Discovery and biochemical characterization of a mannose phosphorylase catalyzing the synthesis of novel ß-1,3-mannosides.

BACKGROUND: Mannoside phosphorylases are frequently found in bacteria and play an important role in carbohydrate processing. These enzymes catalyze the reversible conversion of ß-1,2- or ß-1,4-mannosides to mannose and mannose-1-phosphate in the presence of inorganic phosphate. METHODS: The biochemical parameters of this recombinantly expressed novel mannose phosphorylase were obtained. Furthermore purified reaction products were subjected to ESI- and MALDI-TOF mass spectrometry and detailed NMR analysis to verify this novel type of ß-1,3-mannose linkage. RESULTS: We describe the first example of a phosphorylase specifically targeting ß-1,3-mannoside linkages. In addition to mannose, this phosphorylase originating from the bacterium Zobellia galactanivorans could add ß-1,3-linked mannose to various other monosaccharides and anomerically modified 5-bromo-4-chloro-3-indolyl-glycosides (X-sugars). CONCLUSIONS: An unique bacterial phosphorylase specifically targeting ß-1,3-mannoside linkages was discovered. GENERAL SIGNIFICANCE: Functional extension of glycoside hydrolase family 130.

Synthesis of Non-Natural Heteroaminopolysaccharides by α-Glucan Phosphorylase-Catalyzed Enzymatic Copolymerization: α(1→4)-Linked Glucosaminoglucans.

Thermostable α-glucan phosphorylase-catalyzed enzymatic copolymerization of α-d-glucose 1-phosphate (Glc-1-P) with its analogue monomer, α-d-glucosamine 1-phosphate (GlcN-1-P), from a maltotriose primer was performed to produce non-natural heteroaminopolysaccharides composed of Glc/GlcN units, that is, α(1→4)-linked glucosaminoglucans. The GlcN units in the products were further converted to N-acetyl-d-glucosamine (GlcNAc) units by N-acetylation. The structures of the products were evaluated by the MALDI-TOF MS, (1)H NMR, and (1)H-(1)H COSY NMR measurements, which were completely different from those of the natural glycosaminoglycans. The degrees of polymerization and Glc/GlcN compositional ratios of the products were relatively dependent on the Glc-1-P/Glc-1-P/Glc3 feed ratios. The noncrystalline natures of the present materials were supported by the X-ray diffraction measurement.

Purification and characterization of 1,3-ß-D-glucan phosphorylase from Ochromonas danica.

1,3-ß-D-glucan phosphorylase (BGP) is an enzyme that catalyzes the reversible phosphorolysis of 1,3-ß-glucosidic linkages to form α-D-glucose 1-phosphate (G1P). Here we report on the purification and characterization of BGP from Ochromonas danica (OdBGP). The purified enzyme preparation showed three bands (113, 118, and 124 kDa) on SDS-polyacrylamide gel electrophoresis. The optimum pH and temperature were 5.5 and 25 °C-30 °C. OdBGP phosphorolysed laminaritriose, larger laminarioligosaccharides, and laminarin, but not laminaribiose. In the synthesis reaction, laminarin and laminarioligosaccharides served as good acceptors, but OdBGP did not act on glucose. Kinetic analysis indicated that the phosphorolysis reaction of OdBGP follows a sequential Bi Bi mechanism. The equilibrium of the enzymatic reaction indicated that OdBGP favors the reaction in the synthetic direction. Overnight incubation of OdBGP with laminaribiose and G1P resulted in the formation of precipitates, which were probably 1,3-ß-glucans.

3-O-α-D-glucopyranosyl-L-rhamnose phosphorylase from Clostridium phytofermentans.

