PURIFICATION AND PROPERTIES OF RAT CYSTEINE-RICH INTESTINAL PROTEIN

Cysteine-rich intestinal protein (CRIP) is a zinc-binding protein wher e the binding domain is in the so-called LIM double zinc finger motif. Methods are described for the preparation of CRIP from rat small inte stine. Gel-filtration and ion-exchange chromatography and preparative PAGE gave homogeneous CRIP, based upon analytical PAGE, mass spectrome try and microsequencing. Initial localization of CRIP during chromatog raphy was based on binding of Zn-65 radioisotope introduced into the i ntestine. The stoichiometry of binding by CRIP is less than 2 atoms of zinc per molecule. The metal-binding affinity in vitro is zinc > cadm ium > copper > iron, at low metal concentrations. Zinc is the predomin ant metal bound when these metals are taken up from the intestinal lum en. Zinc binding was not influenced by pH between values of 4.5 to 7.5 . Metallothionein has a much greater zinc-binding affinity than CRIP. The tissue concentration of CRIP is of the order of 15-20 mu g/g of mu cosal tissue, suggesting that the protein is more abundant than zinc-f inger-containing transcription factors. The metal-binding properties o f CRIP are consistent with proposed zinc-related functions for this cy toplasmic protein, which is expressed in the small intestine during th e postnatal period.