Serine Units Research Articles

Identification of the active-site serine in human pancreatic lipase by chemical modification with tetrahydrolipstatin

A chemical modification approach was used in this study to identify the active site serine residue of human pancreatic lipase. Purified human pancreatic lipase was covalently modified by incubation with [ 3H], [ 14C]tetrahydrolipstatin (THL), a potent inhibitor of pancreatic lipase. The radiolabeled lipase was digested with thermolysin, and the peptides were separated by HPLC. A single THL-peptide-adduct was obtained which was identical to that obtained earlier from porcine pancreatic lipase. This pentapeptide with the sequence VIGHS is covalently bound to a THL molecule via the side chain hydroxyl group of the serine unit corresponding to Ser-152 of the lipase. The selective cleavage of the THL-serine bond by mild acid treatment resulted in the formation of the δ-lactone Ro 40–444] in high yield and clearly proves that THL is attached via an ester bond and with retention of stereochemistry at all chiral centers to the side chain hydroxyl group of Ser-152 of the lipase. The results obtained for human pancreatic lipase corroborate the inhibition mechanism of THL found on the porcine enzyme, and are in full agreement with the identification of the Ser-152…His-263…Asp-176 catalytic triad in the X-ray structure of human pancreatic lipase.

Isolation and purification of chondroitin 6-sulfate protein from umbilical cord.

Abstract Intact chondroitin 6-sulfate protein can be extracted from umbilical cord with dilute saline. Hyaluronic acid which is also extracted, is removed by precipitation with cetylpyridinium chloride followed by washing of the precipitate with aqueous sodium chloride. Subsequent purification is effected by passage through cation and anion exchange resins. Elution from the latter with salt solutions of increasing concentration yields chondroitin 6-sulfate proteoglycan in two fractions. The product is isolated from each of the fractions as the calcium salt by fractional precipitation with ethanol. The protein moiety can be cleaved from the mucopolysaccharide either by proteolytic digestion or treatment with alkali. The results obtaired on reaction with alkali and with sodium borohydride indicate that the polysaccharide is covalently linked to the protein through a serine unit.