We found an unreported activity of phosphorylase catalyzed by a protein (Cphy1019) belonging to glycoside hydrolase family 65 (GH65) from Clostridium phytofermentans. The recombinant Cphy1019 produced in Escherichia coli did not phosphorolyze α-linked glucobioses, such as trehalose (α1-α1), kojibiose (α1-2), nigerose (α1-3), and maltose (α1-4), which are typical substrates for GH65 enzymes. In reverse phosphorolysis, Cphy1019 utilized only l-rhamnose as the acceptor among various sugars examined with ß-d-glucose 1-phosphate as the donor. The reaction product was determined to be 3-O-α-d-glucopyranosyl-l-rhamnose, indicating strict α1-3 regioselectivity. We propose 3-O-α-d-glucopyranosyl-l-rhamnose: phosphate ß-d-glucosyltransferase as the systematic name and 3-O-α-d-glucopyranosyl-l-rhamnose phosphorylase as the short name for this novel GH65 phosphorylase.

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

Previous genetic studies have indicated that the type L alpha-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 alpha-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 alpha-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 Pho1 activity, Pho1 likely operates only during the initial stage and not during maturation phase of starch synthesis.

Kinetic studies of HPr, HPr(H15D), HPr(H15E), and HPr(His approximately P) phosphorylation by the Streptococcus salivarius HPr(Ser) kinase/phosphorylase.

HPr is a central protein of the phosphoenolpyruvate:sugar phosphotransferase transport system (PTS). In streptococci, HPr can be phosphorylated at His(15) at the expense of PEP by enzyme I (EI) of the PTS, producing HPr(His approximately P). HPr can also be phosphorylated at Ser(46) by the ATP-dependent HPr(Ser) kinase/phosphorylase (HprK/P), producing HPr(Ser-P). Lastly, HPr can be phosphorylated on both residues, producing HPr(Ser-P)(His approximately P) (HPr-P2). We report here a study on the phosphorylation of Streptococcus salivarius HPr, HPr(H15D), HPr(H15E), and HPr(His approximately P) by HprK/P to assess the involvement of HprK/P in the synthesis of HPr-P2 in streptococcal cells. We first developed a spectrophotometric method for measuring HprK/P kinase activity. Using this assay, we found that the K(m) of HprK/P for HPr at pH 7.4 and 37 degrees C was approximately 110 muM, with a specificity constant (k(cat)/K(m)) of 1.7 x 10(4) M(-1) s(-1). The specificity constants for HPr(H15D) and HPr(H15E) were approximately 13 times lower. Kinetic studies conducted under conditions where HPr(His approximately P) was stable (i.e., pH 8.6 and 15 degrees C) showed that HPr(His approximately P) was a poorer substrate for HprK/P than HPr(H15D), the k(cat)/K(m) for HPr(H15D) and HPr(His approximately P) being approximately 9 and 26 times lower than that for HPr, respectively. Our results suggested that (i) the inefficiency of the phosphorylation of HPr(His approximately P) by HprK/P results from the presence of a negative charge at position 15 as well as from other structural elements and (ii) the contribution of streptococcal HprK/P to the synthesis of HPr-P2 in vivo is marginal.

Simple protein purification through affinity adsorption on regenerated amorphous cellulose followed by intein self-cleavage.

A simple, low-cost, and scalable protein purification method was developed by using a biodegradable regenerated amorphous cellulose (RAC) with a binding capacity of up to 365 mg protein per gram of RAC. The recombinant protein with a cellulose-binding module (CBM) tag can be specifically adsorbed by RAC. In order to avoid using costly protease and simplify purification process, a self-cleavage intein was introduced between CBM and target protein. The cleaved target protein can be liberated from the surface of RAC by intein self-cleavage occurring through a pH change from 8.0 to 6.5. Four recombinant proteins (green fluorescence protein, phosphoglucomutase, cellobiose phosphorylase, and glucan phosphorylase) have been purified successfully.

Identification of galacto-N-biose phosphorylase from Clostridium perfringens ATCC13124.

