Purification and Properties of Mesophyll and Bundle Sheath Cell alpha-Glucan Phosphorylases from Zea mays L. : Equivalence of the Enzymes with the Cytosol and Plastid Phosphorylases from Spinach

Two major α-glucan phosphorylases (I and II) from leaves of the C₄ plant corn (Zea mays L.) were previously shown to be compartmented in mesophyll and bundle sheath cells, respectively (C Mateyka, C Schnarrenberger 1984 Plant Sci Lett 36: 119-123). The two enzymes were separated by chromatography on DEAE-cellulose and purified to homogeneity by affinity chromatography on immobilized starch, according to published procedures, as developed for the cytosol and chloroplast phosphorylase from the C₃ plant spinach. The two α-glucan phosphorylases have their pH optimum at pH 7. The specificity for polyglucans was similar for soluble starch and amylopectin, however, differed for glycogen (K_m = 16 micrograms per milliliter for the mesophyll cell and 250 micrograms per milliliter for the bundle sheath cell phosphorylase). Maltose, maltotriose, and maltotetraose were not cleaved by either phosphorylase. If maltopentaose was used as substrate, the rate was about twice as high with the bundle sheath cell phosphorylase, than with the mesophyll cell phosphorylase. The phosphorylase I showed a molecular mass of 174 kilodaltons and the phosphorylase II of 195 kilodaltons for the native enzyme and of 87 and of 53 kilodaltons for the SDS-treated proteins, respectively. Specific antisera raised against mesophyll cell phosphorylase from corn leaves and against chloroplast phosphorylase from spinach leaves implied high similarity for the cytosol phosphorylase of the C₃ plant spinach with mesophyll cell phosphorylase of the C₄ plant corn and of chloroplast phosphorylase of spinach with the bundle sheath cell phosphorylase of corn.     

Soluble starch synthases and starch branching enzymes from developing seeds of sorghum

Soluble starch synthases and branching enzymes have been partially purified from developing sorghum seeds. Two major fractions and one minor fraction of starch synthase were eluted on DEAE-cellulose chromatography. The minor enzyme eluted first and was similar to the early eluting major synthase in citrate-stimulated activity, faster reaction rates with glycogen primers than amylopectin primers, and in K_m for ADP-glucose (0.05 and 0.08 mM, respectively). The starch synthase peak eluted last had no citrate-stimulated activity, was equally active with glycogen and amylopectin primers, and had the highest K_m for ADP-glucose (0.10 mM). Four fractions of branching enzymes were recovered from DEAE-cellulose chromatography. One fraction eluted in the buffer wash; the other three co-eluted with the three starch synthases. All four fractions could branch amylose or amylopectin, and stimulated α-glucan synthesis catalysed by phosphorylase. Electrophoretic separation and activity staining for starch synthase of crude extracts and DEAE-cellulose fractions demonstrated complex banding patterns. The colour of the bands after iodine staining indicated that branching enzyme and starch synthase co-migrated during electrophoresis.     

Characterization of Pea Chloroplast D-Enzyme (4-alpha-d-Glucanotransferase)

Pea (Pisum sativum L.) chloroplast D-enzyme (4-α-d-glucanotransferase, EC 2.4. 1.25) was purified greater than 750-fold and partially characterized. It is a dimer with a subunit M_r of ca. 50,000. Optimal activity is between pH 7.5 and 8.0 with maltotriose as substrate and the enzyme's K_m for maltotriose is 3.3 millimolar. Chloroplast D-enzyme converts maltotriose to maltopentaose and glucose via the exchange of α-1,4-glycosidic linkages. Maltotriose acts either as a donor or acceptor of a maltosyl group. The enzyme has highest activity with maltotriose as substrate. As initial substrate degree of polymerization is increased to maltoheptaose, D-enzyme activity drops to zero at 10 millimolar substrate concentrations and by 70% at 1 millimolar concentrations. The enzyme cannot use maltose as a substrate. Glucose was found to be a suitable acceptor substrate for this D-enzyme. Addition of glucose to incubation mixtures, or production of glucose by D-enzyme, prevents the synthesis of maltodextrins larger than maltopentaose. Removal of glucose produced by D-enzyme activity with maltotriose as substrate resulted in the synthesis of maltopentaose and maltodextrins with sufficient degrees of polymerization to be suitable substrates for pea chloroplast starch phosphorylase. The possible role of D-enzyme in pea chloroplast starch metabolism is discussed.     

