Liver phosphorylase kinase: characterization of two interconvertible forms and partial purification of phosphorylase kinase α

Summary The presence of two interconvertible forms of phosphorylase kinase has been confirmed in rat liver extracts. The pH optimum of the nonactivated form (PhK b) was lower than the pH optimum of the activated form (PhK ) as reported by others (2). In the absence of calcium the K_m of PhK for phosphorylase b was 53 + 10 U/ ml with a V_m of 17 = 1 U/gm of tissue. The K_m of PhK for phosphorylase b was 20 + 2 U/ml with a V_m of 65 U/gm. Calcium stimulated both forms of phosphorylase kinase(A_0.5 0.03 M). In the presence of 0.1 M calcium the Km for phosphorylase b of both forms of the enzyme was reduced. In addition, calcium increased the V_m of both forms, but the effect was greater for PhK b than for PhK . The K_m of both forms of phosphorylase kinase for ATP was 0.05 mM and was unaffected by calcium. All of these studies were done using liver phosphorylase b as substrate. Conditions for assaying PhK activity virtually independent of PhK b activity also are indicated. This will enable the monitoring of interconversion reactions in tissue extracts.Phosphorylase kinase a was purified to near homogeneity using DEAE-cellulose, Sepharose 4B gel filtration and ATP affinity chromatography. The molecular weight was approximately 1 × 106. The pII profile, calcium requirements and kinetic constants were the same as those for PhK a in the crude extract.     

Properties of α-glucan phosphorylase from pea chloroplasts

A partially purified preparation of α-glucan phosphorylase was obtained from chloroplasts of Pisum sativum by ion-exchange chromatography and gel filtration. The preparation, in which no other enzyme that metabolized starch or glucose 1 -phosphate could be detected, was characterized. The optimum for phosphorolysis was pH 7.2; at pH 8.0 the activity was reduced by 50%. The preparation showed normal hyperbolic kinetics with the substrates, and catalysed the formation of [¹⁴C]glucose 1-phosphate from ¹⁴C-labelled starch grains from pea chloroplasts. None of the following, generally at 5 and 10 mM, significantly altered the rate of phosphorolysis: glucose, fructose, sucrose, fructose 6-phosphate, fructose 1,6-bisphosphate, dihydroxyacetone phosphate, 3-phosphoglycerate, 2-phosphoglycerate, phosphoenolpyruvate, pyruvate, ATP, ADP, AMP, 6-phosphogluconate, 2-phosphoglycollate, Mg²⁺, dithiothreitol. However, phosphorolysis was inhibited by ADPglucose. Measurements of ADPglucose in leaves and in isolated chloroplasts showed that none could be detected in the dark and suggested that the concentration in the light was high enough to cause a modest inhibition of the phosphorylase. The control of the breakdown of chloroplast starch is discussed.     

Purification of a non-chloroplastic α-glucan phosphorylase from spinach leaves

The non-chloroplastic -glucan phosphorylase (EC 2.4.1.1) from spinach leaves has been purified to homogeneity as revealed by dodecylsulfate gel electrophoresis. Both purification and separation from the chloroplastic phosphorylase were achieved by chromatography on Sepharose-bound dextrin. The chloroplastic phosphorylase did not bind to Sepharose-dextrin and was removed from the column by washing with buffer, as verified by polyacrylamide gel electrophoresis of the buffer eluate and by chromatography of a preparation from isolated intact chloroplasts. The non-chloroplastic phosphorylase did bind to a high extent to Sepharose-dextrin and could be eluted by a dextrin gradient. Based on dodecylsulfate gel electrophoresis and pyridoxal phosphate determination, a molecular weight of about 90,000 was found for the monomer. Molecular-weight determination by porosity density gradient electrophoresis and gel filtration on Sephadex G-200 suggested that the native enzyme is a dimer, as are other phosphorylases.Abbreviations DEAE diethylaminoethyl - EDTA ethylenediamine tetraacetic acid - G1P glucose 1-phosphate - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid - PMSF phenylmethyl sulphonyl fluoride - SDS sodium dodecylsulfate - Tris Tris (hydroxymethyl)aminomethane Dedicated to Professor Dr. A. Pirson on the occasion of his 70th birthday     

