Cibacron Blue Research Articles

Neuroprotective effects of modulators of P2 receptors in primary culture of CNS neurones

In previous studies (Volonté and Merlo, 1996. J. Neurosci. Res. 45, 183–193) basilen blue was shown to be a P2 receptor antagonist which abrogated glutamate-mediated cytotoxicity in cerebellar neurones in primary culture. Our work has now been extended to evaluate the neuroprotective action of the compound in additional neuronal systems, as well as in a different paradigm of cell death. We show that basilen blue prevents l-glutamate-mediated neurotoxicity in rat cerebellar (90–100% inhibition), cortical (60–70%) and hippocampal (50%) neurones. Similarly, glutamate-dependent progressive darkening of cell bodies, loss of phase-brightness and rapid cellular swelling are inhibited. Basilen blue is significantly less toxic and more effective at blocking l-glutamate toxicity in mixed cortical/glial cultures, compared to its structural analogue cibacron blue. Moreover, its neuroprotective effect is correlated with the time of incubation with granule neurones. Other purinoceptor ligands, including 2,2′-pyridylisatogen, but not pyridoxal-phosphate-6-azophenyl-2′,4′-disulphonic acid 4-sodium, are also effective in preventing glutamate toxicity. Furthermore, basilen blue prevents serum deprivation- and low potassium-induced apoptotic cell death in cerebellar granule neurones. In summary, our data extend and reinforce the possibility of a potential therapeutic use of P2 receptor modulators as neuroprotective agents for the central nervous system.

Enhanced visualization of parathyroid tissue by infusion of a visible dye conjugated to an antiparathyroid antibody

Background The antiparathyroid antibody BB5-G1 conjugated to cibacron blue, a blue dye, was intravenously infused to enhance parathyroid visualization. Previously, we demonstrated selective staining of human parathyroid implants in athymic nude mice after infusion of the conjugate. Methods Mice possessing implanted parathyroid tissue were randomized into the following: group I, infused with cibacron blue alone; group II, infused with the antibody/dye conjugate; and group III, infused with radiolabeled BB5-G1 alone. Implants were surgically explored. Three blinded observers ranked parathyroid visualization in the operative fields, and corresponding histologic sections were computer analyzed. Results Group II implants were easily visualized; groups I and III showed no staining. Group III showed high gamma counts. Subjective rankings and computer rankings correlated well (p < .05). Group II showed a higher mean staining intensity of 27.45 picogram protein product (PPP)/cell compared with 7.76 PPP/cell of group I (p = .002). Conclusion Cibacron blue/BB5-G1 consistently enhances visualization of implanted parathyroid tissue. © 1999 John Wiley & Sons, Inc. Head Neck 21: 111–115, 1999.

Enhanced visualization of parathyroid tissue by infusion of a visible dye conjugated to an antiparathyroid antibody.

The antiparathyroid antibody BB5-G1 conjugated to cibacron blue, a blue dye, was intravenously infused to enhance parathyroid visualization. Previously, we demonstrated selective staining of human parathyroid implants in athymic nude mice after infusion of the conjugate. Mice possessing implanted parathyroid tissue were randomized into the following: group I, infused with cibacron blue alone; group II, infused with the antibody/dye conjugate; and group III, infused with radiolabeled BB5-G1 alone. Implants were surgically explored. Three blinded observers ranked parathyroid visualization in the operative fields, and corresponding histologic sections were computer analyzed. Group II implants were easily visualized; groups I and III showed no staining. Group III showed high gamma counts. Subjective rankings and computer rankings correlated well (p < .05). Group II showed a higher mean staining intensity of 27.45 picogram protein product (PPP)/cell compared with 7.76 PPP/cell of group I (p = .002). Cibacron blue/BB5-G1 consistently enhances visualization of implanted parathyroid tissue.

