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PubMed1978

Crystallographic studies on the activity of glycogen phosphorylase b.

High resolution studies on the crystal structure of glycogen phosphorylase b have identified the catalytic site to which the substrate glucose-1-phosphate binds strongly with some local conformational changes. The site is situated 8 A (phosphate-to-phosphate distance) from pyridoxal phosphate, an essential cofactor of all glycogen phosphorylases. The catalytic site is 33 A from the site in the N-terminal portion of the molecule to which adenine nucleotides bind. In contrast to phosphorylase a (the active form of the enzyme which is phosphorylated at Ser 14), the positions of the first 19 residues of phosphorylase b are not well defined.

Adenosine Monophosphate↗

PubMed1974

Mobilization of granulose in Clostridium pasteurianum. Purification and properties of granulose phosphorylase.

1. The granulose of Clostridium pasteurianum ATCC 6013 is degraded when the organism is incubated in a medium containing no utilizable source of carbon and energy. 2. Mobilization of the polyglucan does not occur in the presence of exogenous glucose. 3. Breakdown of granulose is effected by a constitutively synthesized alpha-1,4-polyglucan phosphorylase. 4. Partial (530-fold) purification of this granulose phosphorylase was facilitated by its being loosely bound to the native granules of its substrate polyglucan. 5. The enzyme (pH optimum 6.4) was assayed both (a) in the degradative direction, K(m) for P(i)=2.2mm, and (b) in the synthetic direction, K(m) for glucose 1-phosphate=0.05mm. No requirement for bivalent cations was evidenced. 6. Granulose phosphorylase was inhibited by various nucleotide sugars; GDP-glucose, ADP-glucose (K(i)=20mum) and UDP-glucose (K(i)=60mum) were particularly potent competitive inhibitors. ATP, NADP(+) and NADPH (at 1mm) were less effective inhibitors, whereas AMP was slightly stimulatory. 7. It would appear that granulose mobilization is favoured under conditions of low adenylate energy charge, but is prevented under conditions of ;glucose excess' chiefly by ADP-glucose-mediated inhibition of granulose phosphorylase.

Adenosine Diphosphate Glucose↗

PubMed1977

Phosphorylation of the inhibitory subunit of troponin in perfused hearts of mice deficient in phosphorylase kinase. Evidence for the phosphorylation of troponin by adenosine 3':5'-phosphate-dependent protein kinase in vivo.

When hearts from control and phosphorylase kinase-deficient (I strain) mice were perfused with 0.1 micrometer-DL-isoprenaline, there was a parallel increase in contraction, cyclic AMP concentration and troponin I phosphorylation. However, there was no increase in phosphorylase a in the I-strain hearts, whereas the control hearts showed a large increase. Assays of I-strain heart extracts showed a normal cyclic AMP-dependent protein kinase activity but no phosphorylase kinase activity. It is concluded that troponin I is phosphorylated in intact hearts by protein kinase and not phosphorylase kinase.

Animals↗

PubMed1989

Expression of a cDNA for the catalytic subunit of skeletal-muscle phosphorylase kinase in transfected 3T3 cells.

Phosphorylase kinase is a multimeric enzyme of composition (alpha, beta, gamma, delta)4 whose catalytic activity resides in the gamma-subunit. As an approach to understand further its regulation, a cDNA for the gamma-subunit of phosphorylase kinase (gamma PhK) has been cloned into a mammalian expression vector behind the mouse metallothionein-1 promoter. NIH 3T3 cells were co-transfected with this construct (pEV gamma PhK) and pSV2neo, G418-resistant clones were selected, and several were found to have stably incorporated the gamma-subunit cDNA into their genomic DNA. Phosphorylase kinase activity was clearly present in extracts from cultures of pEV gamma PhK-transformed cells and increased several-fold after 24 h of incubation with Zn2+, whereas it was undetectable in the parent 3T3 cells. A significant, but variable, proportion (15-70%) of the activity was Ca2+-dependent. We conclude that the phosphorylase kinase activity expressed by the cells transformed with pEV gamma PhK is due to free gamma-subunit and gamma-subunit associated with cellular calmodulin, which replaces the delta-subunit normally associated with the gamma-subunit in the holoenzyme.

Animals↗

PubMed1992

Deficiency of 5'-deoxy-5'-methylthioadenosine phosphorylase activity in malignancy. Absence of the protein in human enzyme-deficient cell lines.