Simple radioassay for uridine phosphorylase and 5′-nucleotidase

A simple micromethod was developed for the accurate measurement of the activity of uridine phosphorylase in the direction of both ribosylation and phosphorolysis. The technique was applicable also to the measurement of cytoplasmic 5′-nucleotidase. In this micromethod the radioactive products were separated from the substrates by electrophoresis on cellulose acetate membrane in 0.1 m borate buffer, pH 9.0, at 150 V for 40 min. These micromethods are highly sensitive and capable of utilizing small samples (4–30 μg protein); they are valid in crude tissue extracts of rat liver and hepatoma.     

Dephosphorylation of skeletal muscle phosphorylase,glycogen synthase,and phosphorylase kinase β-subunit by a Mn2+-activated protein phosphatase

An Mn²⁺-activated phosphoprotein phosphatase of M_r = 80,000 from rabbit muscle catalyzes the dephosphorylation of skeletal muscle proteins that are phosphorylated by either phosphorylase kinase or cAMP-dependent protein kinase. Phosphorylase or glycogen synthase labeled by phosphorylase kinase at seryl residues 14 or 7, respectively, are both dephosphorylated by the phosphatase. Phosphorylase a and glycogen synthase compete with one another for the phosphatase. The phosphatase discriminates between different sites labeled by the cAMP-dependent protein kinase: glycogen synthase phosphorylated either to 1.0 or 1.8 mol phosphate/mol, or phosphorylase kinase phosphorylated on its β-subunit serve as substrates for the phosphatase, but the phosphorylase kinase α-subunit, the phosphorylated phosphatase inhibitor 1, or casein do not. Histone fraction IIA, phosphorylated by the catalytic subunit, was a poor substrate even at a concentration of 100 μm. Phosphorylation of the α-subunit of phosphorylase kinase had no influence on the kinetics of dephosphorylation of the β-subunit. Thus, the M_r = 80,000 phosphatase meets the functional definition of a protein phosphatase 1 [Cohen, P. (1978) Curr. Top. Cell. Regul.14, 117–196]. Furthermore, from a comparison of the known phosphorylated sites of these proteins, it appears that the phosphatase discriminates between different sites present in the phosphoproteins tested on the basis of the K_m values for the reactions. It displays a preferential activity toward proteins with a primary structure wherein basic residues are two positions amino-terminal from the phosphoserine, AgrLysX-YSer(P) or LysArgX-YSer(P), rather and one residue away, ArgArgX-Ser(P).     

Glycogen phosphorylase ‘b’ in Dictyostelium: stability and endogenous phosphorylation

Summary The slime mold Dictyostelium discoideum has two forms of the enzyme glycogen phosphorylase. The inactive phosphorylase b form requires 5 AMP for activity and is present in early development. The active phosphorylase a form is 5 AMP independent and occurs during later development. We here show that the 92 kd b enzyme subunit exists either as a singlet or a doublet upon SDS-PAGE, depending on the method of sample extraction. In the presence of exogenously added Mn²⁺ and ATP, the phosphorylase b shows apparent conversion into a 5 AMP independent form as measured by enzyme activity. In addition, Mn²⁺ and ATP also support an in vitro phosphorylation of the 92 kd phosphorylase b subunit. We also demonstrate phosphorylation of the b enzyme subunit in vivo by ³²-P incorporation into the enzyme protein. A protein kinase responsible for the observed in vitro phosphorylation of the phosphorylase b subunit is characterized.     

Characterization of d-galactosyl-β1→4-l-rhamnose phosphorylase from Opitutus terrae

We characterized a glycoside hydrolase family 112 protein from Opitutus terrae (Oter_1377 protein). The enzyme phosphorolyzed d-galactosyl-β1→4-l-rhamnose (GalRha) and also showed phosphorolytic activity on d-galactosyl-β1→3-d-glucose as a minor substrate. In the reverse reaction, the enzyme showed higher activity on l-rhamnose derivatives than on d-glucose derivatives. The enzyme was stable up to 45 °C and at pH 6.0–7.0. The values of k_cat and K_m of the phosphorolytic activity of the enzyme on GalRha were 60 s^?1 and 2.1 mM, respectively. Thus, Oter_1377 protein was identified as d-galactosyl-β1→4-l-rhamnose phosphorylase (GalRhaP). The presence of GalRhaP in O. terrae suggests that genes encoding GalRhaP are widely distributed in different organisms.     

