Characterization of calcium binding properties of lithostathine.
The pancreas secretes primarily two types of metabolically important proteins: digestive enzymes and hormones. Lithostathine (LIT) is the only protein excreted from the pancreas that has no known digestive or hormonal activity. Human lithostathine is a 144-amino acid glycoprotein synthesized by the exocrine pancreas that has been implicated in various physiological functions, including inhibition of pancreatic stone formation. To better understand the physiological function of LIT, we expressed the recombinant LIT protein in Escherichia coli and measured its calcium binding properties by equilibrium dialysis and electron paramagnetic resonance (EPR) spectroscopy. Equilibrium dialysis with (45)Ca(2+) showed that LIT binds Ca(2+) with 1:1 stoichiometry. EPR studies using the divalent vanadyl (VO(2+)) ion as a paramagnetic substitute for Ca(2+) also showed that VO(2+) binds to LIT with a metal:protein binding stoichiometry of 1:1 and that VO(2+) competes with Ca(2+) in binding to LIT. Mutations of a cluster of acidic residues on the molecular surface (E30A, D31A, E33A, D37A, D72A, and D73A) resulted in almost complete loss (95-100%) of binding of Ca(2+) and VO(2+), showing that these residues are critical for calcium binding by LIT.
Lee
Byung-In
BI
Department of Chemistry, University of Illinois at Chicago, Chicago, IL 60607, USA.
Mustafi
Devkumar
D
Cho
Wonhwa
W
Nakagawa
Yasushi
Y
D019943:Amino Acid Substitution; D024342:Amino Acids, Acidic / Q000235:genetics; D001667:Binding, Competitive; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003956:Dialysis / Q000379:methods; D004578:Electron Spin Resonance Spectroscopy; D006801:Humans; D051847:Lithostathine; D008958:Models, Molecular; D009419:Nerve Tissue Proteins; D011485:Protein Binding; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D014638:Vanadates / Q000737:chemistry / Q000378:metabolism
D024342:Amino Acids, Acidic; D002135:Calcium-Binding Proteins; D051847:Lithostathine; D009419:Nerve Tissue Proteins; C497617:REG1A protein, human; D011994:Recombinant Proteins; D014638:Vanadates; D002118:Calcium