Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The human hepatoma cell line, HepG2, secreted an activity that degrades platelet-activating factor (PAF) by the hydrolysis of the sn-2 acetyl group. This activity was Ca++ independent, inhibited by diisopropylfluorophosphate but not by p-bromophenacyl bromide, and resistant to treatment with trypsin or pronase. Separation of HepG2-conditioned medium by gel filtration disclosed that the activity was associated with lipoproteins. An antiserum against PAF acetylhydrolase immunoprecipitated this activity. It was not recognized by an antibody against lecithin:cholesterol acyltransferase (LCAT), which also is secreted by HepG2 cells. Therefore the phospholipase A2 activity of LCAT was excluded as a source of the observed activity. PAF added to the culture medium stimulated the secretion of the PAF-degrading activity by HepG2 cells, while lyso-PAF was inactive. Maximal stimulation was observed with 5 ng/ml PAF, which induced a fivefold increase. The presence of 5 ng/ml PAF, enhanced the secretion of [35S]methionine-labeled PAF acetylhydrolase and cycloheximide inhibited both the basal and PAF-stimulated secretion of the labeled enzyme. We conclude that HepG2 cells produce PAF acetylhydrolase. The liver may be a major source of plasma PAF acetylhydrolase, and PAF may induce the production of its inactivating enzyme by the liver.
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PMID:Platelet-activating factor (PAF) stimulates the production of PAF acetylhydrolase by the human hepatoma cell line, HepG2. 184 78

Vibrio species release a lipase which shares many properties with mammalian lecithin-cholesterol acyltransferase. We have studied the action of the enzyme on phospholipid monolayers. At similar surface pressures, reaction velocities were higher with monolayers of dilauroylphosphatidylcholine than with the corresponding phosphatidylglycerol or phosphatidylethanolamine. The dependence of reaction velocity on molecular density was very similar for phosphatidylcholine and phosphatidylethanolamine monolayers. Lag times were shortest with phosphatidylglycerol at low molecular densities, but maximum velocity was reached at considerably lower densities than with the other two lipids. We have found [Hilton, S., McCubbin, N. D., Kay, C., & Buckley, J.T. (1990) Biochemistry 29, 9072-9078] that nicking of the enzyme with trypsin or other proteases results in an increase in its activity against lipids in membranes. Here we show that trypsin treatment results in a large change in the surface activity of the lipase, allowing it to penetrate monolayers at pressures higher than 40 mN.m-1.
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PMID:Action of a microbial lipase/acyltransferase on phospholipid monolayers. 204 45

A fatty acyltransferase with a reaction mechanism similar to that of mammalian lecithin: cholesterol acyltransferase has been purified from culture supernatants of a mutant Aeromonas salmonicida containing the cloned Aeromonas hydrophila structural gene. Typically, more than 35 mg of protein were isolated from 2 L of culture supernatant. The amino-terminal sequence, amino acid composition, and molecular weight of the purified protein corresponded to predictions based on the sequence of the gene, indicating that the signal sequence had been correctly removed during export but that no further processing had occurred. Analysis of the far-UV circular dichroic (CD) spectrum of the enzyme showed that it consists of 31% alpha-helix, 21% beta-sheet, and 16% beta-turn, with 12% of aperiodic form. Treatment of the purified protein with a variety of proteases resulted in nicking near the C-terminus. This led to an increase in enzyme activity against lipids in erythrocyte membranes and increased rate of hydrolysis of p-nitrophenyl butyrate. Activation was accompanied by a change in the CD spectrum and a change in its aggregation state. The trypsin cut site was located between the two cysteines in the enzyme. Evidence is presented that the cysteines are joined by a disulfide bond and therefore cannot participate in acyl transfer. This may distinguish the microbial enzyme from lecithin:cholesterol acyltransferase. This is the second extracellular A. hydrophila protein that we have shown can be activated by proteolysis after it is released.
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PMID:Purification and spectral study of a microbial fatty acyltransferase: activation by limited proteolysis. 227 78

The effect of various chemical and enzymatic modifications of low density lipoprotein (LDL) on its ability to activate the isolated human plasma lysolecithin acyltransferase (LAT) was studied. Removal of all lipids from LDL resulted in the complete loss of LAT activation. Removal of only neutral lipids by extraction with heptane retained up to 50% of the original activity, which was not increased further by reconstitution of the LDL with the extracted lipids. Hydrolysis of the diacylphosphoglycerides of the LDL with phospholipases resulted in complete loss of LAT activation which was partially restored by the addition of egg lecithin. Hydrolysis of more than 4% of LDL protein by trypsin led to a linear decrease in activity with complete loss of activity occurring when about 25% of the LDL protein is hydrolyzed. Modification of the arginine groups of LDL reversibly inhibited the activation of LAT. Modification of lysine residues of LDL by acetylation, acetoacetylation or succinylation also abolished its ability to activate lysolecithin acylation.
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PMID:Role of low density lipoprotein in the activation of plasma lysolecithin acyltransferase activity. Effect of chemical and enzymatic modifications of the lipoprotein on enzyme activity. 392 84