Continuous production of α,α-trehalose by immobilised fungal trehalose phosphorylase

Immobilisation of trehalose phosphorylase from Schizophyllum commune by adsorption onto anion-exchange materials stabilised the enzyme activity at 30°C by approx. 35-fold. Immobilised and free enzymes showed similar pH-dependence of activity but different inactivation behavior above 30°C. A fixed-bed enzyme reactor produced ,-trehalose at a stable substrate conversion of 80% with a productivity of 2.6 g l–1 h–1 for 72 h. Inhibition of trehalose phosphorylase by phosphate limited the productivity of a direct conversion of starch into ,-trehalose.     

Improved histochemical method for the demonstration of the activity of α-glucan phosphorylase

Summary The activity and localization of -glucan phosphorylase in experimental canine glycogen-depleted heart tissue has been investigated with biochemical and histochemical methods using dextran as enzyme acceptor. Only linear, essentially unbranched, dextrans exhibit acceptor properties; highly branched dextrans are not suitable acceptors for the enzyme. Results of Michaelis-Menten constant measurements for the linear essentially unbranched dextran fractions used, indicate that the affinity of the enzyme for the non-reducing end group of the dextran molecule increases with increasing molecular weight of the acceptor.In the glycogen-depleted tissue of anoxic and ischaemic cardiac musculature there is a gradual inactivation of the enzyme during the ischaemic period. Shortly before total inactivation the affinity of the enzyme, especially for the lower molecular dextran fractions, is greatly reduced. Therefore, for the histochemical demonstration of phosphorylase activity in infarcted areas of the heart it is essential to use as acceptor an unbranched dextran fraction with a high average molecular weight.This investigation was partially supported by a grant from the Netherlands Organization for the Advancement of Pure Research (Z.W.O.).     

Formation of α-D-glucose-1-phosphate by thermophilic α-1,4-D-glucan phosphorylase

For the production of α-D-glucose-1-phosphate (G-1-P), α-1,4-D-glucan phosphorylase from Thermus caldophilus GK24 was partially purified to a specific activity of 13 U mg⁻¹ and an enzyme recovery of 15%. The amount of G-1-P reached maximum (18%) when soluble starch was used as substrate, and the smallest substrate for G-1-P formation was maltotriose. The structure of purified G-1-P was confirmed by comparison to ¹³C-NMR data for an authentic sample. In addition to G-1-P, glucose-6-phosphate (12%) was simultaneously produced when 10 mM maltoheptaose was used as substrate. Journal of Industrial Microbiology & Biotechnology (2000) 24, 89–93. Received 12 May 1999/ Accepted in revised form 29 August 1999     

Rice endosperm-specific plastidial α-glucan phosphorylase is important for synthesis of short-chain malto-oligosaccharides

Previous genetic studies have indicated that the type L α-glucan phosphorylase (Pho1) has an essential role during the initiation process of starch biosynthesis during rice seed development. To gain insight into its role in starch metabolism, we characterized the enzymatic properties of the Pho1 recombinant form. Pho1 has significantly higher catalytic efficiency toward both linear and branched α-glucans in the synthesis direction than in the degradation direction with equilibrium constants for the various substrates ranging from 13 to 45. Pho1 activity is strongly inhibited by its own reaction product (Pi) in the synthesis reaction (K_i = 0.69 mM) when amylopectin is the primer substrate, but this inhibition is less pronounced (K_i = 14.2 mM) when short α-glucan chains are used as primers. Interestingly, even in the presence of Pi alone, Pho1 not only degrades maltohexaose but also extends them to synthesize longer MOSs. Production of a broad spectrum of MOSs (G4-G19) was stimulated by both Pi and Glc1P in an additive fashion. Thus, even under physiological conditions of high Pi/Glc1P, Pho1 extends the chain length of short MOSs which can then be used as subsequent primer by starch synthase activities. As ADP-glucose strongly inhibits Pho1activity, Pho1 likely operates only during the initial stage and not during maturation phase of starch synthesis.     

