Sensitivity of lysosomal enzymes to the plant alkaloid sanguinarine: comparison with other SH-specific agents

The influence of the benzo[c]phenanthridine alkaloid sanguinarine on some lysosomal enzyme activities was investigated. Sanguinarine inhibits lysosomal hydrolases in homogenates of cultured mouse fibroblasts. After incubation of mouse fibroblasts in culture with 100 microM sanguinarine an approximately 50% decrease in the activities of N-acetyl-beta,D-glucosaminidase (NAGA), beta-galactosidase (GAL), arylsulfatase and acid lipase was observed. Because the biological activity of sanguinarine might arise from the interaction of its iminium cation with enzyme thiol groups, we compared its effect on NAGA, GAL and acid phosphatase (AcP) activities with the effects of SH-specific reagents p-chloromercuribenzoic acid (CPMA) and N-ethylmaleimide (NEM). Treatment of lysosomal fractions with millimolar concentrations of sanguinarine induces a dose-dependent inhibition of the enzymes; for example, 0.6 mM sanguinarine causes approximately a 40% decrease in AcP and NAGA activities. NEM has similar effects, and increasing the preincubation temperature from 0 degrees C to 37 degrees C intensifies the inhibition due to both agents. CPMA also inhibits the activity of GAL (IC50 0.7 microM), AcP (IC50 12.5 microM) and NAGA (IC50 6.8 microM) in a dose-dependent manner but is more potent than sanguinarine or NEM. Comparative analysis of the primary structures of these enzymes using the program BLAST reveals the presence of highly conserved cysteine residues, which confirms the importance of thiol-groups for their activities. Thus, both the experimental observations obtained in this study and the literature data imply a significant role of redox-based mechanisms in regulating lysosomal functional activity.     

Detection of N-acetyl methionine in human and murine brain and neuronal and glial derived cell lines

Despite the fact that N-acetyl methionine (NAM) supplementation has long been reported as a bioavailable source of methionine in humans, and known to reduce liver toxicity after acetaminophen overdose, its cellular endogenous presence has never been investigated. We demonstrate for the first time that NAM is present in both human and mouse tissues and cells in culture. A wide variety of cultured cells, including a number of brain derived cell types, as well as mouse and human brain tissue all have clearly detectable levels of NAM. Methionine is rapidly acetylated to form NAM in cultured human oligodendroglioma cells with an initial rate of 0.44 ± 0.064 atom percent excess per minute. The presence of measurable quantities of NAM in brain cells in combination with its rapid formation point to a potential physiological role for N-acetylated methionine in the brain. Aminoacylase 1 is responsible for metabolism of NAM to methionine and acetate. Deficiencies in aminoacylase 1 have been linked to a variety of neurological disorders; however, it is unclear whether and how the brain is affected by this defect. The reported presence of NAM in the human brain may provide an invaluable key to discovering the link between aminoacylase 1 mutations and neurological problems.     

Isolation and identification of two novel SDS‐resistant secreted chitinases from Aeromonas schubertii

Two SDS‐resistant endochitinases, designated as ASCHI53 and ASCHI61, were isolated from Aeromonas schubertii in a soil sample from southern Taiwan. MALDI‐TOF mass measurement indicates the molecular weights of 53,527 for ASCHI53 and 61,202 for ASCHI61. N‐terminal and internal amino acid sequences were obtained, and BLAST analysis of the sequences and MS/MS peptide sequencing showed that they were novel proteins. Degradation of chitin by these two endochitinases gave rise to hexameric chitin oligosaccharide, a compound known to have several potent biomedical functions. ASCHI53 and ASCHI61 retained, respectively, 65% and 75%, of their chitinase activity in the presence of 5% SDS and 100% of their activity in the presence of 10% β‐mercaptoethanol. These results demonstrate that they are SDS‐resistant endochitinases and probably have a rigid structure. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Cloning,expression and biocharacterization of OfCht5, the chitinase from the insect Ostrinia furnacalis

Abstract Chitinase catalyzes β‐1,4‐glycosidic linkages in chitin and has attracted research interest due to it being a potential pesticide target and an enzymatic tool for preparation of N‐acetyl‐β‐D‐glucosamine. An individual insect contains multiple genes encoding chitinases, which vary in domain architectures, expression patterns, physiological roles and biochemical properties. Herein, OfCht5, the glycoside hydrolase family 18 chitinase from the widespread lepidopteran pest Ostrinia furnacalis, was cloned, expressed in the yeast Pichia pastoris and biochemically characterized in an attempt to facilitate both pest control and biomaterial preparation. Complementary DNA sequence analysis indicated that OfCHT5 consisted of an open reading frame of 1 665‐bp nucleotides. Phylogenic analysis suggested OfCht5 belongs to the Group I insect chitinases. Expression of OfCht5 in Pichia pastoris resulted in highest specific activity after 120 h of induction with methanol. Through two steps of purification, consisting of ammonium sulfate precipitation and metal chelating chromatography, about 7 mg of the recombinant OfCht5 was purified to homogeneity from 1 L culture supernatant. OfCht5 effectively converted colloidal chitin into chitobiose, but had relatively low activity toward α‐chitin. When chitooligosaccharides [(GlcNAc)_n, n= 3–6] were used as substrates, OfCht5 was observed to possess the highest catalytic efficiency parameter toward (GlcNAc)₄ and predominantely hydrolyzed the second glycosidic bond from the non‐reducing end. Together with β‐N‐acetyl‐D‐hexosaminidase OfHex1, OfCht5 achieved its highest efficiency in chitin degradation that yielded N‐acetyl‐β‐D‐glucosamine, a valuable pharmacological reagent and food supplement, within a molar concentration ratio of OfCht5 versus OfHex1 in the range of 9 : 1–15 : 1. This work provides an alternative to existing preparation of chitinase for pesticides and other applications.     