Lacto-N-biose phosphorylase (LNBP) from bifidobacteria is involved in the metabolism of lacto-N-biose I (Galbeta1-->3GlcNAc, LNB) and galacto-N-biose (Galbeta1-->3GalNAc, GNB). A homologous gene of LNBP (CPF0553 protein) was identified in the genome of Clostridium perfringens ATCC13124, which is a gram-positive anaerobic intestinal bacterium. In the present study, we cloned the gene and compared the substrate specificity of the CPF0553 protein with LNBP from Bifidobacterium longum JCM1217 (LNBPBl). In the presence of alpha-galactose 1-phosphate (Gal 1-P) as a donor, the CPF0553 protein acted only on GlcNAc and GalNAc, and GalNAc was a more effective acceptor than GlcNAc. The reaction product from GlcNAc/GalNAc and Gal 1-P was identified as LNB or GNB. The CPF0553 protein also phosphorolyzed GNB much faster than LNB, which suggests that the protein should be named galacto-N-biose phosphorylase (GNBP). GNBP showed a kcat/Km value for GNB that was approximately 50 times higher than that for LNB, whereas LNBPBl showed similar kcat/Km values for both GNB and LNB. Because C. perfringens possesses a gene coding endo-alpha-N-acetylgalactosaminidase, GNBP may play a role in the intestinal residence by metabolizing GNB that is available as a mucin core sugar.

[Expression, purification and crystallization of a truncated Saccharomyces cerevisiae diadenosion 5',5'''-P1,P4 -tetraphosphate phosphorylase I].

OBJECTIVE: To obtain the crystal of 5',5'''-P1,P4-tetraphosphate phosphorylase I (Apal) of Saccharomyces cerevisiae for X-ray crystal structure and function analysis. METHODS: We amplified the coding region of an N-terminally truncated version of Saccharomyces cerevisiae diadenosion 5',5'''-P1,P4-tetraphosphate phosphorylase I (Apaldnl6) , and cloned it into the pET28 derived expression vector. After having screened the recombinant plasmids by PCR and confirmed them by DNA sequencing, we transformed a positive recombinant plasmid into the Escherichia coli BL21(DE3) cells for efficient expression. Then the expression and solubility of the recombinant Apaldnl6 protein were analyzed by SDS-PAGE after proper concentration of IPTG induction. Following that, we collected the soluble Apaldnl6 protein and purified it to homogeneity by sequential Ni-NTA affinity chromatography and Superdex 75 gel filtration, and then detected the purity and molecular weight of the desired protein by SDS-PAGE and mass spectrometry . In addition, we screened the crystallization conditions of Apaldnl6 with Hampton Research kits using the hanging drop vapor diffusion method. RESULTS: We efficiently expressed an N-terminally truncated Saccharomyces cerevisiae diadenosion 5',5'''-P1, P4-tetraphosphate phosphorylase I in Escherichia coli BL21(DE3). The recombinant protein was partially soluble and was purified to homogeneity with a single band of approximately 36 kDa after SDS-PAGE. Mass spectrometry analysis further confirmed the purity and intactness of the recombinant protein.Moreover, we obtained the needle crystals of Apaldnl6 by hanging drop vapor diffusion method. CONCLUSION: Escherichia coli BL21(DE3) is an efficient expression system for producing enough quantity of Apaldnl6 protein. The purified recombinant Apaldnl6 protein is suitable for crystallization and further structural investigation.

Cloning and characterization of the HPr kinase/phosphorylase gene from Bacillus stearothermophilus No. 236.

