purifi'cation properties ofmesophyll bundle sheath cell a-glucan … ·...

Plant Physiol. (1988) 86, 417-4220032-0889/88/86/0417/06/$0 1.00/0

Purifi'cation and Properties of Mesophyll and Bundle Sheath Cella-Glucan Phosphorylases from Zea mays L.'EQUIVALENCE OF THE ENZYMES WITH THE CYTOSOL AND PLASTID PHOSPHORYLASESFROM SPINACH

Received for publication August 12, 1987 and in revised form October 6, 1987

CHRISTIAN MATEYKA AND CLAUS SCHNARRENBERGER*Institute ofPlant Physiology, Cell Biology, and Microbiology, Free University ofBerlin, Konigin-Luise-Str.12-16a, D-12000 Berlin 33 (West)

ABSTRACT

Two major a-glucan phosphorylases (I and II) from leaves of the C4plant corn (Zea mays L.) were previously shown to be compartmented inmesophyll and bundle sheath cells, respectively (C Mateyka, C Schnar-renberger 1984 Plant Sci Lett 36: 119-123). The two enzymes wereseparated by chromatography on DEAE-cellulose and purified to homo-geneity by affinity chromatography on immobilized starch, according topublished procedures, as developed for the cytosol and chloroplast phos-phorylase from the C3 plant spinach. The two a-glucan phosphorylaseshave their pH optimum at pH 7. The specificity for polyglucans wassimilar for soluble starch and amylopectin, however, differed for glycogen(K. = 16 micrograms per milliliter for the mesophyll cell and 250micrograms per milliliter for the bundle sheath cell phosphorylase).Maltose, maltotriose, and maltotetraose were not cleaved by eitherphosphorylase. If maltopentaose was used as substrate, the rate wasabout twice as high with the bundle sheath cell phosphorylase, than withthe mesophyll cell phosphorylase. The phosphorylase I showed a molec-ular mass of 174 kilodaltons and the phosphorylase II of 195 kilodaltonsfor the native enzyme and of 87 and of 53 kilodaltons for the SDS-treatedproteins, respectively. Specific antisera raised against mesophyll cellphosphorylase from corn leaves and against chloroplast phosphorylasefrom spinach leaves implied high similarity for the cytosol phosphorylaseof the C3 plant spinach with mesophyll cell phosphorylase of the C4 plantcorn and of chloroplast phosphorylase of spinach with the bundle sheathcell phosphorylase of corn.

a-Glucan phosphorylases in leaves ofthe two C3 plants spinachand pea have previously been shown to have a very distinctcompartmentation: one is located in the cytosol and one or twoin the chloroplasts (13, 19, 25). They are part of two sets ofisoenzymes in glycolysis, gluconeogenesis and the oxidative pen-tose phosphate cycle in the cytosol and in the plastids of plantcells, respectively (20). The cytosol and plastid phosphorylasesof C3 plant leaves have many properties in common; however,they differ greatly in their substrate affinity for glycogen, in theireffect on some maltodextrins (17, 22, 28), and in their immu-nological crossreactivity (5, 29).

Similar to C3 plants, multiple forms of phosphorylase can alsobe isolated from leaves of the C4 plant corn (11). It could be

' Part of this work was supported by Deutsche Forschungsgemein-schaft.

shown that one of the two major phosphorylases in corn wasrestricted to mesophyll cells and the other to bundle sheath cells.These findings explain previous (2) and recent (23) reports onphosphorylase activity for mesophyll and bundle sheath cells incorn leaves. Since it may argued that the cytosol and plastidphosphorylases of C3 plants are homologous with the mesophylland bundle sheath cell phosphorylases of C4 plants we havelooked for, and report here on properties of corn leaf phospho-rylases in order to gain evidence for such a conclusion.

MATERIALS AND METHODSEnzyme Sources. Corn (Zea mays L.), type Inrafruh, was

grown from seeds in greenhouses. For enzyme isolation 600 g ofleaf material without midribs was cut with scissors into smallpieces, homogenized in 2.4 L of 100 mM imidazol/HCl (pH 7.0),0.5% polyvinylpyrrolidone, 20 mm 2-ME,2 and 0.1 mm PMSFfor 1 min, first with a Waring Blendor (Waring Products, NewHartfort, CT) and subsequently with an Ultra-Turrax (Janke &Kunkel, Staufen/FRG) at full speed at 0°C. The suspension wassqueezed through two layers of cheesecloth and centrifuged at24,000g for 20 min at 4°C. The pellet was discarded.

