Iron-ascorbate cleavable malic enzyme from hydrogenosomes of Trichomonas vaginalis: purification and characterization
Language English Country Netherlands Media print
Document type Journal Article, Research Support, Non-U.S. Gov't
PubMed
9027755
DOI
10.1016/s0166-6851(96)02777-6
PII: S0166-6851(96)02777-6
Knihovny.cz E-resources
- MeSH
- Chlorides pharmacology MeSH
- Cell Fractionation MeSH
- Intracellular Membranes enzymology MeSH
- Isoenzymes chemistry isolation & purification metabolism MeSH
- Kinetics MeSH
- Hydrogen-Ion Concentration MeSH
- Ascorbic Acid pharmacology MeSH
- Malate Dehydrogenase analysis chemistry isolation & purification metabolism MeSH
- Molecular Sequence Data MeSH
- Molecular Weight MeSH
- NAD metabolism MeSH
- Organelles enzymology MeSH
- Amino Acid Sequence MeSH
- Sequence Analysis MeSH
- Manganese Compounds pharmacology MeSH
- Trichomonas vaginalis enzymology MeSH
- Ferrous Compounds pharmacology MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- Chlorides MeSH
- ferrous sulfate MeSH Browser
- Isoenzymes MeSH
- Ascorbic Acid MeSH
- Malate Dehydrogenase MeSH
- malate dehydrogenase (decarboxylating) MeSH Browser
- manganese chloride MeSH Browser
- NAD MeSH
- Manganese Compounds MeSH
- Ferrous Compounds MeSH
Two isoforms of NAD(P)(+)-dependent malic enzyme (EC 1.1.1.39) were isolated from hydrogenosomes of Trichomonas vaginalis. A positively charged isoform at pH 7 was obtained in a single purification step using cation-exchange chromatography. The second isoform, negatively charged at pH 7.5, was partially purified using a combination of anion-exchange and affinity chromatography. Both isoforms displayed similar physical and kinetic properties. Molecular weight determination of the native enzyme suggested a homotetrameric arrangement of the 60 kDa subunits. The enzyme utilized NAD+ (Km, 6-6.3 microM) preferentially to NADP+ (Km, 125-145 microM). The NAD(+)-dependent activity showed a broad pH optimum with maximum under alkaline conditions (pH 9) likely to be present inside hydrogenosomes. Immunocytochemical studies using a polyclonal rabbit antibody raised against purified T. vaginalis malic enzyme proved hydrogenosomal localization of the enzyme. Subfractionation of hydrogenosomes suggested an association of the malic enzyme with the hydrogenosomal membranes. The 60 kDa malic enzyme subunit was highly sensitive to non-enzymatic cleavage by an iron-ascorbate system resulting in two enzymatically inactive fragments of about 31 kDa. Microsequencing of the fragments revealed that the 60 kDa subunit was cleaved at the metal-binding site between Asp279-Ile280. The enzyme inactivation was inhibited by an excess of manganese. Iron-dependent posttranslational modification might contribute to the regulation of malic enzyme activity in vivo.
References provided by Crossref.org
Arginine deiminase pathway enzymes: evolutionary history in metamonads and other eukaryotes
Iron-induced changes in the proteome of Trichomonas vaginalis hydrogenosomes
Live imaging of mitosomes and hydrogenosomes by HaloTag technology
Flavodiiron protein from Trichomonas vaginalis hydrogenosomes: the terminal oxygen reductase
Alternative pathway of metronidazole activation in Trichomonas vaginalis hydrogenosomes
Giardia mitosomes and trichomonad hydrogenosomes share a common mode of protein targeting
GENBANK
U16836, U16837, U16838, U16839