Actions of glycogen synthase and prosphorylase of rabbit-skeletal muscle on modified glycogens

The high reactivities exhibited by rabbit-muscle synthase and phosphorylase for unmodified glycogen-acceptors decrease progressively, presumably because of a large increase in apparent K_m as the glycogen molecule is converted into its component maltosaccharide chains by the debranching enzyme, isoamylase. Elongation of the outer chains of glycogen acceptor also results in decreased reactivities of the two transglucosylases and this is shown, for phosphorylase acting in the direction of glucan synthesis, to be caused by a decrease in the V_max of the reaction. A partial restoration of the degradative reactivity of phosphorylase by a limited alpha-amylolysis of the long outer-chains of modified glycogen suggests a role of cytoplasmic alpha-amylase in mammalian glycogen metabolism.     

Purification and properties of glutamine synthetase from spinach leaves

The chloroplastic glutamine synthetase of spinach leaves has been purified to homogeneity using affinity chromatography. This involves a tandem `reactive blue A-agarose' and `reactive red-A-agarose' as the final step in the procedure. This procedure results in a yield of 18 milligrams of pure glutamine synthetase per kilogram of starting material. The purity of our enzyme has been demonstrated on both one- and two-dimensional polyacrylamide gels.

Purified glutamine synthetase has a molecular weight of 360,000 daltons and consists of eight 44,000 dalton subunits. The K_m is 6.7 millimolar for glutamate, 1.8 millimolar for ATP (synthetase assay), and 37.6 millimolar for glutamine (transferase assay). The isoelectric point is 6.5 and the pH optima are 7.3 in the synthetase assay and 6.4 in the transferase assay. The irreversible, competitive inhibitors methionine sulfoxamine and phosphinothricin have K_i values of 0.1 millimolar and 6.1 micromolar, respectively. Amino acid analysis has been carried out and the results compared with published analyses for other isoforms of glutamine synthetase.

     

Sucrose synthase of soybean nodules

Sucrose synthase (UDPglucose: d-fructose 2-α-d-glucosyl transferase, EC 2.4.1.13) has been purified from the plant cytosolic fraction of soybean (Glycine max L. Merr cv Williams) nodules. The native enzyme had a molecular weight of 400,000. The subunit molecular weight was 90,000 and a tetrameric structure is proposed for soybean nodule sucrose synthase. Optimum activity in the sucrose cleavage and synthesis directions was at pH 6 and pH 9.5 respectively, and the enzyme displayed typical Michaelis-Menten kinetics. Soybean nodule sucrose synthase had a high affinity for UDP (K_m, 5 micromolar) and a relatively low affinity for ADP (apparent K_m, 0.13 millimolar) and CDP (apparent K_m, 1.1 millimolar). The K_m for sucrose was 31 millimolar. In the synthesis direction, UDPglucose (K_m, 0.012 millimolar) was a more effective glucosyl donor than ADPglucose (K_m, 1.6 millimolar) and the K_m for fructose was 3.7 millimolar. Divalent cations stimulated activity in both the cleavage and synthesis directions and the enzyme was very sensitive to inhibition by heavy metals.     

Interaction between glycogen and glycogen phosphorylase of Tetrahymena

The glycogen phosphorylase of Tetrahymena pyriformis complexes with glycogen as judged by its elution pattern from columns of Sepharose 6B. Complex formation does not occur with starch, amylose, or amylopectin, and neither do these polyglucans serve as primers for the enzyme. To study the association between the phosphorylase and glycogen particles in situ, Tetrahymena were grown under differing physiological conditions, phosphorylase was isolated and chromatographed on a Sepharose 6B column. Phosphorylase activity isolated from cells grown in the absence of glucose was only partially associated with glycogen, while in cells exposed to glucose for 30 min or more all the phosphorylase activity was associated with glycogen. The effects of culture age and anaerobiosis on the relative amounts of free and glycogen-bound enzyme in the cells were also studied. It was concluded from the in vivo experiments that there was no simple relation between the fraction of enzyme bound to glycogen and between cell glycogen content.     

Localization and characteristics of rat liver mitochondrial aldehyde dehydrogenases.