The absence of 5'-deoxy-5'-methylthioadenosine phosphorylase (MTAase) activity in malignant cells, and the putative localization of its gene, suggest that this enzyme deficiency might be due to a genomic alteration also involving a tumour-suppressor gene. We studied the possible occurrence of inactive forms of the protein in two MTAase-negative cell lines, namely K562 and Jurkat, by immunochemical methods. Two highly specific antisera, directed against different epitopes of the phosphorylase [Della Ragione, Oliva, Gragnaniello, Russo, Palumbo & Zappia (1990) J. Biol. Chem. 265, 6241-6246], were used to carry out immunotitration and immunoblotting analyses, as well as to investigate the biosynthesis of the enzyme. No MTAase protein was detected by Western-blotting technique performed under conditions where all the phosphorylase-positive samples gave a clear band at the MTAase subunit molecular mass. No cross-reacting material was observed by a sensitive immunotitration method which permitted the detection of as low as 0.5 ng of protein. Moreover, the results obtained by [35S]methionine-labelling experiments ruled out phosphorylase biosynthesis in the negative cell lines. Altogether, these data suggest that an alteration at the gene level hampering the specific mRNA biosynthesis or resulting in an untranslatable mRNA is the cause of the enzyme deficiency in the MTAase-negative cell lines studied.

Antibody Specificity↗

PubMed1992

A kinetic re-interpretation of the regulation of rabbit skeletal-muscle phosphorylase kinase activity by Ca2+ and phosphorylation.

The regulation of phosphorylase kinase has been proposed to occur physiologically under conditions of zero-order ultrasensitivity [Meinke & Edstrom (1991) J. Biol. Chem. 266, 2259-2266]. This is also one of the conditions that recent theoretical approaches have indicated to be essential in order for an interconvertible enzyme cascade to generate a sensitive response to an effector [Cardenas & Cornish-Bowden (1989) Biochem. J. 257, 339-345]. In contrast, all published kinetic data to date have strongly suggested that activation of phosphorylase kinase by Ca2+ or phosphorylation is attributable solely to a change in affinity for phosphorylase, with no effect on the Vmax. of the reaction. In this study an attempt is made to resolve this conflict. Findings suggest that changes in Vmax. can fully account for the activation of phosphorylase kinase by the physiological mechanisms of cyclic AMP-dependent phosphorylation and increase in Ca2+ concentration.

Animals↗

PubMed1997

Regulation of glycogen synthase and phosphorylase during recovery from high-intensity exercise in the rat.

The aim of this study was to determine the role of the phosphorylation state of glycogen synthase and glycogen phosphorylase in the regulation of muscle glycogen repletion in fasted animals recovering from high-intensity exercise. Groups of rats were swum to exhaustion and allowed to recover for up to 120 min without access to food. Swimming to exhaustion caused substantial glycogen breakdown and lactate accumulation in the red, white and mixed gastrocnemius muscles, whereas the glycogen content in the soleus muscle remained stable. During the first 40 min of recovery, significant repletion of glycogen occurred in all muscles examined except the soleus muscle. At the onset of recovery, the activity ratios and fractional velocities of glycogen synthase in the red, white and mixed gastrocnemius muscles were higher than basal, but returned to pre-exercise levels within 20 min after exercise. In contrast, after exercise the activity ratios of glycogen phosphorylase in the same muscles were lower than basal, and increased to pre-exercise levels within 20 min. This pattern of changes in glycogen synthase and phosphorylase activities, never reported before, suggests that the integrated regulation of the phosphorylation state of both glycogen synthase and phosphorylase might be involved in the control of glycogen deposition after high-intensity exercise.

Animals↗

PubMed1999

Kinetic study on the dimer-tetramer interconversion of glycogen phosphorylase a.

Kinetic theory of dissociating enzyme systems has been applied to a study of the dimer-tetramer interconversion of glycogen phosphorylase a. All kinetic constants for the dissociating-associating reaction of phosphorylase a have been determined. The results indicate that (a) the presence of glucose-1-phosphate has no influence on either the rate of dissociation or the rate of association, and hence does not shift the dimer-tetramer equilibrium of phosphorylase a; (b) the binding og glycogen to the enzyme decreases the association rate of the dimer to form the tetramer, but has no effect on the dissociation rate of the tetramer; (c) both the dimeric and tetrameric form of phosphorylase a can bind glycogen, but the tetrameric form has a lower affinity for glycogen and is catalytically inactive.

Glucosephosphates↗

PubMed1975

Adenine formation from adenosine by mycoplasmas: adenosine phosphorylase activity.

