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

---

Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Rat liver parenchymal cell binding, uptake, and proteolytic degradation of rat 125I-labeled high density lipoprotein (HDL) subfraction, HDL3 (1.10 less than d less than 1.210 g/ml), in which apo-A-I is the major polypeptide, were investigated. Structural and metabolic integrity of the isolated cells was verified by trypan blue exclusion, low lactic dehydrogenase leakage, expected morphology, and gluconeogenesis from lactate and pyruvate. 125I-labeled HDL3 was incubated with 10 X 10(6) cells at 37 degrees and 4 degrees in albumin and Krebs-Henseleit bicarbonate buffer, pH 7.4. Binding and uptake were determined by radioactivity in washed cells. Proteolytic degradation was determined by trichloroacetic acid-soluble radioactivity in the incubation medium. At 37 degrees, maximum HDL3 binding (Bmax) and uptake occurred at 30 min with a Bmax of 31 ng/mg dry weight of cells. The apparent dissociation constant of the HDL3 receptor system (Kd) was 60 X 10(-8) M, based on Mr = 28,000 of apo-A-I, the predominant rat HDL3 protein. Proteolytic degradation showed a 15-min lag and then constant proteolysis. After 2 hours 5.8% of incubated 125I-labeled HDL3 was degraded. Sixty per cent of cell radioactivity at 37 degrees was trypsin-releasable. At 37 degrees, 125I-labeled HDL3 was incubated with cells in the presence of varying concentrations of native (cold) HDL3, very low density lipoproteins, and low density lipoproteins. Incubation with native HDL3 resulted in greatest inhibition of 125I-labeled HDL3 binding, uptake, and proteolytic degradation. When 125I-labeled HDL3 was preincubated with increasing amounts of HDL3 antiserum, binding and uptake by cells were decreased to complete inhibition. Cell binding, uptake, and proteolytic degradation of 125I-labeled HDL3 were markedly diminished at 4 degrees. Less than 1 mM chloroquine enhanced 125I-labeled HDL3 proteolysis but at 5 mM or greater, chloroquine inhibited proteolysis with 125I-labeled HDL3 accumulation in cells. L-[U-14C]Lysine-labeled HDL3 was bound, taken up, and degraded by cells as effectively as 125I-labeled HDL3. These data suggest that liver cell binding, uptake, and proteolytic degradation of rat HDL3 are actively performed and linked in the sequence:binding, then uptake, and finally proteolytic degradation. Furthermore, there may be a specific HDL3 (lipoprotein A) receptor of recognition site(s) on the plasma membrane. Finally, our data further support our previous reports of the important role of liver lysosomes in proteolytic degradation of HDL3.
...
PMID:Rat high density lipoprotein subfraction (HDL3) uptake and catabolism by isolated rat liver parenchymal cells. 18 84

The exposure of apolipoproteins at the surface of human plasma high density lipoproteins (HDL) was assessed by their accessibility to agarose-immobilized forms of trypsin and chymotrypsin. Proteolysis of lipid-free apolipoproteins and the lipoprotein subfractions HDL2 (d = 1.08--1.125 g/ml) and HDL3 (d = 1.125--1.195 g/ml) that differ in lipid-to-protein ratio was compared by polyacrylamide gel electrophoresis and isoelectric focusing of the apolipoproteins and peptide fragments and by quantitation of the various carboxyl-terminal groups formed. Gel filtration of the proteolyzed lipoproteins on Sephadex G-150 column indicated that more than 90% of the apolipoproteins and peptides remain associated with lipoprotein complexes. Proteolysis of lipoproteins occurred more slowly and with less fragmentation of the lipoproteins and apolipoproteins than proteolysis of thelipid-free apolipoproteins or the proteolysis of lipoproteins by soluble proteases reported by other investigators. The difference in lipid content of HDL2 and HDL3 made little difference in their proteolysis. Proteolysis of the lipoproteins by agarose-trypsin was more rapid at 37 degrees C than at 22 degrees C, but the proteolytic products were similar and differed from the products from the lipid free proteins. Peptide fragments from lipoproteins were larger than those from lipid-free proteins, which suggests masking of potentially cleavable groups by lipid. The amounts (mol/g protein) of new carboxyl-terminal tyrosine and phenylalanine released by agarose -chymotrypsin were much greater from the lipid-free proteins, but about 3/4 of the tryptophan residues were inacessible in both lipoproteins and lipid-free proteins. In agarose-trypsin digestion, lysine residues were slightly more masked than arginine in the absence of lipids and much more so in the lipoproteins. However, in the lipoproteins apoA-II, which contains lysine but no arginine, was cleaved more rapidly and extensively by agarose-trypsin than apoA-I.
...
PMID:Surface exposure of apolipoproteins in high density lipoproteins. I. Reactivities with agarose-immobilized proteases. 20 44

