Analysis of the pinocytic process in rat kidney. I. Isolation of pinocytic vesicles from rat kidney cortex

Pinocytosis was induced in rat kidney by exposure to horseradish peroxidase (HRP). Pinocytic vesicle preparations were enriched after homogenization of kidney cortex by differential centrifugation and free-flow electrophoresis with HRP as an exogenous marker. Vesicles were identified by enzymatic analysis and by electron microscopy, including specific staining procedures. Typical brush-border enzymes such as alkaline phosphatase, aminopeptidase, 5'-nucleotidase, lysosomal acid phosphatase, and mitochondrial succinic dehydrogenase were reduced in the vesicular fraction, compared to the kidney cortex homogenate. Glucose-6-phosphatase and Na+-K+-ATPase were only slightly increased in the fraction. These results indicate that preparations of pinocytic vesicles from rat kidney cortex can be enriched. They have biochemical characteristics that differ from those of the cell organelles and membranes previously purified from renal tissue

Morphologische und biochemische Untersuchungen über die Oberflächenstruktur der Bürstensaummembran der Rattenniere /Surface structure of Isolated brushborder of rat kidney: Morphological and biochemical analysis

The action of trypsin (E.C. 3.4.4.4) and papain (E.C. 3.4.4.10) on the surface structure of the microvillus membrane was investigated using isolated brushborder fragments of rat kidney cortex. Negative staining was employed to demonstrate the surface structure. The composition of the membrane was determined by biochemical analysis. Untreated membranes show a smooth surface without any structural arrangement. These membranes contain alkaline phosphatase (E.C. 3.1.3.1), aminopeptidase (E.C. 3.4.1.2), protein and 12 μg hexosamine per mg protein. Treatment with trypsin lowers the equilibrium density of the brushborder fragments from 1.17 to 1.15 and releases glycoproteins from the membranes which contain 54% of the hexosamine present in the starting material. There is no change in the content of alkaline phosphatase and aminopeptidase following trypsin treatment. Negative staining now reveals the presence of particles with a diameter of 40 Å on the surface of the membrane. When brushborder fragments are treated with papain the surface particles as well as glycoproteins are removed. Aminopeptidase activity is no longer present in the brushborder membrane and the equilibrium density is decreased to 1.12. It is proposed that the composition of the surface of kidney microvilli is as follows: particles with aminopeptidase activity are attached to a membrane matrix which contains alkaline phosphatase, other proteins and hexosamine. Both structure are covered by a glycoprotein layer which gives the microvillus surface its smooth appearance