Adaption of a commercial lipase kit to measure bile salt-stimulated lipase in human milk

A highly sensitive,fluorometric method for measuring activities of two major lipases in milk,lipoprotein lipase(LPL)and bile salt-stimulated lipase(BSSL)is described.Human milk LPL showed linear activity from 65 to 347 U/L(U=1μmol/min)and the quantification limit was theoretically about 65 U/L.For human milk BSSL,the linear range was from 10 to 267 U/L and the quantification limit was 18 U/L.The mean LPL and BSSL activities were 409±13 U/L and 5263±132 U/L,respectively.This corresponded well to the values reported in literature.The residual LPL and BSSL activities were reduced dramatically to 21%,and 2%,respectively,when the milk was heated to 55°C;heating to 50°C caused only nominal reductions of LPL and BSSL(activities remaining∼82%and 87%,respectively).No BSSL activity was detected in cow,camel,goat milk or in infant formula.

The Role of Anionic Protein Residues on the Salt Dependence of the Binding of Aminoacyl-tRNA Synthetases to tRNA:A Poisson-Boltzmann Analysis

Long-range electrostatic interactions in proteins/peptides associating to nucleic acids are reflected in the salt-dependence of the binding process.According to the oligocationic binding model,which is based on counterion condensation theory,only the cationic residues of peptides/proteins near the binding interface are assumed to affect the salt dependence in the association of peptides and proteins to nucleic acids.This model has been used to interpret and predict the binding of oligocationic chains-such as oligoarginines/lysines-to nucleic acids,and does an excellent job in these kinds of systems.This simple relationship,which is used to compare or count the number of ionic interactions in protein-nucleic acid complexes,does not hold when acidic residues,i.e.glutamate and aspartate,are incorporated in the protein matrix.Here,we report a combined molecular mechanics(by means of energy-minimization of the structure under the influence of an empirical energy function)and Poisson-Boltzmann(PB)study on the salt-dependence in binding to tRNA of two important enzymes that are involved in the seminal step of peptide formation in the ribosome:Glutamine synthetase(GluRS)and Glutaminyl synthetase(GlnRS)bound to their cognate tRNA.These two proteins are anionic and contain a significant number of acidic residues distributed over the entire protein.Some of these residues are located in the binding interface to tRNA.We computed the salt-dependence in association,SKpred,of these enzyme-tRNA complexes using both the linear and nonlinear solution to the PoissonBoltzmann Equation(PBE).Our findings demonstrate that the SKpred obtained with the nonlinear PBE is in good agreement with the experimental SKobs,while use of the linear PBE resulted in the SKpred being anomalous.We conclude that electrostatic interactions between the binding partners in these systems are less favorable by means of charge-charge repulsion between negatively charged protein residues and phosphateoxygens in the tRNA backbone but also play a significant role in the association process of proteins to tRNA.Some unfavorable electrostatic interactions are probably compensated by hydrogen-bonds between the carboxylate group of the side chain in the interfacial acidic protein residues and the tRNA backbone.We propose that the low experimentally observed SKobs values for both GlnRS-and GluRS-tRNA depend on the distribution and number of anionic residues that exist in these tRNA synthetases.Our computed electrostatic binding free energies were large and unfavorable due to the Coulombic and de-solvation contribution for the GlnRS-tRNA and GluRS-tRNA complexes,respectively.Thus,low SKobs values may not reflect small contributions from the electrostatic contribution in complex-formation,as is often suggested in the literature.When charges are”turned off”in a computer-experiment,our results indicated that”turning off”acidic residues far from a phosphate group significantly influences SKpred.If cationic residues are“turned off”,less impact on SKpred is observed with respect to the distance to the nearest phosphate-group。

Modeling Salt Dependence of Protein-Protein Association:Linear vs Non-Linear Poisson-Boltzmann Equation

Proteins perform various biological functions in the cell by interacting and binding to other proteins,DNA,or other small molecules.These interactions occur in cellular compartments with different salt concentrations,which may also vary under different physiological conditions.The goal of this study is to investigate the effect of salt concentration on the electrostatic component of the binding free energy(hereafter,salt effect)based on a large set of 1482 protein-protein complexes,a task that has never been done before.Since the proteins are irregularly shaped objects,the calculations have been carried out by a means of finite-difference algorithm that solves PoissonBoltzmann equation(PB)numerically.We performed simulations using both linear and non-linear PB equations and found that non-linearity,in general,does not have significant contribution into salt effect when the net charges of the protein monomers are of different polarity and are less than five electron units.However,for complexes made of monomers carrying large net charges non-linearity is an important factor,especially for homo-complexes which are made of identical units carrying the same net charge.A parameter reflecting the net charge of the monomers is proposed and used as a flag distinguishing between cases which should be treated with non-linear Poisson-Boltzmann equation and cases where linear PB produces sound results.It was also shown that the magnitude of the salt effect is not correlated with macroscopic parameters(such as net charge of the monomers,corresponding complexes,surface and number of interfacial residues)but rather is a complex phenomenon that depends on the shape and charge distribution of the molecules.