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PubMed1984

Catalytic reaction of glycogen phosphorylase reconstituted with a coenzyme-substrate conjugate.

The role of pyridoxal 5'-phosphate in the catalytic mechanism of glycogen phosphorylase (EC 2.4.1.1) remains unresolved despite extensive investigation. A previous report from this laboratory (Takagi, M., Fukui, T., and Shimomura, S. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 3716-3719) provided evidence for the direct interaction between the two phosphate groups of the coenzyme and a substrate alpha-D-glucose 1-phosphate. When apophosphorylase is reconstituted with pyridoxal (5')-diphospho(1)-alpha-D-glucose, the enzyme can transfer the glucose moiety to glycogen just as in the normal catalysis. We have studied the kinetics of the glucosyltransfer from this compound to glycogen. The normal and mimic reactions were similar in their kinetic parameters for glycogen and AMP and their activation energies. AMP and other nucleotides that activate the normal reaction could activate the mimic reaction as well. The log k/pH plot for the mimic reaction gave a bell-shaped curve with pKa = 6.90 and pKb = 8.84 at 25 degrees C. The apparent heats of ionization of the corresponding groups having the pKa and pKb were 6.9 and 3.0 kcal/mol, respectively. Reversibility of the glucosyltransfer from the coenzyme-substrate conjugate to glycogen could not be demonstrated, possibly because the equilibrium of this reaction lies further to the polysaccharide synthesis. Based on these and other data which confirm the role of the phosphate group of the coenzyme as an electrophile, we discuss the catalytic mechanism of glycogen phosphorylase. It is suggested that the imidazoyl group of His-376 acts as a nucleophile attacking the anomeric carbon of the substrate glucose 1-phosphate.

Animals↗
PubMed1983

Developmental changes of glycogen phosphorylase b isozymes in rat tissues.

The isozyme distribution of glycogen phosphorylase b was studied in various fetal, neonatal, and adult rat tissues by means of discontinuous polyacrylamide gel electrophoresis in the presence of glycogen in the separating gel. The brain-type isozyme BB being the predominant form in fetal rat tissues is replaced during maturation either partially (brain and kidney) or completely (skeletal muscle and liver) by the isozymes MM or LL, respectively. In the various organs, the developmental formation of the adult phosphorylase isozyme pattern does not proceed simultaneously. The definitive isozyme pattern is expressed in heart muscle at the 19th day of gestation. The isozyme transitions in liver and skeletal muscle are finished about two days after birth, while at this time in the brain just the muscle-type subunit M appears and forms the hybrid MB. The isozyme distribution of the maturated rat brain (BB, MB, MM) is not expressed before the 9th day of postnatal life.

Aging↗
PubMed1984

AMP and IMP binding to glycogen phosphorylase b. A calorimetric and equilibrium dialysis study.

Reaction microcalorimetry and equilibrium dialysis have been used to study the binding of AMP and IMP to glycogen phosphorylase b (EC 2.4.1.1) at 25 degrees C and pH 6.9. The combination of both techniques has enabled us to obtain some of the thermodynamic parameters for these binding processes. Four binding sites were found to be present in the dimeric active enzyme for both AMP and IMP. The binding to two high-affinity sites, which, in our opinion, correspond to the activator sites, seems to be cooperative. The two low-affinity sites, which would then correspond to the inhibitor sites, appear to be independent when the nucleotides bind to the enzyme. The negative delta G0 of binding/site at 25 degrees C is the result in all cases of a balance between negative enthalpy and entropy changes. The large differences in delta H and delta S0 for the binding of AMP to the activator sites (-27 and -70 kJ mol-1; -22 and -150 J X K-1 mol-1) suggest the existence of rather extensive conformational changes taking place in phosphorylase b on binding with the allosteric activator. Whereas the affinity of AMP for the activator sites is about 1 order of magnitude higher than that of IMP, the affinity of both nucleotides, including their delta H and delta S0 values, seems to be the same for the inhibitor sites.

