Enkephallnases A and B Ohno et al 1988

Enkephalinase A (dipeptidyl carboxypeptidase) and enkephalinase B (dipep-tidyl aminopeptidase) are involved in the degradation of enkephalin, which has an opiate-like activity. Enkephalinase A cleaves methionine enkephalin (Tyr-Gly-Gly-Phe-Met) after the second glycine, whereas enkephalinase B cleaves after the first glycine. A postcolumn derivatization system is used to detect N-terminal tyrosine-containing peptides.

02468024680246802468

Serum

Whole Blood

Lung

Kidney

Figure 9.31 Chromatograms obtained from various samples incubated with (upper diagram) or without (lower diagram) Hip-His-Leu (HHL). (A) Standard mixture of 2.7 nmol His-Leu, 2.7 nmol hippuric acid, and 100 nmol Hip-His-Leu. (B) A 50 /¿L aliquot of serum or (C) whole blood was incubated with or without 5 mM Hip-His-Leu. After 30 minutes, 0.75 mL of 3% m-phosphoric acid was added and centrifuged. (D) Lung or (E) kidney was homogenized in 5 volumes of chilled Tris-HCl buffer containing 0.5% Nonidet-P40, and centrifuged. The supernatant was incubated with or without 5 mM Hip-His-Leu. In the case of lung, the supernatant was diluted 20 times with the buffer prior to incubation with Hip-His-Leu. Peaks: 1, His-Leu; 2, hippuric acid; 3, Hip-His-Leu. (From Horiuchi et al., 1982.)

02468024680246802468

Serum

Whole Blood

Lung

Kidney

Figure 9.31 Chromatograms obtained from various samples incubated with (upper diagram) or without (lower diagram) Hip-His-Leu (HHL). (A) Standard mixture of 2.7 nmol His-Leu, 2.7 nmol hippuric acid, and 100 nmol Hip-His-Leu. (B) A 50 /¿L aliquot of serum or (C) whole blood was incubated with or without 5 mM Hip-His-Leu. After 30 minutes, 0.75 mL of 3% m-phosphoric acid was added and centrifuged. (D) Lung or (E) kidney was homogenized in 5 volumes of chilled Tris-HCl buffer containing 0.5% Nonidet-P40, and centrifuged. The supernatant was incubated with or without 5 mM Hip-His-Leu. In the case of lung, the supernatant was diluted 20 times with the buffer prior to incubation with Hip-His-Leu. Peaks: 1, His-Leu; 2, hippuric acid; 3, Hip-His-Leu. (From Horiuchi et al., 1982.)

Substrate and product peptides were separated on a reversed-phase column (200 mm X 4 mm) packed with TSK gel ODS-120T (5 /¿m). The mobile phase was formed from solvents A and B formed from acetonitrile-sodium phosphate buffer (0.3 M, pH 2.3)-water mixed in ratios of 1:20:79 and 3:1:1 (v/v/v), respectively. For detection of N-terminal tyrosines (other tyrosines do not react), the eluate stream was mixed first with 50 /um CoCl2 and 20 mM NH2OH, and then with 0.3 M borate (pH 11.4). The resulting mixture was passed through a reaction coil maintained at 75°C before monitoring at 435 nm (emission) and 335 nm (excitation). Calibration graphs for the products were linear up to at least 5 nmol per assay.

The enzyme reaction ws conducted by adding 30 ju.L of enzyme sample to a mixture containing 50 ¡xL of 0.5 M Tris-HCl (pH 7.4), 50 ju,L of 1 mM bestatin, 20 fiL of 40 mM captopril, and 50 ¡xL of 100 ¡xM methionine enkephalin. Bestatin and captopril are included to inhibit aminopeptidase and angiotensin-coverting enzyme, respectively. Incubation at 37°C proceeded for 30 minutes, and then the reaction was terminated by heating at 100°C for 2

i 200

20 40 60

Incubation time(min)

Figure 9.32 Dependence on incubation time of hippuric acid formation from Hip-His-Leu catalyzed by serum (O), whole blood (•), lung (A), and kidney (A). A 50 ju.L aliquot of sample was used. Each point represents the mean of three determinations. (From Horiuchi et al., 1982.)

o i 200

y 150

20 40 60

Incubation time(min)

Figure 9.32 Dependence on incubation time of hippuric acid formation from Hip-His-Leu catalyzed by serum (O), whole blood (•), lung (A), and kidney (A). A 50 ju.L aliquot of sample was used. Each point represents the mean of three determinations. (From Horiuchi et al., 1982.)

minutes. After 25 minutes of centrifuging at 2000g, a 50 /iL portion of the supernate was injected into the column.

Enzyme preparations were obtained by removing brains from anesthetized rats and separating each brain into the striatum, cortex, pituitary, hypothalamus, hippocampus, and amygdala regions. A portion of each region (ca. 100 mg) was homogenized with 2 mL of water. The homogenate was centrifuged at 800g for 10 minutes and the pellet was extracted with 5 mL of Triton X-100 solution (1 mg/mL). After standing for an hour, the mixture was centrifuged at 2000g for 25 minutes and the supernate was used in the enzyme reaction.

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