Michael B. Martinez

A functional polymorphism of apolipoprotein C1 detected by mass spectrometry

A survey of plasma proteins in approximately 1300 individuals by MALDI‐TOF MS resulted in identification of a structural polymorphism of apolipoprotein C1 (ApoC1) that was found only in persons of American Indian or Mexican ancestry. MS/MS analysis revealed that the alteration consisted of a T45S variation. The methyl group of T45 forms part of the lipid‐interacting surface of ApoC1. In agreement with an impact on lipid contact, the S45 variant was more susceptible to N‐terminal truncation by dipeptidylpeptidase IV in vitro than was the T45 variant. The S45 protein also displayed greater N‐terminal truncation (loss of Thr‐Pro) in vivo than the T45 variant. The S45 variant also showed preferential distribution to the very‐low‐density lipoprotein fraction than the T45 protein. These properties indicate a functional effect of the S45 variant and support a role for residue 45 in lipid contact and lipid specificity. Further studies are needed to determine the effects of the variant and its altered N‐terminal truncation on the metabolic functions of ApoC1.

Steady-state enzyme kinetics in the Escherichia coli periplasm : a model of a whole cell biocatalyst

This study provided analysis of in vivo enzyme kinetics in a model system which consisted of alkaline phosphatase in the periplasm of Escherichia coli. Modeling of complete substrate titration curves was achieved for a wide range of intraperiplasmic enzyme levels and outer membrane permeabilities. The results helped to identify the features most important to optimize in vivo reaction velocity. For many situations, a surprising finding was that maximum enzyme expression was not a major concern. For example, for moderate enzyme expression levels and moderate substrate levels (ca 0-5 mM), the limiting step for the enzyme in the periplasm was substrate (para-nitrophenylphosphate) diffusion through the outer membrane. In vivo reaction velocity was directly proportional to substrate concentration, outer membrane permeability, and the cell concentration. Velocity was also quite insensitive to a potent inhibitor of the enzyme. Even though diffusion-limited, periplasmic reaction velocity was quite sensitive to temperature, suggesting that the conformation of porin proteins in the E. coli outer membrane governed the average size of the pore. This model system therefore defined important features of bacterial whole cell biocatalyst design, which may also apply to other reactors using intact cells as catalysts.