Identification and purification of a calcium-binding protein in hepatic nuclear membranes.
Recent evidence suggests that nuclei possess Ca2+ transport mechanisms to regulate nucleoplasmic/cytosolic Ca2+ gradients. We, therefore, investigated the possibility that Ca(2+)-binding proteins may also exist within the nucleus. Electrophoretic analysis revealed the presence of an acidic 93-kDa protein (p93) in the membranes of isolated nuclei. p93 stained blue with "Stains-All" in SDS-polyacrylamide gels and was the major 45Ca(2+)- and ruthenium red-binding nuclear envelope protein in electroblot overlays. p93 was resistant to extraction by 6 M urea but was solubilized in 2% Triton X-100. Citric acid was highly effective in removing the outer nuclear membrane (ER) with concomitant reduction (< 10-fold) of mannose-6-phosphatase activity, but not p93. 45Ca(2+)-binding assays of purified p93 revealed the presence of high capacity Ca(2+)-binding sites comparable to calreticulin. This evidence strongly suggests that p93 is a major Ca(2+)-binding protein of the inner nuclear envelope membrane. Partial amino acid sequence analysis revealed that p93 was close to 100% homologous with a recently identified ER Ca(2+)-binding protein known as calnexin. It is likely, therefore, that p93 is calnexin. However, mild CHAPS detergent treatment of nuclear envelopes and ER revealed distinctly different solubility properties of each membrane for the extraction of p93. This, together with the citrate data, strongly suggests that p93/calnexin, in isolated nuclear envelopes, is mostly bound to the inner membrane. It is possible that p93 may be involved with the regulation of Ca2+ transients between the nucleoplasm and perinuclear space.
Gilchrist
J S
JS
Division of Cardiovascular Sciences, St. Boniface General Hospital Research Centre, Winnipeg, Manitoba, Canada.
Pierce
G N
GN
D000595:Amino Acid Sequence; D000818:Animals; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification*; D037281:Calnexin; D002467:Cell Nucleus / Q000737:chemistry*; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D007425:Intracellular Membranes / Q000737:chemistry*; D008099:Liver / Q000737:chemistry*; D008297:Male; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D051381:Rats; D017207:Rats, Sprague-Dawley
D002135:Calcium-Binding Proteins; D008565:Membrane Proteins; D037281:Calnexin