Intracellular Localization of Prothrombin. ∗

Abstract

Prothrombin formation and storage are clearly functions of liver parenchymal cells according to information obtained with the fluorescent antibody technique (1,2). The present report extends the information on prothrombin localization to the intracellular level in dogs and cattle.

Materials and methods. Isolated parenchymal cell concentrates were obtained with the technique of St. George (3) on Dowex 50 W resin columns. Evaluation of the homogeneity of the fractions was made by differential cell counts on Leishman stained smears. A typical cell concentrate contained 96.5% parenchymal cells, 3% free nuclei and 0.5% red blood cells. In cell counts up to 600 cells neither Kupffer cells nor platelets were found.

Subcellular fractions were prepared in 0.25 m sucrose by the procedures of Schneider and Hogeboom(4) and de Duve and associates (5). The following fractions were studied: total cell homogenate (TCH), cytoplasmic extract (CE), nuclei (N), mitochondria (M), lysosomes (L), microsomes (Mic) and soluble fraction (S). Further purification of the nuclear fraction was achieved by centrifugation at 345 g for 5 minutes to sediment the associated whole cells and other subcellular particulates. The resulting supernatant containing nuclei was recentrifuged at 750 g for 5 minutes. The nuclei in this precipitate were then resuspended in 0.25 m sucrose and washed twice. All fractions were finally suspended in sucrose so that the stock solutions were 10% by volume.

(TCH) and soluble (S) fraction from a normal dog. B. Identical antigenic substances exist in mierosomes (Mic) and soluble (S) fraction of normal dogs. C. Note the preeipitin band indicative of prothrombin localization in soluble fraction of dicumarolized dogs (S_D) as well as in soluble fraction from Vit. K₁ stimulated (S_K) animals, D. Microsomes obtained from a dicumarolized dog (Mic_D) did not react with antiprothrombin.