Improved histochemical method for the demonstration of the activity of α-glucan phosphorylase

Summary A modified method for the histochemical demonstration of the activity of -glucan phosphorylase is described. In the histochemical system the enzyme catalyses the synthesis of glycogen by transglucosylation from -d-glucosylphosphate. The incubation medium contains dextran as glucosyl acceptor. Therefore, in contrast with the unmodified method, the new technique is able to demonstrate the activity of phosphorylase in ischaemic glycogen-depleted tissues.     

Multiple forms of α-glucan phosphorylase in banana fruits: Properties and kinetics

Multiple forms of α-glucan phosphorylase isolated from mature banana fruit pulp differ from each other in several respects—pH optimun, temperature optimum, energy of activation, primer specificity, kinetics, and sensitivity towards metal ions, nucleotides, sugar mucleotides, amino acids, glycolytic intermediates, sulfhydryl binding agents, sulfhydryl reagents and phenolics.     

The binding of β-d-glucopyranosyl-thiosemicarbazone derivatives to glycogen phosphorylase: A new class of inhibitors

Glycogen phosphorylase (GP) is a promising target for the treatment of type 2 diabetes. In the process of structure based drug design for GP, a group of 15 aromatic aldehyde 4-(β-d-glucopyranosyl)thiosemicarbazones have been synthesized and evaluated as inhibitors of rabbit muscle glycogen phosphorylase b (GPb) by kinetic studies. These compounds are competitive inhibitors of GPb with respect to α-d-glucose-1-phosphate with IC₅₀ values ranging from 5.7 to 524.3 μM. In order to elucidate the structural basis of their inhibition, the crystal structures of these compounds in complex with GPb at 1.95–2.23 Å resolution were determined. The complex structures reveal that the inhibitors are accommodated at the catalytic site with the glucopyranosyl moiety at approximately the same position as α-d-glucose and stabilize the T conformation of the 280s loop. The thiosemicarbazone part of the studied glucosyl thiosemicarbazones possess a moiety derived from substituted benzaldehydes with NO₂, F, Cl, Br, OH, OMe, CF₃, or Me at the ortho-, meta- or para-position of the aromatic ring as well as a moiety derived from 4-pyridinecarboxaldehyde. These fit tightly into the β-pocket, a side channel from the catalytic site with no access to the bulk solvent. The differences in their inhibitory potency can be interpreted in terms of variations in the interactions of the aldehyde-derived moiety with protein residues in the β-pocket. In addition, 14 out of the 15 studied inhibitors were found bound at the new allosteric site of the enzyme.     

Bioprocess development to produce a hyperthermostable S-methyl-5′-thioadenosine phosphorylase in Escherichia coli

Nucleoside phosphorylases are important biocatalysts for the chemo-enzymatic synthesis of nucleosides and their analogs which are, among others, used for the treatment of viral infections or cancer. S-methyl-5′-thioadenosine phosphorylases (MTAP) are a group of nucleoside phosphorylases and the thermostable MTAP of Aeropyrum pernix (ApMTAP) was described to accept a wide range of modified nucleosides as substrates. Therefore, it is an interesting biocatalyst for the synthesis of nucleoside analogs for industrial and therapeutic applications. To date, thermostable nucleoside phosphorylases were produced in shake flask cultivations using complex media. The drawback of this approach is low volumetric protein yields which hamper the wide-spread application of the thermostable nucleoside phosphorylases in large scale. High cell density (HCD) cultivations allow the production of recombinant proteins with high volumetric yields, as final optical densities >100 can be achieved. Therefore, in this study, we developed a suitable protocol for HCD cultivations of ApMTAP. Initially, optimum expression conditions were determined in 24-well plates using a fed-batch medium. Subsequently, HCD cultivations were performed using E. coli BL21-Gold cells, by employing a glucose-limited fed-batch strategy. Comparing different growth rates in stirred-tank bioreactors, cultivations revealed that growth at maximum growth rates until induction resulted in the highest yields of ApMTAP. On a 500-mL scale, final cell dry weights of 87.1–90.1 g L⁻¹ were observed together with an overproduction of ApMTAP in a 1.9%–3.8% ratio of total protein. Compared to initially applied shake flask cultivations with terrific broth (TB) medium the volumetric yield increased by a factor of 136. After the purification of ApMTAP via heat treatment and affinity chromatography, a purity of more than 90% was determined. Activity testing revealed specific activities in the range of 0.21 ± 0.11 (low growth rate) to 3.99 ± 1.02 U mg⁻¹ (growth at maximum growth rate). Hence, growth at maximum growth rate led to both an increased expression of the target protein and an increased specific enzyme activity. This study paves the way towards the application of thermostable nucleoside phosphorylases in industrial applications due to an improved heterologous expression in Escherichia coli.     

