Inactivation during phagocytosis of leucine aminopeptidase, an ecto-enzyme of polymorphonuclear neutrophils

Changes in enzyme activities of the plasma membrane makers were examined during phagocytosis using guinea-pig polymorphonuclear neutrophils. Incubation of neutrophils with fresh serum-opsonized zymosan particles showed a significant reduction in leucine aminopeptidase activity, whereas 5′-nucleotidase and alkaline phosphodieterase activities remained unchanged. Inactivation of leucine aminopeptidase activity was also observed by exposure of neutrophils to complement-opsonized zymosan particles, but not to non-opsonized zymosan, IgG-coated zymosan or polysterene latex particles. Pretreatment of neutrophils with cytochalasin B, which prevents phagocytosis but not surface binding of particles, provoked inactivation to the same degree as when the cells were allowed to phagocytose the particles. However, the inactivation during phagocytosis was protected by serine protease inhibitors. These findings suggest that loss of leucine aminopeptidase activity from phagocytosing cells may be mediated by certain serine protease inhibitor-sensitive factor(s) which are probably activated by the attachment of an opsonized zymosan particle to a specific membrane receptor, probably the C3b receptor.     

Inactivation of phagocytosis-stimulating activity of tuftsin by polymorphonuclear neutrophils: A possible role of leucine aminopeptidase as an ecto-enzyme

The subcellular localization of the tuftsin-inactivating activity was studied using guinea-pig polymorphonuclear neutrophils and the following results were obtained. 1. The tuftsin-inactivating activity was present in the membrane function but not in the cytosol and the granular fractions. 2. Intact neutrophils inactivated tuftsin rapidly. However, when neutrophils were modified chemically by a poorly permeant reagent, diazotized sulfanilic acid, the tuftsin-inactivating activity decreased sifnificantly without any inhibition of marker enzymes of cytosol, microsome, granulesa and mitochondria, suggesting that the tuftsin-inactivating activity is located on the plasma membrane as an ecto-enzyme. 3. When neutrophils were modified by diazotized sulfanilic acid at different concentrations, the tuftsin-inactivating activity of neutrophils was inhibited in proportion to the degree of inhibition of the activity of leucine aminopeptidase, an ecto-enzyme. 4, Hydrolysis of L-leucyl-β-napthylamide, a synthetic substrate of leucine aminopeptidase, was inhibited competitively by tuftsin. 5. Treatmetn of neutrophils with serine protease inhibitors affected neither tuftsin-inactivating nor leucine aminopeptidase activity at all, indicating no involvement of serine proteases, which is said to be located on the cell surface membrane, in the tuftsin-inactivating activity of neutrophils. The possibility was deduced from the above results that leucine aminopeptidase may act as a tuftsin-inactivating enzyme.     

Leucine aminopeptidase as an echo-enzyme of polymorphonuclear neutrophils

Intact polymorphonuclear neutrophils were modified chemically by a poorly permeable reagent, diazotized sulfanilic acid, and the changes in the activity of 5'-nucleotidase, alkaline phosphodiesterase, and leucine aminopeptidase were examined. Among three plasma membrane enzymes, 5'-nucleotidase activity was hardly detected in the human neutrophils. The activity of alkaline phosphodiesterase was observed in all the neutrophils examined, but was not inhibited by diazotized sulfanilic acid in the guinea-pig neutrophils. On the other hand, the activity of leucine aminopeptidase was not only found but also inhibited by diazotized sulfanilic acid without the inhibition of lactate dehydrogenase, a cytosol enzyme, in all the neutrophils, suggesting that leucine aminopeptidase is located generally on the plasma membrane as an ecto-enzyme in the neutrophils.     

Localization of leucine aminopeptidase and vitamin B-12 binding protein in rabbit peripheral blood polymorphonuclear leukocytes

Polymorphonuclear leukocytes were isolated from the peripheral blood of rabbits by Ficoll-Hypaque centrifugation followed by dextran sedimentation. The granulocytes were homogenized in isotonic sucrose and subjected to analytical subcellular fractionation by sucrose density gradient centrifugation. Leucine aminopeptidase, when assayed with L-leucine-7-amido-4-methyl-coumarin as substrate, showed a similar distribution to N-acetyl-ß-glucosaminidase and thus is localized to the tertiary granules. There was no leucine aminopeptidase associated with the plasma membrane of these cells. Further experiments with purified plasma membranes and inhibitor studies using diazotized sulphanilic acid further confirmed that leucine aminopeptidase had a purely intracellular localization. Vitamin B-12 binding protein showed a similar localization to alkaline phosphatase indicating that, as in human polymorphonuclear leukocytes, vitamin B-12 binding protein is located to the specific granules.     

