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PubMed1982

Comparative glucan specificities of two types of spinach leaf phosphorylase.

Two types of alpha-glucan phosphorylase [EC 2.4.1.1] from spinach leaves have been separately purified to near homogeneity. Type I enzyme shows a subunit molecular weight of 92,000 and Km values for amylopectin, glycogen and amylose much smaller than that for maltopentose. Cyclodextrin is a normal competitive inhibitor with respect to maltopentose, while it is a multi-site competitive type inhibitor with respect to amylopectin, glycogen or amylose. Type II enzyme shows a subunit molecular weight of 108,000, and utilizes amylopectin, amylose and maltopentose well, but glycogen very poorly. On affinity electrophoresis, Type II enzyme shows no affinity for glycogen and the dissociation constant for amylopectin is more than a thousand-fold greater than that of Type I enzyme. Type II enzyme has similar characteristics (subunit size, glucan specificity, and mode of cyclodextrin inhibition) to potato phosphorylase, for which cyclodextrin is a normal competitive inhibitor with respect to either maltopentose or amylopectin. Enzyme-glucan binding models have been proposed to explain these different kinetic properties. In spinach Type I phosphorylase, multiple glucan binding sites are located on the same face of the enzyme molecule to enable a single large substrate to be bound by riding on all the site; in spinach Type II and potato phosphorylases, two glucan binding sites are three-dimensionally arranged in a manner that excludes the possibility of the binding of amylopectin by riding on the two sites.

Animals↗
PubMed1989

Desensitization of alpha 1 adrenoceptor-stimulated glycogen phosphorylase activity in vascular smooth muscle.

Desensitization of alpha 1-adrenoceptor-mediated activation of glycogen phosphorylase was investigated in rabbit aorta. Activation of glycogen phosphorylase by epinephrine was antagonized by the alpha 1-receptor selective antagonist prazosin but not by yohimbine (alpha 2-receptor selective) or by propranolol (beta-receptor antagonist). Preincubation of rabbit aortic ring segments for 5 h with norepinephrine (NE, 10(-5) M) led to a 30-fold loss in sensitivity and a 55% decrease in maximal activation of the enzyme by alpha agonists. Preincubation of aortic ring segments with phenylephrine (10(-5) M) in the presence of propranolol (10(-6) M) also caused desensitization of glycogen phosphorylase activation. The desensitization was heterologous since maximal activation of the enzyme by histamine or KCl was also markedly diminished in segments preincubated with NE. In contrast to these results, catecholamine-induced desensitization to alpha 1-adrenoceptor-mediated smooth muscle contraction in aortic ring segments resulted in loss in sensitivity but not maximal force of contraction on subsequent stimulation by alpha 1 agonists. These results suggest that the mechanism responsible for desensitization of glycogen phosphorylase is distal to receptor activation and may involve attenuation of responses to intracellular Ca2+-dependent enzymes which have limited reserve.

Animals↗
PubMed1999

Effects of commonly used cryoprotectants on glycogen phosphorylase activity and structure.

The effects of a number of cryoprotectants on the kinetic and structural properties of glycogen phosphorylase b have been investigated. Kinetic studies showed that glycerol, one of the most commonly used cryoprotectants in X-ray crystallographic studies, is a competitive inhibitor with respect to substrate glucose-1-P with an apparent Ki value of 3.8% (v/v). Cryogenic experiments, with the enzyme, have shown that glycerol binds at the catalytic site and competes with glucose analogues that bind at the catalytic site, thus preventing the formation of complexes. This necessitated a change in the conditions for cryoprotection in crystallographic binding experiments with glycogen phosphorylase. It was found that 2-methyl-2,4-pentanediol (MPD), polyethylene glycols (PEGs) of various molecular weights, and dimethyl sulfoxide (DMSO) activated glycogen phosphorylase b to different extents, by stabilizing its most active conformation, while sucrose acted as a noncompetitive inhibitor and ethylene glycol as an uncompetitive inhibitor with respect to glucose-1-P. A parallel experimental investigation by X-ray crystallography showed that, at 100 K, both MPD and DMSO do not bind at the catalytic site, do not induce any significant conformational change on the enzyme molecule, and hence, are more suitable cryoprotectants than glycerol for binding studies with glycogen phosphorylase.

