Cell-associated Enzyme Research Articles

Molecular Docking Study of Binding of Perylene Di-imide to a Bio Molecular Human Telomeric G-quadruplex

Human telomeres are comprised of d(TTAGGG) repeats involved in the formation of G-quadruplex DNA structures. Ligands stabilizing these G-quadruplex DNA structures are potential inhibitors of the cancer cell-associated enzyme telomerase. In human cells , telomerase adds multiple copies of the 5’-GGTTAG-3’ motif to the end of the G-strand of the telomere and in the majority of tumor cells it results over-expressed. Several structural studies have revealed a diversity of topologies for telomeric quadruplexes, which are sensitive to the nature of the cations present, to the flanking sequences, and probably also to concentration, as confirmed by the different conformations deposited in the Protein Data Bank (PDB). The existence of different polymorphism in the DNA quadruplex and the absence of a uniquely precise binding site give rise to check docking approach . As target we have selected six different experimental models of the human telomeric sequence d[AG3(T2AG3)3] based on three G-tetrads and as ligands the perylene di-imide . We checked out molecular docking simulation of binding of perylene di-imide to a slected G-quadruplex using dock 6.9 to examine whether or not to reproduced the loop binding mode of perylene di-imide. The simulation gave the two highest rank docking pose of perylene di-imide and the binding mode were external stacking on the terminal guanine tetrade and the groove binding.

Silajdan Elde Edilen Lactobacillus sp. ve Lb. rhamnosus,’ un bazı Enzim Aktivitelerinin İncelenmesi

Probiotic lactic acid bacteria are vital microbial group for fermentation of silage, which is crucial importance particularly for dairy animal nutrition. These microorganisms are able to degrade amino acids, carbohydrates and fatty acids, our knowledge, however, about their other enzymatic activities is still limited. To investigate the inulinase and invertase activities of silage originated Lactobacillus sp and Lactobacillus rhamnosus, the isolates were incubated for 24h, 36h and 48h in LB broth and MRS broth. Supernatant and cell associated enzyme activities were determined at pH: 6.0 using inulin and sucrose as substrates, and their specific and total enzyme activities were analysed comparatively. Lactobacillus sp showed the highest invertase activity as 22.1 mg/ml (cell associated) for 24h incubation, whilst the utmost invertase activity was observed as 20 mg/ml at the end of 36h incubation period for supernatants. Same enzyme activities were determined as 20,1 mg/ml and 19,9 mg/ml for 24h incubation, for cell associated and supernatant samples of Lb rhamnosus respectively. The highest cell associated inulinase activity of Lactobacillus sp was recorded as 20,1 mg/ml for 36h incubation time while the maximal activity for supernatant was recorded at 24h incubation. Maximum cell associated and supernatant activities of the isolate was observed as 24,6 mg/ml and 26, mg/ml respectively. Both isolates showed noteworthy amount of invertase and inulinase activities which makes them potential silage inoculants.

Neuraminidase B controls neuraminidase A-dependent mucus production and evasion.

Binding of Streptococcus pneumoniae (Spn) to nasal mucus leads to entrapment and clearance via mucociliary activity during colonization. To identify Spn factors allowing for evasion of mucus binding, we used a solid-phase adherence assay with immobilized mucus of human and murine origin. Spn bound large mucus particles through interactions with carbohydrate moieties. Mutants lacking neuraminidase A (nanA) or neuraminidase B (nanB) showed increased mucus binding that correlated with diminished removal of terminal sialic acid residues on bound mucus. The non-additive activity of the two enzymes raised the question why Spn expresses two neuraminidases and suggested they function in the same pathway. Transcriptional analysis demonstrated expression of nanA depends on the enzymatic function of NanB. As transcription of nanA is increased in the presence of sialic acid, our findings suggest that sialic acid liberated from host glycoconjugates by the secreted enzyme NanB induces the expression of the cell-associated enzyme NanA. The absence of detectable mucus desialylation in the nanA mutant, in which NanB is still expressed, suggests that NanA is responsible for the bulk of the modification of host glycoconjugates. Thus, our studies describe a functional role for NanB in sialic acid sensing in the host. The contribution of the neuraminidases in vivo was then assessed in a murine model of colonization. Although mucus-binding mutants showed an early advantage, this was only observed in a competitive infection, suggesting a complex role of neuraminidases. Histologic examination of the upper respiratory tract demonstrated that Spn stimulates mucus production in a neuraminidase-dependent manner. Thus, an increase production of mucus containing secretions appears to be balanced, in vivo, by decreased mucus binding. We postulate that through the combined activity of its neuraminidases, Spn evades mucus binding and mucociliary clearance, which is needed to counter neuraminidase-mediated stimulation of mucus secretions.

