Ninhydrins inhibit carbonic anhydrases directly binding to the metal ion.

Ninhydrins show extensive application in organic chemistry and agriculture whereas they have been poorly investigated as bioactive molecules for medicinal chemistry purposes. A series of ninhydrin derivatives was here investigated for the inhibition of human carbonic anhydrases (CAs, EC 4.2.1.1), based on earlier evidence that gem diols are able to coordinate the metal ion from the CA active site. Ninhydrins demonstrated a micromolar inhibitory action against CA I and IX (KIs in the range 0.57-68.2 µM) and up to a nanomolar efficacy against CA II and VII (KIs in the range 0.025-78.2 µM), validated isoforms as targets in several CNS-related diseases. CA IV was instead weakly or poorly inhibited. A computational protocol based on docking, MM-GBSA and metadynamics calculations was used to elucidate the putative binding mode of this type of inhibitors to CA II and CA VII. The findings of this study testify that such pharmacologically underestimated ligands may represent interesting lead compounds for the development of CA inhibitors possessing an innovative mechanism of action, i.e., mono- or bis-coordination to the zinc ion through the diol moiety.

Antimicrobial activity and second harmonic studies on organic non-centrosymmetric pure and doped ninhydrin single crystals.

In this paper, we report the successful growth of pure, Cu(2+) ions and Cd(2+) ions doped on ninhydrin single crystals by slow solvent evaporation technique. The presence of Cu(2+) and Cd(2+) ions in the specimen of ninhydrin single crystal has been determined by atomic absorption spectroscopy. The powder X-ray diffraction analysis was done to calculate the lattice parameters of the pure and doped crystals. The percentage of transmittance of the crystal was recorded using the UV-Vis Spectrophotometer. Thermal behaviors of the grown crystals have been examined by the thermal gravimetric/differential thermal analysis. The hardness of the grown crystals was assessed and the results show the minor variation in the hardness value for the pure and doped ninhydrin samples. The value of the work hardening coefficient n was found to be 2.0, 1.0 and 1.06 for pure, copper and cadmium doped ninhydrin crystals respectively. The second harmonic generation efficiency of Cd(2+) and Cu(2+) doped ninhydrin is 8.3 and 6.3 times greater than well known nonlinear crystal of potassium dihydrogen phosphate respectively. The antibacterial and antifungal activities of the title compound were performed by disk diffusion method against the standard bacteria Escherichia coli, Xanthomonas oryzae and against the fungus Aspergillis niger and Aspergillus flavus.

Iterative exposure of clonal BRIN-BD11 cells to ninhydrin enables selection of robust toxin-resistant cells but with decreased gene expression of insulin secretory function.

OBJECTIVES: Prevention of pancreatic beta-cell destruction combined with preservation of insulin secretory function is an important goal for cell-based diabetes therapy. This study describes the generation and characteristics of toxin-resistant beta-cells. METHODS: By using iterative exposures to ninhydrin, a new class of robust ninhydrin-tolerant insulin-secreting BRIN-BD11 ninhydrin-tolerant (BRINnt) cells was generated. Low- and high-passage BRINnt cells were used to evaluate beta-cell function and tolerance against toxins in comparison with native BRIN-BD11 cells. Differences in viability, gene expression, insulin secretory function, antioxidant enzyme activity, DNA damage, and DNA repair efficiency were compared. RESULTS: BRIN-BD11 ninhydrin-tolerant cells exhibited resistance toward ninhydrin and hydrogen peroxide but not streptozotocin (STZ). Both total superoxide dismutase (SOD) and catalase enzyme activities of BRINnt cells were significantly enhanced, and ninhydrin-induced DNA damage was decreased. BRIN-BD11 ninhydrin-tolerant cells also exhibited enhanced DNA repair efficiency. However, this was accompanied by loss of secretagogue-induced insulin release, decreased cellular insulin content, and deficits in insulin and glucose transporter 2 gene expression. Prolonged culture of BRINnt cells in the absence of ninhydrin reversed the degenerated function of BRINnt cells but restored ninhydrin susceptibility. CONCLUSIONS: These data illustrate dissociation between beta-cell toxin resistance and secretory function, indicating difficulties in generation of robust and well-functioning cells using this approach.

