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 noncom- petitive 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-glu- cosyltransferase, 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 glucose- I-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 phos- phorylase was purified from it. The total enzyme activity in- creased about 3-fold after ion exchange chromatography, sug- gesting 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 purifica- tion steps were carried out at 0 to 4°C. Approximately 300 g of 'Supported by the National Science Council Grant NSC-73-0409- B080-01. 2 Present address: Food Industry Research and Development Institute, P.O. Box 246, Hsinchu 300, Taiwan, Republic of China. sweet potato roots (Ipomoea batatas (L.) Lam. cv Tainon 65) were homogenized in an equal volume (w/v) of 50 mM imidazole- HCI 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) equili- brated 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. G- 6875), 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). 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 Downloaded from https://academic.oup.com/plphys/article/80/2/534/6083793 by guest on 06 October 2021
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 ofBiological Chemistry,Academia Sinica, I Roosevelt Road Section 4, Taipei 107, Republic ofChina
ABSTRACT
A protein, starch phosphorylase inhibitor, was purified from the rootof sweet potato (Ipomoea batatas [L.] Lam. cv Tainon 65). It had amolecular weight of 250,000 and could be composed of five identicalsubunits. The isoelectric point of the inhibitor was 4.63. It was a noncom-petitive inhibitor toward the sweet potato enzyme with a K; value of 1.3x 10-' molar when glucose-l-P was the variable substrate. Becausecross-reacting materials of rabbit antiphosphorylase inhibitor of sweetpotato were found in three arbitrarily selected plant materials, viz. potatotuber, spinach leaf, and rice grain, the occurrence of this protein seemeduniversal in higher plants. By an immunofluorescence technique, theinhibitor was located in the amyloplast and cell wall where phosphorylasewas also found. This implies that they may interact in vivo, and theinhibitor 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-glu-cosyltransferase, EC 2.4.1.1) has been found in a variety ofplantsincluding spinach, banana, sweet potato, algae, etc. (for review,see ref. 5). It is generally regarded as a starch degrading enzymealthough the possibility of its playing a synthetic role in someplants is also suggested (21-23). Contrary to the plant enzyme,however, muscle glycogen phosphorylase is assigned a definiterole of a-1,4-glucan breakdown in animals and is well known forbeing under allosteric control. Upon activation through a cascadeof successive enzymic reactions, phosphorylase a is formed andcatalyzes the conversion of glycogen and phosphate to glucose-I-P. However, for decades, plant biochemists and physiologistshave failed to discover a similar regulatory mechanism withrespect to plant phosphorylase (5).As a part ofour effort to elucidate the biochemical mechanism
of rapwid starch accumulation in sweet potato root, starch phos-phorylase was purified from it. The total enzyme activity in-creased about 3-fold after ion exchange chromatography, sug-gesting the presence of a phosphorylase inhibitor. Indeed, wewere able to purify the inhibitor to homogeneity and studied itsproperties, subcellular localization and presence in plants otherthan sweet potato.
MATERIALS AND METHODS
Purification of Starch Phosphorylase Inhibitor. All purifica-tion steps were carried out at 0 to 4°C. Approximately 300 g of
'Supported by the National Science Council Grant NSC-73-0409-B080-01.
2 Present address: Food Industry Research and Development Institute,P.O. Box 246, Hsinchu 300, Taiwan, Republic of China.
sweet potato roots (Ipomoea batatas (L.) Lam. cv Tainon 65)were homogenized in an equal volume (w/v) of50 mM imidazole-HCI buffer (pH 6.4), containing 2 mM DTT, 0.1 mM EDTA, and10% (w/v) sucrose (buffer A). The homogenate was centrifugedat 23,000g for 15 min to remove cell debris. Solid (NH4)2SO4was added to the supematant to 25% saturation (based on 697 gof (NH4)2S04 saturates 1 L of water at 0C). The precipitatewhich formed was removed by centrifugation. The supernatantwas made to 45% (NH4)2S04 and the precipitate collected bycentrifugation. The pellet was dissolved in a minimal amount ofbuffer A, dialyzed against the same buffer, and applied to acolumn of DEAE-Sephacel (Pharmacia) (2.6 x 30 cm) equili-brated with buffer A. After washing with 400 ml of the samebuffer, the column was eluted with a linear gradient of 0 to 0.4M NaCl in buffer A (total volume, 1 L), with a flow rate of 24ml/h (8 ml/fraction) and finally eluted by 1 M NaCl. Thefractions with the inhibitor activity were pooled and concentratedby ultrafiltration (Amicon, YM1O membrane). The concentratewas 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 wasfound to be homogeneous at this stage. Protein was determinedby the method of Lowry et al. (16) using BSA as the standard.