Stabilizing displayed proteins on vegetative Bacillus subtilis cells.

Microbes engineered to display heterologous proteins could be useful biotechnological tools for protein engineering, lignocellulose degradation, biocatalysis, bioremediation, and biosensing. Bacillus subtilis is a promising host to display proteins, as this model Gram-positive bacterium is genetically tractable and already used industrially to produce enzymes. To gain insight into the factors that affect displayed protein stability and copy number, we systematically compared the ability of different protease-deficient B. subtilis strains (WB800, BRB07, BRB08, and BRB14) to display a Cel8A-LysM reporter protein in which the Clostridium thermocellum Cel8A endoglucanase is fused to LysM cell wall binding modules. Whole-cell cellulase measurements and fractionation experiments demonstrate that genetically eliminating extracytoplasmic bacterial proteases improves Cel8A-LysM display levels. However, upon entering stationary phase, for all protease-deficient strains, the amount of displayed reporter dramatically decreases, presumably as a result of cellular autolysis. This problem can be partially overcome by adding chemical protease inhibitors, which significantly increase protein display levels. We conclude that strain BRB08 is well suited for stably displaying our reporter protein, as genetic removal of its extracellular and cell wall-associated proteases leads to the highest levels of surface-accumulated Cel8A-LysM without causing secretion stress or impairing growth. A two-step procedure is presented that enables the construction of enzyme-coated vegetative B. subtilis cells that retain stable cell-associated enzyme activity for nearly 3days. The results of this work could aid the development of whole-cell display systems that have useful biotechnological applications.

Enhancing Gallic Acid Content in Green Tea Extract by Using Novel Cell-Associated Tannase of B acillus massiliensis

Gallic acid content in green tea extract was enhanced by using a novel cell-associated tannase of Bacillus massiliensis. Biomass that contains tannase was used for this study. The activity of the cell-associated tannase was stable during 1 week of storage in the refrigerator. Response surface methodology was applied based on central composite design to determine the effects of three independent variables (pH, temperature and incubation time) and their mutual interactions. A total of 16 experiments were conducted; and a statistical model was developed, which predicted 475.74 mg/L gallic acid production at pH 6.2, 36C and incubation period of 16.71 h. The subsequent verification experiments confirmed the validity of the model. Under optimal conditions, 84.7% of the total hydrolyzable tannins were converted to gallic acid and glucose. This naturally immobilized tannase was stable enough to be used for up to 12 runs. Practical Applications The current study shows that naturally immobilized tannase of Bacillus massiliensis can be used instead of artificially immobilized tannase. Such naturally immobilized tannase has many advantages as it avoids expensive and laborious isolation, purification and immobilization. Ease of separation of cell-associated enzyme from the reaction mixture and absence of any detectable extracellular tannase activity after enzymatic treatment are some of the encouraging facts. Stability during storage up to 7 days, 85% tannic acid hydrolyzing efficiency, activity at pH 3.5–8.0 and operational stability for 12 runs are some of the interesting features of this naturally immobilized enzyme. However, its application for tea treatment will be limited until Bacillus massiliensis gets “Generally Recognized As Safe” status. It can be employed, however, for production of gallic acid from agro residues and production of propyl gallate.

