CCK26–33 Degrading Activity in Brain and Nonneural Tissue: A Metalloendopeptidase

Cholecystokinin octapeptide (CCK26-33) is metabolized by neural membranes with an initial cleavage to CCK29-33 and subsequent breakdown to CCK31-33 and CCK32-33; this pattern of proteolysis occurs on incubation with either P2 or purified lysed synaptosomal membranes. To determine whether the pattern of CCK26-33 proteolysis is unique to the brain and whether regional brain differences in its pathway or rate exist, we analyzed the proteolysis of CCK by synaptic membranes of various brain areas and cellular membranes of peripheral tissue. The pattern of degradation in brain did not differ among the regions studied. The overall proteolysis rate, as measured by the formation of tryptophan, was higher in the striatum than in the cortex, although CCK29-33 was formed at the same rate in both areas. In nonneural tissue, the rate of degradation was highest in liver membranes and lowest in pancreatic acinar cell preparations. Thus, it appears that degradative peptidases are not necessarily colocalized with CCK receptors. The pattern of product formation is the same in peripheral compared with CNS membranes; thus, the degradative pathway does not appear to be unique to brain tissue. The enzyme present in synaptic membranes that is responsible for CCK29-33 formation requires a metal ion and sulfydryl groups for the catalysis and thus is a metalloendopeptidase. Furthermore, its activity is inhibited by Ac-Gly-Phe-Nle-al, a peptide aldehyde whose sequence bears some homology to the amino acid sequence in the region of CCK26-33 that is cleaved by this enzyme.     

Hydrolysis of the Brain Dipeptide N-Acetyl-l-Aspartyl-l-Glutamate: Subcellular and Regional Distribution, Ontogeny, and the Effect of Lesions on N-Acetylated-α-Linked Acidic Dipeptidase Activity

N-Acetylated-alpha-linked acidic dipeptidase (NAALADase) is a Cl- dependent, membrane bound, metallopeptidase that cleaves the endogenous neuropeptide N-acetyl-L-aspartyl-L-glutamate (NAAG) in vitro. To examine the pattern of NAALADase expression in the CNS, subcellular, regional, and developmental studies were conducted. Subcellular fractionation of lysed synaptosomal membranes revealed a substantial enrichment of the peptidase in synaptic plasma membranes as compared to mitochondrial or myelin subfractions. Regional studies reveal an apparent restriction of peptidase activity to kidney and brain. A threefold variation in specific activity was observed among brain regions, with highest specific activity in the cerebellum and lowest in telencephalic structures, a pattern that does not, in general, correlate with NAAG levels. Ontogenetic studies demonstrate a region-dependent, postnatal pattern of expression of NAALADase activity, with adult levels attained earliest in brainstem, as was previously reported for NAAG. Postnatal NAALADase expression would not appear to support a role for the peptidase in constitutive protein processing, but rather suggests that NAALADase may play a role in synaptic peptide degradation. Glutamate (Glu) excised from NAAG by NAALADase could be transported efficiently by uptake processes. Lesion studies, however, do not support a close structural association between NAALADase activity and the corticostriatal sodium-dependent, high-affinity, Glu uptake system. Similar to in vitro data documenting the route of NAAG degradation by NAALADase, after intrastriatal injection, NAAG was rapidly cleaved to two major products, N-acetyl-aspartate and Glu, with a t1/2 of approximately 10 min. Thus, the route of in vivo catabolism of NAAG parallels results from studies on NAALADase activity in vitro. These results are consistent with a role of NAALADase in the synaptic processing of NAAG. However, certain discrepancies in the regional and ontogenetic profiles of NAAG and NAALADase suggest that this relationship is not an exclusive one and may reflect a role for NAALADase on additional N-acetylated acidic peptides in vivo.     

