Pre-steady-state uptake of -glucose by the human erythrocyte is inconsistent with a circulating carrier mechanism

Simulation shows that the four-state mobile carrier model for sugar transport in which the asymmetry arises from unequal rate constants of inward and outward translation of the free-carrier and carrier-sugar complex, does not fit with the observed data for pre-steady-state uptake recently obtained by A.G. Lowe and A.R. Walmsley ((1987) Biochim. Biophys. Acta 903, 547–550). The main reason for this discrepancy is that pre-steady-state fluxes are determined mainly by the dissociation constants K_s of glucose and maltose for the external sites, rather than the K_m (zero-trans_oi) of glucose and the K_i of maltose. The data are also inconsistent with other forms of asymmetric carrier but are fairly consistent with a symmetrical carrier with high-affinity sites for -glucose or with a fixed site carrier model.     

Inhibitions of sugar transport produced by ligands binding at opposite sides of the membrane. Evidence for simultaneous occupation of the carrier by maltose and cytochalasin B

This study examines inhibitions of human erythrocyte D-glucose uptake at ice temperature produced by maltose and cytochalasin B. Maltose inhibits sugar uptake by binding at or close to the sugar influx site. Maltose is thus a competitive inhibitor of sugar uptake. Cytochalasin B inhibits sugar transport by binding at or close to the sugar efflux site and thus acts as a noncompetitive inhibitor of sugar uptake. When maltose is present in the uptake medium, Ki(app) for cytochalasin B inhibition of sugar uptake increases in a hyperbolic manner with increasing maltose. When cytochalasin B is present in the uptake medium, Ki(app) for maltose inhibition of sugar uptake increases in a hyperbolic manner with increasing cytochalasin B. High concentrations of cytochalasin B do not reverse the competitive inhibition of D-glucose uptake by maltose. These data demonstrate that maltose and cytochalasin B binding sites coexist within the glucose transporter. These results are inconsistent with the simple, alternating conformer carrier model in which maltose and cytochalasin B binding sites correspond to sugar influx and sugar efflux sites, respectively. The data are also incompatible with a modified alternating conformer carrier model in which the cytochalasin B binding site overlaps with but does not correspond to the sugar efflux site. We show that a glucose transport mechanism in which sugar influx and sugar efflux sites exist simultaneously is consistent with these observations.     

Modelling of ethanol production by Saccharomyces cerevisiae from a glucose and maltose mixture

Summary A model for ethanol production from a glucose-maltose mixture has been proposed, which includes a term representing the glucose repression effect on maltose consumption. The model parameters were estimated from batch experimental data. Results of sensitivity analysis on the Monod constants for glucose and maltose, and the repression constant, showed that ±10% changes in these three parameters caused no significant effect on data fitting.     

Effect of substrate on the pre-steady-state kinetics of the Na(+)/glucose cotransporter

When measuring Na(+)/glucose cotransporter (SGLT1) activity in Xenopus oocytes with the two-electrode voltage-clamp technique, pre-steady-state currents dissipate completely in the presence of saturating alpha-methyl-glucose (alphaMG, a nonhydrolyzable glucose analog) concentrations. In sharp contrast, two SGLT1 mutants (C255A and C511A) that lack a recently identified disulfide bridge express the pre-steady-state currents in the presence of alphaMG. The dose-dependent effects of alphaMG on pre-steady-state currents were studied for wild-type (wt) SGLT1 and for the two mutants. Increases in alphaMG concentration reduced the total transferred charge (partially for the mutants, totally for wt SGLT1), shifted the transferred charge versus membrane potential (Q-V) curve toward positive potentials, and significantly modified the time constants of the pre-steady-state currents. A five-state kinetic model is proposed to quantitatively explain the effect of alphaMG on pre-steady-state currents. This analysis reveals that the reorientation of free transporter is the slowest step for wt SGLT1 either in the presence or in the absence of alphaMG. In contrast, the conformational change of the fully loaded mutant transporters constitutes their rate-limiting step in the presence of substrate and explains the persistence of pre-steady-state currents in this situation.     

