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PubMed1984

The control of phosphorylase kinase phosphatase activity by polycations and the deinhibitor protein.

The dephosphorylation of phosphorylase beta kinase by the activated ATP, Mg-dependent protein phosphatase, which is highly specific for the beta-subunit, is stimulated by the deinhibitor protein which neutralizes the effect of inhibitor-1 and the modulator protein on the phosphatase. The specific dephosphorylation of the alpha-subunit of phosphorylase beta kinase by a "latent" protein phosphatase isolated from vascular smooth muscle is stimulated by histone H1 but not affected by the deinhibitor protein. These observations show that there is no strict correlation between the insensitivity of a protein phosphatase to inhibitor-1 or modulator protein and the dephosphorylation of the alpha-subunit of phosphorylase beta kinase.

Adenosine Triphosphate↗
PubMed2002

Overexpression of Escherichia coli genes encoding nucleoside phosphorylases in the pET/Bl21(DE3) system yields active recombinant enzymes.

The Escherichia coli genes encoding purine nucleoside phosphorylase, uridine phosphorylase, and thymidine phosphorylase were cloned into pET plasmids to generate highly effective E. coli BL21(DE3) strains producing each of these enzymes. Optimum conditions for biosynthesis of each enzyme as a soluble protein with intact biological activity were found. The crude preparations are approximately 80% pure and can be used immediately for enzymatic transglycosylation. The enzyme preparations were purified to homogeneity by two steps including fractional precipitation with ammonium sulfate and subsequent chromatography on Sephadex G-100 and DEAE-Sephacel.

Cloning, Molecular↗
PubMed1985

Quantitation and significance of 125I-calmodulin binding to myosin light chain kinase and phosphorylase distributed on polyacrylamide gels.

Glycogen phosphorylase (a or b) binds 125I-calmodulin in a Ca2+-dependent manner, in the 125I-calmodulin overlay technique. This binding is quantitatively identical to 125I-calmodulin binding to myosin light chain kinase. In an in vitro assay, calmodulin stimulates phosphorylase activity at limiting concentrations of either glucose-1-phosphate or glycogen, but the Ka is 1000 fold higher than for the kinase, and is not Ca2+-dependent. Activation of phosphorylase, but not myosin light chain kinase, by calmodulin can be mimicked by troponin C or bovine serum albumin. These results demonstrate that the properties of calmodulin interaction with proteins can vary between the 125I-calmodulin technique and a functional assay of calmodulin effect on the same protein.

Animals↗
PubMed1987

Kinetic characterization of rabbit skeletal muscle phosphorylase ab hybrid.

Phosphorylase ab was prepared in vitro by partial phosphorylation of rabbit skeletal muscle phosphorylase b and was isolated by DEAE-Sephacel chromatography. Its phosphorylated and non-phosphorylated subunits could not be distinguished by different affinity to substrates, activators or inhibitors, indicating their coordinated function. In the absence of nucleotide activators, the Km values for Pi and glucose-1-P were 28 mM and 18 mM, respectively. Activity in the presence of 16 mM glucose-1-P was doubled by 10(-4) M AMP or 10(-3) M IMP, mainly by lowering the Km for glucose-1-P. Half-maximum activation was exerted by 2 microM AMP or 0.1 mM IMP. Activation by these nucleotides showed no cooperativity. Glucose exerted competitive inhibition with respect to glucose-1-P, while for the inhibition by glucose-6-P an allosteric mechanism is suggested; the appropriate Ki values were 4.5 mM and 1.5 mM, respectively. The Hill coefficient for glucose-1-P binding was about 1.0, even in the presence of glucose (up to 10 mM), but 10 mM glucose-6-P lowered it to 0.47, indicating a negative heterotropic cooperativity. Effective regulation of the activity of phosphorylase ab by physiological concentrations of Pi, AMP, IMP and glucose-6-P suggests its metabolic control under in vivo condition.

Adenosine Monophosphate↗
PubMed1995

Caffeine inhibition of glycogen phosphorylase from Mytilus galloprovincialis mantle tissue.

A different caffeine inhibition of both phosphorylated and unphosphorylated forms of glycogen phosphorylase from Mytilus mantle has been demonstrated. Caffeine increases the allosteric constant of phosphorylase b 30-fold, acting as an allosteric inhibitor (nH = 2) of mixed type with respect to inorganic phosphate (Pi) and AMP, and of single competitive type with respect to glycogen. The Mytilus phosphorylated form is also caffeine inhibited through competitive inhibition in relation to Pi and glycogen. In this case, the inhibitor does not modify the allosteric constant (near 2), neither does it display allosteric effects (nH = 1). The results demonstrate the notable modification of the nucleotide site promoted by the phosphorylation process and the existence of a functional inhibitory nucleoside site in Mytilus phosphorylase.

