pH-Independent Heat Capacity Changes during Phosphorolysis Catalyzed by the Pyrimidine Nucleoside Phosphorylase from Geobacillus thermoglucosidasius.

Enzyme-catalyzed reactions sometimes display curvature in their Eyring plots in the absence of denaturation, indicative of a change in activation heat capacity. However, the effects of pH and (de)protonation on this phenomenon have remained unexplored. Herein, we report a kinetic characterization of the thermophilic pyrimidine nucleoside phosphorylase from Geobacillus thermoglucosidasius across a two-dimensional working space covering 35 °C and 3 pH units with two substrates displaying different pKa values. Our analysis revealed the presence of a measurable activation heat capacity change ΔCp⧧ in this reaction system, which showed no significant dependence on medium pH or substrate charge. Our results further describe the remarkable effects of a single halide substitution that has a minor influence on ΔCp⧧ but conveys a significant kinetic effect by decreasing the activation enthalpy, causing a >10-fold rate increase. Collectively, our results present an important piece in the understanding of enzymatic systems across multidimensional working spaces where the choice of reaction conditions can affect the rate, affinity, and thermodynamic phenomena independently of one another.

Rat skeletal muscle glycogen degradation pathways reveal differential association of glycogen-related proteins with glycogen granules

Glycogenin, glycogen-debranching enzyme (GDE) and glycogen phosphorylase (GP) are important enzymes that contribute to glycogen particle metabolism. In Long-Evans Hooded rat whole muscle homogenates prepared from extensor digitorum longus (EDL, fast-twitch) and soleus (SOL, oxidative, predominantly slow twitch), it was necessary to include alfa-amylase, which releases glucosyl units from glycogen, to detect glycogenin but not GDE or GP. Up to ∼12 % of intramuscular glycogen pool was broken down using either in vitro electrical stimulation or leaving muscle at room temperature >3 h (delayed, post-mortem). Electrical stimulation did not reveal glycogenin unless alfa-amylase was added, although in post-mortem muscle ∼50 and ∼30 % of glycogenin in EDL and SOL muscles, respectively, was detected compared to the amount detected with alfa-amylase treatment. Single muscle fibres were dissected from fresh or post-mortem EDL muscles, mechanically skinned to remove surface membrane and the presence of glycogenin, GDE and GP as freely diffusible proteins (i.e. cytoplasmic localization) compared by Western blotting. Diffusibility of glycogenin (∼20 %) and GP (∼60 %) was not different between muscles, although GDE increased from ∼15 % diffusible in fresh muscle to ∼60 % in post-mortem muscle. Under physiologically relevant circumstances, in rat muscle and within detection limits: (1) The total cellular pool of glycogenin is always associated with glycogen granules, (2) GDE is associated with glycogen granules with over half the total pool associated with the outer tiers of glycogen, (3) GP is only ever weakly associated with glycogen granules and (4) addition of alfa-amylase is necessary in order to detect glycogenin, but not GDE or GP

Methylthioinosine phosphorylase from Pseudomonas aeruginosa. Structure and annotation of a novel enzyme in quorum sensing.

The PA3004 gene of Pseudomonas aeruginosa PAO1 was originally annotated as a 5'-methylthioadenosine phosphorylase (MTAP). However, the PA3004 encoded protein uses 5'-methylthioinosine (MTI) as a preferred substrate and represents the only known example of a specific MTI phosphorylase (MTIP). MTIP does not utilize 5'-methylthioadenosine (MTA). Inosine is a weak substrate with a k(cat)/K(m) value 290-fold less than MTI and is the second best substrate identified. The crystal structure of P. aeruginosa MTIP (PaMTIP) in complex with hypoxanthine was determined to 2.8 Šresolution and revealed a 3-fold symmetric homotrimer. The methylthioribose and phosphate binding regions of PaMTIP are similar to MTAPs, and the purine binding region is similar to that of purine nucleoside phosphorylases (PNPs). The catabolism of MTA in P. aeruginosa involves deamination to MTI and phosphorolysis to hypoxanthine (MTA → MTI → hypoxanthine). This pathway also exists in Plasmodium falciparum, where the purine nucleoside phosphorylase (PfPNP) acts on both inosine and MTI. Three tight-binding transition state analogue inhibitors of PaMTIP are identified with dissociation constants in the picomolar range. Inhibitor specificity suggests an early dissociative transition state for PaMTIP. Quorum sensing molecules are associated with MTA metabolism in bacterial pathogens suggesting PaMTIP as a potential therapeutic target.

