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)

Development of the cellular slime mould Dictyostelium discoideum strain NC4, in the presence of alpha-chymotrypsin (3 mg/ml) is reversibly arrested at the tight aggregate stage (10/12 h). Pronase has a similar effect, but trypsin only retards normal development by about five hours. Normally developing cells are susceptible to alpha-chymotrypsin if they are transferred into its presence at any time up to the tight aggregate stage (10-12 h). Transfer after this stage does not affect the appearance of fruiting body structures in the normal time (24 h). Electron microscopy showed the ultrastructure of alpha-chymotrypsin-blocked aggregates after starvation for 24 h to be consistent with a block at 10-12 h of normal development. Poorly developed prespore vacuoles, having thin incomplete walls and a paucity of electron-dense material, are present in some cells. No angular vacuolated cells characteristic of stalk cells are visible. Fruiting bodies formed in the presence of a alpha-chymotrypsin, either as minority structures when the enzyme is added before 10-12 h of normal development, or as the majority structures on later enzyme addition, were found to be abnormal. Normal stalks were formed but the spores were immature. Prespore vacuoles were present, though disrupted, and the cells were not encapsulated by spore walls. The electronegativity of intact slime mould amoebae was significantly reduced, and material containing L-[6-3H]-fucose and [1-14C]leucine was removed from the cell surface on alpha-chymotrypsin treatment. Few plasma membrane proteins were affected, however, and staining of polyacrylamide gels for glycopeptides using Con A-peroxide binding also showed little change.
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PMID:The effect of chymotrypsin on the development of Dictyostelium discoideum. 743 Sep 29

The histidine-rich protein hisactophilin is known to be associated with the inner surface of the plasma membrane and to be present as a soluble protein in the cytoplasm of Dictyostelium discoideum cells. Mass spectrometry of hisactophilin from the cytosol or extracted from a membrane fraction showed that none of the hisactophilin purified from D. discoideum cells had the mass predicted from the known cDNA-derived amino acid sequence of the protein. Electrospray mass spectrometry and liquid secondary ion mass spectrometry of tryptic fragments separated by reversed-phase high performance liquid chromatography (HPLC) identified the most hydrophobic peptide as a myristoylated fragment from the N terminus of hisactophilin. Taken together the analytical data, it is concluded that all hisactophilin in D. discoideum cells is N terminally modified by myristoylation. By reversed-phase HPLC, two isoforms of hisactophilin, HsI and HsII, were recovered from the cytosolic as well as the membrane fraction of D. discoideum cells. Whereas the masses of HsI fragments produced by trypsin fit into the previously published sequence of hisactophilin (myristoylation considered), HsII is another protein distinguished from HsI by several amino acid exchanges. HsI and HsII can form homo- and heterodimers by disulfide bridges. Hisactophilin is phosphorylated in vivo. Both isoforms proved to be substrates of membrane-associated threonine/serine kinase from D. discoideum, which may regulate the interaction of hisactophilin with the plasma membrane.
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PMID:The pH-sensitive actin-binding protein hisactophilin of Dictyostelium exists in two isoforms which both are myristoylated and distributed between plasma membrane and cytoplasm. 782 84

The sexual cycle of the cellular slime mold Dictyostelium discoideum offers a suitable system to analyze the mechanism of cell recognition during mating. Sexual cell fusion in D. discoideum typically occurs between complementary heterothallic strains. In addition, several bisexual strains are known which undergo sexual cell fusion with heterothallic strains of either mating type, but cannot do so by themselves. In the present study, trypsin digestion of cell surface molecules was found to induce self-fusion in a bisexual strain WS2162, suggesting the presence on the cell surface of a self-recognition molecule whose homophilic interaction interferes with the cell fusion mechanism.
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PMID:Protease-sensitive component(s) on the cell surface prevents self-fusion in a bisexual strain of Dictyostelium discoideum. 872 72

Two forms of the NAD-dependent glutamate dehydrogenase were partially purified from Dictyostelium discoideum, an activated and a non-activated form. V(max) for the non-activated enzyme was stimulated 88-fold and the activated enzyme 3-fold by 0.1 mM AMP (at their pH optima). Half maximal stimulation by AMP is achieved at 221 +/- 39 microM for the non-activated enzyme and 20 +/- 2 microM for the activated enzyme. We have shown that activation of NAD-GDH in vivo has many similarities to trypsin treatment of non-activated enzyme and that proteolysis is the probable mechanism of activation.
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PMID:Kinetic properties and the mechanism of activation of NAD-dependent glutamate dehydrogenase from Dictyostelium discoideum. 872 2

