Porcine Intestinal Calcium-binding Protein: MOLECULAR PROPERTIES AND THE EFFECT OF BINDING CALCIUM IONS

Abstract

Abstract A calcium-binding protein from porcine intestinal mucosa has been characterized by a variety of physical and chemical techniques. Chromatography on Sephadex G-75 suggests a molecular weight around 12,000, while electrophoresis in sodium dodecyl sulfate-urea-polyacrylamide gel indicates a molecular weight in the region of 7000. Sedimentation equilibrium analyses give evidence for aggregation, with the monomeric form having a molecular weight between 7000 and 9000. The amino acid composition is as follows: Lys11–12, Arg1, Asp7, Thr1, Ser6–7, Glu17, Gly4, Ala5, Val3, Ile3, Leu10, Tyr1, Phe5. There is no histidine, half-cystine, methionine, or tryptophan. The composition requires a minimal molecular weight of 9000. No free terminal amino group was detectable, but digestion with penicillocarboxypeptidases S1 and S2 indicated the following COOH-terminal sequence: .... (Gly, Thr, Asp)Ala-Ile-Val(Phe, Ser)Leu-Lys-GlnOH. The circular dichroism spectrum of the binding protein in the near ultraviolet shows a positive peak at 276 nm with a shoulder at 282 nm characteristic of tyrosine, two positive peaks at 264 nm and 257 nm, and two negative peaks at 268 and 261 nm characteristic of phenylalanine. In the far ultraviolet range two negative bands at 222 nm and 207 nm are observed. Removal of calcium ions causes a marked decrease in the tyrosine band at 276 nm to less than 25% of its initial value, but no difference is observed in the far ultraviolet range. Ultraviolet difference spectra between the calcium-containing and the calcium-free protein show peaks characteristic of tyrosine. No difference in sedimentation rates was observed. It appears that the binding of calcium causes a local perturbance of the environment of the single tyrosine residue, but no gross conformational changes.