BEHAVIOR AND SUPPORTIVE EVIDENCE OF A LARGE XYLANASE-CONTAINING MULTIENZYME COMPLEX OF TEPIDIMICROBIUM XYLANILYTICUM BT14

Cellular behaviors and a xylanolytic-cellulolytic enzyme system of Tepidimicrobium xylanilyticum BT14 towards xylan-rich plant biomass degradation were characterized. During the exponential growth phase, the bacterial cells were bound tightly to the growth substrate where the degradation zones appeared mostly around the cells, indicating that the xylanolytic-cellulolytic enzyme system was linked to the cell surfaces. Interestingly, several cells appeared to secrete extracellular matrix to connect to their neighbors, and the matrix disappeared when cells passed to the stationary growth phase. Cationized-ferritin staining resulted in a dense assembly of bulbs, protuberance-like structures on the growing bacterial cell surfaces. The cell-associated proteins derived by sonication contained predominated xylanase with relatively low carboxymethyl-cellulase (CMCase) activities, suggesting that the xylanolytic-cellulolytic enzyme system occurred as a cell-associated enzyme. By means of gel-filtration chromatography, a high molecular mass protein with the estimated size of 2000 kDa was retrieved from the cell-associated enzymes, and it appeared as a single protein band on non-denaturing gel. However, more bands were obtained after the protein was boiled with sodium dodecyl sulfate and β-mercaptoethanol – which contained 4 xylanases and one CMCase – suggesting that these proteins were organized as a multienzyme complex (MEC) in natural form. Additionally, the predominated xylanolytic MEC preferred binding to cellulose rather than xylan.

The Polymorphisms of DNA G-Quadruplex Investigated by Docking Experiments with Telomestatin Enantiomers

Human telomeres are comprised of d(TTAGGG) repeats involved in the formation of G-quadruplex DNA structures. Ligands stabilizing these G-quadruplex DNA structures are potential inhibitors of the cancer cell-associated enzyme telomerase. In human cells, telomerase adds multiple copies of the 5'-GGTTAG-3' motif to the end of the G-strand of the telomere and in the majority of tumor cells it results over-expressed. Several structural studies have revealed a diversity of topologies for telomeric quadruplexes, which are sensitive to the nature of the cations present, to the flanking sequences, and probably also to concentration, as confirmed by the different conformations deposited in the Protein Data Bank (PDB). The existence of different polymorphisms in the DNA quadruplex and the absence of a uniquely precise binding site prompted us to carefully compare the two different docking approaches: MOLINE and Auto- Dock. As target we have selected six different experimental models of the human telomeric sequence d[AG3(T2AG3)3] based on three Gtetrads and as ligands the telomestatin isomers, whose the S enantomer is experimentally known to recognize the G-quadruplex better than the R one. In this communication we discuss the different binding modes of the well known strong telomestatin G-quadruplex binder form the thermodynamic and the geometrical points of view. With respect to this last issue we propose an easy approach to classify binding modes of G-quadruplex ligands based on a single angle descriptor as tool for the quick analysis of the binding modes.

Conformational studies and solvent-accessible surface area analysis of known selective DNA G-Quadruplex binders

Human telomeres are comprised of d(TTAGGG) repeats involved in the formation of G-quadruplex DNA structures. Ligands that stabilize these G-quadruplex DNA structures are potential inhibitors of the cancer cell-associated enzyme telomerase. In human cells, telomerase adds multiple copies of the 5′-GGTTAG-3′ motif to the end of the G-strand of the telomere and in the majority of tumor cells it results over-expressed. Several structural studies have revealed a diversity of topologies for telomeric quadruplexes, as confirmed by the different conformations deposited in the Protein Data Bank. In recent years an increasing number of chemically diverse telomerase inhibitors have been identified, including both natural and synthetic compounds. Thus telomerase has been regarded as one of the most attractive targets in cancer treatment. In this manuscript, with the aim to rationalize the different experimental activities of known telomerase inhibitors, a computational study was carried out to investigate their conformational properties and the relationships between the target affinity and the ligands solvent-accessible surface area. Among the analyzed different scaffolds of G-quadruplex binders, such a descriptor provided helpful preliminary information to discriminate end-stacking ligand binding affinities, revealing itself as a useful predictive tool in drug design and lead optimization processes.

