Studies in bile salt solutions

Abstract

The pseudo-first-order rate constants of hydrolysis of p-nitrophenylacetate, catalyzed by human milk lipase, have been measured in solutions of 0.01 mol dm-3 Bistris(2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-l,3-diol) buffer at 310.5 K , containing a range of concentrations of sodium taurocholate and sodium cholate, at pH 8.00 and of sodium cholate at pH 6.5. The effect of pH on the activity of the enzyme has been investigated and the stimulation factors of taurocholate and cholate ions and of cholic acid have been calculated to be equal to 5.3. 3.7 and 10.7, respectively. The essential residues for catalytic activity of the enzyme have ionization constants equal to 6.45 -6.46 for pK, and 8.33 -8.40 for pK,. The former value is attributed to the presence of a histidine imidazolium group but the identity of the residue leading to pK, is not proven.Human milk lipase comprises 1 % of the protein in human milk and is inactive against milk fats in the breast. In the presence of bilc salts in the small intestine, its lipolytic activity is stimulated to a degree which is dependent upon the identity and concentration of the substrate [I -121. Because of this property the enzyme has also been called bile-salt-stimulated lipase and bile-activated lipase. Recent studies have implicated human milk lipase as a novel microbicidal agent, capable of rapidly killing Giurdiu lumbilci, a common pathogenic intestinal parasite of humans, by exposure to normal human milk in uitro [13]. Thus human milk may play a protective role in infants exposed to this parasite.Human milk lipase has been shown to survive pH and enzymatic conditions approximating passage through an infant's stomach [l,5]. The optimal activity range has been determined as pH 6 -8 for human milk lipase unstimulated hydrolysis of tributylglyceride and ca. pH 9.5 in the presence of 6 mM sodium taurochloate and between pH 7.5 and 8.5 for both stimulated (3 mM sodium taurocholate) and unstimulated hydrolysis of p-nitrophenylacetate [5], while an optimal pH of ca. 8 has been noted for stimulated (10 mM sodium taurocholate) human milk lipase for both trioleylglyceride and tributylglyceride [I 41.Under favorable circumstances, pH studies can yield useful information to distinguish between the often complex effects of pH variation on enzyme-catalyzed reactions. Such studies led to useful deductions about the nature of the binding between bile salts and porcine pancreatic lipase [I 51. But if the data are to be easily interpreted it is essential that only a limited number of interactions be affected by the change in pH. Previous investigations [5,14] have shown that use of the oilphase-soluble substrate trioleylglyceride has produced two activity/pH profiles of quite difrerent appearance and in the presence of rat pancreatic non-specific lipase [16] it was found that insoluble substrates gave different values of K , and V , for This is part 3 in a series; part 2 appeared elsewhere [l].Abbreuiution. Bistris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl-propane-l,3...

Human Milk Lipase Substrates

“…Since the rate determining step is also insensitive to electronic effects in the presence of bile salt stimulation of the enzyme, it is not necessary to postulate activation of the esterolytic groups in the active site. This conclusion is in agreement with the findings that the bile salts do not alter the shape of the rate-pH profile for the enzyme catalyzed reaction (4) .…”

“…4, No. 4,1985 Under conditions in which we measure the values of k", any variations in rates of hydrolysis should arise only from effects upon the rate determining step, k-; provided that K; remains invariant. For a limited number of substrates we have found that K m lies within the range 1.1 to 3.0 mmol dm-3, although the values of k" varied by three orders of magnitude (7,8).…”

Purification and Characterization of a Novel Cholesterol Esterase fromPseudomonas aeruginosa, with Its Application to Cleaning Lipid-stained Contact Lenses

“…Mammalian cholesterol esterase is strictly dependent on bile salts for hydrolytic activity on insoluble fats and oils, kinetic and biochemical studies have been focused to analyze the activation mechanism. [23][24][25][26][27][28] Wang and Lee have done experiments to probe the accessibility of the active site of the human milk cholesterol esterase, and have indicated that taurocholate was a nonessential activator for the enzyme with water-soluble substrates, but an essential activator with water-insoluble long chain triglycerides. 27) They suggested one of the probable reasons for the diŠerence toward these substrates could be the inaccessibility of the active site of the enzyme to the long chain triglyceride molecule caused by a steric hindrance eŠect.…”

Characterization of the silver-aqueous electrolyte interface by optical second harmonic generation

“…Within the electric dipole approximation, these secondorder processes are restricted to regions where inversion symmetry is broken, such as is atomically present at the interface. 3 It is this feature that has been exploited in several recent studies where SHG has been used to monitor species electrochemically formed on silver,4 5"7 copper, and gold8 electrode surfaces. Molecular adsorption at the interface of the roughened surface has also been observed.9 Shen and co-workers have discussed in detail the SH enhancement effects from roughened surfaces.10 11"12 (1) For reviews, see: Van Duyne, R. P. In "Chemical and Biochemical Application of Lasers"; Moore, c. B., Ed.…”