Purification and characterization of an extracellular lipase from a thermophilic Rhizopus oryzae strain isolated from palm fruit

We have isolated a lipolytic strain from palm fruit that was identified as a Rhizopus oryzae. Culture conditions were optimized and highest lipase production amounting to 120 U/ml was achieved after 4 days of cultivation. The extracellular lipase was purified 1200-fold by ammonium sulfate precipitation, sulphopropyl-Sepharose chromatography, Sephadex G 75 gel filtration and a second sulphopropyl-Sepharose chromatography. The specific activity of the purified enzyme was 8800 U/mg. The lipolytic enzyme has a molecular mass of 32 kDa by SDS-polyacrylamide gel electrophoresis and gel filtration. The enzyme exhibited a single band in active polyacrylamide gel electrophoresis and its isoelectric point was 7.6. Analysis of Rhizopus oryzae lipase by RP-HPLC confirmed the homogeneity of the enzyme preparation. Determination of the N-terminal sequence over 19 amino acid residues showed a high homology with lipases of the same genus. The optimum pH for enzyme activity was 7.5. Lipase was stable in the pH range from 4.5 to 7.5. The optimum temperature for lipase activity was 35 degrees C and about 65% of its activity was retained after incubation at 45 degrees C for 30 min. The lipolytic enzyme was inhibited by Triton X100, SDS, and metal ions such as Fe(3+), Cu(2+), Hg(2+) and Fe(2+). Lipase activity against triolein was enhanced by sodium cholate or taurocholate. The purified lipase had a preference for the hydrolysis of saturated fatty acid chains (C(8)-C(18)) and a 1, 3-position specificity. It showed a good stability in organic solvents and especially in long chain-fatty alcohol. The enzyme poorly hydrolyzed triacylglycerols containing n-3 polyunsaturated fatty acids, and appeared as a suitable biocatalyst for selective esterification of sardine free fatty acids with hexanol as substrate. About 76% of sardine free fatty acids were esterified after 30 h reaction whereas 90% of docosahexaenoic acid (DHA) was recovered in the unesterified fatty acids.     

Proteomic characterization of lipid rafts markers from the rat intestinal brush border

To assess intestinal lipid rafts functions through the characterization of their protein markers, we have isolated lipid rafts of rat mucosa either from the total membrane or purified brush-border membrane (BBM) by sucrose gradient fractionation after detergent treatment. In both membrane preparations, the floating fractions (4-5) were enriched in cholesterol, ganglioside GM1, and N aminopeptidase (NAP) known as intestinal lipid rafts markers. Based on MALDI-TOF/MS identification and simultaneous detection by immunoblotting, 12 proteins from BBM cleared from contaminants were selected as rafts markers. These proteins include several signaling/trafficking proteins belonging to the G protein family and the annexins as well as GPI-anchored proteins. Remarkably GP2, previously described as the pancreatic granule GPI-anchored protein, was found in intestinal lipid rafts. The proteomic strategy assayed on the intestine leads to the characterization of known (NAP, alkaline phosphatase, dipeptidyl aminopeptidase, annexin II, and galectin-4) and new (GP2, annexin IV, XIIIb, Galpha(q), Galpha(11), glutamate receptor, and GPCR 7) lipid rafts markers. Together our results indicate that some digestive enzymes, trafficking and signaling proteins may be functionally distributed in the intestine lipid rafts.     

Isolation and characterization of an extracellular lipase from Mucor sp strain isolated from palm fruit

During our screening of lipolytic fungus which may play a role in the acidification of palm oil, we have recently isolated a Mucor sp strain. Culture conditions were optimized and the highest lipase production amounting to 57 U/ml was achieved after 6 days of cultivation. The extracellular lipase was purified 1050-fold by ammonium sulfate precipitation, carboxymethyl–sephadex chromatography and Sephadex G75 gel filtration to a final specific activity of 6600 IU/mg. The molecular weight of the homogenous lipase was determined about 42 kDa by gel filtration and SDS–polyacrylamide gel electrophoresis. The purified lipase was determined as a glycoprotein with a pI of 6.2. The Nt sequence was determined as AspGluIleGluThrValGlyXPheThrMetAspLeuProProAsnProPro and showed no homology with the sequences of the known lipases suggesting that the enzyme may be a new lipase. The purified lipase hydrolyzed both synthetic and natural triglycerides with the optimal activity recorded on trioctanoin and sunflower oil, respectively. Its activity was strongly inhibited by Triton X-100 and SDS. Metal ions such as Fe³⁺, Fe²⁺ and Hg²⁺ also decreased the lipase activity.     

