High-level expression and characterization of the thermostable leucine aminopeptidase Thelap from the thermophilic fungus Thermomyces lanuginosus in Aspergillus niger and its application in soy protein hydrolysis.

Leucine aminopeptidase (LAP), an exopeptidase that releases amino acid residues, especially leucine, from the N-terminus of polypeptides, is often applied to debitter protein hydrolysate in the food industry. However, there are no thermostable and high activity enzymes that can be used in the food industry. In this study, we obtained the highly active and thermostable leucine aminopeptidases screened from the thermophilic fungi Thermomyces lanuginosus, Talaromyces thermophilus, and Malbranchea cinnamomea. The activity of the recombinant leucine aminopeptidase Thelap was significantly increased to 2771.5 U/mL, as mediated by the CRISPR/Cas9 tool. The recombinant Thelap was easily purified from fermentation broth by Ni-affinity chromatography, and the specific activity of the purified Thelap was increased to 7449.6 U/mg. The recombinant Thelap showed optimal activity at pH 8.5 and 75 °C and remained above 70% of the maximum activity over a wide temperature range (30-80 °C). With regard to temperature stability, Thelap retained more than 90% activity when it was incubated at 65-75 °C for 2 h. K+ and Co2+ increased the enzyme activity of the recombinant Thelap, while Ba2+, Mn2+, Ni2+, Ca2+, Mg2+ and SDS inhibited its enzyme activity, and the inhibition capacity of Mg2+ was the weakest. Upon application in soy protein hydrolysis, Thelap could significantly increase the degree of hydrolysis and remove more hydrophobic amino acids from the N-terminal region of the polypeptide to decrease the bitterness.

High-Level Expression in Escherichia coli, Purification and Kinetic Characterization of LAPTc, a Trypanosoma cruzi M17-Aminopeptidase.

The M17 leucyl-aminopeptidase of Trypanosoma cruzi (LAPTc) is a novel drug target for Chagas disease. The objective of this work was to obtain recombinant LAPTc (rLAPTc) in Escherichia coli. A LAPTc gene was designed, optimized for its expression in E. coli, synthesized and cloned into the vector pET-19b. Production of rLAPTc in E. coli BL21(DE3)pLysS, induced for 20 h at 25 °C with 1 mM IPTG, yielded soluble rLAPTC that was catalytically active. The rLAPTc enzyme was purified in a single step by IMAC. The recombinant protein was obtained with a purity of 90% and a volumetric yield of 90 mg per liter of culture. The enzymatic activity has an optimal pH of 9.0, and preference for Leu-p-nitroanilide (appKM = 74 µM, appkcat = 4.4 s-1). The optimal temperature is 50 °C, and the cations Mg2+, Cd2+, Ba2+, Ca2+ and Zn2+ at 4 mM inhibited the activity by 60% or more, while Mn2+ inhibited by only 15% and addition of Co2+ activated by 40%. The recombinant enzyme is insensitive toward the protease inhibitors PMSF, TLCK, E-64 and pepstatin A, but is inhibited by EDTA and bestatin. Bestatin is a non-competitive inhibitor of the enzyme with a Ki value of 881 nM. The enzyme is a good target for inhibitor identification.

A new method to evaluate the enzyme-suppressing activity of a leucine aminopeptidase 3 inhibitor.

The expression of leucine aminopeptidase 3 (LAP3) is associated with the prognosis for and malignant transformation of many types of tumors. Therefore, a LAP3 inhibitor may represent a new strategy for cancer therapy. Evaluating the suppression of enzyme activity by an LAP3 inhibitor is essential. Right now, leucine aminopeptidases (LAPs) purified from the porcine kidneys are the only enzymes that can be used to evaluate the suppression of enzyme activity by an LAP3 inhibitor. This approach cannot accurately reflect the suppression of human LAP3 by an inhibitor. The current study developed a new method with which to evaluate the suppression of enzyme activity by an LAP3 inhibitor. Total protein from K562 cells seldom catalyzed the LAP3 substrate. A lentivirus was used to induce K562 cells to overexpress LAP3 (K562-LAP3). After puromycin screening, flow cytometry data indicated that 98.8% of cells expressed green fluorescent protein. The expression of LAP3 in K562-LAP3 cells was also assessed using Western blotting. K562-LAP3 cells were lysed with ultrasonication. Total protein was used as an enzyme source and L-leucine p-nitroaniline hydrochloride was used as a substrate to measure enzyme activity. Total protein from K562-LAP3 cells catalyzed the substrate more than that from K562 cells did. The LAP3 inhibitor ubenimex was used as a positive control to evaluate the suppression of LAP3 enzyme activity. Results indicated that ubenimex significantly inhibited the enzyme activity of LAP3. This approach provides a convenient and accurate way to evaluate the suppression of enzyme activity by an LAP3 inhibitor.

