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 properties of three different recombinant hepatitis B virus core proteins expressed in Escherichia coli were compared: an N-terminal fusion protein, a C-terminally truncated protein and a sequence-authentic protein. All three proteins assembled into capsid-like particles with typical HBc-antigenicity, sedimentation behavior and distinctive electron microscopical images. Apart from this, however, variant HBc proteins displayed properties different from sequence-authentic HBc protein p21.4. Unlike p21.4, the particles of the N-terminal fusion protein p22.2 were sensitive to proteolytic attack by trypsin at variable sites within its arginine-rich C-terminus but not in its extended N-terminus. We therefore conclude that the C-terminal region is located on the surface of the p22.2 particle. These particles also showed increased HBe-antigenicity, as did the C-terminally truncated core particles p17.6, and to an even greater extent p18* particles which were derived from p22.2 by tryptic digestion. This might be interpreted as evidence for an--albeit minor--structural change. All variant core particles were less stable and contained less RNA. Electron microscopic indication for DNA binding of C-terminal deleted p17.6 particles was obtained using an aqueous spreading technique.
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PMID:Comparison of three different recombinant hepatitis B virus core particles expressed in Escherichia coli. 819 38

Activation of the superoxide (O2-)-generating NADPH oxidase of phagocytes requires the interaction of membrane-associated cytochrome b559 with three cytosolic components; p47-phox, p67-phox and sigma 1. We proposed that sigma 1 was a heterodimer composed of proteins of 22 kDa and 24 kDa that were tentatively identified as the small GTP-binding protein (G protein) rac1 p21 and GDP-dissociation inhibitor for rho (rho GDI). We now describe a modified procedure for the rapid purification of sigma 1 and demonstrate that the NADPH-oxidase-activating capacity is associated, throughout the purification sequence, with a protein binding 35S-labelled guanosine 5'-[3-O-thio]triphosphate. SDS/PAGE analysis confirmed the absolute association of sigma 1 activity with the presence of both the 22 kDa and 24 kDa proteins. Immunoblotting with a battery of antibodies against the small G proteins demonstrated that the 22-kDa protein was only recognized by antibodies reacting with rac1 p21; no reaction was found with anti-(rac2 p21), anti-[v-ras(H) p21] and anti anti-(rap1 p21). Free rac1 p21 (not in complex with rho GDI) was not detected at any stage of cytosol fractionation. The proteins comprising the sigma 1 heterodimer could be separated by reverse-phase chromatography and amino acid sequencing was performed on peptides derived by trypsin digestion of each of the isolated proteins. This demonstrated the identity of the 22-kDa protein with rac1 p21 and that of the 24-kDa protein with rho GDI. Purified heterodimeric sigma 1 did not require exogenous GTP for activity under conditions that assured the absence of free nucleotides. Treatment of the sigma 1 heterodimer with 1% sodium cholate, followed by gel filtration or anion-exchange chromatography in the presence of 1% sodium cholate, effectively separated rac1 p21 from rho GDI. Monomeric rac1 p21, obtained by these procedures, was able to stimulate cell-free O2- generation. Artificial heterodimeric sigma 1, capable of NADPH oxidase activation, could be reconstituted in vitro by recombining purified monomeric rac1 p21 and rho GDI and removing the sodium cholate used to dissociate the native sigma 1 dimer. Monomeric rac1 p21 exhibited an almost absolute dependence on exogenous GTP following removal of the endogenous nucleotide in low Mg2+ solution. Under similar conditions, heterodimeric sigma 1 was resistant to nucleotide exchange.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Role of the rac1 p21-GDP-dissociation inhibitor for rho heterodimer in the activation of the superoxide-forming NADPH oxidase of macrophages. 822 83

