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 cAMP receptor on the surface of aggregation competent Dictyostelium discoideum cells specifically binds [3H]cAMP in an oscillatory manner with a periodicity of 2 min. The oscillatory cAMP-binding component is developmentallly regulated and has the nucleotide specificity expected for recognition of chemotactic signals. The concentration dependence of the peak amplitudes of cAMP binding exhibit an apparent threshold at 10(-8) M cAMP. The threshold concentration for cAMP binding that we measure is consistent with the concentration dependence of signal relay (cAMP secretion) and the chemotactic response. The kinetic data of binding and dissociation are very rapid, consistent with the time course of oscillations in receptor capacity (affinity). Specific binding oscillations are destroyed by heat or chymotrypsin but are insensitive to trypsin or glycosidase. A plasma membrane localization of receptor is supported by enrichment of cAMP binding in a plasma membrane preparation from differentiated cells. Receptor oscillations with a 2-min period are preserved in the membrane preparations, and the peak amplitudes are increased about 10-fold consistent with the enrichment of other plasma membrane markers. The alternating change in the receptor's binding capacity for cAMP may be the basis of the relay refractory period as well as the primary oscillator involved in the generation of postreceptor events such as stimulation of adenylate cyclase, cAMP secretion, and cellular movement, all of which have been previously shown to oscillate.
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PMID:Properties of the oscillatory cAMP binding component of Dictyostelium discoideum cells and isolated plasma membranes. 22 58

Dictyostelium purpureum S5 and S6, mating type strains, form fruiting-bodies in a monoclonal culture, but product macrocysts in a mix culture. The effects of Concanavalin A (Con A) on both fruiting-body formation and macrocyst formation, and changes of Con A-mediated cell agglutinability during development were studied. It was found that Con A inhibits macrocyst formation but not fruiting-body formation, and that macrocyst-forming cells are much more susceptible to Con A agglutination than are fruiting-body-forming cells during the aggregation stages. When fruiting-body-forming cells are treated with either trypsin or alpha-chymotrypsin, their Con A agglutinability is enhanced to the same extent as that of macrocyst-forming cells. It was also found that when S6 cells are treated with proteases they sometimes produce normal macrocysts even in a monoclonal culture. The results obtained in these experiments showed that the surface properties of fruiting-body-forming cells and macrocyst-forming cells are different, and that the cell surface might play an important role in determining the two developmental courses.
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PMID:Participation of the cell surfaces determining the developmental courses in the cellular slime mould dictyostelium purpureum. 74 45

A method is described for radioiodination to high specific activity of fixed and stained proteins within sodium dodecyl sulfate-polyacrylamide gels, without elution of the proteins from the gel. Following radioiodination, the proteins can be removed from the gel by trypsin treatment and the peptides analyzed. This procedure offers a means to structurally compare the proteins of multicomponent systems when purification of each component to homogeneity is unfeasible. Using this technique, we have compared the tryptic peptides of all the major protein components of Moloney and Rauscher leukemia viruses using only 50 to 100 microgram of total protein from each virus. Additionally, we have analyzed the membrane proteins of Dictyostelium discoideum at various stages in development. The validity of the technique and its value as a tool for comparative studies and identification of precursor-product relationships is discussed.
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PMID:Radioiodination of proteins in single polyacrylamide gel slices. Tryptic peptide analysis of all the major members of complex multicomponent systems using microgram quantities of total protein. 89 22

The glycogen phosphorylase-2 (GP2) activity that appears during the cell differentiation of Dictyostelium was purified to homogeneity. The molecular weight of the nondenatured enzyme was 200,000 as determined by Sephacryl S-300 gel filtration and was 107,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, suggesting that the native enzyme consists of two similar subunits. The intact protein was digested with trypsin and protease V8, and the resulting peptides were purified by microbore high pressure liquid chromatography. The peptides were sequenced, and oligonucleotides were constructed for polymerase chain reaction amplification of the GP2 gene from Dictyostelium genomic DNA template. The resulting polymerase chain reaction products were sequenced directly and were confirmed to encode portions of the GP2 gene. These fragments were used to probe a partial EcoRI genomic library for the remainder of the GP2 gene. The nucleotide sequence of the GP2-selected clones revealed an open reading frame of 2975 base pairs that was interrupted by two introns of 109 and 105 base pairs, respectively. The open reading frame encoded a protein of 992 amino acids with a calculated molecular mass of 112,500 Da and an isoelectric point of 6.4. An unusual sequence within the second exon of GP2, in which the triplet CAA was repeated 11 times, resulted in 11 in-frame glutamine residues of a possible 15 amino acids coded for by this region. The CAA repeat was transcribed, as shown by the sequence of cDNA. Comparison of the amino acid sequence of Dictyostelium GP2 to the phosphorylases from other organisms revealed that the Dictyostelium protein was 50 and 44% identical to yeast and rabbit muscle phosphorylases, respectively. Northern blot analysis showed that GP2 mRNA was absent in amebas and the early stages of development, reached a maximum level of expression at the slug stage, and then decreased in the terminal stages of development. Comparison of the mRNA expression with the appearance of GP2 enzyme protein and enzyme activity revealed that gp2 mRNA and a 113-kDa GP2 enzyme peptide were expressed concurrently at 10 h of development. However, enzyme activity did not appear until 18 h, coincident with a decrease in the level of the 113-kDa peptide and a corresponding increase in the amount of a 106-kDa GP2 peptide. Addition of cAMP to aggregation-competent cells in liquid culture resulted in the induction of GP2 mRNA, GP2 protein, and GP2 enzyme activity.
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PMID:Cloning, structural analysis, and expression of the glycogen phosphorylase-2 gene in Dictyostelium. 131 Mar 12