Energetics and kinetics of maltose transport in Saccharomyces cerevisiae: a continuous culture study.

In Saccharomyces cerevisiae, maltose is transported by a proton symport mechanism, whereas glucose transport occurs via facilitated diffusion. The energy requirement for maltose transport was evaluated with a metabolic model based on an experimental value of YATP for growth on glucose and an ATP requirement for maltose transport of 1 mol.mol-1. The predictions of the model were verified experimentally with anaerobic, sugar-limited chemostat cultures growing on a range of maltose-glucose mixtures at a fixed dilution rate of 0.1 h-1. The biomass yield (grams of cells.gram of sugar-1) decreased linearly with increasing amounts of maltose in the mixture. The yield was 25% lower during growth on maltose than during that on glucose, in agreement with the model predictions. During sugar-limited growth, the residual concentrations of maltose and glucose in the culture increased in proportion to their relative concentrations in the medium feed. From the residual maltose concentration, the in situ rates of maltose consumption by cultures, and the Km of the maltose carrier for maltose, it was calculated that the amount of this carrier was proportional to the in situ maltose consumption rate. This was also found for the amount of intracellular maltose. These two maltose-specific enzymes therefore exert high control over the maltose flux in S. cerevisiae in anaerobic, sugar-limited, steady-state cultures.     

Stop-flow analysis of cooperative interactions between GLUT1 sugar import and export sites.

The human erythrocyte sugar transporter is thought to function either as a simple carrier (sugar import and sugar export sites are presented sequentially) or as a fixed-site carrier (sugar import and sugar export sites are presented simultaneously). The present study examines each hypothesis by analysis of the rapid kinetics of reversible cytochalasin B binding to the sugar export site in the presence and absence of sugars that bind to the sugar import site. Cytochalasin B binding to the purified, human erythrocyte glucose transport protein (GLUT1) induces quenching of GLUT1 intrinsic tryptophan fluorescence. The time-course of GLUT1 fluorescence quenching reflects a second-order process characterized by simple exponential kinetics. The pseudo-first-order rate constant describing fluorescence decay (kobs) increases linearly with [cytochalasin B] while the extent of fluorescence quenching increases in a saturable manner with [cytochalasin B]. Rate constants for cytochalasin B binding to GLUT1 (k1) and dissociation from the GLUT1.cytochalasin B complex (k-1) are obtained from the relationship: kobs = k-1 + k1[cytochalasin B]. Low concentrations of maltose, D-glucose, 3-O-methylglucose, and other GLUT1 import-site reactive sugars increase k-1(app) and reduce k1(app) for cytochalasin B interaction with GLUT1. Higher sugar concentrations decrease k1(app) further. The simple carrier mechanism predicts that k1(app) alone is modulated by import- and export-site reactive sugars and is thus incompatible with these findings. These results are consistent with a fixed-site carrier mechanism in which GLUT1 simultaneously presents cooperative sugar import and export sites.     

Reaction of the glucose carrier of erythrocytes with sodium tetrathionate: Evidence for inward-facing and outward-facing carrier conformations