Plastidial phosphorylase is required for normal starch synthesis in Chlamydomonas reinhardtii.

Among the three distinct starch phosphorylase activities detected in Chlamydomonas reinhardtii, two distinct plastidial enzymes (PhoA and PhoB) are documented while a single extraplastidial form (PhoC) displays a higher affinity for glycogen as in vascular plants. The two plastidial phosphorylases are shown to function as homodimers containing two 91-kDa (PhoA) subunits and two 110-kDa (PhoB) subunits. Both lack the typical 80-amino-acid insertion found in the higher plant plastidial forms. PhoB is exquisitely sensitive to inhibition by ADP-glucose and has a low affinity for malto-oligosaccharides. PhoA is more similar to the higher plant plastidial phosphorylases: it is moderately sensitive to ADP-glucose inhibition and has a high affinity for unbranched malto-oligosaccharides. Molecular analysis establishes that STA4 encodes PhoB. Chlamydomonas reinhardtii strains carrying mutations at the STA4 locus display a significant decrease in amounts of starch during storage that correlates with the accumulation of abnormally shaped granules containing a modified amylopectin structure and a high amylose content. The wild-type phenotype could be rescued by reintroduction of the cloned wild-type genomic DNA, thereby demonstrating the involvement of phosphorylase in storage starch synthesis.

Glycogen stability and glycogen phosphorylase activities in isolated skeletal muscles from rat and toad.

There is increasing evidence that endogenous glycogen depletion may affect excitation-contraction (E-C) coupling events in vertebrate skeletal muscle. One approach employed in physiological investigations of E-C coupling involves the use of mechanically skinned, single fibre preparations obtained from tissues stored under paraffin oil, at room temperature (RT: 20-24 degrees C) and 4 degrees C for several hours. In the present study, we examined the effect of these storage conditions on the glycogen content in three muscles frequently used in research on E-C coupling: rat extensor digitorum longus (EDL) and soleus (SOL) and toad iliofibularis (IF). Glycogen content was determined fluorometrically in homogenates prepared from whole muscles, stored under paraffin oil for up to 6 h at RT or 4 degrees C. Control muscles and muscles stored for 0.5 and 6 h were also analysed for total phosphorylase (Phos(total)) and phosphorylase a (Phos a) activities. No significant change was observed in the glycogen content of EDL and SOL muscles stored at RT for 0.5 h. In rat muscles stored at RT for longer than 0.5 h, the glycogen content decreased to 67.6% (EDL) and 78.7% (SOL) of controls after 3 h and 25.3% (EDL) and 37.4% (SOL) after 6 h. Rat muscles stored at 4 degrees C retained 79.0% (EDL) and 92.5% (SOL) of glycogen after 3 h and 75.2% (EDL) and 61.1% (SOL) after 6 h. The glycogen content of IF muscles stored at RT or 4 degrees C for 6 h was not significantly different from controls. Phos(total) was unchanged in all muscles over the 6 h period, at both temperatures. Phos a was also unchanged in the toad IF muscles, but in rat muscles it decreased rapidly, particularly in EDL (4.1-fold after 0.5 h at RT). Taken together these results indicate that storage under paraffin oil for up to 6 h at RT or 4 degrees C is accompanied by minimal glycogen loss in toad IF muscles and by a time- and temperature-dependent glycogen loss in EDL and SOL muscles of the rat.