Adenosine Monophosphate↗
PubMed1983

The role of cysteinyl residues in the phosphorylase kinase activity as revealed by iodacetamide modification.

In native nonactivated phosphorylase kinase [14C] iodacetamide interacts with 50 cysteinyl residues per enzyme molecule (alpha beta gamma delta)4. According to their reactivity towards iodacetamide these residues can be classified into 3 groups. The most reactive cysteinyl residues are involved in the enzyme activation caused by modification of SH-groups. The enzyme inhibition is biphasic. The fast and slow inactivation reactions follow the pseudo-first order kinetics. The rate of inactivation is increased by Ca2+. Mg-ATP effectively protects the enzyme against the inactivation and chemical modification of three SH-groups per protomer (apha beta gamma delta). The kinetics of inactivation and of the [14C] iodacetamide label incorporation demonstrate that two cysteinyl residues per enzyme protomer (alpha beta gamma delta) are essential for the enzyme activity. These residues are located near the ATP-binding site of the beta and gamma subunits of phosphorylase kinase.

Animals↗
PubMedDate not supplied

[Kinetics of the reaction between muscle glycogen phosphorylase B and glycogen].

Kinetics of glycogen binding by glycogen phosphorylase b has been studied by stopped flow and temperature jump methods. This reaction is followed by increase in light scattering whose amplitude depends upon the enzyme binding sites concentration of glycogen particles occupied by the enzyme. It has been shown that the complex formation has the first order with respect to enzyme and glycogen concentrations. Relaxation kinetics is compatible with proposed bimolecular reaction scheme. Microscopic rate constants of the forward and reverse reactions of glycogen binding by glycogen phosphorylase b are determined in temperature range from 12,7 to 30 degrees C. The possibility of diffusional control of the binding rate is discussed.

Animals↗
PubMed1981

Function of the phosphate group of pyridoxal 5'-phosphate in the glycogen phosphorylase reaction.

To understand the catalytic mechanism of glycogen phosphorylase (EC 2.4.1.1), pyridoxal(5')phospho(1)-beta-D-glucose was synthesized and examined as a hypothetical intermediate in the catalysis. Pyridoxal phosphoglucose bound stoichiometrically to the cofactor site of rabbit muscle phosphorylase b in a similar mode of binding to the natural cofactor, pyridoxal 5'-phosphate. The rate of binding of pyridoxal phosphoglucose was only 1/100 compared with that of pyridoxal phosphate. The enzyme reconstituted with pyridoxal phosphoglucose showed no enzymatic activity at all even after prolonged incubation of the enzyme with substrates and activator. The present data would contradict participation of the phosphate group of pyridoxal phosphate in a covalent glucosyl-enzyme intermediate even if the covalent intermediate was formed during the catalysis.

Animals↗
PubMedDate not supplied

[Kinetics of the phosphorylase reaction under conditions of substrate inactivation].

Electrophoretically homogenous glycogenphosphorylase b was isolated from the Dasyatis pastinaca muscular tissue. The kinetic peculiarities of phosphorylase reaction in time were studied. It is found that the reaction rate substrate concentration real dependences are not described by the Michaelis equation. The fact of the irreversible inactivation of phosphorylase in the process of catalytic reaction is observed. It is supposed that this fact is mechanical destruction in the process of substrate splitting rather than thermoinactivation of the enzyme or its inhibition.

Animals↗
PubMed1982

[Determination of glycogen phosphorylase B activity by the turbidimetric method in the presence of the forward and reverse reaction substrates].

The turbidimetric method based on monitoring changes in the turbidity of glycogen solution at wavelengths above 300 nm has been developed for the measurement of catalytic activity of rabbit skeletal muscle glycogen phosphorylase B. The dependences of the initial rate of enzymatic reaction on molar ratio of inorganic phosphate in the inorganic phosphate-glucose-1-phosphate mixture have been obtained at various concentrations of the allosteric activator, AMP, or in the presence of inhibitors (D-glucose, glucose 6-phosphate). It has been shown that the changes in glycogen phosphorylase B activity in the presence of these modifiers are not affected by variations in the molar ratios of the substrate. A theoretical expression for the dependence of the initial rate of the reversible reaction S in equilibrium P catalyzed by the enzyme has been deduced as a function of the substrate (S) molar ratio.

