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)

It is known that the negatively stained preparations of inner mitochondrial membrane display characteristic approximately 9 nm F1 (ATPase) knobs projecting from the matrix surface. Freeze-etch studies have reported the absence of such knobs from the "etched" surface of the inner mitochondrial membranes. We have demonstrated their presence on the surface of SMP (submitochondrial particles) prepared by freeze-drying for transmission electron microscopy. This identification has been substantiated by comparison with freeze-dried TU particles (trypsin-urea treated SMP) that are devoid of F1 (ATPase). It has been suggested that a layer of water molecules is strongly adsorbed to the surface of SMP and does not sublime during normal freeze-"etching."
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PMID:Visualization of mitochondrial coupling factor F1(ATPase) by freeze-drying. 9 68

Three specimen preparation techniques for electron microscopy were used to investigate the incorporation of the ATPase polypeptide chains in the membranes of fragmented sarcoplasmic reticulum (SR) obtained from rabbit skeletal muscle. Observations were made of both normal vesicles and vesicles exposed to trypsin, which is known to cleave the ATPase protein and to alter the ultrastructure of the vesicles in predictable ways. Freeze-fracture replicas reveal the typical 90-A particles on the concave (PF) faces with a density of 5,730 +/- 520/mum2. On the other hand both negatively stained and deeply etched preparations display outer projections, which are absent on trypsin-incubated vesicles. The etched specimens afford for the first time top views of the vesicles in the absence of any stain. These views reveal outer projections on the PS surface with a density of 21,000 +/- 3,900/mum2, a value nearly approximating the density of the ATPase polypeptide chains (106,000 mol wt) calculated on the basis of protein and membrane area determinations. On the other hand, this value is three to four times higher than that found for the density of the 90-A particles on the concave fracture faces. Since both outer projections and 90-A particles are identified with the ATPase protein, it is suggested that the ATPase polypeptide chains are amphiphilic molecules, with polar ends protruding individually as outer projections on the surface of the vesicles, and hydrophobic ends appearing as 90-A particles on the concave fracture faces. The discrepancy between the densities of the outer projections and the 90-A particles may be attributed either to variable penetration of the polypeptide chains into the membrane bilayer, or to formation of oligomers containing three or four hydrophobic ends and appearing as single 90-A particles. Each ATPase chain forms a complex with 20-30 phospholipid molecules. The remaining phospholipids (approximately 70% of the total SR phospholipids) account for less than half the membrane volume. It is proposed that the outer leaflet of the SR membrane is prevalently composed of the ATPase lipoprotein complex, and the inner leaflet is mostly a phospholipid monolayer.
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PMID:Assembly of ATPase protein in sarcoplasmic reticulum membranes. 13 72

Two fractions of gastric mucosal membranes obtained by Ficoll-sucrose density gradient centrifugation were studied by a variety of techniques to localize the polypeptides. Gel electrophoresis showed the presence of five major polypeptides and several minor ones. Only one of these, 82,000 daltons, was available for iodination in the intact tissue. The two membrane fractions differed in their accessibility to peroxidase. The denser fraction showed two major defined iodination peaks at 82,000 and 102,000 daltons. Freeze-thawing and iodinating with 131-I produced additional labeling of peaks as well as relabeling the 82,000-dalton component, showing it was accessible from both sides of the membrane. The two major components were also sensitive to cross-linking, the 102,000 polypeptide being especially sensitive to --SH oxidation. Proteolysis with trypsin removed both components in the denser membrane fraction, in addition to inhibiting the K+-ATPase and K+-p-nitrophenylphosphatase of that fraction. Phosphorylation with [gamma-32-P]ATP labeled the 102,000-dalton component and K+, HCO3- minus and p-nitrophenylphosphate reduced the level of labeling. Hence the 102,000 region contains a subunit of the ATPase, is readily iodinated in inside-out vesicles, and is the most available for interpeptide S--S cross-linking.
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PMID:Characterization of gastric mucosal membranes. VIII. The localization of peptides by iodination and phosphorylation. 16 6

Histophysiology, ultrastructure, chemical analyses of transplants and implants of Dunn and Ridgway mouse osteosarcomas demonstrate that tumorigenesis is a manifestation of deranged morphogenesis in developing mesenchymal cell populations. The end product of development is defective, incompletely calcified, disorganized bone without any inclusions of bone marrow tissue. When Dunn osteosarcoma is freeze-dried and then implanted, the tumor is resorbed and replaced by deposits of normal cartilage, bone, and bone marrow. Freeze-dried Ridgway osteosarcoma is replaced only by a fibrous connective tissue scar. Disaggregated Dunn tumor osteoblasts synthesize a trypsin-labile collagenase-resistant cell surface localized bone morphogen. Tumor matrix stroma, prepared by sequential chemical extraction of soluble non-collagenous proteins also contains significant quantities of the same bone morphogen. Tumor tissue pulverized to particle size as small as 44 micrometer3 transmitted bone morphogen more rapidly than intact tumor tissue. The total tumor cell and stroma mediated bone morphogen produces three times more normal bone than normal cortical bone matrix. Our working hypothesis is that a normal bone morphogenetic polypeptide (BMP) is synthesized by Dunn osteosarcoma cells and retained by the tumor matrix stroma. Neither the mechanism of transmission nor the mesenchymal cell receptor sites of BMP are known.
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PMID:An osteosarcoma cell and matrix retained morphogen for normal bone formation. 27 29

