APA Activity Research Articles

Long-term application of FYM and fertilizer N improve soil fertility and enzyme activity in 51st wheat cycle under pearl millet-wheat

Our study from an ongoing research experiment initiated in Rabi 1967 at the Research Farm of CCS Haryana Agricultural University, Haryana, India, reports that during the 51st wheat cycle in pearl millet-wheat sequence, adding FYM in both seasons significantly impacted various soil parameters at different wheat growth stages compared to the rabi season. The application of 15 t of FYM ha−1 resulted in a considerable increase in dissolved organic carbon content (9.1–11.2%), available P (9.7–12.1%), and available S (12.6–17.1%), DHA levels by 7.3–22.0%, urease activity (10.1 and 17.0%), β-Glucosidase activity (6.2–8.4%), and APA activity (5.2–10.6%), compared to 10 t FYM ha−1. Application of N120 exhibited a considerable improvement in DHA (11.0–23.2%), β-Glucosidase (9.4–19.2%), urease (13.3–28.3%), and APA (3.3–6.2%) activity compared to control (N0). At stage 3, the box plot revealed that 50% of the available N, P, and S values varied from 223.1 to 287.9 kg ha−1, 53.0 to 98.2 kg ha−1, and 50.0 to 97.6 kg ha−1, respectively. Principal component analysis, with PC1 explaining 94.7% and PC2 explaining 3.15% of the overall variability, and SOC had a polynomial relationship with soil characteristics (R2 = 0.89 to 0.99). Applying FYM15 × N120 treatment during both seasons proved beneficial in sustaining the health of sandy loam soil in North-West India.

Prediction of Antibacterial Peptides against Propionibacterium acnes from the Peptidomes of Achatina fulica Mucus Fractions.

Acne vulgaris is a common skin disease mainly caused by the Gram-positive pathogenic bacterium, Propionibacterium acnes. This bacterium stimulates the inflammation process in human sebaceous glands. The giant African snail (Achatina fulica) is an alien species that rapidly reproduces and seriously damages agricultural products in Thailand. There were several research reports on the medical and pharmaceutical benefits of these snail mucus peptides and proteins. This study aimed to in silico predict multifunctional bioactive peptides from A. fulica mucus peptidome using bioinformatic tools for the determination of antimicrobial (iAMPpred), anti-biofilm (dPABBs), cytotoxic (ToxinPred) and cell-membrane-penetrating (CPPpred) peptides. Three candidate peptides with the highest predictive score were selected and re-designed/modified to improve the required activities. Structural and physicochemical properties of six anti-P. acnes (APA) peptide candidates were performed using the PEP–FOLD3 program and the four previous tools. All candidates had a random coiled structure and were named APAP-1 ori, APAP-2 ori, APAP-3 ori, APAP-1 mod, APAP-2 mod, and APAP-3 mod. To validate the APA activity, these peptide candidates were synthesized and tested against six isolates of P. acnes. The modified APA peptides showed high APA activity on three isolates. Therefore, our biomimetic mucus peptides could be useful for preventing acne vulgaris and further examined on other activities important to medical and pharmaceutical applications.