Cibacron blue allosterically modulates the rat P2X 4 receptor

We have used whole-cell patch clamp electrophysiology to characterise the actions of the P2 antagonist, cibacron blue, on the rat recombinant P2X 4 receptor, stably expressed in human embryonic kidney 293 (HEK293) cells. In single cells, adenosine triphosphate (ATP) evoked inward currents, but the response was subject to considerable run down which precluded obtaining quantitative data. However, when recordings were made from cells that were part of a group of 20–40 electrically coupled cells (cell rafts), run-down of current was not observed and reproducible responses could be obtained. When studied using cell rafts, cibacron blue was a weak antagonist of the rat P2X 4 receptor (IC 50>300 μM) when co-applied with ATP. However, when cell rafts were preincubated with low concentrations of cibacron blue (3–30 μM) for 5 min prior to ATP addition, cibacron blue increased responses to ATP by increasing its potency (up to 4-fold) without affecting the maximum current. Potentiation of ATP-evoked currents was also observed following washout of high, inhibitory concentrations of cibacron blue (300 μM). In contrast to these effects on P2X 4 receptors, cibacron blue inhibited the ATP-induced response in both single cells and rafts of HEK293 cells expressing the P2X 2 receptor (IC 50∼600–800 nM). The effects of cibacron blue on the P2X 4 receptor were quantitatively similar to those of Zn 2+ which also increased ATP-evoked currents by decreasing the EC 50 of ATP (up to 3.5-fold). These data are consistent with the concept that cibacron blue, like zinc, allosterically regulates the function of the P2X 4 receptor.

Aqueous two-phase partition and detergent precipitation of a drug-responsive NADH oxidase from the HeLa cell surface

The partitioning behaviour of a drug (capsaicin)-responsive NADH oxidase (tNOX) activity released from HeLa ceIls by low pH treatment followed by heat and proteinase K was determined. When partitioned in a standard 6.4% PEG 3350/6.4% dextran T-500 two-phase system, the bulk of the tNOX activity was in the dextran-rich lower phase. The activity was inhibited by and bound to the triazine dye, Cibacron blue. Affinity partition, where the Cibacron blue was coupled to amino PEG 5000 and added to the first two-phase separation step, resulted in the partitioning of activity to the upper PEG phase. A second partition with PEG-salts resulted in the release of the tNOX from the Cibacron blue–amino PEG enriched phase into the salt-enriched lower phase. The phase-purified protein exhibited anomalous behavior and tended to multimerize in sodium dodecyl sulphate (SDS) prior to SDS-polyacrylamide gel electrophoresis (PAGE). Multimerization appeared to be enhanced by PEG. The multimerization was enhanced with the reduced protein in the presence of detergent prior to SDS–PAGE. In addition, the activity was precipitated by PEG 8000 at concentrations between 6 and 30% by weight. In the presence of or after exposure to PEG 3350 or PEG 8000, the protein could not be detected by Western blot analysis after SDS–PAGE suggesting that the protein failed to enter the gel even though other HeLa cell surface proteins were unaffected. The anomalous multimerization behavior has thus far precluded the use of phase partition as a practical purification step for the oxidase.

The structures and functions of ATP receptors

Our previous studies have determined the presence of adenosine 5′-triphosphate (ATP) in the cochlear fluids and shown that extracellular ATP introduced into the endolymphatic compartment of the guinea pig cochlea has a significant dose-dependent suppressive effect on both endocochlear potential (EP) and cochlear microphonic (CM), which is mediated via P2 receptors. In the present study, the influence of P2 receptor agonists and antagonists on this suppressive effect was investigated to characterise the subtypes of P2 receptor mediating the ATP-induced effect on cochlear function. Using a double-barreled pipette attached to a pressure injector, small volumes (2–10 nl) of ATP (0.01–1 mM) and P2 receptor agonists or P2 receptor antagonists in artificial endolymph were introduced into the scala media of the first (basal) and third turns of the guinea pig cochlea, while the EP and CM were monitored. ATP and P2 receptor agonists (5×10−14–1×10−11 moles) reversibly decreased the magnitude of EP and CM in a dose-dependent manner, in the order: 2-methylthioATP>ATP>α,β-methyleneATP⪢adenosine 5′-diphosphate. The ATP-induced decline of EP and CM was significantly inhibited by P2 receptor antagonists in the order of potency: pyridoxalphosphate-6-azophenyl-2′,4′-disulphonic acid⪢suramin>cibacron blue. Neither adenosine nor uridine 5′-triphosphate (2×10−13–2×10−11 moles) nor the P2 receptor antagonists on their own had any effect on EP and CM. The ATP effect on the potentials was greater at the third cochlear turn when compared to the first turn. These results provide evidence that in the endolymphatic compartment of the guinea pig, the extracellular ATP effect on cochlear function is likely mediated through an interaction with P2 receptors which assemble as ATP-gated ion channels.