Two NAD-dependent aldehyde dehydrogenase enzymes from rat liver mitochondria have been partially purified and characterized. One enzyme (enzyme I) has molecular weight of 320,000 and has a broad substrate specificity which includes formaldehyde; NADP is not a cofactor for this enzyme. This enzyme has K_m values for most aldehydes in the micromolar range. The isoelectric point was found to be 6.06. A second enzyme (enzyme II) has a molecular weight of 67,000, a K_m value for most aldehydes in the millimolar range but no activity toward formaldehyde. NADP does serve as a coenzyme, however. The isoelectric point is 6.64 for this enzyme. By utilization of the different substrate properties of these two enzymes it was possible to demonstrate a time-dependent release from digitonin-treated liver mitochondria. The high K_m, low molecular weight enzyme (enzyme II) is apparently in the intermembrane space while the low K_m, high molecular weight enzyme (enzyme I) is in the mitochondrial matrix and is most likely responsible for oxidation of acetaldehyde formed from ethanol.     

Studies on phosphorylase isozymes in lower vertebrates. Purification and properties of lamprey phosphorylase.

Skeletal muscle phosphorylase b has been purified from lamprey, Entosphenus japonicus, to a state of homogeneity as judged by the criterion of sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The enzyme was completely dependent on AMP for activity and converted into the a form by rabbit muscle phosphorylase kinase in the presence of ATP and Mg²⁺. The subunit molecular weight determined by SDS-gel electrophoresis was 94,000 ± 1,600 (SE). The enzyme activity was stimulated by Na₂SO₄, but was not affected by mercaptoethanol. The K_m values of the a form for glucose 1-phosphate and glycogen were 3.5 mm and 0.13%, respectively, and those of the b form for glucose 1-phosphate, glycogen, and AMP were 15 mm, 0.4%, and 0.1 mm, respectively. These values were smaller than those reported with lobster phosphorylase and greater than those reported with mammalian skeletal muscle phosphorylases. Electrophoretic and immunological studies have indicated that lamprey phosphorylase b exists as a single molecular form in skeletal muscle, heart, brain, and kidney. Rabbit antibody against lamprey phosphorylase cross-reacted with phosphorylases from skate and shark livers more intensely than with those from skeletal muscles.     

ISOLATION, ULTRASTRUCTURE, AND BIOCHEMICAL CHARACTERIZATION OF GLYCOGEN IN INSECT FLIGHT MUSCLE

Glycogen from flight muscle of the blowfly, Phormia regina, has been characterized ultrastructurally and biochemically. In situ, glycogen is in the form of rosettes, which vary in size with diameters of up to 0.1 µ. Sedimentation analysis of pure glycogen, isolated by mild buffer extraction, reveals a polydisperse molecular weight spectrum, with larger particles having molecular weights of 100 million. Treatment of native glycogen with alkali, under conditions usual for the extraction of the polysaccharide from tissues, results in a 5- to 10-fold reduction in molecular weight, as well as a chemical alteration of the molecule. Flight muscle phosphorylase has a lower affinity for native than for alkali-treated glycogen. The maximum velocity of the enzyme is also lower with native substrate. The apparent K_m for inorganic phosphate is higher with native glycogen as cosubstrate. These kinetic differences with native and partially degraded glycogen demonstrate the importance of using the natural substrate in studies of biochemical control mechanisms.     

Last Step in the Conversion of Trehalose to Glycogen: A MYCOBACTERIAL ENZYME THAT TRANSFERS MALTOSE FROM MALTOSE 1-PHOSPHATE TO GLYCOGEN

