Endogenous Alkaloids Research Articles

LC-MS/MS measurement of alkaloids in alkaline extracts of Areca nut preparations and their physiological effects

Betel quid (BQ) containing Piper betle leaf (L), green unripe Areca catechu nut (AN) and slaked lime (Ca(OH)2) is an addictive carcinogenic stimulant, with no pharmacotherapy, chewed by millions of people in Asia. We measured the effect of the constituents of slaked lime, NaOH and Ca(OH)2, on the levels of endogenous alkaloids in aqueous extracts of AN alone and L+AN (LAN) using UHPLC-MS/MS. Alkaloid levels in AN and LAN extracts were arecoline> >guvacoline>arecaidine>guvacine. In vitro hydrolyzation of extracts of AN and LAN by NaOH or Ca(OH)2, as occurs in vivo when BQ is chewed, dose-dependently decreased levels of muscarinic esters, arecoline (maximum 65%↓) and guvacoline (maximum 69%↓), and increased carboxylic acid GABA uptake inhibitors, arecaidine (maximum 275%↑) and guvacine (maximum 18%↑). In five volunteers, chewing BQ but not LAN, increased heart rate (maximum 19b/min), forehead temperature (maximum 1 °C), facial flushing and sweating, all symptoms of BQ stimulation. Hydrolysis of compounds in LAN is necessary to induce stimulant effects. Levels of arecoline, considered to mediate stimulant and addictive properties of BQ, negatively correlate with the physiological stimulant effects while levels of GABA uptake inhibitors positively correlate. Unripe, green AN warrants investigation as replacement therapy in the treatment of BQ substance use disorder.

Determination of 8 Endogenous Alkaloid Components inBoletusUsing Ultrahigh-Performance Liquid Chromatography Combined with Quadrupole-Time of Flight Mass Spectrometry

A ultrahigh performance liquid chromatography coupled with quadrupole-time of flight mass spectrometry (UPLC-Q-TOF/MS) method was developed for simultaneous determination of 8 endogenous alkaloid compounds inBoletus.Boletussamples were extracted by 50% (V/V) methanol-water solution, then separated by CORTECS UPLC HILIC column using a binary solvent system by gradient elution. The analytes were determined by Q-TOF/MS in TOF MS and information dependent acquisition (IDA)-MS/MS mode. The results showed that mass accuracy error of the 8 endogenous alkaloids were lower than 5.0 × 10−6, good linear relationship was got in range of 0.2–500 μg/L, and correlation coefficient was higher than 0.9990. The limit of detection was in the range of 0.002–0.100 mg/kg and the limit of quantification was in the range of 0.004–0.200 mg/kg. Recovery of the method was in range of 80.1%–101.5% with spike levels of 0.004–2.00 mg/kg, relative standard deviations were lower than 10%. The method was simple, specific, and reliable. It could be used for the rapid screening and quantitative analysis of 8 endogenous alkaloids inBoletus.

Origin of Epilachnapaenulata defensive alkaloids: Incorporation of [1-13C]-sodium acetate and [methyl-2H3]-stearic acid

Ladybird beetles produce a large number of defensive alkaloids. Previous studies suggest that the structural diversity of these endogenous alkaloids can be traced to a common biosynthetic route based on the condensation of several acetate units. In this study, adults of Epilachna paenulata, a phytophagous neotropical species, were fed on diet enriched with potential precursors (sodium acetate, fatty acids and the amino acids lysine and ornithine) labeled with stable isotopes (13C, 2H and 15N). Labeled acetate was incorporated into the structurally related homotropane and piperidine alkaloids. The later also showed incorporation of [methyl-2H3] stearic acid. Our results hence support a fatty acid pathway for the biosynthesis of E. paenulata alkaloids. To our knowledge, this is the first report on the incorporation of a labeled fatty acid into a defensive piperidine alkaloid in insects.

