Abstract
A method that employs gas chromatography-mass spectrometry has been developed to measure N-tau-methylhistidine (3-methylhistidine; 3-MH) synthesis and release from skeletal muscle myotubes in vitro. It shows excellent linearity (0.9999) over the range studied (0-4 nmol), high recovery (92.6%), and low coefficient of variation (1.6%). 3-MH release from myotubes was essentially linear over a 96-h incubation, whereas the loss of 3-MH from cell protein accelerated with increasing time, an effect due, at least in part, to decreasing rates of total protein synthesis. When incubated in either glutamine-free or methionine-free medium for 48 h, 3-MH in cell protein and appearing in the medium were greatly reduced compared with the 48-h controls, suggesting that hypertrophy was greatly reduced. Similar but lesser trends were observed with adenosine 3',5'-cyclic monophosphate. In contrast, 12-O-tetradecanoylphorbol-13-acetate (TPA) appeared to both stimulate 3-MH synthesis and inhibit its release during a 48-h incubation. The development of this method facilitates detailed investigation into the mechanisms through which agents such as TPA regulate myofibrillar protein degradation.
Original language | English |
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Pages (from-to) | C1875-C1879 |
Number of pages | 5 |
Journal | American Journal of Physiology: Cell Physiology |
Volume | 270 |
Issue number | 6 |
Publication status | Published - Jun 1996 |
Keywords
- phorbol ester
- adenosine 3',5'-cyclic monophosphate
- amino acid deletion
- myofibrillar protein degradation
- satellite cells
- gas chromatography mass spectrometry
- myofibrillar protein breakdown
- 3-methylhistidine
- insulin
- stimulation
- metabolism
- myoblasts
- invivo
- acid