Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.24.27 (thermolysin)
 1,894 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Bovine rhodopsin has been reported to be S-palmitylated at cysteines 322 and 323 (Ovchinnikov, Y. A., Abdulaev, N. G., and Bogachuk, A.S. (1988) FEBS Lett. 230, 1-5). Using a combination of enzymatic and chemical cleavage techniques in conjunction with tandem mass spectrometry, the sites of incorporation of the palmityl groups are shown. Bovine rhodopsin in disc membranes was digested with thermolysin to generate the C-terminal fragment (241-327), which was subsequently cleaved with cyanogen bromide to generate the peptide Val-Thr-Thr-Leu-Cys-Cys-Gly-Lys-Asn-Pro (318-327). A bis-S-palmitylated synthetic standard had the same retention time by reversed-phase high performance liquid chromatography as the isolated peptide and the same molecular weight (MH+1511.7) by liquid secondary ion mass spectrometry. Dithiothreitol reduction of both the isolated and the synthetic peptide cleaved the two thioester-linked palmityl groups to produce reduction products of the same appropriately decreased molecular weight (MH+1035.5). Tandem mass spectrometry of the isolated and the synthetic peptide identified the sites of attachment of the palmityl groups on cysteines 322 and 323. These results prove the modification of cysteines 322 and 323 with palmitic acid in bovine rhodopsin, and illustrate the utility of mass spectrometry to characterize the post-translational modifications in G-protein coupled receptors.
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PMID:Palmitylation of a G-protein coupled receptor. Direct analysis by tandem mass spectrometry. 151 31

The amino acid sequence of the thioredoxin isolated from rabbit bone marrow was determined chiefly by high performance tandem mass spectrometry and fast atom bombardment mass spectrometry combined with manual Edman degradation. The sequences of peptides generated by digestion with trypsin alone or in combination with Staphylococcus aureus protease V8 or thermolysin were determined from their collision-induced dissociation mass spectra. Alignment of these sequences and additional sequence information were obtained from the collision-induced dissociation mass spectra of peptides obtained from digestion of the intact protein with S. aureus protease V8 and alpha-chymotrypsin. The resulting sequence of 104 residues is as follows: Val-Lys-Gln-Ile-Glu-Ser-Lys-Ser-Ala-Phe-Gln- Glu-Val-Leu-Asp-Ser-Ala-Gly-Asp-Lys-Leu-Val-Val- Val-Asp-Phe-Ser-Ala-Thr-Trp-Cys-Gly-Pro-Cys-Lys- Met-Ile-Lys-Pro-Phe-Phe-His-Ala-Leu-Ser-Glu-Lys- Phe-Asn-Asn-Val-Val-Phe-Ile-Glu-Val-Asp-Val-Asp- Asp-Cys-Lys-Asp-Ile-Ala-Ala-Glu-Cys-Glu-Val-Lys- Cys-Met-Pro-Thr-Phe-Gln-Phe-Phe-Lys-Lys- Gly-Gln-Lys-Val-Gly-Glu-Phe-Ser-Gly-Ala-Asn-Lys- Glu-Lys-Leu-Glu-Ala-Thr-Ile-Asn-Glu-Leu-Leu.
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PMID:Amino acid sequence of thioredoxin isolated from rabbit bone marrow determined by tandem mass spectrometry. 316 11

The amino acid sequence of the thioredoxin isolated from the photosynthetic green sulfur bacterium Chlorobium thiosulfatophilum was determined chiefly by fast atom bombardment mass spectrometry combined with Edman degradation and tandem mass spectrometry. For this purpose, the protein was digested with trypsin, alpha-chymotrypsin, thermolysin, and Staphylococcus aureus protease or combinations thereof. Chemical cleavage with cyanogen bromide was also used alone or in combination with trypsin. The resulting sequence of 108 amino acids is as follows: Ala-Gly- Lys-Tyr-Phe-Glu-Ala-Thr-Asp-Lys-Asn-Phe-Gln- Thr-Glu-Xle-Xle-Asp-Ser-Asp-Lys-(Ala-Val)-Xle- Val-Asp-Phe-Trp-Ala-Ser-Trp-Cys-Gly-(Pro-Cys)- Met-Met-Xle-Gly-Pro-Val-Xle-Glu-Gln-Xle-Ala-Asp- Asp-Tyr-Glu-Gly-Lys-Ala-Xle-Xle-Ala-Lys-Xle-Asn- Val-Asp-Glu-Asn-Pro-Asn-Xle-Ala-Gly-Gln-Tyr-Gly- Xle-Arg-Ser-Xle-Pro-Thr-Met-Xle-Xle-Xle-Ly s- (Gly-Gly-Lys)-Val-Val-Asp-Gln-Met-Val-Gly-Ala- Xle-Pro-Lys-Asn-Met-Xle-Ala-Lys-Lys-Xle-Asp-Glu-His-Il e-Gly (where Xle represents leucine or isoleucine; sequences in parentheses are based on homology considerations). It exhibits less than 53% homology with Escherichia coli thioredoxin.
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PMID:Mass spectrometrically derived amino acid sequence of thioredoxin from Chlorobium, an evolutionarily prominent photosynthetic bacterium. 329 35