Streptozotocin-resistant BRIN-BD11 cells possess wide spectrum of toxin tolerance and enhanced insulin-secretory capacity.

Since streptozotocin (STZ) exhibits beta-cell toxicity, mediated through diverse mechanisms, multiple toxin resistance can be expected in insulin-secretory cells rendered STZ-resistant. RINm5F, but not all cell lines surviving STZ treatment, possess higher insulin content than native parental cells and additional tolerance against alloxan. To understand the impact of STZ tolerant cell selection on toxin resistance and insulin-secretory function, STZ-resistant BRIN-BD11 cells were generated by iterative acute exposure to 20 mM STZ. These cells, denoted BRINst cells, exhibited resistance to toxic challenges from STZ, H(2)O(2), and ninhydrin. Insulin content and both glucose and arginine-stimulated insulin secretion were significantly enhanced in BRINst cells. The toxin-resistance of BRINst cells was gradually lost during continuous cultivation without STZ challenge. However, enhanced insulin secretory capacity at high passage in BRINst cells persisted. Although total SOD activity was decreased, catalase activity was increased and appeared to be important for the ninhydrin and STZ resistance of BRINst cells. This was associated with reductions of both STZ- and ninhydrin-induced DNA damage, although DNA repair was abolished. Further characterization of cells exhibiting multiple toxin tolerance and an enhanced insulin secretory function could provide useful lessons for understanding of beta-cell survival.

Identification and evaluation of antioxidant, analgesic/anti-inflammatory activity of the most active ninhydrin-phenol adducts synthesized.

Treatment of phenols with ninhydrin in acidic medium afforded 2-hydroxy-2-(ortho-hydroxy-phenyl/naphthyl)-1,3-dioxoindanes, which being unstable were isolated in their hemiketal forms. These synthesized compounds were subjected to TLC screening for radical scavenging and in vitro lipoxgenase and cycloxygenase enzyme inhibition assays. The best compound was identified and studied in detail for steady-state and time-resolved free radical kinetics, viz., DPPH, ABTS(-), *OH and rate constants for these reactions were evaluated. The best compound was also subjected to in vivo anti-inflammatory and analgesic activities in which the compound showed good promise for further structural optimization.

Specificity of a Vibrio vulnificus aminopeptidase toward kinins and other peptidyl substrates.

Recently, phosphoglucose isomerase with a lysyl aminopeptidase (PGI-LysAP) activity was identified in Vibrio vulnificus. In this paper, we demonstrate the proteolytic cleavage of human-derived peptides by PGI-LysAP of V. vulnificus using three approaches: (i) a quantitative fluorescent ninhydrin assay for free lysine, (ii) matrix-assisted laser desorption ionization-two-stage time of flight mass spectrometry (MALDI-TOF-TOF), and (iii) Tricine gel electrophoresis. PGI-LysAP hydrolyzed bradykinin, Lys-bradykinin, Lys-(des-Arg9)-bradykinin, neurokinin A, Met-Lys-bradykinin, histatin 8, and a myosin light chain fragment. We detected the proteolytic release of free L-lysine from peptide digests using a rapid, simple, sensitive, and quantitative fluorescent ninhydrin assay, and results were confirmed by MALDI-TOF-TOF. The use of the fluorescent ninhydrin assay to quantitatively detect free lysine hydrolyzed from peptides is the first application of its kind and serves as a paradigm for future studies. The visualization of peptide hydrolysis was accomplished by Tricine gel electrophoresis. Proteolytic processing of kinins alters their affinities toward specific cellular receptors and initiates signal transduction mechanisms responsible for inflammation, vasodilation, and enhanced vascular permeability. By applying novel approaches to determine the proteolytic potential of bacterial enzymes, we demonstrate that PGI-LysAP has broad exopeptidase activity which may enhance V. vulnificus invasiveness by altering peptides involved in signal transduction pathways.

Forensic applications of chemical imaging: latent fingerprint detection using visible absorption and luminescence.

