X-prolyl Dipeptidyl Aminopeptidase Activity Research Articles

Evaluation of a Commercial Sandwich Enzyme-Linked Immunosorbent Assay for the Quantification of Beta-Casomorphin 7 in Yogurt Using Solid-Phase Extraction Coupled to Liquid Chromatography-Tandem Mass Spectrometry as the "Gold Standard" Method.

This study investigated beta-casomorphin 7 (BCM7) in yogurt by means of LC-tandem MS (MS/MS) and enzyme-linked immunosorbent assay (ELISA) and use LC-MS/MS as the "gold standard" method to evaluate the applicability of a commercial ELISA. The level of BCM7 in milk obtained from ELISA analysis was much lower than that obtained by LC-MS/MS analysis and trended to increase during fermentation and storage of yogurt. Meanwhile, the results obtained from LC-MS/MS showed that BCM7 degraded during stages of yogurt processing, and its degradation may have been caused by X-prolyl dipeptidyl aminopeptidase activity. As a result, the commercial sandwich ELISA kit was not suitable for the quantification of BCM7 in fermented dairy milk.

Influence of fermentation temperature and autolysis on ACE-inhibitory activity and peptide profiles of milk fermented by selected strains of Lactobacillus helveticus and Lactococcus lactis

Bovine milk was fermented with single strains of Lc. lactis or Lb. helveticus at three temperatures around the optimum for each species. Fermentation was stopped at pH 4.6 and 4.3/4.0, and the products were stored at 5 °C for 1 and 7 days. The extent of lysis was examined by release of X-prolyldipeptidyl aminopeptidase activity, and the angiotensin-I-converting enzyme- (ACE-) inhibitory activity and peptide profiles of the products were determined. Fermentation temperature significantly influenced the bacterial growth, extent of lysis, and ACE-inhibitory activity. ACE-inhibitory activity was high at all temperatures, and slightly higher at the optimal growth temperature, whereas the extent of lysis was highest at a suboptimal growth temperature. Peptide profiles were marginally affected by temperature and cell lysis. The major peptides were identified, including a number of known ACE-inhibitory peptides. Our results suggest that the cell wall proteinase was the primary catalyst in release of ACE-inhibitory peptides.

Purification and characterization of a thermosensitive X-prolyl dipeptidyl aminopeptidase (dipeptidyl aminopeptidase yscV) from Saccharomyces cerevisiae

Dipeptidyl aminopeptidase yscV, a heat-labile enzyme with X-prolyl dipeptidyl aminopeptidase activity, was purified about 470-fold from a protoplast lysate of Saccharomyces cerevisiae. The purification procedure included solubilization of tonoplast-bound activity by the non-ionic detergent octyl-β- d-glucopyranoside, glycerol gradient centrifugation and preparative isoelectric focusing. Sodium dodecyl sulfate polyacrylamide gel electrophoresis resulted in a single band for which a molecular weight of 40 000 was calculated. The peptidase was most active at pH 7.0–7.5 with l-alanyl- l-proline -p- nitroanilide as substrate. Substrate specificity studies indicate that the purified enzyme specifically hydrolyzes peptide bonds involving the carboxyl group of prolyl residues penultimate to unprotected termini unless arginine is the N-terminal amino acid. However, X-Ala-arylamide structures are not attacked. The actinomycete inhibitors antipain, chymostatin and pepstatin had no effect on the enzyme activity, but 5 mM phenylmethylsulfonyl fluoride, an inhibitor of serine peptidases, completely inhibited dipeptidyl aminopeptidase yscV activity. Some heavy metals (Ni 2+, Cd 2+, Zn 2+, Hg 2+) at a concentration of 5·10 −4 M were also found to be potent inhibitors of enzyme activity.

Localization of the thermosensitive X-prolyl dipeptidyl aminopeptidase in the vacuolar membrane of Saccharomyces cerevisiae

Most of the X-prolyl dipeptidyl aminopeptidase activity of Saccharomyces cerevisiae was found to be associated with purified vacuolar membranes (specific activity approx. 75-times higher than in the protoplast lysate). The tonoplast-bound enzyme is thermosensitive. Another heat-resistant enzyme was found in the protoplast lysate. The tonoplast-bound thermosensitive enzyme shows an apparent K m of 0.06 mM against L-alanyl-L-prolyl- p-nitroanilide while the heat-resistant enzyme shows an apparent K m of 0.4 mM against the same substrate.

Studies on the regulation of X-prolyl dipeptidyl aminopeptidase activity.

