Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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 interaction of normal and acute-phase high-density lipoproteins of the subclass 3 (N-HDL3 and AP-HDL3) with human neutrophils and the accompanying degradation of HDL3 apolipoproteins have been studied in vitro. The chemical composition of normal and acute-phase HDL3 was similar except that serum amyloid A protein (apo-SAA) was a major apolipoprotein in AP-HDL3 (approx. 30% of total apolipoproteins). 125I-labelled AP-HDL3 was degraded 5-10 times faster than 125I-labelled N-HDL3 during incubation with neutrophils or neutrophil-conditioned medium. Apo-SAA, like apolipoprotein A-II (apo-A-II), was more susceptible than apolipoprotein A-I (apo-A-I) to the action of proteases released from the cells. The amounts of cell-associated AP-HDL3 apolipoproteins at saturation were up to 2.8 times greater than N-HDL3 apolipoproteins; while apo-A-I was the major cell-associated apolipoprotein when N-HDL3 was bound, apo-SAA constituted 80% of the apolipoproteins bound in the case of AP-HDL3. The associated intact apo-SAA was mostly surface-bound as it was accessible to the action of exogenous trypsin. alpha 1-Antitrypsin-resistant (alpha 1-AT-resistant) cellular degradation of AP-HDL3 apolipoproteins also occurred; experiments in which pulse-chase labelling was performed or lysosomotropic agents were used indicated that insignificant intracellular degradation occurred which points to the involvement of cell-surface proteases in this degradation.
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PMID:Neutrophil association and degradation of normal and acute-phase high-density lipoprotein 3. 244 74

The interactions of human apolipoprotein A-I (apo A-I) with dipalmitoylphosphatidylcholine (DPPC) in vesicular complexes at low protein concentrations and in micellar complexes at high protein concentrations are compared. The C-terminal segment of this protein, with a relative molecular weight (Mr) of about 11,000, is protected on trypsin treatment of apo A-I-vesicle complexes. A segment within the sequence from Leu-189 to Arg-215 of apo A-I penetrates the hydrophobic interior of the membrane, as found in a hydrophobic labeling experiment involving 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)-diazirine ([125I]TID). No appreciable stretch of apo A-I in micellar complexes was found to be protected from the tryptic digestion. This indicates that the interactions of apo A-I with lipids in the vesicular and micellar complexes are different. The binding equilibrium of apo A-I as to DPPC vesicles at low protein concentrations, as studied by hydrophobic labeling of the bilayer-penetrating segment, is reached within about 1 h, while the formation of micellar complexes at high protein concentrations takes about 24 h at 42 degrees C. Time-dependent labeling studies involving photoreactive phosphatidylcholine (PC) with high apo A-I concentrations suggested an initial interaction with the head group region of the bilayer followed by interaction with the tail ends of the acyl chains of the lipid. A possible mechanism for the micellization process is discussed.
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PMID:Interaction of human apolipoprotein A-I with dipalmitoylphosphatidylcholine in vesicular and micellar complexes. 251 55

High-density lipoprotein 3 (HDL3) binds to capillary endothelial cells when their lumen surfaces are exposed to 125I-HDL3 by post-mortem perfusion of whole brain. Kinetic studies of binding of HDL3 to isolated membranes show that HDL3 binds only to endothelial membranes with high affinity (Kd = 7 micrograms/ml). Trypsin treatment of membranes abolishes HDL3 binding. High-affinity binding sites for HDL3 were recovered when endothelial cells from bovine brain capillaries were maintained in culture (Kd = 13 micrograms/ml HDL3 protein). The characteristics of the binding were preserved up to the 6th passage. Competition experiments using isolated luminal membranes or cultured endothelial cells indicate that only HDL3 and not LDL or methylated LDL, are able to compete binding of 125I-HDL3. Furthermore, the inhibition of 125I-HDL3 binding by lipoprotein A-I and lipoprotein A-I:A-II strongly suggests that apolipoprotein A-I is implicated in the formation of HDL3-receptor complexes. The binding is increased by loading cells with free cholesterol or LDL cholesterol. In addition, surface-bound 125I-HDL3 remains sensitive to mild trypsin treatment after subsequent incubation of BBCE at 37 degrees C. HDL3 bound to the cell surface is not endocytosed, but rather rapidly released into the medium after binding (t1/2 = 5 min).
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PMID:Interactions of high-density lipoprotein 3 with brain capillary endothelial cells. 255 99

