Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0027960 (mole)
 21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Exploring a new method for the site-specific incorporation of functional groups into proteins, we have studied the combined use of genetic engineering techniques and enzymatic methods. Specifically, a short peptide for use as a substrate of guinea pig liver transglutaminase (TGase) is introduced at the N terminus of human interleukin-2 (hIL-2). The expressed chimeric protein (rTG1-IL-2) is chemically modified at a glutamine site in the appended sequence by TGase-catalyzed transamination with two amines, monodansylcadaverine (MDC), or a constructed derivative of poly (oxyethylene) (POE3). For the TGase-catalyzed modifications with MDC and POE3, 1 mol of donor was incorporated per mole of rTG1-IL-2, respectively. N-Terminal sequence analysis of MDC-modified rTG1-IL-2 (MDC-rTG1-IL-2) showed that the Gln-4 residue in the chimeric protein was site specifically modified with MDC. On the other hand, tryptic mapping of POE3-modified rTG1-IL-2 (POE3-rTG1-IL-2) by matrix-assisted laser desorption/ ionization time-of-flight mass spectrometry (MALDI-TOFMS) suggested that one of the Gln sites in the appended sequence was modified with POE3. The POE3-rTG1-IL-2 retained full bioactivity relative to the unmodified molecule and rhIL-2. This methodology could be a new and general route for the site-specific modification of proteins.
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PMID:Site-specific modification of interleukin-2 by the combined use of genetic engineering techniques and transglutaminase. 885 43

We have prepared an agent possessing both thrombolytic and antiplatelet properties, by conjugating fibrolase, a direct-acting fibrinolytic enzyme isolated from southern copperhead venom, to a peptide which inhibits platelet aggregation. Heterobifunctional coupling reagents, N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) or sulfosuccinimidyl 6-[alpha-methyl-alpha-(2-pyridyldithio)-toluamido]hexanoate (Sulfo-LC-SMPT), were used in a molar ratio of 10:1 (coupling agent/fibrolase). The N-hydroxy-succinimide of the coupling agent reacts with surface epsilon-amino groups of lysine residues on fibrolase and provides a dithio group that is highly reactive with small thiol compounds. The derivatives obtained in the first reaction contain approximately two moles of 2-pyridyl disulphide per mole of enzyme. These derivatives were then reacted with the free thiol group in an antiplatelet peptide at a molar ratio of 2:1 (peptide/fibrolase). The peptide-fibrolase conjugate was purified by cation exchange HPLC and analyzed by amino acid analysis. The conjugate contains one mole peptide per mole of fibrolase and retains approximately 85% fibrinolytic activity. The IC50 for inhibition of platelet aggregation in human PRP is 300 nM for the conjugate and 67 nM for the antiplatelet peptide. These results demonstrate the successful formation of a novel chimeric protein with bifunctional activity.
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PMID:Chimeric fibrolase: covalent attachment of an RGD-like peptide to create a potentially more effective thrombolytic agent. 926 96

Under native conditions, apocytochrome b(5) exhibits a stable core and a disordered heme-binding region that refolds upon association with the cofactor. The termini of this flexible region are in close proximity, suggesting that loop closure may contribute to the thermodynamic properties of the apocytochrome. A chimeric protein containing 43 residues encompassing the cytochrome loop was constructed using the cyanobacterial photosystem I accessory protein E (PsaE) from Synechococcus sp. PCC 7002 as a structured scaffold. PsaE has the topology of an SH3 domain, and the insertion was engineered to replace its 14-residue CD loop. NMR and optical spectroscopies showed that the hybrid protein (named EbE1) was folded under native conditions and that it retained the characteristics of an SH3 domain. NMR spectroscopy revealed that structural and dynamic differences were confined near the site of loop insertion. Variable-temperature 1D NMR spectra of EbE1 confirmed the presence of a kinetic unfolding barrier. Thermal and chemical denaturations of PsaE and EbE1 demonstrated cooperative, two-state transitions; the stability of the PsaE scaffold was found only moderately compromised by the insertion, with a DeltaT(m) of 8.3 degrees C, a DeltaC(m) of 1.5 M urea, and a DeltaDeltaG degrees of 4.2 kJ/mole. The data implied that the penalty for constraining the ends of the inserted region was lower than the approximately 6.4 kJ/mole calculated for a self-avoiding chain. Extrapolation of these results to cytochrome b(5) suggested that the intrinsic stability of the folded portion of the apoprotein reflected only a small detrimental contribution from the large heme-binding domain.
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PMID:Insertion of the cytochrome b5 heme-binding loop into an SH3 domain. Effects on structure and stability, and clues about the cytochrome's architecture. 1545 37