A method is described for isolating glycophorin-enriched vesicles from human erythrocytes by extracting membranes that were incubated for 30 min at 37 degrees C at pH 4.5 and washed at low and high ionic strength with the nonionic detergent Triton X-100. The extracts were 11.8 +/- 2.4 fold enriched in glycophorin and contained 325 +/- 69 microgram sialic acid/mg protein, which represented 61 +/- 16% of the total sialic acid. Upon removal of Triton X-100 one third of the total glycophorin forms glycophorin-enriched vesicles with coextracted, endogenous lipids as shown sedimintation, dextran-density gradient centrifugation, and electron microscopy. Addition of exogenous lipids increased the fraction of glycophorin-enriched vesicles up to 87%. The incorporation of glycophorin in the membrane was shown by hemagglutination inhibition assays using anti-M sera and by the accessibility of glycophorin to trypsin. Freeze-fractured vesicles did not reveal intramembranous particles. The selectivity of the extraction procedure is not simply due to chemical constraints introduced by disulfide cross-linkage of protein component 3, because only 20% of this protein undergo disulfide cross-linking. The selective extraction of glycophorin implies that glycophorin is segregated from protein component 3 and thus from intramembranous particles when erythrocyte membranes have been incubated at pH 4.5. This segregation may precede aggregation of intramembranous particles.
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PMID:Glycophorin-enriched vesicles obtained by a selective extraction of human erythrocyte membranes with a non-ionic detergent. 44 5

The three-dimensional crystal structure of bovine trypsinogen at approximately pH 7.5 was initially solved at 2.6 A resolution using the multiple isomorphous replacement method. Preliminary refinement cycles of the atomic coordinates trypsinogen have been carried out first to a resolution of 2.1 A, and later to 1.9 A, using constrained difference Fourier refinement; During the process, structure factors Fc and phi c were calculated from the trypsinogen structure and final interpretation was based on an electron-density map computed with terms (2 Fo - Fc) and phases phic at a resolution of 1.9 A. Crystals of trypsinogen grown from ethanol-water mixtures are trigonal with space group P3121, and cell dimension a = 55.17 A and c = 109.25 A. The structure is compared with the bovine diisopropylphosphoryltrypsin structure at approximately pH 7.2, oirginally determined from orthohombic crystals by Stroud et al. (Stroud, R.M., Kay L.M., and Dickerson, R.E. (1971), Cold Spring Harbor Symp. Quant. Biol. 36, 125-140; Stroud, R.M., Kay, L.M., and Dickerson, R.E. (1974), J. Mol. Biol. 83, 185-208), and later refined at 1.5 A resolution by Chambers and Stroud (Chambers, J.L., and Stroud, R.M. (1976), Acta Crystallogr. (in press)). At lower pH, 4.0-5.5 diogen, with cell dimensions a = 55.05 A and c = 109.45 A. This finding was used in the solution of the six trypsinogen heavy-atom derivatives prior to isomorphous phase analysis, and as a further basis of comparison between trypsinogen and the low pH trypsin structure. There are small differences between the two diisopropylphosphoryltrypsin structures. Bovine trypsinogen has a large and accessible cavity at the site where the native enzyme binds specific side chains of a substrate. The conformation and stability of the binding site differ from that found in trypsin at approximately pH 7.5, and from that in the low pH form of diisopropylphosphoryltrypsin. The catalytic site containing Asp-102, His-57, and Ser-195 is similar to that found in trypsin and contains a similar hydrogen-bounded network. The carboxyl group of Asp-194, which is salt bridged to the amino terminal of Ile-16 in native trypsin or other serine proteases, is apparently hydrogen bonded to internal solvent molecules in a loosely organized part of the zymogen structure. The unusually charged N-terminal hexapeptide of trypsinogen, whose removal leads to activation of the zymogen, lies on the outside surface of the molecule. There are significant structural changes which accompany activation in neighboring regions, which include residues 142-152, 215-550, 188A-195. The NH group of Gly-193, normally involved in stabilization of reaction intermediates (Steitz, T.A., Henderson, R., and Blow, D.M. (1969), J. Mol. Biol. 46, 337-348; Henderson, R. (1970), J. Mol. Biol. 54, 341-354; robertus, J.D., Kraut, J., Alden, R.A., and Birkoft, J.J. (1972), Biochemistry 11, 4293-4303) in the enzyme, is moved 1.9 A away from its position in trypsin...
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PMID:Structure of bovine trypsinogen at 1.9 A resolution. 55 51