In submerged monolayer culture, Dictyostelium cells can differentiate into prespore and prestalk cells at high cell densities in response to cAMP but not at low cell densities. However, cells at low densities will differentiate in medium taken from developing cells starved at a high density. The putative factor in the medium was designated CMF for conditioned medium factor (Mehdy and Firtel, Molec. cell. Biology 5, 705-713, 1985). In this report, we size-fractionate conditioned medium and show that the activity that allows low density cells to differentiate can be separated into high and low Mr (relative molecular mass) fractions. Interestingly, the two fractions both have the same activity and do not need to be combined to allow differentiation. The large conditioned medium factor is a protein, as determined by trypsin sensitivity, that can be purified to a single 80 x 10(3) Mr band on a silver-stained SDS-polyacrylamide gel, and has CMF activity at a concentration of approximately 4 pM (0.3 ng ml-1). Our results suggest that CMF is a secreted factor that functions in vivo as an indicator of cell density in starved cells. At high cell densities, the concentration of CMF is sufficient to enable cells to enter the multicellular stage of the developmental cycle. When present below a threshold concentration, cells do not initiate the expression of genes required for early development. This factor plays an essential role in the regulatory pathway necessary for cells to obtain the developmental competence to induce prestalk and prespore gene expression in response to cAMP.
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PMID:A secreted 80 x 10(3) Mr protein mediates sensing of cell density and the onset of development in Dictyostelium. 166 29

An in vitro endosome fusion assay using Dictyostelium discoideum is described. The method requires endocytosis of anti-dinitrophenol (DNP) IgG or DNP-derivitized beta-glucuronidase into two sets of cells. After homogenizing the cells, the vesicles were mixed, and fusion was measured by quantitating immune complex formation between DNP-beta-glucuronidase and anti-DNP IgG. Fusion was dependent upon ATP, temperature, pH, ionic strength, and cytosol and sensitive to detergent, dilution, trypsin, N-ethylmaleimide, and guanosine 5'-3-O-(thio)triphosphate. Although weak bases, ionophores, hadacidin, [ethylenebis(oxyethylenenitrilo)]tetraacetic acid, and caffeine inhibit endocytosis in vivo, these reagents had no affect on in vitro endosome fusion. Comparison of Dictyostelium with mammalian cells showed differences in the temperature, pH, and salt requirements for fusion, possibly reflecting differences in the life-styles of various cell types. Like mammalian cells, Dictyostelium required GTP-binding protein(s) and an N-ethylmaleimide-sensitive factor for endosome fusion. Thus, the mechanism driving endosome fusion may have been conserved throughout evolution. Electron microscopic studies confirmed in vitro endosome fusion and revealed endosomes were being engulfed by other endosomes, resulting in formation of multivesicular elements (i.e. autophagic vesicles). This system may be useful for characterizing mutations, evolution, and developmental regulation along the endocytic pathway.
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PMID:Characterization of endosome-endosome fusion in a cell-free system using Dictyostelium discoideum. 173 Jul 25

During Dictyostelium development, the expression of some genes is dependent on cell density. This effect is mediated by soluble factors referred to as conditioned medium factors (CMFs) which the developing cells secrete at very low rates and simultaneously sense. There are at least two classes of CMFs: one is an 80 x 10(3) Mr glycoprotein and the other is a heterogeneous group of molecules, with relative molecular masses between 6.5 x 10(3) and 0.65 x 10(3). Interestingly, the two classes of molecules do not need to be combined for activity. We find that the 80 x 10(3) Mr CMF but not the small CMF is sequestered in vegetative cells. The 80 x 10(3) Mr CMF is then secreted by cells during early development, while the small CMF appears only during late development. Like the 80 x 10(3) Mr CMF, the small CMFs are trypsin-sensitive and contain N- and O-linked glycosylation. The breakdown products of a fraction containing 80 x 10(3) Mr CMF cochromatographed from a Sephadex G-50 column and a reverse-phase HPLC column with small CMFs. The specific activity of CMF increases roughtly 100-fold upon breakdown. The results suggest that, during differentiation, the slowly diffusing 80 x 10(3) Mr CMF is first produced from a precursor pool already present in vegetative cells, allowing differentiation of only those cells in the immediate vicinity of the aggregation center. The breakdown of 80 x 10(3) Mr CMF to a faster-diffusing, higher specific activity form then might enable cells farther from the aggregation center to differentiate.
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PMID:Regulation and processing of a secreted protein that mediates sensing of cell density in Dictyostelium. 181 50

