ANNUAL REPORT OF THE
LABORATORY OF BIOCHEMICAL GENETICS
NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
October 1, 1981 through September 30, 1982

Monoclonal antibodies were obtained that affect transsynaptic
communication, inhibit intercellular adhesion, recognize antigens associated
with the synaptic layers of retina, or identify certain types of cells in
retina. Five antibodies were obtained that bind to antigens that are restricted
to the synaptic layers of retina; three of the antigens are restricted to the
outer synaptic layer, one to the inner synaptic layer, and one to both the inner
and outer synaptic layers of retina. The latter antibody also markedly inhibits
intercellular adhesion of retina cells. Two antibodies are specific for retina
ganglion neurons, seven for rods and cones, and seven for Muller cells. Other
antibodies recognize various sets of neurons; such as horizontal, bipolar, and
-amacrine neurons of the inner nuclear layer of retina.

Monoclonal antibodies also were obtained which recognize
neuroblastoma~hybrid cell antigens. Five antibodies were found that increase
the frequency of miniature endplate potentials of myotubes innervated by the
hybrid cells; whereas, six antibodies markedly decrease the frequency of
Miniature endplate potentials. The antibodies that affect rates of
acetylcholine secretion from hybrid cells do not alter the cell membrane
potential.

In previous studies we showed that a protein in avian retina, termed Top,
is distributed in a large dorsal~ventral gradient. The protein thus defines a
dorsal-ventral axis of the retina and identifies the relative positions of cells
in retina with respect to this axis. During the past year the protein was
purified extensively; the apparent M. of the protein is 47,000 and the
isoelectric point is approximately 4.1. A monoclonal antibody directed against
Top in retina also recognizes a protein in chick cerebral cortex and thalamus.
The protein in brain was purified and characterized; the M, and isoelectric
point of the protein from brain were the same as that found for Top from retina.
At this stage, we know relatively little about the regulatory mechanism which
relates the number of molecules of the protein detected per cell with the
relative positions of the cell in the retina, except that cells dissociated from
retina that were cultured in vitro continue to express the amount of the protein
that would be expected based on the original position of the cell in the intact
retina. Further studies on both the structure of the protein and the mechanism
regulating the expression of the protein are in progress.

 

The species of transmitter synthesized by a neuron determines, at least in
part, the type of neuron and the kinds of synaptic connections that are formed.
A project was initiated to serve as a foundation for future studies on
mechanisms that regulate the expression of choline acetyltransferase genes.
Choline acetyltransferase from rat brain was purified approximately 100,000-fold
and mice then were injected with purified enzyme preparations. Spleen cells
from the immunized mice were fused with P3X63 Ag8 myeloma cells; four of the
hybridoma cell lines that were obtained were found to synthesize antibodies
directed against choline acetyltransferase. The current objective is to use
the antibodies for the purification of mRNA coding for choline acetyltransferase

in order to clone the corresponding molecules of cDNA by recombinant DNA
methods. .

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Sixty five monoclonal antibodies were obtained which bind specifically to
membrane preparations from NG108-15 neuroblastoma-glioma cells. Six antibodies
were found which bind to a greater extent to membranes from differentiated
NG108-15 cells than to membranes from undifferentiated NG108-15 cells.
Two-dimensional gel electrophoresis also revealed several proteins which are
more abundant in membranes from differentiated cells, compared to membranes from
undifferentiated cells.

We previously showed that the acquisition of functional voltage-sensitive
calcium channels by NG108-15 cells enables the cells to form synapses with
striated muscle cells. A 1,4-dihydropyridine analog, {3H]-nitrendipine, which
reportedly binds specifically to voltage-sensitive calcium channels, was used as
a probe for voltage-sensitive calcium channels of NG108-15 cells.
(3H]-Nitrendipine was shown to bind specifically to membranes prepared from
differentiated NG108-15 cells; the dissociation constant, determined by
Scatchard analysis, was approximately 2 x 10710 mM. The number of specific
binding sites for [?H]-nitrendipine per average cell was shown to increase
10-fold, from 1,600 to 16,000, when cells were treated for 3 or more days with
PGE, which activates adenylate cyclase and elevates cellular cAMP levels.

