Table 1.
The specific activity towards glutaryl-Ala-Ala-Phe-MNA cleavage is reduced in NEP mutants.
Table 2.
Rates of hydrolysis of physiological peptides by NEP mutants.
Table 3.
Leu-Enkephalin and Insulin B chain dual substrate assays.
Figure 1.
Time course for NEP mediated hydrolysis of insulin B chain.
Time course assays were conducted by incubation of NEP with 15 µM insulin B chain in 20 mM MES buffer, pH 6.5, at 37°C. A 100 µL aliquot was removed at each time point and 10 µL of 5% trifluoroacetic acid (TFA) was added to stop further hydrolysis. 95 µL were injected into a Vydac C4 column and developed as described in Materials and Methods. Each product was isolated and subjected to mass spectral analysis. Numbers under each peak indicate the identification of the peptide by sequence. Peaks without numbers were not identified.
Table 4.
Products of insulin B chain hydrolysis by NEP.
Figure 2.
Comparison of insulin B chain cleavage between NEP, NEPF563L, and NEPS546E.
A. HPLC profile of insulin B chain cleaved by NEP and NEP mutants at 30% hydrolysis. B. Rates of peak accumulation at each cleavage site normalized to that of NEP for mutant NEPS546E. C. Rates of peak accumulation at each cleavage site normalized to that of NEP for mutant NEPF563L. Dotted lines indicate the overall rate of hydrolysis of insulin B chain from Table 2. Reactions were carried out at 37°C with 15 µM insulin B chain in 20 mM MES, pH 6.5.
Table 5.
Relative rates of accumulation of peaks generated by the NEP and NEP mutant dependent hydrolysis of insulin B chain.
Figure 3.
Time course of NEP mediated hydrolysis of Aß1–40.
Time course measurements were carried out by incubation of NEP with 24 µM Aß1–40 in 20 mM MES, pH 6.5, at 37°. 100 µL aliquots were removed at each time point and 10 µL of 5% TFA was added to stop the reaction. Samples were analyzed as described in Figure 1. Numbers under each peak indicate the identification of the peptide by sequence.
Table 6.
Identification of products resulting from the hydrolysis of Aß1–40 by NEP.
Figure 4.
Comparison of Aß1–40 cleavage between NEP, NEPF563L, and NEPS546E.
A. HPLC cleavage profile of Aß1–40 cleavage by NEP and NEP mutants at ∼30% hydrolysis. B. Rates of peak accumulation at each cleavage site normalized to that of NEP for mutant NEPS546E. C. Rates of peak accumulation at each cleavage site normalized to that of NEP for mutant NEPF563L. Dotted lines indicate the overall rate of hydrolysis of Aß1–40 from Table 2. Reactions were carried out at 37°C with 15 µM Aß1–40 in 20 mM MES, pH 6.5.
Table 7.
Accumulation rates of products of NEP dependent cleavage of Aß1–40.