Correspondingly, 400 g ofderibbed spinach (Spinacia oleraceaL.) was homogenized in 1 L of the same buffer for 1 min with aWaring Blendor. The suspension was processed in the same wayas with corn.Enzyme Purification. All purification steps were performed at

4°C. The buffer used for all subsequent operations was 0.01 MTris/HCl (pH 7.2), 20 mM 2-ME, and 0.1 mM PMSF.Enzymes of the crude extracts were fractionated by adding

solid (NH4)2SO4 to a final concentration of 35% saturation atpH 7.2. After centrifugation at 24,000g in a GSA-rotor of aSorvall RCIIB centrifuge, (NH4)2SO4 was added to the superna-tant to give 60% saturation. Precipitated proteins were dissolvedin buffer, dialyzed overnight, and applied to a DEAE-cellulose(Whatman, Springfield Mill, Springfield, Maidstone, Kent) col-umn of 3 x 40 cm. The enzymes were eluted with a lineargradient of 1.1 L buffer containing 0 to 0.4 M KCI. Fifteen-mlfractions were collected and assayed for phosphorylase activity.The conductivity was measured as an indicator for salt concen-tration. Fractions with phosphorylase activity (peaks I and II)were pooled separately and dialyzed.

Affinity chromatography on immobilized starch was used asthe most important purification step. The gel material was pre-

2Abbreviations: 2-ME, 2-mercaptoethanol; FCS. fetal calf serum;MES, (2(N-morpholino)ethane sulfonic acid); PMSF, phenylmethylsul-fonyl fluoride.

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MATEYKA AND SCHNARRENBERGER

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i

D -

r-

CL-

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FIG. 1. Affinity chromatography ofphosphorylase I (left) and II (right) fromcorn leaves on Sepharose 4B-boundstarch. The elution involved washing withbuffer (A), a salt gradient (B), buffer orsalt in buffer (C), and finally the elutingsolution (D).

Fra c ti on

Table I. Schemefor the Purification ofStarch Phosphorylase I and IIfrom Corn Leaves

Purification Step Volume Protein Activity Spctiitc Purification Recovery

ml mg U U/mg -fold %Crude extract 3200 1600 53 0.033 1 100(NH4)2SO4 precipitation 160 775 36 0.046 1.5 68Phosphorylase I

DEAE-cellulose 148 28 21 0.75 23 40Affinity chromatography (Sepharosebound starch) 31 0.78 16 21 659 30

Phosphorylase IIDEAE-cellulose 115 219 5 0.02 1 9Affinity chromatography 37 0.6 3 5.0 250 6a One unit of enzyme is defined as 1 Amol glucose- I -P formed min-'.

pared by activating Sepharose 4B with cyanogen bromide ac-

cording to Porath et al. (15) and by coupling soluble starchaccording to Schachtele et al. (18). For phosphorylase I thematerial was deactivated by 2 M glycine and for phosphorylaseII by 1.25 mol butylamine dissolved in 50 ml dimethylformamide(24). For chromatography 50 ml starch-bound Sepharose 4B waspacked into a column of 2 cm in diameter and equilibrated withbuffer. For purification of phosphorylase I a column was loadedwith the enzyme, washed first with 50 ml buffer, then with a 100ml gradient of 0 to 1 M KCI in buffer, and finally with 50 mlbuffer. Phosphorylase I was eluted with a 60 ml-gradient of 0 to2% soluble starch in buffer.For purification of phosphorylase II the other column was

loaded with enzyme, washed first with 50 ml buffer, then with a

100 ml gradient of 0 to 1 M NaCl in buffer, and finally with 50ml 1 M NaCl in buffer. Phosphorylase II was eluted with a 60ml-gradient of 0 to 2% starch in 1 M NaCl and buffer. In bothpreparations active fractions were pooled and treated with 2 unitsa-amylase in order to degrade starch. The preparations weredialyzed against buffer overnight and concentrated by ultrafiltra-tion in an Amicon cell. a-Amylase was removed from phospho-rylase by gel filtration on Sephadex G 100.