The N-terminal formic acid fragment (FA1) of the N-[3H]ethylmaleimide-labeled and carboxymethylated bovine mitochondrial phosphate transport protein (PTPN*CM) has been purified and completely sequenced: NH2-Ala-Val-Glu-Glu-Gln-Tyr-Ser-Cys-Asp-Tyr10-Gly-Ser-Gly-Arg-Phe- Phe-Ile-Leu-Cys- Gly20-Leu-Gly-Gly-Ile-Ile-Ser-Cys-Gly-Thr-Thr30-His-Thr -Ala-Leu-Val-Pro-Leu-Asp- -Leu-Val40-Lys-Cys(N-[3H]ethylmaleimide)-Arg-Met-Gln-Val-Asp- COOH. By thermolysin digestion of FA1 and high-performance liquid chromatography isolation of the radioactive subfragment Leu39-Arg43, the sole N-ethylmaleimide-binding residue has been identified as Cys42. FA1 contains a high mole percentage of cysteine (8.5%) and shows silver staining anomaly. Its sequence reveals significant homology in the triplicated gene regions (Pro27,132,229) of the mitochondrial ADP/ATP carrier from beef heart and Neurospora crassa. The hydropathic profile suggests that FA1 contains a transmembrane segment (Phe15-Val40) with only one basic (His31) and one acidic (Asp38) residue. The presence of the phosphate transport protein gene among nuclear genes is suggested from a lack of significant homology between the reverse-translated FA1 (mitochondrial codons) and the bovine mitochondrial genome. The inhibitory action of N-ethylmaleimide on the phosphate transport mechanism is discussed.
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PMID:Sequence of the N-terminal formic acid fragment and location of the N-ethylmaleimide-binding site of the phosphate transport protein from beef heart mitochondria. 406 97

Inhibition of envelysin, a metalloproteinase which dissolves the fertilization envelope of sea urchin embryo, was studied using a synthetic autoinhibitor peptide. Ac-Pro-Arg-Cys-Gly-Val-Pro-Asp-Val-NH2, with a 'cysteine-switch' consensus sequence. Although its effect is reversible, the hatching of sea urchin embryos was effectively delayed by 0.5 mM of the peptide. When alpha 1-proteinase inhibitor was used as the substrate, envelysin was inhibited by the autoinhibitor and an Ala6 analogue, but not by a D-Cys3 analogue. However, envelysin was weakly inhibited by both D- and L-cysteines to the same extent. Snake venom alpha-protease exhibited cleavage and inhibition behavior similar to envelysin with a little weaker stereo-specificity. The results suggest that the coordination of the autoinhibitor Cys residue with the envelysin active site Zn is established only after the amino acid residues on both sides of the Cys residue get into an appropriate interaction with the catalytic site residues, and that the precise orientation of the cysteine SH group is essential. By contrast, thermolysin was weakly inhibited by the three peptide non-stereo-specifically. Furthermore, thermolysin cleaved the autoinhibitor at the Cys3 Gly4 bond when incubated without substrate.
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PMID:Stereo-specific inhibition of sea urchin envelysin (hatching enzyme) by a synthetic autoinhibitor peptide with a cysteine-switch consensus sequence. 846 15

[Cu(2+).Cys-Gly-His-Lys] stimulates thermolysin (TLN) activity at low concentration (below 10 microM) and inhibits the enzyme at higher concentration, with binding affinities of 2.0 and 4.9 microM, respectively. The metal-free Cys-Gly-His-Lys peptide also stimulates TLN activity, with an apparent binding affinity of 2.2 microM. Coordination of copper through deprotonated imine nitrogens, the histidyl nitrogen, and the free N-terminal amino group is consistent with the characteristic absorption spectrum of a Cu(2+)-amino-terminal copper and nickel binding motif (lambda (max) approximately 525 nm). The lack of thiol coordination is suggested by both the absence of a thiol to Cu(2+) charge transfer band and electrochemical studies, since the electrode potential (vs. Ag/AgCl) 0.84 V (DeltaE = 92 mV) for the Cu(3+/2+) redox couple obtained for [Cu(2+).Cys-Gly-His-Lys] was found to be in close agreement with that of a related complex [Cu(2+).Lys-Gly-His-Lys](+) (0.84 V, DeltaE = 114 mV). The N-terminal cysteine appears to be available as a zinc-anchoring residue and plays a critical functional role since the [Cu(2+).Lys-Gly-His-Lys](+) homologue exhibits neither stimulation nor inhibition of TLN. Under oxidizing conditions (ascorbate/O(2)) the catalyst is shown to mediate the complete irreversible inactivation of TLN at concentrations where enzyme activity would otherwise be stimulated. The observed rate constant for inactivation of TLN activity was determined as k (obs) = 7.7 x 10(-2) min(-1), yielding a second-order rate constant of (7.7 +/- 0.9) x 10(4) M(-1) min(-1). Copper peptide mediated generation of reactive oxygen species that subsequently modify active-site residues is the most likely pathway for inactivation of TLN rather than cleavage of the peptide backbone.
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PMID:Stimulation and oxidative catalytic inactivation of thermolysin by copper.Cys-Gly-His-Lys. 1761 68