Human milk bile salt-activated lipase. Further characterization and kinetic studies.

C.S Wang

doi:10.1016/s0021-9258(19)68763-6

Abstract

Further studies on human milk bile salt-activated lipase were performed to provide kinetic and additional chemical characterizations of this enzyme. The enzyme was homogeneous by urea-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing with an isoelectric point of 3.7. A unique feature of the amino acid composition of this enzyme was a high proline content (13 mol %). Results of carbohydrate analyses indicated that the enzyme was a glycoprotein containing fucose, galactose, glucosamine, galactosamine, and sialic acid. Kinetic studies were performed with various water-soluble esters (p-nitrophenyl acetate, 1-monoacetin, 1-monobutyrin, and 1-monocaprylin) as substrate and taurocholate as activator. In the presence of a saturating level of taurocholate, the enzyme reaction was demonstrated to follow a rapid equilibrium random uni bi mechanism. Also, these kinetic studies indicated the formation of an enzyme-activator-substrate ternary complex through a random pathway. The mechanism of the activation by taurocholate was due to its enhancement of the binding of the enzyme to the substrate (6.2-fold) and its enhancement of the rate of conversion from enzyme-substrate transitory complex to the products (1.57-fold) when examined with p-nitrophenyl acetate as substrate.

Highlights

EXPERIMENTALPROCEDURES lipase were performetdo provide kinetic and additional Materials-Unless otherwise stated, all chemicals were purchased chemical characterizations othf is enzyme
Kinetic studies were per- consisted of 36 ml of distilled water, 21 g of sucrose, and 2 ml of 40%
From the electrophoretic and immunochemical studies, it was concluded that the nzyme preparation was homogeneousThe maximal velocity in the presence of a saturating concenwith these criteria, anad further chemical characterization of tration of the activator is p V

Summary

Introduction

EXPERIMENTALPROCEDURES lipase were performetdo provide kinetic and additional Materials-Unless otherwise stated, all chemicals were purchased chemical characterizations othf is enzyme. The enzyme from Sigma.The [14C]taurocholate(60.9pCi/pmol)was obtained from washomogeneousbyurea-sodiumdodecylsulfate-. Kinetic studies were per- consisted of 36 ml of distilled water, 21 g of sucrose, and 2 ml of 40%. Heavy and light solutions were applied phenyl acetate, 1-monoacetin, 1-monobutyrin, and 1-. After reaching 500 V, the focusing late, the enzyme reaction was demonstratedto follow was continued for an additional 24 h. The column content was cola rapid equilibrium random uni bi mechainsm. These kinetic studies indicated the formatioonf an enzyme-activator-substrate ternary complex through a lected in 3 - d fractions for monitoring pH, 280-nm absorbance, and enzymatic activity

Methods

Results

Conclusion