We show that Mycobacterium smegmatis has an enzyme catalyzing transfer of maltose from [¹⁴C]maltose 1-phosphate to glycogen. This enzyme was purified 90-fold from crude extracts and characterized. Maltose transfer required addition of an acceptor. Liver, oyster, or mycobacterial glycogens were the best acceptors, whereas amylopectin had good activity, but amylose was a poor acceptor. Maltosaccharides inhibited the transfer of maltose from [¹⁴C]maltose-1-P to glycogen because they were also acceptors of maltose, and they caused production of larger sized radioactive maltosaccharides. When maltotetraose was the acceptor, over 90% of the ¹⁴C-labeled product was maltohexaose, and no radioactivity was in maltopentaose, demonstrating that maltose was transferred intact. Stoichiometry showed that 0.89 μmol of inorganic phosphate was produced for each micromole of maltose transferred to glycogen, and 56% of the added maltose-1-P was transferred to glycogen. This enzyme has been named α1,4-glucan:maltose-1-P maltosyltransferase (GMPMT). Transfer of maltose to glycogen was inhibited by micromolar amounts of inorganic phosphate or arsenate but was only slightly inhibited by millimolar concentrations of glucose-1-P, glucose-6-P, or inorganic pyrophosphate. GMPMT was compared with glycogen phosphorylase (GP). GMPMT catalyzed transfer of [¹⁴C]maltose-1-P, but not [¹⁴C]glucose-1-P, to glycogen, whereas GP transferred radioactivity from glucose-1-P but not maltose-1-P. GMPMT and GP were both inhibited by 1,4-dideoxy-1,4-imino-d-arabinitol, but only GP was inhibited by isofagomine. Because mycobacteria that contain trehalose synthase accumulate large amounts of glycogen when grown in high concentrations of trehalose, we propose that trehalose synthase, maltokinase, and GMPMT represent a new pathway of glycogen synthesis using trehalose as the source of glucose.     

Purification and characterization of pea chloroplastic phosphoriboisomerase

Pea (Pisum sativum L.) chloroplastic phosphoriboisomerase (EC 5.3.1.6) can be purified to apparent homogeneity in less than 2 days time with a 53% yield. Important steps in the purification include heat treatment and pseudoaffinity chromatography on Red H-3BN Sepharose. The purified isomerase has a subunit molecular mass of 26.4 kD. The N-terminal sequence has been determined through 34 residues. pH optima are 7.8 (ribose-5-phosphate) and 7.7 (ribulose-5-phosphate); K_m values are 0.9 millimolar (ribose-5-phosphate) and 0.6 millimolar (ribulose-5-phosphate). The enzyme is inhibited by erythrose-4-phosphate, sedoheptulosebisphosphate, glyceraldehyde-3-phosphate, and 3-phosphoglycerate at concentrations close to those found in photosynthesizing chloroplasts. Countercurrent phase partitioning experiments indicate that the pea chloroplastic phosphoriboisomerase interacts physically with phosphoribulokinase.     

Partial Purification and Characterization of Uracil-DNA Glycosylase Activity from Chloroplasts of Zea mays Seedlings

A uracil-DNA glycosylase activity has been purified about 750-fold from the chloroplasts of light-grown Zea mays seedlings. This report represents the first direct demonstration of a DNA-glycosylase repair activity in chloroplasts. The activity, in part, was associated with a chloroplast Triton X-100 sensitive membrane. Its apparent K_m was 1.0 micromolar for a poly(dA-dT/U) substrate, and its molecular weight, as determined by gel filtration, was 18,000. The enzyme exhibited optimal activity at pH 7.0 with an atypically narrow pH tolerance. Activity was inhibited greater than 60% by 10 millimolar NaCl, 5 millimolar MgCl₂, or 5 millimolar EDTA. Enzyme activity was inhibited 80% by 10 millimolar N-ethylmaleimide, a sulfhydryl group-blocking agent. The activity removed uracil more rapidly from single-stranded DNA than from double-stranded DNA. With this report, uracil-DNA glycosylase activity has now been attributed to all three DNA-containing organelles of eucaryotic cells.     

Serine hydroxymethyltransferase from soybean root nodules : purification and kinetic properties

Serine hydroxymethyltransferase has been purified 1,550-fold from the plant fraction of soybean (Glycine max [L]. Merr. cv Williams) nodules. The pH optimum for the enzyme was at 8.5. The native molecular weight was 230,000 with a subunit molecular weight of 55,000 which suggested a tetramer of identical subunits. The enzyme kinetics for the enzyme were Michaelis-Menten; there was no evidence for cooperativity in the binding of either substrates or product inhibitors. There were two K_m values for serine at 1.5 and 40 millimolar. The K_m for l-tetrahydrofolate was 0.25 millimolar. l-Methyl-, l-methenyl-, and l-methylene-tetrahydrofolates were all noncompetitive inhibitors with l-tetrahydrofolate with K_i values of 1.8, 3.0, and 2.9 millimolar, respectively. Glycine was a competitive inhibitor with serine with a K_i value of 3.0 millimolar. The intersecting nature of the double reciprocal plots together with the product inhibition data suggested an ordered mechanism with serine the first substrate to bind and glycine the last product released. The enzyme was insensitive to a wide range of metabolites which have previously been reported to affect its activity. These results are discussed with respect to the roles of serine hydroxymethyltransferase and the one-carbon metabolite pool in control of the carbon flow to the purine biosynthetic pathway in ureide biogenesis.     

