Inhibitor binding other sources

Just as an appreciation of the forces involved is essential to comprehending the binding of an inhibitor to an enzyme, so is an understanding of the kinetic analysis of an enzyme-catalyzed reaction essential to any kinetic evaluation of an inhibitor. In this section we provide a brief introduction to the study of enzyme kinetics, particularly steady-state kinetics. Regardless, the reader is advised to refer to other sources for more in-depth reviews of the kinetic equations and mathematical derivations involved (38,60, 67-71). 

Cohen et al. (IS) discovered that fluorodeoxyuridylate (but not fluoro-uridylate) is a potent inhibitor of the phage-induced thymidylate synthetase in E. coll and is competitive with deoxyuridylate. Since this discovery, inhibition of the synthetase from other sources has been studied (see review, reference 8). The inhibition by fluorodeoxyuridylate of the E. coli phage synthetase, or that from chick embryo or Ehrlich ascites cells, is competitive with respect to deoxyuridylate when all of the reaction components are added together. However, noncompetitive kinetics and a stoichiometric binding of the inhibitory nucleotide are obtained with the chick enzyme, if it is first incubated with fluorodeoxymidylate and 5,10-methylene H4-folate. Evidently the eofactor must be present for fluorodeoxyuridylate to bind in the noncompetitive manner (7). Similarly, it has been found that the bacterial enzyme can be virtually titrated with the inhibitor, if enzyme and inhibitor are preincubated prior to assay of the reaction. The Ehrlich cell enzyme differs in that fluorodeoxyuridylate is competitive with deoxyuridylate, in spite of preincubation with the enzyme. 

Mammalian cell culture is the most important source of therapeutic proteins and monoclonal antibodies. Just as mammalian cells are more complicated than most other microorganisms, the media required for their growth is also more complex. The extracellular medium must provide the same nutrients and growth factors that mammalian cells are exposed to in vivo in order for them to survive, proliferate, and differentiate. Serum contains many important components that support the growth of mammalian cells including growth factors, hormones, transport and binding proteins, attachment factors, protease inhibitors, and... 

Biosynthesis has been studied only in the case of the adenine nucleotide exchange carrier, which has been isolated in pure form from several sources [72-74]. Antibodies have been raised to some of these pure proteins [75], and the availability of antibodies as well as the availability of inhibitors [76] which bind specifically to the nucleotide carrier have permitted studies of its biosynthesis not possible for the other carriers. The immunological work is important since the antibodies can detect the carrier in precursor forms, not yet assembled in the membrane. 

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