Summary Sodium tetrathionate reacts with the glucose carrier of human erythrocytes at a rate which is greatly altered in the presence of competitive inhibitors of glucose transport. Inhibitors bound to the carrier on the outer surface of the membrane, either at the substrate site (maltose) or at the external inhibition site (phloretin and phlorizin), more than double the reaction rate. Inhibitors bound at the internal inhibition site (cytochalasin B and androstenedione), protect the system against tetrathionate. After treatment with tetrathionate, the maximum transport rate falls to less than one-third, and the properties of the binding sites are modified in unexpected ways. The affinity of externally bound inhibitors rises: phloretin is bound up to seven times more strongly and phlorizin and maltose twice as strongly. The affinity of cytochalasin B, bound at the internal inhibition site, falls to half while that of androstenedione is little changed. The affinity of external glucose falls slightly. Androstenedione prevents both the fall in transport activity and the increase in phloretin affinity produced by tetrathionate. An inhibitor of anion transport has no effect on the reaction. The observations support the following conclusions: (1) Tetrathionate produces its effects on the glucose transport system by reacting with the carrier on the outer surface of the membrane. (2) The carrier assumes distinct inward-facing and outward-facing conformations, and tetrathionate reacts with only the outward-facing form. (3) The thiol group with which tetrathionate is presumed to react is not present in either the substrate site or the internal or external inhibitor site. (4) In binding asymmetrically to the carrier, a reversible inhibitor shifts the carrier partition between inner and outer forms and thereby raises or lowers the rate of tetrathionate reaction with the system. (5) Reaction with tetrathionate converts the carrier to an altered state in which the conformation at all three binding sites is changed and the rate of carrier reorientation is reduced.     

Correlating Charge Movements with Local Conformational Changes of a Na+-Coupled Cotransporter

To gain insight into the steady-state and dynamic characteristics of structural rearrangements of an electrogenic secondary-active cotransporter during its transport cycle, two measures of conformational change (pre-steady-state current relaxations and intensity of fluorescence emitted from reporter fluorophores) were investigated as a function of membrane potential and external substrate. Cysteines were substituted at three believed-new sites in the type IIb Na⁺-coupled inorganic phosphate cotransporter (SLC34A2 flounder isoform) that were predicted to be involved in conformational changes. Labeling at one site resulted in substantial suppression of transport activity, whereas for the other sites, function remained comparable to the wild-type. For these mutants, the properties of the pre-steady-state charge relaxations were similar for each, whereas fluorescence intensity changes differed significantly. Fluorescence changes could be accounted for by simulations using a five-state model with a unique set of apparent fluorescence intensities assigned to each state according to the site of labeling. Fluorescence reported from one site was associated with inward and outward conformations, whereas for the other sites, including four previously indentified sites, emissions were associated principally with one or the other orientation of the transporter. The same membrane potential change induced complementary changes in fluorescence at some sites, which suggested that the microenvironments of the respective fluorophores experience concomitant changes in polarity. In response to step changes in voltage, the pre-steady-state current relaxation and the time course of change in fluorescence intensity were described by single exponentials. For one mutant the time constants matched well with and without external Na⁺, providing direct evidence that this label reports conformational changes accompanying intrinsic charge movement and cation interactions.     

Correlating Charge Movements with Local Conformational Changes of a Na-Coupled Cotransporter

To gain insight into the steady-state and dynamic characteristics of structural rearrangements of an electrogenic secondary-active cotransporter during its transport cycle, two measures of conformational change (pre-steady-state current relaxations and intensity of fluorescence emitted from reporter fluorophores) were investigated as a function of membrane potential and external substrate. Cysteines were substituted at three believed-new sites in the type IIb Na⁺-coupled inorganic phosphate cotransporter (SLC34A2 flounder isoform) that were predicted to be involved in conformational changes. Labeling at one site resulted in substantial suppression of transport activity, whereas for the other sites, function remained comparable to the wild-type. For these mutants, the properties of the pre-steady-state charge relaxations were similar for each, whereas fluorescence intensity changes differed significantly. Fluorescence changes could be accounted for by simulations using a five-state model with a unique set of apparent fluorescence intensities assigned to each state according to the site of labeling. Fluorescence reported from one site was associated with inward and outward conformations, whereas for the other sites, including four previously indentified sites, emissions were associated principally with one or the other orientation of the transporter. The same membrane potential change induced complementary changes in fluorescence at some sites, which suggested that the microenvironments of the respective fluorophores experience concomitant changes in polarity. In response to step changes in voltage, the pre-steady-state current relaxation and the time course of change in fluorescence intensity were described by single exponentials. For one mutant the time constants matched well with and without external Na⁺, providing direct evidence that this label reports conformational changes accompanying intrinsic charge movement and cation interactions.     