Isoforms of an N-acetyl-β-d-glucosaminidase from the Antarctic krill, Euphausia superba: purification and antibody production

Two forms of the chitinolytic enzyme N-acetyl-β--glucosaminidase (NAGase, EC 3.2.1.52) have been isolated from the Antarctic krill, Euphausia superba, in order to study their potential role in temperature adaptation processes. A chromatographic protocol was developed that allowed complete separation of the two enzyme forms, named NAGase B and NAGase C. The latter was purified to homogeneity with 600-fold enrichment and a yield of 17%. The molecular mass was 150 kDa. NAGase B showed characteristics of a glycoprotein due to affinity towards concanavalin A sepharose, while NAGase C did not. Highly specific polyclonal antibodies to NAGase C [anti-(E. superba-NAGase C)-IgG] showed only negligible cross-reactivity with NAGase B isoforms. A comparison with the Northern krill, Meganyctiphanes norvegica, revealed a corresponding chromatographic pattern with two main activity peaks, for differentiation named NAGase II and NAGase III. Application of the antibody on M. norvegica revealed a high specificity toward NAGase III and a low cross-reactivity with NAGase II. First indication is given that the two forms are no isoenzymes in a strict sense but instead may have different functions in the metabolism of krill.     

A cell‐survival factor (N‐acetyl‐L‐cysteine) alters the in vivo fate of constitutively proliferating subependymal cells in the adult forebrain

The adult mouse brain contains a population of constitutively proliferating subependymal cells that surround the lateral ventricle and are the direct progeny of the neural stem cell. Constitutively proliferating cells divide rapidly; 6 days after labeling, 60% of their progeny undergo cell death, 25% migrate to the olfactory bulbs, and 15% continue to proliferate within the subependyma. We have intraventricularly infused a cell survival factor N‐acetyl‐L ‐cysteine (NAC), which is known to have survival effects without concomitant proliferative effects on cells in vitro, and examined the resulting fate of cells spared from the normally occurring cell death. NAC infusion for 5 days results in a five‐fold increase in the number of retrovirally labeled subependymal cells compared to saline‐infused controls. The increase in the number of subependymal cells is directly proportional to the amount of time during which NAC is present and is not due to increased proliferation. While NAC is able to keep all the normally dying progeny alive, the cells spared from death remain confined to the subependyma lining the lateral ventricles and do not migrate to the olfactory bulbs (one normal fate of constitutively proliferating progeny) or into the surrounding brain parenchyma. When animals survive for an additional 6 days following NAC infusion, the number of retrovirally labeled subependymal cells returns to control values, indicating that the continued presence of NAC is necessary for cell survival. These data suggest that preventing cell death is not sufficient to keep all of the progeny of these cells in a proliferative mode. © 2000 John Wiley & Sons, Inc. J Neurobiol 42: 338–346, 2000     

Purification and characterization of an exo-type β-N-acetylglucosaminidase from Pseudomonas fluorescens JK-0412

Aims: To purify and characterize an exo‐acting chitinolytic enzyme produced from a Gram‐negative bacterium Pseudomonas fluorescens JK‐0412. Methods and Results: A chitinolytic bacterial strain that showed confluent growth on a minimal medium containing powder chitin as the sole carbon source was isolated and identified based on a 16S ribosomal DNA sequence analysis and named Ps. fluorescens JK‐0412. From the culture filtrates of this strain, a chito‐oligosaccharides‐degrading enzyme was purified to apparent homogeneity with a molecular mass of 50 kDa on SDS–PAGE gels. The kinetics, optimum pH and temperature, and substrate specificity of the purified enzyme (named as NagA) were determined. Conclusions: An extracellular chitinolytic enzyme was purified from the Ps. fluorescens JK‐0412 and shown to be an exo‐type β‐N‐acetylglucosaminidase yielding GlcNAc as the final product from the natural chito‐oligosaccharides, (GlcNAc)_n, n = 2–5. Significance and Impact of the Study: As NagA is secreted extracellularly in the presence of colloidal chitin, Ps. fluorescens JK‐0412 can be recognized as a potent producer for industry‐level and cost‐effective production of chitinolytic enzyme. This enzyme appears to have potential applications as an efficient tool for the degradation of chitinous materials and industry‐level production of GlcNAc. To the best of our knowledge, this is the first report on an exo‐type chitinolytic enzyme of Pseudomonas species.