Dipeptidyl peptidase IV (EC 3.4.14.‐) of pig small intestinal brush border membrane was solubilized by Triton X‐100 and purified by immunoadsorption on Sepharose conjugated to antibodies against kidney dipeptidyl peptidase IV. The recovery was around 20% and the purification factor around 500. The enzyme appeared homogeneous in crossed immunoelectrophoresis and migrated in polyacrylamide gel electrophoresis in dodecyl sulphate as a single polypeptide chain corresponding to a molecular weight of 137000. Gel filtration under non‐denaturing conditions indicated a molecular weight around 230000, which suggested a dimeric structure for the isolated form of dipeptidyl peptidase IV.
The enzyme showed a strong tendency to aggregate and its hydrophobic properties were further characterized in studies on detergent binding. Interaction with sodium deoxycholate or cetyltrimethylammonium bromide in charge‐shift crossed immunoelectrophoresis thus resulted in an anodic or cathodic shift in mobility, respectively. Furthermore, by crossed immunoelectrophoresis in the presence of 14C‐labelled Triton X‐100 the detergent binding could be directly visualized by autoradiography. After trypsin treatment the enzyme no longer formed complexes with the detergents.
The intestinal dipeptidyl peptidase IV released the N‐terminal dipeptides from glycyl‐l‐proline‐4‐nitroanilide, glycyl‐l‐proline‐2‐naphthylamide, glycyl‐l‐prolyl‐l‐alanine, L‐alanyl‐l‐alanyl‐l‐alanyl‐l‐alanine, and l‐leucyl‐l‐leucyl‐l‐valyl‐l‐tyrosyl‐l‐serine. It had a very low activity on l‐alanine‐4‐nitroanilide and showed no endopeptidase activity. The activity on all hydrolyzed substrates was sensitive to the serine protease inhibitor diisopropylfuorophosphate. The Km for the hydrolysis of glycyl‐l‐proline‐4‐nitroanilide was 0.24 mM. The dipeptides glycyl‐l‐proline and glycyl‐l‐leucine competitively inhibited the hydrolysis of glycyl‐l‐proline‐4‐nitroanilide with Ki values of 3.7 mM and 1.7 mM, respectively.
The effects of long-term starvation on the activities of sucrase, lactase, and aminopeptidase, and on their respective mRNA were determined in the small intestine of thyroidectomized and sham-operated adult rats. Thyroidectomy reduced the protein loss at the level of the intestinal brush border membranes during starvation. Prolonged fasting caused a significant decrease in sucrase activity, but thyroidectomy partly prevented this effect. However, the amount of the corresponding mRNA dropped during long term starvation without incidence of thyroidectomy. Lactase activity in the brush border membranes was increased by starvation, and thyroidectomy caused a further elevation of the enzyme activity. Simultaneously, lactase mRNA content rose only slightly compared to the enzyme activity. Aminopeptidase activity and mRNA content decreased during starvation and thyroidectomy did not prevent this process. These results indicate that intestinal hydrolases respond non-coordinately to long-term food deprivation. In addition, the thyroid status of the animals has a direct influence on the adaptation of several brush border hydrolases to starvation. This suggests that the drop in plasma thyroid hormones during fasting allows a better maintenance of protein content and of hydrolase activities in the brush border membranes of the small intestine. These adaptive processes seemed to be partly controlled at a post-transcriptional level.
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