The Bacillus stearothermophilus no. 236 gene encoding the bifunctional enzyme HprK/P, the key regulator of carbon catabolite repression/activation (CCR/CCA) in most Gram-positive bacteria, was cloned and the (His)(6)-tagged gene product was characterized in detail. The nucleotide sequence of the hprK/P gene corresponded to an open reading frame of 951 bp that encoded a polypeptide of 316 amino acid residues with a calculated molecular mass of 35,458 Da. The deduced amino acid sequence of the B. stearothermophilus no. 236 HprK/P showed 64.5% identity with the B. subtilis enzyme, allowing us to identify two highly conserved motifs, the nucleotide binding P-loop (Walker motif A) and the HprK/P family signature sequence in the C-terminal half of the protein. Furthermore, complementation experiments showed that the cloned hprK/P gene product was functionally active in the B. subtilis cells. The purified (His)(6)-tagged B. stearothermophilus no. 236 HprK/P migrated on SDS-PAGE gel as a single species with a molecular mass of about 36 kDa, and behaved in gel filtration like a hexameric protein. The recombinant protein catalyzes the pyrophosphate (PPi)-dependent (highest activity at pH 7.0 and 40 degrees C) as well as the ATP-dependent phosphorylation of Ser46 in HPr (maximum activity at pH 8.0 and 45 degrees C). It also catalyzes the inorganic phosphate-dependent dephosphorylation (phosphorolysis) of seryl-phosphorylated HPr, optimally at pH 6.5 and 40 degrees C. BIAcore surface resonance analysis confirmed that a divalent cation, preferentially Mg(2+), was an indispensable cofactor for the three activities of the HprK/P. Fructose-1,6-bisphosphate (FBP) was observed to stimulate ATP-dependent kinase activity, while inorganic phosophate (Pi) inhibited ATP-dependent kinase activity. Mutations in the Walker motif A simultaneously abolished both types of kinase and phosphorylase activities. On the other hand, the conserved signature residues were confirmed to be involved in the PPi-dependent kinase and phosphorylase reactions.

Novel putative galactose operon involving lacto-N-biose phosphorylase in Bifidobacterium longum.

A lacto-N-biose phosphorylase (LNBP) was purified from the cell extract of Bifidobacterium bifidum. Its N-terminal and internal amino acid sequences were homologous with those of the hypothetical protein of Bifidobacterium longum NCC2705 encoded by the BL1641 gene. The homologous gene of the type strain B. longum JCM1217, lnpA, was expressed in Escherichia coli to confirm that it encoded LNBP. No significant identity was found with any proteins with known function, indicating that LNBP should be classified in a new family. The lnpA gene is located in a novel putative operon for galactose metabolism that does not contain a galactokinase gene. The operon seems to be involved in intestinal colonization by bifidobacteria mediated by metabolism of mucin sugars. In addition, it may also resolve the question of the nature of the bifidus factor in human milk as the lacto-N-biose structure found in milk oligosaccharides.

Enzymatic synthesis of a 2-O-alpha-D-glucopyranosyl cyclic tetrasaccharide by kojibiose phosphorylase.

The glucosyl transfer reaction of kojibiose phosphorylase (KPase) from Thermoanaerobacter brockii ATCC35047 was examined using cyclo-{-->6)-alpha-d-Glcp-(1-->3)-alpha-d-Glcp-(1-->6)-alpha-d-Glcp-(1-->3)-alpha-d-Glcp-(1-->} (CTS) as an acceptor. KPase produced four transfer products, saccharides 1-4. The structure of a major product, saccharide 4, was 2-O-alpha-d-glucopyranosyl-CTS, cyclo-{-->6)-alpha-d-Glcp-(1-->3)-alpha-d-Glcp-(1-->6)-[alpha-d-Glcp-(1-->2)]-alpha-d-Glcp-(1-->3)-alpha-d-Glcp-(1-->}. The other transfer products, saccharides 1-3, were 2-O-alpha-kojibiosyl-, 2-O-alpha-kojitriosyl-, and 2-O-alpha-kojitetraosyl-CTS, respectively. These results showed that KPase transferred a glucose residue to the C-2 position at the ring glucose residue of CTS. This enzyme also catalyzed the chain-extending reaction of the side chain of 2-O-alpha-d-glycopyranosyl-CTS.

Purification and characterization of the maize amyloplast stromal 112-kDa starch phosphorylase.

