Characteristics of lysosomes in the rat placental cells

Abstract

Six acid hydrolases, cytochrome oxidase, and alkaline phosphatase were demonstrated in 0.25 m sucrose homogenates of rat chorioallantoic placenta. The acid hydrolases were: acid phosphatase, β-glucuronidase, N-acetyl-β-glucosaminidase, β-galactosidase, and acid deoxyribonuclease, showing optimum activity near pH 4.5; cathepsin, with optimum activity near pH 3.6. The free acid hydrolases present in cytoplasmic extracts expressed 20–40% of their total activity. “Total” activity was defined as the enzyme activity observed in the presence of Triton X-100, while “free” activity denoted enzyme activity measured under similar assay conditions except in the presence of sucrose and absence of Triton X-100. The decreased activity or latency in the assays for the free activity of acid phosphatase, acid deoxyribonuclease, and cathepsin persisted after incubation at pH 5 and 37 ° up to an hour. In contrast, this latency did not persist after incubation of the β-glycosidases. Additionally, the free activity of all the designated enzymes of the cytoplasmic extract was in excess of the nonsedimentable activity observed. When the placental homogenates were fractionated by differential centrifugation according to the scheme developed for the liver, cytochrome oxidase and the acid hydrolases were primarily divided between the two mitochondrial fractions, alkaline phosphatase was localized mainly in the microsome fraction, and catalase activity was almost entirely in the nuclear fraction. The lack of urate oxidase, d-amino acid oxidase and l-lactic acid oxidase activities suggested that peroxisomes do not exist in the placenta. A particulate fraction (M + L + P) was subfractionated by isopycnic equilibration in density gradients of sucrose; cytoplasmic extracts were fractionated in discontinuous gradients of sucrose. In all gradients when tissues from untreated and rats treated with Triton WR-1339 were compared, there was a marked shift in the acid hydrolases to the fractions of lighter density after Triton. The enzyme which showed the greatest shift was cathepsin while the least shift was demonstrated by β-glucuronidase. The observations in this study indicate an important heterogeneity of the lysosomes in this tissue.