These results suggest that prolonged elevation of cellular cAMP levels results
in an increase in the number of voltage-sensitive calcium channels per cell and
that the ability of the cells to form synapses is regulated by the rate of
synthesis. and/or turnover of voltage-sensitive calcium channels.

NG108-15 cells contain large, dense-core vesicles and small, clear
vesicles. Cells with endogenously synthesized [3H]-acetylcholine were lysed
and the vesicles that were released were separated fron cell membranes and then
fractionated by sucrose density centrifugation. Two well-separated peaks of
acetylcholine were found, which comprised greater than 90% of the intracellular

{3H]-acetylcholine found. Similar results were obtained with a mutant cell
line which lacks large dense-core vesicles, but has large vesicles with
amorphous electron-lucent cores. These results suggest that acetylcholine in
NG108-15 cells is stored in large dense-core vesicles and small clear vesicles
and that the clonally inherited change in vesicle morphology in the varient cell
line does not affect the ability of the vesicles to store acetylcholine.

A protein was obtained from bovine brain which markedly stimulates neurite
outgrowth by cultured neurons dissociated from chick embryo cerebral cortex.
The protein was purified approximately 200-fold. The native protein is a dimer
with an apparent M, of 75,000 which on reduction dissociates into subunits.
with an M, of 37,000. The protein, estimated to be 7 90% pure, induces
neurite outgrowth at a concentration of approximately 1 nM. A rabbit antiserum
to the neurite extension factor, termed NEF, was obtained which blocks the
activity of the protein in stimulating neurite extension. Neurite extension
protein was detected by immunohistochemical methods in neurons in some, but not
all, regions of adult rat brain. These results suggest that NEF is required
for neurite extension by some neurons in the central nervous system.

NG108-15 cells release a protein into the medium which induces the
formation of clusters of nicotinic acetylcholine receptors on myotube plasma
membranes. Laminin, a myotube basement membrane glycoprotein, was shown to
potentiate the aggregation of the acetylcholine receptors. Basement membrane
proteins of myotubes such as laminin, collagen types IV and V, and heparin
sulfate proteoglycan were detected on the surface of muscle fibers at early
stages of receptor aggregation in vivo. The basement membrane proteins
associated with acetylcholine receptor aggregates were relatively resistant to
extraction by detergent. Exposure of myotubes to brain extracts resulted in
marked aggregation of acetylcholine receptors on myotubes and the appearance of
cytoskeletal specializations under myotube plasma membranes. These results
suggest that the binding of laminin to the muscle cell surface and the formation
of cytoskeletal structures beneath the cell membrane may promote the formation

- of nicotinic acetylcholine receptor aggregates and/or stabilize the receptor
aggregates.

Polyadenylated mRNA was purified from adrenal medulla and brain and was
used to direct the cell-free synthesis of preproenkephalin, a precursor of
opioid enkephalin pentapeptides. The apparent M, of preproenkephalin was
found to be 30,000. The bovine precursor was show to contain the amino acid
sequences of both methionine- and leucine-enkephalin. Preproenkephalin was
converted to a smaller protein, termed proenkephalin, with an apparent M, of
28,000. Monoclonal antibodies directed against Met-enkephalin and
Met~enkephalin [ArgPhe] were obtained. A rat brain cDNA library was prepared by
recombinant DNA techniques which will be screened for rat preproenkephalin
clones and other clones of interest. cDNA hybridization probes then will be
used to study the regulation of enkephalin gene expression. In a related
project, corticotropin-releasing factor was shown to stimulate the secretion of
beta-endorphin and corticotropin from clonal AtT~20 mouse pituitary tumor cells
and to increase the activity of adenylate cyclase of the cells.

Histidyl-proline diketopiperazine, after injection into rats, was cleared
from the circulation with biphasic kinetics (tis2 = 1.25 and 33 min).
Unmetabolized histidyl-proline diketopiperazine appeared rapidly in the urine.
The longer half-time of clearance of the peptide of 33 min suggested a tissue
reservoir of the peptide. Histidyl-proline diketopiperazine was found to
accumulate in adrenal, liver, and kidney. Therefore, specific binding of the
diketopiperazine to membrane preparations was examined. Such binding was
observed in membrane preparations derived from adrenal and liver.