Studies on the subsite structure of amylases. I. Interaction of glucoamylase with substrate and analogues studied by difference-spectrophotometry.

Studies were made on the ultraviolet difference-spectra of glucoamylase from Rhizopus niveus [EC 3.2.1.3] specifically produced by the substrate maltose and the inhibitors, glucose, glucono-1: 5-lactone (gluconolactone), methyl beta-D-glucoside, cellubiose, and cyclohexa-, and cyclohepta-amyloses. Of these, maltose and gluconolactone produced characteristic difference spectra with a trough near 300 nm. Based on studies with a model compound for a tryptophan residue, Ac-Trp, this trough was attributed to the effect of a negative charge upon the tryptophan residue. From the concentration dependency of the difference spectra, the dissociation constants of the complexes between the enzyme and maltose, glucose, and gluconolactone were evaluated to be 1.2 mM, 51 mM, and 1.5 mM, respectively. These values are in good agreement with the values of Km or K1 obtained from the steady-state kinetics. The difference-spectrophotometric data suggested that referring to the values of subsite affinities of glucoamylase, maltose, and gluconolactone occupy mainly Subsite 1, where the non-reducing-end glucose residue of a substrate is bound in a productive form and that a tryptophan residue with shows a trough near 300 nm in difference spectra is located in this subsite.     

Enzymatic processes for the purification of trehalose

A dual‐enzyme process aiming at facilitating the purification of trehalose from maltose is reported in this study. Enzymatic conversion of maltose to trehalose usually leads to the presence of significant amount of glucose, by‐product of the reaction, and unreacted maltose. To facilitate the separation of trehalose from glucose and unreacted maltose, sequential conversion of maltose to glucose and glucose to gluconic acid under the catalysis of glucoamylase and glucose oxidase, respectively, is studied. This study focuses on the hydrolysis of maltose with immobilized glucoamylase on Eupergit® C and CM Sepharose. CM Sepharose exhibited a higher protein adsorption capacity, 49.35 ± 1.43 mg/g, and was thus selected as carrier for the immobilization of glucoamylase. The optimal reaction temperature and reaction pH of the immobilized glucoamylase for maltose hydrolysis were identified as 40°C and 4.0, respectively. Under such conditions, the unreacted maltose in the product stream of trehalose synthase‐catalyzed reaction was completely converted to glucose within 35 min, without detectable trehalose degradation. The conversion of maltose to glucose could be maintained at 0.92 even after 80 cycles in repeated‐batch operations. It was also demonstrated that glucose thus generated could be readily oxidized into gluconic acid, which can be easily separated from trehalose. We thus believe the proposed process of maltose hydrolysis with immobilized glucoamylase, in conjunction with trehalose synthase‐catalyzed isomerization and glucose oxidase‐catalyzed oxidation, is promising for the production and purification of trehalose on industrial scales. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

Transient kinetic experiments demonstrate the existence of a unique catalytic enzyme form in the peptide-stimulated ATPase mechanism of Escherichia coli Lon protease

Lon is an oligomeric serine protease whose proteolytic activity is mediated by ATP hydrolysis. Although each monomeric subunit has an identical sequence, Lon contains two types of ATPase sites that hydrolyze ATP at drastically different rates. The catalytic low-affinity sites display pre-steady-state burst kinetics and hydrolyze ATP prior to peptide cleavage. The high-affinity sites are able to hydrolyze ATP at a very slow rate. By utilizing the differing Kd's, the high-affinity site can be blocked with unlabeled nucleotide while the activity at the low-affinity site is monitored. Little kinetic data are available that describe microscopic events along the reaction pathway of Lon. In this study we utilize MANT-ATP, a fluorescent analogue of ATP, to monitor the rate constants for binding of ATP as well as the release of ADP from Escherichia coli Lon protease. All of the adenine nucleotides tested bound to Lon on the order of 10(5) M(-1) s(-1), and the previously proposed conformational change associated with nucleotide binding was also detected. On the basis of the data obtained in this study we propose that the rate of ADP release is slightly different for the two ATPase sites. As the model peptide substrate [S2; YRGITCSGRQK(Bz)] [Thomas-Wohlever, J., and Lee, I. (2002) Biochemistry 41, 9418-9425] or the protein substrate casein affects only the steady-state ATPase activity of the low-affinity sites, we propose that Lon adopts a different form after its first turnover as an ATP-dependent protease. Based on the obtained rate constants, a revised kinetic model is presented for ATPase activity in Lon protease in both the absence and presence of the model peptide substrate (S2).     