Adenosine Monophosphate↗
PubMed2003

Immunocytochemical localization of glycogen phosphorylase isozymes in rat nervous tissues by using isozyme-specific antibodies.

Isozyme-specific antibodies were raised against peptides from the low-homology regions of the sequences of rat glycogen phosphorylase BB and MM isozymes by immunization of rabbits and guinea pigs. Immunocytochemical double-labelling experiments on frozen sections of rat nervous tissues were performed to investigate the isozyme localization pattern. Astrocytes throughout the brain and spinal cord expressed both isozymes in perfect co-localization. Ependymal cells only expressed the BB isozyme. Most neurones were not immunoreactive. The rare neurones that contained glycogen phosphorylase only expressed the BB isozyme. Nearly all of these neurones formed part of the afferent somatosensory system. These findings stress the general importance of glycogen in neural energy metabolism and indicate a special role for the glycogen phosphorylase BB isozyme in neurones in the somatosensory system.

Afferent Pathways↗
PubMed1983

Psychological stress activates phosphorylase in the heart of the conscious pig without increasing heart rate and blood pressure.

The present study uses a technique that enables the collection of multiple freeze-biopsy samples from the myocardium of the conscious pig (i.e., through a thoracic window). This technique enables sequential analysis of the metabolic state of the myocardium during different behavioral conditions. The results demonstrate that with daily adaptations to an unfamiliar environment (i.e., stress reduction), the phosphorylase activation ratio (phosphorylase a/total phosphorylase) in the quiescent pig declines steadily from approximately 80% to 30% (r = -0.91, P less than 0.01). This decline occurs with both the mean resting heart rate and left ventricular blood pressure remaining constant. The decline is seen within individual subjects during the whole adaptation sequence as well as between subjects whose samples were taken either early or late in the adaptation series. The dissociation of hemodynamic functional and metabolic activation in the unadapted, psychologically stressed pig may be associated with the occurrence of increased vulnerability of the ischemic heart to ventricular fibrillation, a phenomenon previously observed under the same behavioral conditions.

Animals↗
PubMedDate not supplied

[Phosphorylase and glucose-6-phosphatase activity in rat tissues during ontogenesis].

The activity of phosphorylase and glucose-6-phosphatase was determined in the liver, cerebral hemispheres, musculus gastrocnemius and myocardium of uneven-aged rats. The phosphorylase activity was the highest in rats aged 14-30 days and the glucose-6-phosphatase activity--in rats aged one day. Considerable age changes are observed in the ratio of phosphorylases a and b.

Aging↗
PubMed1982

Properties of glycogen synthase and phosphorylase from Biomphalaria glabrata (mollusca).

Glycogen synthase and phosphorylase were characterized from the cephalopedal region of the snail, Biomphalaria glabrata. Glycogen synthase exhibited increases and decreases in its activity ratio (-G6P/+G6P) under conditions that are known to cause conversion of the two forms of the enzyme from mammalian systems, implying that the snail's synthase also possesses interconvertible forms. Each form had a distinct pH optimum, with the G6P-independent form (synthase alpha) exhibiting maximum activity at pH 7.4, whereas the G6P-dependent form (synthase beta) had optimal activity at pH 8.3. Both synthase alpha and beta displayed classical Michaelis-Menten kinetics for the substrates UDP-glucose and glycogen, and the beta form displayed sigmoidal kinetics for its modulator, G6P. Only UDP-glucose could function as a glucosyl donor in the synthase-catalyzed reaction. ADP, GDP, UDP, and ATP were all competitive inhibitors of synthase alpha, although at varying degrees of efficiency. Glycogen phosphorylase also demonstrated interconversion of its two forms (alpha and beta), as evidenced by changes in its activity ratio (-AMP/+AMP). AMP elicited hyperbolic kinetics from this enzyme. Concentrations of KF above 20 mM were found to inhibit glycogen synthase alpha while stimulating phosphorylase beta, thus causing erroneous activity ratios for both enzymes.

Animals↗
PubMed1975

Polynucleotide phosphorylase from Streptomyces aureofaciens: purification and properties.