Expression, purification and characterization of two leucine aminopeptidases of the blood fluke, Schistosoma mansoni.

Schistosomiasis is a major neglected tropical disease (NTD) and considered the most important of the human helminthiases in terms of morbidity and mortality. Whereas treatment with praziquantel has been effective since the 1980s, the potential for the emergence of drug resistance has propelled the search for new interventions. Studies have revealed key roles of proteases in parasitic helminths during establishment of infection, tissue invasion, immune evasion, parasite feeding and development throughout the different developmental stages, pinpointing them as possible candidates. The leucine aminopeptidases (LAPs), members of the M17 family of Zn-metalloproteases, preferentially cleave leucine (Leu) residues at the N-terminal end of proteins and short peptides. These enzymes display broad proteolytic activities beyond Leu hydrolysis and are involved in processing, maturation, activation and/or degradation of substrates. As a vaccine immunogen, LAP induces protection against infection with the liver fluke Fasciola hepatica. Herein, two LAPs, SmLAP1 (Smp_030000) and SmLAP2 (Smp_083870) of the human blood fluke Schistosoma mansoni were cloned, expressed, purified and biochemically characterized. The enzymes differed in activity against diagnostic substrates, including leucine, methionine and arginine, with an optimal pH of 8.0. The activity increased in the presence of Mg+2 and Mn+2, and was inhibited by bestatin, a specific inhibitor of aminopeptidase. In addition, 1,10-phenanthroline and EDTA inhibited the enzymatic activity of SmLAP2. Finally, immunolocalization using antibodies specific for SmLAP1 and SmLAP2 identified the expression of these proteases in the egg and adult developmental stages of S. mansoni, and in intestinal epithelia, vitelline cells and sub-tegumental regions of the parasite. Characterization of schistosome proteases not only enhances understanding of the biology of schistosomes and schistosomiasis, but may also provide novel intervention approaches.

Characterization of Aspergillus sojae Isolated from Meju, Korean Traditional Fermented Soybean Brick.

Initially, we screened 18 Aspergillus sojae-like strains from Aspergillus spp. isolated from meju (Korean traditional fermented soybean brick) according to their morphological characteristics. Because members of Aspergillus section Flavi are often incorrectly identified because of their phylogenetic similarity, we re-identified these strains at the morphological and molecular genetic levels. Fourteen strains were finally identified as A. sojae. The isolates produced protease and α-amylase with ranges of 2.66-10.64 and 21.53-106.73 unit/g-initial dry substrate (U/g-IDS), respectively, which were equivalent to those of the koji (starter mold) strains employed to produce Japanese soy sauce. Among the isolates and Japanese koji strains, strains SMF 127 and SMF 131 had the highest leucine aminopeptidase (LAP) activities at 6.00 and 6.06 U/g-IDS, respectively. LAP plays an important role in flavor development because of the production of low-molecular-weight peptides that affect the taste and decrease bitterness. SMF 127 and SMF 131 appeared to be non-aflatoxigenic because of a termination point mutation in aflR and the lack of the polyketide synthase gene found in other A. sojae strains. In addition, SMF 127 and SMF 131 were not cyclopiazonic acid (CPA) producers because of the deletion of maoA, dmaT, and pks/nrps, which are involved in CPA biosynthesis. Therefore, A. sojae strains such as SMF 127 and SMF 131, which have high protease and LAP activities and are free of safety issues, can be considered good starters for soybean fermentations, such as in the production of the Korean fermented soybean products meju, doenjang, and ganjang.

