[Enzymatic characterization of lysosome equivalents (spherosomes) in corn seedlings]

Abstract

1. The following acid hydrolases are partially sedimentable from cell-free extracts of corn seedlings: protease (pH-optimum 4,2), phosphatase (pH-optimum 5,0 and 6,5), unspecific esterase (pH-optimum 5,2), RNase (pH-optimum 6,5), arylsulphatase-C (pH-optimum 5,0) and α- and β-amylase (pH-optimum 7,0 and 5,0 respectively). 2. After differential centrifugation of cell-free extracts the sedimentable hydrolases are recovered mainly in the mitochondrial and microsomal fraction. 3. It could be demonstrated that the acid hydrolases protease, phosphatase, RNase, and esterase of the mitochondrial fraction are contained in membrane-bound particles. 4. Isopycnic centrifugation of cell-free extracts in sucrose gradients revealed the presence of three particulate fractions carrying hydrolases. The heaviest fraction has a relative density of 1,138 g\sdcm(-3) and contains acid protease,-phosphatase,-RNase, and acid esterase. A lighter fraction (d=1,105 g\sdcm(-3)) contains the same acid hydrolases. The specifically lightest cell fraction (d=1,070 g\sdcm(-3)) contains the acid hydrolases glucose-6-phosphatase, arylsulphatase-C and small amounts of \ga- and \gb-amylase activity. This fraction also contains NADH-diaphorase activity. 5. By means of enzymatic characterization and staining with fluorochromes the structures carrying hydrolases were identified as two kinds of spherosomes and as fragments of the endoplasmatic reticulum. From these results it is concluded that the spherosomes represent organelles equivalent to the lysosomes of animal cells. 6. β-Glucuronidase, phospholipase-C, lipase, and arylsulphatase A and-B, all of with are typical enzymes of animal lysosomes, are completely absent in cell-free extracts of corn seedlings. 7. The isolated spherosomes do not exhibit indophenoloxydase activity; thus the histochemical demonstration of this enzyme in spherosomes must be considered to be an artifact. 8. Isolated lipid droplets, spherosomes, prospherosomes, and mitochondria incorporate H(-3)-acetate into lipids. Consequently they contain all the nessesary enzymes for lipid synthesis. The high activity of lipid synthesis in the spheroromes points to a possible conversion of these organelles into lipid bodies. 9. Two transaminases are partially bound to the mitochondria; the rest of the activity is probably bound to fragments of membranes less dense than an 18% sucrose solution.