Carboxypeptldase N Grimwood et ai 1988

Carboxypeptidase N (kininase I) cleaves C-terminal lysyl and argininyl residues from peptides. The assay described here is about 1000-fold more sensitive

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Figure 9.35 Typical chromatographic patterns of the hydrolysis of Gly-Pro-Ala with different concentrations (A, 0 fig\ B, 100 fig; C, 200 fig) of rabbit kidney microvillous fractions. Peak 1 (A), Gly-Pro-Ala (3.3 nmol, retention time 6.4 min) decreased; peak 2 (B,C), Gly-Pro (retention time 5.0 min) increased at each protein concentration. Mobile phase, 10.0 mM potassium dihydrogen phosphate containing 1.0 mM, 1-octanesulfonate (pH 2.5). (From Harada et al., 1988.)

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Figure 9.35 Typical chromatographic patterns of the hydrolysis of Gly-Pro-Ala with different concentrations (A, 0 fig\ B, 100 fig; C, 200 fig) of rabbit kidney microvillous fractions. Peak 1 (A), Gly-Pro-Ala (3.3 nmol, retention time 6.4 min) decreased; peak 2 (B,C), Gly-Pro (retention time 5.0 min) increased at each protein concentration. Mobile phase, 10.0 mM potassium dihydrogen phosphate containing 1.0 mM, 1-octanesulfonate (pH 2.5). (From Harada et al., 1988.)

than a spectrophotometric assay. Furylacryloyl-Ala-Lys or furylacryloyl-Ala-Arg is used as substrate. As little as 51 pg of purified carboxypeptidase N can be detected.

The enzyme is assayed by quantifying by HPLC the furylacryloyl-Ala produced from either of the two substrates. The substrate and product are separated on a C]8 RP column (4 fim, Waters) by using 0.1% TFA in 80:20 water-acetonitrile. The eluate is monitored at 280 nm. The formation of product is linear with time and proportional to enzyme concentration when hydrolysis does not exceed 20 to 25%.

The standard assay used by Grimwood et al. (1988) consisted of 10 fiL 2.0 mM substrate in 0.05 M Hepes (pH 7.75) and 10 fiL of enzyme. The reactions proceeded at 37°C and were terminated with 10 fiL 0.4 M H3PO4. Samples of 20 fiL were injected. The lysine-containing peptide is cleaved three times more rapidly than the argininyl peptide. A guard column containing the same chromatographic medium was replaced every 2 weeks.

Applicability of the assay was shown for human carboxypeptidase N purified from plasma as well as enzyme in unpurified plasma and conditioned medium from a human hepatoma cell line (Hep G2).

Microvillous prol«ln (pg)

Figure 9.36 Dependence of microvillus protein concentration of Gly-Pro formation from Gly-Pro-Ala catalyzed dipeptidyl peptidase IV. Each value of the sample concentration represents the mean of three different determinations. Hydrolysis of Gly-Pro-Ala (•) and formation of Gly-Pro (O) were stoichiometrically related. (From Harada et al., 1988.)

Microvillous prol«ln (pg)

Figure 9.36 Dependence of microvillus protein concentration of Gly-Pro formation from Gly-Pro-Ala catalyzed dipeptidyl peptidase IV. Each value of the sample concentration represents the mean of three different determinations. Hydrolysis of Gly-Pro-Ala (•) and formation of Gly-Pro (O) were stoichiometrically related. (From Harada et al., 1988.)

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