Expression of leucine aminopeptidase 3 (LAP3) correlates with prognosis and malignant development of human hepatocellular carcinoma (HCC).

Leucine aminopeptidases (LAPs) were associated with tumor cell proliferation, invasion and/or angiogenesis. LAP3 is one important member of this family. However, its clinical significance and biological function in hepatocellular carcinoma (HCC) remains unknown. In the present study, we demonstrated that LAP3 expression was significantly up-regulated in HCC tissues as well as cells and was closely correlated with lower differentiation, positive lymph node metastasis and high Ki-67 expression, indicating a poor prognosis. Then cell viability assays, flow cytometry assays, wound-healing assays and matrigel invasion assays were performed to demonstrate that LAP3 promoted HCC cells proliferation by regulating G1/S checkpoint in cell cycle and advanced HCC cells migration. Furthermore, we discovered that knockdown LAP3 will enhance the sensitivity of HCC cells to cisplatin, thus promoting the cell death of HCC cells. Collectively, our results indicated that up-regulated expression of LAP3 might contribute to the proliferation and metastasis of HCC. Our data gains greater insight into the cancer-promoting role of LAP3 and its functions in HCC cells, possibly providing potential therapeutic strategies for clinical trials.

Molecular identification and characterization of leucine aminopeptidase 2, an excretory-secretory product of Clonorchis sinensis.

Aminopeptidases serve vital roles in metabolism of hormones, neurotransmission, turnover of proteins and immunological regulations. Leucine aminopeptidases catalyze the hydrolysis of amino-acid residues from the N-terminus of proteins and peptides. In the present study, leucine aminopeptidase 2 (LAP2) gene of Clonorchis sinensis (C. sinensis) was isolated and identified from an adult cDNA library of C. sinensis. Recombinant CsLAP2 was expressed and purified in Escherichia coli BL21. The open reading frame of LAP2 contains 1,560 bp equivalent to 519 amino acids, a similarity analysis showed a relatively low homology with Homo sapiens (19.0 %), Trypanosoma cruzi (18.0 %), Mus musculus (19.3 %), and relatively high homology with Schistosoma mansoni (65.6 %). The optimum condition of rCsLAP2 enzyme activity was investigated using a fluorescent substrate of Leu-MCA at 37 °C and pH 7.5. The K (m) and V (max) values of rCsLAP2 were 18.2 µM and 10.7 µM/min, respectively. CsLAP2 gene expression can be detected at the stages of the adult worm, metacercaria, excysted metacercaria and egg of C. sinensis using real-time PCR, no difference was observed at the stages of the adult worm, metacercaria and egg. However, CsLAP2 showed a higher expression level at the stage of excysted metacercaria than the adult worm (3.90-fold), metacercaria (4.60-fold) and egg (4.59-fold). Histochemistry analysis showed that CsLAP2 was located at the tegument and excretory vesicle of metacercaria, and the tegument and intestine of adult worm. The immune response specific to rCsLAP2 was characterized by a mixed response patterns of Th1 and Th2, indicating a compounded humoral and cellular immune response. The combined results from the present study indicate that CsLAP2 was an important antigen exposed to host immune system, and probably implicated as potential role in interaction with host cells in clonorchiasis.

Production of leucine amino peptidase in lab scale bioreactors using Streptomyces gedanensis.

Studies were conducted on the production of leucine amino peptidase (LAP) by Streptomyces gedanensis to ascertain the performance of the process in shake flask, parallel fermenter and 5-L fermenter utilizing soy bean meal as the carbon source. Experiments were conducted to analyze the effects of aeration and agitation rate on cell growth and LAP production. The results unveiled that an agitation rate of 300 rpm, 50% dissolved oxygen (DO) upholding and 0.15 vvm strategies were the optimal for the enzyme production, yielding 22.72 ± 0.11 IU/mL LAP in parallel fermenter which was comparable to flask level (24.65 ± 0.12 IU/mL LAP) fermentation. Further scale-up, in 5-L fermenter showed 50% DO and 1 vvm aeration rate was the best, producing optimum and the production was 20.09 ± 0.06 IU/mL LAP. The information obtained could be useful to design a strategy to improve a large-scale bioreactor cultivation of cells and production of LAP.