The binding of human 125I-labeled HDL3 (high-density lipoproteins, rho 1.125-1.210 g/cm3) to a crude membrane fraction prepared from bovine liver closely fit the paradigm expected of a ligand binding to a single class of identical and independent sites, as demonstrated by computer-assisted binding analysis. The dissociation constant (Kd), at both 37 and 4 degrees C, was 2.9 micrograms protein/ml (approx. 2.9 X 10(-8) M); the capacity of the binding sites was 490 ng HDL3 (approx. 4.9 pmol) per mg membrane protein at 37 degrees C and 115 at 4 degrees C. Human low-density lipoproteins (LDL) and very-low-density lipoproteins (VLDL) also bound to these sites (Kd = 41 micrograms protein/ml, approx. 6.7 X 10(-8) M for LDL, and Kd = 5.7 micrograms protein/ml, approx. 7.0 X 10(-9) M for VLDL), but this observation must be considered in light of the fact that the normal circulating concentrations of these lipoproteins are much lower than those of HDL. The binding of 125I-labeled HDL3 to these sites was inhibited only slightly by 1 M NaCl, suggesting the presence of primarily hydrophobic interactions at the recognition site. The binding was not dependent on divalent cations and was not displaceable by heparin; the binding sites were sensitive to both trypsin and pronase. Of exceptional note was the finding that various subclasses of human HDL (including subclasses of immunoaffinity-isolated HDL) displaced 125I-labeled HDL3 from the hepatic HDL binding sites with different apparent affinities, indicating that these sites are capable of recognizing highly specific structural features of ligands. In particular, apolipoprotein A-I-containing lipoproteins with prebeta electrophoretic mobility bound to these sites with a strikingly lower affinity (Kd = 130 micrograms protein/ml) than did the other subclasses of HDL.
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PMID:Discrimination between subclasses of human high-density lipoproteins by the HDL binding sites of bovine liver. 300 Apr 54

The amino acid sequence of rabbit apolipoprotein A-I (apo A-I) has been determined by degradation and alignment of two overlapping sets of peptides obtained from tryptic and staphylococcal digestions. All of the peptides of rabbit apo A-I resulting from digestion by staphylococcal protease were isolated and sequenced except residues 33-37. A digestion with trypsin was employed to find overlapping and missing peptides. The N-terminus of rabbit apo A-I was confirmed by sequencing the intact protein up to 20 residues while the C-terminus was identified through its homology with human apo A-I. The protein contains 241 residues in its single chain. Its primary structure is highly homologous to the reported canine apo A-I (80%) and human apo A-I (78%), but exhibits less similarity with rat apo A-I (60%). Like human apo A-I, rabbit apo A-I contains very little histidine (2) and methionine (1); it does however have two residues of isoleucine. Based on a comparison of the hydrophobic-hydrophilic character of apo A-I residues with that of the two synthetic peptides that activated lecithin: cholesterol acyltransferase (Pownall et al. and Yokoyama et al.), we found that the five segments with the highest corresponding homologies on the protein are located within the N-terminal half. This suggests that the N-terminal half of apo A-I contains the major portion of regions activating lecithin: cholesterol acyltransferase.
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PMID:The primary structure of apolipoprotein A-I from rabbit high-density lipoprotein. 309 15

