High-affinity and low-affinity calcium binding and stability of the multidomain extracellular 40-kDa basement membrane glycoprotein (BM-40/SPARC/osteonectin).
Reversible binding of calcium ions to a single high-affinity binding site in the 40-kDa basement membrane protein (BM-40) caused a 33% increase of alpha-helicity, an about 60% change in intrinsic fluorescence and a dramatic increase of the rate of cleavage by alpha-chymotrypsin. All these effects exhibited identical dependencies on calcium concentration from which a dissociation constant Kd = 0.6 microM was determined. Calcium release was accompanied by an increase of the frictional ratio in solution but not by denaturation which occurred at about equal guanidine.HCl concentration for both calcium-saturated and calcium-depleted protein (midpoint 1.5 M). The cleavage sites for alpha-chymotrypsin are located in or near to the EF-hand domain IV of calcium-depleted BM-40 (also known as SPARC, i.e. secreted protein acidic and rich in cysteine, and osteonectin). These and other data indicate that binding occurs in the EF-hand domain from which a large conformational change is transmitted. Low-affinity calcium-binding sites in the N-terminal glutamic-acid-rich domain I of BM-40 were identified by human leukocyte elastase which was found to cleave very specifically in the middle of this domain. From the increase of cleavage rate with increasing calcium concentration a Kd greater than or equal to 10 mM was estimated. It is suggested that variations of calcium levels in the extracellular space in this range may regulate functions of BM-40 such as collagen binding and that high-affinity binding is important for stabilization, folding and secretion during biosynthesis.
Maurer
P
P
Abteilung für Biophysikalische Chemie, Biozentrum, Basel, Switzerland.
Mayer
U
U
Bruch
M
M
Jenö
P
P
Mann
K
K
Landwehr
R
R
Engel
J
J
Timpl
R
R
D000595:Amino Acid Sequence; D000814:Aniline Compounds; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002413:Cations, Divalent; D002918:Chymotrypsin / Q000378:metabolism; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D006868:Hydrolysis; D007700:Kinetics; D019272:Leukocyte Elastase; D051379:Mice; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D015676:Osteonectin / Q000378:metabolism* / Q000648:ultrastructure; D010196:Pancreatic Elastase / Q000378:metabolism; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D014461:Ultracentrifugation; D014966:Xanthenes
D000814:Aniline Compounds; D002413:Cations, Divalent; D015676:Osteonectin; D014966:Xanthenes; C059715:Fluo-3; D002918:Chymotrypsin; D010196:Pancreatic Elastase; D019272:Leukocyte Elastase; D002118:Calcium