Adenosine Monophosphate↗
PubMed1978

Synergistic regulation of phosphorylase a by glucose and caffeine.

Kinetic studies of both liver and muscle phosphorylase a demonstrate that caffeine and glucose inhibit the binding of glucose 1-phosphate to the enzyme in a synergistic competitive and nonexclusive manner. Inhibition studies for numerous other caffeine analogs show that the muscle enzyme has a relaxed specificity for this negative effector. The liver enzyme is more discriminating by preferential binding of methylated oxypurines. Physiological concentrations of AMP and ATP, which affect the enzymic activity at a separate site, prevent glucose from effectively inhibiting the enzyme. The addition of the second synergistic ligand improves the binding of glucose. These data suggest that glucose homeostasis as regulated by phosphorylase may be dependent on a second ligand and that the role of glucose in this physiological process may have been overestimated. A structural rationalization of this synergistic response is discussed with reference to the crystal structure of the muscle enzyme.

Animals↗
PubMed1978

[Structural-functional analysis of the complex between phosphorylase B and adenosine-5'-chloromethylphosphonate].

A complex of phosphorylase B with a tritium-containing AMP analogue, adenosine-5'-chloromethylphosphonate, was obtained. It is found on the basis of the results of the determination of N- and C-terminal amino acids, amino acid composition and sequence, that the peptide 1, modified by adenosine-5'-chloromethylphosphonate, corresponds to the fragment 185-191 in the primary structure of phosphorylase B, and is probably located in the allosteric center of the enzyme. The peptide 2, which is bound with the AMP analogue and corresponds to the fragment 795-798, is suggested to be located at the site of binding the second AMP molecule. The arginine residue 184 is discussed as a possible functional amino acid protein interacting with 5'-phosphate AMP group.

Adenosine Monophosphate↗
PubMed1998

[Correlation between pyrimidine nucleoside phosphorylase (PyNPase)/thymidine phosphorylase/platelet-derived endothelial cell growth factor and histological prognostic factor, and influence of 5'-deoxy-5-fluorouridine (5'-DFUR) administration on PyNPase activities and serum immunosuppressive acidic protein levels. A study group of oral anti-cancer drugs in Seiban/Tajima area].

PURPOSE: Among the pyrimidine nucleoside phosphorylases (PyNPase), thymidine phosphorylase (dThdPase) exists mainly in human tumor tissues, and is an enzyme which converts 5'-deoxy-5-fluorouridine (5'-DFUR) to 5-fluorouracil. Recently, it was reported that dThdPase was identical to platelet-derived endothelial cell growth factor which was an antiogenetic factor, therefore. We think dThdPase may be a prognostic factor. METHODS: We investigated the possible correlation between PyNPase activities in tumor tissues and prognostic factors of histological findings, and examined influences of preoperative oral 5'-DFUR administration at a dose of 1,200 mg/body for 7 days on PyNPase activities and serum immunosuppressive acidic protein levels in patients with gastric cancer. RESULTS: Higher levels of PyNPase activities were especially observed in patients with v+ (p = 0.0161). PyNPase activities (p = 0.1668) and IAP (p = 0.0830) levels showed a decrease by 5'-DFUR administration. CONCLUSION: This study suggests that we must investigate in detail the possibility of PyNPase being prognostic factor, and 5'-DFUR administration may improve the prognosis of patients with gastric cancer.

Antineoplastic Agents↗
PubMed1998

[Correlation between pyrimidine nucleoside phosphorylase (PyNPase)/platelet-derived endothelial cell growth factor and histological prognostic factor, and influences of 5'-deoxy-5-fluorouridine (5'-DFUR) administration on PyNPase levels. 5'-DFUR Joint Research Group in the Osaka Area for Gastric Cancer].

