Abstract
The experiments of Bach and Langley (1) with frog muscle suspensions indicated that the carnosine of this tissue was freely diffusible upon dialysis of homogenates. On the other hand, Reddy and Hegsted (2) observed that the “debris” of rat skeletal muscle homogenates contained 43% of the total carnosine. This debris, representing material sedimented at 650 x g, still retained half of its initial carnosine content after extraction for 7 days in 50% glycerol at -10”. Mitochondria were found to contain over 7% of the total muscle carnosine, and this dipeptide was not removed by suspension of the granules in isotonic sucrose. Reddy and Hegsted concluded that bound carnosine was present within the muscle cell, although it was not clear whether the peptide was attached to myofibrils or whether the microsomes retained any of the carnosine. No results were reported for anserine, the more abundant of the two muscle dipeptides. Inasmuch as a selective binding of carnosine to subcellular components of muscle might offer a clue to the physiological role of this enigmatic peptide, it seemed desirable to further study the intracellular distribution of carnosine, and of anserine as well, in skeletal muscle tissue. We have not been able to confirm the conclusions of Reddy and Hegsted that relatively large concentrations of carnosine are associated with subcellular fractions of rat gastrocnemius muscle. However, by employing more sensitive isotopic labeling methods, it has been possible to demonstrate the retention of very small quantities of carnosine and anserine in certain cellular components of chick pectoral muscle. This tissue was chosen because of its unusually high dipeptide content (3). In addition to normal birds, experiments were performed with genetically dyst,rophic chicks.
Highlights
Carnosine Content of Rat Skeletal dfuuscle FractionsHomogenates of gastrocnemius muscle of adult Sprague-Dawley rats were prepared with 4 parts of 0.88 M sucrose (2); subsequently, debris and mitochondria were sedimented according to Reddy and Hegsted (2)
The debris, after adequate washing with buffer, contained no detectable carnosine, whereas the washed mitochondria accounted for approximately 0.3%
The radioactivity values for carnosine and anserine in chicks, together with our analytical values for carnosine in the rat, indicate that only very small proportions of peptide remain associated with the subcellular fractions of muscle after washing or dialysis
Summary
Carnosine Content of Rat Skeletal dfuuscle FractionsHomogenates of gastrocnemius muscle of adult Sprague-Dawley rats were prepared with 4 parts of 0.88 M sucrose (2); subsequently, debris and mitochondria were sedimented according to Reddy and Hegsted (2). A separate homogenate of muscle with 9 parts of KCI-Tris buffer was used to obtain a 15,000 X g, supernatant extract. This step was performed in order to avoid the use of sucrose, which interfered with carnosine determina-. Separation and Treatment of Chick SubceUular Fractions-The pooled pectoral muscle of two birds (about 20 g of tissue) was subjected to a previously described procedure for the isolation of myofibrils, mitochondria, microsomes, and soluble supernatant phase (4). Half of the effluent was used to measure total i4C, and the other half was employed to determine radioactivity in carnosine (8) 5.8 followed by extraction of the colored imidaeole derivative with butanol
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