Mammalian cells have enzymes to convert adenosine to inosine by deamination and inosine to hypoxanthine by phosphorolysis, but they do not possess the enzymes necessary to form the free base, adenine, from adenosine. Mycoplasmas grown in broth or in cell cultures can produce adenine from adenosine. This activity was detected in a variety of mycoplasmatales, and the enzyme was shown to be adenosine phosphorylase. Adenosine formation from adenine and ribose 1-phosphate, the reverse reaction of adenine formation from adenosine, was also observed with the mycoplasma enzyme. Adenosine phosphorylase is apparently common to the mycoplasmatales but it is not universal, and the organisms can be divided into three groups on the basis of their use of adenosine as substrate. Thirteen of 16 Mycoplasma, Acholeplasma, and Siroplasma species tested exhibit adenosine phosphorylase activity. M. lipophilium differed from the other mycoplasmas and shared with mammalian cells the ability to convert adenosine to inosine by deamination. M. pneumoniae and the unclassified M. sp. 70-159 showed no reaction with adenosine. Adenosine phosphorylase activity offers an additional method for the detection of mycoplasma contamination of cells. The patterns of nucleoside metabolism will provide additional characteristics for identification of mycoplasmas and also may provide new insight into the classification of mycoplasmas.

Acholeplasma laidlawii↗

PubMed1977

Lymphospecific toxicity in adenosine deaminase deficiency and purine nucleoside phosphorylase deficiency: possible role of nucleoside kinase(s).

Inherited deficiencies of the enzymes adenosine deaminase (adenosine aminohydrolase; EC 3.5.4.4) and purine nucleoside phosphorylase (purine-nucleoside:orthophosphate ribosyltransferase; EC 2.4.2.1) preferentially interfere with lymphocyte development while sparing most other organ systems. Previous experiments have shown that through the action of specific kinases, nucleosides can be "trapped" intracellularly in the form of 5'-phosphates. We therefore measured the ability of newborn human tissues to phosphorylate adenosine and deoxyadenosine, the substrate of adenosine deaminase, and also inosine, deoxyinosine, guanosine, and deoxyguanosine, the substrates of purine nucleoside phosphorylase. Substantial activities of adenosine kinase were found in all tissues studied, while guanosine and inosine kinases were detected in none. However, the ability to phosphorylate deoxyadenosine, deoxyinosine, and deoxyguanosine was largely confined to lymphocytes. Adenosine deaminase, but not purine nucleoside phosphorylase, showed a similar lymphoid predominance. Other experiments showed that deoxyadenosine, deoxyinosine, and deoxyguanosine were toxic to human lymphoid cells. The toxicity of deoxyadenosine was reversed by the addition of deoxycytidine, but not uridine, to the culture medium. Based upon these and other experiments, we propose that in adenosine deaminase and purine nucleoside phosphorylase deficiency, toxic deoxyribonucleosides produced by many tissues are selectively trapped in lymphocytes by phosphorylating enzyme(s).

Adenosine Deaminase↗

PubMed1982

Possible metabolic basis for the different immunodeficient states associated with genetic deficiencies of adenosine deaminase and purine nucleoside phosphorylase.

An inherited deficiency of adenosine deaminase (Ado deaminase; adenosine aminohydrolase, EC 3.5.4.4) causes severe combined immunodeficiency disease in humans. A similar deficiency in purine nucleoside phosphorylase (Puo phosphorylase; purine-nucleoside:orthophosphate ribosyltransferase, EC 2.4.2.1) engenders a selective cellular immune deficit. To elucidate the possible metabolic basis for the contrasting immunologic phenotypes, we compared the toxicity toward mature resting human lymphocytes of the Ado deaminase substrates deoxyadenosine and adenosine and the Puo phosphorylase substrate deoxyguanosine. When Ado deaminase was inhibited, micromolar concentrations of deoxyadenosine progressively killed nondividing helper and suppressor-cytotoxic T cells, but not B cells. The toxicity required phosphorylation, with subsequent dATP formation. The deoxyadenosine analogs 2-chlorodeoxyadenosine, 2-fluorodeoxyadenosine, and adenine arabinonucleoside also killed resting T cells. Cell death was unrelated to inhibition of adenosylhomocysteinase (EC 3.3.1.1) but was preceded by a gradual decline in ATP levels. As much as 1 mM deoxyguanosine did not impair resting lymphocyte viability, despite the synthesis of dGTP. The combination of 200 microM adenosine plus 500 microM homocysteine thiolactone killed dividing lymphocytes but had no discernible toxic effect toward resting T cells, which accumulated adenosylhomocysteine over a 4-hr period but thereafter excreted the nucleoside into the culture medium. The different clinical syndromes associated with genetic deficiencies of Ado deaminase and Puo phosphorylase may be explained by the ability of dATP to kill mature resting T lymphocytes by depleting ATP levels.