Mapping of endogenous morphine‐like compounds in the adult mouse brain: Evidence of their localization in astrocytes and GABAergic cells

Endogenous morphine, morphine-6-glucuronide, and codeine, which are structurally identical to vegetal alkaloids, can be synthesized by mammalian cells from dopamine. However, the role of brain endogenous morphine and its derivative compounds is a matter of debate, and knowledge about its distribution is lacking. In this study, by using a validated antibody, we describe a precise mapping of endogenous morphine-like compounds (morphine and/or its glucuronides and/or codeine) in the mouse brain. First, a mass spectrometry approach confirmed the presence of morphine and codeine in mouse brain, but also, of morphine-6-glucuronide and morphine-3-glucuronide representing two metabolites of morphine. Second, light microscopy allowed us to observe immunopositive cell somas and cytoplasmic processes throughout the mouse brain. Morphine-like immunoreactivity was present in various structures including the hippocampus, olfactory bulb, band of Broca, basal ganglia, and cerebellum. Third, by using confocal microscopy and immunofluroscence co-localization, we characterized cell types containing endogenous opiates. Interestingly, we observed that morphine-like immunoreactivity throughout the encephalon is mainly present in γ-aminobutyric acid (GABA)ergic neurons. Astrocytes were also labeled throughout the entire brain, in the cell body, in the cytoplasmic processes, and in astrocytic feet surrounding blood vessels. Finally, ultrastructural localization of morphine-like immunoreactivity was determined by electron microscopy and showed the presence of morphine-like label in presynaptic terminals in the cerebellum and postsynaptic terminals in the rest of the mouse brain. In conclusion, the presence of endogenous morphine-like compounds in brain regions not usually involved in pain modulation opens the exciting opportunity to extend the role and function of endogenous alkaloids far beyond their analgesic functions.

Abstract 938: Discovery of Dendrogenin A as the first endogenous alkylaminooxysterol present in mammals with potent cell differentiation and anticancer activity

Abstract We recently reported that anti-tumor and chemopreventive drugs such as tamoxifen, raloxifene and docasahexaenoic acid are inhibitors at therapeutic doses of cholesterol epoxide hydrolase (ChEH), the enzyme that transforms cholesterol-5,6-epoxides (CE) into cholestane-3β,5α,6β-triol (CT) (de Medina et al, PNAS, 2010). Several lines of evidence point to the existence of an active metabolism centered on CE. We recently reported that the aminolysis of α-CE by biogenic amines under catalytic conditions generated powerful cell differentiating alkylaminooxysterols (de Medina et al, J Med Chem, 2009). Among these active molecules, Dendrogenin A (DDA) was synthesized, based on the hypothesis that it could be an endogenous metabolite formed by the reaction of α-CE with histamine at the level of the ChEH. In the present study, we report the existence of DDA in mammals at concentrations ranging from 70 to 500 pmol/g in tissues and at concentrations 70 to 500 times less in the circulation, while only trace levels of AF17, the regio-isomer of DDA, were detected. DDA was not found in a panel of cancer cells, suggesting a possible deregulation of its synthesis during oncogenesis. In addition, we showed that DDA is the most potent natural inhibitor of ChEH with an IC50 around 100 nM and it exhibits tumor differentiation and anti-tumor activity in cell and animal models. In vivo, these effects were associated with a T cell-mediated anti-tumor response. AF17 was found to be inactive in these different tests, showing a regio-selectivity of action of these compounds. In conclusion, our results shed light on a new metabolic pathway that generates the first endogenous steroidal alkaloid ever described in mammals that may have important functions in maintaining cell integrity and differentiation as well as immune system alert. The discovery of DDA reveals an unexpected cross-talk between cholesterol and histamine metabolism. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 938. doi:10.1158/1538-7445.AM2011-938

Neurotoxic Effects of Tetrahydroisoquinolines and Underlying Mechanisms

Tetrahydropapaveroline (THP), a neurotoxic tetrahydroisoquinoline alkaloid formed by condensation between dopamine and dopaldehyde, has been speculated to cause Parkinson's disease and also to contribute to alcohol dependence. Having two catechol moieties, THP may readily undergo oxidation to form an o-quinone intermediate with concomitant production of reactive oxygen species, which can cause neuronal cell death and DNA damage. This review will deal with the current knowledge of neurotoxic effects of this endogenous alkaloid and underlying biochemical mechanisms.

ChemInform Abstract: Endogenous Alkaloids in Man. Part 9. Potential Tryptophan-Derived Alkaloids in Chloral-Treated Patients: Synthesis and Stereostructure.

ChemInform Abstract: Endogenous Alkaloids in Man. Part 9. Potential Tryptophan-Derived Alkaloids in Chloral-Treated Patients: Synthesis and Stereostructure.

