The Synthesis of Rat Liver Lysosomes1

Abstract

To study the role of the Golgi complex in the formation of lysosomes, a resonably pure Golgi fraction was prepared from rat livers. Enzyme profile and morphology of the Golgi fraction were studied. β-Glucuronidase [EC 3.2.1.31] found in the Golgi fraction was proved to be indigenous to the Golgi complex by hypotonic-shock treatment of various subcellular fractions. Treatment of a low concentration of deoxycholate (DOC) revealed that about 65% of the total β-glucuronidase in the Golgi fraction was in the Golgi contents and about 35% of the enzyme was bound to the Golgi membrane. Detergent-dependent release of β-glucuronidase from the microsomal fraction also indicated that about 60% of the total glucuronicLase in this fraction was in the cisternal iumen and the remaining 40% was bound to the membrane of the endoplasmic reticulum. Three ββ-glucuronidases solubilized from the microsomal, Golgi, and lysosomal fractions were compared by polyacrylamide gel electrophoresis and isoelectric focusing. These techniques indicated the resemblance of the Golgi β-glucuronidase to the lysosomal enzyme and the difference of the microsomal β-glucuronidase from the other two. In particular, gel isoelectric focusing emphasizes the similarity between the Golgi and lysosomal β-glucuronidases. This technique resolved the lysosomal β-glucuronidase into thirteen activity bands in an acidic pH range, in which four bands (L₅ L₇ L₉ and L₁₁) were more intensely stained. The Golgi β-glucuronidase exhibited six activity bands, of which G₁ G₂ G₃ and G₄ predominated and corresponded to L₅ L₇ L₉ and L₁₁, respectively. The microsmal β-glucuronidase showed five activity bands in a neutral pH range. There were no significant differences between the three enzymes with respect to K_m and pH optimum. However, the microsomal β-glucuronidase differed significantly in heat stability from the other two enzymes. From the resemblance of the Golgi β-glucuronidase to the lysosomal enzyme, it is surmised that β-glucuronidase in the Golgi complex may be subsequently transferred into the lysosomes. If the newly synthesized enzyme at the site of membrane-bound ribosomes enters the cisternal iumen of the endoplasmic reticulum and is transferred into the lysosomes via the Golgi complex, the major transformation of the enzyme may occur in the Golgi complex, and the minor one in the lysosomes.