Characterization of the three calcium binding proteins in the human placenta (author's transl)

Abstract

Calcium binding protein (CaBP) has been reported to be involved in absorbing calcium in the duodenum. There is some evidence to show that active transport of calcium ions occurs from mother to fetus in the human placenta. The presence of CaBP in the human placenta was examined according to Taylor and Wasserman's method, extracting the vitamin D-dependent CaBP in the duodenum, and the character of CaBP was then studied. 38,000 X g supernatant of the villous homogenate of the human placenta was heated at 60 degrees C for 5 mins to neutralize the calcium binding activity resulting from the blood. The fractions containing CaBP were separated and partially purified on Sephadex G-100 and DEAE cellulose columns. The CaBP fractions were further purified by preparative agar electrophoresis. The Chelex-100-resin method was used for the detection of calcium binding activity in the eluates and for calculation of Kd values and of calcium binding sites. The molecular weights were determined by electrophoresis of SDS-polyacrylamide gel. Three CaBPs were found in the human placenta. The molecular weight, Kd (micro M) and the binding activity (Ca mol/CaBP mol) of the three placental CaBPs were CaBP-Peak I: 82,000, 0.64, 0.52; Peak II: 12,000, 0.67, 1.10; Peak III: 8,500, 0.75, 2.08, respectively. The tyrosine residue of CaBP has been shown to act as an important binding site in that removal of calcium ions from CaBP generates a blue-shifted phenomenon in the ultraviolet difference spectrum between 270 and 300 nm. However no difference spectrum was observed with the placental CaBPs. This result was further confirmed by amino acid analysis which showed that none of the placental CaBPs contained tyrosine, tryptophan, half-cystine and proline, which are usually found in most proteins. The circular dichronism (CD) spectrum of CaBP-Peak I in the far ultraviolet range showed two negative bands at 222 and 207 nm (alpha-helix structure), and removal of calcium ions caused no difference spectrum. CD spectra of CaBP Peak II and III in the far ultraviolet range revealed random coil structures in the presence and absence of bound calcium ions. These findings indicate that the calcium binding mechanism of the placental CaBPs should be different from that of the others. In this study, three kinds of human placental CaBP newly isolated were characterized according to molecular weights, Kd values and binding activities and clarified as having an amino acid composition quite different from the CaBPs already reported.