The feeding preference of mycophagous Collembola varies with the ectomycorrhizal symbiont

The interactions of the collembolan insect Proisotoma minuta with ectomycorrhizal and/or pathogenic fungi was examined in three experiments: (1) in vitro analysis of feeding patterns, (2) in vitro food preference test, and (3) in situ analysis of ectomycorrhizal colonization in relation to population density. The ectomycorrhizal fungi Laccaria laccata, Pisolithus tinctorius, Suillus luteus, Thelephora terrestris and the pathogenic fungi Rhizoctonia solani were employed in all experiments. In vitro and in situ experiments revealed that Pr. minuta consumed all the ectomycorrhizal fungi tested but the feeding pattern and consumption varied with each isolate. In a comparative in vitro feeding preference test, where Pr. minuta was given a choice, R. solani was grazed more heavily than the ectomycorrhizal fungi. Among the ectomycorrhizal fungi examined, Pi. tinctorius was consumed significantly less than L. laccata, S. luteus or T. terrestris in the presence of R. solani. A 10-week in situ analysis of loblolly pine (Pinus taeda L.) seedling root systems inoculated with Pr. minuta revealed that ectomycorrhizal colonization was significantly less than that of control plants (without Pr. minuta). Collectively, these data suggest that mycophagous Collembola may play a major role in the distribution and biomass of ectomycorrhizal fungi in the rhizosphere of tree seedlings.     

Palmitoylation regulates regulator of G-protein signaling (RGS) 16 function. II. Palmitoylation of a cysteine residue in the RGS box is critical for RGS16 GTPase accelerating activity and regulation of Gi-coupled signalling

Palmitoylation is a reversible post-translational modification used by cells to regulate protein activity. The regulator of G-protein signaling (RGS) proteins RGS4 and RGS16 share conserved cysteine (Cys) residues that undergo palmitoylation. In the accompanying article (Hiol, A., Davey, P. C., Osterhout, J. L., Waheed, A. A., Fischer, E. R., Chen, C. K., Milligan, G., Druey, K. M., and Jones, T. L. Z. (2003) J. Biol. Chem. 278, 19301-19308), we determined that mutation of NH2-terminal cysteine residues in RGS16 (Cys-2 and Cys-12) reduced GTPase accelerating (GAP) activity toward a 5-hydroxytryptamine (5-HT1A)/G alpha o1 receptor fusion protein in cell membranes. NH2-terminal acylation also permitted palmitoylation of a cysteine residue in the RGS box of RGS16 (Cys-98). Here we investigated the role of internal palmitoylation in RGS16 localization and GAP activity. Mutation of RGS16 Cys-98 or RGS4 Cys-95 to alanine reduced GAP activity on the 5-HT1A/G alpha o1 fusion protein and regulation of adenylyl cyclase inhibition. The C98A mutation had no effect on RGS16 localization or GAP activity toward purified G-protein alpha subunits. Enzymatic palmitoylation of RGS16 resulted in internal palmitoylation on residue Cys-98. Palmitoylated RGS16 or RGS4 WT but not C98A or C95A preincubated with membranes expressing 5-HT1a/G alpha o1 displayed increased GAP activity over time. These results suggest that palmitoylation of a Cys residue in the RGS box is critical for RGS16 and RGS4 GAP activity and their ability to regulate Gi-coupled signaling in mammalian cells.     

Palmitoylation regulates regulators of G-protein signaling (RGS) 16 function. I. Mutation of amino-terminal cysteine residues on RGS16 prevents its targeting to lipid rafts and palmitoylation of an internal cysteine residue

Regulators of G-protein signaling (RGS) proteins down-regulate signaling by heterotrimeric G-proteins by accelerating GTP hydrolysis on the G alpha subunits. Palmitoylation, the reversible addition of palmitate to cysteine residues, occurs on several RGS proteins and is critical for their activity. For RGS16, mutation of Cys-2 and Cys-12 blocks its incorporation of [3H]palmitate and ability to turn-off Gi and Gq signaling and significantly inhibited its GTPase activating protein activity toward aG alpha subunit fused to the 5-hydroxytryptamine receptor 1A, but did not reduce its plasma membrane localization based on cell fractionation studies and immunoelectron microscopy. Palmitoylation can target proteins, including many signaling proteins, to membrane microdomains, called lipid rafts. A subpopulation of endogenous RGS16 in rat liver membranes and overexpressed RGS16 in COS cells, but not the nonpalmitoylated cysteine mutant of RGS16, localized to lipid rafts. However, disruption of lipid rafts by treatment with methyl-beta-cyclodextrin did not decrease the GTPase activating protein activity of RGS16. The lipid raft fractions were enriched in protein acyltransferase activity, and RGS16 incorporated [3H]palmitate into a peptide fragment containing Cys-98, a highly conserved cysteine within the RGS box. These results suggest that the amino-terminal palmitoylation of an RGS protein promotes its lipid raft targeting that allows palmitoylation of a poorly accessible cysteine residue that we show in the accompanying article (Osterhout, J. L., Waheed, A. A., Hiol, A., Ward, R. J., Davey, P. C., Nini, L., Wang, J., Milligan, G., Jones, T. L. Z., and Druey, K. M. (2003) J. Biol. Chem. 278, 19309-19316) was critical for RGS16 and RGS4 GAP activity.     

