The intestinal brush-border enzyme lactase splits lactose into its component monosaccharides, glucose and galactose. Relative deficiency of the enzyme during adulthood is a common condition worldwide and is frequently associated with symptoms of lactose intolerance. We studied the synthesis and processing of lactase in normal and adult hypolactasic subjects using human intestinal explants in organ culture. Metabolic labeling experiments in our control subjects with I35Slmethio-nine followed by immunoprecipitation, sodium dodecyl sulfate-polyacrylamide-gel electrophoresis, and fluorography demonstrated that newly synthesized lactase is initially recognized as a precursor molecule with a relative molecular weight (Mr) of 205,000. Over the course of several hours most of the labeled lactase was converted to a mature form of 150,000 Mr. Transiently appearing forms of 215,000 and 190,000 M, were identified and were felt to represent intermediary species generated during intracellular processing.We identified two distinct alterations in lactase biosynthesis accounting for adult hypolactasia. Studies in three deficient subjects demonstrated markedly reduced synthesis of the precursor protein though posttranslational processing appeared identical to normal. Multiple studies in a fourth deficient subject demonstrated synthesis of ample amounts of precursor lactase but reduced conversion to the mature active form of the enzyme. (J. Clin. Invest. 1990Invest. . 86:1338Invest. -1342
A B S T R A C T Past investigation has revealed that the circadian rhythm of intestinal sucrase activity in rats is primarily cued by the time of feeding. We examined the mechanism of the circadian rhythm by methods involving quantitative immunoprecipitation of sucraseisomaltase protein and study of decay of radioactively labeled protein. Rats were placed on a controlled feeding regimen (1000-1500 h) and then sacrificed at 3-h intervals over a 24-h period. Immunotitration experiments indicated that the circadian rhythm was the result ofchanges in the absolute amount of sucrase-isomaltase protein present and not of changes in the enzyme's catalytic efficiency.To study the mechanism of this circadian variation in sucrase-isomaltase mass, [14C]sodium carbonate was injected and, after maximum incorporation into brush border protein, the rats were sacrified at 3-h intervals. Sucrase-isomaltase protein was isolated by immunoprecipitation, and the decrease in total disintegrations per minute over time was used to study degradation of the protein. Enzyme degradation was not constant but exhibited a clear circadian rhythm. The period of increasing enzyme mass was characterized by virtual cessation of enzyme degradation (t1/2 of 38 h), and the period of declining enzyme mass by rapid degradation (t1/2 of 6 h or less). We found similar changes in enzyme degradation in fasted animals, demonstrating that the changes were not the result of decreased isotope reutilization during feeding. We found no evidence of a circadian rhythm in [14C]leucine incorporation into the protein, suggesting that enzyme synthesis was constant.These results indicate that the circadian rhythm of sucrase activity represents changes in the total amount ofenzyme protein that are, at least in large part, secondary to changes in the enzyme's degradation rate.
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