Molecular Assembly of the Extracellular Domain of P2X2, an ATP-Gated Ion Channel

We have produced the putative extracellular domain (ECD) of the ATP-gated ion channel, P2X2, in a bacterial expression system. The hexahistidine-tagged protein was purified by immobilized metal affinity chromatography and refolded by sulfitolysis and dialysis. We demonstrate that P2X2-ECD forms a stable tetramer in solution by gel filtration chromatography, dynamic light scattering and analytical sedimentation centrifugation. [α-32P]ATP has been covalently crosslinked by UV irradiation to the P2X2-ECD and this binding is specific and competable by antagonists suramin and cibacron blue. These results indicate that the binding affinity among P2X2-ECD subunits is appreciably stronger than 3.4 μM (0.1 mg/ml), implying that the extracellular domain of P2X2is primarly responsible for tetramerization of whole P2X2and thus probably plays a role in determining homo- and heteromerization specificity of P2X channel subunits.

Dye-incorporated poly(EGDMA-HEMA) microspheres as specific sorbents for aluminum removal

Aluminum [Al(III)] adsorption onto dye-incorporated poly(ethylene glycol dimethacrylate-hydroxyethyl methacrylate) [poly(EGDMA-HEMA)] microspheres was investigated. Poly(EGDMA-HEMA) microspheres, in the size range of 150–200 μm, were produced by a modified suspension polymerization of EGDMA and HEMA. The reactive dyes (i.e., Congo Red, Cibacron Blue F3GA and Alkali Blue 6B) were covalently incorporated to the microspheres. The maximum dye load was 14.5 μmol Congo Red/g, 16.5 μmol Cibacron Blue F3GA/g and 23.7 μmol Alkali Blue 6B/g polymer. The maximum Al(III) adsorption on the dye microspheres from aqueous solutions containing different amounts of Al(III) ions were 27.9 mg/g, 17.3 mg/g and 12.2 mg/g polymer for the Congo Red, Cibacron Blue F3GA and Alkali Blue 6B, respectively. The maximum Al(III) adsorption was observed at pH 7.0 in all cases. Non-specific Al(III) adsorption was about 0.84 mg/g polymer under the same conditions. High desorption ratios (95%) were achieved in all cases by using 0.1 M HNO 3. It was possible to reuse these dye-incorporated poly(EGDMA-HEMA) microspheres without significant losses in the Al(III) adsorption capacities.

108-pS Channel in Brown Fat Mitochondria Might Be Identical to the Inner Membrane Anion Channel

Single-channel and whole-mitoplast patch-clamp recordings were employed to characterize the 108-pS (Cl-) channel in brown fat mitochondrial mitoplasts. We demonstrated the ability of this channel to conduct di- and trivalent anions, such as sulfate, phosphate, and benzenetricarboxylates, and its blockage by propranolol, 1,4-dihydropyridine-type Ca2+ antagonists, and Cibacron blue. Moreover, we have revealed its pH dependence for the first time. As a basis for the characteristic potential dependence of the whole-mitoplast current, we identified an open probability, increasing with depolarizing (positive) potentials, Eh, and being almost zero in the hyperpolarizing range. Events at negative Eh exhibit a short flickering behavior, whereas at positive Eh, they become much longer. This voltage dependence is influenced by pH in such a way that, at acidic pH, the 108-pS channel possesses a low open probability throughout the observed potential range, whereas at alkaline pH, the channel switches to long openings, even at a negative potential. All these properties lead us to conclude that the inner membrane anion channel, which has been characterized only by light scattering studies, and the 108-pS inner membrane channel, which has been characterized electrophysiologically, are one and the same process.

Electrochemical investigations of reactive dyes; cathodic stripping voltammetric determination of anthraquinone-based chlorotriazine dyes at a hanging mercury drop electrode

Two anthraquinone-based chlorotriazine reactive dyes, Procion Blue MX-R and Cibacron Blue 3GA, have been determined at nanomolar levels using cathodic stripping voltammetry. When the differential pulse voltammetric stripping mode is used no peak is obtained for the reduction of the anthraquinone moiety in these dyes, although the signal is present when the linear sweep mode is used. The absence of this peak in the differential pulse mode is believed to occur because, when this group is reduced in the adsorbed state on application of the potential pulse, it is reduced so rapidly that reduction is complete before the current is monitored at the end of the pulse. The differential pulse signal is present for the reduction of the chlorotriazine active groups. The differential pulse peak for the anthraquinone reduction process does appear when boric acid is present in the supporting electrolyte, and it is also present when the chlorotriazine groups are hydrolysed or reacted with other hydroxy compounds. This means that hydrolysis or similar reactions of these dyes can be monitored by observing both the appearance of the anthraquinone peak and the disappearance of the chlorotriazine peak.

