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)

Angiotensin I-converting enzyme [EC 3.4.15.1] was rapidly and highly purified from a particulate fraction of hog kidney cortex with 13% yield. The procedure, which was rapid, included fractionation on DEAE-cellulose and calcium phosphate gel, chromatographies on DEAE-Sephadex A-50 and hydroxylapatite columns, and gel filtration on a Sephadex G-200 column. The purified enzyme preparation gave two protein bands on standard disc gel electrophoresis, but showed a single protein component on the gel after treatment with neuraminidase [EC 3.2.1.18]. The data strongly suggest that the purified enzyme preparation was a mixture of sialo- and asialo-enzyme. Sialic acid residues apparently do not contribute to the catalytic activity of the enzyme. The enzyme was activated more by chloride ions than by other halide ions tested, using Bz-Gly-Gly-Gly as a substrate. The dissociation constant for chloride ions was determined to be 2.2 mM. Chloride did not protect the enzyme against heat or low pH. The enzyme was resistant to inactivation by trypsin [EC 3.4.21.4] and chymotrypsin [EC 3.4.21.1].
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PMID:Renal angiotensin I-converting enzyme as a mixture of sialo- and asialo-enzyme, and a rapid purification method. 1 Feb 87

Sialic acid has been shown to be part of the antigenic determinant of the Thy-1.2 alloantigen as expressed by the murine cell line S-49.1 TB-2-3 (S-49). This conclusion is based on the loss of cytotoxic inhibitory activity by the action of neuraminidase on the Thy-1.2 alloantigen. Also, sialic acid has the ability to inhibit the cytotoxic assay of AKR anti-C3H Thy-1.2 serum for S-49 cells. Further evidence for the protein nature of the Thy-1.2 alloantigen is apparent by trypsin digestion. Possibly the Thy-1.2 alloantigen as expressed on S-49 cells is a glycoprotein.
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PMID:Studies on the antigenic determinants of the Thy-1.2 alloantigen as expressed by the murine lymphoblastoid line S-49.1 TB-2-3. 5 44

We present evidence for the existence in rat brain of several sialyltransferases able to sialylate sequentially asialofetuin. [14C]Sialylated glycans of asialofetuin were analyzed by gel filtration. Three types of [14C]sialylated glycans were synthesized: N-glycans and monosialylated and disialylated O-glycans. The varying effects of N-ethylmaleimide, lysophosphatidylcholine (lysoPtdCho) and trypsin, were helpful in the identification of these different sialyltransferases. One of them, selectively inhibited by N-ethylmaleimide, was identified as the Neu5Ac alpha 2----3Gal beta 1----3GalNAc-R:alpha 2----6 sialyltransferase previously described [Baubichon-Cortay, H., Serres-Guillaumond, M., Louisot, P. and Broquet, P. (1986) Carbohydr. Res. 149, 209-223]. This enzyme was responsible for the synthesis of disialylated O-glycans. LysoPtdCho and trypsin selectively inhibited the enzyme responsible for the synthesis of monosialylated O-glycan. N-ethylmaleimide, lysoPtdCho and trypsin did not inhibit Neu5Ac transfer onto N-glycans, giving evidence for three different molecular species. To identify the enzyme responsible for monosialylated O-glycan synthesis, we used another substrate: Gal beta 1----3GalNAc--protein obtained after galactosylation of desialylated ovine mucin by a GalNAc-R:beta 1----3 galactosyltransferase from porcine submaxillary gland. This acceptor was devoid of N-glycans and of NeuAc in alpha 2----3 linkages on the galactose residue. When using N-ethylmaleimide we obtained the synthesis of only one product, a monosialylated structure. After structural analysis by HPLC on SAX and SiNH2 columns, we identified this product as Neu5Ac alpha 2----3Gal beta 1----3GalNAc. The enzyme leading to synthesis of this monosialylated O-glycan was identified as a Gal beta 1----3GalNAc-R:alpha 2----3 sialyltransferase. When using lysoPtdCho and trypsin, sialylation was completely abolished, although the Neu5Ac alpha 2----3Gal beta 1----3GalNAc-R:alpha 2----6 sialyltransferase was not inhibited. We provided thus evidence for the interpendence between the two enzymes, the alpha 2----3 sialyltransferase regulates the alpha 2----6 sialyltransferase activity since it synthesizes the alpha 2----6 sialyltransferase substrate.
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PMID:Evidence for an O-glycan sialylation system in brain. Characterization of a beta-galactoside alpha 2,3-sialyltransferase from rat brain regulating the expression of an alpha-N-acetylgalactosaminide alpha 2,6-sialyltransferase activity. 247 71

