S J Fromm | PubMed Health

PubMed1999

A two-step computer-assisted method for deriving steady-state rate equations.

A number of computer-assisted methods have been described for the derivation of enzyme-catalyzed steady-state rate equations [K. R. Runyan and R. B. Gunn (1989) Methods Enzymol. 171, 164-190; R. Varon, F. Garcia-Seville, M. Garvia-Moreno, F. Garcia-Canovas, R. Peyro, and R. G. Duggleby (1997) Comput. Appl. Biosci. 13, 159-167]; however, the required programs are either not readily available or require special software. We present here a two-step computer-assisted procedure for deriving steady-state rate equations using the widely available program Mathematica. In the first step, the differential equations for a particular kinetic mechanism that describe changes in enzyme concentration as a function of time are set equal to zero and entered into Mathematica in matrix form. In the second step, a single command allows for the computation of the distribution equations for the free enzyme and each enzyme-ligand complex.

Computer Simulation↗

PubMed1995

Two-dimensional affinity resolution electrophoresis demonstrates that three distinct heparin populations interact with antithrombin III.

Heparin is a polydisperse, highly sulfated polysaccharide consisting of repeating 1-->4 linked uronic acid and glucosamine sugar residues that binds to coagulation proteins, complement proteins, and growth factors to regulate a variety of biological activities. Heparin is best known as an anticoagulant, an activity that results largely from a specific pentasaccharide sequence in heparin that interacts with a unique site in antithrombin III. Little is known about additional structures within heparin that might interact with antithrombin III or the heparin structures that interact with the myriad of other heparin-binding proteins and peptides. Unfractionated glycosaminoglycan heparin that had been prepared from porcine intestinal mucosa was examined for its capacity to bind antithrombin III using a new technique developed to quantitate that interaction. Two-dimensional affinity resolution electrophoresis is a powerful method that allows assessment of unique species of heparin molecules that bind to protein, allowing determination of heparin molecular weight for each protein-binding heparin species as well as the dissociation constant of each interaction. This study provides the first definitive evidence that glycosaminoglycan heparin contains at least three populations of molecules with affinity for antithrombin III. Furthermore, the affinity of each heparin species for antithrombin III appears to vary inversely with the size of the heparin chain, with some smaller oligosaccharides having greater affinity for antithrombin III than larger oligosaccharides.

Animals↗