Substrate Report for: 4-methylumbelliferyl-oleate

Recombinant human gastric lipase (hGL) was transiently expressed in Nicotiana benthamiana leaves using the CPMV-HT expression system. Expression levels of up to 0.5mg recombinant hGL per gram of infiltrated leaf tissue were obtained. Proteins expressed from two hGL constructs, wild type (wt-hGL) and with a Histidine tag at its C terminal (hGL-His), were purified from leaf tissue using Immobilized Lectin Affinity chromatography and Immobilized Metal Affinity chromatography. Both variants were glycosylated, enzymatically active, and had an apparent molecular weight similar to native hGL (approx. 50kDa). The recombinant hGLs were stable under acidic conditions and in the presence of gastric pepsin. Moreover, as found with the naturally occurring enzyme, the activity of recombinant hGL on the short chain lipid, tributyrin, was higher than on long chain Intralipid 30% emulsion. The maximum specific activity measured on tributyrin was 310 U/mg of protein and the maximum yield was 193 U/g of infiltrated leaf tissue. These results show that transient expression in plants can be used to produce active hGL that could be efficiently purified using established techniques. The approach provides a means of generating large quantities of hGL that could be of use for a number of applications both in vitro and in vivo.

An acid lipase was purified from rabbit liver lysosomes by, in sequence, osmotic treatment of the lysosomal fraction, Sephadex LH-20, DEAE-Sephadex A-50, Bio-Gel A-5m, hydroxyapatite and, finally, Sephadex G-200 column chromatography. The substrate was 4-methylumbelliferyl oleate. The enzyme was solubilized by Sephadex LH-20 column chromatography instead of detergents and organic solvents, to obtain an intrinsic macromolecule. 4-Methylumbelliferyl oleate hydrolase, osmotically released from lysosomal particles, had a very high molecular weight (greater than 800 000) which was reduced by gel filtration on a Sephadex LH-20 column; the final molecular weight of the purified enzyme was 58 000. The specific activity of 4-methylumbelliferyl oleate hydrolase increased at almost the same rate as acid cholesterol esterase and triacylglycerol lipase after Sephadex LH-20 column chromatography; the thermal stability of the activity of the three enzymes was almost identical. We also discuss the properties of the enzyme molecule and the interaction between the enzyme and the lysosomal membrane.

Recombinant human gastric lipase (hGL) was transiently expressed in Nicotiana benthamiana leaves using the CPMV-HT expression system. Expression levels of up to 0.5mg recombinant hGL per gram of infiltrated leaf tissue were obtained. Proteins expressed from two hGL constructs, wild type (wt-hGL) and with a Histidine tag at its C terminal (hGL-His), were purified from leaf tissue using Immobilized Lectin Affinity chromatography and Immobilized Metal Affinity chromatography. Both variants were glycosylated, enzymatically active, and had an apparent molecular weight similar to native hGL (approx. 50kDa). The recombinant hGLs were stable under acidic conditions and in the presence of gastric pepsin. Moreover, as found with the naturally occurring enzyme, the activity of recombinant hGL on the short chain lipid, tributyrin, was higher than on long chain Intralipid 30% emulsion. The maximum specific activity measured on tributyrin was 310 U/mg of protein and the maximum yield was 193 U/g of infiltrated leaf tissue. These results show that transient expression in plants can be used to produce active hGL that could be efficiently purified using established techniques. The approach provides a means of generating large quantities of hGL that could be of use for a number of applications both in vitro and in vivo.

Attempts to engineer enzymes with unique catalytic properties have largely focused on altering the existing specificities by reshaping the substrate binding pockets. Few experiments have aimed at modifying the configuration of the residues essential for catalysis. The difference in the topological location of the triad acids of Geotrichum candidum lipase (GCL) and the catalytic domain of human pancreatic lipase (HPL), despite great similarities in their topologies and 3-D structures, suggest that these are related enzymes whose catalytic triads have been rearranged in the course of evolution (Schrag et al., 1992). In this study we prepared a double mutant GCL in which the catalytic triad acid is shifted to the position equivalent to the location of the triad acid of HPL. The double mutant maintains approximately 10% of the wild type activity against triglycerides and the fluorogenic ester 4-methylumbelliferyl-oleate. The only significant differences between the 3-D structures of the double mutant and wild type GCL are at the mutated sites. Even the water structure in the region of the triad is unchanged. The hydrogen bonding pattern of the catalytic triad of the double mutant is very similar to that of pancreatic lipase. The acid of the double mutant is stabilized by only two hydrogen bonds, whereas three hydrogen bonds are observed in the wild type enzyme. These results strongly support the hypothesis that the pancreatic lipases are evolutionary switchpoints between the two observed arrangements of the catalytic triads supported by the alpha/beta hydrolase fold and suggest that this fold provides a stable protein core for engineering enzymes with unique catalytic properties.

