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 binding of low density lipoprotein (LDL) to fibroblasts occurs through apolipoprotein B, a glycoprotein. The role of the carbohydrate in binding was assessed in two ways: (1) LDL, freed of sialic acid and most of the glucosamine and hexoses by digestion with a mixture of glycosidases, bound to fibroblasts as does native LDL. (2) The glycopeptides liberated from apoprotein B by trypsin and pronase failed to inhibit LDL binding to fibroblasts. Apparently the carbohydrate moiety of LDL does not interact with the plasma membrane receptor.
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PMID:The absence of a role for the carbohydrate moiety in the binding of apolipoprotein B to the low density lipoprotein receptor. 21 98

To explore the process of lipoprotein assembly, plasmids encoding truncated forms of apolipoprotein B (apoB) were transfected into Chinese hamster ovary (CHO) fibroblasts. (One, encoding apoB53, the N-terminal 53% of apoB100, can direct the assembly and secretion of lipoproteins when expressed in hepatoma cells, while the other, encoding the shorter apoB15, does not direct lipoprotein assembly.) Expression of apoB15 in CHO cells resulted in the accumulation of apoB15 protein in both medium and cells. In contrast, apoB was not detectable in medium or within CHO cells transfected with the plasmid encoding apoB53, despite the expression of apoB53 mRNA. ApoB53 did accumulate within transfected cells incubated with the thiol protease inhibitor N-acetylleucylleucylnorleucinal (ALLN), suggesting that it is synthesized but completely degraded in the absence of the inhibitor. ApoB53 was not secreted despite its presence within ALLN-treated cells. Essentially all the apoB53 that accumulated in microsomes from ALLN-treated cells was associated with the membrane and was susceptible to degradation by exogenous trypsin, indicating exposure on the cytoplasmic face of the membrane. Thus, translocation of apoB53 across the endoplasmic reticulum membrane is blocked. However, the apoB53 bound to concanavalin A, suggesting that it is glycosylated and therefore partly exposed to the lumen as well. ApoB requires a unique process, not expressed in CHO fibroblasts, for its complete translocation and entrance into the secretory pathway. This process might account for the inability of abetalipoproteinemic patients to secrete apoB.
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PMID:Translocation of apolipoprotein B across the endoplasmic reticulum is blocked in a nonhepatic cell line. 140 18

Previously, we isolated and characterized unique liposomal-like, cholesterol-rich lipid particles that accumulate in human atherosclerotic lesions. Human plasma low density lipoprotein (LDL) has a molar ratio of total cholesterol to phospholipid (3:1) similar to that of this lesion cholesterol-rich lipid particle. However, LDL is enriched in cholesteryl ester while the lesion lipid particle is enriched in unesterified cholesterol. To examine a possible precursor-product relationship between LDL and the lesion lipid particle, we hydrolyzed the cholesteryl ester core of LDL with cholesterol esterase. Cholesteryl ester hydrolysis occurred only after LDL was treated with trypsin. Trypsin pretreatment was not required for cholesteryl ester hydrolysis of LDL oxidized with copper, a treatment that also degrades apolipoprotein B, the major protein moiety in LDL. In contrast to greater than 90% hydrolysis of cholesteryl ester in trypsin-cholesterol esterase-treated or copper-oxidized LDL, there was only 18% hydrolysis of cholesteryl ester in similarly treated high density lipoprotein. With a limited 10-min hydrolysis of LDL cholesteryl ester, LDL-sized particles and newly formed larger flattened films or discs were present. With complete hydrolysis of LDL cholesteryl ester, LDL particles converted to complex multilamellar, liposomal-like, structures with sizes approximately five times larger than native LDL. These liposomal-like particles derived from LDL were chemically and structurally similar to unesterified cholesterol-rich lipid particles that accumulate in atherosclerotic lesions.
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PMID:Hydrolysis of cholesteryl ester in low density lipoprotein converts this lipoprotein to a liposome. 153 75

Twenty-three of the 25 cysteine residues in apolipoprotein B-100 have been isolated directly from tryptic or peptic peptide mixtures. Sixteen cysteine residues exist in disulfide forms: Cys-1-Cys-3, Cys-2-Cys-4, Cys-5-Cys-6, Cys-7-Cys-8, Cys-9-Cys-10, Cys-11-Cys-12, Cys-13-Cys-14, and Cys-20-Cys-21. All of these except Cys-20-Cys-21 are recently discovered disulfide linkages. In addition to Cys-22 and Cys-24, which have been described as sulfhydryls on low density lipoprotein, Cys-15 to Cys-18 and Cys-23 are in the reduced form. Cys-19 and Cys-25 are not yet confirmed. Our results revealed that all identified disulfide linkages are located in the trypsin-releasable regions and that all except Cys-1-Cys-3 and Cys-2-Cys-4 are linked to the neighboring cysteine. We propose a linear model of apolipoprotein B-100 in low density lipoprotein that wraps around the low density lipoprotein molecule.
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PMID:Isolation and characterization of sulfhydryl and disulfide peptides of human apolipoprotein B-100. 211 73

