Isolation, characterization and seasonal variations in the concentration of N-epsilon-(gamma-glutamyl)-lysine isodipeptide in the blood plasma of the winter flounder (Pseudopleuronectes americanus).
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ABSTRACT:
SUBMITTER: Squires EJ
PROVIDER: S-EPMC1161455 | biostudies-other | 1980 Mar
REPOSITORIES: biostudies-other
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Project description:Gamma-glutamylamine cyclotransferase (GGACT) is an enzyme that converts gamma-glutamylamines to free amines and 5-oxoproline. GGACT shows high activity toward gamma-glutamyl-epsilon-lysine, derived from the breakdown of fibrin and other proteins cross-linked by transglutaminases. The enzyme adopts the newly identified cyclotransferase fold, observed in gamma-glutamylcyclotransferase (GGCT), an enzyme with activity toward gamma-glutamyl-alpha-amino acids (Oakley, A. J., Yamada, T., Liu, D., Coggan, M., Clark, A. G., and Board, P. G. (2008) J. Biol. Chem. 283, 22031-22042). Despite the absence of significant sequence identity, several residues are conserved in the active sites of GGCT and GGACT, including a putative catalytic acid/base residue (GGACT Glu(82)). The structure of GGACT in complex with the reaction product 5-oxoproline provides evidence for a common catalytic mechanism in both enzymes. The proposed mechanism, combined with the three-dimensional structures, also explains the different substrate specificities of these enzymes. Despite significant sequence divergence, there are at least three subfamilies in prokaryotes and eukaryotes that have conserved the GGCT fold and GGCT enzymatic activity.
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Project description:Zero-length isopeptide crosslinks between the side chains of glutamine and lysine are the product of transglutaminase activity. It is generally accepted that transglutaminase activity is dormant under physiological conditions because the calcium concentration inside cells is too low to activate transglutaminase to an open conformation with access to the catalytic triad. Traditional assays for transglutaminase activity measure incorporation of biotin pentylamine or of radiolabeled putrescine in the presence of added calcium. In this report, we identified naturally occurring isopeptide crosslinked proteins using the following steps: immunopurification of tryptic peptides by binding to anti-isopeptide antibody 81D1C2, separation of immunopurified peptides by liquid chromatography-tandem mass spectrometry, Protein Prospector database searches of mass spectrometry data for isopeptide crosslinked peptides, and manual evaluation of candidate crosslinked peptide pairs. The most labor intense step was manual evaluation. We developed criteria for accepting and rejecting candidate crosslinked peptides and showed examples of MS/MS spectra that confirm or invalidate a possible crosslink. The SH-SY5Y cells that we examined for crosslinked proteins had not been exposed to calcium and had been lysed in the presence of ethylenediaminetetraacetic acid. This precaution allows us to claim that the crosslinks we found inside the cells occurred naturally under physiological conditions. The quantity of crosslinks was very low, and the crosslinked proteins were mostly low abundance proteins. In conclusion, intracellular transglutaminase crosslinking/transamidase activity is very low but detectable. The low level of intracellular crosslinked proteins is consistent with tight regulation of transglutaminase activity.
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Project description:N epsilon-(gamma-Glutamyl)-lysine isodipeptide was detected in a protein-free fraction of Chinese-hamster ovary cells and their culture fluid by using radioactive lysine as a tracer. The identity of the isodipeptide was established by its separation on ion-exchange chromatography, analysis by h.p.l.c. after derivatization, recovery of lysine after acidic hydrolysis or after cleavage by a specific enzyme, namely gamma-glutamylamine cyclotransferase. The amount of isodipeptide was raised (460 pmol/10(7) cells and 61 pmol/ml of culture fluid were observed as highest values) as the cell density increased. Effects of inhibitors of intracellular protein degradation have shown that the isodipeptide derives from cross-linking N epsilon-(gamma-glutamyl)-lysine bonds formed by tissue transglutaminase. Estimated half-life values of cross-linked proteins were about 3 h. gamma-Glutamylamine cyclotransferase, which may split the isodipeptide formed during the continuous turnover of cross-linked proteins, was also found in Chinese-hamster ovary cells. Isodipeptide may have been accumulated when either its generated amount is beyond the capacity of gamma-glutamylamine cyclotransferase or it is generated in cell compartments where this enzyme is not present.