Lipases from Geotrichum candidum NRRL Y-553 are of interest because of their unique specificity for cis-9-unsaturated fatty acids relative to both stearic and palmitic acids. The lipases were partially purified by chromatography on Octyl Sepharose, AG MP-1 macroporous anion exchanger, and chromatofocusing resin. The preparation was found to contain multiple, glycosylated lipases varying slightly in pI (pI 4.88, 4.78, 4.65, 4.57 and 4.52) as judged by both activity and silver staining. The molecular mass determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis was 64 kilodaltons for the main species, with minor species of 60 and 57 kilodaltons present as well. The specificity of the crude lipases for hydrolysis of 4-methylumbelliferyl esters of oleic vs. palmitic acid was 20-to-1. The specificity of the purified, partially separated lipases was similar to that of the crude preparation. Thus the lipases could be used even in crude form for the hydrolysis and restructuring of triacylglycerols on a large scale.

The effect of a fungal metabolite, esterastin, on lysosomal acid lipase purified from rabbit liver was studied. Esterastin inhibited the enzyme activity very strongly (IC50, about 80 nM). The inhibition of acid lipase by esterastin was competitive with respect to the substrate and the inhibition constant for esterastin was 90 nM. Esterastin was less inhibitory to other lipolytic enzymes, such as pancreatic lipase and carboxylesterase. Thus esterastin is a potent new inhibitor of lysosomal acid lipase.

An acid lipase was purified from rabbit liver lysosomes by, in sequence, osmotic treatment of the lysosomal fraction, Sephadex LH-20, DEAE-Sephadex A-50, Bio-Gel A-5m, hydroxyapatite and, finally, Sephadex G-200 column chromatography. The substrate was 4-methylumbelliferyl oleate. The enzyme was solubilized by Sephadex LH-20 column chromatography instead of detergents and organic solvents, to obtain an intrinsic macromolecule. 4-Methylumbelliferyl oleate hydrolase, osmotically released from lysosomal particles, had a very high molecular weight (greater than 800 000) which was reduced by gel filtration on a Sephadex LH-20 column; the final molecular weight of the purified enzyme was 58 000. The specific activity of 4-methylumbelliferyl oleate hydrolase increased at almost the same rate as acid cholesterol esterase and triacylglycerol lipase after Sephadex LH-20 column chromatography; the thermal stability of the activity of the three enzymes was almost identical. We also discuss the properties of the enzyme molecule and the interaction between the enzyme and the lysosomal membrane.

Lysosomal acid lipase (LAL) activity was measured using a new fluorometric assay in cultured skin fibroblasts from eight control subjects, two obligate heterozygotes for Wolman's disease (WD), one patient with WD, and one patient with cholesteryl ester storage disease (CESD). The LAL activities (mean+/-SD) were 25.8+/-8.2, 13.2+/-0.1,1.1, and 1.4 nmol 4-methylumbelliferyl oleate (4-MUO) hydrolyzed/min/mg protein, respectively. These results compare favorably with those obtained using standard radioassays. The LAL activities of two cultures of amniotic fluid cells were 12.1 and 10.5. The LAL activity (mean+/-SD) of peripheral leukocytes obtained from 34 laboratory volunteers (19 females, 15 males) was 4.0+/-1.8. Partially purified lymphocytes contained about 25 times as much LAL activity as did granulocytes. Cellogel electrophoresis, followed by staining with 4-MUO, showed at least two bands of LAL (A and B) from normal fibroblasts, amniotic fluid cells, and lymphocytes. Band A was absent from WD and CESD fibroblasts and was reduced in fibroblasts of the WD heterozygotes.