Mechanism of ⁶⁷Ga Accumulation in Normal Rat Liver Lysosomes

Summary

⁶⁷Ga accumulates in various malignant tumors and parenchymatous tissues. It was found to be associated with the soluble fraction of lysosomes (11). The present work investigates the mechanism of ⁶⁷Ga accumulation in normal liver cells.

Lysosomes were isolated from rat liver after intravenous injection of carrier free ⁶⁷Ga. The soluble lysosomal fraction was obtained by sonication followed by centrifugation at 105,000 xg for 2 hrs. Gel filtration on Sephadex G 25 superfine was carried out on the soluble lysosomal fraction in order to investigate the stability of the ⁶⁷Ga-protein complex within the lysosomes under EDTA treatment. After treatment with 1 mM/1 EDTA a considerable amount of the protein bound radioactivity was found to be liberated. In further experiments the ⁶⁷Ga binding lysosomal proteins were fractionated by electrophoresis on 7% Polyacrylamide gels (0.5 cm x 5.5 cm). After staining with Coomassie blue 18 separated protein bands were apparent. ⁶⁷Ga distribution within the gels was assessed by direct counting of radioactivity in gel slices. A considerable amount of the intralysosomal protein bound radioactivity migrated with a relative mobility of 0.36 corresponding to a protein band of molecular weight 85,000 — 90,000. This peak corresponded to the peak of ⁶⁷Ga labeled purified transferrin in control gels. These data were confirmed by Immunoelectrophoresis combined with autoradiography: within the soluble lysosomal fraction a slight transferrin line could be identified.

We conclude that ⁶⁷Ga which is transported in the blood by transferrin (23) and taken up by the hepatic cell through endocytosis (32) is accumulated in the lysosomes associated with transferrin and its degraded fragments.

Trägerfreies ⁶⁷Ga wird in verschiedenen malignen Tumoren sowie in parenchymatösen Organen wie z. B. der Leber angereichert. Innerhalb der Zelle wird es in den Lysosomen gespeichert (11). Wir untersuchten den Medianismus der ⁶⁷Ga-Aufnahme in die normale Leberzelle.

Nach i.v. Applikation des Isotopes wurden Leberlysosomen isoliert und der ⁶⁷Ga-haltige lösliche Lysosomeninhalt durch Ultraschall freigesetzt und mit 105.000 xg abzentrifugiert. Die Stabilität der intralysosomalen ⁶⁷Ga-Bindung wurde vor und nach Zusatz von EDTA (1 mM/1) untersucht. Hierbei zeigten sich nach Gelfiltration über Sephadex G 25 sf ohne EDTA-Zusatz eine Fraktion proteingebundener Radioaktivität sowie eine Fraktion freier Radioaktivität. Nach Zusatz von EDTA (1 mM/1) wurde ein wesentlicher Teil des proteingebundenen ⁶⁷Ga aus seiner Bindung gelöst und erschien zusammen mit dem freien ⁶⁷Ga als ⁶⁷Ga-EDTA-Komplex in einem dritten Gipfel.

Mit Hilfe der Polyacrylamidgelelektrophorese konnte der ⁶⁷ Ga-haltige lösliche Lysosomeninhalt in 18 Proteinbanden aufgetrennt werden. Der überwiegende Teil der proteingebundenen ⁶⁷Ga-Radioaktivität wanderte in einer Proteinbande mit einem Molekulargewicht von 85.000 — 90.000. Diese Bande entsprach der Eichbande von gereinigtem ⁶⁷Ga-markierten Transferrin im Kontrollgel. Der Nachweis und die Identifizierung von intralysosomalem Transferrin gelang mit Hilfe der Immunoelektrophorese kombiniert mit der Autoradiographie.

Wir folgern aus unseren Daten, daß ⁶⁷Ga, das im Blut an Transferrin gekoppelt vorliegt, mit dem Transferrinmolekül durch Endozytose in die Zelle aufgenommen und in die Lysosomen transportiert wird, wo es dann an Transferrin und seine Abbaubruchstücke gebunden vorliegt.

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