Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.64 (proteinase K)
 4,071 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Retinoylation (retinoic acylation) is a posttranslational modification of proteins occurring in a variety of cell types in vitro. This study was done to examine whether retinoylation occurs in vivo. We found that in retinol-deficient rats, radiolabeled retinol or retinoic acid was incorporated into the liver, kidney, and lung in a form that was not removed by extraction with CHCl3:CH3OH. About 98% of the radiolabeled retinoid was acid-soluble after digestion with proteinase K indicating that it was covalently bound to protein. About 50% of the retinoid covalently bound to liver and kidney protein was removed by mild hydrolysis with CH3OH-KOH. Methyl retinoate, all-trans-retinoic acid, and polar metabolites of retinoic acid accounted for essentially all of the retinoids released. We conclude that retinoylation of protein occurs in vivo primarily via the formation of an ester bond.
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PMID:Retinoylation of proteins in rat liver, kidney, and lung in vivo. 889 63

all-trans-Retinoic acid, a highly active form of vitamin A in inducing cellular differentiation, is incorporated covalently into proteins both in vivo and in vitro. The relative rates of incorporation of all-trans-11,12-(3)H-retinoic acid into rat tissue homogenates in the presence of ATP and coenzyme A were testes>>lung> or =brain> or =kidney>liver. Although all studied cellular organelles of the testes incorporated (3)H-retinoic acid into protein, mitochondria were by far the most active; indeed, up to 25% of the added tritiated retinoic acid (RA) became covalently bound to protein in a 90 min incubation period. In the absence of ATP, coenzyme A, or both cofactors, the amount of RA incorporated into the proteins of testes mitochondria fell to 37%, 16%, and 11%, respectively, of that incorporated in their presence. N-Ethylmaleimide (5 mM) strongly inhibited the reaction. Boiled mitochondria were inactive. After extensive extraction with CHCl(3)-CH(3)OH, the protein-bound radioactivity, which proved largely to be retinoic acid, was released by treatment with proteinase K, hydroxylamine, and dilute base. Thus, retinoic acid is most probably linked to protein as a thiol ester. By SDS-polyacrylamide gel electrophoresis, four protein fractions with molecular masses of approx. 20, 24, 29, and 45 kDa, as well as smaller amounts of larger entities, were labeled in testes mitochondria. The possible identities and roles of these retinoylated proteins are currently being explored.
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PMID:Retinoylation of proteins in cell-free fractions of rat tissues in vitro. 1123 17

Lipofuscin accumulates with age in the retinal pigment epithelium (RPE) in discrete granular organelles and may contribute to age-related macular degeneration. Because previous studies suggest that lipofuscin contains protein that may impact pathogenic mechanisms, we pursued proteomics analysis of lipofuscin. The composition of RPE lipofuscin and its mechanisms of pathogenesis are poorly understood in part because of the heterogeneity of isolated preparations. We purified RPE lipofuscin granules by treatment with proteinase K or SDS and showed by light, confocal, and transmission electron microscopy that the purified granules are free of extragranular material and associated membranes. Crude and purified lipofuscin preparations were quantitatively compared by (i) LC MS/MS proteomics analyses, (ii) immunoanalyses of oxidative protein modifications, (iii) amino acid analysis, (iv) HPLC of bisretinoids, and (v) assaying phototoxicity to RPE cells. From crude lipofuscin preparations 186 proteins were identified, many of which appeared to be modified. In contrast, very little protein ( approximately 2% (w/w) by amino acid analysis) and no identifiable protein were found in the purified granules, which retained full phototoxicity to cultured RPE cells. Our analyses showed that granules in purified and crude lipofuscin preparations exhibit no statistically significant differences in diameter or circularity or in the content of the bisretinoids A2E, isoA2E, and all-trans-retinal dimer-phosphatidylethanolamine. The finding that the purified granules contain minimal protein yet retain phototoxic activity suggests that RPE lipofuscin pathogenesis is largely independent of associated protein. The purified granules also exhibited oxidative protein modifications, including nitrotyrosine generated from reactive nitrogen oxide species and carboxyethylpyrrole and iso[4]levuglandin E(2) adducts generated from reactive lipid fragments. This finding is consistent with previous studies demonstrating RPE lipofuscin to be a potent generator of reactive oxygen species and supports the hypothesis that such species, including reactive fragments from lipids and retinoids, contribute to the mechanisms of RPE lipofuscin pathogenesis.
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PMID:Retinal pigment epithelium lipofuscin proteomics. 1843 25

Geranylgeranoic acid (GGA), a 20-carbon acyclic polyprenoic acid (all-trans 3,7,11,15-tetramethyl- 2,4,6,10,14-hexadecatetraenoic acid) and its derivatives were developed as synthetic "acyclic retinoids" for cancer chemoprevention. Previously, we have shown the natural occurrence of GGA in various medicinal herbs and reported enzymatic formation of GGA from geranylgeraniol (GGOH) through geranylgeranial (GGal) by rat liver homogenates. Here, we present several lines of evidence that a putative GGOH oxidase is involved in GGA synthesis by human hepatoma cell lysates. First, conversion of GGOH to GGal did not require exogenous NAD(+), whereas the conversion from GGal to GGA absolutely required additional NAD(+). Second, GGal synthesis from GGOH was coupled with consumption of oxygen from the reaction mixture. Third, GGOH-dependent GGal synthesis activity was proteinase K-resistant and even enhanced by proteinase K treatment; GGOH oxidase activity was enriched in the mitochondrial fraction. Finally, recombinant human monoamine oxidase (MAO)-B, but not MAO-A catalyzed oxidation of GGOH to GGal. These data suggest that a putative mitochondrial GGOH oxidase is involved in the initial step of GGA synthesis from GGOH.
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PMID:Geranylgeraniol oxidase activity involved in oxidative formation of geranylgeranoic acid in human hepatoma cells. 2236 82