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)

As shown in the companion article, tubulin is posttranslationally modified in vivo by palmitoylation. Our goal in this study was to identify the palmitoylation sites by protein structure analysis. To obtain quantities of palmitoylated tubulin required for this analysis, a cell-free system for enzymatic [3H]palmitoylation was developed and characterized in our companion article. We then developed a methodology to examine directly the palmitoylation of all 451 amino acids of alpha-tubulin. 3H-labeled palmitoylated alpha-tubulin was cleaved with cyanogen bromide (CNBr). The CNBr digest was resolved according to peptide size by gel filtration on Sephadex LH60 in formic acid:ethanol. The position of 3H-labeled palmitoylated amino acids in peptides could not be identified by analysis of the Edman degradation sequencer product because the palmitoylated sequencer products were lost during the final derivatization step to phenylthiohydantoin derivatives. Modification of the gas/liquid-phase sequencer to deliver the intermediate anilinothiozolinone derivative, rather than the phenylthiohydantoin derivative, identified the cycle containing the 3H-labeled palmitoylated residue. Therefore, structure analysis of peptides obtained from gel filtration necessitated dual sequencer runs of radioactive peptides, one for sequence analysis and one to identify 3H-labeled palmitoylated amino acids. Further cleavage of the CNBr peptides by trypsin and Lys-C protease, followed by gel filtration on Sephadex LH60 and dual sequencer runs, positioned the 3H-labeled palmitoylated amino acid residues in peptides. Integration of all the available structural information led to the assignment of the palmitoyl moiety to specific residues in alpha-tubulin. The palmitoylated residues in alpha-tubulin were confined to cysteine residues only. The major site for palmitoylation was cysteine residue 376.
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PMID:Posttranslational modification of tubulin by palmitoylation: II. Identification of sites of palmitoylation. 924 44

Previously we demonstrated that specific chromatographically resolvable isoforms of rabbit brain GAPDH catalyze either glycolytic flux or membrane fusion activity (but not both). Moreover, GAPDH membrane fusion activity was latent until it was separated from an endogenous cytosolic inhibitor by anion-exchange chromatography. Herein we demonstrate that the cytosolic inhibitor is nondialyzable, heat-labile, and trypsin-sensitive, thereby identifying it as a cytosolic protein constituent. Chromatographic purification of the rabbit-brain cytosolic protein inhibitor of GAPDH isoform-catalyzed membrane fusion identified a predominant 55-kDa doublet that contained an internal 15-aa peptide identical to a sequence present in alpha-tubulin (residues 65-79). The identity of the 55-kDa doublet as tubulin was substantiated through Western blot analysis and inhibition of GAPDH-catalyzed membrane fusion by authentic tubulin. Stopped-flow kinetic analysis demonstrated the high-affinity, rapid, and direct modulation of GAPDH-catalyzed fusion activity by tubulin. Because GTP-activated Rab 2 recruits GAPDH to membranes about to undergo fusion [Tisdale, E. J. (2001) J. Biol. Chem. 276, 2480-2486] and protein kinase Ciota/lambda phosphorylates GAPDH modulating its interactions with tubulin [Tisdale, E. J. (2001) J. Biol. Chem. 277, 3334-3341], the present study suggests a coordinated mechanism through which membrane trafficking and cellular signaling can be integrated with glycolytic flux.
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PMID:Tubulin is the endogenous inhibitor of the glyceraldehyde 3-phosphate dehydrogenase isoform that catalyzes membrane fusion: Implications for the coordinated regulation of glycolysis and membrane fusion. 1238 82