Implications of a 5′-nucleotidase inhibitor in human leukemic cells for cellular aging and cancer

5′-Nucleotidase activity of normal human embryonic lung fibroblasts (IMR-90) was found to be inhibited by the homogenates of seven different cell lines originated from patients with different kinds of leukemia and of fresh lymphocytes from a patient with Sezary syndrome (circulating T-cell lymphoma). About 97% of the inhibiting activity was found in the soluble fraction of RPMI 8402 cells, a cell line originated from the lymphocytes of a patient with acute lymphocytic leukemia. This inhibiting activity was not destroyed by dialysis, heating at 56°C for 30 min, nor digestion with RNAase or DNAase. About 85% of the inhibiting activity was destroyed by digestion with papain at 37°C for 1 h and it was destroyed completely by heating at 100°C for 30 min. When the heated (56°C for 30 min) soluble fraction of RPMI 8402 cells was mixed with the homogenate of IMR-90 cells, it had no effect on the activities of alkaline, neutral or acid phosphatases, nor of $\text{N-}\text{acetyl}\text{-β-}\text{d}\text{-glucosaminidase}$ or cytochrome c oxidase of IMR-90 cells. Preincubating the mixed samples for 1, 20 and 45 min, respectively, before adding the substrate, the heated soluble fraction of RPMI 8402 cells did not increase the percentage of inhibition for 5′-nucleotidase of the homogenate of IMR-90 cells. No inhibition of other enzyme activities was observed under similar conditions. These data suggest that the inhibiting activity is due to a protein(s) that is not a protease. The inhibiting activity was found in a single peak after the soluble fraction was fractionated by Sephadex G-100 chromatography and sedimentation centrifugation. The molecular weight of the inhibitor was found to be approx. 35 000 by comparing its retention volume and sedimentation rate with those of proteins of known molecular weight. The present study suggests that the previously reported undetectability of 5′-nucleotidase in permanent cell lines could be due to the presence of a protein inhibitor for 5′-nucleotidase in these human leukemic cell lines. It also supports the hypothesis that the increased 5′-nucleotidase activity in normal senescent cells in vitro may be a control in cellular aging that is missing from leukemic cells in vitro.     

Phagocytosis of polymorphonuclear leukocytes by guinea pig peritoneal macrophages: effects of serum and temperature in vitro

Abstract The regulation of phagocytosis of neutrophils by peritoneal macrophages was studied in vitro. Peritoneal exudate cells (PECs) of guinea pigs were lavaged 15 h after the i.p. injection of thioglycollate medium and were cultured in chamberslides. When PECs were cultured in RPMI 1640 medium in the absence of serum, approximately 20% of the macrophages phagocytized autologous neutrophils during 48–72 h of culture. Addition of guinea pig serum to the culture (2.5–20% v/v) suppressed the extent of the phagocytosis. The suppression was induced by globulin-rich ammonium sulfate fractions of the serum. Sera from rat, mouse, hamster, horse or calf also suppressed the phagocytosis, but fetal bovine serum (FBS) supported the phagocytosis, which was inhibited by globulin-rich Cohn fractions of bovine serum. The rate of neutrophil-phagocytosing macrophages was proportional to the rate of the pyknotic change of neutrophils. At a high temperature (42°C), the autophagocytosis took place at 12 h of culture when fresh, but not heat-inactivated, autologous serum was added, implying that complement components may play a role in the hyperthermia-induced phagocytosis of neutrophils by macrophages. At 42°C, ingested neutrophils did not show the pyknotic changes, indicating that intact neutrophils were ingested by macrophages.     

Modulation of the activity of 5′-nucleotidase by the transfer of non-esterified cholesterol to rat-liver microsomal fraction and evidence for regulation of the concentration of non-esterified cholesterol in plasma membranes in vivo