N-Acetylneuraminic acid (NeuNAc) is the terminal sugar residue of the O-linked tetrasaccharide linked to erythrocyte sialoglycoproteins, glycophorins. Erythrocytes lacking NeuNAc have been shown previously to be resistant to invasion by certain isolates of Plasmodium falciparum merozoites. We report here variation between different geographic isolates of P. falciparum in their dependency on NeuNAc for invasion of host erythrocytes. Seven different geographic isolates of P. falciparum were examined for their ability to invade neuraminidase treated erythrocytes. For all isolates invasion was reduced significantly, although considerable variation in NeuNAc dependency was apparent. Three isolates, FCR-3, FVO and It2, exhibited a very high dependence on NeuNAc residues for invasion (invasion reduced greater than 90%), whereas two isolates (Thai-Tn and FC-27) exhibited a moderately high dependence (invasion reduced 75%). Two other isolates (CDC-1 and 7G8) exhibited moderate dependence on NeuNAc (invasion reduced 50%). Cleavage of the complete O-linked tetrasaccharide by O-glycanase removes all carbohydrate from glycophorin A, B and C except the single N-linked oligosaccharide on glycophorin A and C. Invasion of FCR-3 and CDC-1 isolates into O-glycanase treated erythrocytes was not markedly different from that into neuraminidase treated cells indicating that NeuNAc is the important residue of the tetrasaccharide for both isolates. Invasion into endo-beta-galactosidase treated erythrocytes, in which the lactosaminoglycan side chain of band 3 and band 4.5 is cleaved, was not significantly reduced for either the CDC-1 or FCR-3 isolates. Additional results on the trypsin insensitivity of band 3 also suggest that this erythrocyte protein is not important in P. falciparum recognition. The greatest divergence in receptor specificity between FCR-3 and CDC-1 isolates was apparent in invasion into periodate-treated erythrocytes. Periodate oxidation results in cleavage of the exocyclic hydroxyl groups of the terminal NeuNAc but leaves its COOH group unaltered. These experiments also illustrated that the negatively charged COOH group of NeuNAc is not the important group in the interaction of the merozoite with the NeuNAc. Trypsin-treated erythrocytes were almost fully resistant to invasion by CDC-1 as well as the FCR-3 isolates suggesting that the CDC-1 isolate, in addition to interacting with NeuNAc, depends on a trypsin sensitive site for invasion. This site could involve the N-linked saccharide on glycophorin A and C or a protein on the erythrocyte surface unrelated to the glycophorins.
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PMID:Erythrocyte receptor recognition varies in Plasmodium falciparum isolates. 283 May 8

New serotypes were sought among 165 clinical isolates of group-B streptococci that were untypable by antisera for the conventional types Ia, Ib, II and III. The strains were tested for sialic acid, an integral component of the group-B streptococcal type-polysaccharides; trypsin-treated bacteria were tested by slide-agglutination with the sialic-acid-specific lectin from the snail Cepaea hortensis. Sialic acid was detected in 96 of the strains; in 95 of these, new type antigens were identified serologically (type IV, 52; provisional type V, 34; candidate type NT6, seven; provisional type V and candidate type NT6, one; candidate type 7271, one); the remaining strain was found to possess a small amount of Ia antigen. Sialic acid was not detected in 69 strains, and none of these possessed a polysaccharide type-antigen. Chemical measurement of the sialic-acid content of cultures by Aminoff's method gave results in conformity with the lectin-agglutination test and the presence of polysaccharide type-antigens.
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PMID:A search for new group-B streptococcal serotypes. 352 96