Adenosine↗

PubMed1990

Functional and structural similarities between the inhibitory region of troponin I coded by exon VII and the calmodulin-binding regulatory region of the catalytic subunit of phosphorylase kinase.

A sequence homology has been noted between the carboxyl quarter of the catalytic gamma subunit of phosphorylase kinase and the region of troponin I coded by exon VII. Because this portion of troponin I contains the inhibitory region that interacts with actin and troponin C, we have examined whether the gamma subunit of phosphorylase kinase can functionally mimic troponin I by also interacting with actin and troponin C. We have found that troponin C not only activates the isolated gamma subunit of phosphorylase kinase but also binds with approximately the same affinity as calmodulin. Although actin had no effect on the activity of the gamma subunit alone, it did inhibit the activity of gamma-calmodulin and gamma-troponin C complexes. Conversely, the gamma subunit was able to inhibit actomyosin ATPase in a process that could be overcome by calmodulin. These results suggest that actin and calmodulin (or troponin C) compete for binding to the gamma subunit. Moreover, the structural and functional similarities between the gamma subunit and troponin I suggest that the gamma subunit of phosphorylase kinase may have evolved from the fusion of a protein kinase protogene with a progenitor of exon VII of troponin I.

Actins↗

PubMed1995

Purification, cloning, and expression of murine uridine phosphorylase.

Uridine phosphorylase was purified 10,300-fold from tumors of the murine colorectal adenocarcinoma cell line, Colon-26. Degenerate DNA probes were synthesized corresponding to partial amino acid sequences and used to screen a Colon-26 cDNA library. A cDNA clone of 1327 base pairs that contains a 5' untranslated region, a coding region of 933 base pairs, and a 3' nontranslated region with a polyadenylated tail was identified. The cDNA was confirmed to be uridine phosphorylase by 1) sequence comparison to uridine phosphorylase of Escherichia coli, 2) substrate specificity studies with recombinant protein expressed in COS-7 cells that demonstrated relatively high enzyme activity with uridine as substrate compared low levels when thymidine was used, and 3) inhibition of enzyme activity by the competitive inhibitor 2,2'-anhydro-5-ethyluridine. Northern blot analysis using the cDNA as a probe, demonstrated high levels of mRNA expression in Colon-26. Expression was low in NIH3T3 cells, but high in DMBA-3 and PH-1 cells, which are NIH3T3-derived cells that have been transformed with mutated murine Ha-ras and viral Ha-ras, respectively. Expression of uridine phosphorylase mRNA in these cell lines was further enhanced by treating the cells with the inflammatory cytokines, tumor necrosis factor-alpha, interleukin 1 alpha, and interferon gamma.

Adenocarcinoma↗

PubMed2006

Peptide microarray analysis of substrate specificity of the transmembrane Ser/Thr kinase KPI-2 reveals reactivity with cystic fibrosis transmembrane conductance regulator and phosphorylase.

Human lemur (Lmr) kinases are predicted to be Tyr kinases based on sequences and are related to neurotrophin receptor Trk kinases. This study used homogeneous recombinant KPI-2 (Lmr2, LMTK2, Cprk, brain-enriched protein kinase) kinase domain and a library of 1,154 peptides on a microarray to analyze substrate specificity. We found that KPI-2 is strictly a Ser/Thr kinase that reacts with Ser either preceded by or followed by Pro residues but unlike other Pro-directed kinases does not strictly require an adjacent Pro residue. The most reactive peptide in the library corresponds to Ser-737 of cystic fibrosis transmembrane conductance regulator, and the recombinant R domain of cystic fibrosis transmembrane conductance regulator was a preferred substrate. Furthermore the KPI-2 kinase phosphorylated peptides corresponding to the single site in phosphorylase and purified phosphorylase b, making this only the second known phosphorylase b kinase. Phosphorylase was used as a specific substrate to show that KPI-2 is inhibited in living cells by addition of nerve growth factor or serum. The results demonstrate the utility of the peptide library to probe specificity and discover kinase substrates and offer a specific assay that reveals hormonal regulation of the activity of this unusual transmembrane kinase.

Amino Acid Sequence↗

PubMed1975

Phosphorylase activity in chronic erythremic myelosis.