Polysaccharidase and glycosidase production of avicel grown rumen fungusOrpinomycessp. GMLF5

Extracellular and cell-associated enzyme preparations were obtained from ruminal anaerobic fungi Orpinomyces sp. GMLF5 grown in culture containing microcrystalline cellulose (avicel) as sole energy source and degradation capacities of the preparations towards several polysaccharides and glycosides were studied. Fungus showed substantial increases in xylanase, carboxymethyl cellulase (CMCase), lichenase, amylase, beta-xylosidase, beta-glucosidase and alpha-L-arabinofuranosidase activities between 72 and 168 hours. High amounts of cell associated beta-xylosidase were noted in 4 and 5 days old cultures. Optimum temperature and pH of the polysaccharidases were found at 50 degrees C and 6.0-6.5, respectively. Xylanase was found to be virtually stable at 50 degrees C, CMCase and lichenase were stable at 40 degrees C for 200 min, however amylase was found more sensitive to heat treatment. The fibrolytic enzymes of the isolate GMLF5 were observed to be capable of hydrolyze the avicel.

Possible Roles of Phospholipase A 2 in the Biological Activities of Acanthamoeba castellanii (T4 Genotype)

Using phospholipases A(2)-specific spectrophotometric assays, it was shown thatA. castellaniilysates and their conditioned medium exhibit phospholipase activities. The extracellular levels of PLA(2)detected were significantly reduced compared with the cell-associated enzyme (P<0.05). Sphinganine, a PLA(2)inhibitor showed robust amoebistatic properties but had no effect on the viability ofA. castellanii. The potency of sphinganine was demonstrated effectively towards purified PLA(2)derived from porcine pancreas. Using sphinganine, it was observed that PLA(2)is involved in neither binding nor cytotoxicity of the human brain microvascular endothelial cells due toA. castellanii. Unlike as was the case forDictyosteliumamoebae, PLA(2)appeared to be involved inA. castellaniiphagocytosis of the fluorescently-labelled polystyrene beads. Horseradish peroxidase was used as a tracer molecule to develop assays to study pinocytosis inA. castellanii. The findings revealed that sphinganine impedes phagocytosis but augments pinocytosis inA. castellaniisuggesting distinct nature of processes. A complete understanding of the role of phospholipases in the biology and pathogenesis ofA. castellaniiinfections will determine their potential as therapeutic targets.

A modified spectrophotometric assay to estimate deglycosylation of steroidal saponin to sapogenin by mixed ruminal microbes

The lack of a method for measuring deglycosylation of saponins in ruminal fluid has limited the ability to investigate the impact of these compounds on rumen microorganisms. A simple spectrophotometric assay was adapted and a protocol developed to enable measurement of steroidal saponin and sapogenin in ruminal fluid. The procedure was used for in vitro determination of deglycosylation activity of rumen bacteria obtained from cattle fed or not fed Yucca schidigera saponin, and to determine the relative deglycosylase activities of extracellular and cell-associated enzymes from ruminal content. Modifications to the spectrophotometric assay (i.e. heating time shortened to 10 min and 0.5 mL dH(2)O added to the reaction mixture) improved the stability of the optical density (425 nm) of the chromophore for up to 24 h post-reaction. Centrifugation (12 000 x g, 20 min) enabled differential estimations of steroidal saponin and sapogenin in ruminal fluid. Steroidal saponin added to defaunated ruminal fluid (dRF) or clarified ruminal fluid (cRF) was recovered completely from the mixture as saponin + sapogenin (99.1% and 100.6%, respectively), whereas saponin recovery from the supernatant of dRF was greatly reduced (P < 0.001) compared to that from supernatant of cRF (58.5 vs. 98.7%). Saponin recoveries from the supernatants of dRF and cRF did not differ between donor cattle fed or not fed Yucca schidigera saponin (59.2 vs. 57.3% and 98.4 vs. 99.3%, respectively). The majority (89-90%) of saponin added to a ruminal extracellular enzyme preparation was recoverable in supernatant after 24 h, compared with only 26-32% remaining in supernatant from incubation with a cell-associated enzymes fraction. Mixed rumen bacteria deglycosylate steroidal saponin to sapogenin, at activity levels unaffected by prior exposure to saponin, but they were unable to degrade the sapogenin core structure. Deglycosylation activity occurred primarily in the cell-associated enzyme fraction of ruminal content.