Hippocampal glycogen metabolism, EEG, and behavior.

Glycogen phosphorylase a (GPa) is correlated with metabolic activation, suggesting its potential use as a marker for neuronal activity. In dentate gyrus, GPa patches are induced by glutamate infusion. Hippocampal electroencephalogram (EEG) and neuronal firing rates are modulated by behavioral state, and cell discharge is suppressed by restraint. In rats, under conditions of free exploration, passive movement under loose or secure restraint, quiet wakefulness, and anesthesia, GPa activity and 6-10-Hz theta power were inversely related: The more active the animal, the stronger the theta rhythm and the lower the GPa activity. Thus, GPa was least under conditions in which the hippocampus processes external information, and at intermediate levels during restraint, when neuronal firing is lowest. This dissociation raises doubts about the use of metabolic activity as an indicator of changes in neuronal activity or of information processing per se.

Histocytochemical and immunohistochemical studies related to the role of glycogen in human developing digestive organs.

To elucidate the role of glycogen in the epithelium of developing digestive organs, we investigated the appearance of glycogen and glycogen phosphorylase (GP) in these organs. We studied 64 externally normal human embryos at Carnegie stages 13-23 (5.1-28.0 mm in crown-rump length, 4-8 weeks of gestation) by histocytochemical staining for glycogen and immunohistochemical staining with antibodies against two isoenzymes of GP: brain-type (BGP) and muscle-brain-type (MBGP) GP. At stage 13, glycogen appeared in the epithelium of the digestive tract and the parenchyma of the pancreas. As development advanced, glycogen granules increased in number and size in these tissues, and they became evenly distributed in the epithelium of the digestive tract as either single particles or aggregates, as deduced by electron microscopy at late embryonic stages. Immunoreactivity specific both for BGP and for MBGP was detected in the digestive tract and the pancreas from stage 13. As development advanced, both BGP- and MBGP-immunoreactive cells increased in number and in immunoreactivity, and the number of MBGP-immunoreactive cells became larger than that of BGP-immunoreactive cells. By contrast, in hepatic cells, which serve as a major storage site for glycogen in adults, glycogen was detected only from stage 20, in smaller amounts, without formation of aggregates, and no immunoreactivity specific for BGP or MBGP was apparent throughout the embryonic stages examined. Thus, in the epithelium of the digestive tract and the parenchyma of the pancreas, but not in hepatic cells, the appearance and localization of GP coincided almost exactly with that of glycogen. These observations suggest that glycogen in the epithelium of the digestive tract and the parenchyma of the pancreas has not only been synthesized but also degraded from an early embryonic period and may, thus, be related to active cellular metabolism that is specific for embryonic development, including proliferation of the epithelium and interactions between epithelium and mesenchyme.

Multiple metabolic effects of CGRP in conscious rats: role of glycogen synthase and phosphorylase.

Calcitonin gene-related peptide (CGRP) is a neuropeptide that is released at the neuromuscular junction in response to nerve excitation. To examine the relationship between plasma CGRP concentration and intracellular glucose metabolism in conscious rats, we performed insulin (22 pmol.kg-1.min-1) clamp studies combined with the infusion of 0, 20, 50, 100, 200, and 500 pmol.kg-1.min-1 CGRP (plasma concentrations ranging from 2 x 10(-11) to 5 x 10(-9) M). CGRP antagonized insulin's suppression of hepatic glucose production at plasma concentrations (approximately 10(-10) M) that are only two- to fivefold its basal portal concentration. Insulin-mediated glucose disposal was decreased by 20-32% when CGRP was infused at 50 pmol.kg-1.min-1 (plasma concentration 3 x 10(-10) M) or more. The impairment in insulin-stimulated glycogen synthesis in skeletal muscle accounted for all of the CGRP-induced decrease in glucose disposal, while whole body glycolysis was increased despite the reduction in total glucose uptake. The muscle glucose 6-phosphate concentration progressively increased during the CGRP infusions. CGRP inhibited insulin-stimulated glycogen synthase in skeletal muscle with a 50% effective dose of 1.9 +/- 0.36 x 10(-10) M. This effect on glycogen synthase was due to a reduction in enzyme affinity for UDP-glucose, with no changes in the maximal velocity. In vitro CGRP stimulated both hepatic and skeletal muscle adenylate cyclase in a dose-dependent manner. These data suggest that 1) CGRP is a potent antagonist of insulin at the level of muscle glycogen synthesis and hepatic glucose production; 2) inhibition of glycogen synthase is its major biochemical action in skeletal muscle; and 3) these effects are present at concentrations of the peptide that may be in the physiological range for portal vein and skeletal muscle. These data underscore the potential role of CGRP in the physiological modulation of intracellular glucose metabolism.