Adenosine Monophosphate↗
PubMed2006

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.

Algal Proteins↗
PubMed1988

The relationship between the two forms of glycogen phosphorylase in Dictyostelium discoideum.

The cellular slime mold, Dictyostelium disoideum, provides an ideal model system to study eukaryotic cell differentiation. In D. discoideum, glycogen degradation provides precursors for the synthesis of developmentally regulated structural products. The enzyme responsible for glycogen degradation, glycogen phosphorylase, exists in active and inactive forms. The active, or 'a' form, is independent of 5'adenosine monophosphate (5'AMP) while the inactive, or 'b' form, is 5'AMP-dependent. The activity of the 'b' form predominates early in development, while the activity of the 'a' form peaks in mid-late development; their combined specific activities remain constant at any point. Polyclonal antibodies raised to the purified forms of this enzyme showed low cross-reactivity. The anti-'a' serum reacted with a 104-kDa protein that was associated with phosphorylase 'a' activity; the anti-'b' serum reacted with a 92-kDa protein that was associated with phosphorylase 'b' activity and weakly cross-reacted with the 104-kDa protein. Immunoblots of peptide maps of the purified enzyme forms showed that each antibody was specific for the proteolytic fragments of its respective antigen. We also demonstrated in vitro phosphorylation of the 'b' form by an endogenous protein kinase. Cyclic AMP perturbation of intact cells caused induction of both phosphorylase-'a' activity and the 104-kDa protein. Immunotitration data suggested that the 'a' form accumulates due to de novo protein synthesis, although this result must be interpreted with caution.

Cyclic AMP↗
PubMed1976

Heterotropic interactions of ligands with phosphorylase b.

1. The interaction of rabbit muscle glycogen phosphorylase b with pairs of ligands has been examined. 2. The electron spin resonance spectrum of a spin label, covalently attached to the protein, provided information about dissociation constants, formation of ternary complexes and both negative and positive interactions between different ligand pairs. 3. AMP competes with a series of nucleotides (ADP, ATP, CMP aand cytosine) but with adenosine a ternary enzyme - AMP - adenosine complex can be formed. 4. ADP binding is tight and ADP inhibits the AMP activation of phosphorylase b in a physiologically important concentration range. 5. The substrates glucose 1-phosphate and glycogen tighten AMP binding in the ternary complex as does the competitive inhibitor UDPG. Inorganic phosphate is different in this respect. Gluconolactone, a transition state analogue, competes with glucose 1-phosphate (but not with glycogen) but does not prevent completely the binding of the sugar phosphate. 6. The effect of glucose b-phosphate on phosphorylase is rather complex as it 'formally competes' with both AMP and UDPG probably mediated by a conformational changes and not by 'direct' interactions with these two ligands. Glycerol 2-phosphate, a commonly used buffer for phosphorylase, also shows complex interactions.

Adenosine Monophosphate↗
PubMed1978

Phosphorylase kinase isoenzymes in deficient ICR/IAn mice.

ICR/IAn mice present a deficiency in phosphorylase kinase activity; the extent of this deficiency is less in some tissues [Lyon, S.B. Biochem. Genet. 4, 169--185 (1970)] than in skeletal muscle, where enzyme activity is 0.3% of normal [Cohen, P.T. W & Cohen, P. FEBS Lett. 29, 113--115 (1973)]. New-born mice of this strain were also reported (Lyon, 1970) to reveal a small amount of skeletal muscle enzyme activity. The properties of these residual phosphorylase kinases were compared to those of control C57 BL mice, with reference to control muscle and liver enzymes which were shown to be of different molecular species [Daegelen-Proux et al. Biochim. Biophys Acta, 452, 398--405 (1976)]. The properties investigated were the immunological reactivity against an antiserum raised against muscle phosphorylase kinase, the thermal stability and the Ca2+ dependency. The results suggest that the muscle enzyme from the new-born ICR/IAn mice and the heart enzyme from adult deficient mice are different to the muscle enzyme from adult normal mice, but they have properties in common with normal adult liver enzyme. These results lead to the conclusion that there exists in the muscle of I strain a "foetal form" of phosphorylase kinase, the activity of which decreases progressively after birth. Out work also confirmed the observations made by Cohen et al. [Eur. J. Biochem. 66, 347--356 (1976)] which showed that there is no evidence for the existence of a cross-reacting material in the muscle of adult deficient mice.