Polyacrylamide Gelelectrophoresis. Analytical SDS-PAGEwas performed in slab gels of 10% polyacrylamide according toLaemmli (9). Proteins were visualized with Coomassie brilliantblue R-250. Preparative SDS-PAGE was performed in 2 mmthick gels and with one large slot for protein application. Proteinswere visualized by 4 M sodium acetate as described by Higgins

and Dahmus (7).Molecular Weight Determination. The mol wt of the two

native phosphorylases was determined by ultracentrifugationaccording to Martin and Ames (10). Gradients of 5 to 20%sucrose in 10 mM Tris/HCl (pH 7.2) were prepared in 11.5 mltubes of a SW 41 Ti rotor (Beckman) and loaded with 0.3 ml ofphosphorylase and markers. The gradients were centrifuged for20 h in a Sorvall OTD2 ultracentrifuge and fractionated fromthe bottom into 140 ,l fractions. Markers were catalase (mol wt240,000), aldolase (mol wt 160,000), lactate dehydrogenase (molwt 140,000), glucose 6-P dehydrogenase (mol wt, 116,000) andhexokinase (mol wt 60,000). In addition, the mol wt ofthe nativeenzymes was determined by molecular sieving in Sephadex G200 replacing the markers lactate dehydrogenase and hexokinaseby BSA (mol wt 67,000). The mol wt of the subunits wasdetermined by SDS-PAGE with rabbit phosphorylase (mol wt95,000). BSA (mol wt 67,000), ovalbumin (mol wt 45,000),carbonic anhydrase (mol wt 31,000), and Cyt c (mol wt 12,500)as markers.

Antisera. Prior to immunization of rabbits the nearly purephosphorylase I from corn leaves was subjected to preparativeSDS-PAGE. The major protein band of phosphorylase I was cutout, homogenized with a Potter-Elvejhem tissue grinder andeluted with 0.4% NH4CO3, 0.1% SDS and 0.2% 2-ME. Theenzyme was dialyzed against enzyme buffer (see above). Forphosphorylase II from spinach leaves this step was omitted.About 100 jig ofpure protein was mixed with complete Freund'sadjuvant and injected intramuscularly into a rabbit. After 3

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Fra ction

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a-GLUCAN PHOSPHORYLASES

1 2 3 4 5 kD

_ 95

-- 67

FIG. 2. SDS-PAGE of phosphorylase I(left) and II (right) from corn leaves. Lane 1,

45 crude extract (20 ,g protein); lane 2, DEAE-cellulose (10 ,Ag); lane 3, affinity chromatog-raphy (4 pg); lane 4: preparation SDS-PAGE(2.5 gg); lane 5, markers; lane 6, affinitychromatography (4.5 ,g); lane 7, markers.

-- 31

-- 12.5

4-

100 Phosphorylase IIE

N

50A

5 6 7 8 9pH

FIG. 3. pH-dependence of corn leaf phosphorylase I (top) and II(bottom). Buffers used were MES (0), imidazol (-), and Tris (A).

weeks the animals were immunized again in weekly intervalsthree more times with the same amount of protein and incom-plete Freund's adjuvant. Four d after the last injection theanimals were bled and after 4 h the antisera collected by centrif-ugation. The antisera were tested in the Ouchterlony double

Table II. Km and V Values ofPurified Phosphorylase I and! ofCornLeaves

Substrate Phosphorylase I Phosphorylase II

Km V Km VPhosphate 1.9 mm 2.2 U/mg 1.5 mM 1.2 U/mgStarch 6.5 gg/ml 2.0 U/mg 9.3 Asg/ml 1.0 U/mgAmylopectin 26.3 gg/ml 0.8 U/mg 14.5 ,ug/ml 1.9 U/mgGlycogen 16.4 ,g/ml 1.8 U/mg 250 ug/ml 0.9 U/mg

Table III. Activity ofPhosphorylase I and I ofCorn and SpinachLeaves with Different Maltodextrins

The activity was measured in the phosphorolytic direction. All con-centrations were 1 mm except for starch (I mg/ml).