Leucine aminopeptidase in the ixodid tick Haemaphysalis longicornis: endogenous expression profiles in midgut.

We previously identified a cDNA from the ixodid tick Haemaphysalis longicornis that encodes leucine aminopeptidase, HlLAP. Functionally, recombinant HlLAP effectively hydrolyzed synthetic amino acid derivatives. Here, we investigated the temporal expression profiles of midgut HlLAP in adult H. longicornis parthenogenetic ticks from the starting of blood feeding until just before the onset of oviposition. Midgut HlLAP transcript expression level was higher during post-engorgement period than that during feeding period. Endogenous HlLAP in the midgut was also observed with higher expression level during post-engorgement period. Histological localization of HlLAP was in the cytosol of midgut epithelial cells, notably the newly differentiated basophilic cells at post-engorgement. Our data suggested that HlLAP was dominantly localized in basophilic cells, where it may play regulatory roles in protein biosynthesis and degradation.

Production of L-leucine aminopeptidase by selected Streptomyces isolates.

AIMS: To screen various Streptomyces cultures producing L-leucine aminopeptidase (LAP). METHODS AND RESULTS: Twenty-one Streptomyces strains were screened for LAP production. The best three producers were found to be Streptomyces mobaraensis NRRL B-3729, Streptomyces gedanensis IFO 13427, and Streptomyces platensis NRRL 2364. pH optima of the three enzymes were in the range of 8.0-8.5 and the temperature optima varied between 50 and 65 degrees C. LAP of S. mobaraensis was stable at 60 degrees C and pH 8.5 for 60 min. Metal ion salts, CoCl(2).6H(2)O and ZnSO(4).7H(2)O in 0.7 mmol l(-1) concentration enhanced the relative enzyme activity in all three enzymes. Molecular mass of LAP of S. mobaraensis was found to be approx. 37 kDa. CONCLUSIONS: Streptomyces mobaraensis NRRL B-3729, S. gedanensis IFO 13427, and S. platensis NRRL 2364 were found to be good producers of extracellular LAP. The approx. 37 kDa enzyme of S. mobaraensis is considerably thermostable. SIGNIFICANCE AND IMPACT OF THE STUDY: A good number of Streptomyces were screened and the ability of the aminopeptidases to release a particular N-terminal amino acid along with its good thermal stability makes them interesting for controlling the degree of hydrolysis and flavour development for a wide range of substrate.

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.

Protein, leucine aminopeptidase, esterase, acid phosphatase and photosynthetic responses of oleander (Nerium oleander L.) during cold acclimation and freezing treatments.

Six-month-old oleander (Nerium oleander L.) pot plants, derived from vegetative propagation by cuttings, were tested for their ability to cold hardening. Damage of the non-acclimated (NA) plants was visible when treated by low freezing temperatures (below -2 degrees C). The responses of total proteins, leucine aminopeptidase (LAP), esterase (EST) and acid phosphatase (ACP) isoforms of NA and cold-acclimated (CA; 4 degrees C for 14 days) plants were compared using polyacrylamide gel electrophoresis. These molecular markers were also compared in NA and CA plants which received for 2h temperatures of 0, -2, -4, -6 and -8 degrees C. A new 38-kDa polypeptide appeared from day 7 to 14 during the acclimation treatment in the bark extracts and on day 14 in the leaf extracts. The above-mentioned polypeptide band (38 kDa) strongly appeared in all freezing treatments (0, -2, -4, -6 and -8 degrees C) in both bark and leaf extracts of the CA plants. Alterations in the number and the intensity of LAP and EST isoforms as well as in the intensity of ACP isoforms were observed in both bark and leaf of the CA oleander plants. A newly expressed EST isoform is proposed as biochemical marker for the cold acclimation treatment. CO2 assimilation rates (A) as well as transpiration rates (E) in NA plants were positive in 0 degrees C and negative in all temperatures below zero in the freezing treatments. In contrast, CO2 assimilation rates (A) and transpiration rates (E) were positive in CA plants in all temperatures of freezing treatment. A significant decrease (P<0.05) in chlorophyll (Chl) a, Chl a+b concentration and Chl a/b ratio were noticed in oleander plants during the acclimation treatment (from day 0 to 14), while Chl b concentration was unchanged at the respective time. On the other hand, no significant (P<0.05) differences were observed in the freezing treatments.