Kinetic properties of two starch phosphorylases from pea seeds

Both of the starch phosphorylase fractions from Victory Freezer pea seeds, that can be separated by DEAE—cellulose chromatography and purified by Sepharose 4B-starch affinity chromatography, contain pyridoxal 5′-phosphate. The addition of further quantities of pyridoxal 5′-phosphate causes inactivation. Both enzymes showed similar bi-substrate kinetics with d-Glc-1-P and varying amounts of amylopectin and also with P_i and varying amounts of amylopectin. In the direction of glucan sythesis the K_m for amylopectin with phosphorylase II was much higher than with phosphorylase I. However, the two enzymes differed in their behaviour on glucan degradation at varying concentrations of P_i. With phosphorylase II the K_m for amylopectin was dependent on the concentration of P_i but that for phosphorylase I was constant. Phosphorylase II was strongly inhibited by ADPG in the direction of glucan degradation but only slightly in the direction of glucan synthesis by both ADPG and UDPG. Phosphorylase I was only slightly inhibited by ADPG in both directions and by UDPG in synthesis. UDPG inhibited both enzymes moderately in glucan degradation,     

Mode of glucan degradation by purified phosphorylase forms from spinach leaves

The glucan specifity of the purified chloroplast and non-chloroplast forms of -1,4-glucan phosphorylase (EC 2.4.1.1) from spinach leaves (Steup and E. Latzko (1979), Planta 145, 69–75) was investigated. Phosphorolysis by the two enzymes was studied using a series of linear maltodextrins (degree of polymerization 11), amylose, amylopectin, starch, and glycogen as substrates. For all unbranched glucans (amylose and maltodextrins G₅–G₁₁), the chloroplast phosphorylase had a 7–10-fold higher apparent affinity (determined by initial velocity measurements) than the non-chloroplast phosphorylase form. For both enzyme forms, the minimum chain length required for a significant rate of phosphorolysis was five glucose units. Likewise, phosphorolysis ceased when the maltodextrin was converted to maltotetraose. With the chloroplast phosphorylase, maltotetraose was a linear competitive inhibitor with respect to amylose or starch (K _i-0.1 mmol 1^-1); the inhibition by maltotetraose was less pronounced with the non-chloroplast enzyme. In contrast to unbranched glucans, the non-chloroplast phosphorylase exhibited a 40-, 50-, and 300-fold higher apparent affinity for amylopectin, starch, and glycogen, respectively, than the chloroplast enzyme. With respect to these kinetic properties the chloroplast phosphorylase resembled the type of maltodextrin phosphorylase.Abbreviations G1P Glucose 1-phosphate - MES 2(N-morpholino)ethane sulphonic acid - P_i orthophosphate - Tris Tris(hydroxymethyl)aminomethane     

Hexose kinases from the plant cytosolic fraction of soybean nodules

The enzymes responsible for the phosphorylation of hexoses in the plant cytosolic fraction of soybean (Glycine max L. Merr cv Williams) nodules have been studied and a hexokinase (ATP:d-hexose 6-phosphotransferase EC 2.7.1.1) and fructokinase (ATP:d-fructose 6-phosphotransferase EC 2.7.1.4) shown to be involved. The plant cytosolic hexokinase had optimum activity from pH 8.2 to 8.9 and the enzyme displayed typical Michaelis-Menten kinetics. Hexokinase had a higher affinity for glucose (K_m 0.075 millimolar) than fructose (K_m 2.5 millimolar) and is likely to phosphorylate mainly glucose in vivo. The plant cytosolic fructokinase had a pH optimum of 8.2 and required K⁺ ions for maximum activity. The enzyme was specific for fructose (apparent K_m 0.077 millimolar) but concentrations of fructose greater than 0.4 millimolar were inhibitory. The native molecular weight of fructokinase was 84,000 ± 5,000. The roles of these enzymes in the metabolism of glucose and fructose in the host cytoplasm of soybean nodules are discussed.     