Corn leaf Spinach leafSugar phosphorylase phosphorylase

I II I II

Starch 100 100 100 100Maltose 0 0 0 0Maltotriose 0 0 0 0Maltotetraose 0 0 0 0Maltopentaose 76 182 69 163Maltotetraose + starch 100 54 100 36

diffusion test (14) and in immunotitration tests by immunopre-cipitation with protein-A-coated Staphylococcus aureus Cowan Icells (8). Immunoblotting was performed according to Towbinet al. (31). Proteins from SDS-PAGE were transferred electro-phoretically to nitrocellulose in 25 mm Tris/HCl (pH 8.3), 0.19M glycine, and 20% (v/v) methanol. The nitrocellulose wassaturated twice with FCS 5% (v/v) in PBS and incubated with

6 7 kD

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45

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FIG. 4. Sedimentation velocity analysis of corn leaf phosphorylase I(top) and II (bottom) (0- - -0) in gradients of 5 to 20% sucrose. Markerswere Catalase (A A), aldolase (O-O ), lactate dehydrogenase(0-C O), glucose 6-P dehydrogenase (x x), and hexokinase(A-A).

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x

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FIG. 5. Subunit mol wt determination of the phosphorylase I (top)and II (bottom) from corn leaves by SDS-PAGE.

a 50-

C-C 25

C 100 - i

75-

50-

25 -

1 2 4 8 16 32 64 128258 -foldSe rU m d l u t o n

FIG. 6. Immunotitration of phosphorylases with antisera against thephosphorylase I from corn leaves (top) and the phosphorylase II fromspinach leaves (bottom). Phosphorylase I from corn (0), phosphorylaseII from corn (0), phosphorylase I from spinach (A), phosphorylase IIfrom spinach (A).

a-phosphorylase antiserum (1:1000 dilution with 5% FCS in PBSand 0.02% (w/v) sodium azide). After five washings with 5%FCS in PBS for 5 min each the nitrocellulose was incubated in a

protein-A-horseradish-peroxidase solution (1:1000 dilution with5% FCS in PBS) for 1 to 2 h, washed 5 times in PBS for 5 mineach, and incubated in PBS containing 0.06% (w/v) 4-chloro-1-naphthol, 20% methanol and 0.018% H202. The peroxidasereaction was stopped by rinsing the nitrocellulose with water.

Assay. a-Glucan phosphorylase (EC 2.4.1.1.) was assayed bymeasuring glucose 1-P formation from soluble starch and cou-

pling the reaction to phosphoglucomutase and glucose 6-P de-hydrogenase (1). Catalase (EC 1.1 1.1.6.), aldolase (EC 4.1.2.13.),and glucose 6-P dehydrogenase (EC 1.1.1.49.) were assayed ascited in Ref. (8). Lactate dehydrogenase was tested according toBergmeyer et al. (1) and hexokinase according to Turner et al.(32). Protein was determined with the Coomassie blue method(21).

RESULTS

Purification of Phosphorylases. Corn leafphosphorylases were

separated by anion-exchange chromatography on DEAE-cellu-lose as described by Mateyka and Schnarrenberger (11). Themajor purification was achieved by subsequent affinity chroma-tography on immobilized starch as documented in Figure 1 andTable I. Phosphorylase I was purified 659-fold up to a specificactivity of 21 units mg-' protein and phosphorylase II 250-foldup to a specific activity of 5.0 units mg-' protein. PhosphorylaseI was far more stable (recovery 30%) than phosphorylase II(recovery 6%). The preparations showed only small contamina-tions (Fig. 2). The high mol wt band in the phosphorylase Ipreparation (Fig. 2, lane 3) was eliminated by preparative SDS-PAGE. By the same procedure, phosphorylase I and II fromspinach leaves were purified to homogeneity with specific activityof 9.3 and 8.0 units mg-' protein (data not shown) and used for

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a-GLUCAN PHOSPHORYLASES

and of 195,000 for phosphorylase II by both methods. Occasion-ally, during velocity sedimentation analyses an additional phos-phorylase activity was observed with a mol wt of270,000. DuringSDS-PAGE (Fig. 5) mol wt of 87,000 and 53,000 were obtainedfor the subunits of phosphorylase I and II, respectively. Thiswould imply a dimeric structure for phosphorylase I and atetrameric structure for phosphorylase II.Immunochemical Properties. Antisera raised against phospho-

