Messenger RNAs expressed in intestine of adult but not baby rabbits. Isolation of cognate cDNAs and characterization of a novel brush border protein with esterase and phospholipase activity.
Using a subtractive hybridization method, we have cloned cDNAs corresponding to 10 different mRNAs which share the property of being expressed in the intestine of adult but not baby rabbits. Four could be identified as coding for previously known gene products (sucrase-isomaltase, a glutathione S-transferase, a cytochrome P450, and a long form of ferritin mRNA), while six code for previously unknown proteins. One clone, AdRab-B, codes for a protein of 1458 amino acids, including (i) a putative signal sequence at the NH2 terminus, (ii) four internal repeats, 308-346 amino acids in length, (iii) a hydrophobic stretch near the COOH terminus, which represents a potential membrane anchor, and (iv) a short hydrophilic stretch at the very COOH terminus. The corresponding protein was studied with the aid of antibodies prepared against polypeptides expressed from segments of the cDNA in Escherichia coli. The protein was shown to be proteolytically processed in the intestine (but not when expressed in COS cells) and to be targeted to the brush border membrane of the enterocytes. Finally, the protein was found to have esterase and phospholipase A/lysophospholipase activity.
Department of Biochemistry, Swiss Federal Institute of Technology, ETH-Zentrum, Zürich.
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Boll
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Schmid-Chanda
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Semenza
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Mantei
D000375:Aging / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000302:isolation & purification*; D004950:Esterases / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D015870:Gene Expression; D007082:Ileum / Q000254:growth & development; D007413:Intestinal Mucosa / Q000201:enzymology* / Q000254:growth & development; D007700:Kinetics; D008565:Membrane Proteins / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D008871:Microvilli / Q000201:enzymology*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010740:Phospholipases / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism*; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014162:Transfection
D008565:Membrane Proteins; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D011994:Recombinant Proteins; D004247:DNA; D004950:Esterases; D010740:Phospholipases