Invertases of Lilium Pollen : Characterization and Activity during In Vitro Germination

Two different forms of invertase are found in pollen of lily (Lilium auratum). One form is cytoplasmic (Invertase 1) and the other is bound to the pollen wall (Invertase 2). Invertase 1 has been partially purified and is a glycoprotein (apparent molecular weight, 450 kilodaltons) with a K_m of 0.65 millimolar for sucrose. The two invertases differ in pH optimum and thermal stability. Invertases of lily pollen are β-fructofuranosidases which can hydrolyze sucrose but not melizitose. The mature pollen grains have enzyme activity in both cytoplasmic and wall fractions, and no increase in the activity of either occurs during germination. The wall-bound enzyme could not be released by treatments with detergents or high salt concentrations.     

Properties of Citrate-stimulated Starch Synthesis Catalyzed by Starch Synthase I of Developing Maize Kernels

Chromatography of extracts of maize on diethylaminoethyl-cellulose resolves starch synthase activity into two fractions (Ozbun, Hawker, Preiss 1971 Plant Physiol 48: 785-769). Only starch synthase I is capable of synthesis in the absence of added primer and the presence of 0.5 molar citrate. This enzyme fraction has been purified about 1,000-fold from maize kernels homozygous for the endosperm mutant amylose-extender (ae). Because ae endosperm lacks the starch-branching enzyme which normally purifies with starch synthase I, the final enzyme fraction was free of detectable branching enzyme activity. This allowed a detailed characterization of the citrate-stimulated reaction. The citrate-stimulated reaction was dependent upon citrate concentrations of greater than 0.1 molar. However, the reaction is not specific for citrate and malate also stimulated the reaction. Branching enzyme increased the velocity of the reaction about 4-fold but did not replace the requirement for citrate. Citrate reduced the K_m for the primers amylopectin and glycogen from 122 and 595 micrograms per milliliter, respectively, to 6 and 50 micrograms per milliliter, respectively. The enzyme was found to contain 1.7 milligrams of anhydroglucose units per enzyme unit. Thus reaction mixtures contained 1 to 5 micrograms (5 to 25 micrograms per milliliter) of endogenous primer. The citrate-stimulated reaction could be explained by an increased affinity for this endogenous primer. The starch synthase reaction in the absence of primer is dependent upon several factors including endogenous primer concentration, citrate concentration as well as branching enzyme concentration.     

Multiple forms of (1 → 4)-α-d-glucan, (1 → 4)-α-d-glucan-6- glycosyl transferase from developing zea mays L. Kernels

Two major forms of branching enzyme from developing kernels of maize have been detected after DEAE-cellulose chromatography. Branching-enzyme I, which contained 24% of the activity based on a phosphorylase-stimulation assay, but 74% of the activity based on the branching of amylose as monitored by change in spectra of the iodine-glucan complex, eluted with the column wash and was unassociated with starch-synthase activity. Branching-enzyme II was bound to DEAE-cellulose and was coeluted with both primed and unprimed starch-synthase activities. Both fractions were further purified by chromatography on aminoalkyl-Sepharose columns. Single peaks were observed for both fractions by gel filtration on BioGel A_1.5m columns and native molecular weights were estimated at 70,000–90,000 for both enzymes. Subunit molecular weights of branching-enzymes I and II were estimated by dodecyl sodium sulfate-gel electrophoresis at 89,000 and 80,000, respectively. Thus both enzymes are primarily monomeric. Branching-enzymes I and II could be distinguished by chromatography on DEAE-cellulose or 4-aminobutyl-Sepharose, and by disc-gel electrophoresis with activity staining. Branching-enyme I had a lower ratio of activity (phosphorylase stimulation-amylose branching; based on enzyme units). The ratio varied from 30–60 as compared to about 300–500 for branching-enzyme II. Likewise, branching-enzyme I had a lower K_m value for amylose than branching- enzyme II, the values being 160 and 500 μg/ml, respectively. Both enzymes could introduce further branches into amylopectin, as decreases in the overall absorption and wavelength maxima of the iodine complexes were observed. Combined action of the branching enzymes and rabbit-muscle phosphorylase a (12:1 ratio based on enzyme units) resulted in similar patterns of incorporation of d-glucose into the growing α-d-glucan and the synthesis of high molecular-weight polymers. However, the α-d-glucans differed, as shown by spectra of iodine complexes and average unit-chain length. Branching-enzyine II was separated into two fractions (IIa and IIb) by chromatography on 4-aminobutyl-Sepharose. These Fractions differed only in the branching of amylopectin, fractional IIb being more active than IIa.