Actobindin is a protein from Acanthamoeba castellanii with bivalent affinity for monomeric actin. Because it can bind two molecules of actin, actobindin is a substantially more potent inhibitor of the early phase of actin polymerization than of F-actin elongation. The complete amino acid sequence of 88 residues has been deduced from the determined sequences of overlapping peptides obtained by cleavage with trypsin, Staphylococcus V8 protease, endoproteinase Asp-N, and CNBr. Actobindin contains 2 trimethyllysine residues and an acetylated NH2 terminus. About 76% of the actobindin molecule consists of two nearly identical repeated segments of approximately 33 residues each. This could explain actobindin's bivalent affinity for actin. The circular dichroism spectrum of actobindin is consistent with 15% alpha-helix and 22% beta-sheet structure. A hexapeptide with sequence LKHAET, which occurs at the beginning of each of the repeated segments of actobindin, is very similar to sequences found in tropomyosin, muscle myosin heavy chain, paramyosin, and Dictyostelium alpha-actinin. A longer stretch in each repeated segment is similar to sequences in mammalian and amoeba profilins. Interestingly, the sequences around the trimethyllysine residues in each of the repeats are similar to the sequences flanking the trimethyllysine residue of rabbit reticulocyte elongation factor 1 alpha, but not to the sequences around the trimethyllysine residues in Acanthamoeba actin and Acanthamoeba profilins I and II.
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PMID:The covalent structure of Acanthamoeba actobindin. 237 77

The endoplasmic reticulum-localized enzyme alpha-glucosidase II is responsible for removing the two alpha-1,3-linked glucose residues from N-linked oligosaccharides of glycoproteins. This activity is missing in the modA mutant strain, M31, of Dictyostelium discoideum. Results from both radiolabeled pulse-chase and subcellular fractionation experiments indicate that this deficiency did not prevent intracellular transport and proteolytic processing of the lysosomal enzymes, alpha-mannosidase and beta-glucosidase. However, the rate at which the glucosylated precursors left the rough endoplasmic reticulum was several-fold slower than the rate at which the wild-type precursors left this compartment. Retention of glucose residues did not disrupt the binding of the precursor forms of the enzymes with intracellular membranes, indicating that the delay in movement of proteins from the ER did not result from lack of association with membranes. However, the mutant alpha-mannosidase precursor contained more trypsin-sensitive sites than did the wild-type precursor, suggesting that improper folding of precursor molecules might account for the slow rate of transport to the Golgi complex. Percoll density gradient fractionation of extracts prepared from M31 cells indicated that the proteolytically processed mature forms of alpha-mannosidase and beta-glucosidase were localized to lysosomes. Finally, the mutation in M31 may have other, more dramatic, effects on the lysosomal system since two enzymes, N-acetylglucosaminidase and acid phosphatase, were secreted much less efficiently from lysosomal compartments by the mutant strain.
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PMID:Biogenesis of lysosomal enzymes in the alpha-glucosidase II-deficient modA mutant of Dictyostelium discoideum: retention of alpha-1,3-linked glucose on N-linked oligosaccharides delays intracellular transport but does not alter sorting of alpha-mannosidase or beta-glucosidase. 250 71

The electrophoretic pattern of cysteine proteinases in axenically grown myxamoebae of Dictyostelium discoideum can be altered by the addition of either Gram-negative (Klebsiella aerogenes, Escherichia coli) or Gram-positive (Micrococcus lysodeikticus, Bacillus subtilis) bacteria to the culture. No changes occurred, however, if either yeast or latex beads were used in place of bacteria. The changes involved the simultaneous loss of proteinases characteristic of the axenic cells (the A-forms) and the acquisition of those found in cells which have been grown on bacteria (the B-forms). Using K. aerogenes the conversion was complete within 4 h. Extracellular proteinase activity was unaffected during this period. After the D. discoideum cells had been lysed, no equivalent change in proteinase band pattern could be produced either by prolonged incubation of cell extracts or by treatment with proteinases. An identical conversion could be induced in cultures of myxamoebae by a factor, cysteine proteinase converting factor (CPCF), present in the 15,000 g supernatant of a sonicated suspension of K. aerogenes. CPCF was macromolecular, as demonstrated by both ultrafiltration and gel filtration, acid-precipitable, but was soluble in ethanol or alkali. Its activity was unaffected by treatment with trypsin. The results suggested that CPCF might be a component of the bacterial cell wall, and since its activity was affected by lysozyme treatment, peptidoglycan is implicated. The results can be interpreted in terms of a novel nutrient-dependent post-translational change which affected most of the cysteine proteinases present in D. discoideum myxamoebae.
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PMID:A bacterial factor induces changes in cysteine proteinase forms in the cellular slime mould Dictyostelium discoideum. 305 31


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