Limited proteolysis was used to study the domain structure and to produce a large N-terminal fragment of human apolipoprotein AI (apoAI). Digestion of reconstituted high density lipoprotein (rHDL) prepared with apoAI and dipalmitoyl phosphatidylcholine or palmitoyloleoyl phosphatidylcholine by chymotrypsin, trypsin, elastase, and subtilisin generated a major fragment of 22 kDa. Under milder conditions proteolysis of lipid-free apoAI produced a fragment of similar size. The fragments shared the same N terminus as intact apoAI, and the chymotryptic fragment had a molecular weight of 22,384 as determined by electrospray ionization mass spectrometry. Thus the fragment consists of the N-terminal 192 amino acid residues of apoAI, and the region around Tyr192 seems to be especially accessible to proteases. In aqueous solution the fragment, apoAI-(1-192), had an alpha-helix content similar to that of apoAI (approximately 52%) but existed only as monomers and dimers. ApoAI-(1-192) lysed dimyristoyl phosphatidylcholine liposomes slowly compared with apoAI but did form rHDL complexes with palmitoyloleoyl phosphatidylcholine or dipalmitoyl phosphatidylcholine when prepared by the sodium cholate dialysis method. ApoAI-(1-192) rHDL exhibited sizes and size distributions distinct from apoAI rHDL but displayed similar stability against denaturation. The isolated apoAI-(1-192) rHDLs retained a high ability to activate lecithin-cholesterol acyltransferase, comparable with the most effective apoAI rHDL. The results suggest that the C-terminal domain of apoAI is crucial for self-association and initial lipid binding but is not involved in specific lecithin-cholesterol acyltransferase activation.
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PMID:Properties of an N-terminal proteolytic fragment of apolipoprotein AI in solution and in reconstituted high density lipoproteins. 774 65

Vibrio and Aeromonas spp. secrete an unusual 35-kDa lipase that shares several properties with mammalian lecithin-cholesterol acyltransferase. The Aeromonas hydrophila lipase contains two cysteine residues that form an intramolecular disulfide bridge. Here we show that changing either of the cysteines to serine does not reduce enzymatic activity, indicating that the disulfide bond is not required for correct folding. However, when either of the cysteines is replaced, the enzyme is more readily denatured by urea and more sensitive to degradation by trypsin than is the wild-type enzyme, evidence that the bridge has an important role in stabilizing the protein's structure. The two mutant proteins with serine-for-cysteine replacements were secreted by Aeromonas salmonicida containing the cloned genes, although the levels of both in the culture supernatants were lower than the level of the wild-type enzyme. When the general secretory pathway was blocked with carbonyl cyanide chlorophenylhydrazone, the cell-associated pools of the mutant enzymes appeared to be degraded, whereas the wild-type pool remained stable. We conclude that reduced extracellular levels of the mutant proteins are the result of their increased sensitivities to proteases encountered inside the cell during export.
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PMID:The disulfide bond in the Aeromonas hydrophila lipase/acyltransferase stabilizes the structure but is not required for secretion or activity. 915 Feb 3

The major N-linked carbohydrate structures were determined for recombinant human plasma lecithin:cholesterol acyltransferase (LCAT). The analysis of the structure of oligosaccharides by fast atom bombardment mass spectrometry (FAB-MS) and linkage analysis was preceded by reduction and carboxymethylation of the intact glycoproteins and digestion with trypsin and proline specific endopeptidase. The N-glycans were subsequently released from the glycopeptides by PNGase F digestion and the oligosaccharides were separated using a C18 Sep-pak cartridge. The data from the combination of FAB spectrometry and linkage analysis show that the N-linked glycans present on recombinant LCAT (rLCAT) were composed primarily of triantennary and tetraantennary structures with and without core fucosylation. A minor population of glycans (less than 5%) contained up to three repeats of N-acetyllactosamine in one or more antennae. The LCAT activities of both recombinant and circulating forms of plasma LCAT were determined using low molecular weight and lipoprotein substrates. The catalytic behavior of these two enzyme forms were found to be very similar if not identical. These findings validate the concept that the recombinant enzyme can serve as an appropriate model for structure/function studies of LCAT and provide the foundation for subsequent structural studies.
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PMID:Characterization of recombinant human plasma lecithin: cholesterol acyltransferase (LCAT): N-linked carbohydrate structures and catalytic properties. 955 45