Reaction of an exofacial sulfhydryl group on the erythrocyte hexose carrier with an impermeant maleimide. Relevance to the mechanism of hexose transport

The presence of a reactive exofacial sulfhydryl on the human erythrocyte hexose carrier was used to test several predictions of the alternating conformation or one-site model of transport. The cell-impermeant glutathione-maleimide-I (GS-Mal) irreversibly inhibited hexose entry by decreasing the transport Vmax. This effect was potentiated by phloretin and maltose but decreased by cytochalasin B, indicating that under the one-site model the external sulfhydryl is on the outward-facing carrier but that it does not overlap with the exofacial substrate-binding site. Incubation of erythrocytes with maltose competitively inhibited the binding of [3H]cytochalasin B to the inward-facing carrier (Ki = 40 mM). Furthermore, both equilibrium cytochalasin B binding and its photolabeling of the band 4.5 carrier protein were decreased in ghosts prepared from GS-Mal-treated cells. Thus induction of an outward-facing carrier conformation with either maltose or GS-Mal caused the endofacial substrate-binding site to disappear. Dose-response studies of GS-Mal treatment of intact cells suggested that some functional carriers lack a reactive external sulfhydryl, which can be partially regenerated by pretreatment with excess cysteine. These data provide direct support for the one-site model of transport and further define the role of the external sulfhydryl in the transport mechanism.     

Improved specificity of reagentless amperometric PQQ-sGDH glucose biosensors by using indirectly heated electrodes

Indirectly heated electrodes operating in a non-isothermal mode have been used as transducers for reagentless glucose biosensors. Pyrroloquinoline quinone-dependent soluble glucose dehydrogenase (PQQ-sGDH) was entrapped on the electrode surface within a redox hydrogel layer. Localized polymer film precipitation was invoked by electrochemically modulating the pH-value in the diffusion zone in front of the electrode. The resulting decrease in solubility of an anodic electrodeposition paint (EDP) functionalized with Osmium complexes leads to precipitation of the redox hydrogel concomitantly entrapping the enzyme. The resulting sensor architecture enables a fast electron transfer between enzyme and electrode surface. The glucose sensor was operated at pre-defined temperatures using a multiple current-pulse mode allowing reproducible indirect heating of the sensor. The sensor characteristics such as the apparent Michaelis constants K(M)(app) and maximum currents I(max)(app) were determined at different temperatures for the main substrate glucose as well as a potential interfering co-substrate maltose. The limit of detection increased with higher temperatures for both substrates (0.020 mM for glucose, and 0.023 mM for maltose at 48 degrees C). The substrate specificity of PQQ-sGDH is highly temperature dependent. Therefore, a mathematical model based on a multiple linear regression approach could be applied to discriminate between the current response for glucose and maltose. This allowed accurate determination of glucose in a concentration range of 0-0.1mM in the presence of unknown maltose concentrations ranging from 0 to 0.04 mM.     

