Rat RN22
schwannoma cells in vitro release into their growth medium a macromolecular factor that, when bound to
polyornithine-coated culture substrata, will stimulate neuritic regeneration from axotomized peripheral and central neurons. During the purification of this factor, the neurite-promoting activity co-purifies with
laminin immunoreactivity as measured by an
enzyme-linked
immunoadsorbent assay. The purified factor has an immunoreactivity per milligram of
protein similar to that of purified rat
yolk sac tumor laminin. After
sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions, the purified factor exhibits a major band at 200 kilodaltons (kD) and two minor ones at about 130 and 35 kD. The 200-kD band comigrates with the 200-kD band of purified rat
laminin. After SDS-PAGE under non-reducing conditions, the rat
schwannoma factor and rat
laminin both exhibit a band in the 900-kD range with the
schwannoma factor band migrating slightly faster than the
laminin one. The 200-kD (reducing conditions) and 900-kD (non-reducing conditions) bands of both the
schwannoma factor and
laminin are stained by immunoblotting with
antisera raised against rat and human
laminin and against a partially purified preparation of the
schwannoma factor. On immunoblots the 400-kD band of
laminin (a band not seen in the
schwannoma factor preparation) also stains with all three
antisera. When the
antibodies from each of the three
antisera are immobilized on
protein A-
agarose beads, the beads will completely remove from
solution the neurite-promoting activities of both the
schwannoma factor and
laminin.
Antibodies raised against rat
laminin fail to block the neurite-promoting activity of the purified
schwannoma factor but totally block that of rat
laminin. In contrast,
antibodies raised against the
schwannoma factor will block the neurite-promoting activities of both the
schwannoma factor and
laminin. By rotary shadowing electron microscopy the
schwannoma factor preparation exhibits cross-shaped images similar but not identical to those previously reported for rat and mouse
laminin. In addition, the
schwannoma factor preparation contains images resembling
proteoglycans.