Functional characterization and specific effects of various peptides on enzymatic activity of a DPP-III homologue from goat brain

The purified dipeptidyl aminopeptidase from goat brain showed several characteristics similar to DPP-III although it possesses a dissimilar molecular weight and different inhibition behavior. The enzyme was found to be inhibited by metallochelators and thiol inhibitors which could be reversed by introducing metals and thiols, respectively. The enzyme activity is also significantly affected by DMSO and ethanol. It was found to be highly sensitive to even very low concentration of urea. The inhibitory potency of several dipeptides and bioactive peptides on this enzyme was investigated to characterize its active site. The highest potency was observed for the dipeptides having aromatic and bulky side chains such as Phe-Met, Leu-Arg, Met-Arg, Trp-Met and Leu-Trp.     

Neuron-specific aminopeptidase and puromycin-sensitive aminopeptidase in rat brain development

Neuron-specific aminopeptidase (NAP) and the ubiquitous puromycin-sensitive aminopeptidase (PSA) were compared in the rat hippocampus during early development. Hippocampus contains the highest amount of NAP determined by a fast-protein liquid chromatography-aminopeptidase analyzer using Leu -naphthylamide as substrate. Both enzymes were found in the hippocampus in all ages. NAP was lower in immature rat; the 19th embryonic-day fetus contained the least. It increased steeply during the prenatal through the early postnatal period, 9-fold by the first month. The rate of increase diminished subsequently, increasing 20% in the second month and 13% in the third. The age-dependent increase in NAP activity was parallel to its protein expression as determined by Western blot. The specific molecular activity (hydrolytic activity/NAP antigenicity) in newborn, 15-day-old, and 30-day-old rats were 1.00, 0.88, and 1.00, respectively. The PSA developmental profile without linear increase in activity was distinct from NAP. PSA activity was higher than NAP in decreasing order, 100–4 times, during the same development span. Similarly, different growth profiles for NAP and PSA were also found in the primary culture of developing cerebellar granule cells. Puromycin (1–5 M) blocked neurite outgrowth and caused apoptosis by nonantibiotic effects. Our data suggest that the synaptosome-enriched NAP plays a role in neuron growth, differentiation, and information programming.     

Proteolytic enzymes in the post-translational processing of polypeptide hormone precursors

Selective and limited proteolysis is a key step in the post-translational modification of peptide hormone precursors. This process appears to involve a proteolytic machinery including highly specific endoproteases. Some of the enzyme systems possibly involved in the processing of pro-neuropeptides will be described and their mechanism of action discussed. Special emphasis will be on the following: i) the physico-chemical characteristics of proteolytic enzymes which are believed to be involved in the processing of some of these polypeptide hormone precursors; ii) the bio-specificity of these enzymes toward the substrates; iii) the importance of both secondary and tertiary structures of the cleavage domain in recognition by the selective proteases. These properties will be discussed in connection with the possible importance of the maturation enzymes in the in vivo regulation of hormone biosynthesis.Special issue dedicated to Dr. E. M. Shooter and Dr. S. Varon.     

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 m M ) reduced activity by 28%, and 36 and 65% inhibition was noted with phenylmethylsulphonyl fluoride and puromycin (both at 1 m M ), 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 K _i 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.     

Isolation of an extracellular Mn-dependent enzyme mineralizing melanoidins from the white rot fungus Trametes versicolor

Abstract Morphological and physiological properties of Tetrahymena thermophila immobilized by encapsulation in calcium-alginate hollow spheres were found to be substantially different from those of suspended cells. Immobilized T. thermophila reached lengths of 70–100 μm, whereas the average cell of suspension cultures was about 40 μm long. Suspended cells appeared typically pear-shaped while immobilized cells developed a proboscis-like anterior end. Contrary to suspended T. thermophila , encapsulated cells were functionally deficient in phagocytosis although developing an oral apparatus. The diameter of the macronucleus of immobilized cells was about two times larger than the macronucleus of suspended cells and contained twice as much DNA, while the DNA content of the micronucleus remained unchanged. High cell density fermentations of suspended cells indicated that the alterations observed in immobilized cells were not due to close physical contacts between the cells.     

