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)

Gq alpha and G11 alpha differ from other G protein alpha subunits in that they have unique, conserved 6 residue amino-terminal extensions. Wild-type and amino-terminal mutants of Gq alpha expressed in COS cells were analyzed for their ability to functionally couple with co-expressed neurokinin NK2 receptor. Wild-type, T2A and delta 2-7 Gq alpha were able to stimulate agonist driven phospholipase C (PLC) activity in identical manners. Other activities of these two amino-terminal mutants including aluminum fluoride stimulated PLC activity, palmitoylation, interaction with G beta gamma subunits and GTP gamma S-induced trypsin resistance are also similar to the wild-type alpha subunit. This demonstrates that the NK2 receptor is able to functionally interact with the alpha subunit of Gq and that the first seven amino-acids of Gq alpha are not required for any of the alpha subunit functions tested. In contrast to the T2A and delta 2-7 mutants, a C9,10A Gq alpha mutant was not able to couple to either the NK2 receptor or PLC, as assessed by high-affinity agonist binding and activation of PLC either in intact cells or in vitro. The C9,10A protein was able to assume a GTP gamma S-induced trypsin-resistant conformation and partitioned primarily to the pelletable fraction in a manner similar to the wild-type protein. However, it was not labeled with [3H]palmitic acid. This suggests that blocking palmitoylation at the amino-terminus of Gq alpha results in a loss of functional activity which reflects an inability to interact with both the receptor and downstream signaling targets.
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PMID:Palmitoylation but not the extreme amino-terminus of Gq alpha is required for coupling to the NK2 receptor. 795 23

A tightly membrane-associated form of spectrin (TMA-spectrin) was labeled when human red blood cells were incubated with [3H]palmitic acid. About 90% of spectrin was not fatty acid-acylated and was extracted from membranes by low salt buffers. The 3H-palmitoylated TMA-spectrin, however, resisted low and even high salt extraction and remained associated with inside-out vesicles that were generated in the process of spectrin-actin extraction from membranes. TMA-spectrin was preferentially extracted from KCl-stripped vesicles by 5 M urea at low ionic strength. TMA-spectrin was purified by gel filtration and by ion exchange chromatography in the presence of urea and a non-ionic detergent. Purified TMA-spectrin was 3H-palmitoylated exclusively in the beta subunit to 0.28 mol/mol after a 12-h incubation of red cells. The labeled palmitate may be bound as an ester or thioester, since hydroxylamine (1 M, pH 7.5) released the label completely. Peptide maps of 3H-palmitoylated TMA-spectrin showed three or two labeled peptides from the beta subunit, when generated by V8 protease and trypsin, respectively. Two types of antibodies to spectrin reacted with purified TMA-spectrin, and TMA-spectrin contained the same antigenic peptides as low salt-extractable spectrin. Rabbit anti-ankyrin antibodies did not bind to TMA-spectrin. The substoichiometric incorporation of [3H]palmitic acid into TMA-spectrin could result from the slow turnover of endogenously bound fatty acids. Generation of the tightly membrane-associated and 3H-palmitoylated subpopulation of spectrin cannot be due to entrapment of an unmodified residual fraction of spectrin in right-side-out vesicles. Instead, the data suggest the existence of a subpopulation of spectrin molecules that undergo a covalent fatty acid modification and thereby alter their binding properties. This may offer a new, metabolically dependent mechanism for dynamic interactions between spectrin and the membrane lipid bilayer.
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PMID:A tightly membrane-associated subpopulation of spectrin is 3H-palmitoylated. 850 31

The major outer surface protein, OspA, of Borrelia burgdorferi is a lipoprotein which is a particular interest because of its potential as a vaccine candidate. However, serotypic and genetic analysis of OspA from both European and North American strains have demonstrated antigenic and structural heterogeneities. We purified OspA to homogeneity by exploiting its resistance to trypsin digestion. By treating spirochetes with trypsin and then using Triton X-114 extraction and ion-exchange chromatography, we obtained a yield of 2 mg of pure OspA protein per liter of culture. INtrinsic labeling with [14C]palmitic acid confirmed that OspA was lipidated, and partial digestion established lipidation at the amino-terminal end of the molecule. The reactivity of five anti-OspA murine monoclonal antibodies to nine different isolates of B. burgdorferi was ascertained by Western blot (immunoblot) analysis. Purified OspA was fragmented by enzymatic or chemical cleavage, and the monoclonal antibodies were able to define four distinct immunogenic domains. Further resolution of the epitope specificity to determine humoral and cellular immune responses to OspA has implications for vaccine development and for the utility of this protein as a reagent in diagnostic testing for Lyme borreliosis.
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PMID:Purification of Borrelia burgdorferi outer surface protein A (OspA) and analysis of antibody binding domains. 855 77

