Phosphatides of pig heart cell fractions
Journal of Biological Chemistry
In a previous publication (1) an analysis of the phosphatides of pig heart ventricle was reported. This report is an extension of this work and deals with the phosphatide composition of pig heart ventricle mitochondria, microsomes, and the cytoplasmic supernatant fluid which is obtained upon centrifugation of the microsomes. Significant differences were found in these cell fractions, especially with regard to an acidic glycerophosphatide fraction which is very active metabolically and which
... cally and which occurs nearly exclusively in the mitochondria. METHODS AND REAGENTS Pig Heart Cell Fractions-49 gm. of pig heart ventricle (obtained as fresh as possible and freed from excess fat) were chopped into small pieces with scissors and homogenized at 0" in portions in 0.25 M sucrose by use of a ball type homogenizer (2). 10 ml. of sucrose solution were used for each gram of heart muscle. The resulting homogenate was adjusted to pH 7.0 by the addition of solid sodium bicarbonate' and then centrifuged at 1000 r.p.m. for 20 minutes. If necessary, centrifugation was repeated at 1000 to 2500 r.p.m. for 5 to 10 minutes until nearly all the myofibrils were removed (determined by microscopic examination) and only the mitochondria and microsomes were left in the supernatant fluid. The supernatant fluid was decanted off and centrifuged at 15,000 r.p.m. for 7 minutes. The precipitated mitochondria (Fraction I) were saved for lipide extraction. The supernatant fluid containing the microsomes was diluted to 400 ml. with 0.25 M sucrose and centrifuged at 25,000 r.p.m. for 1 hour in a Spinco model L preparative centrifuge (Rotor No. 30). The precipitated microsomes (Fraction II) and the supernatant fluid (Fraction III) were separated and saved for lipide extraction. Lipide Extraction The mitochondria (Fraction I) were homogenized in 20 ml. of cold 0.15 M KC1 and centrifuged at 3000 r.p.m. for 40 minutes. The precipitate was extracted three times with chloroformmethanol, l:l, for 10 minutes, each at 40". The combined extracts were filtered through a sintered glass funnel and the filtrate evaporated to dryness under nitrogen in vacua at 40'. The residue thus obtained was extracted four times with warm chloroform (40") to obtain the total lipides.