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Project description:We report on the discovery of interactions of porcine endometrial 17 beta-oestradiol dehydrogenase with actin. The 17 beta-oestradiol dehydrogenase of porcine uteri is an essentially unidirectional enzyme compounded in specialized organelles. The enzyme activity in Brij 35 extracts of the particulate fraction of epithelial cells sedimenting between 1800 and 11,000 g(av). was collected by immunoadsorption and eluted at low pH. The eluate contained three proteins of 32, 45 and 80 kDa as shown by SDS/PAGE and silver staining. They were identified by amino acid sequencing and immunotyping as oestradiol dehydrogenase (32 kDa), actin (45 kDa) and a covalent dehydrogenase-actin complex (80 kDa). Disulphides, aldimines, periodate-degradable bonds and hydrophobic interactions were excluded as linkages in the 80 kDa protein. The epsilon-(gamma-glutamyl)-lysine nature of the covalent cross-link was recognized by narrow-bore h.p.l.c. analysis of enzymic digests of electro-eluted 80 kDa material. An involvement of the actin anchor in positioning of the oestradiol dehydrogenase-containing organelles according to metabolic requirements is discussed.
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Project description:We isolated from a tomato cDNA library the tomPRO1 locus, which encodes gamma-glutamyl kinase (GK) and gamma-glutamyl phosphate reductase (GPR). This locus is unusual among eukaryotic genetic elements because it contains two open reading frames, and thus resembles prokaryotic polycistronic operons. The first open reading frame, specifying GK, is terminated by a TAA codon, which is followed by five nucleotides, an ATG translation initiation codon, and the second open reading frame, encoding GPR. DNA sequence analysis of fragments obtained by PCR amplification confirmed that the internal TAA and neighboring sequences are present in the endogenous tomPRO1 sequence in tomato. We demonstrated with RNase protection assays that the tomPRO1 locus is transcribed in tomato tissue culture cells, into a product that contains the internal stop codon. In Escherichia coli, tomPRO1 directed the synthesis of two proteins, a 33-kDa GK and a 44-kDa GPR. Antibodies against the 44-kDa GPR purified from E. coli recognized a 70-kDa product in tomato tissue culture cells and a 60-kDa product in leaves and roots. These results suggest that in tomato tissues, GPR is made as part of a longer polypeptide by some translational mechanism that enables bypass of the internal stop codon, such as frameshifting or ribosome hopping. The tomPRO1 locus may be the first example of a nuclear genetic element in plants that encodes two functional enzymes in two distinct open reading frames.
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Project description:The biosynthesis of the antibiotic epsilon-poly-lysine (ε-PL) in Streptomyces albulus is performed by polylysine synthase (pls); however, the regulatory mechanism of this process is still unknown. Here, we first obtained the complete genome sequence of S. albulus ZPM, which consists of 9,784,577 bp and has a GC content of 72.2%. The genome houses 44 gene clusters for secondary metabolite biosynthesis, in which 20 gene clusters are involved in the biosynthesis of polyketides and nonribosomally synthesized peptides. High-throughput sequencing was further performed, and genetic variants were identified from pooled libraries consisting of the 30 highest-yield mutants or 30 lowest-yield mutants. More than 350 genetic variants associated with ε-PL yield have been identified. One hundred sixty-two affected proteins, from important metabolic enzymes to novel transcriptional regulators, were identified as being related to ε-PL synthesis. HrdD, one of the affected genes, is a sigma factor that shows the most sensitive response to pH change and contains a non-synonymous mutation (A132V) in mutant strains with lower ε-PL yields. Electrophoretic mobility shift assays showed that the pls gene is likely regulated by transcriptional activator HrdD. The data obtained in this study will facilitate future studies on ε-PL yield improvement and industrial bioprocess optimization.
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