Chemical imaging technology is a rapid examination technique that combines molecular spectroscopy and digital imaging, providing information on morphology, composition, structure, and concentration of a material. Among many other applications, chemical imaging offers an array of novel analytical testing methods, which limits sample preparation and provides high-quality imaging data essential in the detection of latent fingerprints. Luminescence chemical imaging and visible absorbance chemical imaging have been successfully applied to ninhydrin, DFO, cyanoacrylate, and luminescent dye-treated latent fingerprints, demonstrating the potential of this technology to aid forensic investigations. In addition, visible absorption chemical imaging has been applied successfully to visualize untreated latent fingerprints.

Amino acid and protein targets of 1,2-diacetylbenzene, a potent aromatic gamma-diketone that induces proximal neurofilamentous axonopathy.

The gamma-diketone analogs 1,2-diacetylbenzene (1,2-DAB) and 2,5-hexanedione (2,5-HD), but not the delta-diketone 1,3-diacetylbenzene (1,3-DAB) or the beta-diketone 2,4-hexanedione, induce neuropathological changes in the rodent central and peripheral nervous systems. The molecular targets of these neurotoxic aromatic and aliphatic gamma-diketones, and of their nonneurotoxic structural analogs and ninhydrin, are examined by assessing their differential reactivity with neural and nonneural amino acids and proteins in vitro and in vivo. Whereas 1,2-DAB is chromogenic and forms polymers with amino acids (notably lysine) and proteins (especially lysine-rich proteins), 1,3-DAB lacks these properties. Ninhydrin forms a chromophore without evidence of protein polymerization. 1,2-DAB preferentially targets neurofilament over microtubule protein in vitro and in situ. Based on protein reactivity, 1,2-DAB is three orders of magnitude more reactive than 2,5-HD. Lysine-rich neurofilament protein subunits NF-H and NF-M are more susceptible than lysine-poor NF-L and beta-tubulin to 1,2-DAB. These observations correlate with the development of proximal (1,2-DAB) and distal (2,5-HD) neurofilament-filled axonal swellings and segregated intact microtubules observed during systemic treatment with aromatic and aliphatic gamma-diketones.

Effects of cytotoxic agents on functional integrity and antioxidant enzymes in clonal beta-cells.

Effects of cytotoxic agents and hydrogen peroxide were examined using pancreatic BRIN-BD11 cells and the parental insulinoma RINm5F cell line. Cell viability was determined using the MTT colorimetric assay and the TUNEL assay was used to assess apoptosis and acridine orange assay was used to determine levels of apoptosis versus necrosis. RT-PCR studies were employed to investigate the effects of the toxins on the expression of antioxidative enzymes, superoxide dismutase (SOD), glutathionine peroxidase (GPX) and catalase (CAT). Streptozotocin, hydrogen peroxide, alloxan and ninhydrin exerted time- and concentration-dependent toxic effects on BRIN-BD11 and RINm5F cells. RT-PCR showed that 90 minutes exposure of BRIN-BD11 cells or RINm5F cells to 5 mM ninhydrin down regulates SOD, GPX and CAT antioxidative enzymes. Glutathionine peroxidase gene expression was also down regulated in both types of cell by hydrogen peroxide. There were no significant differences in antioxidant gene expression after exposure to the other toxins under the conditions employed. TUNEL assay revealed that streptozotocin (8 mM) and hydrogen peroxide (125 microM) had no significant effect on the number of cells undergoing apoptosis. However after exposure to ninhydrin (5 mM) almost 100% of the non-viable BRIN-BD11 cells and around 50% of the RINm5F cells were dying by apoptosis. With the BRIN-BD11 cells there was around a 30% increase in the number of apoptotic cells compared with 50% in the RINm5F cells after exposure to alloxan (16 mM). The results indicate multiple effects of cytotoxic agents on functional integrity and antioxidant enzyme gene expression in clonal beta-cells.

1,2-diacetylbenzene, the neurotoxic metabolite of a chromogenic aromatic solvent, induces proximal axonopathy.

