Gerard O'Cuinn

Purification and characterization of a post-proline dipeptidyl aminopeptidase from Streptococcus cremoris AM2

The present study describes the purification of a post-proline dipeptidyl aminopeptidase from the cytoplasm of Streptococcus cremoris AM2. On the basis of its elution from a calibrated Sephadex G200 column, the enzyme had a molecular weight of 117000 and exhibited a broad pH optimum activity between 6·0 and 9·0. The activity was most comprehensively inhibited by phenylmethylsulphonylfluoride and more modestly inhibited by 1,10-phenanthroline and 8-hydroxyquinoline but not by EDTA. A range of peptides containing either proline or alanine as the penultimate amino acid residue could act as substrates. The presence of proline on the carboxy side of the scissile bond prevented hydrolysis. However the enzyme could release Pro-Pro from Pro-Pro-Gly-Phe-Ser-Pro. The significance of this substrate specificity is considered in the context of removal of either single proline residues or prolylproline sequences from oligopeptides during cheese ripening.

Physicochemical and sensory characteristics of whey protein hydrolysates generated at different total solids levels

Whey protein hydrolysates were generated at different total solids (TS) levels (50-300 g/l) using the commercially available proteolytic preparation Debitrase HYW20, while enzyme to substrate ratio, pH and temperature were maintained constant. Hydrolysis proceeded at a faster rate at lower TS reaching a degree of hydrolysis (DH) of 16.6% at 300 g TS/l, compared with a DH of 22.7% at 50 g TS/l after 6 h hydrolysis. The slower breakdown of intact whey proteins at high TS was quantified by gel-permeation HPLC. Reversed-phase (RP) HPLC of hydrolysate samples of equivalent DH (approximately 15%) generated at different TS levels indicated that certain hydrophobic peptide peaks were present at higher levels in hydrolysates generated at low TS. Sensory evaluation showed that hydrolysates with equivalent DH values were significantly (P < 0.0005) less bitter when generated at 300 g TS/l (mean bitterness score = 25.4%) than hydrolysates generated at 50 g TS/l (mean bitterness score = 39.9%). A specific hydrophobic peptide peak present at higher concentrations in hydrolysates generated at low TS was isolated and identified as beta-lactoglobulin f(43-57), a fragment having the physical and chemical characteristics of a bitter peptide.

Prolidase activity of Lactococcus lactis subsp. cremoris AM2: partial purification and characterization

Prolidase activity from cytoplasm of Lactococcus lactis subsp. cremoris (Streptococcus cremoris) AM2 was partially purified. The enzyme had M r 42000 and optimum activity between pH 7·35 and 8·25 in citrate, phosphate and borate buffers while in a universal buffer system an optimum pH between 8·3 and 9·0 was observed. The activity was strongly inhibited by the chelating agents E.DTA, 1,10-phenanthroline and 8-hydroxyquinoline. Inhibition was also noted with dithio-threitol, N -ethylmaleimide and bacitracin. The enzyme was active on all amino-acylproline substrates tested except Gly-Pro and Gip-Pro and also showed activity against Pro-Pro. While most prolyl amino acids tested were not hydrolysed, hydrolysis was noted with Pro-Ala and Pro-Val. K m values of 20 mM and 10 mM were obtained with Phe-Pro and Met-Pro respectively; however, substrate inhibition was observed with Ile-Pro and Leu-Pro.

Purification and characterisation of an aminotripeptidase from cytoplasm of Lactococcus lactis subsp. cremoris AM2

Abstract An aminopeptidase was purified 120-fold with a 78% recovery from the cytoplasm of Lactococcus lactis subsp. cremoris AM2. The purified enzyme exhibited no activity on dipeptides, dipeptideamides, tripeptideamides or tetrapeptides. As activity was observed only with tripeptides, from which it released the N -terminal amino acid, the enzyme was adjudged to be a strict aminotripeptidase. The enzyme had a Mr of 105 000 and showed one band, corresponding to an Mr of 55 000 on SDS-PAGE electrophoresis. Inhibition by EDTA. 8-hydroxyquinoline and 1.10 phenanthroline indicated that the peptidase was a metallo enzyme. Dithiothreitol, bestatin and amastatin caused total inhibition, whereas p -chloromercuribenzoate, bacitracin and phenyl methyl sulphonyl fluoride were without effect. K m values for tripeptides were within the range 0·18 (Leu-Leu-Leu) to 0·38 mM (Trp-Gly-Gly). Substrate inhibition was noted with some tripeptides at concentrations above 1·5 mM.