All Streptococcus pneumoniae isolates tested to date express a species-common lipoprotein designated as pneumococcal surface adhesin A (PsaA). This protein is cell-associated, hydrophobic, immunogenic, and genetically conserved. It is currently under investigation as a potential component in third-generation pneumococcal vaccine formulations. To overcome the problem of low-level expression of native hydrophobic PsaA in S. pneumoniae, and also of the recombinant PsaA (rPsaA) in Escherichia coli, we generated a stable E. coli construct expressing functional palmitoylated rPsaA ( approximately 10 mg/l of fermentation culture) using Borrelia burgdorferi outer surface protein A (OspA, a hydrophobic lipoprotein) signal peptide. By Western blot analysis, the chimeric rPsaA ( approximately 34 kDa) was detected in the cell lysate using anti-PsaA antibodies. It was partially purified by extracting the cell pellet with PBS/Triton X(R)-114 buffers, followed by anion exchange filter chromatography. A trypsin digestion profile of rPsaA closely resembled that of the native protein, as revealed by SDS-PAGE/silver staining. Lipidation of rPsaA was confirmed by labeling recombinant E. coli cells with [(3)H] palmitic acid and analyzing the labeled E. coli cells by Western blotting coupled with autoradiography. Further, analysis of purified rPsaA by mass spectrometry (MALDI-TOF) revealed a heterogenous spectrum with a major peak (M+H)(+1) of mass 33,384 Da (theoretical mass of palmitoylated rPsaA=33,361 Da). Purified rPsaA was immunogenic in CBA/NCAHN-XID female mice following intranasal immunization with or without adjuvant, as determined by measurement of anti-PsaA serum IgG levels. These anti-PsaA antibodies reacted with both native and rPsaA polypeptides. Our data strongly suggest that E. coli-expressed rPsaA is palmitoylated and closely resembles the native protein in structure and immunogenicity. It was also observed to elicit measurable protection against nasopharyngeal carriage with S. pneumoniae.
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PMID:Purification and characterization of Streptococcus pneumoniae palmitoylated pneumococcal surface adhesin A expressed in Escherichia coli. 1069 29

Data obtained with the neutral red cytotoxicity assay reveal that human lens epithelial cells in culture are highly sensitive to low micromolar concentrations of unsaturated, cis-configured fatty acids in the following order: arachidonic acid>linolenic acid=linoleic acid=oleic acid, whereas the saturated fatty acids are much less effective. Though the cytotoxic effects of the unsaturated fatty acids could not be discerned from effects of their oxidation products, the fact that oleic acid is equally cytotoxic as linoleic acid or linolenic acid as well as previously reported findings with bovine lens epithelial cells support the idea that the unsaturated fatty acid molecules directly account for the cytotoxicity and not their products of lipid peroxidation. Bleb formation and cell retraction are early morphological signs of fatty acid-induced lens cell damage. These cellular alterations are accompanied by an aggregation of intermediate filaments in a first step, whereas the disorganization of microfilaments occurs at a later time and only at higher fatty acid concentrations. Measurements of protein-, RNA- and DNA-synthesis turned out to be much less sensitive parameters for the fatty acid-induced damage of lens cells. The uptake rate of linoleic acid by human lens cells is relatively high (4.35 fmol sec(-1) per 1000 cells), 30 and 50% higher as compared with diploid human embryonal lung fibroblasts and chemically transformed mouse fibroblasts, respectively. Saturation kinetics in combination with competition between linoleic acid, oleic acid and palmitic acid on one hand and ineffectiveness of trypsin and DIDS treatment on the other hand hint at cytoplasmic fatty acid binding proteins as receptors with high binding affinity (5.55 micromol l(-1), calculated for the linoleic acid-albumin complex) to be involved in the fatty acid uptake in human lens cells. Cellular fatty acid uptake is mainly influenced by the albumin concentrations present in physiological solutions. Albumin determinations in aqueous humor from 177 cataract patients reveal an age-dependent, statistically significant albumin rise with average values below 2 micromol l(-1) up to the age of 40 years to about 4 micromol l(-1) at the age between 80 and 90 years with single values up to 10 micromol l(-1). Using physiological fatty acid mixtures it is demonstrated that fatty acid-induced lens cell damage is strongly increased by elevated albumin concentrations found in aqueous humor of the elderly, who already have cataracts. Free fatty acid induced lens cell damage as a possible cause for age-dependent cataracts as well as a molecular link between systemic diseases such as diabetes and cataract formation is discussed.
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PMID:Fatty acid cytotoxicity to human lens epithelial cells. 1550 Aug 27


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