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 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

Cerebral amyloid angiopathy (CAA) is caused by the cerebrovascular deposition of Alzheimer amyloid beta protein (Abeta) and shows an increased incidence in carriers of the apolipoprotein E (APOE) epsilon4 genotype. To study the pathogenesis of CAA, primary cultures of human and canine smooth muscle cells from leptomeningeal vessels were incubated with fluorescein- and biotin-conjugated amyloid beta-protein. In the presence of human serum or cerebrospinal fluid, A beta1-40 and Abeta1-42 were rapidly internalized and appeared within endosomal and lysosomal vesicles. The accumulation of intracellular Abeta was enhanced by chloroquine and blocked by cycloheximide and brefeldin A and pretreatment with trypsin, suggesting that the internalization of Abeta occurs by receptor-mediated endocytosis. The internalization of Abeta was also inhibited by lipoprotein-deficient serum or by incubation with the 39-kd receptor-associated protein, indicating that Abeta is internalized via a receptor of the low-density lipoprotein receptor family. A lipoprotein pathway was confirmed by colocalization of cell surface-bound or internalized Abeta with APOE and low-density lipoprotein receptor-related protein. We propose a pathogenetic model of CAA, in which Abeta-APOE-complexes contained within the cerebrospinal fluid or the extracellular fluid of the brain are internalized and accumulated in cerebrovascular smooth muscle cells. Such a model could explain the preferential localization of CAA to the outer and middle layers of cortical and leptomeningeal arterioles, while indicating a mechanism by which the APOE genotype might determine the risk of CAA.
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PMID:Cerebrovascular smooth muscle cells internalize Alzheimer amyloid beta protein via a lipoprotein pathway: implications for cerebral amyloid angiopathy. 927 58

A novel lectin (CAA-II) was isolated and purified from the seeds of Cicer arietinum by ammonium sulphate fractionation and affinity chromatography on an N-acetyl-D-galactosamine-linked agarose column. The lectin is composed of four identical subunits of 30 kDa and the molecular mass of the native lectin was estimated to be 120 kDa by gel filtration chromatography and confirmed by mass spectrometry. The lectin showed agglutination activity against rabbit erythrocytes (trypsin-treated and untreated) as well as against human erythrocytes. Haemagglutination inhibition assays showed that the lectin is a galactose-specific protein having a high affinity for N-acetyl-D-galactosamine. The molecular weight, haemagglutination pattern, carbohydrate specificity and N-terminal amino acid sequence indicated that the lectin is clearly distinct from the previously reported chickpea lectin CAA-I.
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PMID:Purification and characterization of an N-acetyl-D-galactosamine-specific lectin from seeds of chickpea (Cicer arietinum L.). 1701 37

Bacillus thuringiensis has been successfully used for agricultural pest and medical insect control with its significant benefits based on environmental and safety considerations. However, the deficiency of this pesticide, such as limited spectrum of insecticidal activity, low toxicity to the targets and the inducement of insect resistance, results in the urgent need to exploit new resources of B. thuringiensis or to modify known strains by genetic engineering. In this study, six recombinant Bacillus thuringiensis BT-ACC, BT-AAC, BT-ACA, BT-CAA, BT-CCA and BT-CAC were constructed through the domain swapping between crystal protein CrylAa and CrylCa. SDS-PAGE and Western blot revealed that only the recombinant BT-CAA and BT-CCA produced a 135kDa chimeric protein CrylCAA and CrylCCA respectively, but the production level was lower than the native protein CrylAa and CrylCa. These chimeric crystal proteins could be activated by trypsin, giving a 65kDa protease-resistant core toxin as the native crystal proteins CrylAa and CrylCa. Electron microscopy study indicated that the two chimeric proteins could be produced and accumulated as spherical or granules crystals during sporulation of BT-CAA and BT-CCA, whereas native CrylAa and CrylCa were accumulated as bipyramidal crystals. Unexpectedly, the toxicity of purified chimeric crystal proteins Cryl CAA and Cryl CCA was 3 - 5 times lower to Spodoptera exigua, and 190 - 260 times lower to Helicoverpa armigera than that of native CrylAa and CrylCa. These data suggested that the domain swapping of different crystal proteins might influence the formation and toxicity to targets of chemeric crystal proteins.
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PMID:[Domain swapping of Cry1Aa and Cry1Ca from Bacillus thuringiensis influence crystal formation and toxicity]. 1730 52