rylase I of corn leaves showed a strong reaction in an immuno-titration test with phosphorylase I of corn and spinach leaves(Fig. 6, top). In order to precipitate 50% of 4.7 ,ug phosphorylaseI in this immunoprecipitation test a 2.4-fold larger amount ofantiserum was needed for the spinach leaf enzyme as comparedto the corn leaf enzyme. This corresponds to 40% cross-reactionof the spinach leaf phosphorylase I with the corn leaf phospho-rylase I. With the same antiserum no cross-reaction was observedwith either spinach or corn leafphosphorylase II. In the reciprocalexperiment with the antiserum against phosphorylase II of spin-ach leaves (Fig. 6, bottom), 35% cross-reaction was observedwith the corn leaf phosphorylase II as compared with the spinachleaf phosphorylase II; however, no reaction occurred with eitherspinach or corn leaf phosphorylase I.

Since titration by immunoprecipitation is only of limitingsensitivity, we have also applied immunoblotting to determinecross-reactions (Fig. 7). The antiserum against corn leaf phos-phorylase I gave good signals with either corn or spinach leafphosphorylase I, but only poor signals with corn leaf phospho-rylase II. Similarly, the antiserum against the spinach leaf phos-phorylase II, reacted with both the spinach and corn leaf phos-phorylase II; however, not with corn leaf phosphorylase I. Inaddition, the data show that the corn and spinach leaf phospho-rylases I have the same subunit size, while the subunit of spinachleaf phosphorylase II is about twice as large as the subunit ofcorn leaf phosphorylase II.

DISCUSSION

In a previous study it was shown that the corn leaf phospho-1 2 3 4 5 6 7 rylase I is a mesophyll cell and the com leaf phosphorylase II a

bundle sheath cell enzyme (1 1). The properties of the two phos-phorylases, as described in this paper, indicate that the two corn

FIG. 7. Immunoblotting of the punified phosphorylase I and II from leaf phosphorylases are homologous to the spinach phosphoryl-corn and spinach leaves with antisera against the phosphorylase I from ase I and II, which have previously been shown to be compart-corn (left) and phosphorylase II from spinach (rght). Lanes 1 and e, mented in the cytosol and the chloroplasts ofthe C3 plant spinachphosphorylase I from corn; lane 2, phosphorylase I from spinach; lanes (13, 25). It is, therefore, suggestive, that the corn leaf phospho-3, 6, and 7, phosphorylase II from corn; lane 5, phosphorylase II from rylase I is a cytosolic enzyme in the mesophyll cells and the cornspinach. leaf phosphorylase II occurs in chloroplasts of the bundle sheath

cells.comparisons with the corn leaf phosphorylases. The above conclusion is furthermore supported by the follow-

Catalytic Properties. Corn leaf phosphorylase I and II showed ing facts:broad pH activity profiles with an optimum around pH 7 (Fig. (a) Phosphorylase I and II from corn leaves can be separated3). Substrate affinity was tested for phosphate, soluble starch, in a similar fashion by ion exchange chromatography as theiramylopectin, and glycogen (Table II). K,,,- and V-values for all counterparts in spinach (1 1, 26).substrates were largely the same for both phosphorylases. How- (b) The substrate affinity of phosphorylase I and II is identicalever, the Km for glycogen was 15 times smaller for phosphorylase for phosphate and soluble starch and differs for glycogen 20 toI than for phosphorylase II. Maltose, maltotriose, and malto- 300-fold in spinach leaves (17, 28) and 15-fold in corn leavestetraose were ineffective substrates for both phosphorylases (Ta- (this paper). Differences exist in the affinity for amylopectinble III). The maximal velocity with maltopentaose was 2.5-fold which is a better substrate for the corn leaf phosphorylase II thanhigher for phosphorylase II than for phosphorylase I. On the for spinach phosphorylase II.other hand, starch phosphorolysis activity of phosphorylase II (c) The effects of maltodextrins are virtually the same for thewas inhibited by 50% with maltotetraose while the activity of two phosphorylases of spinach and corn leaves. This includesphosphorylase I was not affected. These effects were similar to that maltotriose and maltotetraose are not substrates for eitherthe two spinach leaf phosphorylases I and II. spinach or corn phosphorylases and that maltopentaose is

Molecular Weight. The mol wt ofthe corn leaf phosphorylases cleaved with the same maximal velocity by the phosphorylaseswas estimated by velocity sedimentation in sucrose gradients II of either spinach or corn, but only with about 40% of this(Fig. 4). An additional analysis was performed by gel filtration velocity by the respective phosphorylases I.on Sephadex G 200 (data not shown). By regression analysis with (d) Immunochemical properties imply high structural homol-markers, a mol wt of 174,000 was obtained for phosphorylase I ogy between the two phosphorylases I of spinach and corn and

421

A




between the two phosphorylases II (this paper). However, highstructural diversity was found between phosphorylase I and II ofspinach leaves on the one side (5, 29) and phosphorylase I andII of corn leaves on the other side (this paper).