Construction and one-step purification of Bacillus kaustophilus leucine aminopeptidase fused to the starch-binding domain of Bacillus sp. strain TS-23 alpha-amylase.

The starch-binding domain of Bacillus sp. strain TS-23 alpha-amylase was introduced into the C-terminal end of Bacillus kaustophilus leucine aminopeptidase (BkLAP) to generate a chimeric enzyme (BkLAPsbd) with raw-starch-binding activity. BkLAPsbd, with an apparent molecular mass of approximately 65 kDa, was overexpressed in Escherichia coli M15 cells and purified to homogeneity by nickel-chelate chromatography. Native PAGE and chromatographic analyses revealed that the purified fusion protein has a hexameric structure. The half-life for BkLAPsbd was 12 min at 70 degrees C, while less than 20% of wild-type enzyme activity retained at the same heating condition. Compared with the wild-type enzyme, the 60% decrease in the catalytic efficiency of BkLAPsbd was due to a 91% increase in K (m) value. Starch-binding assays showed that the K (d) and B (max) values for the fusion enzyme were 2.3 microM and 0.35 micromol/g, respectively. The adsorption of the crude BkLAPsbd onto raw starch was affected by starch concentration, pH, and temperature. The adsorbed enzyme could be eluted from the adsorbent by 2% soluble starch in 20 mM Tris-HCl buffer (pH 8.0). About 49% of BkLAPsbd in the crude extract was recovered through one adsorption-elution cycle with a purification of 11.4-fold.

Possible involvement of adipocyte-derived leucine aminopeptidase via angiotensin II in endometrial carcinoma.

OBJECTIVE: It has recently been appreciated that a local autocrine or paracrine renin-angiotensin system (RAS) may exist in a number of tissues. Angiotensin II (AngII) is a potent RAS-derived vasoconstrictor peptide, and it is involved in tumor angiogenesis. We have cloned human adipocyte-derived leucine aminopeptidase (A-LAP), which degrades Ang II. This study investigated whether the expression of A-LAP, Ang II, angiotensin type I receptor (AT1R) and vascular endothelial growth factor (VEGF) correlates with clinicopathologic factors and prognosis in patients with endometrial endometrioid adenocarcinoma. METHODS: Histologic sections of formalin-fixed, paraffin-embedded specimens from 94 primary endometrial carcinomas were stained for A-LAP, AngII, AT1R and VEGF using each antibody. Disease-free survival (DFS) and other clinicopathologic characteristics were analyzed according to the intensity of each staining. RESULTS: Of 94 cases, 91 (96.8%) showed specific A-LAP immunostaining. A-LAP expression demonstrated negative correlations with myometrial invasion (p = 0.01) and vascular infiltration (p = 0.01). Of 94 cases, 77 (81.9%) showed specific AngII immunostaining. We found a positive correlation between AngII expression and surgical stage (p = 0.01). Of 94 cases, 56 (59.6%) showed specific AT1R immunostaining and 73 (77.7%) specific VEGF immunostaining. We found a positive correlation between VEGF expression and lymph node metastasis (p = 0.05). AngII and AT1R expression predicted a significantly poorer prognosis. Contrarily, A-LAP expression indicated a significantly more favorable prognosis in endometrial endometrioid adenocarcinoma patients. Multivariate analysis demonstrated that A-LAP expression (odds ratio, 0.12; 95% confidence interval, 0.025-0.618; p = 0.01) was an independent prognostic factor. CONCLUSIONS: In this study, we demonstrated the existence of local RAS and A-LAP in endometrial endometrioid adenocarcinoma as prognostic predictors of clinical outcome. These findings suggest that the assessment of RAS and A-LAP status provides clinically useful prognostic information in patients with endometrial carcinoma.