Bloodstream forms of Trypanosoma brucei brucei are unable to synthesize cholesterol but appear to bind and take up plasma low-density lipoproteins (LDL) from their host. Whether cholesterol homeostasis of this unicellular parasite also requires interactions with host high-density lipoprotein (HDL) particles is unknown. Equilibrium binding of radioiodinated apolipoprotein E-depleted human HDL3 (d = 1.125-1.21 g/ml) and bovine HDL (d = 1.063-1.21 g/ml) by T.b.brucei was rapid (less than 30 min) at 4 degrees C and was characterized by a saturable, specific component. There were five times the number of high-affinity binding sites for human HDL3 as for bovine HDL (64,000 vs. 11,500 per trypanosome) and their binding affinity was greater with an equilibrium dissociation constant (Kd) of 157 nM compared to 315 nM for bovine HDL). Binding of rat and rabbit HDL3 was similar to bovine HDL. By contrast, equilibrium binding of human LDL was slower (approximately 6 h) and the number of high-affinity binding sites (Kd = 23 nM) was much lower for this ligand (660 per trypanosome). Total binding of HDL3 was independent of divalent cations and was only slightly inhibited by heparin, but when the trypanosomes were preincubated with trypsin or pronase the binding was markedly reduced. After 30 min at 37 degrees C, binding of bovine HDL and human HDL3 was 10-20% higher than at 4 degrees C; after 45 min trypanolysis occurred with human HDL3 but not with bovine HDL. Chemical modification of HDL3 by treatment with cyclohexanedione, by acetylation or by reductive alkylation had little effect on its ability to compete with [125I]labelled HDL3 for binding by the parasite. Nitrosylation of HDL3 with tetranitromethane increased its binding ability, suggesting that trypanosomes might possess scavenger receptors, and native HDL3 was less effective than nitrosylated HDL3 in displacing bound [125I]labelled nitrosylated HDL3. These findings suggest that, in addition to a receptor for LDL, T.b.brucei has other lipoprotein binding sites which separately recognize HDL from permissive host species such as bovine, trypanolytic HDL such as human HDL3, and more negatively charged HDL particles such as nitrosylated HDL3.
...
PMID:Characteristics of the binding of human and bovine high-density lipoproteins by bloodstream forms of the African trypanosome, Trypanosoma brucei brucei. 153 61

The characteristics of HDL3 binding sites on cultured smooth muscle cells originally isolated from rabbit aorta were studied with 125I-labelled rabbit HDL3. The results indicated that specific and high-affinity HDL3 binding sites were present on the surface of smooth muscle cells (Bmax = 0.321 micrograms/mg cell protein; KD = 5.6 x 10(-8) mol/L). The presence of a high concentration of non-labelled apo A1 blocked the 125I-HDL3 binding rate by 50%. 125I-HDL3 pretreated (blocked) with rat antirabbit apo A1 IgG lost up to 70% of its original binding activity. Additionally, rabbit apo A1 was isolated, purified and its amino acid composition analyzed. The binding rate of 125I-HDL3 to smooth muscle cells was not affected by the presence of various concentrations of cholesterol. It was also relatively unaffected by trypsin or pronase treatment and independent of Ca2+ concentration. The optimal temperature and pH were 4 degrees C and 2 respectively. All of these characteristics differ from those of the LDL receptors. These results suggest that apo A1 is a major ligand of HDL3 receptors.
...
PMID:[Characterization of HDL receptor and its ligand in cultured smooth muscle cells isolated from rabbit aorta]. 165 83

Cultured cells possess high-affinity binding sites (receptors) for high density lipoprotein (HDL) that appear to mediate removal of excess intracellular cholesterol from cells. To examine the role of intact HDL apoproteins in receptor-mediated cholesterol removal, HDL3 apoproteins were digested with the proteolytic enzymes trypsin and pronase, and the residual particles were used in sterol efflux experiments. Protease treatment abolished the interaction of HDL3 with the 110-kd cell membrane protein postulated to represent the HDL receptor molecule, indicating that this interaction is mediated by HDL apoproteins rather than lipids. Compared with native HDL3 protease-modified HDL3 had a markedly reduced ability to selectively remove sterol from intracellular pools, even though modified particles promoted greater cholesterol efflux from the plasma membrane than did native particles. These results indicate that whereas sterol efflux from plasma membranes is mediated by HDL lipids, removal of excess intracellular sterol from cells is mediated by HDL apoproteins. These findings are consistent with the hypothesis that receptor binding of HDL apoproteins stimulates translocation of excess intracellular sterol to the cell surface where it becomes accessible for removal by HDL or other lipid-rich acceptor particles.
...
PMID:High density lipoprotein apolipoproteins mediate removal of sterol from intracellular pools but not from plasma membranes of cholesterol-loaded fibroblasts. 199 58

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.
...
PMID:Neutrophil association and degradation of normal and acute-phase high-density lipoprotein 3. 244 74