Purification and characterization of tripeptidylpeptidase-II from post-mortem human brain

A soluble tripeptidylaminopeptidase has been isolated from human post-mortem cerebral cortex by anion exchange, hydrophobic interaction and size-exclusion chromatography. From gel filtration studies the active enzyme can exist in both high molecular weight (Mr>10⁶) and smaller forms. The enzyme hydrolyses Ala-Ala-Phe-7-amino-4-methylcoumarin with a pH optimum of around 7.5 and K_m of 148 M. It did not hydrolyse N-succinyl-Ala-Ala-Phe-7-amino-4-methylcoumarin, aminoacyl- or dipeptidyl-7-amino-methylcoumarins and was not inhibited by bestatin. The enzyme was inhibited by phenylmethylsulphonyl-fluoride, 3,4-dichloroisocoumarin, N-hydroxymercuriphenyl-sulphonic acid and N-ethylmaleimide showing that its activity is serine and cysteine dependent. The purified enzyme released tripeptides from several naturally occurring neuropeptides with quite broad specificity. Cholecystokinin octapeptide, angiotensin III and neurokinin A were the most rapidly hydrolysed. Peptides with Pro residues arount the point of cleavage were not hydrolysed.     

Release of a plasma membrane-bound triaminopeptidase activity from mammalian cells by thermolysin.

Aminopeptidase and other enzyme activities on cellular surface were determined in the presence and absence of endopeptidases. Gly-Pro-Leu-AP activity was specifically released into the medium by thermolysin treatment, while the other activities retained on the cellular surface were markedly decreased. A similar phenomenon was also observed in rat liver membrane, mouse FM3A, spleen lymphocyte and other cells. Structural rearrangement of some protein components in the cell plasma membrane was suggested.     

Purification and characterization of tripeptide aminopeptidase from bovine dental follicles

Tripeptide aminopeptidase (EC 3.4.11.4) was purified from bovine dental follicles by a series of chromatographies. Purified enzyme had a specific activity of 59.5 units per mg protein with L-prolyl-glycylglycine as substrate. The pH optimum was 8.0. The purified native enzyme had a Mr of 230,000 and was shown to be a tetramer of subunit Mr of 58,000. The isoelectric point was pH 7.0. The enzyme was inhibited 5-5-dithio-bis (2-nitrobenzoic acid),o-phenanthroline, and bestatin. Substrate specificity studies indicated that the enzyme specifically hydrolyzes the N-terminal amino acid residue from tripeptides only.     

Human brain aminopeptidase A: biochemical properties and distribution in brain nuclei

Aminopeptidase A (APA) generated brain angiotensin III, one of the main effector peptides of the brain renin angiotensin system, exerting a tonic stimulatory effect on the control of blood pressure in hypertensive rats. The distribution of APA in human brain has not been yet studied. We first biochemically characterized human brain APA (apparent molecular mass of 165 and 130 kDa) and we showed that the human enzyme exhibited similar enzymatic characteristics to recombinant mouse APA. Both enzymes had similar sensitivity to Ca(2+). Kinetic studies showed that the K(m) (190 mumol/L) of the human enzyme for the synthetic substrate-l-glutamyl-beta-naphthylamide was close from that of the mouse enzyme (256 mumol/L). Moreover, various classes of inhibitors including the specific and selective APA inhibitor, (S)-3-amino-4-mercapto-butyl sulfonic acid, had similar inhibitory potencies toward both enzymes. Using (S)-3-amino-4-mercapto-butyl sulfonic acid, we then specifically measured the activity of APA in 40 microdissected areas of the adult human brain. Significant heterogeneity was found in the activity of APA in the various analyzed regions. The highest activity was measured in the choroids plexus and the pineal gland. High activity was also detected in the dorsomedial medulla oblongata, in the septum, the prefrontal cortex, the olfactory bulb, the nucleus accumbens, and the hypothalamus, especially in the paraventricular and supraoptic nuclei. Immunostaining of human brain sections at the level of the medulla oblongata strengthened these data, showing for the first time a high density of immunoreactive neuronal cell bodies and fibers in the motor hypoglossal nucleus, the dorsal motor nucleus of the vagus, the nucleus of the solitary tract, the Roller nucleus, the ambiguus nucleus, the inferior olivary complex, and in the external cuneate nucleus. APA immunoreactivity was also visualized in vessels and capillaries in the dorsal motor nucleus of the vagus and the inferior olivary complex. The presence of APA in several human brain nuclei sensitive to angiotensins and involved in blood pressure regulation suggests that APA in humans is an integral component of the brain renin angiotensin system and strengthens the idea that APA inhibitors could be clinically tested as an additional therapy for the treatment of certain forms of hypertension.     