Cell-associated β-xylosidase from Aureobasidium pullulans ATCC 20524: Purification, properties, and characterization of the encoding gene

A cell-associated enzyme exhibiting beta-xylosidase activity was purified from the cell extract of the dimorphic fungus, Aureobasidium pullulans strain ATCC 20524, grown on oat spelt xylan. The purified enzyme was homogeneous as judged by SDS-PAGE, which showed an apparent M(r) of 88.5 kDa. beta-Xylosidase activity was optimal at pH 3.5 and 70 degrees C. The enzyme exhibited apparent K(m) and V(max) values of 3.5 mM and 263 micromol/mg/min, respectively, for p-nitrophenyl-beta-d-xylopyranoside. The enzyme also showed some alpha-l-arabinofuranosidase activity. Southern blot analysis indicated that the beta-xylosidase gene (xylI) was present as a single copy in the genome. The genomic DNA and cDNA encoding this protein were cloned and sequenced. The open reading frame of 2415 bp was interrupted by two introns of 54 and 52 bp, and it encoded a presumed signal peptide of 20 residues and a mature protein of 785 residues with a calculated M(r) of 85,045 Da and a deduced isoelectric point of 4.57. The protein was N-glycosylated and possessed 16 potential N-glycosylation sites. Two distinct transcription start points of the xylI gene were present at nt -32 (A) and -26 (A) from the start codon. The xylI cDNA was functionally expressed in the yeast Pichia pastoris. The deduced amino acid sequence of the xylI gene product was 49% identical to the Talaromyces emersonii beta-xylosidase Bxl1, which belongs to the glycoside hydrolase family 3.

Characterization of a unique class C acid phosphatase from Clostridium perfringens.

Clostridium perfringens is a gram-positive anaerobe and a pathogen of medical importance. The detection of acid phosphatase activity is a powerful diagnostic indicator of the presence of C. perfringens among anaerobic isolates; however, characterization of the enzyme has not previously been reported. Provided here are details of the characterization of a soluble recombinant form of this cell-associated enzyme. The denatured enzyme was approximately 31 kDa and a homodimer in solution. It catalyzed the hydrolysis of several substrates, including para-nitrophenyl phosphate, 4-methylumbelliferyl phosphate, and 3' and 5' nucleoside monophosphates at pH 6. Calculated K(m)s ranged from 0.2 to 0.6 mM with maximum velocity ranging from 0.8 to 1.6 micromol of P(i)/s/mg. Activity was enhanced in the presence of some divalent cations but diminished in the presence of others. Wild-type enzyme was detected in all clinical C. perfringens isolates tested and found to be cell associated. The described enzyme belongs to nonspecific acid phosphatase class C but is devoid of lipid modification commonly attributed to this class.

Establishment of immortalized Schwann cells from Fabry mice and their low uptake of recombinant α-galactosidase

Peripheral neuropathy is one of the important manifestations of Fabry disease. Enzyme replacement therapy with presently available recombinant alpha-galactosidases does not always improve the Fabry neuropathy. But the reason has not been determined yet. We established a Schwann cell line from Fabry mice, characterized it, and then examined the uptake of alpha-galactosidase by cells and its effect on the degradation of accumulated substrate. The cells exhibited a distinct Schwann cell morphology and biochemical phenotype (alpha-Galactosidase activity was deficient, and numerous cytoplasmic inclusion bodies were present in the cells). A recombinant alpha-galactosidase added to the culture medium was incorporated into the cultured Fabry Schwann cells dose dependently. But the increase in cell-associated enzyme activity was less than that in the cases of human and mouse Fabry fibroblasts. The administration of a high dose of the enzyme improved the pathological changes in cells, although a low dose of it did not. Cellular uptake of the enzyme was strongly inhibited in the presence of mannose 6-phosphate. This suggests that the enzyme is incorporated via cation-independent mannose 6-phosphate receptors in Schwann cells. The low expression of cation-independent mannose 6-phosphate receptors in Schwann cells must be one of the reasons their uptake of the present enzymes was low. The administration of a high dose of the enzyme or the development of an enzyme containing many mannose 6-phosphate residues is required to improve Fabry neuropathy.