The transfer of non-esterified cholesterol to rat-liver microsomal fraction resulted in a considerable decrease in the activity of 5′-nucleotidase and in changes in the characteristics of the Arrhenius plots of the enzyme. The decrease in the activity of 5′-nucleotidase and the increase in the concentration of non-esterified cholesterol in the serum-treated preparations were serum-concentration-dependent and incubation-time-dependent. The enzyme in serum-treated preparations with high non-esterified cholesterol content showed Arrhenius plots with a constant activation energy between 37 and 19°C, whereas the enzyme in the non-treated microsomal fraction or the lipoprotein-deficient serum-treated preparations showed a break at about 28°C, with activation energies higher below and lower above the break. These changes in the temperature-induced kinetics are consistent with an increase in the concentration of non-esterified cholesterol in the plasma membrane vesicles of the serum-treated preparations. The Arrhenius plots of 5′-nucleotidase in liver microsomal fraction from rats fed cholesterol-supplemented diet showed constant activation energy between 37 and 19°C and had similar characteristics with the plots for 5′-nucleotidase in serum-treated preparations. Since the changes in the characteristics of Arrhenius plots of the enzyme in microsomal fraction from rats that had been denied food for 36 h were in the opposite direction to those produced by feeding cholesterol, these results are consistent with a lower concentration of non-esterified cholesterol in hepatic plasma membranes from fasted rats relative to that in plasma membranes from fed rats. The isolation of a plasma membrane preparation with negligible contamination of endoplasmic reticular membranes from rats fed the standard or cholesterol-supplemented diet and from fasted rats showed that the ratio of cholesterol to phospholipid has increased in the preparation from rats fed cholesterol and decreased in that from rats that had been denied food relative to the ratio in the preparation from rats fed the standard diet. The Arrhenius plots of 5′-nucleotidase in these preparations showed characteristics similar to the corresponding plots of the enzyme in the microsomal fraction from the rats in the three experimental conditions.     

Isolation and kinetic properties of 5′-nucleotidase from guinea-pig skeletal muscle

5′-Nucleotidase (EC 3.1.3.5) has been solubilized and purified 1200-fold from guinea-pig skeletal muscle, to a specific activity of 40 U/mg protein. The purified enzyme yields a single protein band on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Guinea-pig skeletal muscle 5′-nucleotidase is extremely sensitive to inhibition by nucleoside di- and triphosphates. The inhibition is of the competitive type, and can be reversed only by strong excess of Mg²⁺. Nucleoside diphosphates are more powerful inhibitors than nucleoside triphosphates. The K_i values for ADP and ATP are 0.036 and 0.28 μM, respectively. The purified enzyme does not require exogenous cations for maximal activity and is inhibited by EDTA. This inhibition is reversed by divalent cations. This indicates that the enzyme contains a tightly bound metal cation.     

Acyl group composition of lipids and the activities of (Na+ + K+)-ATPase, 5′-nucleotidase and γ-glutamyltranspeptidase in salivary glands and kidneys of rats fed diets containing different dietary fats

Two nutritional models, an essential fatty acid deficiency model and the feeding of saturated versus unsaturated fats, were used in a feeding study in order to assess the relationship between tissue fatty acid composition and the activities of some membrane-associated enzymes. Purified diets containing 7% hydrogenated coconut, oil, 7% corn oil, 10% safflower oil or butter were fed to rats for a total of 49 weeks (1 week of pregnancy, 3 weeks of lactation and 45 weeks post-weaning). Tissue homogenates from submandibular salivary glands and kidneys were analyzed for fatty acid composition of total lipids and phospholipids. Changes in fatty acid patterns typical of essential fatty acid deficiency such as an increase in the levels of 16:1 and 18:1, a decrease in 18:2 and 20:4 and an accumulation of 20:3ω9 were observed in salivary glands and kidneys of rats fedd the deficient diet. Tissues of rats fed 10% butter also showed fatty acid compositional changes which were somewhat similar to those in essential fatty acid deficiency, but to a lesser degree. The activities of ouabain-sensitive (Na⁺ + K⁺)-ATPase were higher in homogenates of salivary glands and kidneys of the deficient rats and those fed butter as compared with their controls. The results suggest a relationship between the double bond index of fatty acids as an indication of membrane lipid fluidity and allosteric modification of (Na⁺ + K⁺)-ATPase activity. However, other explanations for the observed changes in (Na⁺ + K⁺-ATPase activity cannot be ruled out. There were no diet-related differences in the activities of γ-glutamytranspeptidase or 5′-nucleotidase.     

Elevated specific activity of 5′-nucleotidase in a spinal cord myelin fraction from shiverer mice comparison with other myelin-associated enzymes and myelin proteins

Meylin partially purified from spinal cords of dysmyelinating mutant (shiverer) mice had almost three-fold the specific activity of 5′-nucleotidase found in the respective myelin fraction from normal mice. The specific activities of two other normally myelin-associated enzymes, 2′,3′-cyclic nucleotide-3′-phosphohydrolase and carbonic anhydrase, were only slightly higher in the myelin membranes from shiveres, compared to those from controls. In the mutants, the three enzymes probably occur in oligodendrocyte processes. Hhypothetically, the 5′-nucleotidase in the myelin sheath in shiverer and normal mice may be localized in specialized structures.     

