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Incorporation in vitro of [ 14 C]Leucine into the Mitochondrial Protein of Lucilia cuprina

1971; Wiley; Volume: 22; Issue: 1 Linguagem: Inglês

10.1111/j.1432-1033.1971.tb01518.x

1432-1033

Keith L. Williams, L.M. Birt,

Neurobiology and Insect Physiology Research

The basic requirements for incorporation in vitro of [ 14 C]leucine into sterile mitochondria isolated from thoracic flight muscles of newly emerged Lucilia cuprina (the Australian sheep blowfly) have been studied in detail. Particular attention has been given to the influence of bacterial contamination on the incorporation. The incubation medium developed has the following composition: (final concentrations) 18 mM MgSO 4 ; 1 mM EDTA; 10 mM potassium phosphate; 50 μM each of 19 amino acids (excluding leucine); 65 mM KCl; 10mM HEPES ( N ‐2‐hydroxyethylpiperazine‐ N ′‐2‐ethane sulfonic acid) buffer; pH optimum7.4 (ATP‐generating system) or 8.0 ( dl ‐α‐glycerophosphate as substrate); and either ATP‐generating system (4 mM ATP, 5 mM phosphoenolpyruvate, 20 μg pyruvate kinase) or substrate‐supported system (20 mM dl ‐α‐glycerophosphate + 4 mM ADP). This medium is similar to that used for investigation of amino acid incorporation by mitochondria from the tobacco hornworm, but different from that used with locust mitochondria, especially in having a higher Mg 2+ content, higher pH optimum and lower amino acid mixture content.