Several widely used aromatic hydrocarbon solvents reportedly induce blue-green discoloration of tissues and urine in animals and humans. The chomophore has been proposed to result from a ninhydrin-like reaction with amino groups in proteins. The present study examines the neurotoxic property of 1,2-diacetylbenzene (1,2-DAB), the active metabolite of the chromogenic and neurotoxic aromatic solvent 1,2-diethylbenzene. Rats treated with 1,2-DAB, but not with the nonchromogenic isomer 1,3-DAB or with ninhydrin developed blue discoloration of internal organs, including the brain and spinal cord. Only 1,2-DAB induced limb weakness associated with nerve fiber changes, which were most prominent in spinal cord and spinal roots. Changes began with the formation of proximal, neurofilament-filled axonal swellings of the type seen after treatment with 3,4-dimethyl-2,5-hexanedione, a potent derivative of the active metabolite of the neurotoxic aliphatic hydrocarbon solvents n-hexane and methyl n-butyl ketone. These compounds are metabolized to a gamma-diketone that forms pyrroles with target proteins, such as neurofilament proteins. A comparable mechanism is considered for 1,2-DAB, an aromatic gamma-diketone.

Effect of ninhydrin on the biochemical and histopathological changes induced by ethanol in gastric mucosa of rats.

Studies on the effect of ninhydrin in the normal gastric mucosa and against the ethanol induced gastric injury were undertaken in rats in view of the presence of a carbonyl function as well as hydroxyl groups in its chemical structure. In spite of its potentials to generate hydroxyl radicals, it is deemed to possess antioxidant property by virtue of its electrophilic nature. Recent studies have shown gastro-protection to mediate through a reaction between the electrophilic compounds and sulfhydryl groups of the mucosa. Hence it was found worthwhile to evaluate the interaction between the oxidant and antioxidant functions in the structure of the same compound. The effects of ninhydrin pretreatment on gastric mucosal injuries caused by 80% ethanol, 25% NaCl and 0.2M NaOH were investigated in rats. The gastric tissue in ethanol-treated rats was analyzed for different histopathological lesions. In addition, the effects on ethanol-induced changes in the gastric levels of proteins, nucleic acids, non-protein sulfhydryl (NP-SH) and malondialdehyde (MDA) were also evaluated. Ninhydrin, as such, failed to induce any significant changes in normal gastric mucosa, while its pretreatment at oral doses of 5, 10 and 20 mg/kg was found to provide a dose-dependent protection against the ulcers induced by ethanol, NaOH and NaCl. The results of histopathological evaluation revealed a protective effect of ninhydrin on congestion, hemorrhage, edema, erosions and necrosis caused by ethanol. Furthermore, the pretreatment afforded a dose-dependent inhibition of the ethanol-induced depletion of proteins, nucleic acids, NP-SH and increase of MDA in the gastric tissue. The results obtained clearly demonstrate the anti-ulcerogenic activity of ninhydrin. The exact mechanism of action is not known. However, the carbonyl function in ninhydrin appears to achieve antioxidant balance and protect the gastric mucosa from the ethanol-induced gastric injury. Further studies are warranted to investigate the toxicity and detailed mechanism of action of this potent compound before any clinical trials, especially at the effective lower doses.

Studies on the cytotoxic, biochemical and anti-carcinogenic potentials of ninhydrin on Ehrlich ascites carcinoma cell-bearing Swiss albino mice.

Ninhydrin (2,2-dihydroxy-1,3-indane dione) was evaluated for its antitumor and cytotoxic properties in Ehrlich ascites carcinoma cell (EAC Cell)-bearing mice. The rationale behind this study has been mainly the literature reports of its characteristic interference with DNA synthesis and calcium homeostasis. Antitumor activity was evaluated from the total count and viability of EAC cells in addition to their nucleic acid, protein, non-protein sulfhydryls (NP-SH) and malondialdehyde (MDA) contents. The EAC cell-bearing animals were also observed for the effect on their survival and body weight variations. In addition, the tumors grown at the site of injection were evaluated for histopathological changes. Ninhydrin treatments (5, 10 and 20 mg/kg/day) abate the increase in body weight and advanced the duration of survival in EAC cell-bearing mice. The results on histopathological investigations show retardation in tumor growth, decreased frequency of mitotic figures and hair follicles and an increased necrosis in the tumor by ninhydrin treatment. Our results on cytotoxicity, which demonstrated compression in the number of EAC cells and their viability substantiate these data. The results of biochemical studies on EAC cells exhibit a reduction in the levels of DNA, RNA, proteins and NP-SH with a subsequent increase in the concentrations of MDA after ninhydrin treatment. Inhibition in tumor growth was dose dependently significant with the same dose regimen. The observed cytotoxic and antitumor activity of ninhydrin was comparable to cyclophosphamide. The possible mode of action of ninhydrin-induced cytotoxic and antitumor activity appear to be due to its interference with mitochondrial function resulting in inhibition of DNA synthesis, an effect that is being investigated further.

