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At least 811 records · Page 45Linked to original sources
PubMed1995

A second L-type isozyme of potato glucan phosphorylase: cloning, antisense inhibition and expression analysis.

In potato tubers two starch phosphorylase isozymes, types L and H, have been described and are believed to be responsible for the complete starch breakdown in this tissue. Type L has been localized in amyloplasts, whereas type H is located within the cytosol. In order to investigate whether the same isozymes are also present in potato leaf tissue a cDNA expression library from potato leaves was screened using a monoclonal antibody recognizing both isozyme forms. Besides the already described tuber L-type isozyme a cDNA clone encoding a second L-type isozyme was isolated. The 3171 nucleotide long cDNA clone contains an uninterrupted open reading frame of 2922 nucleotides which encodes a polypeptide of 974 amino acids. Sequence comparison between both L-type isozymes on the amino acid level showed that the polypeptides are highly homologous to each other, reaching 81-84% identity over most parts of the polypeptide. However the regions containing the transit peptide (amino acids 1-81) and the insertion sequence (amino acids 463-570) are highly diverse, reaching identities of only 22.0% and 29.0% respectively. Northern analysis revealed that both forms are differentially expressed. The steady-state mRNA levels of the tuber L-type isozyme accumulates strongly in potato tubers and only weakly in leaf tissues, whereas the mRNA of the leaf L-type isozyme accumulates in both tissues to the same extent. Constitutive expression of an antisense RNA specific for the leaf L-type gene resulted in a strong reduction of starch phosphorylase L-type activity in leaf tissue, but had only sparse effects in potato tuber tissues. Determination of the leaf starch content revealed that antisense repression of the starch phosphorylase activity has no significant influence on starch accumulation in leaves of transgenic potato plants. This result indicated that different L-type genes are responsible for the starch phosphorylase activity in different tissues, but the function of the different enzymes remains unclear.

Amino Acid Sequence↗
PubMed1984

Glucose stimulation of heart phosphorylase phosphatase activity in vitro and in vivo.

To determine the mechanism of the glucose stimulation, glucose or glucose-6-phosphate was added to dilute heart extracts in the presence or absence of AMP. The intracellular glucose, tissue glucose-6-phosphate, and tissue AMP concentrations were also determined in 24-h starved animals given glucose; 24-h starved animals given insulin as well as diabetic starved and diabetic starved insulin-treated animals were also studied. The A0.5 for glucose stimulation of cardiac phosphorylase phosphatase activity was approximately 1.2 mM. The A0.5 for glucose-6-phosphate was approximately 0.02 mM. The glucose-6-phosphate concentration in all animals exceeded the A0.5 by 10-fold. However, the intracellular glucose concentration in the glucose-treated, insulin-treated, diabetic, and diabetic insulin-treated rats was in the range of the A0.5 for stimulation of phosphorylase phosphatase activity. AMP completely inhibited phosphorylase phosphatase activity at a concentration of 0.2 mM. Physiological concentrations of glucose and glucose-6-phosphate partially reversed this inhibition. Administration of glucose or insulin resulted in an increase in intracellular glucose concentration, an increase in tissue glucose-6-phosphate and a decrease in tissue AMP concentrations. These data suggest that glucose may be a physiological regulator of phosphorylase phosphatase in heart muscle as it is in liver.

Adenosine Monophosphate↗
PubMed1992

Differential scanning calorimetry study of glycogen phosphorylase b-detergent interactions.