Cibacron Blue F3G-A anchored monolayers with biospecific affinity for NAD(H)-dependent lactate dehydrogenase: characterization by FTIR-spectroscopy and atomic force microscopy

Fourier transform infrared reflection absorption spectroscopy (FT-IRAS) and atomic force microscopy have been used to analyze several alkanethiol self-assembled monolayers derivatized with the triazine dye Cibacron Blue F3G-A. This compound, when in solution, works as a competitive inhibitor for NAD(H)-dependent lactate dehydrogenase in building a complex by a site-specific affinity binding at the enzyme's NAD +-binding pocket. Therefore, the chemisorption of alkanethiol self-assembled monolayers bearing Cibacron Blue 3FG-A as affinity ligand should provide a way to prepare gold surfaces with a high biospecific affinity for lactate dehydrogenase. The results presented in this work, together with previously reported data from electroenzymatic studies of lactate dehydrogenase-modified gold electrode surfaces, show that the feasibility of such monolayers to bind enzyme strongly depends on the structure and composition of the alkanethiol underlayer used to anchor Cibacron Blue. Infrared spectra of the Cibacron Blue-anchored monolayers show that a sufficiently high amount of dye can be succesfully attached to functionalized alkanethiol self-assembled monolayers with a certain degree of disorder in their structure. Atomic force images of lactate dehydrogenase-modified gold surfaces confirm that higher and more homogeneous surface coverage of the enzyme can be obtained from poorly ordered mixed short- and long-alkanethiol self-assembled monolayers. These results suggest that the higher electroenzymatic activities shown by lactate dehydrogenase-modified gold surfaces prepared from poorly ordered Cibacron Blue-anchored mixed alkanethiol self-assembled monolayers can be correlated with a higher loading of enzyme bound to the metal surface as a result of the higher number of affinity sites (Cibacron Blue) available on the alkanethiol monolayer.

Cibacron blue F3GA-incorporated macroporous poly(2-hydroxyethyl methacrylate) affinity membranes for heavy metal removal

Macroporous poly(2-hydroxyethyl methacrylate), poly(HEMA), membranes were prepared by UV-initiated photopolymerization of HEMA in the presence of an initiator (azobisisobutyronitrile, AIBN). An affinity dye, i.e., Cibacron Blue F3GA was then incorporated covalently. These affinity membranes with a swelling ratio of 58%, and carrying 10.67 mmol Cibacron Blue F3GA/m 2 membrane were used in the adsorption/desorption of some selected heavy metal ions [i.e., As(III), Cd(II) and Pb(II)] from aqueous media. Very high adsorption rates were observed and adsorption equilibria were reached in about 30 min. The maximum adsorptions of heavy metal ions onto the dye-incorporated affinity membranes from their single solutions were 12.6 mmol/m 2 for As(III), 61.0 mol/m 2 for Cd(II) and 79.0 mol/m 2 for Pb(II). However, when the heavy metal ions competed (in the case of the adsorption from their mixture) the amounts of adsorption for As(III), Cd(II) and Pb(II) were quite close. Desorption of heavy metal ions was carried out by using 0.1 M HNO 3 (pH 1.0). Up to 95% of the adsorbed heavy metal ions were desorbed in 60 min. Repeated adsorption/desorption cycles showed the feasibility of this novel affinity membrane for heavy metal removal.

Comparison of albumin binding capacities of three different reactive dye-derivatized poly(ethylene glycol dimethacrylate-hydroxyethyl methacrylate) microbeads

Bovine serum albumin (BSA) adsorption onto dye-derivatized poly(ethylene glycol dimethacrylate-hydroxyethyl methacrylate) [poly(EGDMA-HEMA)] microbeads carrying three different reactive dye ligands (i.e. Congo Red, Cibacron Blue F3GA, and Alkali Blue 6B) was investigated. Swellable poly(EGDMA-HEMA) microbeads, in the size range of 150-200 μm, were produced by a modified suspension copolymerization of EGDMA and HEMA. The dyes were covalently attached to the microbeads. The maximum amounts of dye loadings were 14.5, 16.5, and 23.7 μmol g-1 for Congo Red, Cibacron Blue F3GA, and Alkali Blue 6B, respectively. The maximum BSA adsorption on the dye-derivatized microbeads from aqueous solutions containing different amounts of BSA were 90, 60.5, and 40 mg g-1 for the Congo Red, Cibacron Blue F3GA, and Alkali Blue 6B carrying microbeads, respectively. The maximum BSA adsorptions were observed at pH 6.0 in all cases. Desorption of albumin molecules were achieved by using 1.0 M NaSCN (pH 8.0). High desorption ratios (more than 85% of the adsorbed BSA) were observed in all cases. It was possible to reuse these novel sorbents without significant losses in the adsorption capacities.