BRAIN-PENETRATING AMINOPEPTIDASE A INHIBITORS FOR NEW TREATMENT OF HYPERTENSION AND HEART FAILURE

Objective: Brain renin-angiotensin system (RAS) hyperactivity is involved in the pathophysiology of hypertension and heart failure (HF). Angiotensin III/(AngIII), one of the main effector peptides of the brain RAS, exerts tonic stimulatory control over blood pressure/(BP) in hypertensive rats and plays a pivotal role in sympathetic hyperactivity and left ventricular/(LV) dysfunction in rats post-myocardial infarction/(MI). Thus, aminopeptidase A/(APA), the enzyme generating brain AngIII represents a potential therapeutic target for the treatment of these pathologies. Our aim was to evaluate the mode of action of firibastat, a brain-penetrating prodrug of the APA inhibitor, EC33, to reduce BP in hypertensive rats and prevent LV dysfunction in mice post-MI. Design and method: Firibastat and MLN4760, an angiotensin-converting enzyme2/(ACE2) inhibitor were administered by central route in hypertensive DOCA-salt rats and mean arterial BP (MABP) was recorded following insertion of a catheter into the femoral artery. Brain APA and ACE2 activities were measured 30 min post-treatment. Two days post-MI induced by the left anterior descending artery ligation, mice were randomized to receive during 4–8 weeks oral treatment with vehicle, firibastat (150 mg/kg) or the ACE inhibitor, enalapril (1 mg/kg). Cardiac function was evaluated by echocardiography Results: 1-Firibastat, when given centrally in hypertensive rats, is immediately cleaved by brain reductases, generating EC33 which inhibits brain APA activity, blocks brain AngIII formation and decreases BP. At the same time, firibastat treatment increases the activity of brain ACE2, majoring the conversion of AngII to Ang1-7, which by acting on the Mas-receptor, participate to the firibastat-induced BP decrease. 2-Chronic firibastat treatment in mice post-MI normalizes brain APA activity (normalizing brain RAS activity) and prevents sympathetic hyperactivity. It reduced LV end-diastolic pressure, LV end-systolic diameter and volume, and increased LV ejection fraction. It decreased the expression of HF and fibrosis biomarkers. Firibastat treatment prevents cardiac dysfunction and attenuates cardiac hypertrophy and fibrosis. Conclusions: Following these data, firibastat was selected for clinical development. Phase IIa/IIb clinical trials provided pharmacological proof-of-principle of firibastat efficacy for decreasing BP in hypertensive patients. The ongoing phase II study/QUORUM will determine if firibastat treatment may constitute a new therapeutic approach for preventing HF post-MI.

Aminopeptidase A initiates tumorigenesis and enhances tumor cell stemness via TWIST1 upregulation in colorectal cancer.

Metastasis accounts for the high mortality rate associated with colorectal cancer (CRC), but metastasis regulators are not fully understood. To identify a novel gene involved in tumor metastasis, we used oligonucleotide microarrays, transcriptome distance analyses, and machine learning algorithms to determine links between primary and metastatic colorectal cancers. Aminopeptidase A (APA; also known as ENPEP) was selected as our focus because its relationship with colorectal cancer requires clarification. Higher APA mRNA levels were observed in patients in advanced stages of cancer, suggesting a correlation between ENPEP and degree of malignancy. Our data also indicate that APA overexpression in CRC cells induced cell migration, invasion, anchorage-independent capability, and mesenchyme-like characteristics (e.g., EMT markers). We also observed TWIST induction in APA-overexpressing SW480 cells and TWIST down-regulation in HT29 cells knocked down with APA. Both APA silencing and impaired APA activity were found to reduce migratory capacity, cancer anchorage, stemness properties, and drug resistance in vitro and in vivo. We therefore suggest that APA enzymatic activity affects tumor initiation and cancer malignancy in a TWIST-dependent manner. Results from RT-qPCR and the immunohistochemical staining of specimens taken from CRC patients indicate a significant correlation between APA and TWIST. According to data from SurvExpress analyses of TWIST1 and APA mRNA expression profiles, high APA and TWIST expression are positively correlated with poor CRC prognosis. APA may act as a prognostic factor and/or therapeutic target for CRC metastasis and recurrence.

Effects of UV-B and heavy metals on nitrogen and phosphorus metabolism in three cyanobacteria.

Cyanobacteria sp. (diazotrophic and planktonic) hold a major position in ecosystem, former one due to their intrinsic capability of N2-fixation and later because of mineralization of organic matter. Unfortunately, their exposure to variety of abiotic stresses is unavoidable. Comparative analysis of interactive effect of UV-B and heavy metals (Cd/Zn) on nitrogen and phosphorus metabolism of three cyanobacteria (Anabaena, Microcystis, Nostoc) revealed additive inhibition (χ(2) significant p < 0.05) of NH4(+) and PO4(3-) uptake whereas increase in nitrate uptake upon UV-B + heavy metal exposure. Glutamine synthetase and Alkaline phosphatase activity was reduced after all treatments whereas Nitrate reductase activity showed slight stimulation in UV-B and UV-B + heavy metals treatment. Combination of UV-B and metals seems more detrimental to the NH4(+), PO4(3-) uptake, GS and APA activity. A significant stimulation in NO3(-) uptake and NR activity following exposure to UV-B in all the three cyanobacteria suggests UV-B-induced structural change(s) in the enzyme/carriers. Metals seem to compete for the binding sites of the enzymes and carriers; as noticed for Anabaena and Microcystis showing change in Km while no change in the Km value of Nostoc suggests non-competitive nutrient uptake. Higher accumulation and more adverse effect on Na(+) and K(+) efflux proposes Cd as more toxic compared to Zn.