The overall thermal denaturation of glycogen phosphorylase b is irreversible and our results conform to the theoretical prediction of a reversible process followed by a slower irreversible process. The basic thermodynamic parameters of glycogen phosphorylase b denaturation have been worked out and found to be: critical temperature 57.0 +/- 0.5 degrees C, transition half-width 8 +/- 1 degrees C, and calorimetric enthalpy change and Van't Hoff enthalpy change of the denaturation process 450 +/- 50 and 105 +/- 15 kcal/mol of enzyme monomer, respectively, at pH 7.4. These parameters have been found to be largely altered by the detergents octylglucoside, cholate, and deoxycholate at or below their critical micelle concentration, but not by Triton X-100 nor by lecithin liposomes. Organic solvents, such as dimethyl sulfoxide and methanol, and the presence of sarcoplasmic reticulum membranes produces an alteration of the denaturation thermogram of glycogen phosphorylase b similar to that produced by the above-mentioned detergents. These results allow us to hypothesize that hydrophobic domains of glycogen phosphorylase b are involved in its association to sarcoplasmic reticulum membranes in the sarcoplasmic reticulum/glycogenolytic complex of mammalian skeletal muscle.

Calorimetry, Differential Scanning↗
PubMedDate not supplied

Histochemical detection of glycogen phosphorylase activity as parameter for early ischemic damage in rat heart.

In the present study we have investigated whether enzyme histochemical parameters can be applied to detect early ischemic damage in rat heart after ischemia without restoration of the blood flow. Ischemia was induced by incubating heart fragments for 0, 10, 20, 30, 60, 120 and 240 min at 37 degrees C. The activity and localization of the following enzymes was studied in unfixed cryostat sections using quantitative histochemical methods: lactate dehydrogenase, creatine kinase, succinate dehydrogenase, phosphofructokinase, acid phosphatase, 5'-nucleotidase and glycogen phosphorylase. Moreover, the ultrastructure of the tissue was studied with special attention to the appearance of flocculent densities in mitochondria, which can be seen as a sign of irreversible cell damage. It was shown that glycogen phosphorylase activity in rat heart decreased after short periods (30 min) of in vitro ischemia, whereas all other enzymes studied were not decreased up to 240 min, with the exception of lactate dehydrogenase and phosphofructokinase activities which were diminished only at 240 and 120 min of ischemia, respectively. Some reaction product was found after incubating for 5'-nucleotidase activity in the absence of substrate, indicating the presence of endogenous substrate(s). This endogenous substrate disappeared from the myocytes after 20 min of ischemia. It is assumed that AMP and/or other phosphate-containing compounds play an essential role in the activation of glycogen phosphorylase. Significant reduction of glycogen phosphorylase activity is correlated with the irreversible stage of damage of myocytes as judged from the ultrastructure.

5'-Nucleotidase↗
PubMed1984

The effect of exercise training on glycogen, glycogen synthase and phosphorylase in muscle and liver.

It is thought that exercise training in both man and the rat results in a protective effect against the depletion of carbohydrate stores during exercise (glycogen-sparing). However there has been no comprehensive study of the effects of training on glycogen anabolic and catabolic enzymes with liver or muscle. The aim of this study was to examine whether changes in these enzymes occur and whether these changes may provide an explanation for the glycogen-sparing which results from exercise training. Male rats were trained by a treadmill running program at three different workloads. In addition, there were three control groups: free eating (SF), food restricted (SR), and one SF with a single bout of exercise prior to sacrifice. Exercise training was associated with a 60-150% increase in glycogen synthase and phosphorylase and a 50-70% increase in glycogen content in soleus, an intermediate muscle, but not in extensor digitorum longus (EDL), a white muscle nor in liver. The increase in glycogen synthase and phosphorylase in intermediate muscle was proportional to the degree of training and there was a significant correlation between glycogen content, glycogen synthase, and phosphorylase activity in intermediate muscle. Cytochrome c oxidase activity, an indicator of respiratory capacity, increased 50% in gastrocnemius of trained rats and was significantly correlated with glycogen synthase and phosphorylase in soleus. These results indicate a significant effect of exercise training on glycogen anabolic and catabolic enzymes in intermediate muscle, with no significant effects in white muscle or liver. The changes do not provide an explanation for glycogen-sparing, but are consistent with improved capacity of intermediate muscle for rapid glycogen mobilisation and repletion.

Animals↗
PubMed1995

Purification and isoenzymic composition of glycogen phosphorylase b from normal and abnormal (PSE) muscles.