Activity Assay. Phosphorylase inhibitor was assayed in thedirection of glucan synthesis in a 0.25 ml reaction mixturecontaining 1 Mg purified sweet potato phosphorylase, 40 mMMes-NaOH buffer (pH 5.9), 10 mm glucose-I-P (Sigma, No. G-6875), 0.3% (w/v) soluble starch, and 50 to 10 Mul of columneffluent. After incubation at 37°C for 3 min, the Pi released wasdetermined (3). One IU3 was defined as the amount of inhibitorwhich caused a decrease of 1 unit (1 ,umol Pi/min) of sweetpotato starch phosphorylase.
Polyacrylamide Gel Electrophoresis. All gels used were 0.8mm thick. For mol wt determination, gradient polyacrylamide(pore-limit) gel was used (17, 24). SDS-PAGE was carried outaccording to the method of Laemmli (13). After electrophoresis,gels were stained with Coomassie brilliant blue and dried by thesandwich method (12).
Preparation and Testing of Antibody. The purified inhibitor(0.3 mg) was emulsified with a complete Freund's adjuvant andinjected subcutaneously into several sites on the back ofan adultNew Zealand white rabbit. The animal was boosted three timesat 10 to 14 d intervals in the same manner, except that anincomplete Freund's adjuvant was used and the protein dosagewas reduced to 0.15 mg each. Ten d after the final injection, 25ml of blood were drawn from the ear vein. The y-globulinfraction was isolated by DEAE chromatography as described byLinn et al. (15). Ouchterlony (19) double diffusion test andimmunoelectrophoresis were performed on microscope slidescoated with 2% agarose in a barbital buffer (40 mm, pH 8.2).
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STARCH PHOSPHORYLASE INHIBITOR FROM SWEET POTATO
Immunoelectrophoresis was conducted at 10 V/cm for 2 h withthe same buffer in the electrode chambers. After electrophoresis,anti-inhibitor y-globulin was put into the central trough. Theslides (either for electrophoresis or double diffusion test) wereincubated at4°C for 20 h, then washed overnight in PBS, air-dried, and stained with Coomassie brilliant blue.
Isoelectric Focusing. Analytical IEF was performed accordingto the method of Hudson and Hay (9). Casting of thin-layerpolyacrylamide gel of 0.5 mm thickness containing1% Ampho-line (LKB, pH range 3.5-10, or 4-6), and all subsequent stepsafter IEF, namely fixing, staining, and preservation, were referredto the'LKB pamphlet 1818-P (1l).H-Type Amino Acid Analysis. The purified inhibitor in buffer
A was dialyzed against an acetate buffer to remove sucrose,lyophilized, and hydrolyzed in an evacuated tube with 6 N HCiat 108 to 1 10°C for 24 h. The hydrolysate was analyzed by aShimadzu HPLC-amino acid analyzing system (10).
Fluorescence Immunohistochemistry. Antibodies and preim-mune serum were diluted with PBS containing 1% BSA. Forindirect immunofluorescence observations, paraffin sections (8,gm) on slides were first incubated with rabbit antiphosphorylaseinhibitor y-globulin (0.3 mg/ml) at 37°C for 1 h. The slides wererinsed in PBS and then incubated with FITC-conjugated goatanti-rabbit IgG diluted 1:30 (Cappel Lab., Malvern, PA, No.1212-0081) at room temperature for 30 min. Control experi-ments were performed in the same manner, except that preim-mune rabbit serum (1 mg/ml) was used as the primary antibody.After extensive washing with PBS to remove nonspecific fluores-cence, the sections were mounted in glycerol/PBS (9:1) andobserved with an Olympus BH-2 fluorescence microscope. Pho-tomicrographs were recorded on Kodak Tri-X film rated at ASA400.Crude Extracts from Other Plants. Spinach (Spinacia oleracea
L.), potato (Solanum tuberosum L.), and rice (Oryza sativa L.)were purchased form the local market (varieties unspecified).About 25 g of each sample were homogenized with 25 ml ofbuffer A and centrifuged (23,000g, 15 min), and the supernatantwas brought to 65% saturation with solid (NH4)2SO4. The pre-cipitate was collected by centrifugation, dissolved in 1 ml ofbuffer A, and dialyzed against the same buffer. The dialyzedsolutions were used directly for the detection of cross-reactingmaterials of rabbit antiphosphorylase inhibitor of sweet potatoby double diffusion test.