Dipeptidyl Aminopeptidase III of Guinea-Pig Brain: Specificity for Short Oligopeptide Sequences

Abstract: A dipeptidyl aminopeptidase III‐type activity has been purified from the cytoplasm of guinea‐pig brain using arginyl‐arginyl‐7‐amido‐4 methylcoumarin as substrate. The enzyme was purified 754‐fold relative to the crude homogenate and with a 12.7% recovery. The purified enzyme was found to have a relative molecular weight of 85,000 and consists of one polypeptide chain of relative molecular weight 80,000, on the basis of its migration on calibrated sodium dodecyl sulphate‐polyacrylamide gel electrophoresis gel. It is highly sensitive to the presence of chelating agents, sulphydryl reactive agents, and the dipeptide Tyr‐Tyr. Dithiothreitol (1 mM) reduced activity by 28%, and 36 and 65% inhibition was noted with phenylmethylsulphonyl fluoride and puromycin (both at 1 mM), respectively. Little or no inhibition was observed with bestatin, bacitracin, captopril, amastatin, and arphamenine B. The purified enzyme released dipeptide moieties from a wide range of peptides including enkephalin sequences and also angiotensin sequences up to the octapeptide angiotensin II. These sequences inhibited the hydrolysis of arginyl‐arginyl‐7‐amido‐4‐methylcoumarin by dipeptidyl aminopeptidase III with Ki values in the micromolar range. No hydrolysis was observed with angiotensin I or with peptide sequences containing more than 10 amino acids. No hydrolysis was observed also with peptide sequences containing a Pro residue on either side of the sissile bond. Peptides containing less than four amino acids were not hydrolysed.

Purification and characterization of aminopeptidase P from Lactococcus lactis subsp. cremoris

Aminopeptidase P was purified 65.3-fold from the cytoplasm of Lactococcus lactis subsp. cremoris AM2 with a 5.8% yield. The purified enzyme was found to consist of one polypeptide chain with a relative molecular mass of 41,600. Metal chelating agents were found to be inhibitory and Mn2+ and Co2+ stimulated activity 7-fold and 6-fold respectively. The purified enzyme removed the N-terminal amino acid from peptides only where proline (and in one case alanine) was present in the penultimate position. No hydrolysis was observed either with dipeptides even when proline was present in the C-terminal position or when either N-terminal proline or pyroglutamate was present preceding a proline residue in the penultimate position of longer peptides. On the basis of this substrate specificity either aminopeptidase P or post-proline dipeptidyl aminopeptidase are necessary along with a broad specificity aminopeptidase to effect complete hydrolysis of casein-derived peptides containing a single internally placed proline residue. However, both aminopeptidase P and post-proline dipeptidyl aminopeptidase would be required together with a broad specificity aminopeptidase in order to completely hydrolyse casein-derived peptides that contain two internally placed consecutive proline residues. As bitter casein-derived peptides are likely to contain either single prolines or pairs of prolines, aminopeptidase P appears to be an important enzyme for debittering.

Dipeptidyl aminopeptidase activities of guinea-pig brain

1. The subcellular distribution of dipeptidyl aminopeptidase activities in guinea-pig brain was investigated. Our studies show that DAP I (Gly-Arg-NH-Mec hydrolase) type activity was found to have an acidic optimum and was associated with the nuclear pellet. 2. No DAP II (Lys-Ala-NH-Mec hydrolase) type activity could be detected. Apparent hydrolysis was mainly due to aminopeptidase activity. 3. DAP III (Arg-Arg-NH-Mec hydrolase) type activity is largely cytoplasmic, but there was evidence of a membrane form associated with the synaptosomes. 4. DAP IV (Gly-Pro-NH-Mec hydrolase) type activity is present on the synaptosomal membrane, and also enriched in the microsomes. A soluble form of Gly-Pro-NH-Mec hydrolase activity is also present in the cytoplasm. Whether this activity is a DAP II or IV type activity is still yet to be determined.