Beta very low density lipoprotein (VLDL) was isolated from a patient with hepatic lipase deficiency. The particles were found to contain apolipoprotein B-100 (apoB) and apolipoprotein E (apoE) and were rich in cholesterol and cholesteryl ester relative to VLDL with pre beta electrophoretic mobility. These particles were active in displacing human low density lipoprotein (LDL) from the fibroblast apoB,E receptor and produced a marked stimulation of acyl-CoA:cholesterol acyltransferase. Treatment of intact beta-VLDL with trypsin abolished its ability to displace LDL from fibroblasts. Incubation of trypsin treated beta-VLDL with fibroblasts resulted in a significant stimulation of acyl-CoA:cholesterol acyltransferase activity. beta-VLDL isolated from a patient with Type III hyperlipoproteinemia and an apoE2/E2 phenotype had a higher cholesteryl ester/triglyceride ratio than the beta-VLDL of hepatic lipase deficiency and contained apoB48. It displaced LDL from fibroblasts to a small but significant extent. The Type III beta-VLDL stimulated acyl-CoA:cholesterol acyltransferase to a level similar to that of trypsin-treated beta-VLDL isolated from the hepatic lipase-deficient patient. These results demonstrate that the cholesterol-rich beta-VLDL particles present in patients with hepatic lipase deficiency are capable of interacting with fibroblasts via the apoB,E receptor and that this interaction is completely due to trypsin-sensitive components of the beta-VLDL. These particles were very effective in stimulating fibroblast acyl-CoA:cholesterol acyltransferase. This stimulation was due to both trypsin-sensitive and trypsin-insensitive components.
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PMID:The beta very low density lipoprotein present in hepatic lipase deficiency competitively inhibits low density lipoprotein binding to fibroblasts and stimulates fibroblast acyl-CoA:cholesterol acyltransferase. 317 May 42

Methodology for determining amino acid sequences of proteins by tandem mass spectrometry is described. The approach involves enzymatic and/or chemical degradation of the protein to a collection of peptides which are then fractionated by high-performance liquid chromatography. Each fraction, containing as many as 10-15 peptides, is then analyzed directly, without further purification, by a combination of liquid secondary-ion/collision-activated dissociation mass spectrometry on a multianalyzer instrument. Interpretation of collision-activated dissociation mass spectra is described, and results are presented from a study of soluble peptides produced by treatment of apolipoprotein B with cyanogen bromide and trypsin.
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PMID:Protein sequencing by tandem mass spectrometry. 346 91

Apolipoprotein(a) [apo(a)] is a glycoprotein with Mr approximately equal to 280,000 that is disulfide linked to apolipoprotein B in lipoprotein(a) particles. Elevated plasma levels of lipoprotein(a) are correlated with atherosclerosis. Partial amino acid sequence of apo(a) shows that it has striking homology to plasminogen. Plasminogen is a plasma serine protease zymogen that consists of five homologous and tandemly repeated domains called kringles and a trypsin-like protease domain. The amino-terminal sequence obtained for apo(a) is homologous to the beginning of kringle 4 but not the amino terminus of plasminogen. Apo(a) was subjected to limited proteolysis by trypsin or V8 protease, and fragments generated were isolated and sequenced. Sequences obtained from several of these fragments are highly (77-100%) homologous to plasminogen residues 391-421, which reside within kringle 4. Analysis of these internal apo(a) sequences revealed that apo(a) may contain at least two kringle 4-like domains. A sequence obtained from another tryptic fragment also shows homology to the end of kringle 4 and the beginning of kringle 5. Sequence data obtained from two tryptic fragments show homology with the protease domain of plasminogen. One of these sequences is homologous to the sequences surrounding the activation site of plasminogen. Plasminogen is activated by the cleavage of a specific arginine residue by urokinase and tissue plasminogen activator; however, the corresponding site in apo(a) is a serine that would not be cleaved by tissue plasminogen activator or urokinase. Using a plasmin-specific assay, no proteolytic activity could be demonstrated for lipoprotein(a) particles. These results suggest that apo(a) contains kringle-like domains and an inactive protease domain.
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PMID:Partial amino acid sequence of apolipoprotein(a) shows that it is homologous to plasminogen. 347 6