Partially phosphorylated glycogen phosphorylase in the lugworm Arenicola marina, its regulatory function during hypoxia.

Glycogen phosphorylase (GPase) from the body wall of the lugworm Arenicola marina (Annelida, Polychaeta) probably exists as a phospho-dephospho hybrid (GPase ab). The hybrid was identified by phosphorylation of purified lugworm GPase b (unphosphorylated form) with rabbit muscle GPase kinase and [gamma-32P]ATP. The completeness of phosphorylation was checked on DEAE-Sephacel. Only one GPase form was eluted. Its 32P incorporation was determined to 0.52 +/- 0.08 mol 32P/100,000 x g protein (n = 4). This GPase ab produced by in vitro phosphorylation has shown similar dependences on AMP and caffeine as GPase extracted from the body wall of the lugworm. Its reversible conversion with endogenous phosphatase and kinase to GPase b has also been demonstrated while a completely phosphorylated form (GPase a) was not detected neither in vivo nor in vitro. Lugworm GPase ab has shown a 2.4-fold higher specific activity as GPase b. The Km for P(i) was 16 mmol/l in absence and 13 mmol/l in presence of AMP. Half maximum activation by AMP was reached at 9 mumol/l. IMP up to 10 mmol/l did not activate and ATP up to 4 mmol/l did not inhibit GPase ab in absence of AMP.

Phosphorylase: a biological transducer.

A transducer is a device that receives energy from one system and transmits it, often in a different form, to another. Glycogen phosphorylase receives information from the cell or organism in the form of metabolic signals. The energy associated with the binding of these ligand signals is integrated and transmitted at an atomic level, allowing precise adjustment of the enzymatic activity. Understanding this elegant allosteric control has required several different approaches, but the structural requirements of allostery are being defined.

The allosteric transition of glycogen phosphorylase.

The crystal structure of R-state glycogen phosphorylase b has been determined at 2.9 A resolution. A comparison of T-state and R-state structures of the enzyme explains its cooperative behaviour on ligand binding and the allosteric regulation of its activity. Communication between catalytic sites of the dimer is provided by a change in packing geometry of two helices linking each site with the subunit interface. Activation by AMP or by phosphorylation results in a quaternary conformational change that switches these two helices into the R-state conformation.

The family of glycogen phosphorylases: structure and function.

Glycogen phosphorylase plays a central role in the mobilization of carbohydrate reserves in a wide variety of organisms and tissues. While rabbit muscle phosphorylase remains the most studied and best characterized of phosphorylases, recombinant DNA techniques have led to the recent appearance of primary sequence data for a wide variety of phosphorylase enzymes. The functional properties of rabbit muscle phosphorylases are reviewed and then compared to properties of phosphorylases from other tissues and organisms. Tissue expression patterns and the chromosomal localization of mammalian phosphorylases are described. Differences in functional properties among phosphorylases are related to new structural information. Evolutionary relationships among phosphorylases as afforded by comparative analysis of proteins and gene sequences are discussed.

Hormonal regulation of fat body glycogen phosphorylase activity in larval Manduca sexta during starvation.