Reagents for the chemical development of latent fingerprints: scope and limitations of benzo[f]ninhydrin in comparison to ninhydrin.

Benzo[f]ninhydrin was compared to ninhydrin for fingerprint development on paper. Overall, the performance of ninhydrin on exhibits was slightly better than that of benzo[f]ninhydrin. The significant advantages of the benzo[f]ninhydrin over ninhydrin were the much stronger fluorescence it gave after treatment with zinc salts and a slightly quicker reaction under ambient conditions. This fluorescence is, however, similar to that obtained with other reagents, such as DFO or ninhydrin analogs. These advantages apparently are not sufficient to justify regular usage of benzo[f]ninhydrin, especially when one considers its low solubility and high cost.

Modulation of human glucokinase intrinsic activity by SH reagents mirrors post-translational regulation of enzyme activity.

The low-affinity glucose phosphorylating enzyme glucokinase plays a key role in the process of glucose recognition in pancreatic B-cells. To evaluate mechanisms of intrinsic regulation of enzyme activity human pancreatic B-cell and liver glucokinase and for comparison rat liver glucokinase were expressed in E. coli bacteria. A one-step purification procedure through metal chelate affinity chromatography revealed 58 kDa proteins with high specific activities in the range of 50 U/mg protein and K(m) values around 8 mM for the substrate D-glucose with a preference for the alpha-anomer. There were no tissue specific differences, no species differences in the electrophoretic mobility, and no differences of the kinetic properties of these well conserved enzymes. The deletion of the 15 tissue-specific NH2-terminal amino acids of the human glucokinase resulted in a catalytically active enzyme whose kinetic properties were not significantly different from those of the wild-type enzymes. The human and rat glucokinase isoforms were non-competitively inhibited by the sulfhydryl group reagents alloxan and ninhydrin with Ki values in the range of 1 microM. The inhibition of glucokinase enzyme activity was reversed by dithiothreitol with an EC50 value of 9 microM for alloxan and of 50 microM for ninhydrin. D-Glucose provided protection against alloxan-induced inhibition of human and rat glucokinase isoenzymes with half-maximal effective concentrations between 11 and 16 mM. The enzyme inhibition by alloxan was accompanied by a change in the electrophoretic mobility with a second lower molecular 49 kDa glucokinase band which can be interpreted as a compact glucokinase molecule locked by disulfide bonds. Quantification of free sulfhydryl groups revealed an average number of 3.6 free sulfhydryl groups per enzyme molecule for the native human glucokinase isoforms. Alloxan decreased the average number of free sulfhydryl groups to 1.9 per enzyme molecule indicating that more than one SH side group is oxidized by this compound. The extraordinary sensitivity of the SH side groups of the glucokinase may be a possible mechanism of enzyme regulation by interconversion of stable (active) and unstable (inactive) conformations of the enzyme. In pancreatic B-cells the glucose-dependent increase of reduced pyridine nucleotides may stabilize the enzyme in the 58 kDa form and provide optimal conditions for glucose recognition and glucose-induced insulin secretion.

Arginyl residues are involved in the transport of Fe2+ through the plasma membrane of the mammalian reticulocyte.