PURPOSE: Pyrimidine nucleoside phosphorylase (PyNPase), among which thymidine phosphorylase (dThdPase) exists mainly in human tumor tissues, is an enzyme to convert 5'-deoxy-5-fluorouridine (5'-DFUR) to 5-fluorouracil. Recently, it was reported that dThdPase was identical to platelet-derived endothelial cell growth factor, angiogenetic factor. Therefore, we expect that there is possibility of dThdPase being a prognostic factor. METHODS: We investigated for a possible correlation between PyNPase activities in tumor tissues and prognostic factors of histological findings, examined the influences of preoperative oral 5'-DFUR administration to PyNPase levels and investigated for a correlation between HPLC methods and ELISA methods in patients with gastric cancer. RESULTS: Higher levels of PyNPase were observed in patients with advanced t,n,v, and ly factors. PyNPase levels decreased by 5'-DFUR in patients with differentiated cases. A high correlation was found between HPLC and ELISA methods. CONCLUSION: This study suggests that we must investigate possibility of PyNPase being a prognostic factor in more detail.

Antineoplastic Agents↗
PubMed2006

Tissue microarray analysis of methylthioadenosine phosphorylase protein expression in melanocytic skin tumors.

BACKGROUND: Using tissue microarrays, we investigated whether methylthioadenosine phosphorylase (MTAP) protein expression is associated with clinicopathologic variables in benign and malignant melanocytic skin tumors. OBSERVATIONS: Cytoplasmic MTAP expression was detected in 227 (72.1%) of 315 informative cases. Expression was significantly reduced in primary malignant melanomas and in melanoma metastases compared with benign nevi (P<.001 for both). No difference was noted in MTAP expression between primary malignant melanomas and melanoma metastases. In primary malignant melanomas, a Ki67-labeling index less than 5% was associated with MTAP expression (P = .04), suggesting that loss of MTAP expression is associated with proliferation. No other variables had significant associations with MTAP expression. Lymph node metastases demonstrated significantly higher MTAP expression compared with skin metastases (P = .01). In the overall cohort, MTAP expression was not associated with prognosis. Among 26 patients with MTAP-positive melanomas and tumor recurrence, 18 patients who received interferon therapy had a significant benefit compared with 8 patients who did not receive interferon therapy (P = .009). This was not seen in the patients with MTAP-negative tumors. Conclusion Methylthioadenosine phosphorylase protein expression may be a predictive marker of interferon therapy resistance in patients with melanoma and disease progression.

Antineoplastic Agents↗
PubMed1993

Fanconi's syndrome with hepatorenal glycogenosis associated with phosphorylase b kinase deficiency.

OBJECTIVE: To describe two patients with Fanconi's nephropathy secondary to glycogen storage disease and speculate on the possible etiology. DESIGN: Convenience sample. SETTING: Tertiary care, referral center. PATIENTS: Two related children referred for failure to thrive, rickets, and hepatomegaly. INTERVENTION: Dietary and therapeutic measures for rickets and renal tubular acidosis. MEASUREMENTS AND RESULTS: The main laboratory findings were fasting hypoglycemia and massive glucosuria, with evidence of multiple renal tubular dysfunction characteristic of the Fanconi syndrome. Liver and kidney biopsy specimens were consistent with glycogen storage disease. Enzymatic assay of liver homogenates revealed marked deficiency of phosphorylase b kinase in one patient and absent activity in the other. CONCLUSION: Phosphorylase b kinase deficiency may be causally related to hepatorenal glycogenosis with the Fanconi syndrome. More patients with this syndrome need to be studied before a definitive causal role is implicated.

Biopsy↗
PubMed1996

Ultrastructural localization of glycogen phosphorylase predominantly in astrocytes of the gerbil brain.

