Localization of a sulphate-activating system within Euglena mitochondria.
Intact mitochondria, obtained from Euglena gracilis Klebs var. bacillaris Cori mutant W10BSmL, which lacks plastids, and purified on Percoll density gradients, form adenosine 3'-phosphate 5'-phosphosulphate from sulphate. The optimal conditions include addition of 17 mM-Tricine/KOH, pH 7.6, 18 mM-MgCl2, 250 mM-sucrose, 5.66 mM-sodium ADP (or 0.94 mM-sodium ATP), 1 mM-K2SO4, carrier-free 35SO4(2-) (32.1 microCi) and 1.0 mg of mitochondrial protein in a total volume of 2.65 ml and incubation at 30 degrees C. Experiments with the inhibitor of adenylate kinase P1, P5-di(adenosine 5'-)pentaphosphate indicate that ATP is the preferred substrate for sulphate activation; ADP is utilized by conversion into ATP via adenylate kinase. ATP sulphurylase, adenylylsulphate kinase (APS kinase) and inorganic pyrophosphatase constitute the sulphate-activating system; ADP sulphurylase is undetectable. Fractionation of Euglena mitochondria with digitonin and centrifugation allowed the separation of outer-membrane vesicles and mitoplasts as judged by electron microscopy and selected enzymic markers. The detergent-labile association of the sulphate-activating system with the mitoplasts (similar to that of adenylate kinase), the fact that most of the adenosine 3'-phosphate 5'-phosphosulphate formed by intact mitochondria is found in the surrounding medium, and the ease with which nucleotide substrates reach the activating system in intact organelles, suggest that the enzymes of sulphate activation are located on the outer surface of the mitochondrial inner membrane.
D000227:Adenine Nucleotides / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000037:antagonists & inhibitors; D002499:Centrifugation, Density Gradient; D004072:Digitonin / Q000494:pharmacology; D015226:Dinucleoside Phosphates; D004590:Electrophoresis, Paper; D005056:Euglena gracilis / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D008854:Microscopy, Electron; D008928:Mitochondria / Q000378:metabolism*; D010724:Phosphoadenosine Phosphosulfate / Q000378:metabolism; D013431:Sulfates / Q000378:metabolism*
D000227:Adenine Nucleotides; D015226:Dinucleoside Phosphates; D013431:Sulfates; D010724:Phosphoadenosine Phosphosulfate; C012275:P(1),P(5)-di(adenosine-5'-)pentaphosphate; D000255:Adenosine Triphosphate; D004072:Digitonin