Protease-activated receptor 2 (PAR2) is upregulated by Acanthamoeba plasminogen activator (aPA) and induces proinflammatory cytokine in human corneal epithelial cells.

Acanthamoeba plasminogen activator (aPA) is a serine protease elaborated by Acanthamoeba trophozoites that facilitates the invasion of trophozoites to the host and contributes to the pathogenesis of Acanthamoeba keratitis (AK). The aim of this study was to explore if aPA stimulates proinflammatory cytokine in human corneal epithelial (HCE) cells via the protease-activated receptors (PARs) pathway. Acanthamoeba castellanii trophozoites were grown in peptone-yeast extract glucose for 7 days, and the supernatants were collected and centrifuged. The aPA was purified using the fast protein liquid chromatography system, and aPA activity was determined by zymography assays. Human corneal epithelial cells were incubated with or without aPA (100 μg/mL), PAR1 agonists (thrombin, 10 μM; TRAP-6, 10 μM), and PAR2 agonists (SLIGRL-NH2, 100 μM; AC 55541, 10 μM) for 24 and 48 hours. Inhibition of PAR1 and PAR2 involved preincubating the HCE cells for 1 hour with the antagonist of PAR1 (SCH 79797, 60 μM) and PAR2 (FSLLRY-NH2, 100 μM) with or without aPA. Human corneal epithelial cells also were preincubated with PAR1 and PAR2 antagonists and then incubated with or without PAR1 agonists (thrombin and TRAP-6) and PAR2 agonists (SLIGRL-NH2 and AC 55541). Expression of PAR1 and PAR2 was examined by quantitative RT-PCR (qRT-PCR), flow cytometry, and immunocytochemistry. Interleukin-8 expression was quantified by qRT-PCR and ELISA. Human corneal epithelial cells constitutively expressed PAR1 and PAR2 mRNA. Acanthamoeba plasminogen activator and PAR2 agonists significantly upregulated PAR2 mRNA expression (1- and 2-fold, respectively) (P < 0.05). Protease-activated receptor 2 antagonist significantly inhibited aPA, and PAR2 agonists induced PAR2 mRNA expression in HCE cells (P < 0.05). Protease-activated receptor 1 agonists, but not aPA, significantly upregulated PAR1 mRNA expression, which was significantly inhibited by PAR1 antagonist in HCE cells. Acanthamoeba plasminogen activator and PAR2 agonists stimulated IL-8 mRNA expression and protein production, which is significantly diminished by PAR2 antagonist (P < 0.05). Protease-activated receptor 1 antagonist did not alter aPA-stimulated IL-8 mRNA expression and protein production in HCE cells. Flow cytometry and immunocytochemistry showed that aPA and SLIGRL-NH2 (PAR2 agonist) upregulated PAR2 surface protein as compared to that in unstimulated HCE cells. Thrombin, but not aPA, stimulated PAR1 surface protein in HCE cells. Acanthamoeba plasminogen activator specifically induces expression and production of IL-8 in HCE cells via PAR2 pathway, and PAR2 antagonists may be used as a therapeutic target in AK.