Studies on the morphology, cell biology, and immunology of invasion have characterized events that are now being studied at the molecular level. The initial events of invasion are receptor-specific. A determinant associated with Duffy blood group antigens is involved in the invasion of human erythrocytes by P. knowlesi and P. vivax. The Duffy Fya antigen has recently been identified and further characterization of its role in reception and invasion should now be possible. P. falciparum utilizes erythrocyte ligands that differ from those of P. knowlesi and P. vivax. Sialic acid and a trypsin-sensitive erythrocyte membrane component are important for invasion by P. falciparum parasites. There is evidence that at least two ligands are involved in invasion. For P. knowlesi there is a ligand for attachment, common to both Duffy-negative and Duffy-positive human erythrocytes, and a second ligand for invasion, which is found only on Duffy-positive human erythrocytes. P. vivax also appears to utilize two ligands, a Duffy-associated ligand and a ligand specific for reticulocytes. P. falciparum binds to sialic acid-dependent and sialic acid-independent trypsin-sensitive ligands. P. falciparum merozoites require erythrocyte sialic acid to varying degrees in order to invade; this indicates heterogeneity of the receptor mechanism. Monoclonal antibodies and recombinant DNA technology have greatly facilitated the identification, isolation, and characterization of proteins that may be involved in invasion. Molecules that may have invasion-related functions include those whose antibodies block invasion, those that bind to erythrocyte ligands important for invasion, those that appear on the merozoite surface, and those that appear to be inserted into the erythrocyte membrane at the time of invasion. It has not been possible to identify a definite function for any of the molecules identified thus far. No monoclonal or polyclonal monospecific antibody has been identified that reacts specifically over the surface of the apical region of the merozoite where junction formation occurs. Identification of molecules responsible for apical attachment and junction formation will be important for our understanding of invasion. In terms of vaccine development, it is not yet known whether any of the molecules discussed here will prove to be effective immunogens. It is clear from the data obtained with the 140-kd protein of P. knowlesi that antigenic variation poses a potential problem for vaccine development. As the molecular events responsible for invasion become better understood, novel ways may be devised to interfere with the process and prevent the disease.
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PMID:Invasion of erythrocytes by malaria parasites: a cellular and molecular overview. 353 49

The regression and turnover of the surface glycoconjugates of trypsin-prepared pig and human cultured epidermal cells have been determined using the glycoprotein precursors N-acetyl-D-(I-3H) glucosamine (3H-NAG) and N-(3H)-acetyl-D-mannosamine (3H-NAM). Sialic acid assays have been performed on similar unlabelled cells. The major points which emerged from this study were: (1) Trypsin-damaged cell surfaces are rapidly repaired, probably by normal membrane turnover. There was a 12% regeneration of sialic acid within 2 h and total resynthesis occurred within 24 h. (2) The presence of an internal membrane system, part of which also demonstrates turnover, probably contributed to the speed of surface membrane repair. Some of the glycoprotein/sialic acid of this internal membrane system (30%) remains bound for a considerable length of time. (3) The membrane turnover maintains the cell in equilibrium so that total loss equals the synthesis of glycoprotein. (4) The equilibration of 3H-NAG or 3H-NAM uptake between 24 and 48 h is limited by the relative concentrations of glucose and labelled sugar in the medium at this time. (5) 3H-NAm was a more specific marker of glycoprotein than 2H-NAG. (6) The results for human epidermal cells closely matched those for pig epidermal cells, indicating that pig cells can be used as a model for human cells.
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PMID:Synthesis and turnover of membrane glycoconjugates in monolayer culture of pig and human epidermal cells. 724 76