A membrane-associated creatine kinase (EC 2.7.3.2) identified as an acidic species of the non-receptor,peripheral v-proteins in Torpedo acetylcholine receptor membranes

Human liver mRNA isolated from subjects phenotyped as homozygous PiMM or PiZZ α₁-antitrypsin, was translated in a reticulocyte cell-free system, and α₁-antitrypsin identified by immunoprecipitation. In the presence of dog pancreas membranes the translated α₁-antitrypsin appeared as a larger product. Treatment with endo-β-N-glucosaminidase yielded a protein smaller than the reticulocyte translated product, presumably due to removal of the N-terminal signal sequence by membranes and sugar residues by endo-β-N-glucosaminidase. Quantitation of α₁-antitrypsin translated from PiMM and PiZZ livers suggests that both mRNA species were present at the same cellular concentration, and that processing to the core glycosylation stage proceeded at identical rates.     

Function of the signal peptide and N- and C-terminal propeptides in the leucine aminopeptidase from Aeromonas proteolytica

The leucine aminopeptidase from Aeromonas proteolytica (also known as Vibrio proteolyticus) (AAP) is a metalloenzyme with broad substrate specificity. The open reading frame (ORF) for AAP encodes a 54 kDa enzyme, however, the extracellular enzyme has a molecular weight of 43 kDa. This form of AAP is further processed to a mature, thermostable 32 kDa form but the exact nature of this process is unknown. Over-expression of different forms of AAP in Escherichia coli (with AAP's native leader sequence, with and without the N- and/or C-terminal propeptides, and as fusion protein) has allowed a model for the processing of wild-type AAP to be proposed. The role of the A. proteolytica signal peptide in protein secretion as well as comparison to other known signal peptides reveals a close resemblance of the A. proteolytica signal peptide to the outer membrane protein (OmpA) signal peptide. Over-expression of the full 54 kDa AAP enzyme provides an enzyme that is significantly less active, due to a cooperative inhibitory interaction between both propeptides. Over-expression of AAP lacking its C-terminal propeptide provided an enzyme with an identical kcat value to wild-type AAP but exhibited a larger Km value, suggesting competitive inhibition of AAP by the N-terminal propeptide (Ki approximately 0.13 nM). The recombinant 32 kDa form of AAP was characterized by kinetic and spectroscopic methods and was shown to be identical to mature, wild-type AAP. Therefore, the ease of purification and processing of rAAP along with the fact that large quantities can be obtained now allow new detailed mechanistic studies to be performed on AAP through site-directed mutagenesis.     

The dual effects of aluminum as activator and inhibitor of adenylate cyclase in the liver fluke Fasciola hepatica

The liver fluke, $\text{Fasciola hepatica}$, has a very active adenylate cyclase which can be stimulated by NaF or by serotonin and guanine nucleotides. Micromolar amounts of AlCl₃ augment the activation by F-. In contrast, when the enzyme is activated with serotonin and guanine nucleotides, AlCl₃ inhibits the activation. Aluminum also inhibits the activation by forskolin. Gallium mimics the effects of aluminum.     

Resonance raman spectroscopy of chemically modified and isotopically labelled purple membranes. II. Kinetic studies

Kinetic resonance Raman spectra of native and isotopically labelled purple membranes are compared. Using these data and the assignments of the previous paper in this sequence, we have confirmed that the Schiff base is deprotonated at times that are short in comparison to M₄₁₂ evolution. In addition, by monitoring the kinetic resonance Raman spectra in ²H₂O with 488.0 nm excitation we have been able to characterize in more detail the vibrational features associated with this unprotonated intermediate that precedes M₄₁₂. Furthermore, the kinetic spectra of fully deuterated purple membranes in H₂O have allowed us to assign the 1465 cm⁻¹ band in these spectra to the C=C stretching frequency of BR₅₇₀ and the 1512 cm⁻¹ band to the C=C stretching frequency of M₄₁₂. These spectra have also provided an indication of a Raman spectral feature associated with O₆₄₀ and, finally, our kinetic spectra have provided evidence that there is a significant alteration in the rate constants for the evolution of the various intermediates when the non-exchangeable protons on the membrane are replaced by deuterons.