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 nature of the polypeptide backbone of human apolipoprotein (apo) E present in the very low density lipoproteins (VLDL) of normal and homozygous type III hyperlipoproteinemic patients was investigated by tryptic cleavage fingerprinting and specific chemical modification studies. Apo E from normal subjects was resolved on polyacrylamide isoelectric focussing gels into five bands (apo E-I', E-I, E-II, E-III, and E-IV), whereas apo E from type III patients was resolved into three bands (apo E-I3', E-I3, and E-II3). The apo E isoforms, contained within unstained polyacrylamide gel slices, were washed to remove ampholytes, desialylated, and digested with L-1-tosylamido-2-phenylethylchloromethyl ketone treated trypsin. Autoradiography of 125I-labelled tryptic apo E peptides showed complete identity between all isoforms from normal subjects. High performance liquid chromatographic (HPLC) analysis showed that complete peptide identity exists between apo E-I', E-I, and E-II and between apo E-I3', E-I3, and E-II3. Distinct HPLC peptide profiles were found for apo E-II, E-III, E-IV, and E-II3. These resolved peak differences were reproducible between runs, between digests, and between apo E isolations, suggesting that the distinct profiles were neither a result of artifacts nor of contamination. Specific chemical modification studies revealed that human apo E isomorphism is due, in part, to differences in arginine and cysteine residues but not to lysine residues. These findings indicate that human apo E isomorphism results from differences in the primary amino acid sequence of the individual isoforms in addition to charged carbohydrate heterogeneity. Furthermore, the apo E isomorphic profile observed in homozygous type III hyperlipoproteinemic patients reflects both a deficiency of apo E-III and E-IV and the presence of the altered apo E-II isoprotein (apo E-II3).
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PMID:Tryptic peptide analysis of the human apolipoprotein E isomorphs. 717 90

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

Due to the great length of apolipoprotein (apo) B-100, the localization of lipid-associating domains in this protein has been difficult. To address this question, we developed a computer program called Locate that searches amino acid sequences to identify potential amphipathic alpha-helixes and beta-strands by using sets of rules for helix and strand termination. A series of model chimeric protein test datasets were created by tandem linking of amino acid sequences of multiple proteins containing four different secondary structural motifs: motif A (exchangeable plasma apolipoproteins); motif G (globular alpha-helical proteins); motif C (coiled-coil alpha-helical proteins); and motif B (beta pleated-sheet proteins). These four test datasets, as well as randomly scrambled sequences of each dataset, were analyzed by Locate using increasingly stringent parameters. Using intermediately stringent parameters under which significant numbers of amphipathic helixes were found only in the unscrambled motif A, two dense clusters of putative lipid-associating amphipathic helixes were located precisely in the middle and at the C-terminal end of apoB-100 (a sparse cluster of class G* helixes is located at the N-terminus). The dense clusters are located between residues 2103 through 2560 and 4061 through 4338 and have densities of 2.4 and 2.2 amphipathic helixes per 100 residues, respectively; under these conditions, motif A has a density of 1.4 amphipathic helixes per 100 residues. These two domains correspond closely to the two major apoB-100 lipid-associated domains at residues 2100 through 2700 and 4100 through 4500 using the principle of releasability of tryptic peptides from trypsin-treated intact low-density lipoprotein. The classes of amphipathic helixes identified within these two putative lipid-associating domains are considerably more diverse than those found in the exchangeable plasma apolipoproteins. Interestingly, apoB-48 terminates at the N-terminal edge of the middle cluster. By using a similar strategy for analysis of amphipathic beta-strands, we discovered that the two gap regions between the three amphipathic helix clusters are highly enriched in putative amphipathic beta-strands, while the three amphipathic helical domains are essentially devoid of this putative lipid-associating motif. We propose, therefore, that apoB-100 has a pentapartite structure, NH2-alpha 1-beta 1-alpha 2-beta 2-alpha 3-COOH, with alpha 1 representing a globular domain.
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PMID:apoB-100 has a pentapartite structure composed of three amphipathic alpha-helical domains alternating with two amphipathic beta-strand domains. Detection by the computer program LOCATE. 791 18