Sealed reticulocyte ghosts were treated with reagents that modify a variety of amino acid residues. Only ninhydrin and phenylglyoxal, both modifiers of arginyl residues, produced inhibition of the initial rate of 59Fe2+ uptake. The inhibition (i) was dependent on the concentration of ninhydrin or phenylglyoxal, (ii) increased from pH 7 to 9, a feature of the modification of arginine by ninhydrin or phenylglyoxal, and (iii) was blocked when Fe2+ was present during the modification step. A23187, an effective membrane Fe2+ transporter, diminished the inhibitory effect of ninhydrin and phenylglyoxal, indicative that the transport of iron through the membrane, and not a secondary process, was selectively inhibited. We conclude that the iron transporter from the plasma membrane of erythroid cells has one or more arginyl residues in a segment accessible to ninhydrin or phenylglyoxal, and that this residue is involved in the transmembrane transport of iron.

Tumour-promoting activity of ninhydrin on mouse skin.

Ninhydrin (2,2-dihydroxy-1,3-indanedione; CAS No. 485-47-2) is widely used as a reagent for the detection of free amino and carboxyl groups in proteins and peptides. It is an irritant to mammalian skin. Various toxic effects of ninhydrin have been reported in laboratory animals; however, so far there has been no evaluation of its carcinogenic and co-carcinogenic potential in laboratory animals by long-term in vivo bioassay. Ninhydrin was found to induce the activity of gamma-glutamyl transpeptidase (GGT) in mouse skin but it failed to alter the activity of the enzyme ornithine decarboxylase when compared with animals treated with standard tumour promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA). In the present investigations, the tumour-promoting activity of ninhydrin (including both stage I and stage II of tumour promotion) was tested on Swiss albino mice in a multistage mouse skin model of carcinogenesis. The animals were initiated with a single topical application of 7,12-dimethylbenz-anthracene followed by four topical applications of ninhydrin biweekly as stage I promoter for 2 wk. Stage II promotion was twice weekly through topical application of mezerein. The results revealed that ninhydrin is a strong stage I tumour promoter and its efficacy was comparable with that of TPA at the dose level used in the experiment. However, ninhydrin failed to produce tumours when tested as a stage II or complete tumour promoter on mouse skin.

[The effect of middle-molecular ninhydrin-positive substances isolated from the rat myocardium on rat serum arylamidase activity after administration of ethanol]. / Vplyv strednemolekulových ninhydrínpozitívnych látok izolovaných z myokardu potkanov po etanole na aktivitu arylamidáz v krvnom sére potkanov.

Protein-free extract from the myocardium of control rats and the majority of its fractions were found to stimulate arylamidase activity. Ethanol administration to rats for 1, 3 or 7 weeks increased the amount of fractions which displayed an inhibitory effect. The observed changes were probably due to enhanced protein degradation and/or to decreased amino acid incorporation into proteins following ethanol administration. (Tab. 4, Fig. 2, Ref. 19.).

Effects of alloxan and ninhydrin on mitochondrial Ca2+ transport.

Alloxan at millimolar concentrations slightly inhibited the velocity of Ca2+ uptake by isolated rat liver mitochondria irrespective of the free Ca2+ concentration between 1 and 10 microM and was an effective concentration-dependent stimulator of mitochondrial Ca2+ efflux. Ninhydrin also slightly inhibited the velocity of mitochondrial Ca2+ uptake but only at free Ca2+ concentrations above 5 microM. However, ninhydrin was a strong stimulator of mitochondrial Ca2+ efflux even at micromolar concentrations, 10-50 times more potent than alloxan. The mitochondrial membrane potential was reduced 10-20% at most by alloxan and ninhydrin. Alloxan and ninhydrin also stimulated Ca2+ efflux from isolated permeabilized liver cells. When isolated intact liver cells had been pre-incubated with alloxan or ninhydrin before permeabilization of the cells the ability of spermine to induce mitochondrial Ca2+ uptake was abolished. Glucose provided the typical protection against the effects of alloxan on mitochondrial Ca2+ transport only in experiments with intact cells but not in experiments with permeabilized cells or isolated mitochondria. Therefore glucose protection is apparently due to inhibition of alloxan uptake into the cell. Glucose provided no protection against effects of ninhydrin under any of the experimental conditions. Thus both alloxan and ninhydrin are potent stimulators of Ca2+ efflux by isolated mitochondria but very weak inhibitors of the velocity of mitochondrial Ca2+ uptake. The direct effects of ninhydrin on mitochondrial Ca2+ efflux may contribute to the cytotoxic action of this agent whereas the direct effects of alloxan on mitochondrial Ca2+ transport require concentrations which are too high to be of relevance for the induction of the typical pancreatic B-cell toxic effects of alloxan. However, the effects on mitochondrial Ca2+ transport during incubation of intact cells which may result from the generation of cytotoxic intermediates during alloxan xenobiotic metabolism may well contribute to the pancreatic B-cell toxic effect of alloxan.