Effect of Immunization with the Mannose-InducedAcanthamoebaProtein andAcanthamoebaPlasminogen Activator in MitigatingAcanthamoebaKeratitis

The mannose-induced cytopathic protein (MIP-133) and Acanthamoeba plasminogen activator (aPA) play key roles in the pathogenesis of Acanthamoeba keratitis by inducing a cytopathic effect on the corneal epithelial and stromal cells and by production of proteolytic enzymes that facilitate the invasion of trophozoites through the basement membrane. The goal of the present study was to gain insight into the pathogenicity of Acanthamoeba infection as well as to determine whether oral immunization with aPA and MIP-133 produce an additive protection against Acanthamoeba keratitis. MIP-133 and aPA were isolated by chromatography. The purity of the concentrated MIP-133 and aPA was confirmed by SDS-PAGE and fibrinolytic activity, respectively. aPA activity of Acanthamoeba cultures was quantitated by radial diffusion in fibrin-agarose gel. The capacity of aPA and MIP-133 to induce cytolysis of corneal epithelial cells was tested in vitro. Chinese hamsters were orally immunized with four weekly doses of aPA or MIP-133 conjugated with cholera toxin. The animals were immunized before infection to determine the prophylactic effect of oral immunization. The therapeutic effect of oral immunization with aPA and MIP-133 was determined after corneal infection had been established. The animals were then infected via Acanthamoeba castellanii-laden contact lenses. aPA was characterized in pathogenic and nonpathogenic strains of Acanthamoeba spp. Oral immunization with MIP-133 before and after infection with Acanthamoeba significantly reduced the severity of corneal infection which includes infiltration and ulceration (P < 0.05) and shortened the duration of the disease. Immunization with aPA alone did not significantly affect the course of disease (P > 0.05). These data suggest that once trophozoites invade the cornea, MIP-133 production is necessary to initiate corneal disease and plays an important role in the subsequent steps of the pathogenic cascade of Acanthamoeba keratitis.

Response of cyanobacterial mats to nutrient and salinity changes

Cyanobacterial mats (CBM), complex assemblages of cyanobacteria, bacteria and algae, are important ecosystem components of oligotrophic marshes in limestone-based regions of the Caribbean. We conducted a mesocosm experiment and evaluated the response of CBM to factorial combinations of low, medium and high phosphorus, nitrogen and salinity. Changes in composition of the main species groups of cyanobacteria and algae, primary production, cellular nutrients and enzymatic activities were recorded as response variables. The redundancy analysis with concentrations of P, N and salinity as explanatory variables showed that the primary production of CBM and the amount of phytoplankton expressed as Chl a were best explained by concentration of P, with less significant positive effect of N and a negative effect of salinity. Abundance of green algae and Chroococcales was positively correlated with increasing concentrations of P and N and reached 27.6% and 21.9%, respectively, in high P and high N treatment at the end of experiment. N 2-fixation averaged 75 and 175 nmol C 2H 4 cm −2 min −1, at low nitrogen and medium or high P, respectively, and it was negatively correlated with nitrogen concentration and positively correlated with abundance of a group of heterocytous cyanobacteria from genus Nostoc. At low N concentrations, increasing P concentrations supported higher N 2-fixation. Activity of the alkaline phosphatase, APA, was negatively correlated with P and salinity and positively with N. We also found a significant negative correlation between the APA activity and the P content of the mat. At high P and N concentrations, the mats were impacted by grazing, had a tendency to disintegrate and become shaded out by a massive growth of phytoplankton. We confirmed an overall negative effect of nutrient increase on CBM.

A Rae1-Containing Ribonucleoprotein Complex Is Required for Mitotic Spindle Assembly

Centrosome-independent microtubule polymerization around chromosomes has been shown to require a local gradient of RanGTP, which discharges mitotic cargoes from the nuclear import receptor importin beta. Here, we have used an activity-based assay in Xenopus egg extracts to purify the mRNA export protein Rae1 as a spindle assembly factor regulated by this pathway. Rae1 is a microtubule-associated protein that binds directly to importin beta. Depletion of Rae1 from extracts or cells severely inhibits mitotic spindle assembly. A purified Rae1 complex stabilizes microtubules in egg extracts in a RanGTP/importin beta-regulated manner. Interestingly, Rae1 exists in a large ribonucleoprotein complex, which requires RNA for its activity to control microtubule dynamics in vitro. Furthermore, we provide evidence that RNA associates with the mitotic spindle and that it plays a direct, translation-independent role in spindle assembly. Our studies reveal an unexpected function for RNA in spindle morphogenesis.