Specificity of Neurotensin Metabolism by Regional Rat Brain Slices

Regional differences in neurotensin metabolism and the peptidases involved were studied using intact, viable rat brain microslices and specific peptidase inhibitors. Regional brain slices (2 mm x 230 microns) prepared from nucleus accumbens, caudate-putamen, and hippocampus were incubated for 2 h in the absence and presence of phosphoramidon, captopril, N-[1(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Phe-p-aminobenzoate, and o-Phenanthroline, which are inhibitors of neutral endopeptidase 24.11, angiotensin-converting enzyme, metalloendopeptidase 24.15, and nonspecific metallopeptidases, respectively. Neurotensin-degrading proteolytic activity varied by brain region. Significantly less (35.0 +/- 1.6%) neurotensin was lost from hippocampus than from caudate-putamen (45.4 +/- 1.0%) or nucleus accumbens (47.8 +/- 1.1%) in the absence of inhibitors. Peptidases responsible for neurotensin metabolism on brain slices were found to be predominantly metallopeptidases. Metalloendopeptidase 24.15 is of major importance in neurotensin metabolism in each brain region studied. The relative contribution of specific peptidases to neurotensin metabolism also varied by brain region; angiotensin-converting enzyme and neutral endopeptidase 24.11 activities were markedly elevated in the caudate-putamen as compared with the nucleus accumbens or hippocampus. Interregional variation in the activity of specific peptidases leads to altered neurotensin fragment formation. The brain microslice technique makes feasible regional peptide metabolism studies in the CNS, which are impractical with synaptosomes, and provides evidence for regional specificity of neurotensin degradation.     

Partial purification and characterization of glycylprolyl dipeptidyl aminopeptidase in porcine pancreas

This study reports the presence of glycylprolyl dipeptidyl aminopeptidase in porcine pancreas, and its partial purification and some properties. Crude enzyme preparation was obtained by extraction from acetone-dried powder of the pancreas at pH 7.6. For solubilization of enzyme, freezing and thawing were carried out. Crude enzyme extract was fractionated with ammonium sulfate precipitation, gel filtration on Sephadex G-200 column and ion-exchange chromatography on DEAE-cellulose. Partially purified enzyme showed 2897-folds purification. The enzyme activity on polyacrylamide gel electrophoresis showed good agreement with a main protein band stained with Coomassie brilliant blue. Molecular weight of this enzyme from the pancreas was estimated to be 300 000 by gel filtration on Sephacryl S-300 column. Optimum pH was between 8.5 and 9.0, and K_m value for glycylproline-p-nitroanilide tosilate was 0.33 mM. This enzyme from the pancreas was a serine enzyme and was relatively stable to heat at 60°C for 10 min.     

Activation and Inhibition of Cerebral Prolidase

Purified of prolidase from calf brain (acetone and [NH4]2SO4 fractionation) separated this enzyme from proteases, leucine aminopeptidase, master dipeptidase, and Gly-Gly dipeptidase. Prolidase was tested with peptidase and protease inhibitors, used at higher levels (35 times or more) than their ID50 for peptidases and proteases. Bacitracin, leupeptin, chymostatin, and antipain had no effect; pepstatin slightly increased activity, and only bestatin was inhibitory. Antibiotics that affect protein synthesis did not inhibit prolidase. Peptides with proline at the NH2 end activated prolidase, whereas those with proline at the carboxyl end inhibited it. Di, tri, and tetra-Pro peptides increased prolidase activity. Thyrotropin-releasing hormone had no effect on prolidase; its analog Pro-His-Pro-NH2 gave high activation and decreased the Km from 20 mM to 1.54 mM. Pro-peptide inhibitors and activators were not themselves split by prolidase. The results indicate influences of specific peptides, for both inhibition and activation, on prolidase activity.     

Determination of the Solution Conformation of HIV-1 Tat(1–9) Peptides by Means of Molecular Dynamics Simulations Considering NMR Data and Docking Studies into an Active Site Model of DP IV