Model building and quantitative analysis of a tandem immuno-capturing assay as a screening tool for breast cancer.

The onset of breast cancer appears to occur, on average, a decade earlier in Mexican women in comparison to American or European women. Early detection and prevention of breast cancer are of crucial importance to increase survival and improve quality of life. Based on the molecular elucidation of critical events leading to breast carcinogenesis, a tandem immuno-capturing blood test was developed as a quantitative population screening assay in view of providing a cost-effective and non-invasive alternative to population screening. Clinical analysis of 63 Mexican women within an age group of 35-70, revealed that Interstron activity increases from 800+/-65 IUJPA (Interstron Units) in the asymptomatic normal women to 994+/-100 IUJPA in the symptomatic/benign group, reaching 1289+/-81 IUJPA in the cancerous group. Accordingly, activity thresholds were established at 800 and 1200 IUJPA respectively, encompassing three risk groups: (i) Healthy Otherwise Normal (<800 IUJPA); (ii) Grey Risk Area (>800 and <1200 IUJPA), and (iii) At Risk group (>1200 IUJPA). Taking into account both baseline and clinical case reports, the Healthy Otherwise Normal group and the At Risk group were mostly homogeneous in nature, comprising a population of normal and cancer patients respectively. The Grey Risk group is heterogeneous, likely reflecting a transitional nature towards a potential early stage of breast disease development. Based on these results, a screening algorithm was developed as the underlining principle for population surveillance encompassing over 30,000 Mexican women. The current screening results have enabled us to objectively prioritize medical attention to approximately 1 in 8 women out of the general population mapped within the At Risk group. Overall, our findings suggest that monitoring Interstron activity units provides a valuable quantitative screening analysis as to selectively streamline the population of women in need of early medical counseling and/or mammography, thereby enhancing both the quality and cost-effectiveness of preventative population surveillance programs targeting breast cancer.

Transcriptional regulation of angiogenesis-related puromycin-insensitive leucyl-specific aminopeptidase in endothelial cells.

Puromycin-insensitive leucyl-specific aminopeptidase (PILSAP) was expressed in endothelial cells (ECs) and played an important role in angiogenesis. Here, we characterized its transcriptional regulation. Mouse PILSAP gene contained 19 exons and located in the chromosome 13C1-C2. We identified two transcripts; one transcribed from exon 1 and the other from exon 2. Mouse ECs expressed dominantly the one from exon 1. The promoter analysis using 5' upstream region of exon 1 revealed that -1868 to -1812 was critical for its transcription in mouse ECs. We identified a motif of the transcription factor PEBP2 in this region, and the deletion or mutation of this motif decreased promoter activity. Protein extracted from mouse ECs bound specifically to this motif. AML1/Runx1/PEBP2alphaB increased PILSAP mRNA in mouse ECs, whereas dominant interfering chimerical PEBP2beta-MYH11 decreased it. These results indicate that the expression of PILSAP in mouse ECs is regulated, at least in part, by PEBP2.

High cell density cultivation of Brevibacterium linens and formation of proteinases and lipase.

Brevibacterium linens forms hydrolytic enzymes which can be used to accelerate the ripening of cheese without causing bitterness. B. linens ATCC 9172 was grown to a high cell density (50 g dry wt l-1 after 60 h) in a mineral medium containing lactic acid, soy-peptone and ammonium sulphate by applying a continuous feed of nutrients. The maximal activities of L-leucine aminopeptidase and cell-associated proteinase were 286 U l-1 and 202 U l-1, respectively. The cell-associated lipolytic activity exhibited a strong and sudden increase at 46 h, resulting in a maximum of 9.5 U g-1 dry wt; thus the volumetric productivity of proteolytic and lipolytic activity was 4220 U l-1 h-1 and 7.3 U l-1 h-1, respectively.

