Biosynthesis and axoplasmic transport of neurophysin in the hypothalamo-neurohypophysial system of the grass frog Rana pipiens

摘要

Neurons of the preoptic nucleus (PON) are capable of synthesizing cysteine-rich proteins. The time course study reveals the presence in the neural lobe of a (\u2735)S-cysteine labeled protein (Np), which first appeared at 4 hours after the isotope injection and persisted throughout the entire observation period of 5 days. The Np protein is tentatively identified as the neurophysin protein of Rana pipiens. The minimum rate of transport for neurophysin is calculated as 0.9 mm/hr (22 mm/day). The Np protein can be further resolved by SDS-PAGE into 2 proteins, Np I (M.W. 23,000) and Np II (M.W. 20,100); or by isoelectric focusing in polyacrylamide gel into two proteins with pI = 4.9, and pI = 4.6. Also, two (\u2735)S-cysteine labeled proteins (pI = 5.8 and pI = 5.2) are present in the preoptic nucleus and infundibulum within one hour isotope injection. Preliminary evidence suggests a putative precursor role for the pI = 5.2 protein;Axoplasmic transport in the hypothalamo-neurohypophysial system of the frog has been effectively blocked by microiontophoretic ejection of vinblastine into the median eminence. Paracrystalline structures, presumably formed by the binding of vinblastine to tubulin, fill many of the neurosecretory axons in the median eminence 1 and 3 days after the ejection. These paracrystals are thought to be the major cause for the blockade. Large axon dilatations (Herring bodies) are induced proximal to the ejection site at day 3, local disposal of NGVs by lysosomes is seen within these Herring bodies at day 8, and to a lesser extent at day 15. Herring bodies are no longer seen at day 30 after the ejection. The sequence of fine structural changes observed suggests that the blockade is reversible. The early (day 3-8) responses in the distal neural lobe are marked by the degeneration of some neurosecretory axons. Whereas fine structure recovery in both the hypothalamo-neurohypophysial tract and the neural lobe at later stages (day 15-30) imply a functional recovery;Microtubule protein, tubulin was purified to apparent homogeneity from porcine brain. The purity of the PC-tubulin was checked by SDS-PAGE and the molecular weight was determined to be about 52,000. PC-tubulin was unable to assemble into microtubules under conditions where in vitro assembly from 2xMT is readily accomplished. However, the addition of the MAP fraction to the PC-tubulin greatly facilitate in vitro assembly of the latter into microtubules with an average diameter of 21 nm. Monospecific antisera against glutaraldehyde cross-linked PC-tubulin or SDS-PAGE purified tubulin were obtained and used to stain the in vitro assembled microtubules by the unlabeled peroxidase-anti-peroxidase (PAP) method. The resulting PAP complex-decorated microtubules attain an average diameter of 53 nm.