Apolipoprotein B-100 (apoB-100) is the major protein in low-density lipoprotein (LDL) and contains the ligand for binding LDL to its cell surface receptor. Lipoprotein [a] (Lp[a]) is a lipoprotein that consists of LDL and apolipoprotein [a] (apo[a]). The primary structure of apoB-100 has been determined by a combination of recombinant DNA and protein sequencing methods. Using high-performance liquid chromatographic techniques, we have identified sulfhydryl and disulfide groups of apoB-100 from LDL. Sixteen of the 25 cysteine residues in apoB-100 exist in disulfide form. All 14 cysteine residues within the N terminal end of apoB-100 are linked in disulfide bridges. Using the fluorescent sulfhydryl probe, 5-iodoacetoamidofluoresceine, two free sulfhydryls of apoB-100 on LDL were identified at positions 3734 and 4190. Based on its differential susceptibility to trypsin, apoB-100 can be divided into five domains: domain 1 (residues 1-1000), largely trypsin-releasable (TR); domain 2 (residues 1001-1700), alternating TR and trypsin non-releasable (TN); domain 3 (residues 1701-3070), largely TN; domain 4 (residues 3071-4100), mainly TR and mixed; and domain 5 (residues 4101-4536), almost exclusively TN. Based on our data, we propose that the structure of apoB-100 in LDL is probably an elongated form that wraps around the LDL particle, and that Cys3734 of apoB-100 may be the cysteine residue linked to a cysteine of apo[a].
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PMID:Primary structure of apoB-100. 818 50

Apolipoproteins share a common structural feature, their interaction with phospholipids. It is believed that amphipathic helical sequences enable apolipoproteins to bind to lipid bilayer and to form discoidal particles of defined dimensions. While the knowledge of the apo A-I sequence and secondary structure has been used to make predictions about its mode of association with lipids, the available experimental data necessary to propose a precise model of these discoidal structures are still limited. An important step in our understanding of these structures would be to identify the apolipoprotein lipid-associated domains. Proteolysis of apo A-I-DMPC reconstituted HDL (rHDL) and free apo A-I is used here to identify lipid-protected domains of apo A-I. Free cleaved peptides were separated from rHDL associated peptides by density gradient centrifugation. The lipid-associated peptides were further analyzed by SDS-PAGE and transferred by Western blot to a ProBlott membrane for sequencing. Cleavage occurred at residue 43 with proteinase K, 46 with trypsin and residue 47 or 48 with pronase. A large domain from about residue 45 to the C-terminal remains highly protected against hydrolysis eventhough it contains several bonds susceptible to proteolytic cleavage. No protected fragments were detected by SDS-PAGE after enzymatic cleavage of free apo A-I in identical experimental conditions.
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PMID:Enzymatic hydrolysis of reconstituted dimyristoylphosphatidylcholine-apo A-I complexes. 837 88

Involvement of cellular surface factors in cellular lipid efflux mediated by lipid-free apolipoprotein has been investigated. Lipid-free human apolipoprotein (apo) A-I generated net efflux of cholesterol and phospholipid from mouse peritoneal macrophages and rat aorta smooth muscle cells. Ratio of cholesterol to phospholipid was much lower in the lipid released by this mechanism from the smooth muscle cells than that from the macrophages, in agreement with our previous observation (Li, Q., Komaba, A. and Yokoyama, S. (1993) Biochemistry 32, 4597-4603). On the other hand, free apoA-I did not cause any lipid efflux from human erythrocytes. In contrast, apparent efflux of cellular cholesterol to HDL was similarly observed from all of these three cellular membranes. Trypsin treatment of the cultured macrophages completely inhibited apoA-I-mediated efflux of cholesterol and phospholipid. Smooth muscle cells also showed complete inhibition of the apoA-I-mediated cellular lipid efflux by trypsin treatment except that it required longer incubation with the enzyme. The same cellular treatment with trypsin even by prolonged incubation had only a limited effect on apparent cellular cholesterol efflux to HDL and apolipoprotein-free lipid microemulsions. Thus, free apolipoprotein-mediated cellular lipid efflux seems to depend on a trypsin-susceptible cellular surface factor(s) that erythrocytes may lack, being distinct from physicochemical cholesterol exchange reaction between cell and lipoprotein.
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PMID:Involvement of a cellular surface factor(s) in lipid-free apolipoprotein-mediated cellular cholesterol efflux. 854 29