Aging↗
PubMed1979

1,4-alpha-Glucan phosphorylase from the slime mold Physarum polycephalum. Purification, physico-chemical and kinetic properties.

Glycogen phosphorylase from macroplasmodia of Physarum polycephalum was purified 76-fold to homogeneity. The native enzyme migrated as a single protein band on analytical disc gel electrophoresis coinciding with phosphorylase activity. After reduction in the presence of sodium dodecylsulfate one protein band was detectable which corresponded to an Mr of 93 000. The molecular weight of the native enzyme determined by gel sieving or gradient-polyacrylamide gel electrophoresis was 172000 and 186000, respectively. The enzyme contained about 1 mol pyridoxal 5'-phosphate and less than 0.1 mol covalently bound phosphate per mol subunit. The amino acid composition of the enzyme was determined. In the direction of phosphorolysis the kinetic data were determined by initial velocity studies, assuming a rapid equilibrium random mechanism. Glucose 1-phosphate and GDP-glucose were competitive inhibitors toward phosphate and noncompetitive to glycogen. 5'-AMP, a weak activator of the enzyme, counteracted the glucose-1-phosphate inhibition completely. Physarum phosphorylase was compared with phosphorylases from other sources on the basis of chemical and kinetic properties. No evidence for the presence of phosphorylated forms has yet been found.

Adenosine Monophosphate↗
PubMed1981

Latent phosphorylase phosphatases from rat liver: relationship with the heat-stable inhibitory protein.

A high-speed supernatant from rat liver contains at least two latent phosphorylase phosphatases the activities of which are revealed by treatment with ethanol, urea, mercaptoethanol or trypsin. This fraction also contains at least one protein which, after heating, inhibits to various degrees the activated form(s) of the two phosphatases. The two latent enzymes can be separated by cellulase-phosphate chromatography and can be differentiated by their preferential activation by ethanol or trypsin and by their different sensitivity to the inhibitory protein after ethanol activation. Activation of the latent phosphorylase phosphatases by ethanol, urea or mercaptoethanol is not accompanied by the destruction of the precursor of the inhibitory protein whereas activation by trypsin is. However, trypsin treatment of fractions previously activated by ethanol decreases their activity and also increases their sensitivity to the inhibitory protein in a way which is unrelated to the destruction of this inhibitor. Furthermore, some protein fractions, almost free of the precursor of the inhibitory protein can be readily activated by trypsin. In is concluded that the activation of the latent phosphorylase phosphorylase phosphatases is unrelated to the destruction of the inhibitory protein.

Animals↗
PubMed2004

Relationships between structure, function and stability for pyridoxal 5'-phosphate-dependent starch phosphorylase from Corynebacterium callunae as revealed by reversible cofactor dissociation studies.

Using 0.4 m imidazole citrate buffer (pH 7.5) containing 0.1 mm l-cysteine, homodimeric starch phosphorylase from Corynebacterium calluane (CcStP) was dissociated into native-like folded subunits concomitant with release of pyridoxal 5'-phosphate and loss of activity. The inactivation rate of CcStP under resolution conditions at 30 degrees C was, respectively, four- and threefold reduced in two mutants, Arg234-->Ala and Arg242-->Ala, previously shown to cause thermostabilization of CcStP [Griessler, R., Schwarz, A., Mucha, J. & Nidetzky, B. (2003) Eur. J. Biochem.270, 2126-2136]. The proportion of original enzyme activity restored upon the reconstitution of wild-type and mutant apo-phosphorylases with pyridoxal 5'-phosphate was increased up to 4.5-fold by added phosphate. The effect on recovery of activity displayed a saturatable dependence on the phosphate concentration and results from interactions with the oxyanion that are specific to the quarternary state. Arg234-->Ala and Arg242-->Ala mutants showed, respectively, eight- and > 20-fold decreased apparent affinities for phosphate (K(app)), compared to the wild-type (K(app) approximately 6 mm). When reconstituted next to each other in solution, apo-protomers of CcStP and Escherichia coli maltodextrin phosphorylase did not detectably associate to hybrid dimers, indicating that structural complementarity among the different subunits was lacking. Pyridoxal-reconstituted CcStP was inactive but approximately 60% and 5% of wild-type activity could be rescued at pH 7.5 by phosphate (3 mm) and phosphite (5 mm), respectively. pH effects on catalytic rates were different for the native enzyme and pyridoxal-phosphorylase bound to phosphate and could reflect the differences in pK(a) values for the cofactor 5'-phosphate and the exogenous oxyanion.

