Summary. An intermediate‐ and a high‐purity factor‐VIII concentrate for clinical use have been prepared on a large scale by cryoethanol precipitation, extraction of the precipitate with tris buffer, and fractionation with polyethylene glycol. With bench‐scale fractionation, the intermediate material is 22‐fold purified on the average and the mean yield is 63%, while the high‐purity factor VIII is 274‐fold purified with a mean yield of 62%. With fractionation of 100 1. or more of fresh frozen plasma, the intermediate material shows a 30% yield and 14‐30‐fold purification; the high‐purity factor VIII shows an 18% yield and 125–350‐fold purification using 5.8 g/100 ml polyethylene glycol (PEG). A yield of nearly 30% should be possible with PEG, 4–5 g/100 ml. Both factor‐VIII preparations are stable for over a year in the lyophilized state at 4°C. Other plasma proteins can be fractionated from the residual plasma by routine Cohn procedures.

Two molar urea (pH 7.5) and column chromatography on Sepharose 4B were used to separate clathrin (coat protein) from the membrane of coated vesicles from bovine brain. Lytron (polystyrene) particles were used for study of the interaction of ciathrin with contractile proteins. Muscle G-actin, F-actin, and a-actinin were bound by clathrin-coated Lytron particles, while no interaction was found when muscle tropomyosin and serum albumin were tested. Clathrin molecules dispersed in a solution of 20 mM Tris*HCl (pH 7.5) were found to e elongated. When the pH was adjusted from 7.5 to 6.5, clathrin molecules associated into basketlike or cage structures similar in size and shape to those observed in enriched preparations of coated vesicles. Below pH 6.0, cages or baskets became amorphous aggregates. Raising the pH from 6.5 to 8.0, addition of 5-10 mM ATP or EDTA, or addition of 200 mM KCI resulted in the disassembly of baskets and the formation of filamentous arrays of various widths. Because of clathrin's biochemical and biophysical properties, its interaction with contractile proteins, and its presence in the membrane of vesicles of various cell types, we classified clathrin in the group of mechanochemical proteins.

A protein kinase activity was observed in coated vesicles, prepared from bovine brain, that had clathrinassociated protein 2 (CAP2, also known as clathrin light chain 2) as its principal substrate. Coated vesicles were purified by sucrose density gradient centrifugation followed by Sephacryl