RESULTS AND DISCUSSION
Purification of Phosphorylase Inhibitor. Through steps of(NH4)2SO4 fractionation (25-45% saturation), DEAE-Sephacelion exchange chromatography and Sephadex G-100 gel filtration(Fig. 1), phosphorylase inhibitor was purified to homogeneity. Inthe DEAE-Sephacel step, phosphorylase was eluted at 0.3 M NaCl(arrow, Fig. la). The enzyme was purified to homogeneity byfiltration through Sepharose CL-6B and rechromatography onDEAE-Sephacel. The inhibitor emerged at the void volume ofthe gel filtration step, indicating that a molecular sieve with apore size larger than that of Sephadex G-100, such as SepharoseCL-6B, would be a better choice. The purification procedure wassimple and about 5 mg of inhibitor were obtained from 300 g ofsweet potato roots with a recovery of 66% and a purification of140-fold (Table I). Single bands were obtained on gradient andSDS-PAGE (Fig. 2). No spurs or additional arcs were noted byeither immunodiffusion or immunoelectrophoresis (Fig. 3).These observations and the single band obtained on IEF (Fig. 5)suggest that the inhibitor has been purified to homogeneity.The purified inhibitor completely lost its activity when heated
at 80°C for 30 s, or digested with papain or chymotrypsin; thelatter treatment yielded peptide fragments as revealed by SDS-PAGE.
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FIG. 1. Chromatographic elution patterns of sweet potato root phos-phorylase inhibitor. A, DEAE-Sephacel chromatogram of the 25 to 45%(NH4)2SO4 precipitate of crude extract. The linear NaCl gradient elutedthe main activity at about 0.15 M. The arrow indicates where phospho-rylase emerged. B, Sephadex G-100 chromatogram. The first peak whichemerged at the void volume contained the inhibitor activity. A at 280nm (0); phosphorylase inhibitor activity (0); NaCI gradient(--).
Molecular Weight and Subunit Molecular Weight. The molwt of the inhibitor was estimated as 250,000 by gradient PAGE(Fig. 2A). A value of 245,000 was obtained when a gel filtrationcolumn (1.6 x 82 cm) of Sepharose CL-6B calibrated againstthyroglobulin (669K), ferritin (440K), catalase (232K), and al-dolase (158K) was used (Fig. 4). By relating the elution volumeto those of standard proteins, the inhibitor was estimated to havea Stoke's radius of 52.5 A. SDS-PAGE (Fig. 2b) showed that theinhibitor was composed of only one kind of subunit with a molwt of 51,000. Thus the native inhibitor appeared to be penta-meric.
Isoelectric Point. The value estimated by isoelecric focusing inthin-layer polyacrylamide gel was either 4.62 (pH range 3.5-10)or 4.63 (pH range 4-6) (Fig. 5).
Inhibition Pattern. The double reciprocal transformation ofthe initial velocity data (Fig. 6) obtained in the direction ofglucan synthesis with glucose- 1-P as the variable substrate re-vealed that the proteinaceous effector was a typical noncom-petitive inhibitor against the sweet potato starch phosphorylase;in the presence of the inhibitor, the Vmax decreased while theapparent Km for glucose- 1-P (0.6 mM) remained unchanged. Thedissociation constant of the enzyme-inhibitor complex (Ki) wasestimated to be 1.3 x 10- M in the presence of 0.3% solublestarch (pH 5.9, 37°C). The inhibitor appears to be more potentthan several small molecules (such as ATP, UDP-glc and ADP-glc) which are competitive inhibitors of starch phosphorylasewith K, values in the order of 10-' M (3 and TC Chang, JC Su,
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CHANG AND SU Plant Physiol. Vol. 80, 1986
Table I. Purification ofPhosphorylase Inhibitorfrom Sweet Potato RootThe results were based on 300 g of sweet potato. See "Materials and Methods" for details.