Human plasma low density lipoproteins (LDL) are the major carriers of cholesterol and cholesteryl esters in the circulation. Their increased levels correlate positively with increased risk of coronary artery disease. LDL contain a single major apolipoprotein of apparent molecular weight (Mr) = 550,000, designated apolipoprotein B-100 (apoB-100), and in some LDL preparations, minor components termed apoB-74 (410,000) and apoB-26 (145,000). The structural relationship of the apoB-74 and -26 proteins to the apoB-100 has remained obscure and their roles in cholesterol metabolism are unknown. In the present study, we show that the addition of kaolin to plasma anticoagulated with EDTA induces the proteolytic cleavage of apoB-100. As a result, two apoB peptides are produced with Mr indistinguishable from plasma apoB-74 and -26. The specific cleavage of apoB-100 was mimicked in vitro by purified human plasma and tissue kallikreins. In contrast, thrombin, factor Xa, plasmin, trypsin, and chymotrypsin did not produce these peptides when incubated with LDL. The findings of the study suggest that apoB-74 and -26 are proteolytic fragments of apoB-100 and that the endogenous protease has a kallikrein-like specificity for DLD-apoB-100. The role of plasma and tissue kallikreins in cholesterol metabolism remains to be determined.
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PMID:Processing of apolipoprotein B-100 of human plasma low density lipoproteins by tissue and plasma kallikreins. 364 5

Apolipoprotein B-100, the major protein constituent of human plasma low-density lipoproteins (LDL), was carboxyamidomethylated, digested with trypsin and the water-soluble tryptic peptides were coincubated with liposomes of dimyristoylphosphatidylcholine (DMPC). At 24.3 degrees C the peptides induced lipid solubilization as evidenced by optical clearing of the lipid-peptide mixture. Lipid-peptide complexes were isolated by density-gradient ultracentrifugation in KBr and had the following properties: DMPC/peptide ratio of 5.6 (w/w); buoyant density of 1.07-1.09 g/ml; discoidal morphology (51 +/- 4 X 260 +/- 28 A) as determined by electron microscopy; and molecular weight of 1.5 X 10(6) as determined by nondenaturing polyacrylamide gel electrophoresis. Compared to liposomes and sonicated vesicles of DMPC, the lipid-peptide complexes had a more rigid structure as assessed by fluorescence polarization. Whereas intact LDL had 42% alpha-helix and 15% beta-pleated sheet, the lipid-peptide complexes contained 70% alpha-helix and less than 5% beta-pleated sheet. The lipid-peptide complexes did not bind to the fibroblast high-affinity LDL receptor. These results show that specific regions in apolipoprotein B-100 which interact with phospholipid have an amphipathic character and may represent primary sites for lipid-protein interaction in LDL.
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PMID:Interaction of tryptic peptides of apolipoprotein B-100 with dimyristoylphosphatidylcholine. 373 Apr 6

The effect of trypsin treatment on the heparin- and receptor-binding properties of human plasma low-density lipoproteins (LDL) was examined. LDL were treated with trypsin (2% by weight) for 16 h at 37 degrees C, and the trypsinized core particles (T-LDL) were isolated by gel permeation chromatography on Sepharose CL-4B. Trypsin degraded the apolipoprotein B moiety (Mr = 550,000) of LDL into numerous peptides of Mr less than 110,000, resulting in the release of 25% +/- 5% (n = 6) of its surface-associated protein. Relative to LDL, T-LDL had an increased phospholipid/protein ratio, decreased flotation density and alpha-helical structure, and increased fluidity of the surface and core constituents. Compared to LDL, T-LDL showed a 60% decreased capacity to suppress [1-14C]acetate incorporation into cellular sterols consistent with decreased binding to the LDL receptor. In contrast, T-LDL showed an enhanced capacity to form soluble complexes with heparin in the absence and presence of 2 mM Ca2+. Between 5 and 25 mM Ca2+, both LDL and T-LDL were maximally precipitated by heparin; the stoichiometry of the insoluble complexes (uronic acid/phospholipid, w/w) was 0.054 +/- 0.004 and 0.055 +/- 0.005 (n = 18) for LDL and T-LDL, respectively. Thus, trypsin treatment significantly diminished the lipoprotein's interaction with cells but not with heparin. This finding suggests that proteolysis may decrease receptor-mediated uptake of LDL without diminishing the lipoprotein's reactivity with acellular components of the arterial wall.
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PMID:Effect of trypsin treatment on the heparin- and receptor-binding properties of human plasma low-density lipoproteins. 376 46


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