Ion exchange chromatography and preparative isoelectric focusing allowed the identification of five isoenzymes of glycogen phosphorylase b from the longissimus dorsi muscle of normal pigs and those prone to having pale, soft and exudative (PSE) muscle. The isoelectric point of the isoenzymes varied in the pH range from 6.29 to 6.55. One of them, with an isoelectric point at about a pH of 6.49, accounts for 65% of the total glycogen phosphorylase b activity. No significant differences between normal and PSE-prone pigs were observed in the total glycogen phosphorylase b activity and in the isoenzyme distribution pattern. It is concluded that the fast glycogen turnover in PSE-prone pigs is not due to a different isoenzyme pattern of phosphorylase b.

Animals↗
PubMed1990

Phosphorylase b kinase deficiency in man: a review.

Phosphorylase b kinase is involved in the activation of glycogen phosphorylase and is thus involved in the breakdown of glycogen. The enzyme exists as several tissue specific isoenzymes of which the muscle enzyme (rabbit) has been most characterized. It is a multimeric protein composed of four subunits, alpha, beta, gamma and delta. The four subunits are coded on different chromosomes, the alpha, beta and gamma subunit genes being on the X, 16 and 17 chromosomes respectively. The delta subunit is a calmodulin and confers calcium sensitivity on phosphorylase b kinase. Tissue specificity of the enzyme is conferred, at least in some cases, by variation in the gamma subunit. Seven different clinical types of phosphorylase b kinase deficiency have been described. The most common type is X-linked and affects the liver only; other types affect liver, muscle and liver, muscle or heart and have an autosomal recessive mode of inheritance, while in some types the mode of inheritance is not clear. Diagnosis based on the study of erythrocytes or leukocytes can be misleading due to the tissue specific nature of the enzyme, and liver or muscle biopsies may be required.

Diagnosis↗
PubMed1987

Methylthioadenosine phosphorylase in human breast cancer.

Methylthioadenosine (MTA) phosphorylase activity was measured in 47 biopsies from primary breast cancers (n = 34) and metastatic tumors (n = 13). Most specimens were also evaluated by DNA flow cytometry and determination of estrogen and progesterone receptor contents. Median MTA phosphorylase activity was 317 pmol/mg protein/min (range 50-1312 pmol/mg protein/min), but great variations were observed. Samples from four individuals had very low MTA phosphorylase activity (less than or equal to 70 pmol/mg protein/min). No correlation with aneuploidy, receptor status, or the presence of metastases in the lymph nodes could be demonstrated. However, MTA phosphorylase activity showed a significant (p = 0.009) negative correlation with the fraction of cells in the S-phase of the cell cycle.

Adult↗
PubMed1994

Effect of vitamin B6 deficiency on the expression of glycogen phosphorylase mRNA in rat liver and skeletal muscle.

The effect of vitamin B6 deficiency on the expression of glycogen phosphorylase mRNA in rat liver and skeletal muscle was investigated. The level of phosphorylase mRNA in the muscle of vitamin B6-deficient rats was reduced to 40% of that in the control rats. By contrast, the phosphorylase mRNA level was increased 5-fold in the liver of the deficient animals. It was also found that the expression of the beta-actin gene, generally regarded as a 'housekeeping' gene, was unaffected by B6 deficiency in the muscle but was enhanced in the liver of the deficient animals. These observations suggest that vitamin B6 may modulate the transcriptional activation of the phosphorylase gene in a tissue-specific manner.

Actins↗
PubMed1983

Purification and partial characterization of bovine heart phosphorylase kinase.