ChemInform Abstract: Endogenous Alkaloids in Man. Part 33. Dimethyl (2S,4S)- and (2R,4S)-5,5-Dimethyl-1,3-thiazolidine-2,4-dicarboxylates, Two Diastereomeric Glyoxylate-Derived Heterocycles.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Abnormal Nociception and Opiate Sensitivity of STOP Null Mice Exhibiting Elevated Levels of the Endogenous Alkaloid Morphine

Background-Mice deficient for the stable tubule only peptide (STOP) display altered dopaminergic neurotransmission associated with severe behavioural defects including disorganized locomotor activity. Endogenous morphine, which is present in nervous tissues and synthesized from dopamine, may contribute to these behavioral alterations since it is thought to play a role in normal and pathological neurotransmission.Results-In this study, we showed that STOP null brain structures, including cortex, hippocampus, cerebellum and spinal cord, contain high endogenous morphine amounts. The presence of elevated levels of morphine was associated with the presence of a higher density of mu opioid receptor with a higher affinity for morphine in STOP null brains. Interestingly, STOP null mice exhibited significantly lower nociceptive thresholds to thermal and mechanical stimulations. They also had abnormal behavioural responses to the administration of exogenous morphine and naloxone. Low dose of morphine (1 mg/kg, i.p.) produced a significant mechanical antinociception in STOP null mice whereas it has no effect on wild-type mice. High concentration of naloxone (1 mg/kg) was pronociceptive for both mice strain, a lower concentration (0.1 mg/kg) was found to increase the mean mechanical nociceptive threshold only in the case of STOP null mice.Conclusions-Together, our data show that STOP null mice displayed elevated levels of endogenous morphine, as well as an increase of morphine receptor affinity and density in brain. This was correlated with hypernociception and impaired pharmacological sensitivity to mu opioid receptor ligands.

Pain, stress and relaxation: Involvement of basic biological principles and healthy autoregulation

Trauma and stress often involve the expression of pain. In the course of traumatic and stressful events, an ‘orchestra’ of neurobiological mechanisms and signaling molecules get activated with the primary goal of ensuring survival and fighting against the perturbating event, i.e., stressor. Pain can be a major process in this phenomenon, directing attention to the initial trigger situation. It is now known that pain processes also involve local and/or systemic immune as well as cardiovascular and neuronal pathways, including proinflammtory endogenous opioid peptides (e.g., endorphins) and down-regulatory opiate alkaloids (e.g., morphine). Usually, relaxation is a result of the systemic down-regulation that occurs after stress response pathways get shut-down, following their initial induction. As pain occurs in the primary phase of this process, it gets an inhibitory push-back under the influence of endorphins and other analgetic endogenous signaling molecules in this still early, i.e., stress response-associated stage. The rationale of this phase would be that the ‘stressed’ organism chiefly fights against the stressor and then, subsequently, cares about the possible wounds and traumas acquired. These secondary processes include endogenous morphine signaling, allowing the pain to come back again, yet in an altered and more ‘distant’ way, enabling the organism to face it and decide upon necessary actions for recovery and learning. This secondary phase also reduces over-stimulation, e.g., of the immune system, involving anti-inflammation. Hence, down-regulation serves recovery, i.e., systemic back-up. Stress induction and subsequent termination follow each other automatically and naturally, i.e., ideally, therefore activating, among others, endogenous limbic reward and motivation circuitries. However, little is known about the interplay between pain perception and its relationship with catecholamine molecules other than dopamine serving as an endogenous morphine precursor. We believe that the perception of pain and the body's self-attempt to alleviate it, utilizing conventional homeostatic mechanisms, is mediated by key catecholamines, and that this effect is further modulated by nitric oxide. We further propose a paradigm which biologically links pain, autoregulation, endogenous morphine and the catecholamines together, demonstrating a complex symbiotic signalling system. Integrative, complementary and mind–body medicine would, by their nature, include this paradigm in their underlying concepts and therapeutic strategies.