We studied the role of microsomal triglyceride transfer protein (MTP) in the synthesis, secretion, and cotranslational degradation of apolipoprotein (apo) B using nonhepatic COS-7 cells that expressed C-terminally truncated forms of apoB (from apoB15 to apoB94) with or without the large subunit of human MTP. With the exception of apoB15 and apoB18, secretion of all of the apoB forms was stimulated by expression of MTP, even though a small amount of short apoB forms (</=apoB48) could be secreted by cells transfected with apoB alone. The majority of the apoB protein, including apoB72 and apoB94, was secreted as high density lipoprotein (1.08-1.17 g/ml). Pulse-chase experiments revealed that the secretion efficiency of apoB94 and apoB72 was low (ranging from 2 to 12%). The failure to secrete buoyant lipoproteins and the low secretion efficiency were associated with insufficient lipid synthesis by the cells. The incorporation of [3H]oleate into cellular triglyceride and phosphatidylcholine by COS cells over a 2-h period was 28 and 38%, respectively, of that by rat hepatoma (McA-RH7777) cells. In addition to the desired full-length apoB, cells transfected with large constructs (>/=apoB60) also produced smaller species with a size of approximately220 kDa (designated B48-like protein). Coexpression with MTP decreased formation of the B48-like proteins by 40-60%. The reduction in B48-like protein formation was specific to MTP expression; coexpression with other proteins (e.g. apoA-I or apoB15) did not alter B48-like protein production. Kinetic analysis suggested that B48-like proteins were produced concurrently (cotranslational) with the full-length apoB94 and apoB72 and were not products of post-translational degradation. Although some of the B48-like proteins might be derived from truncated species (approximately 7 kb in size) of apoB mRNA that were found in cells transfected with large apoB constructs, MTP coexpression did not affect the relative levels of the aberrant 7-kb RNA with respect to the full-length mRNA. However, coexpression of MTP decreased the accessibility of apoB to exogenous trypsin by 2-fold for apoB72 and by 10-fold for apoB94 in isolated microsomes. Thus, the reduced B48-like protein formation by MTP may be a consequence of attenuated cotranslational degradation during apoB translocation across the ER membrane. Formation of B48-like proteins was insensitive to N-acetyl-leucyl-leucyl-norleucinal, a cysteine protease inhibitor known to block post-translational degradation of apoB. These results indicate that MTP facilitates the assembly and secretion of lipoproteins containing apoB and also attenuates the formation of B48-like proteins, probably by assisting apoB translocation across the ER membrane.
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PMID:The microsomal triglyceride transfer protein facilitates assembly and secretion of apolipoprotein B-containing lipoproteins and decreases cotranslational degradation of apolipoprotein B in transfected COS-7 cells. 866 86