Placental Aminopeptidase A as a Possible Barrier of Angiotensin II between Mother and Fetus

Aminopeptidase A (AP-A EC 3.4.11.7), which is a membrane-bound zinc metalloprotease, is present in the placenta. AP-A selectively hydrolyzes N-terminal glutamyl and aspartyl residues and cleaves angiotensin II to form angiotensin III. To determine the role of placental aminopeptidase A under physiological and pathological conditions, we evaluated its immunolocalization and enzymatic activities in the placenta. AP-A was localized in the basal zone of the syncytiotrophoblast, in the membranes of the cytotrophoblast, and in fetal arterioles and venules within the stem villi. AP-A activity in the microsomal fraction of placental villi seemed to be remained essentially constant throughout gestation. The renin–angiotensin system is considered to be accelerated in pre-eclampsia. This AP-A activity was higher in pre-eclampsia (2.86±0.30nmol β NA/mg protein/h) than in uncomplicated pregnancy from 28 to 41 weeks of gestation (2.08±0.18nmol β NA/mg protein/h). Angiotensin II evoked AP-A activity in first trimester trophoblast, and Losartan and PD 123177 in combination significantly inhibited this induction of AP-A activity. The results of immunohistochemical evaluation and enzymatic activity suggested that placental aminopeptidase A may play a role as a component of the barrier of angiotensin II between mother and fetus.

Histidine 450 plays a critical role in catalysis and, with Ca2+, contributes to the substrate specificity of aminopeptidase A.

Aminopeptidase A (EC 3.4.11.7, APA) is a 130 kDa membrane-bound protease that contains the HEXXH consensus sequence found in the zinc metalloprotease family, the zincins. In addition to the catalytic zinc atom, APA contains a Ca2+ ion that increases its enzymatic activity. Aligning the sequences of the mouse APA, APN, and other monozinc aminopeptidases led to the identification of a conserved histidine (His 450 in mouse APA). Replacing this residue with a phenylalanine (Phe 450) by site-directed mutagenesis resulted in markedly lower levels of APA activity and in a change in the sensitivity of APA to Ca2+ (the EC50 for Ca2+ was 25 microM in the wild type and only 279 microM in the mutant). Kinetic studies, with a supramaximal Ca2+ concentration (4 mM), showed that the Km of the mutant enzyme for the substrate alpha-L-glutamyl-beta-naphthylamide was 25 times higher than that of the wild type, whereas the kcat value was much lower (factor of 22). Thus, overall, the wild-type enzyme had a cleavage efficiency that was 571 times higher than that of the mutant. The inhibitory potencies of two different classes of inhibitors, a glutamate thiol and a glutamate phosphonate compound, were significantly lower (factors of 19 and 22, respectively) for the mutated enzyme than for the wild-type enzyme. In contrast, inhibition by lysine thiol was unaffected. These data strongly suggest that His 450 is critical for catalytic activity and is involved in substrate binding via interaction with the P1 carboxylate side chain of the substrate. Furthermore, His 450, together with Ca2+, may contribute to the substrate specificity of APA for N-terminal acidic amino acid residues.

Aminopeptidase a is a constituent of activated pericytes in angiogenesis.

Monoclonal antibody (MAb) RC38 recognizes a human renal antigen of 160 kD recently identified as human aminopeptidase A (APA; EC 3.4.11.7). This ectoenzyme is able to hydrolyse selectively N-terminal glutamyl and aspartyl residues from oligopeptides. By enzyme histochemistry, APA activity has also been localized in the microvessels of all organs in animals and man. The purpose of this study was to investigate the distribution of human APA as recognized by MAb RC38 in the microvasculature of normal human tissues and pathological conditions associated with neovascularization. Unexpectedly, in normal tissues vascular staining with MAb RC38 was generally weak and often absent, while in tumours, granulation tissue, and chronic synovitis, marked microvascular staining was demonstrated. By immuno-electron microscopy, the antigen was found on the cell membrane of activated pericytes and their processes in the tumour vasculature. RC38 expression could not be detected on cultured human endothelial cells or pericytes. These observations suggest that pericyte expression of a subtype of APA (as recognized by MAb RC38) is markedly enhanced in the vasculature of tumours and wound healing tissue as compared with normal resting tissues. This provides further evidence of the altered state of pericytes in these conditions. Pericyte APA may be involved in the metabolism of biologically active oligopeptides during neovascularization, supporting a regulatory role of pericytes in this process. In addition, MAb RC38 may be useful as a marker of pericyte activation in tissue sections.