Construction, exploitation and evolution of a new peptide library displayed at high density by fusion to the major coat protein of filamentous phage.

The amino-terminus of the major coat protein (PVIII) of filamentous phage can be extended, up to 6-7 residues, without interfering with the phage life cycle. We have constructed a library of approximately ten millions different phage each displaying a different octapeptide joined to the amino-terminus of the 2700 copies of PVIII. Most of the resulting clones are able to produce infective particles. This molecular repertoire constituted by the periodic regular decoration of the phage filament surface, can be utilized to search elements that bind proteins or relatively small organic molecules like the textile dye Cibacron blue. By sequential growth cycles we have performed a library evolution experiment to select phage clones that have a growth advantage in the absence of any requirement for binding a specific target. The consensus of the best growers reveals a Pro rich sequence with large hydrophobic residues at position 7 and Asn at position 1 of the random peptide insert. We propose that the assembly secretion process is favoured in phages displaying this family of peptides since they fit the groove between two adjacent PVIII subunits by making advantageous molecular contacts on the phage surface.

1,4-Benzoquinone Reductase from the BasidiomycetePhanerochaete chrysosporium:Spectral and Kinetic Analysis

The reaction mechanism of a 1,4-benzoquinone reductase from the wood-rotting basidiomycetePhanerochaete chrysosporiumwas investigated. The native, oxidized, FMN-containing enzyme was reduced quantitatively by NADH and the resulting reduced enzyme was reoxidized in the presence of one equivalent of 2,6-dimethoxy-1,4-benzoquinone (DMBQ). The stoichiometry of NADH oxidation versus DMBQ reduction is 1:1. The enzyme catalyzes the reduction of quinones to hydroquinones by a ping-pong steady-state mechanism. However, inhibition is observed at low NADH concentrations. Quinone products derived from the autooxidation of the unstable compounds 1,2,4-trihydroxybenzene and 5-chloro-2,3,4-trihydroxybenzene also appear to be substrates for the quinone reductase. The enzyme reduces the one-electron acceptors ferricyanide and ferricytochrome c (Cc3+) with rates of 58.4 and 0.08%, respectively, compared to DMBQ. The stoichiometry of NADH oxidation versus ferricyanide reduction is 1:2. In the presence of quinones the rates of Cc3+and ferricyanide reduction are increased, owing to the nonenzymatic reduction of these acceptors by enzyme-generated hydroquinone products. Dicumarol and Cibacron blue are competitive inhibitors with respect to NADH, withKivalues of 2.1 and 0.30 μM, respectively. Reconstitution of the apoprotein with FMN yields a fully active enzyme at an FMN-to-protein ratio of 2:1, suggesting that the flavin content of the enzyme is two molecules of FMN per dimer.

Effect of purinergic blockers on outward current in isolated smooth muscle cells of the sheep bladder.

Freshly dispersed cells from sheep urinary bladder were voltage clamped using the whole cell and inside-out patch-clamp technique. Cibacron and Basilen blue increased outward current in a dose-dependent manner with a half-maximal response at 10(-5)M. Suramin, in concentrations to 10(-3)M, had no such effect. The Cibacron blue response was abolished in Ca2+ -free physiological salt solution, suggesting that it was acting on a Ca2+ -dependent current. Similarly, the Cibacron blue-sensitive current was significantly attenuated by charybdotoxin. Cibacron blue did not modulate inward current nor were its effects modified by caffeine or heparin, suggesting that its effect on outward current was not secondary to an increase in intracellular Ca2+. Application of 10(-4)M Cibacron blue to the inside membrane of excised patches caused a rapid increase in open probability of a large conductance (300 pS) K+ channel. These results suggest that Cibacron blue is a potent activator of a Ca2+ -dependent outward current in bladder smooth muscle cells in addition to its action as a purinergic blocker.

