MACROMOLECULAR AGGREGATES IN CALCIFICATION 83 



Dr. Barker (University of Alabama Medical Center): Should we not be 

 as critical of FDNB's blocking free amino groups other than e-amino groups 

 of lysine or hydroxylysine of reconstituted collagen as we are of the lack of 

 specific freeing of e-amino groups upon decalcification of bony structures? 



Dr. Glimcher: I would be the first to agree that we must be extremely 

 cautious in interpreting any group-blocking experiments, particularly if only 

 one blocking agent such as FDNB is used. Because of the extremely large size 

 of the collagen macromolecule, the number of alpha amino groups is relatively 

 small; thus, we can be reasonably sure that the major effect of using FDNB is 

 to block the €-NH2 groups. As I pointed out, however, it is quite possible 

 that the actual sites involved in nucleation are adjacent to the e-NHo groups, 

 and that when one blocks the €-NH2 groups by substituting such large radicals 

 as FDNB, the really important adjacent groups are sterically (or electrostat- 

 ically) shielded and prevented from interacting with the mineral ions. We 

 would then be misled and erroneously assign a role to the 6-NH2 groups. It is 

 for these reasons, and not the lack of the specificity of FDNB (and other 

 blocking agents), that I am most concerned about in the interpretation of the 

 results of experiments such as the ones that I described. 



Dr. Beck (University of Pittsburgh): Is it too early to speculate on the 

 role(s) of parathormone, Vitamin D, and dihvdroergosterol? 



Dr. Glimcher: Yes! 



Dr. Prosser (University of Illinois): Is there any correspondence between 

 apatite crystal formation in bone and calcification of chitin in crustaceans? 

 Also calcification in clams, where the protein does not show the periodicity of 

 collagen? 



Dr. Glimcher: Although we have only studied the collagen hydroxyapa- 

 tite system in vitro, we believe that the underlying basic principle, wherein 

 the organic matrix nucleates the inorganic crystals as a result of a specific 

 stereochemical array of certain reactive groups, may apply to biological min- 

 eralization in general. In the case of oyster shells, for instance, calcium 

 carbonate in the form of either calcite or argonite is deposited in an intimate 

 relationship to the organic matrix, conchiolin, which is composed of conchiolin, 

 an unidentified protein. The fact that there is no repeating structure visible 

 with the electron microscope in the organic matrix of such material does not 

 mean that the matrix is not a well-organized and well-ordered structure at the 

 molecular level. In the case of collagen we are fortunate in being able to "see" 

 this ordering by electron microscopy and by low-angle x-ray diffraction be- 

 cause of certain characteristics dependent on the periodic concentration of cer- 

 tain reactive polar amino acids. 



Dr. Richards (University of Minnesota): Do the apatite microcrystals in 

 bone have the typical crystal faces of larger crystals grown from solution in 

 vitro? If so, do you have any idea why in some cases, such as the subcutaneous 

 anchors of holothurian worms, one finds normal calcite crystals in weird shapes 

 such as (anchors? 



Dr. Glimcher: Unfortunately, the apatite crystals of bone are so small 

 (15-30 A X 200-400 A) that it is impossible to be sure either of their gross 

 external habit or of their individual crystal faces. 