Changes in the actin-myosin interface are thought to play an important role in microfilament-linked cellular movements. In this study, we compared the actin binding properties of the motor domain of Dictyostelium discoideum (M765) and rabbit skeletal muscle myosin subfragment-1 (S1). The Dictyostelium motor domain resembles S1(A2) (S1 carrying the A2 light chain) in its interaction with G-actin. Similar to S1(A2), none of the Dictyostelium motor domain constructs induced G-actin polymerization. The affinity of monomeric actin (G-actin) was 20-fold lower for M765 than for S1(A2) but increasing the number of positive charges in the loop 2 region of the D. discoideum motor domain (residues 613-623) resulted in equivalent affinities of G-actin for M765 and for S1. Proteolytic cleavage and cross-linking approaches were used to show that M765, like S1, interacts via the loop 2 region with filamentous actin (F-actin). For both types of myosin, F-actin prevents trypsin cleavage in the loop 2 region and F-actin segment 1-28 can be cross-linked to loop 2 residues by a carbodiimide-induced reaction. In contrast with the S1, loop residues 559-565 of D. discoideum myosin was not cross-linked to F-actin, probably due to the lower number of positive charges. These results confirm the importance of the loop 2 region of myosin for the interaction with both G-actin and F-actin, regardless of the source of myosin. The differences observed in the way in which M765 and S1 interact with actin may be linked to more general differences in the structure of the actomyosin interface of muscle and nonmuscle myosins.
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PMID:Differences in the ionic interaction of actin with the motor domains of nonmuscle and muscle myosin II. 1010 95

Under starvation conditions, amoebae of Dictyostelium discoideum aggregate to form multicellular masses; the aggregates are then initiated to differentiate. We have reported previously that a signal substance exists in conditioned medium of D. discoideum, and we named it prespore-cell-inducing factor (psi, Psi factor) [Oohata, Nakagawa, Tasaka, and Fujii (1997) Development 124, 2781-2787]. The factor can induce isolated amoebae to differentiate into prespore cells. Moreover, we suggested that it caused not only cell differentiation but also cell division. In the present study, we have purified Psi factor from the conditioned medium and characterized it. The purified Psi factor induced both prespore cell differentiation and cell division of prespore cells. Its apparent molecular mass was 180 kDa by gel filtration and 106 kDa by SDS/PAGE. Based on these results, Psi factor exists as a dimer in normal conditions. Periodic acid/Schiff staining showed that Psi factor was a glycoprotein. It was ascertained by Edman degradation that Psi factor is blocked at the N-terminal. Treatment with pyroglutamate aminopeptidase removed the N-terminal block and allowed determination of the amino-acid sequence of Psi factor. Moreover, three internal amino-acid sequences were determined in limited proteolysis experiments using trypsin and endoproteinase Lys-C. The homology search for these sequences supports the fact that Psi factor is a novel differentiation factor.
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PMID:A prespore-cell-inducing factor in Dictyostelium discoideum: its purification and characterization. 1049 38

When Dictyostelium cells starve, they begin secreting a glycoprotein called conditioned medium factor (CMF). When there is a high density of starved cells, as indicated by a high concentration of CMF, the cells begin expressing some genes and aggregate using pulses of cAMP as a chemoattractant. CMF regulates gene expression via a G protein-independent pathway, whereas CMF regulates cAMP signal transduction via a G protein-dependent pathway. To elucidate receptors mediating cell density sensing, we used CMF-Sepharose to isolate membrane proteins that bind CMF. We identified a 50-kDa protein, CMFR1, that is sensitive to trypsin treatment of whole cells. We obtained partial amino acid sequence of CMFR1 and isolated the cDNA encoding it. The derived amino acid sequence has no significant similarity to known proteins and has two or three predicted transmembrane domains. Expression of CMFR1 in insect cells caused an increase in CMF binding. Repression of CMFR1 in Dictyostelium by gene disruption resulted in a approximately 50% decrease of the CMF binding and a loss of CMF-induced G protein-independent gene expression. The G protein-dependent CMF signal transduction pathways appear to be functional in cmfr1 cells, suggesting that cells sense the density-sensing factor CMF using two or more different receptors.
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PMID:A putative receptor mediating cell-density sensing in Dictyostelium. 1056 29