Purification Protein Total Activity Specific Activity Purification RecoverySteps
FIG. 3. Ouchterlony double diffusion test (A) and immunoelectro-phoresis (B) ofphosphorylase inhibitor. A, The central well (A) contained3 Mg of purified inhibitor and the surrounding wells 0.1 mg (2 td) ofpreimmune rabbit serum (B), 20 gg (C and D) or 10 jig (E) of y-globulinfraction of anti-sweet potato phosphorylase inhibitor serum. B, Wells Aand B contained 4.5 Mg of phosphorylase inhibitor each. After electro-phoresis, antiphosphorylase inhibitor y-globulin (2.5 mg/ml) was addedto 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 wtstandards (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 (3.5Ag 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-1261) was also tested, but no inhibition wasfound. This result could be expected. The sequence of the N-terminal 104 residues of potato phosphorylase was determinedby Nakano et al. (18). A comparison of this sequence with thecomplete sequence of the rabbit muscle enzyme (25) shows thatthe N-terminal 33-residue regions are completely different fromeach other. As this region is responsible for both allosteric andcovalent controls in the rabbit enzyme (4), the dissimilarity mayexplain the difference in regulatory properties between the twoenzymes (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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ose CL-6B gel filtration. Standard proteins are: 1, thyroglobulin (669K);2, ferritin (440K); 3, catalase (232K); 4, aldolase (158K). Standardproteins (0); phosphorylase inhibitor (-).
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STARCH PHOSPHORYLASE INHIBITOR FROM SWEET POTATO
Table II. H-Type Amino Acid Composition ofPhosphorylase InhibitorResults 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
FIG. 5. IEF of phosphorylase inhibitor in 0.5 mm thick polyacryl-amide gel layers. Lane 1, 6 gg inhibitor, pH range 4 to 6. Lane 2, 3.6 uginhibitor, pH range 3.5 to 10.
O 0.4 0.8 1.2 1.6 2.0
1/(Glucose-1-P) , ( mM)1FIG. 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 inhibitorincluded (-).
potato is derived from the proto- and metaxylem during rootdevelopment. Parenchymal cells ofthis part contain a lot of largeamyloplasts full of starch. Tissue sections of this part specificallybound the antibody at the surface as well as the inside of starchgranules, as revealed by fluorescence (Fig. 7A). In addition, thecell wall also highly fluoresced (Fig. 7A). When the sections of avery young root in size no more than 1 cm in diameter weretested, distinct fluorescence was also shown by the same subcel-lular sites (figures not shown). In control experiments, whichwere performed with preimmune rabbit serum as the primaryantibody, the fluorescence of amyloplast and cell wall couldhardly be detected (Fig. 7B). Schneider et al. (21) located starchphosphorylase of potato tuber in the amyloplast, and now inaddition to this locus, we found the enzyme in the cell wall ofroot parenchymal cell of sweet potato (TC Chang et al., unpub-lished data). These results indicate that both phosphorylase andits inhibitor are present in the same subcellular loci from a veryearly stage of root development and thus their interaction in vivoseems unavoidable.
Presence of the Inhibitor in Other Plants. Figure 8 shows thatcross-reacting materials ofrabbit anti-sweet potato phosphorylaseinhibitor were present in three arbitrarily selected plant materials,viz. potato tuber, spinach leaf, and rice grain. The precipitin linesformed with the three plant extracts completely fused, whilespurs toward wells containing the crude extracts (wells B and C)
were found; this means that the sweet potato inhibitor hasadditional unique antigenic determinants detected by the anti-serum. In addition, two precipitin lines were observed for eachof these samples, indicating that there could be two forms ofphosphorylase inhibitors in these plant tissues with one dominantover the other. Providing the diverse occurrence ofphosphorylaseinhibitor in different species (mono- and di-cotyledons) andorgans (root, tuber, leaf and grain), it may be reasonable toconclude that this inhibitor is of universal occurrence in higherplants. Although starch phosphorylase has been purified from avariety of sources (1, 2, 14, 20, 21), no report of its inhibitor ofprotein nature has appeared. Why it has escaped detection for solong is an intriguing question.Hammond and Preiss (7) reported an ATP-stimulated inacti-
vating factor(s) of starch phosphorylase. However, the inactiva-tion appeared to be due to proteinase action. The discovery ofstarch phosphorylase inhibitor implies the presence ofa new typeof regulatory mechanism of the enzyme in plants, which may becompletely different from that of the glycogen phosphorylasesystem.