A plastidic 112-kDa starch phosphorylase (SP) has been identified in the amyloplast stromal fraction of maize. This starch phosphorylase was purified 310-fold from maize endosperm and characterized with respect to its enzymological and kinetic properties. The purification procedure included ammonium sulfate fractionation, Sephacryl 300 HR chromatography, affinity starch adsorption, Q-Sepharose, and Mono Q chromatography. The procedure resulted in a nearly homogeneous enzyme preparation as determined by native and SDS-polyacrylamide gel electrophoresis. Anti-SP antibodies recognized the purified 112-kDa SP enzyme and N-terminal amino acid sequence analysis confirmed that the purified enzyme is the amyloplast stromal 112-kDa SP. Analysis of the purified enzyme by Superose 6 gel filtration chromatography indicated that the native enzyme consisted of two identical subunits. The pH optimum for the enzyme was 6.0 in the synthetic direction and 5.5 in the phosphorolytic direction. SP activity was inhibited by thioreactive agents, diethyl pyrocarbonate, phenylglyoxal, and ADP-glucose. The activation energies for the synthetic and phosphorolytic reactions were 11.1 and 16.9 kcal/mol, respectively, and the enzyme was thermally labile above 50 degrees C. Results of kinetic experiments indicated that the enzyme catalyzes its reaction via a sequential Bi Bi mechanism. The Km value for amylopectin was eight-fold lower than that of glycogen. A kinetic analysis indicated that the phosphorolytic reaction was favored over the synthetic reaction when malto-oligosaccharides (4 to 7 units) were used as substrates. The specificity constants (Vmax/Km) of the enzyme measured in either the synthetic or the phosphorolytic directions increased with increasing chain length.

The glycogen phosphorylase-2 promoter binding protein in Dictyostelium is replication protein A.

During Dictyostelium development, glycogen degradation is a crucial event that provides glucose monomers used in the synthesis of the essential structural components for cellular differentiation. The product of the developmentally regulated glycogen phosphorylase-2 gene (gp2) catalyzes the degradation. DNA-binding proteins were found to bind to a regulatory site of the gp2 gene in a stage-dependent pattern. Gel-shift analysis of undifferentiated amoebae cell extract revealed a protein migrating at 0.40 Rf, while 17 hour differentiated cell extract produced a species migrating at 0.32 Rf. Both the 0.32 and 0.40 Rf proteins were purified and found to consist of three subunits of 18, 35 and 62 kDa (for 0.40 Rf) or 81 kDa (for 0.32 Rf). Data base searches identified the protein as the Dictyostelium homologue of replication protein A (DdRPA). Amino acid sequence analysis showed identity between the 62 and 81 kDa subunits. Incubation of cell-free extracts under appropriate conditions at low pH, resulted in conversion of the 81 kDa to the 62 kDa subunit. Northern blot analysis revealed that the levels of expression of the large subunit of DdRPA were constant throughout differentiation and the size of the mRNA was the same at all stages of development. The results raise the possibility that pH induced post-translational modifications of DdRPA are involved in events that halt cell proliferation and induce differentiation in Dictyostelium.

alpha-1,4-D-glucan phosphorylase of gram-positive Corynebacterium callunae: isolation, biochemical properties and molecular shape of the enzyme from solution X-ray scattering.

The alpha-1,4-D-glucan phosphorylase from gram-positive Corynebacterium callunae has been isolated and characterized. The enzyme is inducible approx. 2-fold by maltose, but remarkably not repressed by D-glucose. The phosphorylase is a homodimer with a stoichiometric content of the cofactor pyridoxal 5'-phosphate per 88-kDa protein subunit. The specificity constants (kcat/Km, glucan) in the directions of glucan synthesis and degradation are used for the classification of the enzyme as the first bacterial starch phosphorylase. A preference for large over small substrates is determined by variations in the apparent binding constants rather than catalytic-centre activities. The contribution of substrate chain length to binding energy is explained assuming two glucan binding sites in C. callunae phosphorylase: an oligosaccharide binding site composed of five subsites and a high-affinity polysaccharide site separated from the active site. A structural model of the molecular shape of the phosphorylase was obtained from small-angle solution X-ray scattering measurements. A flat, slightly elongated, ellipsoidal model with the three axes related to each other as 1:(0.87-0.95):0.43 showed scattering equivalence with the enzyme molecule. The model of C. callunae phosphorylase differs from the structurally well-characterized rabbit-muscle phosphorylase in size and axial dimensions.

