COLCHICINE‐BINDING ACTIVITY IN PARTICULATE FRACTIONS OF MOUSE BRAIN¹

Abstract

Abstract— Both particulate and soluble fractions of brain homogenates bound [3H]colchicine. Approximately one‐half of the total colchicine‐binding activity in mouse brain was found in the particulate fraction. Of the particulate fractions, the microsomal and nerveending subfractions which sediment at the 1·0–1·2 m interface on sucrose gradients were richest in colchicine‐binding activity. Intact microtubules were not found in these fractions, but colchicine‐binding activity of these fractions may be related to the presence of microtubular protein.

The subcellular distribution of colchicine‐binding protein (‘microtubule protein’) in rat brain

“…The possibility that membrane components which bind colchicine represent stabilized forms of microtubular protein is not inconsistent with the observation that colchicine-binding activity of synaptic membranes is largely retained after repeated washings with phosphate-buffered sucrose solutions containing 0.1% Triton Xl 00, or after storage of the membranes at -10" for up to two weeks (Lyons and Lagnado, paper in preparation; cf. also [3,4]). This is in contrast with the rapid decay of colchicinebinding activity observed with soluble preparations under similar conditions [IO] .…”

Characterization of the Colchicine Binding of Membrane Fractions From Rat and Mouse Liver

“…The colchicine-binding capability is different in the various membranes and seems to be especially high, for example, in nuclear membranes from mammalian liver and microsomes and synaptic membranes from mammalian brain . The occurrence of colchicine-binding proteins in such a particle-bound form has been alternatively explained as due to (a) the association of tubulin with such membranes (8,36) or (b) that colchicine and related drugs can also be bound by nontubu-(d) incubation temperature, from 0•C to 90,•C, with lin membrane components . The present study or without preincubation at 90 6640r, 30 mina Bound extends our earlier investigations on the localizacolchicine was determined '(sy`the "filter technique tion and distribution of colchicine-binding sites in according to Weisenberg et' al .'…”

“…Moreover it has been implied in most of these studies that these "colchicine-binding proteins" are recovered in a soluble form, i .e ., in the 100,000 g supernate from a cellular homogenate . However, recently, colchicine-binding has been described for particulate cellular subfractions from various tissues, THE JOURNAL OF CELL BIOLOGY • VOLUME 60,1974 • pages 297-303 especially in membrane fractions from mammalian brain and liver (6,8,25,26,36,42) . The colchicine-binding capability is different in the various membranes and seems to be especially high, for example, in nuclear membranes from mammalian liver and microsomes and synaptic membranes from mammalian brain .…”

“…that of the repeated washes by ultracentrifugation (36) and with the method of Feit and Barondes (8), which uses a centrifugation through 10% sucrose (in our experiments this step was followed by a centrifugation for 30 min at 160,000 g) . If not otherwise indicated, the determinations of the bound drug represent the differences of the amounts bound in the specific probe and that in the corresponding zero time and 0 • C "blank ."…”

“…Radiochemical purity and possible chemical alterations of the radioactive drugs were checked by scanning the thin-layer chromatograms with a gasflow-counter and comparing the mobilities with that of unlabeled colchicine (35) . After the assay the bound radioactivity was reisolated (3,8) and identified in the same way . The incubation was carried out in the "assay medium" under gentle magnetic stirring .…”