Possible involvement of placental peptidases that degrade gonadotropin-releasing hormone (GnRH) in the dynamic pattern of placental hCG secretion via GnRH degradation.

The presence of an extrahypothalamic gonadotropin releasing hormone (GnRH) in human placenta is well known and this decapeptide is presumed to play an important role in the regulation of the function and growth of human placenta. Immunohistochemistry showed that neutral endopeptidase 24.11 (NEP), a candidate of the responsible enzyme of GnRH degradation, is highly expressed on the cell surface of trophoblasts. Hydrolysis of GnRH by human villi was studied by measuring liberated amino acids using high performance liquid chromatography. The GnRH degrading activity was 1.53 times higher after incubation with the membrane fraction of first trimester villi than that after incubation with the membrane fraction of term villi. Phosphoramidon, a potent inhibitor of NEP, reduced the liberated amino acids to about a half, suggesting that NEP is a responsible enzyme for GnRH degradation. Ubenimex, which can inhibit several aminopeptidases, also reduced the liberated amino acids to about 50 per cent. O-phenanthroline, EDTA, and thiorphan could inhibit GnRH degradation but inhibitors of post proline endopeptidase could not. Furthermore, GnRH degrading activity of the membrane fraction was reduced remarkably after the membrane fraction was immunotitrated by anti NEP and anti placental leucine aminopeptidase (P-LAP) IgG. In conclusion, NEP and P-LAP are responsible enzymes for GnRH degradation in human villi.

Identification of residues critical for activity of the wound-induced leucine aminopeptidase (LAP-A) of tomato.

The importance of two putative Zn2+-binding (Asp347, Glu429) and two catalytic (Arg431, Lys354) residues in the tomato leucine aminopeptidase (LAP-A) function was tested. The impact of substitutions at these positions, corresponding to the bovine LAP residues Asp255, Glu334, Arg336, and Lys262, was evaluated in His6-LAP-A fusion proteins expressed in Escherichia coli. Sixty-five percent of the mutant His6-LAP-A proteins were unstable or had complete or partial defects in hexamer assembly or stability. The activity of hexameric His6-LAP-As on Xaa-Leu and Leu-Xaa dipeptides was tested. Most substitutions of Lys354 (a catalytic residue) resulted in His6-LAP-As that cleaved dipeptides at slower rates. The Glu429 mutants (a Zn2+-binding residue) had more diverse phenotypes. Some mutations abolished activity and others retained partial or complete activity. The E429D His6-LAP-A enzyme had Km and kcat values similar to the wild-type His6-LAP-A. One catalytic (Arg431) and one Zn-binding (Asp347) residue were essential for His6-LAP-A activity, as most R431 and D347 mutant His6-LAP-As did not hydrolyze dipeptides. The R431K His6-LAP-A that retained the positive charge had partial activity as reflected in the 4.8-fold decrease in kcat. Surprisingly, while the D347E mutant (that retained a negative charge at position 347) was inactive, the D347R mutant that introduced a positive charge retained partial activity. A model to explain these data is proposed.

Expression of placental leucine aminopeptidase/oxytocinase in neuronal cells and its action on neuronal peptides.

The placental leucine aminopeptidase (P-LAP)/oxytocinase whose serum level increases with gestation is thought to contribute to the maintenance of normal pregnancy. P-LAP mRNAs are expressed in various tissues other than the placenta. In this study, we identified P-LAP protein in the brain. In contrast with the placenta where a significant portion of P-LAP is released, the enzyme was localized in the membrane fraction in brain and PC12 cells and no soluble form of the enzyme was detected. When PC12 cells were differentiated into neuronal cells by nerve growth factor (NGF), a significant increase in the expression level of P-LAP in the cell was observed. As in the case of insulin treatment of 3T3-L1 adipocytes, treatment of PC12 cells with forskolin caused the translocation of the enzyme from intracellular vesicle to the cell surface plasma membrane. In addition, P-LAP was shown to degrade several bioactive neuropeptides such as Met-enkephalin and dynorphin A (1-8). These results suggest that P-LAP plays an important role in the regulation of neuronal cell function in the brain.