We studied the structural requirements of apolipoprotein (apo) B for assembly of very low density lipoproteins (VLDL) using rat hepatoma McA-RH7777 cells expressing human apoB (h-apoB). Recombinant h-apoB48, like endogenous rat apoB48 (r-apoB48), was secreted as VLDL in addition to high density lipoproteins (HDL) by transfected cells, indicating that the N-terminal 48% of apoB contains sequences sufficient for VLDL assembly. Truncation of the C terminus of h-apo-B48 to -B42 or -B37 had little effect on the ability of apoB to assemble VLDL, whereas truncation to -B34 or -B29 markedly diminished or abolished VLDL formation. None of the truncations affected the integration of apoB into HDL. To determine whether the ability to assemble VLDL is governed by apoB length or by sequences beyond apoB29, we created chimeric proteins that contained human apoA-I and a segment derived from between the C-terminal 29 and 34%, 34 and 37%, or 37 and 42% of apoB100. The resulting chimeras, namely AI/B29-34, AI/B34-37, and AI/B37-42, were secreted by the transfected cells as lipoproteins with buoyant density (d < 1.006 g/ml), electrophoretic mobility (pre-beta), and size characteristics of human plasma VLDL. The chimeras could assemble discrete VLDL particles devoid of endogenous r-apoB100, and could actively recruit triglycerides and phospholipids into the lipoproteins. However, these chimeras were secreted inefficiently. Pulse-chase analysis showed that less than 5% of the newly synthesized AI/B proteins were secreted, and more than 70% was degraded intracellularly. Degradation of the chimeras could be blocked by the cysteine protease inhibitor N-acetyl-leucyl-leucyl-norleucinal, but the treatment did not enhance their secretion. Protease protection analysis of microsomes isolated from transfected cells indicated that >65% of AI/B chimeras (compared with <25% of r-apoB100) were inaccessible to exogenous trypsin. These data suggest that the recruitment of large quantities of triglycerides during VLDL formation is not governed simply by apoB length, but is mediated by short hydrophobic sequences ranging from 152 to 237 amino acids (3-5%) of apoB. The existence of multiple such hydrophobic sequences within apoB48 may facilitate efficient assembly of hepatic VLDL particles.
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PMID:Apolipoprotein B sequence requirements for hepatic very low density lipoprotein assembly. Evidence that hydrophobic sequences within apolipoprotein B48 mediate lipid recruitment. 870 89

Density gradient ultracentrifugation and anion-exchange chromatography combination were effective for the purification of the eel vitellogenin from the plasma of estradiol-treated eels. The vitellogenin was very high density glycolipoprotein (P = 1.27 g/ml) and its apolipoprotein was M(r) 196 k in both reduced and non-reduced conditions by SDS-PAGE. The major lipid component was phospholipid. The N-terminal amino-acid sequence of the vitellogenin was as follows: (Ac)Thr-Pro-Ala-Leu/Ala-Asp-Tyr. Amino-acid composition of the eel vitellogenin was similar to those of other teleosts. The protease activity appeared in the trypsinized vitellogenin, but was not detected in the purified vitellogenin. The protease was separated from the used trypsin and the other cleaved vitellogenin by a dextran sulfate cellulose column. The molecular weight of the protease was determined by zymogram using SDS-polyacrylamide gel containing casein and was 50 k. It was concluded that the eel vitellogenin possesses the protease activity as a latent form.
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PMID:Protease activity appeared after trypsin treatment of the purified vitellogenin from eel Anguilla japonica. 882 6

Neomycin therapy reduces plasma levels of low density lipoprotein and lipoprotein[a] (Lp[a]). To determine whether neomycin directly alters the biogenesis of Lp[a], we have examined the effect of neomycin on apolipoprotein[a] (apo[a]) synthesis and secretion in primary cultures of baboon hepatocytes. Using this system, we have previously shown that apo[a] is synthesized as a lower molecular weight precursor that upon maturation becomes associated with the cell surface before release into the culture medium. Treatment of hepatocytes with 10 mM neomycin reduced levels of apo[a] in the culture medium by as much as 12-fold. Although a portion of the reduced secretion could be accounted for by a reduction in total protein synthesis, the greatest effect of neomycin on apo[a] secretion was to decrease the release of mature apo[a] from the hepatocyte cell surface into the culture medium. Treatment of hepatocyte cultures with trypsin confirmed that mature apo[a] in neomycin-treated cells was still transported to the cell surface. Examination of related antibiotics demonstrated that inhibition of apo[a] secretion is a general property shared by the deoxystreptamine antibiotics. The mechanism by which neomycin affects the apo[a]-cell surface interaction is not known, but neomycin is known to perturb cell surface membranes, inhibit the interaction of some ligands with their cell surface receptors, and inhibit the metabolism of phosphatidylinositol 4,5 biphosphate. These studies suggest that cell surface association of apo[a] may play a role in Lp[a] biogenesis in vivo.
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PMID:Neomycin inhibits secretion of apolipoprotein[a] by increasing retention on the hepatocyte cell surface. 890 83


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