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Kinetic data for the hydrolysis of potassium pacetylphenyl sulphate by fresh rat liver suspensions suggested that the arylsulphatase activity so observed could be attributed to a single enzyme (Dodgson, Spencer & Thomas, 1953) which was later found to be localized mainly in the microsomes of the liver cell (Dodgson, Spencer & Thomas, 1954a). Preliminary attempts to purify the microsome enzyme (unpublished data) were handicapped by its insolubility in various buffers and salt solutions of widely differing pH even after treatment of the liver by acetone-drying, alternate freezing and thawing, or use of the tissue disintegrator (Mickle, 1948). Sulphate and phosphate ions had little effect on the enzyme. These results contrast sharply with the arylsulphatase pattern reported for ox liver (Roy, 1953a, b; 1954). Using dipotassium 2-hydroxy-5nitrophenyl sulphate as the assay substrate, Roy was able to separate two arylsulphatases from the soluble material of acetone-dried ox liver and both enzymes were strongly inhibited by sulphate and phosphate ions. With the same substrate the arylsulphatase activity of mouse liver was found to be mainly concentrated in the mitochondria (Roy, 1953a). The present work, a preliminary report of which has already appeared (Dodgson, Spencer & Thomas, 1954 b), shows how the occurrence of three different arylsulphatases in mammalian livers enables these contrasting findings to be reconciled. METHODS Arylsulphates. Potassium p-acetylphenyl sulphate (APS) and potassium p-nitrophenyl sulphate (NPS) were prepared by the method of Burkhardt & Lapworth (1926) and dipotassium 2-hydroxy-5-nitrophenyl sulphate (nitrocatechol sulphate, NCS) according to Roy (1953a). Measurement of arylnulphata8e activity. The method previously used for APS (Dodgson, Lewis & Spencer, 1953) was also adopted for NPS but using the Am.. and e,,. determined for p-nitrophenol (Dodgson & Spencer, 1953). The use of NPS was restricted to liver extracts, as recoveries of p-nitrophenol from fresh liver suspensions are low (Dodgson & Spencer, 1953). The faint turbidity of the final solutions was the same in both test and controls and duplicate determinations were satisfactory. Turbidity only inter

b-Arrestin 1-GFP or b-arrestin 2-GFP were coexpressed transiently with G protein-coupled receptor kinase 2 within cells stably expressing the orexin-1, apelin or melaninconcentrating hormone (MCH), receptors. In response to agonist ligands both the orexin-1 and apelin receptors were able to rapidly translocate both b-arrestin 1-GFP and b-arrestin 2-GFP from cytoplasm to the plasma membrane. For the MCH receptor this was only observed for b-arrestin 2-GFP. b-Arrestin 1-GFP translocated by the apelin receptor remained at the plasma membrane during prolonged exposure to ligand even though the receptor became internalized. By contrast, for the orexin-1 receptor, internalization of b-arrestin 1-GFP within punctate vesicles could be observed for over 60 min in the continued presence of agonist. Co-internalization of the orexin-1 receptor was observed by monitoring the binding and traf®cking of TAMRA-(5-and 6-carboxytetramethylrhodamine) labelled orexin-A. Subsequent addition of an orexin-1 receptor antagonist resulted in cessation of incorporation of b-arrestin 1-GFP into vesicles at the plasma membrane and a gradual clearance of b-arrestin 1-GFP from intracellular vesicles. For the melaninconcentrating hormone receptor the bulk of translocated b-arrestin 2-GFP was maintained at concentrated foci close to, or at, the plasma membrane. These results demonstrate very distinct features of b-arrestin±GFP interactions and traf®cking for three G protein-coupled receptors for which the natural ligands have only recently been identi®ed and which were thus previously considered as orphan receptors.