Apoproteins↗
PubMed1983

Absence of functional messenger RNA for glycogen phosphorylase in the muscle of two patients with McArdle's disease.

(1) Two unrelated cases of McArdle's disease (glycogen storage disease type V, deficiency in muscle-type glycogen phosphorylase, EC.2.4.1.1) with no detectable inactive protein, and two heterozygous relatives of one patient, have been investigated for the presence or absence of functional messenger RNA. Methods were developed and scaled down to be compatible with clinical-size biopsies. (2) Total muscle RNA was prepared and translated in a rabbit reticulocyte cell-free system. Neosynthesized products were isolated by immunoaffinity microchromatography. Phosphorylase was compared with control enzymes neosynthesized and purified under the same conditions. (3) No functional mRNA for phosphorylase could be detected in the muscles of the two patients. A decreased amount of messenger for phosphorylase, compared with the control enzymes, was found in the muscles of the two heterozygotes. (4) Exploration of functional messenger RNA in clinical enzymopathies should be readily adaptable to a number of enzymatic diseases.

Adolescent↗
PubMed1990

Purification of glycogen phosphorylase from bovine brain and immunocytochemical examination of rat glial primary cultures using monoclonal antibodies raised against this enzyme.

The physiological function in brain of glycogen and the enzyme catalyzing the rate-limiting step in glycogenolysis, glycogen phosphorylase (EC 2.4.1.1), is unknown. As a first step toward elucidating such a function, we have purified bovine brain glycogen phosphorylase isozyme BB 1,700-fold to a specific activity of 24 units/mg protein. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subsequent silver staining, a single major protein band corresponding to an apparent molecular mass of 97 kDa was observed. Mouse monoclonal antibodies raised against the enzyme were purified and shown to be monospecific as indicated by immunoblotting. Immunocytochemical examination of astroglia-rich primary cultures of rat brain cells revealed a colocalization of glycogen phosphorylase with the astroglial marker glial fibrillary acidic protein in many cells. The staining for the enzyme appeared at two levels of intensity. There were other cells in the culture showing no specific staining under the experimental conditions employed. Neurons in neuron-rich primary cultures did not show positive staining. The data suggest that glycogen phosphorylase may be predominantly an astroglial enzyme and that astroglia cells play an important role in the energy metabolism of the brain.

Animals↗
PubMed1985

Effects of adrenaline infusion on cAMP and glycogen phosphorylase in fast-twitch and slow-twitch rat muscles.

This study investigated the effect of adrenaline infusion on the cAMP content, glycogen phosphorylase activity and the rate of glycogen breakdown in rat extensor digitorum longus (EDL) and soleus muscles. Adrenaline was constantly infused in a dose of 0.15 micrograms kg-1 body wt min-1. The cAMP content increased approximately 2.8-fold in both muscles after 2 min of infusion. Phosphorylase a + b activity was six times higher in fast-twitch muscle (EDL) than in slow-twitch (soleus) and remained unchanged during the infusion. Phosphorylase a activity increased by 8.4-fold in EDL and 2.4-fold in soleus muscles during the infusion period. Glycogen content decreased in EDL muscle by 10% whereas no change was observed in soleus. It is concluded that beta-adrenergic stimulation by adrenaline results in a similar cAMP increase in both muscles. The low rate of glycogen breakdown in EDL and the unchanged content of glycogen in soleus muscle suggest that cAMP mediated transformation of phosphorylase b to a in itself is not adequate for a rapid glycogenolysis in muscle.

Animals↗
PubMed1980

Calcium control of muscle phosphorylase kinase through the combined action of calmodulin and troponin.