Determination of netropsin-DNA binding constants from footprinting data

A theory for deriving drug-DNA site binding constants from footprinting data is presented. Plots of oligonucleotide concentration, as a function of drug concentration, for various cutting positions on DNA are required. It is assumed that the rate of cleavage at each nucleotide position is proportional to the concentration of enzyme at that nucleotide and to the probability that the nucleotide is not blocked by drug. The probability of a nucleotide position not being blocked is calculated by assuming a conventional binding equilibrium for each binding site with exclusions for overlapping sites. The theory has been used to evaluate individual site binding constants for the antiviral agent netropsin toward a 139 base pair restriction fragment of pBR-322 DNA. Drug binding constants, evaluated from footprinting data in the presence of calf thymus DNA and poly(dGdC) as carrier and in the absence of carrier DNA, were determined by obtaining the best fit between calculated and experimental footprinting data. Although the strong sites on the fragment were all of the type (T.A)4, the value of the binding constant was strongly sequence dependent. Sites containing the dinucleotide sequence 5'-TA-3' were found to have significantly lower binding constants than those without this sequence, suggesting that an adenine-adenine clash produces a DNA structural alteration in the minor groove which discourages netropsin binding to DNA. The errors, scope, and limitations associated with the method are presented and discussed.     

The kinetics of glucose transport in human red blood cells

A quenched-flow apparatus and a newly developed automated syringe system have been used to measure initial rates of D-[14C]glucose transport into human red blood cells at temperatures ranging from 0 degrees to 53 degrees C. The Haldane relationship is found to be obeyed satisfactorily at both 0 and 20 degrees C, but Arrhenius plots of maximum D-[14C]glucose transport rates are non-linear under conditions of both equilibrium exchange and zero trans influx. Fitting of the data by non-linear regression to the conventional model for glucose transport gives values at 0 degrees C of 0.726 +/- 0.0498 s-1 and 12.1 +/- 0.98 s-1 for the rate constants governing outward and inward movements of the unloaded carrier molecule and 90.3 +/- 3.47 s-1 and 1113 +/- 494 s-1 for outward and inward movements of the carrier-glucose complex. Activation energies for these four rate constants are respectively 173 +/- 3.10, 127 +/- 4.78, 88.0 +/- 6.17 and 31.7 +/- 5.11 kJ X mol-1. These parameters indicate that at low temperatures the marked asymmetry of the transport mechanism arises mainly from the high proportion of inward-facing carriers and carrier-glucose complexes, and that there is a relatively small difference between the affinities of the carrier for glucose in the inward and outward-facing conformations. At high (physiological) temperatures the carrier is fairly evenly distributed between outward- and inward-facing conformations and the affinity for glucose is about 2.5-times greater outside than inside.     

On porcine pancreatic alpha-amylase action: kinetic evidence for the binding of two maltooligosaccharide molecules (maltose, maltotriose and o-nitrophenylmaltoside) by inhibition studies. Correlation with the five-subsite energy profile

Hydrolysis of small substrates (maltose, maltotriose and o-nitrophenylmaltoside) catalysed by porcine pancreatic alpha-amylase was studied from a kinetic viewpoint over a wide range of substrate concentrations. Non-linear double-reciprocal plots are obtained at high maltose, maltotriose and o-nitrophenylmaltoside concentrations indicating typical substrate inhibition. These results are consistent with the successive binding of two molecules of substrate per enzyme molecule with dissociation constants Ks1 and Ks2. The Hill plot, log [v/(V-v)] versus log [S], is clearly biphasic and allows the dissociation constants of the ES1 and ES2 complexes to be calculated. Maltose and maltotriose are inhibitors of the amylase-catalysed amylose and o-nitrophenylmaltoside hydrolysis. The inhibition is of the competitive type. The (apparent) inhibition constant Kiapp varies with the inhibitor concentration. These results are also consistent with the successive binding of at least two molecules of maltose or maltotriose per amylase molecule with the dissociation constants Ki1 and Ki2. These inhibition studies show that small substrates and large polymeric ones are hydrolysed at the same catalytic site(s). The values of the dissociation constants Ks1 and Ki1 of the maltose-amylase complexes are identical. According to the five-subsite energy profile previously determined, at low concentration, maltose (as substrate and as inhibitor) binds to the same two sites (4,5) or (3,4), maltotriose (as substrate and as inhibitor) and o-nitrophenyl-maltoside (as substrate) bind to the same three subsites (3,4,5). The dissociation constants Ks2 and Ki2 determined at high substrate and inhibitor concentration are consistent with the binding of the second ligand molecule at a single subsite. The binding mode of the second molecule of maltose (substrate) and o-nitrophenylmaltoside remains uncertain, very likely because of the inaccuracy due to simplifications in the calculations of the subsite binding energies. No binding site(s) outside the catalytic one has been taken into account in this model.     