The hormonal regulation of fat body glycogen phosphorylase activity in Manduca sexta larvae was studied. During the first 3 hr of starvation the corpora cardiaca (CC) release a glycogen phosphorylase-activating hormone (GPAH). The haemolymph of 24-hr-starved larvae seems to contain increased levels of GPAH, but after 48 hr the titre can be assumed to be as low as prior to starvation. Abdominal stretch receptors do not appear to be involved in the regulation of GPAH release from the CC. Phosphorylase activation can be prevented by the injection of glucose or by feeding the animals with agar containing various carbohydrates. These treatments seem to prevent the release of GPAH from the CC rather than the action of GPAH on the fat body. The physiological signal which initiates peptide release remains unclear.

Influence of plating density on individual cell growth, cell division and differentiation of neonatal rat heart primary cultures.

The influence of plating cell density of an originally enriched myocardial cell population has been studied in neonatal rat heart cells in culture. Low density (LDM) is defined as a density (24 h after plating) of 209 +/- 44 cells/mm2 (mean +/- SEM) and is compared with high density (HDM), 419 +/- 67 cells/mm2. Cell growth is evaluated by the total cell number, the percentage of myocardial cells (M) in culture (PAS method) and the protein content per cell. Some differentiation parameters such as beating rates, glycogen concentration, enzymatic activities (cytochrome C oxidase and glycogen phosphorylase) are studied with time in culture (48, 96 and 192 hr). High density was designed to yield a complete confluency of the cells within 24 hr after plating and to minimize cell division of the non-muscle cells (F). At high density, cell division of F cells is effectively limited, thus leading to a more stable model regarding the cell density per plate and the percentage of M cells: 85.7 +/- 4% and 33.4 +/- 6% in LDM cultures compared with 86.5 +/- 4.7% and 51.7 +/- 9.8% in HDM cultures at 24 and 192 hr (mean +/- SEM). Heart cells increase similarly in size with age in culture in both groups. In HDM cultures the spontaneous contractions begin sooner (24 hr) than in LDM cultures and are more rapidly synchronized. The beating rate is higher in HDM cultures between 48 and 96 hr; however, after this time it falls in HDM and does not fall in LDM. Thus the overgrowth of muscle cells by non-muscle cells is not responsible for loss of beating with time in culture but more likely high density could be a limiting factor for isotonic contraction. There is more glycogen per myocyte in LDM than in HDM cultures. The cell density influences the enzymatic activities of cytochrome C oxidase and glycogen phosphorylase. The cytochrome oxidase activity is higher in HDM cultures than in LDM cultures at 96 hr whereas glycogen phosphorylase activity is higher in LDM cultures at time 96 and 192 hr. In LDM cultures, the ratio cytochrome C oxidase/glycogen phosphorylase decreases with time in culture from 1.685 +/- 0.680 at 48 hr to 0.780 +/- 0.290 at 192 hr but not in HDM cultures (2.13 +/- 0.36 and 1.64 +/- 0.34 respectively). Thus plating density influences properties of heart cell cultures with regard to the overgrowth of the F-cell population and the differentiated state of M cells.

[Mechanisms of involvement of Na+(Li+) in the process of intracellular transmission of the trigger signal in skeletal muscles]. / O mekhanizmakh vovlecheniia Na+(Li+) v protsess vnutrikletochnoi peredachi puskovogo signala v skeletnykh myshtsakh.

The data obtained suggest that Li+ can replace Na+ in transmission of triggering signal from excited surface membrane of muscle fibers onto intracellular effector systems. The participation of extracellular calcium in transmission of activation into the cell being excluded, the Ca2+-activation of the contractile apparatus and kinase-phosphorylase system in electrically stimulated muscles presumably proceeds from the induction of Ca2+ release from sarcoplasmic reticulum (SR) by transmembrane Na+- or Li+-current. The chemical specific features of Na+ and Li+ displayed at that, corroborate the hypothesis of Na+-trigger mechanism of Ca2+ release from the SR in skeletal muscle fibers of vertebrates.