1. Polynucleotide phosphorylase from a chlortetracycline-producing strain of Streptomyces aureofaciens was isolated by Polymin P fractionation. Using chromatography on DEAE-cellulose and Sephadex G-150 the enzyme, which appears homogeneous in gel chromatography and sedimentation analysis, was purified 2000-fole giving a final yield of 15%. 2. The sedimentation coefficient (s-o 20, w) of the native enzyme in 0.2 M NaCl is 9.15 S and its molecular weight is 210 000 plus or minus 15 000. Molecular weight estimated by sodium dodecylsulfate gel electrophoresis was about 100 000. 3. We have determined the optimal conditions for nucleoside 5'-diphosphate polymerization, their phosphate exchange and phosphorolysis of polyribonucleotides catalysed by polynucleotide phosphorylase from S. aureofaciens. 4. Chlortetracycline is a competitive inhibitor of S. aureofaciens polynucleotide phosphorylase. 5. Polynucleotide phosphorylase is activated in the polymerization reaction by ionic strength (K+, Na+, NH4+) while polyribonucleotide phosphorolysis is activated only by NH4+.

Ammonia↗
PubMed1978

[Quaternary structure and proteolysis of the polynucleotide phosphorylase from C. perfringens].

This report describes structural studies on purified polynucleotide phosphorylase from C. perfringens. A method is described for the purification of the enzyme which yields a product equivalent in activity to the native polynucleotide phosphorylase from E. coli. These studies revealed a molecular heterogeneity arising from successive stages of proteolysis, to which this enzyme is especially sensitive; unusally, the enzyme is obtained as a mixture of variable proportions of the native and proteolysed forms. We found in all cases a trimeric basic structure composed of the native (alpha) or proteolysed (lapha) or proteolysed (alpha', alpha") catalytic sub-units, However, the enzyme is rather easily dissociated into its sub-units, a phenomenon which seems to accompany proteolysis (Table). Under the action of either endogenous proteases or trypsin, two enzymatic forms are obtained: their quaternary structures seem analogous, but they differ in their catalytic properties from each other and from the initial enzyme. With some care at each step of purification, the polynucleotide phosphorylase of E. coli can be obtained exclusively in its native form. The greater susceptibility to proteolysis of the enzyme from C. perfrigens and the relationship between such degradation and quaternary structure seem to be at the origin of the peculiar behavior of this polynucleotide phosphorylase.

Chemical Fractionation↗
PubMed2001

Crystal structure of maltose phosphorylase from Lactobacillus brevis: unexpected evolutionary relationship with glucoamylases.

BACKGROUND: Maltose phosphorylase (MP) is a dimeric enzyme that catalyzes the conversion of maltose and inorganic phosphate into beta-D-glucose-1-phosphate and glucose without requiring any cofactors, such as pyridoxal phosphate. The enzyme is part of operons that are involved in maltose/malto-oligosaccharide metabolism. Maltose phosphorylases have been classified in family 65 of the glycoside hydrolases. No structure is available for any member of this family. RESULTS: We report here the 2.15 A resolution crystal structure of the MP from Lactobacillus brevis in complex with the cosubstrate phosphate. This represents the first structure of a disaccharide phosphorylase. The structure consists of an N-terminal complex beta sandwich domain, a helical linker, an (alpha/alpha)6 barrel catalytic domain, and a C-terminal beta sheet domain. The (alpha/alpha)6 barrel has an unexpected strong structural and functional analogy with the catalytic domain of glucoamylase from Aspergillus awamori. The only conserved glutamate of MP (Glu487) superposes onto the catalytic residue Glu179 of glucoamylase and likely represents the general acid catalyst. The phosphate ion is bound in a pocket facing the carboxylate of Glu487 and is ideally positioned for nucleophilic attack of the anomeric carbon atom. This site is occupied by the catalytic base carboxylate in glucoamylase. CONCLUSIONS: These observations strongly suggest that maltose phosphorylase has evolved from glucoamylase. MP has probably conserved one carboxylate group for acid catalysis and has exchanged the catalytic base for a phosphate binding pocket. The relative positions of the acid catalytic group and the bound phosphate are compatible with a direct-attack mechanism of a glycosidic bond by phosphate, in accordance with inversion of configuration at the anomeric carbon as observed for this enzyme.

Binding Sites↗
PubMed1987

Yeast diadenosine 5',5'''-P1,P4-tetraphosphate alpha,beta-phosphorylase behaves as a dinucleoside tetraphosphate synthetase.