Purification and characterisation of an alpha-glucan phosphorylase from the thermophilic bacterium Thermus thermophilus.

An alpha-glucan phosphorylase has been purified 4500-fold from the thermophilic bacteria Thermus thermophilus. In contrast to other bacterial phosphorylases the thermophilic enzyme seems neither to be inducible by maltose nor repressed by glucose. T. thermophilus phosphorylase shares major properties with known mesophilic phosphorylases such as pyridoxal 5'-phosphate content (1 M pyridoxal-P/M subunit), subunit molecular mass (about 90 kDa) and inhibitor constants. The optimum temperature of T. thermophilus phosphorylase was observed at 70 degrees C in the pH range 5.5-6.5. While at 25 degrees C the subunit composition of the thermophilic enzyme is an octameric form, the preferential form at the optimum temperature of 70 degrees C seems to be a dimer. Most remarkably, in the direction of synthesis and degradation the limiting size of the oligosaccharide substrate is shorter by one glucose residue than the minimum size of substrate degraded by other alpha-glucan phosphorylases. Maltotetraose and glycogen are degraded with rates similar to that observed with maltoheptaose (Vmax = 18 U/mg). Correspondingly, maltotriose functions as primer in the synthesis direction. Differences in fluorescence and absorption spectra of the cofactor and the failure of arsenate acting as a substrate indicate that the active site structure of T. thermophilus phosphorylase differs from that of known alpha-glucan phosphorylases.

Chimeric muscle and brain glycogen phosphorylases define protein domains governing isozyme-specific responses to allosteric activation.

Muscle and brain glycogen phosphorylases differ in their responses to activation by phosphorylation and AMP. The muscle isozyme is potently activated by either phosphorylation or AMP. In contrast, the brain isozyme is poorly activated by phosphorylation and its phosphorylated a form is more sensitive to AMP activation when enzyme activity is measured in substrate concentrations and temperatures encountered in the brain. The nonphosphorylated b form of the brain isozyme also differs from the muscle isozyme b form in its stronger affinity and lack of cooperativity for AMP. To identify the structural determinants involved, six enzyme forms, including four chimeric enzymes containing exchanges in amino acid residues 1-88, 89-499, and 500-842 (C terminus), were constructed from rabbit muscle and human brain phosphorylase cDNAs, expressed in Escherichia coli, and purified. Kinetic analysis of the b forms indicated that the brain isozyme amino acid 1-88 and 89-499 regions each contribute in an additive fashion to the formation of an AMP site with higher intrinsic affinity but weakened cooperativity, while the same regions of the muscle isozyme each contribute to greater allosteric coupling but weaker AMP affinity. Kinetic analysis of the a forms indicated that the amino acid 89-499 region correlated with the reduced response of the brain isozyme to activation by phosphorylation and the resultant increased sensitivity of the a form to activation by saturating levels of AMP. This isozyme-specific response also correlated with the glycogen affinity of the a forms. Enzymes containing the brain isozyme amino acid 89-499 region exhibited markedly reduced glycogen affinities in the absence of AMP compared to enzymes containing the corresponding muscle isozyme region. Additionally, AMP led to greater increases in glycogen affinity of the former set of enzymes. In contrast, phosphate affinities of all a forms were similar in the absence of AMP and increased approximately the same extent in AMP. The potential importance of a number of isozyme-specific substitutions in these sequence regions is discussed.

Quantification and removal of glycogen phosphorylase and other enzymes associated with sarcoplasmic reticulum membrane preparations.