Controlled mixed-mode interaction chromatography on membrane adsorbers

Membrane adsorbers (MAs) are used for protein separation in controlled mixed-mode interaction chromatography. The strong anion- and cation-exchange MAs used are made either from a synthetic copolymer or from modified cellulose membranes. The affinity MAs (Cibacron Blue) are also made from the copolymer membranes. Standard protein mixtures, whey proteins, and biotechnological culture supernatants are separated. The influence of the flow-rate, the ratio of cation- and anion-exchange MAs inserted in the stack, and the pattern, i.e. the comparative worth of an alternating vs. a two-consecutive-stacks arrangement, on the separation is investigated. While the flow-rate shows no influence, both the pattern of arrangement and the ratio of the two types of ion exchanger do. Compared to single-mode MA chromatography, a broader range of proteins, e.g. in terms of the isoelectric points, can be separated in a single chromatographic procedure. Whey proteins (β-lactoglobulin, α-lactalbumin, BSA, IgG) are separated at pH 6, using a mixed-mode ion-exchange system. Here however, a two-stack approach is used to allow for module-uncoupling before elution, to prevent IgG and α-lactalbumin from coeluting. By using a mix of anion-exchange and Cibacron Blue affinity MAs, recombinant human antithrombin III (rh-AT III) can be separated in a single run from the major protein impurities present in the fermenter supernatant, namely transferrin and BSA.

Inhibition kinetics of camel lens zeta-crystallin: multiple inhibition studies.

The inhibition of camel lens zeta-crystallin by nitrofurantoin (NF) was uncompetitive with respect to co-factor NADPH, (Ki = 90 microM) and competitive with respect to the substrate 9,10-phenanthrenequinone (PQ), (Ki = 50 microM). Inhibition at micromolar concentrations was also observed with dicoumarol, NADP+ and cibacron blue (CB). Theorell-Yonetani double-inhibition analysis showed that NF and dicoumarol were mutually exclusive inhibitors against PQ. However, analysis of NF and NADP+ by a double-inhibition plot showed that they simultaneously bind to the enzyme molecule. These studies demonstrate that NF and dicoumarol share the same site so that both molecules are prevented from binding at the same time, while NF and NADP+ can bind simultaneously to different sites on the enzyme. Although CB was noncompetitive with respect to PQ, double inhibition analysis showed that CB and dicoumarol or NF were mutually exclusive inhibitors against PQ, implying a distinct mode of inhibition for CB.

Inhibition of camel lens zeta-crystallin/NADPH:quinone oxidoreductase activity by Cibacron blue.

Camel lens zeta-crystallin/NADH:quinone oxidoreductase activity was inhibited by Cibacron blue 3GA (CB) with 9.10-phenanthrenequinone (PQ) as an electron acceptor and NADPH as an electron donor in a time-independent and concentration dependent manner. The IC50 value of CB was 50 nM. The Lineweaver-Burk plots and the secondary plots indicated that the inhibition was linear mixed type (partial competitive and pure noncompetitive) with respect to NADPH and noncompetitive with respect to PQ. The estimated inhibition constant (Ki) values were 26.0 nM for NADPH and 55.0 nM for PQ respectively, suggesting that CB has high affinity towards the NADPH binding site. The secondary plots of inhibition with respect to NADPH, also indicate a dissociation constant (Ki) value of 68.0 nM for the zeta-crystallin-NADPH-CB complex. This Ki being greater than the Ki value suggests that noncompetitive inhibition is predominant over competitive inhibition at the NADPH binding site.

Active-site-directed inhibition of sucrose-phosphate synthase by Cibacron blue F3G-A and 1-deoxynojirimycin

Sucrose-phosphate synthase (SPS, E.C. 2.4.1.14) from spinach (Spinacia oleracea L.) was partially purified and the inhibition of the enzyme reaction by 1-deoxynojirimycin and Cibacron blue F3G-A analyzed. Cibacron blue was a high-affinity competitive inhibitor with respect to the substrate UDPglucose (Ki = 80 nM) and a mixed-type inhibitor with respect to fructose-6-phosphate. 1-Deoxynojirimycin was a mixed-type inhibitor of SPS with respect to UDPglucose [Ki(EI) = 5.8 mM] and a uncompetitive inhibitor with respect to fructose 6-phosphate. These results are discussed in relation to the mechanism of the reaction catalysed by SPS and the secondary structure of the enzyme.