Effects of the sodium ionophore, monensin, were examined on the passage from neuronal cell body to axon of materials undergoing fast intracellular transport. In vitro exposure of bullfrog dorsal root ganglia to concentrations of drug <1 .0 UM led to a dose-dependent depression in the amount of fast-transported [3H]leucine-or [3H]glycerol-labeled material appearing in the nerve trunk. Incorporation of either precursor was unaffected . Exposure of a desheathed nerve trunk to similar concentrations of monensin, while ganglia were incubated in drug-free medium, had no effect on transport. With [3H]fucose as precursor, fast transport of labeled glycoproteins was depressed to the same extent as with [3H]leucine; synthesis, again, was unaffected . By contrast, with [3 H]galactose as precursor, an apparent reduction in transport of labeled glycoproteins was accounted for by a marked depression in incorporation . The inference from these findings, that monensin acts to block fast transport at the level of the Golgi apparatus, was supported by ultrastructural examination of the drug-treated neurons. An extensive and selective disruption of Golgi saccules was observed, accompanied by an accumulation of clumped smooth membranous cisternae.Quantitative analyses of 48 individual fast-transported protein species, after separation by two-dimensional gel electrophoresis, revealed that monensin depresses all proteins to a similar extent . These results indicate that passage through the Golgi apparatus is an obligatory step in the intracellular routing of materials destined for fast axonal transport.

The presence of a requirement for calcium during the fast transport of [3H]protein in axons was assessed in desheathed spinal nerves of bullfrog. The nerves were desheathed locally along 4 mm of their length, and desheathing was judged effective on the basis of an enhanced uptake of [3H]leucine into that region of nerve trunk. Desheathing per se had a slight inhibitory effect on transport. Incubation of desheathed nerve trunks in calcium‐free medium reduced transport by 60‐80% relative to that in desheathed nerves incubated in normal medium. Addition of Mg2+ or Sr2+ to the calcium‐free medium allowed transport to proceed normally. Addition of Co2+ or Mn2+ to normal medium did not affect transport in desheathed isolated nerve trunks. When ganglia and nerve trunks were both incubated in medium containing 0.18 mM‐CoCl2, transport was depressed to a similar extent proximal and distal to the desheathed region. This again indicates that Co2+ does not inhibit transport in desheathed nerves, whereas it does inhibit transport in the ganglia. Additive inhibitory effects were observed when ganglia were incubated in medium containing 0.018 mM‐CoCl2, and desheathed nerves were incubated in calcium‐free medium. Differences in the divalent cation specificities of the axonal and ganglionic calcium requirements suggest that calcium supports transport in nerves in a manner distinct from its role in maintaining transport in spinal ganglia. It is concluded that the ganglionic calcium requirement involves initiation of axonal transport in the soma rather than translocation in the intraganglionic region of axon.

Metalloendoprotease activity that was sensitive to the metal chelator 1,10-phenanthroline and to synthetic dipeptide substrates of the enzyme was detected in homogenates of dorsal root ganglia (DRG) and spinal nerve from the bullfrog. Exposure of an intact in vitro preparation of DRG and spinal nerves to 1,10-phenanthroline led to a dose-dependent depression in the accumulation of fast-transported 3H-labeled protein proximal to a nerve ligature. In nonligated preparations, the chelator treatment reduced the amount of transported protein entering the nerve; no marked effect on the transport rate was observed. Exposure of a desheathed region of spinal nerve to 1,10-phenanthroline, while DRG were maintained in control medium, resulted in a slight depression of fast transport. This effect was not dose dependent over the range that produced a dose response when both DRG and spinal nerve were exposed to the drug. Treatment of DRG and spinal nerve with the metalloendoprotease substrate analogues carbobenzoxy (CBZ)-Ser-Leu-amide or CBZ-Gly-Leu-amide inhibited fast axonal transport, whereas treatment with CBZ-Gly-Gly-amide, which is not a substrate, had no detectable effect on transport. Selective exposure of desheathed nerve trunk to CBZ-Ser-Leu-amide inhibited fast transport, but the effect was less marked than when DRG and nerve trunk were treated. Although previous studies have focused on the role of metalloendoprotease activity in exocytosis, the present data suggest that the enzyme may also be involved in earlier stages of intracellular transport.

Several fast-transported proteins that appear as single bands after sodium dodecyl sulfate-polyacrylamide gel electrophoresis resolve into multiple spots during isoelectric focusing. A method was devised for determining if such microheterogeneity in net charge indicates that individual polypeptides have been posttranslationally modified to differing extents. Dorsal root ganglia were pulse-labeled with [35S]methionine and either [3H]leucine or [3H]proline, proteins fast-transported into peripheral sensory axons were separated by two-dimensional gel electrophoresis, and isotope incorporation ratios of proteins associated with individual gel spots were determined. When four microheterogeneous glycoproteins were analyzed, each protein "family" showed markedly similar isotope ratios for its three to seven characteristic spots. Such ratios differed between families by almost twofold. In addition, a group of nonglycosylated, sulfate-containing proteins was identified as a family on the basis of the similar isotope incorporation ratios of its component spots. These results suggest that protein microheterogeneity can result from variable sulfation of tyrosine residues as well as from variation in sialic acid-containing oligosaccharide side-chains. More generally, the method can be utilized to test for protein microheterogeneity in cases where the amounts of protein are too low to permit peptide mapping analysis and where the nature of the charge-altering modification is unknown.