Purification and properties of rat cysteine-rich intestinal protein.
Cysteine-rich intestinal protein (CRIP) is a zinc-binding protein where the binding domain is in the so-called LIM double zinc finger motif. Methods are described for the preparation of CRIP from rat small intestine. Gel-filtration and ion-exchange chromatography and preparative PAGE gave homogeneous CRIP, based upon analytical PAGE, mass spectrometry and microsequencing. Initial localization of CRIP during chromatography was based on binding of 65Zn radioisotope introduced into the intestine. The stoichiometry of binding by CRIP is less than 2 atoms of zinc per molecule. The metal-binding affinity in vitro is zinc > cadmium > copper > iron, at low metal concentrations. Zinc is the predominant metal bound when these metals are taken up from the intestinal lumen. 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 micrograms/g of mucosal tissue, suggesting that the protein is more abundant than zinc-finger-containing transcription factors. The metal-binding properties of CRIP are consistent with proposed zinc-related functions for this cytoplasmic protein, which is expressed in the small intestine during the postnatal period.
Khoo
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Food Science and Human Nutrition Department, University of Florida, Gainesville 32611.
Cousins
R J
RJ
D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003545:Cysteine; D004591:Electrophoresis, Polyacrylamide Gel; D007413:Intestinal Mucosa / Q000378:metabolism*; D007421:Intestine, Small / Q000378:metabolism; D060588:LIM Domain Proteins; D008297:Male; D013058:Mass Spectrometry; D008668:Metallothionein / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D051381:Rats; D017207:Rats, Sprague-Dawley; D015032:Zinc / Q000378:metabolism*; D016335:Zinc Fingers; D015037:Zinc Radioisotopes
D002352:Carrier Proteins; C071434:Crip1 protein, rat; D060588:LIM Domain Proteins; D015037:Zinc Radioisotopes; D008668:Metallothionein; D015032:Zinc; D003545:Cysteine