Although it has been believed for several years that Ca2+ are the means by which glycogenolysis and muscle contraction are synchronized, it is only over the past two years that this concept has started to be placed on a firm molecular basis. The current evidence suggests that the regulation of phosphorylase kinase by Ca2+ in vivo is achieved through the interaction of this divalent cation with calmodulin (the delta subunit) and troponin C, and that the relative importance of these two calcium binding proteins depends on the state of phosphorylation of the enzyme [FIGURE 1]. In the low-activity dephosphorylated b form, increasing Ca2+ from 0.1 microM to concentrations in the microM range produces a 5-10-fold activation through the binding of Ca2+ to the delta subunit, and a further 15-25-fold activation through the binding of Ca2+ to troponin C [TABLE 1]. Troponin C rather than the delta subunit is therefore the dominant calcium dependent regulator of the b form, providing an attractive mechanism for coupling glycogenolysis and muscle contraction. On the other hand, the high-activity phosphorylated a form is only activated very slightly by troponin [Section 7]. The delta subunit is therefore the dominant calcium dependent regulator of the hormonally activated state of the enzyme. It has recently become clear that phosphorylase kinase not only phosphorylates and activates phosphorylase, but also phosphorylates glycogen synthase, decreasing its activity. The regulation of phosphorylase kinase by Ca2+ may therefore also provide a mechanism for achieving synchronous control of the pathways of glycogenolysis and glycogen synthesis.

Animals↗
PubMed2001

Altered glycogen synthase and phosphorylase activities in skeletal muscle of tetraplegic patients.

Despite marked differences in both the extent of physical activity and in muscle metabolism and structure between tetraplegic and control subjects, the glycogen content in the skeletal muscle of both groups is similar. We determined whether this similarity could be explained by the activities of key enzymes of glycogen metabolism. Muscle biopsies were analysed for glycogen synthase (GS) and glycogen phosphorylase (GP) activities, as well as for metabolites. Glycogen content did not differ significantly between the two groups. Total glycogen synthase activity was reduced by almost 60 % in tetraplegics (P < 0.01), whereas total phosphorylase activity did not differ between groups. GS fractional activity did not differ between groups, whereas phosphorylase fractional activity (-/+ AMP) was significantly higher in the tetraplegics (0.08 +/- 0.01, control; 0.25 +/- 0.02, tetraplegics; P < 0.001). Neither uridine diphosphate (UDP)-glucose nor glucose 6-phosphate (G-6-P) content in muscle differed significantly between groups. These data demonstrate that, in tetraplegics, muscle glycogen content is preserved despite decreases in GS activity and increases in phosphorylase fractional activity. Muscle paralysis has differential effects on the activities of GS and GP. Experimental Physiology (2001) 86.2, 205-209.

Adult↗
PubMed2002

The temporal relationship between glycogen phosphorylase and activation of the pyruvate dehydrogenase complex during adrenaline infusion in resting canine skeletal muscle.

The present study examined the effect of adrenaline infusion on the activation status of glycogen phosphorylase and the pyruvate dehydrogenase complex (PDC) and on the accumulation of glucose-6-phosphate (G-6-P) and acetylcarnitine in resting canine skeletal muscle. The study was performed in an effort to gain some insight into the temporal relationship between glycogen phosphorylase and PDC activation in vivo in skeletal muscle, which is currently unresolved. Multiple muscle samples were obtained from canine brachial muscle (n = 10) before and during (1, 3, 7 and 15 min) adrenaline infusion (0.14 microg (kg body mass)(-1) min(-1), I.V.). Adrenaline infusion increased glycogen phosphorylase "a" by > 2-fold above basal levels after 3 min (pre-infusion = 9.2 +/- 1.1 vs. 3 min = 22.3 +/- 4.0 mmol glucosyl units (kg dry muscle)(-1) min(-1), P < 0.05). The concentration of G-6-P increased transiently from its basal concentration at 1 min (pre-infusion = 1.5 +/- 0.2 vs. 1 min = 4.4 +/- 0.9 mmol kg dry muscle)(-1), P < 0.01), declined to its pre-infusion concentration at 3 min (P < 0.05), and then increased again after 7 min of infusion (P < 0.05). The PDC was activated following 7 min of adrenaline infusion (pre-infusion = 0.22 +/- 0.04 vs. 7 min = 1.04 +/- 0.15 mmol acetyl-CoA (kg wet muscle)(-1) min(-1), P < 0.01), and this degree of activation was maintained for the duration of infusion. During the first 3 min of infusion, the concentration of acetylcarnitine declined (pre-infusion = 3.8 +/- 0.3 vs. 3 min = 1.6 +/- 0.2 mmol (kg dry muscle)(-1), P < 0.05), before transiently increasing at 7 min above the 3 min concentration (3 min = 1.6 +/- 0.2 vs. 7 min = 5.1 +/- 1.0 mmol (kg dry muscle)(-1), P < 0.01). This is the first study to demonstrate that adrenaline can indirectly activate the PDC in skeletal muscle in vivo at rest. The results demonstrate that adrenaline increased glycogen phosphorylase activation and glycolytic flux within 3 min of infusion, but took several more minutes to activate the PDC. This temporal relationship, combined with a probable adrenaline-induced increase in metabolic rate (and thereby resting ATP demand), resulted in the biphasic changes in G-6-P and acetylcarnitine with infusion time.