Improved accumulation of ajmalicine and tetrahydroalstonine in Catharanthus cells expressing an ABC transporter

The biosynthetic pathway of monoterpenoid indole alkaloids in Catharanthus roseus is located throughout various membranes at both the cellular and intercellular levels. ATP-binding cassette (ABC) transporters are known to export vincristine and vinblastine from human cancer cells. It has recently been shown that ABC transporters are also involved in the transport of various monoterpenoid alkaloids in Catharanthus roseus cells. Over-expression of an ABC transporter in this plant might therefore affect the regulation of the alkaloid biosynthetic pathway. CjMDR1, an ABC transporter gene originally isolated from Coptis japonica, was expressed in Catharanthus roseus cell cultures. Cells showing a positive PCR signal of the transgene in both cDNA and genomic DNA samples were subject to transport studies using selected substrates. Unexpectedly, transport of the isoquinoline alkaloid berberine, the main substrate of CjMDR1 transporter in Coptis japonica, was not affected as compared with control and wild-type Catharanthus cells. On the other hand, the endogenous alkaloids ajmalicine and tetrahydroalstonine were accumulated significantly more in Catharanthus roseus cells expressing CjMDR1 in comparison with control lines after feeding these alkaloids.

Endogenous Morphine in SH-SY5Y Cells and the Mouse Cerebellum

BackgroundMorphine, the principal active agent in opium, is not restricted to plants, but is also present in different animal tissues and cell types, including the mammalian brain. In fact, its biosynthetic pathway has been elucidated in a human neural cell line. These data suggest a role for morphine in brain physiology (e.g., neurotransmission), but this hypothesis remains a matter of debate. Recently, using the adrenal neuroendocrine chromaffin cell model, we have shown the presence of morphine-6-glucuronide (M6G) in secretory granules and their secretion products, leading us to propose that these endogenous alkaloids might represent new neuroendocrine factors. Here, we investigate the potential function of endogenous alkaloids in the central nervous system.Methodology and Principal FindingsMicroscopy, molecular biology, electrophysiology, and proteomic tools were applied to human neuroblastoma SH-SY5Y cells (i) to characterize morphine and M6G, and (ii) to demonstrate the presence of the UDP-glucuronyltransferase 2B7 enzyme, which is responsible for the formation of M6G from morphine. We show that morphine is secreted in response to nicotine stimulation via a Ca2+-dependent mechanism involving specific storage and release mechanisms. We also show that morphine and M6G at concentrations as low as 10−10 M are able to evoke specific naloxone-reversible membrane currents, indicating possible autocrine/paracrine regulation in SH-SY5Y cells. Microscopy and proteomic approaches were employed to detect and quantify endogenous morphine in the mouse brain. Morphine is present in the hippocampus, cortex, olfactory bulb, and cerebellum at concentration ranging from 1.45 to 7.5 pmol/g. In the cerebellum, morphine immunoreactivity is localized to GABA basket cells and their termini, which form close contacts on Purkinje cell bodies.Conclusions/SignificanceThe presence of morphine in the brain and its localization in particular areas lead us to conclude that it has a specific function in neuromodulation and/or neurotransmission. Furthermore, its presence in cerebellar basket cell termini suggests that morphine has signaling functions in Purkinje cells that remain to be discovered.

The endogenous alkaloid harmane: Acidifying and activity-reducing effects on hippocampal neurons in vitro

Rationale The endogenous alkaloid harmane is enriched in plasma of patients with neurodegenerative or addictive disorders. As harmane affects neuronal activity and viability and because both parameters are strongly influenced by intracellular pH (pH i), we tested whether effects of harmane are correlated with altered pH i regulation. Methods and results Pyramidal neurons in the CA3 field of hippocampal slices were investigated under bicarbonate-buffered conditions. Harmane (50 and 100 μM) reversibly decreased spontaneous firing of action potentials and caffeine-induced bursting of CA3 neurons. In parallel experiments, 50 and 100 μM harmane evoked a neuronal acidification of 0.12 ± 0.08 and 0.18 ± 0.07 pH units, respectively. Recovery from intracellular acidification subsequent to an ammonium prepulse was also impaired, suggesting an inhibition of transmembrane acid extrusion by harmane. Conclusion Harmane may modulate neuronal functions via altered pH i-regulation. Implications of these findings for neuronal survival are discussed.

Application of vanadate-induced nucleotide trapping to plant cells for detection of ABC proteins.