Direct photoaffinity labeling of proteins of longitudinal sarcoplasmic reticulum (LSR) of rabbit skeletal muscle with [32P]GTP revealed GTP-binding proteins of about 52, 45 and 30 kDa. ADP-ribosylation with [32P]NAD in the presence of cholera toxin (CTX) or pertussis toxin (PTX) indicates the existence of a CTX substrate (about 45 kDa); no PTX substrates were observed. Western blots of LSR probed with RM/1, an antiserum against a decapeptide from the C-terminus of Gs alpha, showed an immunoreactive band at about 45 kDa. [32P]GTP overlays of Western blots of LSR showed a heavily-labeled protein of about 29 kDa and one or more additional slightly smaller proteins that were more weakly labeled. When LSR was subjected to mild trypsin hydrolysis, the Western blot overlay revealed three bands at about 23, 25 and 29 kDa. Western blots of LSR proteins showed no significant immunoreactivity with the anti-(pan)-ras monoclonal antibodies 142-24E05 and Ras 11. ADP-ribosylation of LSR with [32P]NAD in the presence of C3 exoenzyme of Clostridium botulinum yielded a labeled band at about 23 kDa. Our results indicate the presence in rabbit LSR of a Gs alpha, the absence of Gi and G(o), and the presence of several low molecular weight GTP-binding proteins, distinct from p21 ras, one of which belongs to the rho or rac family.
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PMID:Identification of heterotrimeric and low molecular weight GTP-binding proteins in rabbit skeletal muscle longitudinal sarcoplasmic reticulum. 841 93

For comparison of the physiological potential of single thyroid cells versus cells integrated into follicles it would be ideal to work with suspensions consisting exclusively of single cells instead of a mixture of single cells and follicle fragments. In this study, various techniques for the isolation of single cells have been tested for their effect on cell viability, the ultrastructure of the isolated cells, the percentage of single cells and the ability of these cells to form follicles in culture. In addition, the cells were characterized for the preservation of their morphology and the ability to respond to TSH by comparing their immunocytochemical staining pattern with anti-vimentin and anti-ras p21 antibody to that of the intact thyroid tissue. Dispase treatment of thyroid tissues alone produced suspensions with a relatively small proportion of single cells. These cells stained with anti-vimentin and anti-ras p21 antibody to a similar percentage as thyroid cells in the intact gland. A combination of dispase treatment with either filtration or trypsin treatment severely compromised the viability of the cells. A high proportion of single cells with a good viability could be obtained either by centrifugation of dispase treated tissues or by culturing of dispase treated tissues as monolayers and subsequent detachment from the culture vessels with trypsin. Whereas the immunological staining with anti-vimentin and anti-ras oncogene antibody in the centrifuged cells resembled that of intact tissue, cells cultured as monolayers reacted differently. The differences in the immunological staining were still observed when the cells which had been grown as monolayers were stimulated with TSH. Differential centrifugation appeared to be the ideal method for the isolation of unaltered and viable single cells but is a rather laborious method to obtain larger amounts of single thyroid cells.
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PMID:Efficiency of various dissociation methods for the preparation of thyroid single cell suspensions. 853 60

Acanthamoeba myosin I heavy chain (MIHC) kinase is a monomeric 97-kDa protein that is activated by binding to acidic phospholipids or by autophosphorylation. Activation by phospholipids is inhibited by Ca2+-calmodulin. In the accompanying paper (Brzeska, H., Martin, B., and Korn, E. D. (1996) J. Biol. Chem. 271, 27049-27055), we identified the catalytic domain as the COOH-terminal 35 kDa produced by trypsin digestion of phosphorylated MIHC kinase. In this paper, we report the cloning and sequencing of the corresponding cDNA and expression of fully active catalytic domain. The expressed catalytic domain has substrate specificity similar to that of native kinase and resistance to trypsin similar to that of fully phosphorylated MIHC kinase. MIHC kinase catalytic domain has only 25% sequence identity to the catalytic domain of protein kinase A and similarly low sequence identity to the catalytic domains of protein kinase C- and calmodulin-dependent kinases, but 50% sequence identity and 70% similarity to the p21-activated kinase (PAK) and STE20 family of kinases. This suggests that MIHC kinase is (at least) evolutionarily related to the PAK family, whose activities are regulated by small GTP-binding proteins. The homology includes the presence of a potential MIHC kinase autophosphorylation site as well as conserved Tyr and Ser/Thr residues in the region corresponding to the P+1 loop of protein kinase A. A synthetic peptide corresponding to this region of MIHC kinase is phosphorylated by both the expressed catalytic domain and native MIHC kinase.
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PMID:The catalytic domain of acanthamoeba myosin I heavy chain kinase. II. Expression of active catalytic domain and sequence homology to p21-activated kinase (PAK). 890 Jan 96