Differential expression of sialylglycoconjugates and sialidase activity in distinct morphological stages of Fonsecaea pedrosoi.

The expression of sialoglycoconjugates in Fonsecaea pedrosoi conidia, mycelia, and sclerotic cells was analyzed using influenza A and C virus strains, sialidase treatment, and lectin binding. Conidium and mycelium whole cells were recognized by Limax flavus (LFA), Maackia amurensis (MAA), and Sambucus nigra (SNA) lectins, denoting the presence of surface sialoglycoconjugates containing alpha 2,3- and alpha 2,6-sialylgalactosyl sequences. Sialidase-treated conidia reacted more intensively with peanut agglutinin (PNA), confirming the occurrence of sialyl-galactosyl linkages. Conidial cells agglutinated in the presence of influenza A and C virus strains, which confirmed the results obtained from lectin-binding experiments and revealed the presence of sialoglycoconjugates bearing 9-O-acetyl-N-acetylneuraminic acid (Neu5,9Ac(2)) surface structures. Western blotting analysis with peroxidase-labeled LFA demonstrated the occurrence of sialylglycoproteins in protein extracts from conidia and mycelia, with molecular masses corresponding to 56 and 40 kDa. An additional band of 77 kDa was detected in conidial extracts, suggesting an association between sialic acid expression and morphogenesis. Synthesis of sialic acids was correlated with sialidase expression, since both conidial and mycelial morphological stages presented secreted and cell-associated enzyme activity. Sialoglycoconjugates were not detected in F. pedrosoi sclerotic cells from in vitro and in vivo sources, which also do not express sialidase activity. The surface sialyl residues in F. pedrosoi are apparently involved in the fungal interaction with immune effector cells, since sialidase-treated conidia were less resistant to phagocytosis by human neutrophils from healthy individuals. These findings suggest that sialic acid expression in F. pedrosoi varies according to the morphological transition and may protect infecting propagules against immune destruction by host cells.

The relationship between ligand aggregation and G-quadruplex DNA selectivity in a series of 3,4,9,10-perylenetetracarboxylic acid diimides.