Bovine heart phosphorylase kinase has been isolated by a procedure involving precipitation with polyethylene glycol, DEAE-Sephacel chromatography and calmodulin-Sepharose affinity chromatography. The isolated enzyme had a specific activity of 8.3 IU/mg of protein at pH 8.2 at 30 degrees C in the presence of 1% glycogen. The native enzyme had a sedimentation coefficient of 23 S and the Mr of the alpha', beta, gamma, and delta subunits, were 140,000, 130,000, 46,000, and 18,000, respectively. Activation of the phosphorylase kinase by the catalytic subunit of bovine heart cAMP-dependent protein kinase increases the pH 6.8/8.2 activity ratio from 0.01 to 0.32-0.38. Glycogen (1%) decreased the Km of the activated phosphorylase kinase at pH 6.8 for phosphorylase b from 5.5 to 1.25 mg/ml. Trypsin treatment increased the pH 6.8 activity but decreased the pH 8.2 activity. During this process the alpha' subunit was converted to a Mr 110,000 polypeptide and the enzyme activity was converted essentially to a 5.9 S species having an apparent Mr of 100,000 as determined by gel filtration. On extended trypsin treatment only one major polypeptide corresponding to the beta subunit remained. The same polypeptide was present in the active fractions following gel filtration of the trypsinized kinase.

Animals↗
PubMed1986

Identification of two forms of glycogen phosphorylase in Dictyostelium.

It has been known for 20 years that during cellular differentiation of Dictyostelium discoideum, glycogen is degraded to provide the glucose precursors that are required for the synthesis of the end-products of development. Because this pathway provided a distinct developmentally regulated event, a number of laboratories have investigated the regulation of the first step in glycogen degradation, glycogen phosphorylase. Of particular interest was the possible regulation of this enzyme by cAMP. Cyclic AMP is know to act as a signal in this organism for both chemotaxis and cell differentiation. The phosphorylase activity was found to increase during development and, therefore, it has been used in many studies as a marker for late stage development. However, only one form of the phosphorylase was found, and therefore it was concluded that cAMP was not involved in regulation of this key step in the developmental pathway. Here we report the discovery of a second form of the enzyme. This form is completely dependent on AMP for activity and is found only in the undifferentiated stage. This second form contains several of the properties of the nonphosphorylated enzyme that occurs in systems that are regulated by cAMP. This result and the recent discovery of a cAMP-dependent protein kinase has rekindled the possibility that at least one of the effects of cAMP in this organism occurs via a cAMP-dependent cascade of phosphorylation; that is, the activation of glycogen phosphorylase and subsequent production of the precursors for the end-products of development.

Adenosine Triphosphate↗
PubMed1987

Phosphorylase-mediated mobilization of the amino group of adenine in Bacillus cereus.

Mobilization of the ribose moiety of purine nucleosides as well as of the amino group of adenine may be realized in Bacillus cereus by the concerted action of three enzymes: adenosine phosphorylase, adenosine deaminase, and purine nucleoside phosphorylase. In this pathway, ribose-1-phosphate and inorganic phosphate act catalytically, being continuously regenerated by purine nucleoside phosphorylase and adenosine phosphorylase, respectively. As a result of such a metabolic pathway, adenine is quantitatively converted into hypoxanthine, thus overcoming the lack of adenase in B. cereus.

Adenine↗
PubMed1975

Photooxidation of alpha-glucan phosphorylases from rabbit muscle and potato tubers.

Photooxidation of alpha-glucan phosphorylases from rabbit muscle and potato tubers in the presence of rose bengal leads to a rapid loss of enzymatic activity which follows first-order kinetics. The process is pH dependent, being more rapid at higher pH. The inactivation is closely related to the destruction of histidine residues in the enzyme. It is suggested that histidine residues are largely responsible for the loss of enzymatic activity in the photooxidation. The inactivation of potato phosphorylase is retarded by substrates, whereas that of the muscle enzyme is not. The rate of photoinactivation of muscle phosphorylase b is increased with AMP, and decreased with ATP, ADP, IMP and glucose-6-P. This finding is considered to be closely related to the allosteric transition of phosphorylase.

Adenosine Monophosphate↗
PubMed1983

Evidence that the heat-stable protein activator of phosphorylase phosphatase is histone H1.

