin sections.

cence techniques and

LITERATURE CITED 1. ARIKI M. T FUIKU 1975 a-Glucan phosphorylase from sweet potato: isolation and properties of the partially degraded enzyme. Biochim Biophys Acta 386: 301-308 2. BURR B. OE NELSON 1975 Maize a-glucan phosphorylase. Eur J Biochem 56: 539-546 3. FISKE CH, Y SUBBAROW 1925 The colorimetric determination of phosphorus. J Biol Chem 66: 375-400 4. FLETTERICK RJ, NB MADSEN 1980 The structures and related functions of phosphorylase a. Annu Rev Biochem 49: 31-61 5. FUKUi T 1983 Plant phosphorylases: structure and function. In T Akazawa, T Asahi, H Imaseki, eds, The New Frontiers in Plant Biochemistry. Japan Scientific Societies Press, Tokyo, pp 71-82 6. FUKUi T, S SHIMOMURA, K NAKANO 1982 Potato and rabbit muscle phosphorylases: comparative studies on the structure, function and regulation of regulatory and nonregulatory enzymes. Mol Cell Biochem 42: 129-144 7. HAMMOND JBW, J PREISS 1983 ATP-dependent proteolytic activity from spinach leaves. Plant Physiol 73: 902-905 8. HANES CS 1940 The breakdown and synthesis of starch by an enzyme system from pea seeds. Proc R Soc Lond B Biol 128: 421-450

Publications. London, pp 184-187 10. Instruction for High-Performance Analytical Electrofocusing in 0.5 MM ThinLayer Polyacrylamide Gels, 1818-P. LKB, Bromma, Sweden 11. ISHIDA Y, T FUJITA, K ASAI 1981 New detection and separation method for amino acid by high performance liquid chromatography. J Chromatogr 204: 143-148 12. JUANG RH, YD CHANG, HY SUNG, JC SU 1984 Oven-drying method for polyacrylamide gel slab packed in cellophane sandwich. Anal Biochem 141: 348-350 13. LAEMMLI UK 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685 14. LEE EYC, JJ BRAUN 1973 Sweet corn phosphorylase: purification and properties. Arch Biochem Biophys 156: 276-286 15. LINN TG, AL GREENLEAF, RG SHORENSTEIN, R LosiCK 1973 Loss ofthe sigma activity of RNA polymerase of Bacillus subtilis during sporulation. Proc Natl Acad Sci USA 70: 1865-1869 16. LOWRY OH, NJ ROSEBROUGH, AL FARR, Rl RANDALL 1951 Protein measurement with the Folin phenol reagent. J Biol Chem 193: 265-275 17. MARGOLIS J, KG KENDRICK 1968 Polyacrylamide gel electrophoresis in a continuous molecular sieve gradient. Anal Biochem 25: 347-362 18. NAKANO K, T FUKUI, H MATSUBARA 1980 Structural basis for the difference of the regulatory properties between potato and rabbit muscle phosphorylases. The NHrterminal sequence of the potato enzyme. J Biol Chem 225: 9255-9261 19. OUCHTERLONY 0 1949 Antigen antibody reactions in gels. Acta Pathol Microbiol Scand 26: 507-515 20. PREISS J, TW OKITA, E GREENBERG 1980 Characterization of the spinach leaf phosphorylases. Plant Physiol 66: 864-869 21. SCHNEIDER EM, JU BECKER, D VOLKMANN 1981 Biochemical properties of potato phosphorylase change with its intracellular localization as revealed by immunological methods. Planta 151: 124-134 22. SIVAK MN, JS TANDECARZ, CE CARDINI 1981 Studies on potato tuber phosphorylase-catalyzed reaction in the absence of an exogenous acceptor. I. Characterization and properties of the enzyme. Arch Biochem Biophys 212: 525-536 23. SLABNIK E, RB FRYDMAN 1970 A phosphorylase involved in starch biosynthesis. Biochem Biophys Res Commun 38: 709-714 24. SLATER GG 1969 Stable pattern formation and determination of molecular size by pore-limit electrophoresis. Anal Chem 41: 1039-1041 25. TITANi K, A KOIDE, J HERMANN, LH ERICSSON, S KUMAR, RD WADE, KA WALSH, H NEURATH, EH FISCHER 1977 Complete amino acid sequence of rabbit muscle glycogen phosphorylase. Proc Natl Acad Sci USA 74: 47624766