"Increased" sensory stimulation leads to changes in energy-related enzymes in the brain.

The facial whiskers of mice project through several synapses to anatomically distinct structures called barrels in the contralateral cerebral cortex. With appropriate illumination, individual barrels can be recognized and dissected from unfixed, freeze-dried tissue sections taken parallel to the plane of layer IV. The tissue then can be analyzed using quantitative microhistochemical techniques to determine the level of various substances of biological importance (W.D. Dietrich, D. Durham, O. H. Lowry, and T. A. Woolsey (1981) J. Neurosci. 1: 929-935). The present paper describes results obtained in this way from adult mice subjected to a chronic "sensory deprivation" by repeatedly clipping all of the whisker hairs on one side of the face and during the recovery from this deprivation in which the whisker hairs were allowed to grow back. Sensory deprivation for 60 days leads to significant changes in the levels of the three energy-related enzymes studied--citrate synthase, malate dehydrogenase, and glycogen phosphorylase. surprisingly, during clipping, the enzyme levels in the barrels of the contralateral cortex are essentially normal, whereas enzyme levels in the barrels of the ipsilateral cortex are increased significantly. Specifically, activities expressed as a percentage of levels in normal animals were: citrate synthase, 135%; malate dehydrogenase, 130%; and glycogen phosphorylase, 170%. Forty-five days after the deprivation is reversed, the levels return to normal. These significant changes occurred in adult mice several synapses away from the sensory periphery. The data are in contrast to our earlier results in which damage to the primary afferents reduced the levels of the enzymes citrate synthase and malate dehydrogenase contralateral to the manipulation. A possible explanation for the enzymatic changes observed in the cortex ipsilateral to the clipped whiskers is an increased utilization of the intact sensory periphery by the animals; this has some behavioral support.

Potato and rabbit muscle phosphorylases: comparative studies on the structure, function and regulation of regulatory and nonregulatory enzymes.

Phosphorylases (EC 2.4.1.1) from potato and rabbit muscle are similar in many of their structural and kinetic properties, despite differences in regulation of their enzyme activity. Rabbit muscle phosphorylase is subject to both allosteric and covalent controls, while potato phosphorylase is an active species without any regulatory mechanism. Both phosphorylases are composed of subunits of approximately 100 000 molecular weight, and contain a firmly bound pyridoxal 5'-phosphate. Their actions follow a rapid equilibrium random Bi Bi mechanism. From the sequence comparison between the two phosphorylases, high homologies of widely distributed regions have been found, suggesting that they may have evolved from the same ancestral protein. By contrast, the sequences of the N-terminal region are remarkably different from each other. Since this region of the muscle enzyme forms the phosphorylatable and AMP-binding sites as well as the subunit-subunit contact region, these results provide the structural basis for the difference in the regulatory properties between potato and rabbit muscle phosphorylases. Judged from CD spectra, the surface structures of the potato enzyme might be significantly different from that of the muscle enzyme. Indeed, the subunit-subunit interaction in the potato enzyme is tighter than that in the muscle enzyme, and the susceptibility of the two enzymes toward modification reagents and proteolytic enzymes are different. Despite these differences, the structural and functional features of the cofactor, pyridoxal phosphate, site are surprisingly well conserved in these phosphorylases. X-ray crystallographic studies on rabbit muscle phosphorylase have shown that glucose-1-phosphate and orthophosphate bind to a common region close to the 5'-phosphate of the cofactor. The muscle enzyme has a glycogen storage site for binding of the enzyme to saccharide substrate, which is located away from the cofactor site. We have obtained, in our reconstitution studies, evidence for binding of saccharide directly to the cofactor site of potato phosphorylase. This difference in the topography of the functional sites explains the previously known different specificities for saccharide substrates in the two phosphorylases. Based on a combination of these and other studies, it is now clear that the 5'-phosphate group of pyridoxal phosphate plays a direct role in the catalysis of this enzyme. Information now available on the reaction mechanism of phosphorylase is briefly described.