The enzymatic characterization of sarcoplasmic reticulum membrane fragments from rabbit skeletal muscle presented in this paper shows that glycogen phosphorylase, as well as other enzymes (e.g., creatine kinase, myokinase, phosphorylase kinase, glycosidase, AMP-deaminase, phosphoglucomutase) are associated with these membrane preparations. Amongst these enzymes, the highest activity associated with sarcoplasmic reticulum membranes is that of glycogen phosphorylase, which is mostly (at least 95%) in its b form (dephosphorylated form), since its activity in sarcoplasmic reticulum membranes is largely dependent upon AMP. A protocol is presented to quantify the amount of phosphorylase bound to sarcoplasmic reticulum membranes from fluorimetric measurements of the content of its coenzyme, pyridoxal 5'-phosphate. The content of phosphorylase ranged from 0.03 to 0.37 mg phosphorylase per mg of membrane protein, in sarcoplasmic reticulum membrane preparations made following several of the protocols most commonly used and also depending upon the length of the starvation period of the animal before killing. We also show that dilution of sarcoplasmic reticulum membranes to 0.1-0.2 mg protein per ml in a buffer containing 50 mM Tes-KOH (pH 7.4), 0.1 M KCl and 0.25 M sucrose removes at least 95% of glycogen phosphorylase from these membrane fragments, as well as other enzymes like myokinase and glycosidase. On these grounds, we suggest to introduce a final dilution step as indicated above in protocols of sarcoplasmic reticulum membrane preparations.

Purification of two forms of bovine liver glycogen phosphorylase b with distinct subunit composition.

The procedures for the purification of two forms of bovine liver glycogen phosphorylase b are described. Both forms showed a single band in nondenaturing gel electrophoresis. Gel electrophoresis in the presence of sodium dodecyl sulfate produced a single-band pattern for one of the enzyme forms (phosphorylase b1) and a triple-band pattern for the other (phosphorylase b3). Molecular weights associated with these bands were 97 kDa in the first case and 97, 55, and 40 kDa in the second. The yield from 1 kg of liver was approximately 10 mg for phosphorylase b1 and 140 mg for phosphorylase b3. The specific activity was 40-44 U/mg in both cases. As phosphorylase b1 is composed of just one kind of monomer, it is a novel bovine liver phosphorylase b structure.

Control of glycogen synthase and phosphorylase by amylin in rat skeletal muscle. Hormonal effects on the phosphorylation of phosphorylase and on the distribution of phosphate in the synthase subunit.

The effects of amylin and insulin on the phosphorylation of glycogen synthase and phosphorylase were investigated using rat diaphragms incubated with 32Pi. Muscles were incubated with insulin (200 nM) or amylin (200 nM) for 30 min before extracts were prepared. The 32P contents of the enzymes were determined after immunoprecipitation and SDS-polyacrylamide gel electrophoresis. Amylin increased both the activity ratio (-AMP/+AMP) and the 32P content of phosphorylase by approximately 2-fold. Insulin alone was without significant effect on phosphorylase, but insulin blocked the effect of amylin on increasing the phosphorylation of phosphorylase. Insulin increased the glycogen synthase activity ratio (low glucose-6-P/high glucose-6-P) by approximately 80%. Amylin decreased this ratio from 0.14 to 0.08 and increased the phosphorylation of synthase by approximately 40%. To investigate changes in phosphorylation of different sites in the synthase, the enzyme was subjected to exhaustive proteolysis with trypsin, and 32P-labeled fragments were separated by reverse phase high performance liquid chromatography. Insulin decreased the 32P contents of sites 3(a+b+c) and 2(a+b), which appears to account for the increase in synthase activity. Amylin increased phosphorylation of sites 1a, 1b, and 3(a+b+c), but not sites 2(a+b). With insulin plus amylin, phosphorylation of none of the sites was significantly changed. The results indicate that the effects of amylin on glycogen synthase must involve more than activation of cAMP-dependent protein kinase, as this kinase phosphorylates site 2 and does not phosphorylate sites 3(a+b+c).