The diadenosine 5',5'''-P1,P4-tetraphosphate alpha,beta-phosphorylase (Ap4A phosphorylase), recently observed in yeast [Guaranowski, A., & Blanquet, S. (1985) J. Biol. Chem. 260, 3542-3547], is shown to be capable of catalyzing the synthesis of Ap4A from ATP + ADP, i.e., the reverse reaction of the phosphorolysis of Ap4A. The synthesis of Ap4A markedly depends on the presence of a divalent cation (Ca2+, Mn2+, or Mg2+). In vitro, the equilibrium constant K = ([Ap4A][Pi])/[(ATP][ADP]) is very sensitive to pH. Ap4A synthesis is favored at low pH, in agreement with the consumption of one to two protons when ATP + ADP are converted into Ap4A and phosphate. Optimal activity is found at pH 5.9. At pH 7.0 and in the presence of Ca2+, the Vm for Ap4A synthesis is 7.4 s-1 (37 degrees C). Ap4A phosphorylase is, therefore, a valuable candidate for the production of Ap4A in vivo. Ap4A phosphorylase is also capable of producing various Np4N' molecules from NTP and N'DP. The NTP site is specific for purine ribonucleotides (N = A, G), whereas the N'DP site has a broader specificity (N' = A, C, G, U, dA). This finding suggests that the Gp4N' nucleotides, as well as the Ap4N' ones, could occur in yeast cells.

Acid Anhydride Hydrolases↗
PubMed1999

The crystal structure of the Escherichia coli maltodextrin phosphorylase-acarbose complex.

Acarbose is a naturally occurring pseudo-tetrasaccharide. It has been used in conjunction with other drugs in the treatment of diabetes where it acts as an inhibitor of intestinal glucosidases. To probe the interactions of acarbose with other carbohydrate recognition enzymes, the crystal structure of E. coli maltodextrin phosphorylase (MalP) complexed with acarbose has been determined at 2.95 A resolution and refined to crystallographic R-values of R (Rfree) = 0.241 (0.293), respectively. Acarbose adopts a conformation that is close to its major minimum free energy conformation in the MalP-acarbose structure. The acarviosine moiety of acarbose occupies sub-sites +1 and +2 and the disaccharide sub-sites +3 and +4. (The site of phosphorolysis is between sub-sites -1 and +1.) This is the first identification of sub-sites +3 and +4 of MalP. Interactions of the glucosyl residues in sub-sites +2 and +4 are dominated by carbohydrate stacking interactions with tyrosine residues. These tyrosines (Tyr280 and Tyr613, respectively, in the rabbit muscle phosphorylase numbering scheme) are conserved in all species of phosphorylase. A glycerol molecule from the cryoprotectant occupies sub-site -1. The identification of four oligosaccharide sub-sites, that extend from the interior of the phosphorylase close to the catalytic site to the exterior surface of MalP, provides a structural rationalization of the substrate selectivity of MalP for a pentasaccharide substrate. Crystallographic binding studies of acarbose with amylases, glucoamylases, and glycosyltranferases and NMR studies of acarbose in solution have shown that acarbose can adopt two different conformations. This flexibility allows acarbose to target a number of different enzymes. The two alternative conformations of acarbose when bound to different carbohydrate enzymes are discussed.

Acarbose↗
PubMed1976

Thermostable polynucleotide phosphorylases from Bacillus stearothermophilus and Thermus aquaticus.

Polynucleotide phosphorylase from Bacillus stearothermophilus has been purified to homogeneity. Polyacrylamide gel electrophoresis run under denaturing conditions indicates that the enzyme is a tetramer with subunits of apparent molecular weight 51,000 daltons. A partial purification of polynucleotide phosphorylase from Thermus aquaticus has also been effected. The two enzymes show similar catalytic properties, which differ little from those of mesophilic polynucleotide phosphorylases. The use of thermostable polynucleotide phosphorylases for in vitro nucleic acid synthesis is discussed.

Geobacillus stearothermophilus↗
PubMed1980

Characterization of the spinach leaf phosphorylases.

