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.     

Leucine aminopeptidase as an ecto-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.     

Subcellular localization and properties of adenosine diphosphatase activity in human polymorphonuclear leukocytes

Adenosine diphosphatase (ADPase) activities were studied in human polymorphonuclear leukocytes with a recently developed radio-assay. The neutrophils were homogenized in isotonic sucrose and subjected to analytical subcellular fractionation. The sucrose density gradient fractions were assayed for ADPase activity and for principal organelle marker enzymes. ADPase activity was distributed between the plasma membrane, specific granule and soluble fractions. The plasma membrane and specific granule activities had similar kinetic and inhibitor properties but the cytosolic enzyme was clearly different. Studies with the non-penetrating inhibitor diazotized sulphanilic acid and measurements of latent activity indicate that plasma membrane ADPase activity is located on the external aspect to the cell. Its possible role in inhibiting platelet aggregation is discussed. Neutrophils were isolated from control subjects, patients with chronic granulocytic leukaemia and patients in the third trimester of pregnancy. The specific activities (mU/mg protein) of ADPase activity, in contrast to those of alkaline phosphatase, were similar in all three groups. This result, together with fractionation experiments and inhibition studies strongly suggests that ADPase activity is not attributable to neutrophil alkaline phosphatase.     

Glucocorticoid hormones increase the activity of gamma-glutamyltransferase in a highly differentiated hepatoma cell line

Gamma-Glutamyltransferase activity was detected in the plasma membrane of the highly differentiated hepatoma cell line Fao, (0.93 mU/mg cell protein). Dexamethasone (1 microM) provoked a 2-3-fold increase in the activity of the enzyme in the presence of fetal calf serum. Maximal induction occurred 48-72 h after addition of the glucocorticoid to the cell culture medium. The hormonal specificity was demonstrated by the relative potencies of several glucocorticoids and sex steroids: hydrocortisone and corticosterone increased gamma-glutamyltransferase activity while tetrahydrocorticosterone and all sex steroids tested were ineffective. The effect of dexamethasone on gamma-glutamyltransferase activity wa specific since the activities of several other plasma membrane enzymes were not modified. The mechanism of the dexamethasone-induced increase in gamma-glutamyltransferase activity was neither by modification of the affinity of the enzyme for its substrates nor by alteration of the subcellular distribution of the enzyme. This increase was prevented by cycloheximide and actinomycin D. The data presented are consistent with a specific glucocorticoid receptor-mediated induction of gamma-glutamyltransferase activity in Fao cells. The kinetic parameters of the induction process by glucocorticoids are very similar to those found in adult rat liver. These results suggest that the Fao cell line is a very convenient system for the study of the molecular mechanisms of glucocorticoid effects on differentiated cells.     

Purification and characterization of α-l-fucosidase from the liver of a fucosidosis patient

Intact viable 13762 mammary-adenocarcinoma ascites cells hydrolyse added ATP. The localization of hydrolysis product and inactivation by the slowly penetrating chemical reagent diazotized sulphanilic acid indicate that this ATPase is at the external surface of the cell. A number of features differentiate this enzyme from mitochondrial, myosin and cation-transport ATPases. It is stimulated by either Ca2+ or Mg2+ and has little or no activity in their absence. It is insensitive to ouabain, oligomycin and azide. It is the major ATPase activity found in homogenates of gently disrupted 13762 cels. The ATPase activity is inhibited at high substrate concentrations and shows an apparent stimulation by concanavalin A in isolated membranes, but not in intact cells. The stimulation by concanavalin A results predominantly from a release from substrate inhibition.     

Evidence that Mg2+- or Ca2+-activated adenosine triphosphatase in rat pancreas is a plasma-membrane ecto-enzyme.

Preparations of enzymically dispersed rat pancreatic cells hydrolyse externally added nucleoside triphosphates and diphosphates at high rates in the presence of Mg2+ or Ca2+. The lack of response to specific inhibitors and activators differentiates this hydrolytic activity from that of other well-characterized ion-transporting ATPases. Studies based on inactivation of this hydrolytic activity by the covalently reacting, slowly permeating probe diazotized sulphanilic acid indicated that this nucleoside tri- and di-phosphatase is primarily a plasma-membrane ecto-enzyme. It is the major ATPase activity associated with intact cells, homogenates and isolated plasma-membrane fractions. Concanavalin A stimulates this ATPase activity of intact cells and isolated plasma-membrane fractions. The insensitivity of this ATPase activity to univalent ions and inhibitors of pancreatic electrolyte secretion, taken together with the evidence that the active site is externally located, suggests that this enzyme is not directly involved in HCO3- secretion in the pancreas. Its actual function remains unknown.     