Synthesis and structure–activity relationships of C-glycosylated oxadiazoles as inhibitors of glycogen phosphorylase

A series of per-O-benzoylated 5-β-d-glucopyranosyl-2-substituted-1,3,4-oxadiazoles was prepared by acylation of the corresponding 5-(β-d-glucopyranosyl)tetrazole. As an alternative, oxidation of 2,6-anhydro-aldose benzoylhydrazones by iodobenzene I,I-diacetate afforded the same oxadiazoles. 1,3-Dipolar cycloaddition of nitrile oxides to per-O-benzoylated β-d-glucopyranosyl cyanide gave the corresponding 5-β-d-glucopyranosyl-3-substituted-1,2,4-oxadiazoles. The O-benzoyl protecting groups were removed by base-catalyzed transesterification. The 1,3,4-oxadiazoles were practically inefficient as inhibitors of rabbit muscle glycogen phosphorylase b while the 1,2,4-oxadiazoles displayed inhibitory activities in the micromolar range. The best inhibitors were the 5-β-d-glucopyranosyl-3-(4-methylphenyl- and -2-naphthyl)-1,2,4-oxadiazoles (K_i = 8.8 and 11.6 μM, respectively). A detailed analysis of the structure–activity relationships is presented.     

Thermophilic α-glucan phosphorylase from Clostridium thermocellum: Cloning,characterization and enhanced thermostability

ORF Cthe0357 from the thermophilic bacterium Clostridium thermocellum ATCC 27405 that encodes a putative α-glucan phosphorylase (αGP) was cloned and expressed in Escherichia coli. The protein with a C-terminal His-tag was purified by Ni²⁺ affinity chromatography; the tag-free protein obtained from a cellulose-binding module–intein–αGP fusion protein was purified through affinity adsorption on amorphous cellulose followed by intein self-cleavage. Both purified enzymes had molecular weights of ca. 81,000 and similar specific activities. The optimal conditions were pH 6.0–6.5 and 60 °C for the synthesis direction and pH 7.0–7.5 and 80 °C for the degradation direction. This enzyme had broad substrate specificities for different chain length dextrins and soluble starch. The thermal inactivation of this enzyme strongly depended on temperature, protein concentration, and certain addictives that were shown previously to benefit the protein thermostability. The half lifetime of 0.05 mg αGP/mL at 50 °C was extended by 45-fold to 90 h through a combined addition of 0.1 mM Mg²⁺, 5 mM DTT, 1% NaCl, 0.1% Triton X-100, and 1 mg/mL BSA. The enzyme with prolonged stability would work as a building block for cell-free synthetic enzymatic pathway biotransformations, which can implement complicated biocatalysis through assembly of a number of enzymes and coenzymes.     

Characterization of multiple forms of α-glucan phosphorylase from Musa paradisiaca fruits

Three forms of α-glucan phosphorylase from mature banana fruit pulp separated by ammonium sulfate fractionation and DEAE-cellulose chromatography were anodic at pH 8·6 on starch gel electrophoresis. The three forms differed in sensitivity to the phenolics extracted from immature and mature banana fruit pulp. Only two forms of the enzyme were detected in immature banana fruit pulp.