During a previous study of the opsonic requirements for neutrophil (polymorphonuclear leukocyte [PMN])-mediated killing of enterococci, we identified two strains of Enterococcus faecium (TX0015 and TX0016) that were resistant to PMN-mediated killing. To better define the mechanism of this resistance, we examined phagocytosis with a fluorescence assay and found that TX0016 was completely resistant to phagocytosis by PMNs; this finding was confirmed by electron microscopy. Examination of multiple strains of enterococci revealed that all 20 strains of Enterococcus faecalis tested were readily phagocytosed (mean, 18 intracellular organisms per PMN; range, 7 to 28). In contrast, only 13 (50%) of 26 strains of E. faecium tested were susceptible to phagocytosis (> or = 7 organisms per PMN); the other 13 strains showed < or = 3 organisms per PMN. Enterococcus casseliflavus ATCC 25788 and one strain of Enterococcus hirae were also resistant to phagocytosis, while two strains of Enterococcus durans, Enterococcus mundtii ATCC 43186, and one strain each of Enterococcus raffinosus and Enterococcus solitarius were readily phagocytosed. Exposure of E. faecium TX0016 to sodium periodate, but not to the protease trypsin or pronase or to phospholipase C, eliminated resistance to phagocytosis. Sialic acid, a common periodate-sensitive structure used by microorganisms to resist opsonization, could not be demonstrated in E. faecium TX0016 by the thiobarbituric acid method, nor was phagocytosis of TX0016 altered by neuraminidase treatment. This study suggests that there is a difference in susceptibility to phagocytosis by PMNs between different species of enterococci and that a carbohydrate-containing moiety which is not sialic acid may be involved in the resistance of E. faecium TX0016 to phagocytosis.
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PMID:Resistance of Enterococcus faecium to neutrophil-mediated phagocytosis. 796 Jan 41

The distribution and internalization of anionic sites in heart muscle cells (HMC) were studied by direct measurements of their zeta potential (ZP) and by ultrastructural cytochemistry. Our data showed that HMC are negatively charged and that their anionic sites are distributed over the entire sarcolemma. Treatments with neuraminidase and trypsin altered the ZP value and also reduced binding of cationized ferritin (CF) to the sarcolemma. Sialic acid was shown to be an important component on the surface of HMC, since its removal reduced the cell surface negative charge by 25%. Phospholipase C did not significantly change the surface charge, nor did it alter HMC reactivity to CF particles when compared with control cells. Endocytosis of anionic sites was investigated using two different protocols that allow follow-up of this dynamic process. Incubation of HMC with cationized ferritin particles at 37 degrees C induced a redistribution of ligand-bound anionic sites, followed by their internalization or detachment. The clustering of anionic sites on the surface of HMC indicates that these cells are characterized by a high level of membrane fluidity. CF particles were localized inside early and late endosomes, lysosomes, and also in ferritin-enriched vesicles near the sarcolemma. An endocytic pathway for anionic sites in HMC is discussed.
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PMID:The nature of anionic sites and the endocytic pathway in heart muscle cells. 814 29

Recombinant human lymphotoxin (rhLT) produced by CHO cells transfected with human LT genomic DNA was purified to homogeneity, but approximately 5% of the molecules were devoid of the last two amino terminal residues. A peptide N-glycosylated at Asn62 (Tr-45) and one partially O-glycosylated at Thr7 (Tr-14) on cleavage with trypsin were separated by reverse phase HPLC. The N-linked sugar chains of Tr-45 were released quantitatively as oligosaccharides on hydrazinolysis (100 degrees C, 8 h), followed by N-acetylation. After being reduced with either NaB3H4 or NaB2H4, their structures were determined by a combination of serial lectin affinity chromatography, exoglycosidase digestion, and methylation analysis: 82.7% of the sugar chains occur as biantennary complex-type sugar chains, the remainder being C-2 and C-2,4/C-2,6 branched triantennary, and C-2,4 and C-2,6 branched tetraantennary complex-type sugar chains with a fucosylated mannose core. Their sialic acid residues occur only as the Neu5Ac alpha 2-->3Gal group. The clearance velocity from the bloodstream dramatically increased with desialylation, and rhLT tends to have accumulated in the kidney, indicating that there may exist other mechanisms for clearance from the circulation besides the galactose-binding protein in hepatocytes and the filtration system of the kidney. Desialylated rhLT showed a lectin-like binding character to uromodulin similar to that of tumor necrosis factor, although intact rhLT did not. The interaction between desialylated rhLT and uromodulin was inhibited by N,N'-diacetylchitobiose and [Man alpha 1-->6(Man alpha 1-->3)Man alpha 1-->6](Man alpha 1-->2Man alpha 1-->3)Man beta 1-->4GlcNAc beta 1-->4GlcNAc-->Asn. These results indicate that the lectin-like domain of rhLT is exposed on its desialylation.
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PMID:N-linked sugar chain structure of recombinant human lymphotoxin produced by CHO cells: the functional role of carbohydrate as to its lectin-like character and clearance velocity. 832 80


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