Identification of Glutamate Residues Essential for Catalytic Activity and Zinc Coordination in Aminopeptidase A

Aminopeptidase A (EC 3.4.11.7, APA) is a homodimeric membrane-bound glycoprotein that contains the consensus sequence HEXXH(385-389) found in zinc metallopeptidases such as thermolysin. The x-ray structure of the latter enzyme revealed that the two histidines of this motif are two of the three zinc-coordinating ligands and that the glutamate is a crucial amino acid involved in catalysis. Alignment of the sequence of mouse APA with those of the already characterized metallopeptidases showed the presence of several conserved amino acids such as a glutamate residue in position 408 which may constitute the putative third zinc ligand. The functional implication of this residue and the role of glutamate 386 in the HELVH(385-389) motif of APA have been investigated by replacing these residues with an aspartate (Asp-386, Asp-408) or an alanine (Ala-386, Ala-408) by site-directed mutagenesis. Expressed mutated proteins in position 386 showed no APA activity. Ala-408 was also inactive, and Asp-408 had 5% of the wild type enzyme activity and a similar Km. 65Zn incorporation measurements indicated that Ala-386 binds the zinc ion as well as the wild type enzyme, whereas the Ala-408 mutant did not. These results provide evidence that Glu-408 is the third zinc-coordinating residue of APA, confirm the presumed involvement of Glu-386 in the catalytic process of the enzyme, and identify APA as a zinc metallopeptidase functionally similar to thermolysin.

Histidine residue in the zinc-binding motif of aminopeptidase A is critical for enzymatic activity.

The murine BP-1 antigen (also called 6C3) is a homodimeric, phosphorylated cell surface glycoprotein that is expressed on immature B-lineage cells, bone marrow stromal cell lines, thymic cortical epithelial cells, endothelial cells, enterocytes, and renal proximal tubular cells. The amino acid sequence deduced from a BP-1 cDNA predicted a type II integral membrane protein with a zinc-binding motif (His-Glu-Xaa-Xaa-His) found in zinc-dependent metallopeptidases, and functional analysis suggested that BP-1 is aminopeptidase A [APA; L-alpha-aspartyl(L-alpha-glutamyl)-peptide hydrolase, EC 3.4.11.7]. Here we constructed an expression vector in which the BP-1 cDNA was placed downstream from the SR alpha promoter and used this construct to transfect COS-7 and Ltk- cells. Both transfectants expressed the BP-1 antigen on the cell surface and APA activity. The enzymatic activity of recombinant APA was increased by Ca2+ and inhibited by Zn2+, consistent with previous reports with purified APA. Point mutation of one of the histidine residues in the zinc-binding motif to phenylalanine completely abolished APA enzymatic activity, suggesting the structure of the zinc-binding motif of APA is critical for catalytic activity. Both wild-type and mutant BP-1 were glycosylated, transported to the cell surface, and possessed molecular weights similar to native BP-1 molecules on the murine 18.81 pre-B-cell line. The successful expression of both wild-type and mutant APA should allow more precise analysis of the diverse physiological roles of this ectoenzyme.

Aminopeptidase A activity of the murine B-lymphocyte differentiation antigen BP-1/6C3.

The predicted amino acid sequence of the cDNA encoding the murine B-lymphocyte differentiation antigen BP-1/6C3 suggested that it is a member of the zinc-dependent metalloprotease family, possibly an aminopeptidase related to aminopeptidase N [microsomal aminopeptidase; alpha-aminoacyl-peptide hydrolase (microsomal), EC 3.4.11.2]. In the present studies, we examined the enzymatic activity of this antigen. From brush border preparations of the small intestine, a rich source of many endopeptidases and exopeptidases, the BP-1 antibody selectively removed aminopeptidase A [APA; L-alpha-aspartyl(L-alpha-glutamyl)-peptide hydrolase, EC 3.4.11.7] activity. The APA activity of a panel of cell lines correlated in linear fashion with cell-surface levels of the BP-1/6C3 antigen. APA activity was demonstrated for the BP-1/6C3 antigen immunopurified from the pre-B-cell membrane. This activity was enhanced by alkaline earth metals such as Ca2+ and was abrogated by amastatin and angiotensin, which are known competitive inhibitors of APA. The data indicate that the murine BP-1/6C3 antigen is active APA, an enzyme that catalyzes specifically the removal of unsubstituted, N-terminal glutamic acid and aspartic acid residues from peptides.