Inhibition of aconitase by alloxan and the differential modes of protection of glucose, 3-O-methylglucose, and mannoheptulose.

Alloxan inhibited aconitase with a half maximal inhibitory concentration of 0.5 mM in sonically disrupted and 2.3 mM in intact isolated liver mitochondria. For dialuric acid the half maximal inhibitory concentrations were 1.1 mM and 2.5 mM, respectively. Ninhydrin and N-ethylmaleimide (NEM) also inhibited aconitase with half maximal inhibitory concentrations in the submillimolar range and t-butylhydroperoxide (BuOOH) in the millimolar range, which, however, were not different for disrupted and intact mitochondria. Only the aconitase substrate citrate, but not glucose provided protection of the enzyme against inhibition. In intact liver cells the half maximal inhibitory concentration for alloxan was 6.8 mM. Again, dialuric acid and BuOOH were less potent inhibitors while ninhydrin and NEM were more potent inhibitors of aconitase in intact liver cells. In intact liver cells, glucose and 3-O-methylglucose, but not mannoheptulose and citrate provided protection against alloxan inhibition. The results show that aconitase is not an enzyme particularly sensitive towards alloxan inhibition and thus apparently not a primary site for mediation of alloxan toxicity as it is the glucokinase. This makes a primary site of alloxan action in the mitochondria extremely unlikely. On the other hand the results demonstrate that both the intact mitochondrial and plasma membrane as uptake barriers provide protection against alloxan toxicity. In addition the results clearly show, that 3-O-methylglucose provides protection against alloxan action only at the level of the plasma membrane through inhibition of alloxan uptake into the cell, while the site of protection of mannoheptulose is only the sugar binding site of the glucokinase. In contrast, glucose is shown here to be the only sugar with a dual protective effect both through inhibition of alloxan uptake through the plasma membrane like 3-O-methylglucose and through protection of the glucokinase sugar binding site against alloxan inhibition of the enzyme like mannoheptulose. In the light of these results the unique protective potency of glucose as compared to that of other sugars is not surprising.

Alloxan and ninhydrin inhibition of hexokinase from pancreatic islets and tumoural insulin-secreting cells.

Alloxan inhibited hexokinase activity in cytoplasmic fractions of transplantable radiation-induced rat islet cell tumours, ob/ob mouse pancreatic islets, rat liver and rat kidney. Half maximal inhibitory concentrations of alloxan were greater than those previously found for half maximal inhibition of pancreatic islet or liver glucokinase. D-glucose, preferentially the alpha-anomer, and D-mannose protected hexokinase activity against alloxan inhibition. 1,4-Dithiothreitol completely protected against and partially reversed the alloxan inhibition of hexokinase. The ability of various dithiols to reverse the inhibition of hexokinase by alloxan was dependent on the spacing between the SH (thiol) groups. Only dithiols with intermediate spacing between the SH groups were effective. Dithiols with two vicinal SH groups such as 1,2-dimercaptoethane and 2,3-dimercaptopropanol (BAL) and dithiols with more widely spaced SH groups such as 1,5-dimercaptopentane were ineffective. Thus a reaction of alloxan with two SH groups in the sugar binding site of the hexokinase with the formation of a disulfide bond may be involved in the reversible inhibition of the enzyme. Ninhydrin also inhibited hexokinase from all four tissues studied. The half maximal inhibitory concentrations of ninhydrin were lower than those of alloxan. Inhibition of hexokinase may be an important factor in the general cytotoxic action of ninhydrin. However, inhibition of pancreatic islet hexokinase is unlikely to be the initial event in the pancreatic B-cell toxic action of alloxan, even if inhibition of hexokinase by high concentrations of alloxan may contribute to the B-cell toxic action.(ABSTRACT TRUNCATED AT 250 WORDS)