The platelet-activating factor PAF (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a potent lipid first messenger active in general cell activation, fertilization, inflammatory and allergic reactions, asthma, HIV pathogenesis, carcinogenesis, and apoptosis. There is substantial evidence that PAF is involved in intracellular signalling, but the pathways are poorly understood. Inactivation of PAF is carried out by specific intra- and extracellular acetylhydrolases (PAF-AHs), a subfamily of phospholipases A2 that remove the sn-2 acetyl group. Mammalian brain contains at least three intracellular isoforms, of which PAF-AH(Ib) is the best characterized. This isoform contains a heterodimer of two homologous catalytic subunits alpha1 and alpha2, each of relative molecular mass 26K, and a non-catalytic 45K beta-subunit, a homologue of the beta-subunit of trimeric G proteins. We now report the crystal structure of the bovine alpha1 subunit of PAF-AH(Ib) at 1.7 A resolution in complex with a reaction product, acetate. The tertiary fold of this protein is closely reminiscent of that found in p21(ras) and other GTPases. The active site is made up of a trypsin-like triad of Ser 47, His 195 and Asp 192. Thus, the intact PAF-AH(Ib) molecule is an unusual G-protein-like (alpha1/alpha2)beta trimer.
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PMID:Brain acetylhydrolase that inactivates platelet-activating factor is a G-protein-like trimer. 898 54

Myosin regulatory light chain (RLC) phosphorylation has been implicated in Rho-mediated stress fibre formation. The recent observation that Rho kinase phosphorylates RLC in vitro suggests that serine/threonine kinases other than those in the myosin light chain kinase (MLCK) family have the potential to activate myosin II. In this study we report that gamma-PAK, which is activated by the GTP-binding proteins Cdc42 and Rac, catalyses phosphorylation of intact non-muscle myosin II and isolated recombinant RLC. gamma-PAK phosphorylated endothelial cell myosin II to 0.85 +/- 0.02 mol PO4 per mol RLC. Phosphorylation is Ca2+/calmodulin-independent and the enzyme has a K(m) and Vmax for myosin II regulatory light chain of 12 microM and 180 nmol/min/mg respectively. No myosin II heavy chain phosphorylation was detected. Phosphopeptide maps and phosphoamino acid analysis revealed that gamma-PAK phosphorylates Ser-19 but does not phosphorylate Thr-18. A panel of recombinant RLC mutants was used to confirm that Ser-19 is the only phosphorylation site modified by gamma-PAK. On substitution of both Ser-19 and Thr-18 with Ala or Glu, no phosphorylation of other Ser/Thr residues in the RLC was detected. Similar to MLCK, Arg-16 is required for interaction of gamma-PAK with the substrate, since converting Arg-16 to Ala significantly reduced RLC phosphorylation. Endothelial cell monolayers permeabilized with saponin retract upon exposure to either Cdc42 or trypsin-activated gamma-PAK and ATP. Activation of gamma-PAK is required to initiate Ca2+/calmodulin-independent cell retraction and actin rearrangement. Taken together, these data suggest that myosin II activation by the p21-activated family of kinases may be physiologically important in regulating cytoskeletal organization.
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PMID:Phosphorylation of non-muscle myosin II regulatory light chain by p21-activated kinase (gamma-PAK). 1004 84

We cloned and sequenced a new cytolysin gene from Bacillus thuringiensis subsp. medellin. Three IS240-like insertion sequence elements and the previously cloned cyt1Ab and p21 genes were found in the vicinity of the cytolysin gene. The cytolysin gene encodes a protein 29.7 kDa in size that is 91.5% identical to Cyt2Ba from Bacillus thuringiensis subsp. israelensis and has been designated Cyt2Bc. Inclusions containing Cyt2Bc were purified from the crystal-negative strain SPL407 of B. thuringiensis. Cyt2Bc reacted weakly with antibodies directed against Cyt2Ba and was not recognized by an antiserum directed against the reference cytolysin Cyt1Aa. Cyt2Bc was hemolytic only upon activation with trypsin and had only one-third to one-fifth of the activity of Cyt2Ba, depending on the activation time. Cyt2Bc was also mosquitocidal against Aedes aegypti, Anopheles stephensi, and Culex quinquefasciatus, including strains resistant to the Bacillus sphaericus binary toxin. Its toxicity was half of that of Cyt2Ba on all mosquito species except resistant C. quinquefasciatus.
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PMID:Characterization of Cyt2Bc toxin from Bacillus thuringiensis subsp. medellin. 1187 72