The specific activity of X-prolyl-dipeptidyl aminopeptidase in Saccharomyces cerevisiae grown on glucose-containing medium remains constant during exponential growth and increases less than twofold when cells reach the stationary phase. In cells harvested from exponential growth on glucose-containing medium the specific activity of the enzyme is found to be 20-30% lower than the specific activity observed in media without glucose, containing acetate or ethanol as the carbon source. X-Prolyl-dipeptidyl aminopeptidase is not inactivated after the addition of glucose to stationary phase cells. Growth of the yeast on poor nitrogen sources or under nitrogen-starvation results in a three- to fourfold increase in the level of the enzyme.

Changes of X-prolyl dipeptidyl-aminopeptidase activity in developing rat brain.

We found X-prolyl dipeptidyl-aminopeptidase activity in rat brain and examined the developmental changes at various ages. The total enzyme activity per brain increased until 4 weeks of age, and then decreased during maturation. Specific activity in young rat brain was higher than that in adult rat brain. The properties of the brain enzyme were different from those of pituitary and other tissues.

A new assay of X-prolyl dipeptidyl-aminopeptidase activity in human serum with glycylproline p-phenylazoanilide as substrate.

A new assay procedure for X-prolyl dipeptidyl-aminopeptidase activity in human serum was developed with glycylproline p-phenylazoanilide tosylate as substrate. p-Phenylazoaniline liberated by the enzyme reaction was measured photometrically at 493 nm after stopping the reaction with acid. This assay was simple and sensitive, and less than 50 microliter of human serum was required for the assay. Km value was 2.5 mM and the optimum pH was 8.7. After disc gel electrophoresis of human serum, the enzyme activity could be distinctly observed as a reddish band on the gel when the gel was incubated with this substrate.

Reduction of serum X-prolyl dipeptidyl-aminopeptidase activity in tumour-bearing mice and reversal of reduced enzyme activity by lentinan, an anti-tumour polysaccharide

Serum X-prolyl dipeptidyl-aminopeptidase activity which had been shown to be depressed in cancer patients was clearly reduced in mice with Ehrlich carcinoma and Sarcoma 180, and slightly reduced in mice with methylcholanthrene-induced sarcomas. The reduced enzyme activity was completely reversed during tumour regression of sarcoma 180 by administration of lentinan, which causes regression of sarcoma 180.

Comparison of X-prolyl dipeptidyl-aminopeptidase activity in human cerebrospinal fluid with that in serum

X-Prolyl dipeptidyl-aminopeptidase activities in cerebrospinal fluid and serum from the same patients without neurological diseases, undergoing surgery under lumbar anesthesia, were assayed fluorometrically with a newly synthesized fluorogenic substrate, 7-glycylproline-4-methylcoumarinamide; the values were 129.1 +/- 19.5 nmoles/min/l and 54.17 +/- 3.11 mumoles/min/l (mean +/- SEM, n = 23), respectively, and there was no correlation between both activities (r = 0.0894).

Fluorescence assay of X-prolyl dipeptidyl-aminopeptidase activity with a new fluorogenic substrate

We have synthesized a new fluorogenic substrate, 7-(Gly-Pro)-4-methylcoumarinamide, for X-prolyl dipeptidyl-aminopeptidase. Release of 7-amino-4-methylcoumarin by the enzymatic hydrolysis was determined fluorometrically. This new assay was highly sensitive; the enzyme activity in as little as 0.01 μl of human serum was measured, and it was discovered in human cerebrospinal fluid. The specific activity in human parotid saliva was about 17-fold higher than that in serum.

X-Prolyl dipeptidyl-aminopeptidase activity, with X-proline p-nitroanilides as substrates, in normal and pathological human sera.

X-Prolyl dipeptidyl-aminopeptidase (no EC no. assigned) activity in normal and pathological human sera was assayed with several newly synthesized X-proline p-nitroanilides as chromogenic substrates. Normal values for 88 healthy subjects (15 to 81 years old), with glycylproline p-nitroanilide as substrate at pH 8.7, were 54.9 +/- 1.5 (SE) (range, 25.7 - 96.0) mumol/min per liter of serum at 37 degrees C. The results suggest that the enzyme activities with all X-proline p-nitroanilides were increased in patients with hepatitis and decreased in patients with gastric cancer. On Sephadex G-200 column chromatography, normal human sera showed a single peak of enzyme activity with glycylproline p-nitroanilide as the substrate, which coincided with the peak with glycylproline beta-naphthylamide but was different from the peaks with leucine beta-naphthylamide. Sera from patients with hepatitis or liver cirrhosis showed an increase in the normal peak and the appearance of another new peak with glycylproline p-nitroanilide as substrate.