Transcriptional Regulation of Rat Calbindin Expression during Development Determined by Bacterially Expressed Protein.

Abstract

SummaryCalbindin-D9k expression in intestinal mucosal cells reveals a specific pattern during development in rats. It shows a low basal level in suckling and adult rats, but after weaning at 21 d of age, increases to three times that of the basal level for several days only, around 24 d. We attempted to clarify whether the regulation of developmental change was at the transcriptional or post-transcriptional level. The calbindin-D9k protein and mRNA concentrations during pre-and postweaned de velopment were determined by Western blot and Northern blot analysis, respectively, and compared with calcium binding activity by 45Ca. For Western blot analysis, a corresponding antibody was raised in rabbit using a bacterially expressed fusion protein, glutathione S-transferase (GST, EC 2.5.1.18), and calbindin-D9k. Calbindin-D9k cDNA was linked to a GST gene within a molecule of vector plasmid and a fusion protein was expressed in Escherichia coll. There were significant (p<0.001) correlations between calbindin-D9k protein, mRNA concentrations and calcium-binding ac tivity: r=0.90 for protein vs. mRNA, r=0.93 for protein vs. binding activity and r=0.95 for mRNA vs. binding activity. These results indicate that calbindin-D9k expression during postnatal development is regulated at the transcriptional level. Key Words rat calbindin-D9k, fusion protein, postnatal development, Northern blot analysis, Western blot analysis Calbindin-D9k is an intestine-specific, calcium-binding protein found in rats. It has a molecular weight of 9,000 and its expression depends on the level of 1, 25-dihydroxyvitamin D3 (1, 2). The nucleotide sequence (3, 4) and amino acid sequence of calbindin-D9k (5) have already been determined. However, the mo lecular mechanism of its exact function in intestinal mucosal cells and how its

Development of Intestinal Transport Function in Mammals

“…The nonsaturable diffusional pathway operates also as a single Ca 2ϩ absorptive pathway in preterm low-birth-weight infants (42). The appearance of saturable active Ca 2ϩ absorption is associated with a substantial increase in the capacity for other steps involved in transcellular Ca 2ϩ absorption: 1) entry along a steep electrochemical gradient (129), 2) translocation through the cell (123,249), and 3) extrusion at the basolateral membrane against a steep electrochemical gradient by Ca 2ϩ pump, i.e., Ca 2ϩ -ATPase (130, 249). E) PHOSPHATE ABSORPTION.…”