Study on aminopeptidase A.

A comparative biochemical and histochemical investigation of aminopeptidase A (APA, E.C. 3.4.11.7) was carried out. α-Glu-1NA, α-Glu-2NA, α-Glu-MNA and Asp-2NA were used as substrates, Fast Blue B (FBB), hexazotized new fuchsin (HNF) and hexazonium-p-rosaniline (HPR) as coupling agents. Biochemical determination of APA activity was performed in whole homogenates as well as in homogenized freeze-dried cryostat sections of many rat, guinea-pig and human organs. α-Glu-2NA proved to be the best substrate for the biochemical determination of APA activity. It displays favourable kinetic properties and abilities for spectrophotometric as well as fluorometric measurements.K m of rat kidney enzyme amounts to 0.15–0.43 mM, of rat jejunum enzyme 0.35 mM, of myocardial enzyme 0.2 mM and of rat brain enzyme 0.25 mM. The enzyme is activated by Ca2+ and inhibited by EDTA (1 mM) and 1,10-phenanthroline (1 mM). E600, DFP and PCMB (1 mM) did not influence APA activity. The activity was detected in every organ examined. Great organ and species differences were demonstrated. The highest APA activity resides in the kidney and in the small intestinal mucosa. The structural association of APA is not absolutely firm and up to 30% (depending on the organ and eventual pretreatment of sections with chloroform-acetone which impedes the leakage to some extent) escapes into the aqueous incubation solution. The highest values were recorded in phosphate and cacodylate buffers followed by citrate phosphate, citrate, Tris-HCl and acetate buffers. There were no great differences in activities measured at pH 6.5 and 7.2. For the histochemical demonstration of APA activity α-Glu-MNA is the substrate of choice.K m estimated biochemically amounts to 0.3–0.43 mM for the kidney enzyme, 0.35 mM for the rat jejunal enzyme and 0.5 mM for rat brain enzyme.K m estimated by microdensitometric measurements of the reaction product in the brush border of cells of proximal tubules and of jejunal enterocytes at the sites of villi amount to 0.6 and 0.4 mM respectively. The best results were obtained using 2–4 mM concentration of α-Glu-MNA, and satisfactory results using 0.36–0.72 mM concentration. FBB appears to be the best coupling agent in the routine. The most sensitive way to depict APA in sites with low and moderate activity is to use chloroform-acetone pretreated cryostat sections adherent to semipermeable membranes. Sites with high enzyme activity are best revealed upon incubation of chloroform-acetone pretreated cryostat sections adherent to slides. Of the unsubstituted naphthylamine derivative of α-Glu or Asp α-Glu-2NA is superior to α-Glu-1NA or Asp-2NA. In this case HPR or HNF are to be used as coupling agents in cryostat sections adherent to slides. Due to a higher decomposition rate and higher inhibitory influence of diazonium salts on APA activity in the alkaline pH range it is advisable to carry out the reaction at pH 6.5–7. The most important binding sites of APA are: The functional significance of APA is discussed in relation to the degradation of angiotensin II (endothelium, kidney) and to other brush border peptidases releasing amino acids which are absorbed (enterocytes). The histochemical demonstration is indispensable for a correct interpretation of biochemical data.

Comparative Effectiveness of Vitamin B12, Whole Liver Substance and Extracts High in Apa Activity, as Growth Promoting Materials for Hyperthyriod Animals

Comparative Effectiveness of Vitamin B12, Whole Liver Substance and Extracts High in Apa Activity, as Growth Promoting Materials for Hyperthyriod Animals