Organ and intracellular localization of short-chain acyl-CoA synthetases in rat and guinea-pig.

ABSTRACT

1. Homogenates of rat epididymal fat pad, heart, kidney, lactating mammary gland, liver, skeletal muscle and small intestinal mucosa have been partitioned into a particulate and supernatant fraction. With reliable marker enzymes for the mitochondrial matrix and the cytosol propionyl-CoA carboxylase and pyruvate kinase, the distributions of the acyl-CoA synthetase activities measured at 1 and 10 mM C2, C3 and C4 over mitochondria and cytosol have been calculated. From these values an estimate was made of the K0.5 of the fatty acids. 2. A distinct fatty acid-activating enzyme was assumed to be present in one of the compartments when that fatty acid was activated with a K0.5 less than or equal to 1.5 mM in an amount of greater than 13% of the total cellular activity. Adipose tissue, gut, liver and mammary gland, all organs of a high lipogenetic capacity, contained a cytosolic acetyl-CoA synthetase. At 1 mM acetate 60, 31, 77 and 83% of the total cellular activities in these organs were cytosolic in nature, with activities of 0.021, 0.32, 0.37 and 1.16 mumol C2 activated per min per g wet weight, respectively. 3. Mitochondrial acetyl-CoA and butyryl-CoA synthetases were found in adipose tissue, gut, heart, kidney, mammary gland and muscle. They were absent in liver. Adipose tissue and liver contained a mitochondrial propionyl-CoA synthetase with activities at 1 mM C3 of 0.014 and 1.50 mumol C3 activated per min per g wet weight, respectively. 4. At 1 mM, C2 was activated with decreasing rates by kidney, heart, mammary gland and gut (7.6-1.0 mumol C2 activated per min per g wet weight). C3 (1 mM) activation was about equal (1.6-1.9 mumol C3 activated per min per g wet weight) in liver, kidney and heart. C4 (1 mM) was activated with decreasing rates by heart, liver, kidney and gut (4.0-0.5 mumol C4 activated per min per g wet weight) in the order given. 5. The influence of the isolation method and the diet on fatty acid activation in small intestinal mucosal scrapings have been studied. To demonstrate the existence of cytosolic acetyl-CoA synthetase in fed animals a pre-treatment of everted intestine by low amplitude vibration has been found essential. Also C16 activation was highly (95%) decreased in a non-pre-vibrated preparation. 24 h starvation lowered cytosolic C2 and total C16 activation by 90 and 80%, respectively. Refeeding of starved rats with a balanced fat-free diet, and not with sucrose only, gave the same cytosolic C2 and total C16 activation as normally fed rats. 6. In guienea-pig heart, kidney, liver and muscle about the same partitions have been found as in the respective rat organs. The acetate activation in liver was factor 6 lower. Acetate and butyrate activation in guinea-pig muscle was much higher (6 and 37 times, respectively).