Acetylcarnitine↗
PubMed1974

Regulation of maltodextrin phosphorylase synthesis in Escherichia coli by cyclic adenosine 3', 5'-monophosphate and glucose.

Cyclic adenosine 3', 5'-monophosphate (AMP) stimulates maltodextrin phosphorylase synthesis in Escherichia coli cells induced with maltose. A maximal effect occurs at 2 to 3 mM cyclic AMP. The action of cyclic AMP is specific, inasmuch as adenosine triphosphate, 3'-AMP, 5'-AMP, adenosine, and dibutyryl cyclic AMP are inactive. Glucose, alpha-methyl glucoside, 2-deoxyglucose, and pyridoxal 5'-phosphate repress maltodextrin phosphorylase synthesis. This repression is reversed by cyclic AMP. The action of cyclic AMP appears to be at the transcriptional level, since cyclic AMP fails to stimulate phosphorylase production in induced cells in which messenger ribonucleic acid synthesis has been arrested by rifampin or by inducer removal. The two other enzymes involved in the metabolism of maltose, amylomaltase and maltose permease, are also induced in this strain of E. coli and affected by glucose and cyclic AMP in a manner similar to phosphorylase.

Adenosine↗
PubMed1976

Regulation of purine utilization in bacteria. VII. Involvement of membrane-associated nucleoside phosphorylase in the uptake and the base-mediated loss of the ribose moiety of nucleosides by Salmonella typhimurium membrane vesicles.

Although uridine and adenosine are converted by membrane-associated nucleoside phosphorylases to ribose-1-phosphate (ribose-1-P) and the corresponding bases (uracil and adenine), only ribose -1-P is accumulated within Salmonella typhimurium LT2 membrane vesicles. In accordance with these observations, no uptake is observed when the vesicles are incubated with the bases or nucleosides labeled in their base moieties. The vesicles lack a transport system for ribos-1-P, since excess ribose-1-P does not inhibit the uptake of the ribose moiety of uridine. In addition, there is no exchange with preaccumulatedribose-1-P. Thus, uridine, rather than ribose-1-P, must serve as the initially transported substrate. The uptake of the ribose portion of uridine is coupled to electron transport, and the levels to which ribose-1-P are accumulated may be reduced by adding various bases to the reaction mixtures. The bases appear to inhibit the uridine phosphorylase reaction and/or cause an efflux of ribose-1-P from the vesicles. This loss of ribose-1-P reflects the accumulation of nucleosides in the external medium after being synthesized within the membranes. Synthesis of the nucleosides from intravesicular ribose-1-P and exogenous base proceeds even though the bases are not accumulated by the vesicles. Furthermore, ribose-1-P cannot significantly inhibit uridine phosphorylase activity unless the membranes are disrupted. These observations indicate that the membrane-associated nucleoside phosphorylases may have a transmembranal orientation with their base and ribose-1-P binding sites on opposite sides of the membranes. Such an asymmetric arrangement of these enzymes may facilitate the uptake of the ribosyl moiety of nucleosides by a group translocation mechanism. Thus, nucleosides may be cleaved during the membrane transport process, with the resultant bases delivered to the external environment while ribose-1-P is shunted to the intravesicular space.

Adenosine↗