The human immunodeficiency virus 1 Tat protein suppresses antigen-, anti-CD3-and mitogen-induced activation of human T cells when added to T cell cultures. This activity is important for the development of AIDS because lymphocytes from HIV-infected individuals exhibit a similar antigen-specific dysfunction. Moreover, Tat was found to interact with dipeptidyl peptidase IV (DP IV). To find out the amino acid sequence important for the inhibition of the DP IV enzymatic activity we investigated N-terminal Tat(1–9) peptide analogues with amino acid substitutions in different positions. Interestingly, the exchange of Pro⁶ with Leu and Asp⁵ with Ile strongly diminished the DP IV inhibition by Tat(1–9). Based on data derived from one-and two-dimensional ¹H NMR investigations the solution conformations of the three nonapeptides in water were determined by means of molecular dynamics simulations. These conformations were used for studies of the docking behavior of the peptides into a model of the active site of DP IV. The results suggest that several attractive interactions between the native Tat(1–9) and DP IV lead to a stable complex and that the reduced affinity of both L⁶-Tat(1–9) and I⁵-Tat(1–9) derivatives might be caused by conformational alterations in comparison to the parent peptide.Supplementary material to this paper is available in electronic form at http://dx.doi.org/10.1007/s0089480040200     

The MEROPS batch BLAST: a tool to detect peptidases and their non-peptidase homologues in a genome

Many of the 181 families of peptidases contain homologues that are known to have functions other than peptide bond hydrolysis. Distinguishing an active peptidase from a homologue that is not a peptidase requires specialist knowledge of the important active site residues, because replacement or lack of one of these catalytic residues is an important clue that the homologue in question is unlikely to hydrolyse peptide bonds. Now that the rate at which proteins are characterized is outstripped by the rate that genome sequences are determined, many genes are being incorrectly annotated because only sequence similarity is taken into consideration. We present a tool called the MEROPS batch BLAST which not only performs a comparison against the MEROPS sequence collection, but also does a pair-wise alignment with the closest homologue detected and calculates the position of the active site residues. A non-peptidase homologue can be distinguished by the absence or unacceptable replacement of any of these residues. An analysis of peptidase homologues in the genome of the bacterium Erythrobacter litoralis is presented as an example.     

Oligopeptidase B: a processing peptidase involved in pathogenesis

Oligopeptidase B is a "processing peptidase" from the prolyl oligopeptidase family of serine peptidases present in Gram negative bacteria, protozoa and plants. Unlike the prototype prolyl oligopeptidase, oligopeptidase B hydrolyses peptides on the carboxyl side of pairs of basic amino acid residues. Molecular modelling and mutation studies have identified carboxyl dyads in the C-terminal catalytic domain that mediate substrate and inhibitor binding. The peptidase is efficiently inhibited by non-peptide irreversible serine peptidase inhibitors, peptidyl-chloromethylketones, -phosphonate alpha-aminoalkyl diphenyl esters with basic residues at P1, and tripeptide aldehydes, but not by proteinaceous host plasma inhibitors such as alpha2-macroglobulin and serpins. Access of these large molecular mass inhibitors and substrates larger than approximately 30 amino acid residues to the catalytic cleft is restricted by the N-terminal beta-propeller domain. The physiological role of oligopeptidase B from various sources has not yet been elucidated. However, the peptidase has been identified as an important virulence factor and therapeutic agent in animal trypanosomosis. This review highlights the structure-function properties of oligopeptidase B in context with its physiological and/or pathological roles which make the enzyme a promising drug target.     

The use of proteolysis to study the structure of nardilysin

Treatment of a 128 kDa mouse nardilysin with trypsin initially produced an active 105 kDa N-terminally cleaved form. Continued trypsin digestion occurred at the C-terminus, producing inactive core species of approximately 92, 76.5, and 62 kDa. Protease V8 digestion generated a stable approximately 105 kDa form, nardilysin(V8), that was cleaved near the N-terminal trypsin site. The approximately 105 kDa nardilysin(V8) exhibited the same K(m) as did the uncleaved enzyme for substrates of the type Abz-GGFX(1)X(2)X(3)VGQ-EDDnp, where X residues were varied. However, k(cat) for nardilysin(V8) was 5-6 times greater. Both uncleaved nardilysin and nardilysin(V8) are inhibited by NaCl; however, nardilysin(V8) exhibits an IC(50) of approximately 2 mM compared to an IC(50) of approximately 50 mM for uncleaved nardilysin. Nardilysin(V8) is more sensitive to inhibition by phosphate buffer. Treatment of nardilysin(V8) with trypsin generated primarily the 92 kDa form which was inactive. Attempts to express nardilysin as a 105 kDa truncated N-terminal form or as a C-terminally truncated form led to inactive proteins.