Activity cycles and the control of four digestive proteinases in the posterior midgut of Rhodnius prolixus Stål (Hemiptera: Reduviidae)

Proteinase levels in the posterior midgut of fifth-instar and adult Rhodnius prolixus follow a cyclic pattern after ingestion of the bloodmeal. In the fifth instar, cathepsin B showed two peaks: the first occurred 6 days after ingestion and the second at the time of ecdysis. Cathepsin D, cathepsin B and lysosomal carboxypeptidase B reached maximal levels 6 days after ingestion. At this time the highest levels of these proteinases were found in mated females, the lowest in males and intermediate levels in virgin females. Maximal levels of aminopeptidase occurred later than catheptic enzymes, and the decline, after maximal levels were achieved, was much more gradual.Catheptic-proteinase levels within the posterior midgut in fifth-instar larvae and adults correlated positively with the amount of protein contained in this gut region. This indicates that production of these proteinases is controlled by a secretagogue mechanism. Aminopeptidase levels were controlled in a different manner. The mated state or sex of adults altered the proteinase levels by changing the amount of protein that was passed into the digestive midgut from the crop.     

Direct N-terminal sequencing of polypeptides using a thermostable bacterial aminopeptidase and MALDI-TOF mass spectrometry

Mass spectrometry-based amino acid sequencing is currently based almost entirely on collision-induced peptide fragmentation and analyses. Here, we describe a single-stage MS-based technique for amino acid sequencing involving partial, heterogenous digestion of a peptide by a processive, non-specific, heat-loving Bacillus subtilis-derived aminopeptidase (BsuAP), which acts optimally at 70 °C and allows ‘single-shot’ sequencing to be carried out through simultaneous accumulation, and detection of sub-populations of peptides of progressively reducing length.     

Methionyl aminopeptidase from rat liver: distribution of the membrane-bound subcellular enzyme

The selective distribution of methionyl aminopeptidase (MAP) among rat liver mitochondria (heavy and light) and microsomes is reported. Several properties of MAP from the three subcellular fractions showed that the enzyme is a typical aminopeptidase able to remove N-terminal methionine from oligopeptides and methionyl-2-naphthylamide but not from Met-Ala-Ser. MAP is a membrane-bound enzyme sensitive to SH-group oxidants and inhibitable by L-methionine but not by usual arylaminopeptidase inhibitors. It is suggested that, MAP may play an important role during protein synthesis in rat liver.Abbreviations AANA Aminoacyl-2-Naphthylamides (MetNA, AlaNA, etc...) - AApNA Aminoacyl-pNitroanilides (MetpNA, AlapNA, etc...) - AANH₂ L-Aminoacylamides (MetNH₂, AlaNH₂, etc...) - APase Acid Phosphatase - BSA Bovine Serum Albumin - DEAF Diethylaminoethyl - EDTA Ethylenediaminetetraacetic Acid - GDH Glutamate Dehydrogenase - MLBK Methionyl-Lysyl-Bradykinin (Met-Lys-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg) - MAP Methionyl Aminopeptidase - pOHMB Sodium p-Hydroxymercuribenzoate - SDS Sodium Dodecyl Sulfate - SRA Specific Relative Activity     

Secretory vesicle aminopeptidase B related to neuropeptide processing: molecular identification and subcellular localization to enkephalin- and NPY-containing chromaffin granules

Biosynthesis of peptide hormones and neurotransmittters involves proteolysis of proprotein precursors by secretory vesicle cathepsin L. Cathepsin L generates peptide intermediates with basic residues at their NH(2)-termini, indicating that Arg/Lys aminopeptidase is needed to generate the smaller biologically active peptide. Therefore, this study identified the Arg/Lys aminopeptidase that is present in secretory vesicles of adrenal medulla and neuroendocrine tissues, achieved by molecular cloning and localization in 'model' neuropeptide-containing secretory vesicles (bovine). Molecular cloning of the bovine aminopeptidase B (AP-B) cDNA defined its primary sequence that allowed selection of antisera for immunolocalization studies. AP-B was present in secretory vesicles that contain cathepsin L with the neuropeptides enkephalin and neuropeptide Y. The AP-B in several neuroendocrine tissues was detected by western blots. Recombinant bovine AP-B showed preference for Arg-methylcoumarinamide substrate. AP-B was inhibited by arphamenine, an inhibitor of aminopeptidases. Bovine AP-B showed similar activities for Arg-(Met)enkephalin (ME) and Lys-ME neuropeptide substrates to generate ME, while rat AP-B preferred Arg-ME. Furthermore, AP-B possesses an acidic pH optimum of 5.5-6.5 that is similar to the internal pH of secretory vesicles. The significant finding of the secretory vesicle localization of AP-B with neuropeptides and cathepsin L suggests a role for this exopeptidase in the biosynthesis of neuropeptides.     