Proteomic of lipid rafts in the exocrine pancreas from diet-induced obese rats

In the present work, we induced obesity in rats with high-energy-starch diet and studied exocrine pancreas response. The zymogen granule (ZG) or purified plasma membrane (PM) from the exocrine pancreas was used for the isolation of the detergent-resistant membranes (DRMs). Based on high content of cholesterol, GM1, the bile salt dependent lipase (BSDL), and GP2 enrichment, the low-density fractions were defined as lipid rafts. Additionally, the rafts vesicles were determined by immunogold labeling with anti BSDL. By combining MALDI-TOF/MS and nano-LC ESI Q-TOF MS/MS proteomic identification we have selected 33 proteins from the lipid rafts which were classified into at least four functional families. Our data suggest that the acinar PM from the diet-induced obesity rats may be organized into lipid rafts, and characterization of rafts proteome can contribute to improve our understanding of food digestion under obesity.     

Association of PepT1 with lipid rafts differently modulates its transport activity in polarized and nonpolarized cells

The transporter PepT1, apically expressed in intestinal epithelial cells, is responsible for the uptake of di/tripeptides. PepT1 is also expressed in nonpolarized immune cells. Here we investigated the localization of PepT1 in lipid rafts in small intestinal brush border membranes (BBMs) and polarized and nonpolarized cells, as well as functional consequences of the association of PepT1 with lipid rafts. Immunoblot analysis showed the presence of PepT1 in low-density fractions isolated from mouse intestinal BBMs, polarized intestinal Caco2-BBE cells, and nonpolarized Jurkat cells by solubilization in ice-cold 0.5% Triton X-100 and sucrose gradient fractionation. PepT1 colocalized with lipid raft markers GM1 and N-aminopeptidase in intestinal BBMs and Caco2-BBE cell membranes. Disruption of lipid rafts with methyl-beta-cyclodextrin (MbetaCD) shifted PepT1 from low- to high-density fractions. Remarkably, we found that MbetaCD treatment increased PepT1 transport activity in polarized intestinal epithelia but decreased that in intestinal BBM vesicles and nonpolarized immune cells. Mutational analysis showed that phenylalanine 293, phenylalanine 297, and threonine 281 in transmembrane segment 7 of the human di/tripeptide transporter, hPepT1, are important for the targeting to lipid rafts and transport activity of hPepT1. In conclusion, the association of PepT1 with lipid rafts differently modulates its transport activity in polarized and nonpolarized cells.     

Concentrates of DHA from fish oil by selective esterification of cholesterol by immobilized isoforms of lipase from Candida rugosa

Two lipases (Lip A and Lip B), were purified from a commercial lipase preparation produced by Candida rugosa and partially characterized. The purified lipases were immobilized on Duolite A 568 and used in the selective esterification of cholesterol with free fatty acids from sardine fish oil. The results showed that Lip A and Lip B preferentially esterified saturated and monounsaturated fatty acids allowing a 3.4-fold (Lip B, 24 h) and 4-fold (Lip A, 10 h) enrichment of docosahexaenoic acid in the remaining free fatty acid fraction. Selectivity towards eicosapentaenoic acid was less pronounced. By this selective esterification docosahexaenoic acid was concentrated from 7.4 to 32% with a recovery of 95% of its initial content in sardine fish oil.     

Metabolomic and Lipidomic Analysis of Serum Samples following Curcuma longa Extract Supplementation in High-Fructose and Saturated Fat Fed Rats

We explored, using nuclear magnetic resonance (NMR) metabolomics and fatty acids profiling, the effects of a common nutritional complement, Curcuma longa, at a nutritionally relevant dose with human use, administered in conjunction with an unbalanced diet. Indeed, traditional food supplements have been long used to counter metabolic impairments induced by unbalanced diets. Here, rats were fed either a standard diet, a high level of fructose and saturated fatty acid (HFS) diet, a diet common to western countries and that certainly contributes to the epidemic of insulin resistance (IR) syndrome, or a HFS diet with a Curcuma longa extract (1% of curcuminoids in the extract) for ten weeks. Orthogonal projections to latent structures discriminant analysis (OPLS-DA) on the serum NMR profiles and fatty acid composition (determined by GC/MS) showed a clear discrimination between HFS groups and controls. This discrimination involved metabolites such as glucose, amino acids, pyruvate, creatine, phosphocholine/glycerophosphocholine, ketone bodies and glycoproteins as well as an increase of monounsaturated fatty acids (MUFAs) and a decrease of n-6 and n-3 polyunsaturated fatty acids (PUFAs). Although the administration of Curcuma longa did not prevent the observed increase of glucose, triglycerides, cholesterol and insulin levels, discriminating metabolites were observed between groups fed HFS alone or with addition of a Curcuma longa extract, namely some MUFA and n-3 PUFA, glycoproteins, glutamine, and methanol, suggesting that curcuminoids may act respectively on the fatty acid metabolism, the hexosamine biosynthesis pathway and alcohol oxidation. Curcuma longa extract supplementation appears to be beneficial in these metabolic pathways in rats. This metabolomic approach highlights important serum metabolites that could help in understanding further the metabolic mechanisms leading to IR.