Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Dimethylglycine dehydrogenase (EC 1.5.99.2) and sarcosine dehydrogenase (EC 1.5.99.1) are the folate binding proteins of rat liver mitochondria. These two enzymes contain covalently bound flavin and catalyze similar oxidative demethylation reactions (Wittwer, A. J., and Wagner, C. (1981) J. Biol. Chem. 256, 4102-4108). Flavin-peptides have been purified from these two enzymes after proteolytic digestion by trypsin and chymotrypsin. The spectral and chromatographic properties of these flavin peptides changed after treatment with nucleotide pyrophosphatase in a manner consistent with the conversion of an FAD-peptide to an FMN-peptide. The pKa for pH-dependent fluorescence quenching of the purified flavin-peptides was not affected by borohydride reduction which, in conjunction with the pKa values, indicated that the flavin was covalently linked via the 8 alpha position of the isoalloxazine ring to an imidazole N(3) of a histidine residue. Peptides from both enzymes showed histidylflavin at the N terminus. Amino acid composition and sequence analysis showed that the flavin-peptide from dimethylglycine dehydrogenase was His(flavin)-Ala-Ala-Gly-Leu. Amino acid composition and N-terminal analysis suggested the sequence of the flavin-peptide of sarcosine dehydrogenase was His(flavin)-(Ala, Gly,Thr)-Leu.
 ...
PMID:Identification of the covalently bound flavin of dimethylglycine dehydrogenase and sarcosine dehydrogenase from rat liver mitochondria. 649 Jun 27

We analyzed the folding, covalent flavinylation, and mitochondrial import of the rabbit reticulocyte lysate-translated bacterial 6-hydroxy-D-nicotine oxidase (6-HDNO) fused to the mitochondrial targeting sequence of rat liver dimethylglycine dehydrogenase. Translation of 6-HDNO in FAD-supplemented reticulocyte lysate resulted in a protein that contained covalently incorporated FAD, exhibited enzyme activity, and was trypsin-resistant, a characteristic of the tight conformation of the holoenzyme. The attached mitochondrial presequence did not prevent folding, binding of FAD, or enzyme activity of the 6-HDNO moiety of the fusion protein (pre-6-HDNO). Pre-6-HDNO was imported into rat liver mitochondria and processed by the mitochondrial processing peptidase. Incubation of the trypsin-resistant pre-holo-6-HDNO protein with deenergized rat liver mitochondria demonstrated that upon contact with mitochondria, the protein was unfolded and became trypsin sensitive. Mitochondrial import assays showed that the unfolded pre-holo-6-HDNO with covalently attached FAD was imported into rat liver mitochondria. Inside the mitochondrion the holo-6-HDNO was refolded into the trypsin-resistant conformation. However, when pre-apo-6-HDNO was imported only part of the protein became trypsin resistant (approximately 20%). Addition of FAD and the allosteric effector glycerol 3-phosphate to apo-6-HDNO containing mitochondrial matrix was required to transform the protein into the trypsin-resistant conformation characteristic of holo-6-HDNO.
 ...
PMID:Folding, flavinylation, and mitochondrial import of 6-hydroxy-D-nicotine oxidase fused to the presequence of rat dimethylglycine dehydrogenase. 771 2

Rat dimethylglycine dehydrogenase (Me2GlyDH) was used as model protein to study the biogenesis of a covalently flavinylated mitochondrial enzyme. Here we show that: 1) enzymatically active holoenzyme correlated with trypsin resistance of the protein; 2) folding of the reticulocyte lysate-translated protein into the trypsin-resistant, holoenzyme form was a slow process that was stimulated by the presence of the flavin cofactor and was more efficient at 15 degrees C than at 30 degrees C; 3) the mitochondrial presequence reduced the extent but did not prevent holoenzyme formation; 4) covalent attachment of FAD to the Me2GlyDH apoenzyme proceeded spontaneously and did not require a mitochondrial protein factor; 5) in vitro only the precursor, but not the mature form, of the protein was imported into isolated rat liver mitochondria; in vivo, in stably transfected HepG2 cells, both the precursor and the mature form were imported into the organelle; 6) holoenzyme formation in the cytoplasm did not prevent the translocation of the proteins into the mitochondria in vivo; and 7) lack of vitamin B2 in the tissue culture medium resulted in a reduced recovery of the precursor and the mature form of Me2GlyDH from cell mitochondria, suggesting a decreased efficiency of mitochondrial protein import.
 ...
PMID:Biogenesis of the covalently flavinylated mitochondrial enzyme dimethylglycine dehydrogenase. 862 65

The involvement of rat liver mitochondria in the flavinylation of the mitochondrial matrix flavoenzyme dimethylglycine dehydrogenase (Me2GlyDH) has been investigated. Me2GlyDH was synthesized as an apoenzyme in the rabbit reticulocyte lysate (RL) transcription/translation system and its flavinylation was monitored by virtue of the trypsin resistance of the holoenzyme. The rate of holoenzyme formation in the presence of FAD was stimulated with increasing efficiency by the addition of solubilized mitoplasts, mitochondrial matrix and DEAE-purified matrix fraction. Apo-Me2GlyDH was also converted into holoenzyme when the solubilized mitoplasts were supplemented with FMN and ATP. This observation is consistent with the existence of a mitochondrial FAD synthetase generating the FAD needed for holoenzyme formation from its precursors. Holoenzyme formation in the presence of FAD increased linearly with the concentration of matrix protein in the assay, and depended on the amount of externally added Me2GlyDH with saturation characteristics. These findings suggest the presence of a protein factor in the mitochondrial matrix which stimulates Me2GlyDH flavinylation. This factor was different from both mitochondrial heat shock protein (Hsp)70, as shown by immunodepletion experiments, and mitochondrial Hsp60, as demonstrated by the capability of a DEAE-purified matrix fraction devoid of Hsp60 to accelerate flavinylation of both RL translated and purified Me2GlyDH.
 ...
PMID:A protein factor of rat liver mitochondrial matrix involved in flavinylation of dimethylglycine dehydrogenase. 1088 Sep 57