DOI Seite / Zitierlink:
https://doi.org/10.11588/diglit.48120#0036
18
Molecular and Cell Biology of Autoantibodies and Autoimmunity
80, 64, 47, 46, 40, 37, 33, 30, 27, 26, 22, 20, and 18 kDa. This complex was iden-
tified with the PM/Scl antigen by its electrophoretic pattern equal to that given
by a control reference serum kindly provided by Eng Tan. Of these proteins, those
of 64 and 40 kDa were in a phosphorylated form.
When studied by IIF on Hep-2 and HeLa cells cultivated on slides, each serum
showed a fluorescent pattern mainly nucleolar and weakly granular in the
nucleoplasm. The cells’ cytoplasm also showed a weak fluorescence preferentially
with a perinuclear localization.
These sera were studied by following the immunoblotting technique on HeLa
and FLC cell extracts and on isolated nucleolus and nucleolar chromatine extracts
of these same cells. All the sera, including the reference serum, recognized the
110 kDa molecular weight polypeptide. This was the same protein that manifested
the nucleolar and nucleoplasmic fluorescent pattern, since the antibodies affinity
purified by elution from the nitrocellulose paper (IB), afforded the same
fluorescence pattern as the sera. When the antibodies were studied by IB on
nucleoli extracts and on total HeLa and FLC cell extracts, they recognized the
same reactive 110 kDa peptide. Finally, we demonstrated that the 110 kDa
polypeptide, carrier of the epitopes recognized by the anti-PM/Scl antibodies is
the same one that, with this same molecular weight, forms part of the
macromolecular complex immunoprecipitated by these antibodies.
In the study of the snRNAs precipitated by these sera, only small quantities
of rRNA were detected in each case.
The enzymatic digestions with DNase, RNase and trypsin of the cells
employed for the IIF, demonstrated that this antigen was resistant to digestion
with DNase and RNase and sensitive to treatment with trypsin. On the other
hand, after treating the cultivated cells with Actinomycin D, with concentrations
capable of inhibiting the action of the RNA pol. I, the antigen appeared localized
in the nucleoplasm of the cell.
Upon treating the extracts employed for the IB studies of this antigen with
DNase, RNase, and trypsin, it was observed that the reaction of the sera with the
110 kDa protein is sensitive to the action of DNase and of trypsin. On the other
hand, after treating with DNase the complex precipitated by the anti-PM/Scl an-
tibodies, the 110 kDa protein did not appear in the SDS-PAGE gel employed for
the electrophoretic analysis of these proteins.
Finally, the results obtained by immunoprecipitation of total cell extracts and
isolated nucleoli extracts allow the hypothesis of the existance of two
macromolecular forms of the antigen to be postulated; one constituted by the
aforementioned 13 polypeptides, and the other, enriched in the 110 kDa protein
with a preferential nucleolar localization.
The results of the redistribution of the antigen, after being treated with ac-
tinomycin D, suggested that this antigen is found associated with the synthesis or
maturation machinery of the rRNA.
Molecular and Cell Biology of Autoantibodies and Autoimmunity
80, 64, 47, 46, 40, 37, 33, 30, 27, 26, 22, 20, and 18 kDa. This complex was iden-
tified with the PM/Scl antigen by its electrophoretic pattern equal to that given
by a control reference serum kindly provided by Eng Tan. Of these proteins, those
of 64 and 40 kDa were in a phosphorylated form.
When studied by IIF on Hep-2 and HeLa cells cultivated on slides, each serum
showed a fluorescent pattern mainly nucleolar and weakly granular in the
nucleoplasm. The cells’ cytoplasm also showed a weak fluorescence preferentially
with a perinuclear localization.
These sera were studied by following the immunoblotting technique on HeLa
and FLC cell extracts and on isolated nucleolus and nucleolar chromatine extracts
of these same cells. All the sera, including the reference serum, recognized the
110 kDa molecular weight polypeptide. This was the same protein that manifested
the nucleolar and nucleoplasmic fluorescent pattern, since the antibodies affinity
purified by elution from the nitrocellulose paper (IB), afforded the same
fluorescence pattern as the sera. When the antibodies were studied by IB on
nucleoli extracts and on total HeLa and FLC cell extracts, they recognized the
same reactive 110 kDa peptide. Finally, we demonstrated that the 110 kDa
polypeptide, carrier of the epitopes recognized by the anti-PM/Scl antibodies is
the same one that, with this same molecular weight, forms part of the
macromolecular complex immunoprecipitated by these antibodies.
In the study of the snRNAs precipitated by these sera, only small quantities
of rRNA were detected in each case.
The enzymatic digestions with DNase, RNase and trypsin of the cells
employed for the IIF, demonstrated that this antigen was resistant to digestion
with DNase and RNase and sensitive to treatment with trypsin. On the other
hand, after treating the cultivated cells with Actinomycin D, with concentrations
capable of inhibiting the action of the RNA pol. I, the antigen appeared localized
in the nucleoplasm of the cell.
Upon treating the extracts employed for the IB studies of this antigen with
DNase, RNase, and trypsin, it was observed that the reaction of the sera with the
110 kDa protein is sensitive to the action of DNase and of trypsin. On the other
hand, after treating with DNase the complex precipitated by the anti-PM/Scl an-
tibodies, the 110 kDa protein did not appear in the SDS-PAGE gel employed for
the electrophoretic analysis of these proteins.
Finally, the results obtained by immunoprecipitation of total cell extracts and
isolated nucleoli extracts allow the hypothesis of the existance of two
macromolecular forms of the antigen to be postulated; one constituted by the
aforementioned 13 polypeptides, and the other, enriched in the 110 kDa protein
with a preferential nucleolar localization.
The results of the redistribution of the antigen, after being treated with ac-
tinomycin D, suggested that this antigen is found associated with the synthesis or
maturation machinery of the rRNA.