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The enzyme nagarse modifies parameters associated with function of mechanically‐liberated mitochondria (1159.7)
Author(s) -
Kras Katon,
Willis Wayne,
Barker Natalie,
Czyzyk Traci,
Katsanos Christos
Publication year - 2014
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.28.1_supplement.1159.7
Subject(s) - centrifugation , differential centrifugation , chemistry , enzyme , suspension (topology) , mitochondrion , nuclear chemistry , chromatography , biochemistry , mathematics , homotopy , pure mathematics
Nagarse (N) is traditionally used to isolate mitochondria (mito) from skeletal muscle (SM). We investigated the effects of N on functional parameters of mechanically liberated mito. Mito from mouse (n = 7) gastrocnemius muscle (119‐175 mg tissue) was isolated using a glass on glass homogenizer followed by differential centrifugation. The 800g supernatant from the first centrifugation, containing mito, was divided into two equal volumes. One was exposed to N and the other was not. In each volume, mito were isolated by standard differential centrifugation. State 3 max ADP‐stimulated O 2 consumption rates (J o ) were measured using polarography with malate + pyruvate + glutamate. N increased state 3 J o , 16.1 ± 1.1 vs. 11.9 ± 1.4 nmol O 2 / ml mito suspension / min; P < 0.004, while it decreased protein concentration in the final mito suspension (typically used to adjust final J o values), 1.9 ± 0.1 vs. 2.5 ± 0.1 mg / ml; P < 0.004. Citrate synthase activity in the final mito suspension, indicative of the available mito, was not different in the preparation treated with N vs. that not treated with N (0.067 ± 0.01 vs. 0.065 ± 0.01μmol / min; P = 0.8). These findings suggest that N treatment: 1) hydrolyzes non‐mito contaminating proteins in the final mito pellet, and 2) induces apparent qualitative changes in mito function. Therefore, calculated functional parameters of mito treated with N are not directly comparable to mito not treated with N. Grant Funding Source : Supported by NIH/NIDDK RO1 DK094062