Year

2016

Season

Summer

Paper Type

Master's Thesis

College

College of Arts and Sciences

Degree Name

Master of Science (MS)

Department

Biology

NACO controlled Corporate Body

University of North Florida. Department of Biology

First Advisor

Dr. Greg Ahearn

Second Advisor

Dr. John Hatle

Third Advisor

Dr. Julie Avery

Department Chair

Dr. Cliff Ross

College Dean

Dr. Daniel Moon

Abstract

In animals, the accepted model of carbohydrate digestion and absorption involves reduction of disaccharides into the simple sugars glucose, fructose and galactose. Previous studies have shown the presence of disaccharides maltose and trehalose in the blood of several crab species, the crayfish and the American lobster. In 2011, a gene for a distinct disaccharide sucrose transporter (SCRT) was first found in Drosophila melanogaster and characterized using a yeast expression system. The purpose of the current study was to identify and characterize a putative disaccharide transporter analog in crustaceans using the American lobster, Homarus americanus. Brush border membrane vesicles purified from the hepatopancreas were utilized. After identification of a sucrose transporter in the brush border membrane of the hepatopancreas, transport kinetics experiments were used to characterize it using 14C radio-labeled sucrose and a Millipore filter isolation technique. Lack of glycyl-sarcosine inhibition of sucrose uptake into vesicles indicated that the highly non-specific dipeptide transporter PEPT1 was not the functional transporter of sucrose. A more acidic pH of 4 was shown to drive sucrose transport in the absence of sodium. Sodium was then shown to also significantly stimulate sucrose uptake, which resulted in an overshoot at 1 minute over a hyperbolic potassium uptake curve, suggesting that both sodium and acidic pH were capable of driving disaccharide transport. Experiments that used a variety of monosaccharides and polysaccharides indicated that the disaccharides maltose and trehalose were the only sugars to significantly inhibit carrier-mediated sucrose transport (maltose P = 0.017, trehalose P = 0.023 using a one-way ANOVA) (Km = 0.1951 ± 0.0630 mM sucrose, Jmax = 0.5884 ± 0.0823 nmol/mg protein x 1 minute), suggesting specificity of the transporter. Sucrose in the presence of 20 mM maltose had a Km of 0.5847 ± 0.1782 mM sucrose (P = 0.030) and a Jmax of 0.6536 ± 0.1238 nmol/mg protein x 1 minute (P = 0.006). ANOVA P-values indicate the difference between the sucrose control curve and the maltose curve. The highly significant reduction between the Km values of the control sucrose curve and the maltose curve suggests competitive inhibition between the two sugars. These two disaccharides could utilize the same transporter, and are appropriate for the physiology of the animal in this case, as lobsters commonly digest glycogen and chitin, polymers of maltose and trehalose, respectively. These findings suggest there is a brush-border proton-, or sodium-dependent, hepatopancreatic carrier process, shared by sucrose, maltose, and trehalose, that may function to absorb disaccharides that occur from digestion of naturally-occurring dietary constituents.

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