The initial rate of filipin association with unesterified cholesterol in high density lipoproteins (HDL) was measured by stopped-flow spectrophotometry to assess the roles played by apolipoproteins and phospholipids in modulating the surface exposure of cholesterol. The initial rate of filipin-unesterified cholesterol association was enhanced upon hydrolysis of the glycerophospholipids of human HDL3 by phospholipase A2. Rate enhancements were also observed following trypsin-catalyzed hydrolysis of apolipoprotein A-I in canine HDL and of apolipoproteins A-I and A-II in human HDL3. However, the initial rate of filipin-unesterified cholesterol association was not altered upon incubation of HDL3 with polymorphonuclear cells, which causes hydrolysis of apolipoprotein A-II but leaves apolipoprotein A-I intact. These results are consistent with the general structural model of HDL in which unesterified cholesterol, apolipoproteins and glycerophospholipids are presumed to be localized at the surface of the HDL particle. From these studies and from results indicating that the initial rate of filipin-unesterified association was enhanced in canine HDL hybrids in which 50% of the apolipoprotein A-I had been replaced by apolipoprotein A-II, we also conclude that apolipoprotein A-I in HDL is in closer proximity to unesterified cholesterol than apolipoprotein A-II. Thus, it appears that rapid kinetic measurements of filipin-cholesterol association may be useful in assessing the organization of unesterified cholesterol in serum lipoproteins.
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PMID:Organization of unesterified cholesterol in high density lipoproteins probed by filipin. 681 43

The effect of n-propanol on the secondary and tertiary structure of human apolipoprotein A-I (apoA-I), an interfacial protein, was investigated using near and far ultraviolet (UV)-circular dichroism (CD) and fluorescence spectroscopy, as well as limited proteolytic digestion with trypsin, and cross-linking with bis(sulfosuccinimidyl) suberate. The structure of apoA-I in n-propanol (30%, v/v) was compared with that in Tris buffer and in reconstituted, spherical or discoidal, high density lipoproteins (rHDL). Addition of n-propanol to apoA-I in Tris buffer induces major changes in its near and far CD spectra: alpha-helical content increases by 27% and the near UV-CD spectrum becomes very similar to that of apoA-I in rHDL particles. Fluorescence spectral, lifetime, and polarization results, and quenching by KI confirm that major structural changes occur in the N-terminal half of apoA-I as n-propanol is added: the Trp residues become more exposed to solvent than in buffer alone or in rHDL. Higher concentrations of guanidine hydrochloride or urea are required to denature apoA-I in n-propanol than in buffer alone, but a similar free energy of unfolding is observed. The N-terminus of apoA-I is relatively resistant to trypsin digestion and the C-terminus has equivalent digestion sites for apoA-I in the three states, but the kinetics of digestion are much slower in n-propanol and in rHDL compared to apoA-I in Tris buffer. Cross-linking experiments reveal that dimers of apoA-I exist in n-propanol, in contrast to dimers plus multimeric aggregates in Tris buffer. From these results we conclude that in 30% n-propanol the structure of apoA-I approaches that of 'native' lipid-bound apoA-I, in contrast to its structure in the aqueous Tris buffer.
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PMID:Native-like structure and self-association behavior of apolipoprotein A-I in a water/n-propanol solution. 819 99

We prepared discoidal and spherical model high density lipoprotein (HDL) with apolipoprotein A-I and 1-palmitoyl-2-oleoyl phosphatidylcholine at various lipid:protein ratios and compared their reactivity with exo- and endopeptidases to that of human HDL2 and HDL3. Limited proteolysis with trypsin, Staphylococcus V8 protease, and elastase yielded a major stable peptide of 11,000-11,500 daltons under conditions which completely degraded lipid-free A-I. By Western blotting this protease-resistant fragment was shown to consist of the amino-terminal 90-100 residues of the A-I protein; the residues on the carboxyl side of this peptide are therefore protease-sensitive and appear to correlate with the putative "hinge" region, which is especially reactive with antibodies. The amino terminus was very resistant to digestion with a variety of aminopeptidases, whereas carboxypeptidases could remove up to 70 residues from the lipid-free A-I protein or 12-24 residues from A-I in various HDL. When these truncated forms of A-I, in combination with lipid, were used to examine binding interactions with rat hepatic plasma membranes, it was found that removal of up to 20-24 residues from the carboxyl terminus had no significant effect on binding, whereas removal of 70 residues completely eliminated specific binding to the membranes. Taken together, our data indicate that there is a protease-resistant domain constituted by the first 90 residues of A-I, which, in HDL, contain little of the class of amphipathic helix characteristic of the rest of the molecule and most likely form a structure dominated by protein-protein interactions. At the carboxyl end of the protein, there is a functional domain constituted by residues 149-219 that possesses the capacity to bind to proteins on hepatic membranes.
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PMID:Structural and functional domains of apolipoprotein A-I within high density lipoproteins. 836 78