Mg2+-ATPase as a membrane ecto-enzyme of human granulocytes. Inhibitors, activators and response to phagocytosis.

(1) The Mg2+-ATPase of purified human granulocytes is located at the plasma membrane. Thus, no additional enzyme activity was detected when the cells were disrupted. Moreover, the Mg2+-ATPase activity of intact cells was inhibited by such poorly permeant reagents as diazotized sulfanilic acid and suramin. Finally, the enzyme activity of cell homogenates was recovered in particulate fractions. (2)The surface Mg2+-ATPase of human granulocytes had an apparent Km of 50 microns for ATP and displayed substrate inhibition. (3) The enzyme was not affected by ouabain, but was inhibited by N-ethyl malemide, sodium meta-periodate, suramin and diazotized sulfanilic acid. The enzyme was activated by cytochalasins B and D and by UDP. Activation by UDP was characterized by changes in the enzyme's apparent Km and V and by belief of substrate inhibition. (4)Internalization of surface membranes subsequent to phagocytosis of suitable particles did not result in depletion of Mg2+-ATPase from the cell surface. The enzyme activity did not decrease after exposure to several varieties of paraffin oil emulsion particles, even if the challenged cells had been pretreated with colchicine of cytochalasin B. (5) Since suramin, which inhibited Mg2+-ATPase, had no effect upon other granulocyte functions such as chemotaxis, superoxide anion generation, or phagocytosis, it is unlikely that the enzyme plays a major role in these functions.     

Enzyme analysis and subcellular fractionation of human peripheral blood lymphocytes with special reference to the localization of putative plasma membrane enzymes

Human lymphocytes were isolated from defibrinated blood by Ficoll-Hypaque centrifugation with erythrocyte hypotonic lysis. Homogenates of mixed lymphocytes were subjected to analytical subcellular fractionation by sucrose gradient centrifugation in a Beaufay automatic zonal rotor. The principal organelles were characterized by their marker enzymes: cytosol (lactate dehydrogenase), plasma membrane (5′-nucleotidase), endoplasmic reticulum (neutral α-glucosidase), mitochondria (malate dehydrogenase), lysosomes (N-acetyl-β-glucosaminidase), peroxisomes (catalase). γ-Glutamyl transferase was exclusively localized to the plasma membrane. Leucine amino-peptidase, especially when assayed in the presence of Co²⁺, was also partially localized to the plasma membrane. Experiments with diazotized sulphanilic acid, a non-permeant enzyme inhibitor, showed that these plasma membrane enzymes are present on the cell surface. No detectable alkaline phosphatase was found in the lymphocytes. Acid phosphatase and β-glucuronidase were localized to lysosomes and there was some evidence for lysosomal heterogeneity. Leucine amino peptidase, optimal at pH 8.0, showed a partial localization to intracellular vesicles, possibly lysosomes, especially when assayed in the presence of EDTA. These studies provide a technique for determining the intracellular distribution of hitherto unassigned lymphocyte constituents and serve as a basis for investigating the cell pathology of lymphocytic disorders.     

Effect of chronic ethanol consumption on the cellular and subcellular distribution of gamma-glutamyltransferase in rat liver

After 6 weeks of chronic ethanol consumption hepatic gamma-glutamyl-transferase and -hydrolase activities increased compared with pair-fed controls. There was no change in 5'-nucleotidase activity. It was found that the increase in gamma-glutamyltransferase activity occurred exclusively in the parenchymal cells although the principal cellular localisation for this enzyme is the biliary tract in both control and ethanol-fed rats. In both groups of animals the gamma-glutamyltransferase activities were localised by analytical subcellular fractionation techniques to soluble, plasma membrane and canalicular fractions, but the plasma membrane activity was selectively increased in the ethanol-fed rats.     

Gamma-glutamyltransferase from azo dye induced hepatoma and fetal rat liver. Similarities in their kinetic and immunological properties.

Some properties of gamma-glutamyltransferase ((gamma-glutamyl)-peptide: amino-acid gamma-glutamyltransferase EC 2.3.2.2) from azo dye induced hepatoma and fetal rat liver were studied using kinetic and immunological criteria. There was no significant difference between the hepatoma enzyme and fetal rat liver enzyme in some of their catalytic properties. Antisera against the purified hepatoma enzyme also reacted to the fetal rat liver enzyme in the inhibition test and the precipitin reaction. A structural similarity between the hepatoma enzyme and fetal rat liver enzyme was observed and the acquirement of fetal characteristics in hepatoma was discussed.     

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.     

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.     