(e) The size of the native phosphorylases I and II of C3 plantsand of the C4 plant corn is very similar. Phosphorylase I has adimeric structure in the C3 plant spinach (17, 22, 27) and in theC4 plant corn (this paper). The same situation applies to thesubunit structure of phosphorylase II in C3 plants (22, 24). Forphosphorylase II in corn leaves, however, we observed a subunitsize only about half of the size of the other phosphorylases. Thisleads to the conclusion that the corn leaf phosphorylase II mustbe a tetrameric enzyme. The reason for a different subunitstructure of phosphorylase II in C3 and C4 plants is unknown.

In summary, the majority of properties of phosphorylase I andII from corn leaves support the view that they are indeed acytosolic enzyme in mesophyll cells and a chloroplast enzyme inbundle sheath cells of C4 plants. The function of the cytosolicphosphorylase I in mesophyll cells is yet unclear as has alreadybeen stated for the cytosolic phosphorylases in C3 plants (27).The present findings on the two corn leaf phosphorylases con-tribute further evidence for the concept that cytosolic functionsof C3 plants are mainly compartmented in mesophyll cells of C4plants. This different compartmentation includes, e.g. sucroseand starch metabolism (2, 3, 16, 23), regulatory enzymes offructose 1,6-bisP/fructose 2,6-bisP/fructose 6-P metabolism (30),and enzymes of nitrate fixation and ammonia fixing enzymes (6,12). It appears that Kranz anatomy, as described by Haberlandt(4) in 1881, implies many more problems of intra- and intercel-lular compartmentation than those related to C4 metabolism.

LITERATURE CITED

1. BERGMEYER HU, K GAWEHN, M GRABL 1974 Enzyme als biochemischeReagentien. In HU Bergmeyer, ed, Methoden der enzymatischen Analyse,Vol 1. Verlag Chemie, Weinheim/BergstraBe, pp 454-558

2. DOWNTON WJS, JS HAWKER 1973 Enzymes of starch and sucrose metabolismin Zea mays leaves. Phytochemistry 12: 155 1-1556

3. FURBANK RF, M STITr, CH FOYER 1985 Intercellular compartmentation ofsucrose synthesis in leaves of Zea mays L. Planta 164: 172-178

4. HABERLANDT G 1881 Vergleichende Anatomie des assimilatorischen Gewe-besystems von Pflanzen. Engelmann Verlag, Leipzig

5. HAMMOND JBW, J PREISS 1983 Spinach leaf intra and extra chloroplastphosphorylase activities during growth. Plant Physiol 73: 709-712

6. HAREL E, PJ LEA, BJ MIFLIN 1977 The localisation of enzymes of nitrogenassimilation in maize leaves and their activities during greening. Planta 134:195-200

7. HIGGINs RC, ME DAHMUS 1979 Rapid visualization of protein bands inpreparative SDS-polyacrylamide gels. Anal Biochem 93: 257-260

8. KRUGER I, C SCHNARRENBERGER 1983 Purification, subunit structure andimmunochemical comparison of fructose-bisphosphate aldolases from spin-ach and corn leaves. Eur J Biochem 136: 101-106

9. LAEMMLI UK 1970 Cleavage of structural proteins during the assembly of thehead of bacteriophage T4. Nature 227: 680-685

10. MARTIN RG, BN AMES 1961 A method for determining the sedimentationbehavior of enzymes: Application to protein mixtures. J Biol Chem 236:1372-1379

11. MATEYKA C, C SCHNARRENBERGER 1984 Starch phosphorylase isoenzymes inmesophyll and bundle sheath cells ofcorn leaves. Plant Sci Lett 36: 119-123