Several studies have demonstrated that lipid-free apolipoproteins can promote cholesterol and phospholipid efflux from cells; however, the mechanisms and the role of cell-mediated pathways involved remain incompletely elucidated. We have recently demonstrated that brefeldin A or monensin, agents that disrupt Golgi apparatus structure and function, inhibit intracellular cholesterol efflux from cells to high density lipoproteins. In the present study we examined the effects of those agents on cell cholesterol and phospholipid efflux to purified apolipoprotein A-I (apoA-I) and apolipoprotein-depleted acceptors from cholesterol-loaded fibroblasts. Brefeldin A or monensin treatment of cells during incubation with apoA-I inhibited efflux of cellular cholesterol by greater than 80% compared with control cells, measured by changes in cellular cholesterol radioactivity, mass, and the substrate pool of cholesterol available for esterification by acyl coenzyme A:cholesterol acyltransferase. Inhibition of cholesterol efflux by these agents could not be overcome by increasing the apoA-I concentration and persisted during incubations up to 24 h. Similarly, brefeldin A and monensin inhibited up to 80% of apoA-I-mediated efflux of labeled phospholipids from cholesterol-loaded cells relative to controls. In contrast, lipid efflux mediated by apolipoprotein-depleted acceptors (trypsin-modified HDL and sonicated phospholipid vesicles) was not sensitive to these drugs. On the basis the known effects of brefeldin A and monensin on Golgi apparatus structure and function, these results are consistent with the notion that efflux of cell lipids by apolipoprotein-dependent mechanisms, but not by apolipoprotein-independent mechanisms, require active cellular processes involving an intact and functional Golgi apparatus.
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PMID:Apolipoprotein-mediated cellular cholesterol and phospholipid efflux depend on a functional Golgi apparatus. 901 4

It is becoming increasingly accepted that removal of cellular cholesterol occurs by at least two pathways, one involving the well-described aqueous diffusion mechanism and another promoted by lipid-free apolipoproteins. We compared the contribution of apolipoprotein-dependent and -independent pathways, taking into consideration the influence of cellular metabolism, on cholesterol efflux promoted by different extracellular acceptor types. The acceptors used were assumed to participate in only passive efflux by lipid-dependent mechanisms (phospholipid vesicles and trypsin-modified high density lipoproteins) or to stimulate efflux by apolipoprotein-dependent pathways (purified apolipoprotein A-I and high density lipoproteins). Apolipoprotein-mediated cholesterol efflux was only apparent in growth-arrested or cholesterol-enriched cells and required metabolic energy. In contrast, cholesterol efflux by apolipoprotein-depleted acceptors did not depend on cell growth state, cholesterol enrichment, or metabolic energy. Apolipoprotein-mediated efflux was not observed at temperatures below 22 degrees C, while apolipoprotein-independent efflux was only reduced by 50% at 4 degrees C compared with incubations at 37 degrees C. Additionally, apolipoproteins promoted a more rapid and larger decrease in intracellular cholesteryl esters when measured by changes in cholesteryl ester radioactivity, mass, or the pool of cholesterol available for esterification by acyl coenzyme A:cholesterol acyltransferase. Efflux of excess cellular cholesterol by an apolipoprotein-dependent pathway appears to involve specific cellular events consistent with the properties of an active transport pathway and distinguishable from cholesterol efflux by apolipoprotein-depleted acceptors through passive mechanisms.
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PMID:Cholesterol efflux mediated by apolipoproteins is an active cellular process distinct from efflux mediated by passive diffusion. 932 90


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