The chloroplastic and the cytoplasmic phosphorylases were purified and their kinetic properties characterized. The cytoplasmic enzyme was purified to homogeneity via affinity chromatography on a glycogen-Sepharose column. Subunit molecular weight studies indicated a value of 92,000, whereas a native molecular weight value of 194,000 was obtained by sucrose density gradient centrifugation. The chloroplast enzyme's native molecular weight was determined to be 203,800. The cytoplasmic enzyme shows the same V(max) for maltopentaose, glycogen, amylopectin, amylose, and debranched amylopectin but is only slightly active toward maltotetraose. The K(m) for phosphate at pH 7.0 is 0.9 millimolar and for glucose-1-phosphate, 0.64 millimolar. The K(m) values for phosphorolysis of amylopectin, amylose, glycogen, and debranched amylopectin are 26, 165, 64, and 98 micrograms per milliliter, respectively. In contrast, the relative V(max) values for the chloroplast enzyme at pH 7.0 are debranched amylopectin, 100, amylopectin, 63.7, amylose, 53, glycogen, 42, and maltopentaose, 41. K(m) values for the above high molecular weight polymers are, respectively, 82, 168, 122 micrograms per milliliter, and 1.2 milligrams per milliliter. The K(m) value for inorganic phosphate is 1.2 millimolar. The chloroplastic phosphorylase appears to have a lower apparent affinity for glycogen than the cytoplasmic enzyme. The results are discussed with respect to previous findings of multiple phosphorylase forms found in plant tissues and to possible regulatory mechanisms for controlling phosphorylase activity.

Journal Article↗
PubMed1988

Starch Phosphorylase Inhibitor Is beta-Amylase.

The proteinaceous noncompetitive inhibitor of starch phosphorylase isolated from the root of sweet potato (Ipomoea batatas [L.] Lam.) (TC Chang, JC Su 1986 Plant Physiol 80: 534-538) has been identified as a beta-amylase. The starch phosphorylase inhibitor and beta-amylase activities copurified to give a protein indistinguishable from commercial beta-amylase by electrophoretic and immunological methods, and the two activities showed parallel responses in pH, temperature, and inhibitor sensitivity tests. The amylolytic pattern of the inhibitor corresponded to that of beta-amylase and its inhibitory effect toward starch phosphorylase was due to neither deprivation of starch, the primer for the phosphorylase assay, nor the inhibitory effect of amylolytic products.

Journal Article↗
PubMed1995

Glucose analogue inhibitors of glycogen phosphorylase: from crystallographic analysis to drug prediction using GRID force-field and GOLPE variable selection.

Several inhibitors of the large regulatory enzyme glycogen phosphorylase (GP) have been studied in crystallographic and kinetic experiments. GP catalyses the first step in the phosphorylysis of glycogen to glucose-l-phosphate, which is utilized via glycolysis to provide energy to sustain muscle contraction and in the liver is converted to glucose. alpha-D-Glucose is a weak inhibitor of glycogen phosphorylase form b (GPb, K(i) = 1.7 mM) and acts as a physiological regulator of hepatic glycogen metabolism. Glucose binds to phosphorylase at the catalytic site and results in a conformational change that stabilizes the inactive T state of the enzyme, promoting the action of protein phosphatase 1 and stimulating glycogen synthase. It has been suggested that in the liver, glucose analogues with greater affinity for glycogen phosphorylase may result in a more effective regulatory agent. Several N-acetyl glucopyranosylamine derivatives have been synthesized and tested in a series of crystallographic and kinetic binding studies with GPb. The structural results of the bound enzyme-ligand complexes have been analysed together with the resulting affinities in an effort to understand and exploit the molecular interactions that might give rise to a better inhibitor. Comparison of the N-methylacetyl glucopyranosylamine (N-methylamide, K(i) = 0.032 mM) with the analogous beta-methylamide derivative (C-methylamide, K(i) = 0.16 mM) illustrate the importance of forming good hydrogen bonds and obtaining complementarity of van der Waals interactions. These studies also have shown that the binding modes can be unpredictable but may be rationalized with the benefit of structural data and that a buried and mixed polar/non-polar catalytic site poses problems for the systematic addition of functional groups. Together with previous studies of glucose analogue inhibitors of GPb, this work forms the basis of a training set suitable for three-dimensional quantitative structure-activity relationship studies. The molecules in the training set are void of problems and potential errors arising from the alignment and bound conformations of each of the ligands since the coordinates were those determined experimentally from the X-ray crystallographic refined ligand-enzyme complexes. The computational procedure described in this work involves the use of the program GRID to describe the molecular structures and the progam GOLPE to obtain the partial least squares regression model with the highest prediction ability. The GRID/GOLPE procedure performed using 51 glucose analogue inhibitors of GPb has good overall predictivity [standard deviation of error predictions (SDEP) = 0.98 and Q(2) = 0.76] and has shown good agreement with the crystallographic and kinetic results by reliably selecting regions that are known to affect the binding affinity.

Journal Article↗