Lipid rafts are glycolipid- and cholesterol-enriched membrane microdomains implicated in membrane signaling and trafficking. The highly hydrophobic nature of lipid raft proteins pose significant problems of solubilization and recovery that hinder analysis by mass spectrometry (MS) and may under-report the composition of lipid rafts. In a previous investigation of the monocyte lipid raft in which proteins were digested with trypsin following polyacrylamide gel electrophoresis we identified 52 proteins. Here we report the development of a sodium dodecyl sulfate (SDS)-aided approach in which proteins are digested in solution and examined by high-performance liquid chromatography-matrix-assisted laser desorption/ionization-tandem mass spectrometry (HPLC-MALDI-MS/MS) using a novel LC-MALDI interface thereby circumventing the need to separate proteins on gels. Using this approach we identified 71 proteins in the lipid raft, 45 of which were not detected using in-gel digestion. Among the new proteins are alpha- and beta-tubulin, tubulinspecific chaperone A, a folding protein involved in tubulin dimer assembly, and KIF13, a microtubule motor protein indicating that proteins involved in microtubule assembly and trafficking are more readily detected using an in-solution approach. To investigate why tubulin was not identified by in-gel digestion, we compared the distribution of alpha-tubulin and the raft marker flotillin-2 in buoyant density gradients before and after separation on SDS-gels. Both proteins were present in the raft fractions, but tubulin was selectively lost following separation on SDS-gels. Assemblies of cytoskeletal proteins with lipid rafts may therefore be resolved using in-solution digestion that would be missed using gel-based approaches.
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PMID:Lipid raft proteomics: analysis of in-solution digest of sodium dodecyl sulfate-solubilized lipid raft proteins by liquid chromatography-matrix-assisted laser desorption/ionization tandem mass spectrometry. 1537 91

The quartz crystal microbalance (QCM) was used to create piezoelectric whole-cell biosensors utilizing either living endothelial cells (ECs) or the metastatic human mammary cancer cell line MDA-MB-231 adhering to the gold QCM surface under in vitro growth conditions. We utilized the whole-cell QCM biosensors for the detection of the effects of varying concentrations of the microtubule binding drugs taxol and nocodazole by measuring changes in the QCM steady state frequency (Deltaf) and motional resistance (DeltaR), shift values. Using 0.11-50 microM nocodazole, we observed the Deltaf shift values of the biosensors, consisting of 20,000 ECs, to decrease significantly in magnitude (nearly 100%) to a limiting value, in a dose-dependent fashion, over a 5- to 6-h incubation period following drug addition. This effect is consistent with nocodazole's known disruption of intracellular microtubules. On the other hand, 10 microM taxol caused little alteration in Deltaf over the same time period, consistent with its microtubule hyperstabilization effect. When the EC QCM biosensor Deltaf shift values were normalized by the number of ECs found firmly attached to the QCM surface via trypsin removal and electronic counting, the dose curve was shifted to lower nocodazole concentrations, resulting in a more sensitive drug biosensor. The kinetics of the Deltaf decrease with increasing nocodazole concentrations measured by the EC QCM biosensor was found to be similar at all drug concentrations and was well fit by a single first-order exponential decay equation. For all nocodazole doses, t(0.5) was invariant, averaging t(0.5)=0.83+/-0.14 h. These data demonstrate that a single dynamic sensing system within the cell, the microtubules, is disrupted by the addition of nocodazole and this process is sensed by the cell QCM biosensor. This interpretation of the data was confirmed by a fluorescence light microscopy investigation of ECs undergoing treatment with increasing nocodazole doses using a fluorescent antibody to alpha-tubulin. These studies revealed a corresponding loss of the spread morphology of the cells, concomitant with a rearrangement of the extended native microtubules into increasingly large aggregates with the cells eventually lifting from the surface in significant numbers at 50 microM. At 6 microM nocodazole, partial reversibility of the EC QCM biosensor was demonstrated. These results indicate that the EC QCM biosensor can be used to detect and study EC cytoskeleton alterations and dynamics. We suggest the potential of this cellular biosensor for the real-time identification or screening of all classes of biologically active drugs or biological macromolecules that affect cellular attachment and cellular spreading, regardless of their molecular mechanism of action.
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PMID:A comparative study of the cytoskeleton binding drugs nocodazole and taxol with a mammalian cell quartz crystal microbalance biosensor: different dynamic responses and energy dissipation effects. 1716 75


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