Abstract
Sucrase-isomaltase (SI) expression along the longitudinal and vertical axis of the small intestine was studied by sequentially isolating enterocytes from villus to crypt of rat proximal jejunum and distal ileum. Gradients of sucrase activity were observed with greatest activity occurring in jejunal and villus regions. Along the villus-to-crypt axis, gradients of SI mRNA abundance corresponded with activity. However, along the longitudinal axis no differences in SI mRNA levels were observed, thus not accounting for the observed 3-5-fold difference in SI activities between jejunum and ileum. Comparison of SI immunoprecipitates from jejunal and ileal mucosal scrapings showed significant differences in gel mobilities of the more mature forms, which did not appear to affect SI functional activities. When relative rates of de novo SI protein synthesis were compared, [35S]methionine incorporation into all SI forms was observed to be 3-5-fold greater in jejunum than in ileum at all time points. Because these results suggested differences in regional translational regulation, subcellular distribution of SI mRNA in jejunal and ileal epithelial cells was compared. A greater proportion of jejunal SI mRNA was found to be associated with membrane-bound polyribosomes. We conclude 1) sucrase expression along the villus-to-crypt axis correlates with SI mRNA abundance, 2) post-translational processing of SI differ in ileum and jejunum, but appear not to determine SI expression, and 3) differences in translational processing in distal ileum and proximal jejunum may determine sucrase activity along the longitudinal axis of rat small intestine.
Highlights
Our findings suggest that sucraseisomaltase expression along the villus-to-crypt axis correlates with abundance of SI mRNA, but that differences in transfold greater injejunum than inileum at all timepoints. lational processes in distalileum and proximal jejunum deter
Because these results suggested differences in regionmaline sucrase activity along the longitudinal axis of the gut
We conclude 1)sucrase expression along the villus-to-crypt axis correlates with SI mRNA abun
Summary
Identification and Quantitation of SI rnRNA-RNA was immediately extracted from fresh cell isolates using a modification of the guanidinium isothiocyanate method [18]. Equal loading of total RNA in each lane was confirmed by hybridization to a cDNA probe for the. Slot-blot analyseswere performed using serial dilutions of 0.25 and 1.0 pg of total RNA. As for Northern blots, equivalent loading of RNA in all blots was confirmed by later reprobing with the cDNA to the. Laser densitometry of the autoradiographs provided quantitation of theamount of bound radiolabeled DNA in arbitrary absorbance units/microgram of total RNA. Homogenates were centrifuged througha discontinuous sucrose gradient to separate membrane-bound polyribosomes from messenger ribonucleoprotein (mRNP) complexes and non-membrane-bound ribosomes as previously described [20].Twelve fractions were collected and slot-blots made using 10 pg of total RNA from each fraction. Membranes were probed with SI and 18S cDNAs as described above
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