12. NEYRA CA, RH HAGEMAN 1978 Pathway for nitrate assimilation in corn (Zeamays L.) leaves. Cellular distribution of enzymes and energy sources fornitrate reduction. Plant Physiol 62: 618-621

13. OKITA THW, E GREENBERG, DN KUHN, J PREISS 1979 Subcellular localizationof the starch degradative and biosynthetic enzymes of spinach leaves. PlantPhysiol 64: 187-192

14. OUCHTERLONY 0 1949 Antigen-antibody reactions in gels. Ark Mineral Geol26B: 1-9

15. PORATH J, K ASPBERG, H DREVIN, R AXEN 1973 Preparation of cyanogenbromide-activated agarose gels. J Chromatogr 86: 53-56

16. PREISS J, C LEVI 1980 Starch biosynthesis and degradation. In MD Hatch, NKBoardman, eds. Biochemistry of Plants, Vol. 3. Academic Press, New York,pp 371-423

17. PREISS J, TW OKITA, E GREENBERG 1980 Characterization of the spinach leafphosphorylases. Plant Physiol 66: 864-869

18. SCHACHTELE KH, E SCHILTZ, D PALM 1978 Amino-acid sequence of thepyridoxal-phosphate-binding site in Escherichia coli maltodextrin phospho-rylase. Eur J Biochem 92: 427-435

19. SCHACHTELE C, M STEUP 1986 a-1,4-Glucan phosphorylase from leaves ofspinach (Spinacia oleracea L.) I. In situ localization by indirect immunoflu-orescence. Planta 167: 444-451

20. SCHNARRENBERGER C, M HERBERT, I KRUGER 1983 Intracellular compart-mentation of isozymes of sugar phosphate metabolism in green leaves. InMC Rattazzi, JG Scandalios, GS Whitt, eds, Isozymes: Current Topics inBiological and Medical Research, Vol 8. Alan R. Liss, New York, pp 23-51

21. SEDMAK JJ, SE GROSSBERG 1977 A rapid, sensitive, and versatile assay forprotein using Coomassie Brilliant Blue G250. Anal Biochem 79: 544-552

22. SHIMOMURA S, M NAGAI, T FUKUI 1982 Comparative glucan specificities oftwo types of spinach leaf phosphorylase. J Biochem 91: 703-717

23. SPILATRO SR, J PREISS 1987 Regulation of starch synthesis in the bundle sheathand mesophyll of Zea mays L. Intercellular compartmentation of enzymesof starch metabolism and the properties of the ADPglucose phosphorylases.Plant Physiol 83: 621-627

24. STEUP M 1981 Purification of chloroplast a- 1,4-glucan phosphorylase fromspinach leaves by chromatography on sepharose-bound starch. BiochimBiophys Acta 659: 123-131

25. STEUP M, E LATZKO 1979 Intracellular localization of phosphorylases inspinach and pea leaves. Planta 145: 69-75

26. STEUP M, C SCHACHTELE, E LATZKO 1980 Separation and partial characteriza-tion of chloroplast and non-chloroplast a-glucan phosphorylases from spin-ach leaves. Z Pflanzenphysiol 96: 365-374

27. STEUP M, C SCHACHTELE, E LATZKO 1980 Purification of a non-chloroplast a-glucan phosphorylase from spinach leaves. Planta 148: 168-173

28. STEUP M, C SCHACHTELE 1981 Mode of glucan degradation by purifiedphosphorylase forms from spinach leaves. Planta 153: 351-361

29. STEUP M, C SCHACHTELE 1986 a- 1,4-Glucan phosphorylase from leaves ofspinach. II. Peptide patterns and immunological properties. A comparisonwith other phosphorylase forms. Planta 168: 222-231

30. STI-r M, HW HELDT 1985 Control of photosynthetic sucrose synthesis byfructose-2,6-bisphosphate. Intercellular metabolite distribution and proper-ties of the cytosolic fructose bisphosphatase in leaves of Zea mays L. Planta164: 179-188

31. ToWBIN, H, T STAEHELIN, J GORDON 1979 Electrophoretic transfer of proteinsfrom polyacrylamide gels to nitrocellulose sheets: Procedure and someapplications. Proc Natl Acad Sci USA 76: 4350

32. TURNER JF, DD HARRISON, L COPELAND 1977 Fructokinase of pea seeds.Plant Physiol 60: 666-669




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