The heat-stable protein activator of phosphorylase phosphatase [FEBS Lett. 146, 331-334 (1982)] was located in the nuclear fraction. The activator was isolated from the nuclear fraction of various tissues. The largest amount was obtained from calf thymus and swine renal cortex. The activator protein from swine renal cortex was purified to apparent homogeneity. The activator had the same mobility as authentic histone H1 on sodium dodecyl sulfate or acetic acid/urea polyacrylamide gel electrophoresis. Phosphorylase phosphatase was activated by histone H1 or purified activator in an identical manner. Other histones were without effect or inhibited phosphorylase phosphatase activity. It is concluded that the heat-stable protein activator of phosphorylase phosphatase is histone H1.

Animals↗
PubMed1984

Mg2+ counteracts the inhibitory effect of spermine on liver phosphorylase kinase.

When the effect of polyamines on pig liver phosphorylase kinase was examined, spermine was found to be the most inhibitory. Although putrescine stimulated the reaction slightly, the spermidine effect was dependent on the phosphorylase b concentration. The inhibitory effect of spermine was counteracted by increasing the Mg2+ concentration. At 0.3 mM Mg2+, the apparent Km for phosphorylase b was increased 9-fold by the addition of 5 mM spermine. However, increasing Mg2+ to 3 mM decreased the value to the initial level obtained at 0.3 mM Mg2+ alone. These results suggest that a possible role of Mg2+ in the regulation of liver phosphorylase kinase is to protect the enzyme from the inhibitory action of a polyamine such as spermine.

Animals↗
PubMed1984

Synergistic activation of rat hepatocyte glycogen phosphorylase by A23187 and phorbol ester.

The combination of 1.6 microM 4 beta phorbol, 12 beta myristate, 13 alpha acetate (PMA) and 1 microM A23187 produced a five-fold greater stimulation of rat hepatocyte glycogen phosphorylase activity than was seen with PMA alone. Vasopressin activation of glycogen phosphorylase was comparable to that seen with PMA plus A23187. Glycogen phosphorylase activity due to PMA plus A23187 was increased significantly after 30 sec, maximal at 120 and sustained at elevated levels for 240 sec. In contrast, activation due to vasopressin was maximal at 30 sec followed by a decrease. The addition of PMA 5 min prior to the A23187 abolished the synergism between these two agents. These data are compatible with the hypothesis that diacylglycerol and Ca2+ synergistically increase glycogen phosphorylase activity in rat hepatocytes.

Animals↗
PubMed1985

Separation of rabbit liver latent and spontaneously active phosphorylase phosphatases by chromatography on heparin-sepharose.

Latent and spontaneously active forms of phosphorylase phosphatase were separated by heparin-Sepharose chromatography of rabbit liver extract. The latent enzyme had an absolute polycation (histone H1, polybrene) requirement for the activity assayed with phosphorylase a and phosphorylase kinase substrates. Ethanol treatment resulted in the activation of both phosphatases by dissociating of 150-180 kDa holoenzymes to 33-38 kDa catalytic subunits as judged by gel filtration. The latent and spontaneously active phosphatases were differentiated according to their abilities to dephosphorylate the alpha and the beta subunits of phosphorylase kinase and sensitivities to inhibition by inhibitor-2 or heparin, and were classified as type-2A and type-1 phosphatases, respectively.

Animals↗
PubMed1990

K252a is a potent and selective inhibitor of phosphorylase kinase.

The inhibition of phosphorylase kinase by a number of protein kinase inhibitors was examined. Both K252a and staurosporine are potent inhibitors of phosphorylase kinase with IC50 values of 1.7 nM and 0.5 nM respectively. K252a shows a 300-fold selectivity for this enzyme over protein kinase C whereas staurosporine shows only a 20-fold selectivity for phosphorylase kinase. In contrast, the Roche bis-indolyl maleimides inhibit phosphorylase kinase with IC50 values of approximately 1 microM and are highly selective for protein kinase C.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