Galline, a protamine of domestic fowl, was obtained by two preparation procedures from the semen of a strain of White Plymouth Rock and submitted to fractionation by column chromatography on Bio-Gel CM-30. In the first procedure the specimen prepared from sperm heads was purified by the use of distilled water and dilute acetic acid and fractionated into almost eight fractions (G-I-G-VIII) in the same way as the specimen from a strain of New Hampshire (1,2). No difference could be found between galline specimens from the two different strains based on the amino acid and terminal analyses of each fraction. The specimen of galline from sperm heads purified with 1% citric acid (the second procedure) was composed of only one component, which was isolated as a single peak. The smaller fractions, G-I-G-VII, were found to be derived from G-VIII by the action of trypsin-like protease contained in the extract of sperm heads with 1% citric acid. This enzyme seems to originate in the acrosome of fowl spermatozoa. Consequently, it is concluded that intact galline is composed of only one molecular species and its total amino acid sequence is represented by the completed formula of G-VIII as shown in the preceding paper (4).
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PMID:Studies on a protamine (galline) from fowl sperm. 4. Degradation of galline by trypsin-like protease of fowl sperm heads. 99 42

The amino acids of Lady Amherst's pheasant and golden pheasant egg-white lysozymes have been sequenced. The carboxymethylated lysozymes were digested with trypsin followed by sequencing of the tryptic peptides. Lady Amherst's pheasant lysozyme proved to consist of 129 amino acid residues, and a relative molecular mass of 14,423 Da was calculated. This lysozyme had 6 amino acids substitutions when compared with hen egg-white lysozyme: Phe3 to Tyr, His15 to Leu, Gln41 to His, Asn77 to His, Gln 121 to Asn, and a newly found substitution of Ile124 to Thr. The amino acid sequence of golden pheasant lysozyme was identical to that of Lady Amherst's phesant lysozyme. The phylogenetic tree constructured by the comparison of amino acid sequences of phasianoid birds lysozymes revealed a minimum genetic distance between these pheasants and the turkey-peafowl group.
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PMID:The amino acid sequence of Lady Amherst's pheasant (Chrysolophus amherstiae) and golden pheasant (Chrysolophus pictus) egg-white lysozymes. 136 78

A Kunitz-type trypsin inhibitor was purified from Leaf-nosed viper venom and the primary structure determined by peptide analysis. In relation to other trypsin inhibitors, the protein has an extended C-terminal segment and a distinct pattern of residue alterations at the functionally important contact sites with proteases.
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PMID:Purification and characterization of a Kunitz-type trypsin inhibitor from Leaf-nosed viper venom. 174 83

Factor IX is the zymogen of the serine protease factor IXa involved in blood coagulation. In addition to a catalytic domain homologous to the chymotrypsin family, it has Ca2+, phospholipid, and factor VIIIa binding regions needed for full biologic activity. We isolated a nonfunctional factor IX protein designated factor IXEagle Rock (IXER) from a patient with hemophilia B. The variant protein is indistinguishable from normal factor IX (IXN) in its migration on sodium dodecyl sulfate-gel electrophoresis, isoelectric point in urea, carbohydrate content and distribution, number of gamma-carboxyglutamic acid residues, and beta-OH aspartic acid content, and in its binding to an anti-IXN monoclonal antibody which has been shown previously to inhibit the interaction of factor VIIIa with factor IXaN. Further, IXER is cleaved to yield a factor IXa-like molecule by factor XIa/Ca2+ at a rate similar to that observed for IXN. However, in contrast to IXaN, IXaER does not bind to antithrombin-III (specific inhibitor of IXaN) and does not catalyze the activation of factor X (substrate) to factor Xa. To identify the mutation in IXER, all eight exons of IXN and IXER gene were amplified by the polymerase chain reaction technique and cloned. A single point mutation (G----T) which results in the replacement of Val for Gly363 in the catalytic domain of IXER was identified. Gly363 in factor IXa corresponds to the universally conserved Gly193 in the active site sequence of the chymotrypsin serine protease family. X-ray crystallographic data in the literature demonstrate a critical role of this Gly in stabilizing the active conformation of chymotrypsin/trypsin in two major ways: 1) in the formation of the substrate binding site; and 2) in the development of the oxyanion hole. Our computer structural data support a concept that the Gly363----Val change prevents the development of the active site conformation in factor IXa such that the substrate binding site and the oxyanion hole are not formed in the mutated enzyme.
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PMID:Experimental and theoretical evidence supporting the role of Gly363 in blood coagulation factor IXa (Gly193 in chymotrypsin) for proper activation of the proenzyme. 230 34


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