The proteinase-activated receptors (PAR) PAR1 and PAR2 mediate responses to thrombin and trypsin-like proteases, respectively. Both receptors are expressed on endothelial cells where they have been reported to transduce a similar set of intracellular responses. In cultured human umbilical vein endothelial cells (HUVEC), we observed a marked difference in shape changes induced by PAR-activating peptides (PAR-APs); unlike PAR1-AP, PAR2-AP failed to stimulate cell rounding. Objectives were to shed light on the mechanisms underlying PAR-mediated cytoskeletal responses. We examined the activation of the Rho family GTPases in HUVEC using highly selective PAR1- and PAR2-APs to do this. Both peptides induced a robust and transient activation of RhoA, with the time course of activation being more sustained for the PAR1-AP. Interestingly, divergent effects on Rac activity were observed. Addition of PAR1-AP inhibited basal Rac activity as well as the phosphorylation of the Rac effector, p21-activated kinase (PAK). In contrast, PAR2-AP induced a modest activation of Rac, phosphorylation of PAK and translocation of cortactin from the cytosol to membrane ruffles, a Rac-dependent event. In vivo, only PAR1-AP rapidly enhanced vascular permeability in a mouse skin assay. We conclude that the differential regulation of the Rac/PAK pathway by PAR1 and PAR2 agonists in endothelial cells points toward distinct roles for these receptors in the control of vascular permeability and blood vessel remodeling.
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PMID:Modulation of Rho GTPase activity in endothelial cells by selective proteinase-activated receptor (PAR) agonists. 1287 83

Both endocrine and exocrine pancreatic cells arise from pancreatic-duodenal homeobox 1 (pdx1)-positive progenitors. The molecular mechanisms controlling cell fate determination and subsequent proliferation, however, are poorly understood. Unlike endocrine cells, less is known about exocrine cell specification. We report here the identification and characterization of a novel exocrine cell determinant gene, exocrine differentiation and proliferation factor (exdpf), which is highly expressed in the exocrine cell progenitors and differentiated cells of the developing pancreas in zebrafish. Knockdown of exdpf by antisense morpholino caused loss or significant reduction of exocrine cells due to lineage-specific cell cycle arrest but not apoptosis, whereas the endocrine cell mass appeared normal. Real-time PCR results demonstrated that the cell cycle arrest is mediated by up-regulation of cell cycle inhibitor genes p21(Cip), p27(Kip), and cyclin G1 in the exdpf morphants. Conversely, overexpression of exdpf resulted in an overgrowth of the exocrine pancreas and a severe reduction of the endocrine cell mass, suggesting an inhibitory role for exdpf in endocrine cell progenitors. We show that exdpf is a direct target gene of pancreas-specific transcription factor 1a (Ptf1a), a transcription factor critical for exocrine formation. Three consensus Ptf1a binding sites have been identified in the exdpf promoter region. Luciferase assay demonstrated that Ptf1a promotes transcription of the exdpf promoter. Furthermore, exdpf expression in the exocrine pancreas was lost in ptf1a morphants, and overexpression of exdpf successfully rescued exocrine formation in ptf1a-deficient embryos. Genetic evidence places expdf downstream of retinoic acid (RA), an instructive signal for pancreas development. Knocking down exdpf by morpholino abolished ectopic carboxypeptidase A (cpa) expression induced by RA. On the other hand, exdpf mRNA injection rescued endogenous cpa expression in embryos treated with diethylaminobenzaldehyde, an inhibitor of RA signaling. Moreover, exogenous RA treatment induced anterior ectopic expression of exdpf and trypsin in a similar pattern. Our study provides a new understanding of the molecular mechanisms controlling exocrine cell specification and proliferation by a novel gene, exdpf. Highly conserved in mammals, the expression level of exdpf appears elevated in several human tumors, suggesting a possible role in tumor pathogenesis.
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PMID:Exdpf is a key regulator of exocrine pancreas development controlled by retinoic acid and ptf1a in zebrafish. 1906 90


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