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A reversed-phase gradient elution system is described for the simultaneous separation of the type I and type III isomers of 8-, 7-, 6-, 5- and 4-carboxylated porphyrins and isocoproporphyrins. The method, adaptable for isocratic and stepwise separation of individual groups of isomers, is also suitable for preparative isolation of pure porphyrins. The analyses of porphyrin isomers in the urine and faeces of porphyric patients are examples of applications.

A reversed-phase system is described for the simultaneous isocratic separation of coproporphyrin I, II, III and IV isomers. The retention behaviour of coproporphyrin I and III is studied in detail. The method is suitable for both analytical and semi-preparative separation.

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The effect of selected weak bases on the subcellular distribution and processing of internalized insulin by the liver has been studied. The effect of these bases on both the degradation products formed and on the kinetics of degradation have also been studied.1. Methylamine, ammonium chloride and dansyl cadaverine but not chloroquine reduce the total amount of insulin endocytosed.2. Ammonium chloride, dansyl cadaverine and chloroquine but not methylamine inhibit subsequent degradation and/or translocation of degradation products.3. None of the weak bases changed the species of the degradation products found within the endocytic vesicles. 4. Kinetic analysis of intravesicular degradation indicates that dissociation from the receptor is the rate- 5.Chloroquine and dansyl cadaverine but not methylamine or ammonium chloride showed specific inhibition 6. The effect of chloroquine and dansyl cadaverine on the kinetics of degradation suggest that they are acting limiting process in degradation.of insulin degradation in isolated endocytic vesicles.by switching the receptor into a tight-binding conformation thereby slowing dissociation.The liver is a major target organ for insulin, and over 50% of insulin may be cleared in a single pass through the portal circulation [l]. It is now established that insulin binds rapidly and reversibly to specific receptors in a number of tissues including the liver [2], and is then internalized [3]. The subsequent subcellular processing pathway remains uncertain although degradation seems to be the ultimate fate for most of the internalized insulin [4].Although it is still not clear whether or not degradation is initiated by a plasma-membrane protease [5], it was formerly believed that the major site of degradation was the lysosome [6]. Evidence for this was based on electron-micrographic studies [6] and from the effects of weak bases such as chloroquine which cause an accumulation of insulin in the liver [7]. It is suggested that weak bases concentrate in lysosomes by means of proton trapping [8] and thus lysosomal involvement in the degradation was inferred. However, the Golgi apparatus has been shown to be altered morphologically by chloroquine [9] and it is known that some endocytic compartments are also maintained at a low pH [lo], and thus will also trap weak bases. In addition, evidence is accumulating that in the adipocyte [ll] This study examines the effect of selected weak bases on the subcellular distribution and processing of internalized insulin in the perfused rat liver. It also examines kinetically and by HPLC analysis their effect on the in vitro degradation of internalized insulin contained in isolated vesicles. MATERIALS AND METHODS MaterialsChloroquine, methylamine, dansylcadaverine and bovine serum albumin (radio-immunoassay grade) were purchased from Sigma Chemical Co. Ltd (Poole, Dorset, UK). [1251-tyr~sine~'~]Insulin was labelled and purified as described previously [18] from a zinc-stabilized porcine insulin (Monotard MC, Novo, Basingstoke, UK). The specific ac...