Phosphorylase activity was detected in the cytoplasm of erythroid precursors of 6 of 7 patients with chronic erythremic myelosis (Di Guglielmo syndrome), in proerythroblasts and megaloblasts from 3 patients with pernicious anemia and in 2 patients with severe folate deficiency in neoplastic lymphocytes from 2 patients with acute lymphoblastic leukemia, and in 1 patient with leukemic lymphosarcoma. In all of these patients, most of the erythroid precursors and/or neoplastic lymphocytes contained increased amounts of glycogen when stained with the PAS reagent. Phosphorylase activity was not detected in erythroid precursors obtained from 6 presumed normal individuals or from 3 of 7 patients with a variety of other types of anemia in which the erythroid precursors were PAS-negative. Similarly, phosphorylase activity was absent in lymphocytes obtained from presumed normal individuals. Although the mechanisms responsible for the pathogenesis of PAS positivity are unclear, it is possible that the increased phosphorylase activity found in cells that are PAS-positive may reflect a disorder in the biosynthetic pathway of glycogen.

Anemia, Hemolytic↗

PubMed1985

Effect of deficient and excess dietary vitamin B-6 on amino transaminase and glycogen phosphorylase activity and pyridoxal phosphate content in two muscles from postpubertal gilts.

Postpubertal gilts averaging 111 kg and gaining 2.7 kg/wk were fed daily 1.9 kg/d of a diet providing 0.45, 2.1 or 83 mg of vitamin B-6/d. An additional group of animals were fed the high vitamin B-6 diet providing 83 mg of vitamin B-6/d for the initial 57 d of the experiment and then switched to 0.45 mg of vitamin B-6/d for the remainder of the 121-d experiment (61 gilts total). On d 0, 57 and 121, animals from each treatment were killed, and samples of the semitendinosus (ST) and semimembranosus (SM) were removed. Glutamic-oxaloacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT), glycogen phosphorylase and pyridoxal phosphate (PLP) were measured in muscle tissues. The erythrocyte GOT activity coefficient indicated that gilts consuming 0.45 or 2.1 mg of vitamin B-6/d developed a vitamin B-6 deficiency. A vitamin B-6 deficiency resulted in the loss of whole-muscle transaminase activity (enzyme activity X muscle weight) with little effect on whole-muscle total phosphorylase or total PLP content. Excess dietary vitamin B-6 increased whole-muscle total PLP and total phosphorylase content with small decreases in whole-muscle transaminase. Under these conditions, muscle tissue acts as a nonmobile reservoir of PLP. Sixty to 95% of muscle PLP was bound to muscle glycogen phosphorylase.

Alanine Transaminase↗

PubMed1978

Amino acid sequence around the pyridoxal 5'-phosphate binding site in potato phosphorylase.

The amino acid sequence around the pyridoxal 5'-phosphate binding site in potato phosphorylase was determined in order to compare it with those in phosphorylases from other sources having different regulatory properties. The potato enzyme was reduced by NaBH4 in the presence of urea, carboxymethylated, and digested with chymotrypsin and trypsin. Pyridoxyl peptides were isolated by the differential procedure using paper electrophoresis or DEAE-cellulose column chromatography. In Edman degradation of these peptides, pyridoxyllysine was identified as the phenylthiohydantoin derivative of pyridoxyllysine using a combination of thin-layer chromatography and the Pauli reaction. The sequence around pyridoxyllysine, comprising 57 amino acid residues, was determined except for a region with 6 amino acid residues. The pyridoxal 5'-phosphate binding site in potato phosphorylase showed a high homology with those of the rabbit muscle and yeast enzymes. This finding suggests that the cofactor should be directly related to the essential process of phosphorylase action.

Amino Acid Sequence↗

PubMed1982

Structural similarities in the active-site region between potato and rabbit muscle phosphorylases: a lysyl residue located close to the pyridoxal 5'-phosphate.

P1,P2-bis(5'-pyridoxal)diphosphate crosslinks between the original cofactor (pyridoxal 5'-phosphate) linking residue and Lys-573 in rabbit muscle phosphorylase (Shimomura, S., Nakano, K., & Fukui, T. (1978) Biochem. Biophys. Res. Commun. 82, 462-468). We have applied the same technique to potato phosphorylase to compare the structures of the active-site regions of the two enzymes, which have different regulatory properties. The reagent was bound to the potato enzyme in the same binding mode as the rabbit muscle enzyme. A sequence study on the potato enzyme labeled with this reagent revealed that it crosslinks between the original cofactor-linking lysyl residue and another lysyl residue, respectively corresponding to Lys-679 and Lys-573 in the rabbit muscle enzyme, and that the sequence Lys-573 to Leu-577 in the rabbit muscle enzyme is conserved in the potato enzyme. These findings indicate structural similarities in the active-site region between the phosphorylases, and suggest the importance of a lysyl residue in the catalytic mechanism of the phosphorylase reaction.

Amino Acid Sequence↗