Human telomeres are comprised of d(TTAGGG) repeats that are capable of forming G-quadruplex DNA structures. Ligands that bind to and stabilize these G-quadruplex DNA structures are potential inhibitors of the cancer cell-associated enzyme telomerase. Other potential biological uses of G-quadruplex targeting ligands have been proposed. One particularly challenging aspect of the contemplated uses of G-quadruplex targeting ligands is their selectivity for G-quadruplex DNA versus double-stranded DNA structures. We have previously reported the observation that two structurally related 3,4,9,10-perylenetetracarboxylic acid diimide-based G-quadruplex DNA ligands, PIPER [N,N'-bis(2-(1-piperidino)ethyl)-3,4,9,10-perylenetetracarboxylic acid diimide] and Tel01 [N,N'-bis(3-(4-morpholino)propyl)-3,4,9,10-perylenetetracarboxylic acid diimide], have different levels of G-quadruplex DNA binding selectivity at pH 7 as determined by absorbance changes in the presence of different DNA structures [Kerwin, S. M., Chen, G., Kern, J. T., and Thomas, P. W. (2002) Bioorg. Med. Chem. Lett. 12, 447-450]. Here we report that the less G-quadruplex DNA selective ligand PIPER can unwind double-stranded, closed circular plasmid DNA, as determined by a topoisomerase I assay. A model for the interaction of Tel01 with the G-quadruplex DNA structure formed by d(TAGGGTTA) was determined from NMR experiments. This model is similar to the previously published model for PIPER bound to the same G-quadruplex DNA and failed to provide a structural basis for the observed increased selectivity of Tel01 interaction with G-quadruplex DNA. In contrast, investigation into the aggregation state of Tel01 and PIPER as well as other 3,4,9,10-perylenetetracarboxylic acid diimide analogues bearing basic side chains demonstrates that ligand aggregation is correlated with G-quadruplex DNA binding selectivity. For all six analogues examined, those ligands that were aggregated at pH 7 in 70 mM potassium phosphate, 100 mM KCl, 1 mM EDTA buffer also demonstrated G-quadruplex DNA binding selectivity under these buffer conditions. Ligands that were not aggregated under these conditions display much lower levels of G-quadruplex DNA selectivity. The aggregation state of these ligands is extremely sensitive to the buffer pH. Tel01, which is aggregated at pH 7, is not aggregated at pH 6.4, where it demonstrates only modest G-quadruplex DNA binding selectivity, and PIPER in pH 8.5 buffer is both aggregated and highly G-quadruplex DNA-selective. To our knowledge, these studies demonstrate the first DNA structure selectivity as achieved through pH-mediated ligand aggregation. The potential impact of these findings on the selectivity of other classes of G-quadruplex DNA ligands is discussed.

N-linked glycosylation of macrophage-derived PAF-AH is a major determinant of enzyme association with plasma HDL

Human plasma PAF-AH (platelet-activating factor-acetylhydrolase) is a Ca2+-independent phospholipase A2 of hematopoietic origin associated with LDL and HDL; it degrades PAF and oxidizes phospholipids. We show that human macrophages synthesize PAF-AH as a premedial Golgi precursor containing high mannose N-linked glycans. Secreted PAF-AH possesses a molecular mass of ~55 kDa and contains mature N-linked glycans. Secreted PAF-AH activity (90 ± 4% of the total) bound to a wheat germ lectin column and could be eluted with N-acetylglucosamine, whereas digestion with N-acetylneuraminidase II completely abolished enzyme absorption. Tunicamycin significantly reduced cell-associated PAF-AH activity and inhibited enzyme secretion; but it did not alter the ratio of secreted to cell-associated enzyme (1.8 at 6 h and 3.1 at 24 h), suggesting that glycosylation is not essential for PAF-AH secretion. Digestion of cell-associated PAF-AH or secreted PAF-AH with peptide N-glycosidase F affected neither catalytic activity nor its resistance to proteolysis with trypsin or proteinase K; in addition, it did not affect PAF-AH association with LDL, but significantly increased its association with HDL.We suggest that macrophage-derived PAF-AH contains heterogeneous asparagine-conjugated sugar chain(s) involving sialic acid, which hinders its association with HDL but does not influence the secretion, catalytic activity, or resistance of PAF-AH to proteases.

Platelet-associated and secreted PAF-acetylhydrolase activity in patients with stable angina: sequential changes of the enzyme activity after angioplasty.

Platelet-activating factor (PAF), the lipid mediator of inflammation and potent platelet agonist, can be hydrolysed and inactivated by PAF-acetylhydrolase (PAF-AH). We investigated the PAF-AH activity in relation to PAF formation in platelets from patients with stable angina undergoing elective percutaneous transluminal coronary angioplasty (PTCA). Twenty-seven patients with stable angina, undergoing PTCA, and 30 age- and sex-matched controls were studied. The platelet-associated and secreted PAF-AH activity was measured, before PTCA, as well as at 4 h, 48 h and 6 months afterwards. PAF formation by thrombin-stimulated platelets and the platelet aggregation responses to PAF and ADP were also determined. The PAF-AH activity secreted by thrombin-stimulated platelets before PTCA (in pmol/10(9) cells/h) was significantly higher compared to controls (892 +/- 222 vs. 624 +/- 144, P < 0.001). The enzyme activity was not altered at 4 h after PTCA, but was significantly increased at 48 h (1284 +/- 312, P < 0.005) to return to the levels observed in the control group 6 months afterwards. Detectable levels of PAF in thrombin-stimulated platelets were found only at 6 months after PTCA. Furthermore, the cell-associated enzyme activity in resting platelets before PTCA was significantly lower compared with controls. Unlike in controls, the platelet-associated enzyme activity in the patient group was not increased after stimulation with thrombin and it was associated by a platelet hyperaggregability to PAF. Both the intact cell-associated activity and the platelet hyper-reactivity to PAF were restored at 6 months after PTCA. Alterations in the platelet PAF-AH activity, which affect the PAF formation in thrombin-stimulated platelets and are associated by an increased aggregatory response to PAF, are observed in patients with stable angina and are completely restored after PTCA.