Hydrolysis of αs1- and β-casein-derived peptides with a broad specificity aminopeptidase and proline specific aminopeptidases from Lactococcus lactis subsp. cremoris AM2

Aminopeptidase hydrolysis of αs1‐ and β‐casein‐derived synthetic peptides containing non‐consecutive and consecutive proline residues was characterised. Aminopeptidase P (Pep P) (EC 3.4.11.9) or post‐proline dipeptidyl aminopeptidase (PPDA) (EC 3.4.14.5) along with lysine‐paranitroanilide hydrolase (KpNA‐H) (EC 3.4.11.1) activities are required in the degradation of peptides containing non‐consecutive proline residues. However, both Pep P and PPDA along with KpNA‐H are required for hydrolysis of peptides containing consecutive proline residues. The results demonstrate the mechanism by which combinations of purified general and proline specific aminopeptidases from Lactococcus lactis subsp. cremoris AM2 hydrolyse peptides containing proline residues.

Purification and characterization of a lysine-p-nitroanilide hydrolase, a broad specificity aminopeptidase, from the cytoplasm of Lactococcus lactis subsp. cremoris AM2

A hydrolase activity that cleaves lysyl-p-nitroanilide (Lys-pNA) has been purified from the cytoplasm of Lactococcus lactis subsp. cremoris AM2 by chromatography on DE52, DEAE Affi-Gel Blue Gel, Hydroxyapatite Bio-Gel HTP and Phenyl Sepharose. The purified aminopeptidase was found to have a native M(r) of 50,000-55,000 by gel filtration chromatography and by FPLC gel filtration on Superose 12 and to be composed of a single polypeptide chain following SDS-PAGE. Enzyme activity was almost completely inhibited by EDTA, amastatin, puromycin and bestatin, while the sulphydryl-reactive agents p-chloromercuribenzoate and iodoacetamide were inhibitory. The enzyme was found to be very unstable during the purification procedures at 4 degrees C and its stability was greatly improved when 10 ml glycerol/l and 2 mM-dithiothreitol were included in the purification buffers. The purified enzyme was found to hydrolyse a wide range of dipeptides, tripeptides and longer peptides provided that proline was not present in the penultimate position from the N-terminus or that a pyroglutamyl residue was not present at the N-terminus. While neither Asp-pNA nor Pro-pNA was hydrolysed by the purified enzyme, the release of N-terminal acidic residues from peptides was observed in addition to the release of N-terminal proline from Pro-Leu-Gly-NH2, Pro-Leu-Gly-Gly and Pro-His-Pro-Phe-His-Leu-Phe-Val-Tyr. This ability of Lys-pNA hydrolase to release N-terminal proline residues was employed in concert with a purified aminopeptidase P preparation to release alternate N-terminal amino acids from Tyr-Pro-Phe-Pro-Gly. The complementary action of these enzymes represents an alternative mechanism to that of post-proline dipeptidyl aminopeptidase for metabolism of proline-containing peptides.

Alanine aminopeptidase of guinea-pig brain: a broad specificity cytoplasmic enzyme capable of hydrolysing short and intermediate length peptides.

Alanine aminopeptidase is reported to be a broad specificity aminopeptidase acting on peptides of different lengths. In this study we wish to define the properties of the activity from guinea-pig brain and compare these properties with previous findings. Alanine amino-peptidase was purified from cytoplasm of guinea-pig brain by a four-step procedure involving chromatography on DE-52, hydroxylapatite, Sephacryl S-200 and DEAE-Sephacryl. Relative molecular mass was determined by chromatography on Sephacryl S-200 column and subunit size determined by SDS-PAGE under denaturing conditions. Cations which reactivate the enzyme were determined with EDTA treated enzyme. Substrate specificity was determined by TLC and kinetic parameters were derived from Lineweaver-Burk plots. A 216-fold purification was achieved by the above procedures. The purified enzyme was found to consist of one polypeptide chain with a relative molecular mass of 104,000. Its activity was inhibited by chelating agents, sulphydryl reactive agents, puromycin, bestatin and amastatin but stimulated over 6-fold by dithiothreitol. Some dipeptides and all tripeptides and longer peptides containing up to 16 amino acids tested were hydrolysed provided neither Glp or Pro occurred at the N-terminus or that Pro did not occur in the penultimate position from the N-terminus. The enzyme preferred bulky non-polar residues at the N-terminal and penultimate positions and was found to hydrolyse three dipeptidyl methyl coumarin amides used in detecting dipeptidyl aminopeptidases. Alanine aminopeptidase is thus a broad specificity amino-peptidase acting on short and intermediate length peptides whose affinity for substrates increases with increasing peptide length. Its properties are well suited to a role in peptide turnover in brain cytoplasm.