The localization of glycogen phosphorylase in gerbil brain was determined by immunoelectron microscopy using the pre-embedding peroxidase technique. Electron-dense granular reaction product of peroxidase activity was observed in astrocytes of all brain regions examined (cerebral cortex, striatum, cerebellar cortex, hippocampal formation, corpus callosum, mesencephalic trigeminal nucleus). The reaction product was distributed in a diffuse pattern throughout the cytoplasmic matrix of perikarya and processes; sometimes the nucleus of labeled astrocytes also contains immunopositive material. The light microscopically visible glycogen phosphorylase immunoreactivity associated with capillaries could be characterized as a staining of astrocytic endfeet ensheathing capillaries. Endothelial cells and pericytes were never labeled. In addition to astrocytes, ependymal cells also presented immunopositive material in their cytoplasm. On the other hand, no reaction product was observed in cells identified as oligodendroglia or microglia. Neurons (with the exception of neurons of the mesencephalic trigeminal nucleus), their processes, and their synaptic endings were free of reaction product. In the neuropil we frequently observed immunopositive glial processes adjacent to synaptic structures. This intimate spatial relationship may be interpreted as a morphological sign of a metabolic interaction. The data support the hypothesis that astroglia play a key role in glycogen metabolism and energization of the brain.

Animals↗
PubMed2005

Myopathy and phosphorylase kinase deficiency caused by a mutation in the PHKA1 gene.

Phosphorylase kinase (PhK) deficiency is the underlying cause of variable clinical symptoms depending on the tissues involved. Until today, only a few cases of myopathy associated with muscle PhK deficiency caused by a mutation in the gene encoding the alpha subunit of phosphorylase kinase (PHKA1) have been reported. We describe a male patient with myopathy and absent muscle PhK activity caused by a frameshift mutation in the gene encoding the alpha subunit of PhK on chromosome Xq12-q13.

Adult↗
PubMed2004

Increased blood-brain barrier permeability with thymidine phosphorylase deficiency.

Mitochondrial neurogastrointestinal encephalomyopathy is an autosomal recessive multisystemic disorder caused by thymidine phosphorylase deficiency. Whereas the pathomechanism of the secondary mitochondrial dysfunction has been extensively studied, that of the leukoencephalopathy has not been elucidated. We hypothesized that the white matter hyperintensities on T2-weighted magnetic resonance images reflect disturbance of blood-brain barrier function. Albumin immunohistochemistry disclosed quantitative (p < 0.01) and qualitative differences between the mitochondrial neurogastrointestinal encephalomyopathy and control brains, indicating that loss of thymidine phosphorylase function impairs the integrity of the blood-brain barrier.

Adolescent↗
PubMed1987

Glucose 6-phosphatase and glycogen phosphorylase activities in chondrocytes in epiphyseal cartilage of growing rats.

Glycogen, glycogen phosphorylase, and glucose 6-phosphatase (G6Pase) activities were examined cytochemically in chondrocytes of femoral epiphyseal cartilages and cartilaginous ribs of 3- and 7-day-old rats. G6Pase activity was also examined biochemically. Glycogen was abundant in chondrocytes of the reserve zone, while it became scarce in the cells of the proliferative zone. From the upper part (adjoining the proliferative zone) to the lower part of the hypertrophic zone, glycogen accumulated in chondrocytes and decreased in the cells of the degenerative zone. Inversely, glycogen phosphorylase a and G6Pase activities were relatively high in chondrocytes of the proliferative zone and upper hypertrophic zone and were low in the cells of the reserve zone, lower hypertrophic zone, and degenerative zone. The reaction product for G6Pase was present in the endoplasmic reticulum and nuclear envelope of all types of chondrocytes composing the cartilages, although the amounts of reaction product varied with the cell types in parallel with the histochemical results. Biochemical G6Pase activity was higher in epiphyseal cartilages than in cartilaginous ribs. The possible mechanism and significance of the accumulation and decrease of glycogen in chondrocytes of the epiphyseal cartilage were discussed.

Animals↗