Environmental regulation of glycosidase and peptidase production by Streptococcus gordonii FSS2.

The synthesis of cell-associated and secreted proteins by Streptococcus gordonii FSS2, an infective endocarditis (IE) isolate, was influenced by both environmental pH and carbon source. Controlling the pH at 7.5 in stirred batch cultures showed that cell-associated and secreted protein concentrations were increased during late exponential and stationary phase by 68% and 125%, respectively, compared with similar cultures without pH control. The expression of five glycosidase and eight peptidase activities were examined using fluorogen-labelled synthetic substrates. Enzyme activities were significantly down-regulated during exponential growth, increasing during stationary phase (P<0.01) whether the culture pH was controlled at pH 7.5 or allowed to fall naturally to pH 4.4. Culture-supernatant activities were significantly increased (P<0.05) when the pH was maintained at 6.0 or 7.5, indicating modulation of enzyme activity by pH. Growth under nitrogen-limitation/glucose-excess conditions resulted in a significant repression of cell-associated glycosidase activities (P<0.01), whilst in the supernatant, activities were generally reduced. The expression of peptidase activities in the culture supernatant did not significantly change. The results suggest a possible role for catabolite repression by glucose in regulating enzyme expression. When S. gordonii FSS2 was cultured with 50% (v/v) added heat-inactivated foetal bovine serum, several cell-associated enzyme activities increased initially but were then reduced as the culture time was extended to 116 h. Culture-supernatant enzyme activities (N-acetyl-beta-D-glucosaminidase, N-acetyl-beta-D-galactosaminidase, thrombin, Hageman factor, collagenase and chymotrypsin), however, were significantly increased (P<0.01) over the same time period. The findings indicated that most of the important glycosidases synthesized by S. gordonii FSS2 were down-regulated by acid growth conditions and may also be subject to catabolite repression by glucose but conversely may be up-regulated by growth in serum. These results may have implications for streptococcal growth in an IE vegetation and in the mouth between meals or during sleep.

Immobilization of Cell-Associated Enzyme by Entrapping in Gluten Matrix

Gluten is advantageous for use as a new base material for cell and enzyme immobilization because it is biodegradable, cheaper than other natural polymers and readily available. In this work, a procedure for immobilization of cell-associated enzymes by entrapping within gluten matrices was developed. According to this method, cells of E. coli containing penicillin G acylase were mixed with gluten solution during the formation of gel, and the resultant gel was hardened by the addition of oxidized starch or glutaraldehyde. Scanning electron micrographs of the cell-immobilized preparations indicated that they were porous and the pore size decreased with increasing the dosage of cross-linking agent. Oxidized starch was superior to glutaraldehyde as the crosslinker, since the gel matrices hardened by the former were more accessible to the substrate and less harmful to the enzyme. The immobilized preparations in the form of either a single sheet or small pieces containing a biomass concentration up to 10%, w/w were effective for catalyzing the hydrolysis of penicillin G. Neither the optimal temperature nor optimal pH for the activity of cell-associated enzymes was changed by immobilization. In order to establish a membrane reactor for large-scale production, a stainless steel net could be used as the support for formulating a cell-immobilized gluten sheet.