The vanadate-induced nucleotide trapping technique, which has been conventionally used to characterize mammalian ATP-binding cassette (ABC) proteins, was applied to berberine-producing plant cell cultures, Thalictrum minus and Coptis japonica. One membrane protein at ca. 180 kDa was photoaffinity-labeled with 8-azido-[alpha-(32)P]ATP in the T. minus cells in the presence of vanadate, which was specifically induced by the addition of benzyladenine in a similar manner as the induction of berberine biosynthesis in these cell cultures, whereas three bands were observed in the C. japonica cells in the size region between 120 and 150 kDa corresponding to full-sized ABC protein. The benzyladenine-induced band in T. minus showed properties similar to those of human MDR1, including the recognition of berberine, which suggests that the ABC protein detected in T. minus takes this endogenous alkaloid as a putative substrate for transport. This is the first application of this technique to plant cells.

Cytotoxicity of dopamine-derived tetrahydroisoquinolines on melanoma cells

Tetrahydroisoquinolines (TIQs) are endogenous alkaloid compounds detected in urine, central nervous system and some peripheral tissues in Mammalia. No data are at present available on TIQ levels in skin, although in vitro biochemical evidences indicate that they may undergo auto-oxidation with production of reactive oxygen species or may be enzymatically converted into melanin pigments. The effect of two catechol-bearing TIQs, salsolinol (SAL) and tetrahydropapaveroline (THP), on the viability of melanotic or amelanotic melanoma cell lines was investigated. Both SAL and THP were well tolerated up to roughly 30 μM and became overtly toxic at higher concentrations, with SAL being better tolerated than THP. Intracellular activity of some antioxidant enzymes, tyrosinase and α-ketoglutarate dehydrogenase was also evaluated to assess the cell response to oxidative and metabolic challenge of TIQs treatment. Catalase and superoxide dismutase pre-treatment only partially prevented TIQs toxicity while a complete protection was obtained with N-acetylcysteine and GSH. TIQs were able to provoke upregulation of the scavenging enzymes catalase and DT-diaphorase and to determine a decrease of the α-ketoglutarate dehydrogenase activity. SAL and THP enhanced tyrosinase activity and melanin production, suggesting that they were indeed tyrosinase substrates leading to melanin formation. The results support the evidence that TIQs were toxic toward melanoma cells, leading to their death by necrosis. TIQs toxicity was likely due to increased oxidative stress by generation of reactive oxygen species and oxidative metabolites. Our study represents an intent to furnish an additional contribution for the comprehension of catechol cytotoxicity.

Tetrahydroisoquinoline derivatives of enkephalins: synthesis and properties

Tetrahydroisoquinolines (TIQs) are endogenous alkaloid compounds deriving from the non-enzymatic Pictet–Spengler condensation of catecholamines with aldehydes. These compounds are able to unsettle catecholamines uptake and release from synaptosomes and have been detected in urine and in post-mortem Parkinsonian brains. We have obtained in vitro, by the reaction of dopa-enkephalin (dopa-Gly–Gly–Phe–Leu) with acetaldehyde in the presence of rameic ions, a TIQ derivative of Leu-enkephalin. The isolation and the recovery of the peptide was obtained by HPLC. The acid hydrolysis and the subsequent analysis of the peptide lysate by the Amino acid analyser clearly revealed the absence of dopa, while the electrospray ionisation mass spectrometry showed that the sequence of the enkephalin derivative was the following: 3-carboxy-salsolinol-Gly–Gly–Phe–Leu (TIQ-enkephalin). This compound was not a good substrate for microsomal aminopeptidase and pronase with respect to Leu-enkephalin. Tested in the binding assay, the TIQ-enkephalin exhibited a very poor affinity toward the enkephalin receptors. When the TIQ-enkephalin was incubated with tyrosinase or peroxidase/H 2O 2, the formation of TIQ-opio-melanins occurred.

Endogenous alkaloids in man: XXXVIII. “Chiral” and “achiral” determination of the neurotoxin TaClo (1-trichloromethyl-1,2,3,4-tetrahydro-β-carboline) from blood and urine samples by high-performance liquid chromatography–electrospray ionization tandem mass spectrometry