Evolutionary analysis of the mammalian M1 aminopeptidases reveals conserved exon structure and gene death

The members of the M1 aminopeptidase family share conserved domains, yet show functional divergence within the family as a whole. In order to better understand this family, this study analyzed the mammalian members in depth at exon, gene, and protein levels. The twelve human members, eleven rat members, and eleven mouse members were first analyzed in multiple alignments to visualize both reported and unreported conserved domains. Phylogenetic trees were then generated for humans, rats, mice, and all mammals to determine how closely related the homologs were and to gain insight to the divergence in the family members. This produced three groups with similarity within the family. Next, a synteny study was completed to determine the present locations of the genes and changes that had occurred. It became apparent that gene death likely resulted in the lack of one member in mouse and rat. Finally, an in-depth analysis of the exon structure revealed that nine members of the human family and eight in mouse, are highly conserved within the exon structure. Taken together, these results indicate that the M1 aminopeptidase family is a divergent family with three subgroups and that genetic evidence mirrors categorization of the family by enzymatic function.     

Immobilization in polyelectrolyte complex capsules: Encapsulation of a gluconate-oxidizing Serratia marcescens strain

In previous papers, it was shown that eukaryotic microbial systems can be encapsulated in polyelectrolyte complexes (PEC) prepared from sodium cellulose sulfate and poly(dimethyldiallylammonium chloride) with maintainance of vitality. In the present study, prokaryotic cells were successfully encapsulated in these PEC. Serratia marcescens B345 (IMET 11312) was chosen as a model organism. This strain converts gluconic acid to 2-ketogluconic acid. Since the 2-ketogluconic acid produced has very strong complexing properties, the number of applicable immobilization methods is restricted. Due to the high stability of PEC towards complexing agents, these problems can be overcome by the described method.

As already described in previous papers, a preimmobilization of cells in a PEC coprecipitate prior to capsule formation proved to be advantageous also for encapsulation of bacilli. The mean productivity of the encapsulated S. marcescens cells was 1–4.4 g l⁻¹ h⁻¹ in comparison to 5 g l⁻¹ h⁻¹ for free cells. The productivity was highly dependent on the flow rate of the reactor. The encapsulated cells were used for 1,200 h in a continuous biotransformation process for the production of 2-ketogluconic acid.     

l-leucine aminopeptidase production by filamentous Aspergillus fungi

AIMS: To screen various filamentous fungi belonging to Aspergillus spp. producing leucine and methionine aminopeptidases. METHODS AND RESULTS: Twenty-eight Aspergillus strains representing 14 species within the genus were screened for L-leucine aminopeptidase (LAP) production in two media in shake flask fermentation. Two Aspergillus sojae (NRRL 1988 and NRRL 6271) and one Aspergillus oryzae (NRRL 6270) strains were selected as the best producers for further studies. The peak LAP activities were 2.61, 2.59 and 1.30 IU ml(-1) for the three fungi on days 2, 5 and 4 respectively. In addition to LAP, L-methionine aminopeptidase (MAP) activity was also detected. Apart from submerged fermentation, the highest LAP yields by solid-state fermentation were 11.39, 17.40 and 13.02 IU g(-1) dry matter for the above fungi. The temperature and pH optimum of the enzyme was found to be in the range of 65-75 degrees C at pH 8.0-9.0 for all three fungi. Metal ions, Co(2+) and Fe(2+) in 2 mmol l(-1) concentration apparently enhanced the relative enzyme activity and heat stability. CONCLUSIONS: Two A. sojae (NRRL 1988 and NRRL 6271) and one A. oryzae (NRRL 6270) strains were found to be the best producers of LAP and MAP. The preliminary characterization studies revealed that the enzyme is considerably thermostable and belongs to the class metalloenzymes. SIGNIFICANCE AND IMPACT OF THE STUDY: A good number of aspergilli were screened and the ability of the fungal aminopeptidase to release a particular N-terminal amino acid along with its high thermal stability, makes them interesting for controlling the degree of hydrolysis and flavour development for a wide range of substrate.