Effects of ATP depletion on the mechanism of hexose transport in intact human erythrocytes

Depletion of ATP is known to inhibit glucose transport in human erythrocytes, but the kinetic mechanism of this effect is controversial. Selective ATP depletion of human erythrocytes by 10 micrograms/ml A23187 in the presence of extracellular calcium inhibited 3-O-methylglucose influx noncompetitively and efflux competitively. ATP depletion also decreased the ability of either equilibrated 3-O-methylglucose or extracellular maltose to inhibit cytochalasin B binding in intact cells, whereas neither total high-affinity cytochalasin B binding nor its Kd was affected. Under the one-site model of hexose transport these data indicate that ATP depletion decreases both the affinity of the inward-facing glucose carrier for substrate and its ability to reorient outwardly in intact cells.     

Apparent half-lives of sugar transport proteins inSaccharomyces cerevisiae

Using incubation in the presence of 0.4 mM cycloheximide the half-lives of the principal membrane transport proteins in baker's yeast were found to be: more than 24 h for the constitutive glucose carrier, 2.2 h for the inducible galactose carrier, 1.2 h for the inducible maltose carrier and 0.8 h for the inducible alpha-methyl-D-glucoside carrier. The distinct nature of the two last-named carriers was thus supported. De-induction of the galactose carrier was enhanced in the presence of glucose plus cycloheximide but not of either substance alone. Chloramphenicol suppressed all effects of cycloheximide. In contrast to the enzymes of galactose metabolism, the induction of the glactose carrier was not under the control of a mitochondrial factor and took place in a rho-mutant. The system induced by maltose but not the one induced by alpha-methyl-D-glucoside was de-induced rapidly by the intervention of a cytoplasm-synthesized protein.     

The maltase, glucoamylase and transglucosylase activities of acid α-glucosidase from rabbit muscle

1. The maltase and glucoamylase activities of acid alpha-glucosidase purified from rabbit muscle exhibited marked differences in certain physicochemical properties. These included pH stability, inactivation by thiol-group reagents, inhibition by alphaalpha-trehalose, methyl alpha-d-glucoside, sucrose, turanose, polyols, glucono-delta-lactone and monosaccharides, pH optimum and the kinetics and pH-dependence of cation activation. 2. The results are interpreted in terms of the existence of at least two specific substrate-binding sites or sub-sites. One site is specific for the binding of maltose and probably other oligosaccharides. The second site binds polysaccharides such as glycogen. 3. The sites appear to be in close proximity, since glycogen and maltose are mutually inhibitory substrates and interact directly in transglucosylation reactions. 4. Acid alpha-glucosidase exhibited intrinsic transglucosylase activity. The enzyme catalysed glucosyl-transfer reactions from [(14)C]maltose (donor substrate) to polysaccharides (glycogen and pullulan) and to maltose itself (disproportionation). The pH optimum was 5.1, with a shoulder or secondary activity peak at pH5.4. The glucose transferred to glycogen was attached by alpha-1,4- and alpha-1,6-linkages. Three major oligosaccharide products of enzyme action on maltose (disproportionation) were detected. 5. The kinetics of enzyme action on [(14)C]maltose showed that the rate of transglucosylation increased in a sigmoidal fashion as a function of substrate concentration, approximately in parallel with a decrease in the rate of glucose release. 6. The results are interpreted to imply competitive interaction at a specific binding site between maltose and water as glucosyl acceptors. 7. The results are discussed in terms of the possible existence of multiple subgroups of glycogen-storage disease type II.