Nucleolin is a calcium-binding protein.
We have purified a prominent 110-kDa protein (p110) from 1.6 M NaCl extracts of rat liver nuclei that appears to bind Ca2+. p110 was originally identified by prominent blue staining with 'Stains-All' in sodium dodecyl sulfate-polyacrylamide gels and was observed to specifically bind ruthenium red and 45Ca2+ in nitrocellulose blot overlays. In spin-dialysis studies, purified p110 saturably bound approximately 75 nmol Ca2+/mg protein at a concentration of 1 mM total Ca2+ with half-maximal binding observed at 105 microM Ca2+. With purification, p110 became increasingly susceptible to proteolytic (likely autolytic) fragmentation, although most intermediary peptides between 40 and 90 kDa retained "Stains-All", ruthenium red, and 45Ca2+ binding. N-terminal sequencing of intact p110 and a 70-kDa autolytic peptide fragment revealed a strong homology to nucleolin. Two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)/IEF revealed autolysis produced increasingly acidic peptide fragments ranging in apparent pI's from 5.5 for intact p110 to 3.5 for a 40 kDa peptide fragment. Intact p110 and several peptide fragments were immunostained with a highly specific anti-nucleolin antibody, R2D2, thus confirming the identity of this protein with nucleolin. These annexin-like Ca2+-binding characteristics of nucleolin are likely contributed by its highly acidic argyrophilic N-terminus with autolysis apparently resulting in largely selective removal of its basic C-terminal domain. Although the Ca2+-dependent functions of nucleolin are unknown, we discuss the possibility that like the structurally analogous HMG-1, its Ca2+-dependent actions may regulate chromatin structure, possibly during apoptosis.
Gilchrist
James S C
JS
Department of Oral Biology and Physiology, Division of Stroke and Vascular Disease, University of Manitoba, Winnipeg, Manitoba, Canada.
Abrenica
Bernard
B
DiMario
Patrick J
PJ
Czubryt
Michael P
MP
Pierce
Grant N
GN
D000818:Animals; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002155:Calsequestrin / Q000378:metabolism; D002232:Carbocyanines; D002467:Cell Nucleus / Q000378:metabolism*; D004396:Coloring Agents; D008099:Liver / Q000737:chemistry; D009687:Nuclear Proteins / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry; D010750:Phosphoproteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D016601:RNA-Binding Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D012430:Ruthenium Red / Q000378:metabolism; D020539:Sequence Analysis, Protein; D000097796:Nucleolin
D002118:Calcium; D002135:Calcium-Binding Proteins; D002155:Calsequestrin; D002232:Carbocyanines; D004396:Coloring Agents; D009687:Nuclear Proteins; D010446:Peptide Fragments; D010750:Phosphoproteins; D016601:RNA-Binding Proteins; D012430:Ruthenium Red; D000097796:Nucleolin; C012040:stains-all