An improved sensitive assay for the determination of the dopaminergic and serotonergic neurotoxin 1-trichloromethyl-1,2,3,4-tetrahydro-β-carboline (TaClo) is presented, based upon on-line coupling of high-performance liquid chromatography with electrospray ionization tandem mass spectrometry (HPLC–ESI-MS–MS). Applying synthetic [D 4]TaClo as a fourfold deuterated internal standard, TaClo was detected and reliably quantified as a trace constituent of blood samples (0.5 up to 70 ng g −1 of clot) obtained from six patients orally treated with the hypnotic chloral hydrate. Unambiguous identification of this tricyclic “endogenous alkaloid” was achieved by selected reaction monitoring (SRM) experiments. The molecular ion peaks of TaClo, m/ z 289 (for [ 35Cl 3]TaClo) and m/ z 291 (for its [ 37Cl 35Cl 2]isotopomer), were both monitored to undergo a retro-Diels–Alder fragmentation by loss of a CH 2NH portion (−29 u) as typical of a tetrahydropyrido ring system of tetrahydro-β-carbolines. Detection of the resulting fragments, m/ z 260 and m/ z 262, with the expected statistical chlorine isotopic intensities of 100:96 confirmed the identity of the TaClo molecule. In addition, an enantiomer-specific device was developed for TaClo, by employing a chiral reversed-phase HPLC column in combination with circular dichroism (CD) spectroscopy and MS–MS analysis (LC–CD and LC–MS–MS coupling). In a human clot sample, both TaClo enantiomers were found in equimolar concentration (i.e., as a racemate) corroborating a spontaneous, non-enzymatic formation of TaClo from biogenic tryptamine and therapeutically administered chloral. In urine samples of TaClo-treated rats, by contrast, the ( S)-antipode was found to predominate, hinting at an enantiomer-differentiating metabolism of the compound.

Endogenous morphine and codeine: Possible role as endogenous anticonvulsants

Exogenously administered morphine can have both convulsive or anticonvulsive effects, depending on the dose and species. The levels of the endogenous opiate alkaloids morphine and codeine were significantly elevated in specific rat brain regions by the convulsive drug, pentylenetetrazole, as well as by the anticonvulsant drugs, carbamazepine and phenytoin. Morphine and codeine levels in peripheral tissues (heart, lung, spleen and adrenal) were unaffected by these drugs. Maximal increases in morphine levels were seen in the hypothalamus and striatum (2–10-fold), while lesser increases occurred in the midbrain and brain stem (2–4-fold). Codeine levels were also markedly increased in hypothalamus (5–10 fold), In contrast to morphine, codeine levels were also increased in the hippocampus (2–10-fold), but were unchanged in the striatum. These studies suggest that the endogenous alkaloids morphine and codeine are involved in the modulation of convulsions and that morphine and/or codeine may act as an endogenous anticonvulsant.

Endogenous Alkaloids in Man, Part 33. Dimethyl (2S,4S)-and (2R,4S)-5,5-Dimethyl-1,3-thiazolidine-2,4-dicarboxylates, Two Diastereomeric Glyoxylate-Derived Heterocycles

Both title compounds were obtained in stereochemically pure form by refluxing 5,5-dim ethyl-1 ,3-thiazolidine-2,4-dicarboxylic acid (3) with methanolic HCl, followed by a separation of the two diastereomers 4a and 4b on silica gel. The X-ray structure analyses of these stereoisomers confirm the relative configuration to be cis for 4a and trans for 4b with respect to the two carboxymethyl substituents at C(2) and C(4). For both molecules, an envelopetype conformation of the thiazolidine ring was found with C(5)-S(1)-C(2)-N(3) being located in a plane showing a small ring torsion angle of -11.2° and -3.1° for the cis- and the rrafts-epimer, respectively. The trans-configured compound 4b adopts a nearly ideal envelope conformation with C(4) being the flap atom, while the cis-isomer 4a. apparently because of steric interactions of the C(2) and C(4) substituents, shows a twisted envelope conformation.

Nicotiana glauca invertase: characterization and effects of endogenous alkaloids

An acid-soluble invertase from Nicotiana glauca has been studied for the first time. The plant was selected because it is known that the genus Nicotiana has vacuolar alkaloids; the same localization of the acid-soluble invertases in higher plants. The enzyme was partially purified and the optimum pH, Km, Mr, activation energy, fructose effect, activation by protein, sugar specificity, lectin activation were determined. The effects on invertase activity of the alkaloids, nicotine, anabasine and nornicotine present in the plant were also studied. These alkaloids produced a cumulative inhibition of the invertase activity. Other substances like TRIS hydrochloride, pyridoxal hydrochloride and pyridoxine hydrochloride also produced inhibition. All these substances have in common the presence of a quaternary nitrogen atom in their structure. The individual effect of these compounds on the invertase activity appears without meaning as regulators of the enzyme, but their general occurrence may have a role in the functional behaviour of the invertase.