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Plant Physiol. Vol. 80, 1986
FIG. 7. Immunofluorescence micrographs of the tissue sections of sweet potato root. A, Paraffin sections were first treated with rabbitantiphosphorylase inhibitor ay-globulin, followed by FITC-conjugated goat anti-rabbit IgG. Starch granules (G) and cell walls (arrow head) highlyfluoresced (x200). B, Control experiment. Sections were processed as in (a) except that preimmune rabbit serum was used as the primary antibody(x200).
B
FIG. 8. Presence of inhibitor in other plants. The central wells con-
tained 20 jsg of y-globulin fraction of antiphosphorylase inhibitor serum(A) or 0.1 mg ofpreimmune rabbit serum protein (H), and the surround-ing wells contained 3 gg phosphorylase inhibitor (B), 2 Ml each of extractsfrom 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 immunofluores-cence techniques and Miss Y. D. Chang for the preparation of paraffin sections.
LITERATURE CITED
1. ARIKI M. T FUIKU 1975 a-Glucan phosphorylase from sweet potato: isolationand 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 ofphosphorylase a. Annu Rev Biochem 49: 31-61
5. FUKUi T 1983 Plant phosphorylases: structure and function. In T Akazawa, TAsahi, H Imaseki, eds, The New Frontiers in Plant Biochemistry. JapanScientific Societies Press, Tokyo, pp 71-82
6. FUKUi T, S SHIMOMURA, K NAKANO 1982 Potato and rabbit muscle phospho-rylases: comparative studies on the structure, function and regulation ofregulatory and nonregulatory enzymes. Mol Cell Biochem 42: 129-144
8. HANES CS 1940 The breakdown and synthesis of starch by an enzyme systemfrom pea seeds. Proc R Soc Lond B Biol 128: 421-450
9. HUDSON L, FC HAY 1980 Practical Immunology, Ed 2. Blackwell ScientificPublications. London, pp 184-187
10. Instruction for High-Performance Analytical Electrofocusing in 0.5 MM Thin-Layer Polyacrylamide Gels, 1818-P. LKB, Bromma, Sweden
11. ISHIDA Y, T FUJITA, K ASAI 1981 New detection and separation method foramino acid by high performance liquid chromatography. J Chromatogr 204:143-148
12. JUANG RH, YD CHANG, HY SUNG, JC SU 1984 Oven-drying method forpolyacrylamide gel slab packed in cellophane sandwich. Anal Biochem 141:348-350
13. LAEMMLI UK 1970 Cleavage of structural proteins during the assembly of thehead of bacteriophage T4. Nature 227: 680-685
14. LEE EYC, JJ BRAUN 1973 Sweet corn phosphorylase: purification and prop-erties. Arch Biochem Biophys 156: 276-286
15. LINN TG, AL GREENLEAF, RG SHORENSTEIN, R LosiCK 1973 Loss ofthe sigmaactivity of RNA polymerase of Bacillus subtilis during sporulation. ProcNatl Acad Sci USA 70: 1865-1869
16. LOWRY OH, NJ ROSEBROUGH, AL FARR, Rl RANDALL 1951 Protein measure-ment with the Folin phenol reagent. J Biol Chem 193: 265-275
17. MARGOLIS J, KG KENDRICK 1968 Polyacrylamide gel electrophoresis in acontinuous molecular sieve gradient. Anal Biochem 25: 347-362
18. NAKANO K, T FUKUI, H MATSUBARA 1980 Structural basis for the differenceof the regulatory properties between potato and rabbit muscle phosphoryl-ases. The NHrterminal sequence of the potato enzyme. J Biol Chem 225:9255-9261
20. PREISS J, TW OKITA, E GREENBERG 1980 Characterization of the spinach leafphosphorylases. Plant Physiol 66: 864-869
21. SCHNEIDER EM, JU BECKER, D VOLKMANN 1981 Biochemical properties ofpotato phosphorylase change with its intracellular localization as revealed byimmunological methods. Planta 151: 124-134
22. SIVAK MN, JS TANDECARZ, CE CARDINI 1981 Studies on potato tuber phos-phorylase-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 biosyn-thesis. Biochem Biophys Res Commun 38: 709-714
24. SLATER GG 1969 Stable pattern formation and determination of molecularsize by pore-limit electrophoresis. Anal Chem 41: 1039-1041
25. TITANi K, A KOIDE, J HERMANN, LH ERICSSON, S KUMAR, RD WADE, KAWALSH, H NEURATH, EH FISCHER 1977 Complete amino acid sequence ofrabbit muscle glycogen phosphorylase. Proc Natl Acad Sci USA 74: 4762-4766
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