The soluble N-ethylmaleimide-sensitive-factor-attachment proteins (SNAP) are eukaryotic soluble proteins required for membrane fusion. Based on their initial identification in bovine brain cytosol, they are divided in alpha/beta and gamma subfamilies. SNAPs act as adapters between N-ethylmaleimide-sensitive factor (NSF), a hexameric ATPase, and membrane SNARE proteins (SNAP receptors). Within the NSF/SNAP/SNARE complex, SNAPs contribute to the catalysis of an ATP-driven conformational change in the SNAREs, resulting in dissociation of the complex. We have constructed a Dictyostelium discoideum strain overexpressing a c-myc-tagged form of D. discoideum NSF (NSF-myc). Its immunoprecipitation from detergent-solubilized membrane extracts reveals two associated polypeptides with apparent molecular masses of 33 and 36 kDa (p33 and p36) that are absent in NSF-myc immunoprecipitates from cytosol. Analysis of trypsin-digested peptides by microsequencing and mass spectrometry and comparison with cDNA sequences identify p33 and p36 as the D. discoideum homologues of alpha- and gamma-SNAP, respectively. The alpha-/gamma-SNAP molar ratio is close to 3 in vegetative amoebae from this organism. The molecular identification of gamma-SNAP in plants (Arabidopsis thaliana) and insects (Drosophila melanogaster) documents, for the first time, the wide distribution of the gamma subtype. Altogether, these results suggest a specific role for gamma-SNAP, distinct from that of alpha-SNAP.
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PMID:Functional and molecular identification of novel members of the ubiquitous membrane fusion proteins alpha- and gamma-SNAP (soluble N-ethylmaleimide-sensitive factor-attachment proteins) families in Dictyostelium discoideum. 1072 46

The social amoeba Dictyostelium discoideum exhibits high activities of phospholipase and lysophospholipase [Ferber, Munder, Fischer and Gerisch (1970) Eur. J. Biochem. 14, 253-257]. We assayed Dictyostelium lysates to demonstrate the presence of a highly active phospholipase B (PLB) enzyme that removed both fatty-acid chains from phosphatidylcholine and produced the water-soluble glycerophosphorylcholine. We purified the PLB activity from Dictyostelium cytosol using standard agarose media (size exclusion and ion exchange), and combined this with an affinity purification step using myristoylated ARF1 (ADP-ribosylation factor 1), a protein which has a single fatty acid at its N-terminus. Two proteins co-purified (48 kDa and 65 kDa), and the 48 kDa protein was digested with trypsin, peptide fragments were separated by reverse-phase chromatography, and the resultant peptides were sequenced by Edman degradation. From the peptide sequences obtained, database searches revealed a gene which encodes a protein of 65 kDa with unknown function. The 48 kDa protein therefore appears to be a fragment of the full-length 65 kDa product. Expression of the gene in Escherichia coli confirmed that it encodes a PLB. Characterization of its substrate specificity indicated that, in addition to phosphatidylcholine deacylation, the enzyme also hydrolysed phosphatidylinositol and phosphatidylethanolamine. The PLB identified in the present study is not related to existing PLBs found in bacteria, fungi or mammals. There are, however, genes similar to Dictyostelium PLB in mammals, flies, worms and Giardia, but not in yeast. We therefore have identified a novel family of intracellular PLBs.
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PMID:Identification of phospholipase B from Dictyostelium discoideum reveals a new lipase family present in mammals, flies and nematodes, but not yeast. 1519 48

The nuclear proteasome activator REGgamma/PA28gamma is an ATP- and ubiquitin-independent activator of the 20S proteasome and has been proposed to degrade and thereby regulate both a key human oncogene, encoding the coactivator SRC-3/AIB1, and the cyclin-dependent kinase inhibitor p21 (Waf/Cip1). We report the identification and characterization of a PA28/REG homolog in Dictyostelium. Association of a recombinant Dictyostelium REG with the purified Dictyostelium 20S proteasome led to the preferential stimulation of the trypsin-like proteasome peptidase activity. Immunolocalization studies demonstrated that the proteasome activator is localized to the nucleus and is present in growing as well as starving Dictyostelium cells. Our results indicate that the Dictyostelium PA28/REG activator can stimulate both the trypsin-like and chymotrypsin-like activities of the 20S proteasome and supports the idea that the REGgamma-20S proteasome represents an early unique nuclear degradation pathway for eukaryotic cells.
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PMID:Characterization of a REG/PA28 proteasome activator homolog in Dictyostelium discoideum indicates that the ubiquitin- and ATP-independent REGgamma proteasome is an ancient nuclear protease. 1941 24


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