Cultured smooth muscle cells of rabbit aorta were studied by 125I labelled rabbit HDL3. Saturation curves, measured at 4 C, showed the presence of two different components: the low-affinity non-saturable binding portion and the high-affinity binding portion (Kd about 5.6 x 10(-8) mol/L and Bmax about 0.321 micrograms/mg cell protein). Scatchard analyses of the high-affinity binding portion suggest the presence of single class binding sites. Binding of rabbit HDL3 to cultured smooth muscle cells was relatively resistant to trypsin or pronase, and independent to Ca2+. The binding rate of 125I-HDL3 to the smooth muscle cells was highest at 4 C and the optimal pH was 2. Additionally, presence of high concentration apoAI reduced 50% of the binding rate of 125I-HDL3, and 125I-HDL3, being pretreated (blocked) with rat anti-rabbit apoAI IgG of different concentrations lost 70% of its original binding rate with smooth muscle cells. The results suggest that rabbit aorta smooth muscle cells possess a specific binding sites for apoE-free HDL which recognizes apoAI as a ligand.
...
PMID:Characterization of HDL binding sites and its ligand in cultured smooth muscle cells of rabbit's aorta. 251 72

High density lipoprotein (HDL3) binds with high affinity to many types of cells, but controversy exists concerning the nature and biological significance of the binding. We have recently demonstrated that HDL and apoproteins (apo)-AI, -AII, and -CI stimulate a specific and dose-dependent increase in placental lactogen (hPL) release from human trophoblast cells. To examine the possible relationship between HDL3 binding and stimulation of hPL release, we have characterized the binding of [125I]HDL3 to an enriched fraction of hPL-producing trophoblast cells. Binding studies were performed on trophoblast cells isolated by isopycnic centrifugation of collagenase/hyaluronidase-dispersed placental tissue and apo-E free-HDL3 (density, 1.125-1.215 g/ml). Scatchard analysis of binding studies performed at 37 C for 2 h revealed two classes of binding sites: 1) high affinity binding sites with a Kd of 9.7 +/- 2.2 micrograms/ml (1.3 x 10(-7) M) and 9.8 +/- 3.2 x 10(5) binding sites/trophoblast cell, and 2) low affinity binding sites with a Kd of 172.8 +/- 64.8 micrograms/ml (2.3 x 10(-6) M) and an estimated 3.2 x 10(6) sites/cell. As has been found in hepatocytes and other cells, the number of HDL3-binding sites per trophoblast cell (but not the binding affinity) decreased at lower incubation temperatures. In addition, HDL3 binding to trophoblasts cells did not require calcium and was not affected by prior treatment of the cells with pronase or trypsin. HDL3-binding sites on trophoblast cells, however, were not specific for HDL3. Low density lipoprotein (density, 1.063-1.055 g/ml), which does not stimulate hPL release, was nearly as potent on a molar basis as HDL3 in binding to the high and low affinity binding sites on trophoblast cells. Furthermore, nitrated HDL3, which does not compete for high affinity binding to trophoblast cells, stimulated hPL release. Although the characteristics of HDL3 binding to trophoblast cells are similar to those of other cells, these results strongly suggest that the binding of HDL3 to high affinity binding sites is not essential for HDL-mediated hPL release.
...
PMID:High density lipoprotein3 binding and biological action: high affinity binding is not necessary for stimulation of placental lactogen release from trophoblast cells. 258 47

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.
...
PMID:Discrimination between subclasses of human high-density lipoproteins by the HDL binding sites of bovine liver. 300 Apr 54

Cultured extrahepatic cells possess a specific high affinity receptor for high density lipoprotein (HDL) that is induced by cholesterol delivery to cells. Current results suggest that HDL receptors on cultured human fibroblasts and mouse peritoneal macrophages promote reversible binding of HDL to the cell surface without internalization of lipoprotein particles. When 125I-HDL3 was bound to cultured cells at 0 degrees C and then warmed to 37 degrees C after removal of unbound lipoprotein, most of the cell surface-bound HDL was released rapidly (t1/2 = 3 min) into the medium without entering a cellular pool that was inaccessible to digestion by trypsin at 0 degrees C. This lack of internalization of HDL was evident under conditions where internalization of 125I-low density lipoprotein and 125I-transferrin were readily detected. When cells were exposed to 125I-HDL3 at 37 degrees C, only a trace amount of iodinated apoprotein remained associated with cells after treatment of cells with trypsin. Fibroblasts treated with medium containing increasing concentrations of cholesterol exhibited a dose-dependent increase in reversible, trypsin-sensitive binding of 125I-HDL3 at 37 degrees C without an attendant increase in trypsin-resistant binding. These results suggest that reversible binding of HDL to its cell-surface receptor without subsequent endocytosis of receptor-HDL complexes is the mechanism by which HDL receptors facilitate cholesterol transport from cells.
...
PMID:Interaction of high density lipoprotein with its receptor on cultured fibroblasts and macrophages. Evidence for reversible binding at the cell surface without internalization. 302 79


1 2 Next >>

---