Latent form of glutathione reductase in the rat liver

The existence of membrane-bound forms of glutathione reductase in rat liver and transplantable hepatoma G-27 was demonstrated, using differential centrifugation techniques. The activity of the sedimentable form of the liver enzyme was detected only in the presence of detergents. Conditions for the manifestation of the latent glutathione reductase activity in whole liver homogenates and in the 105000 g pellet were determined. Solubilization of the latent form of the enzyme in the presence of sodium deoxycholate increases 2-fold the glutathione reductase activity in liver homogenates (but not in hepatoma). Simultaneous determination of the disulfidereductase, nonspecific NADPH-oxidase and gamma-glutamyltransferase (membrane-bound enzyme of glutathione metabolism) activities was performed.     

Labelling of the cyclitol permease in Klebsiella aerogenes.

The protein component(s) of the cyclitol-transport system in Klebsiella aerogenes has been labelled by using three different procedures. One method is based on differential alkylation with N-ethylmaleimide, and another on incorporation of amino acids during the induction process. A protein of mol.wt. 34 000 was labelled by both procedures; by alkylation with N-ethylmaleimide two other proteins of mol.wts. 55 000 and 67 000 were also labelled. The third uses diazotized [35S]sulphanilic acid after protection by substrate and the comparison of labelling of induced cells with non-induced cells; the label was also concentrated in a mol.wt.-33 000 peak. The labelled protein is, from the evidence, the cyclitol carrier.     

Studies on the leukotriene D4-metabolizing enzyme of rat leukocytes, which catalyzes the conversion of leukotriene D4 to leukotriene E4

Leukotriene D4-metabolizing enzyme was studied using rat neutrophils, lymphocytes and macrophages. These leukocyte sonicates converted leukotriene D4 to leukotriene E4. However, the leukotriene D4-metabolizing activity varied with cell type, and macrophages showed the highest activity among these leukocytes. The subcellular localization of the leukotriene D4-metabolizing enzyme of macrophages was examined, and the leukotriene D4-metabolizing activity was found to be present in the membrane fraction, but not in the nuclear, granular and cytosol fractions. When macrophages were modified chemically with diazotized sulfanilic acid, a poorly permeant reagent which inactivates cell-surface enzymes selectively, the leukotriene D4-metabolizing activity of macrophages decreased significantly (about 95%) without any inhibition of marker enzymes of microsome, cytosol, lysosome and mitochondria. When neutrophils and lymphocytes were modified with diazotized sulfanilic acid, the leukotriene D4-metabolizing activity was also inhibited about 90% by the modification. Among various enzyme inhibitors used, o-phenanthroline, a metal chelator, remarkably inhibited the leukotriene D4-metabolizing activity of leukocytes, and the o-phenanthroline-inactivated enzyme activity was fully reactivated by Co2+ and Zn2+. These findings seem to indicate that rat neutrophils, lymphocytes and macrophages possess the leukotriene D4-metabolizing metalloenzyme which converts leukotriene D4 to leukotriene E4, on the cell surface, although macrophages have a higher enzyme activity than the other two.     

Light form of Morris hepatoma gamma-glutamyltransferase

Using solubilization with bromelain, the light form of gamma-glutamyltransferase was purified from Morris hepatoma 5123D. Some properties of this enzyme were compared to those of the light form rat kidney GGT. Anthglutin and its isomer inhibit competitively the former enzyme but non-competitively the latter. On zymograms of rat control sera, five GGT fractions were noted, but in sera of rats with hepatoma 5123D also the light form and the increase of GGT activity ain region of fraction II were observed. Only these two enzyme fractions react with antibody anti heavy form of Morris hepatoma GGT.     

Gamma-glutamyltransferase activity in the human red blood cell membrane (author's transl)]

A gamma-glutamyltransferase activity is found in the human red blood cell membrane. Membrane isolation was carried out according to the method of Dodge et al. (Dodge, J. T., Mitchell, C. and Hanahan, J. (1963) Arch. Biochem. Biophys. 100, 119-130) (modified) and proteins were solubilized either with 1% sodium deoxycholate or 5 mM EDTA or 10 mM of its disodium salt, under various conditions of time and temperature. The gamma-glutamyltransferase activity of the membrane preparations was investigated using two substrates, gamma-L-glutamyl-p-nitroanilide and gamma-L-glutamyl-alpha-naphthylamide. The specific enzymatic activities of the various preparations, expressed in m units per mg of protein, were found to have similar values under similar technical conditions. The chelating agents seem to allow a more specific isolation than the detergent. The presence of a gamma-glutamyltransferase activity in the erythrocyte membrane is discussed in relation to the membrane association of this enzyme in other tissues.     

Purification and immunological characterization of a new form of gamma-glutamyltransferase of human semen.

A new form of gamma-glutamyltransferase was purified from human seminal plasma. The purified enzyme was composed of two non-identical subunits with apparent molecular masses of 150 and 95 kDa on polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS), and showed a molecular mass of 500 and 250 kDa on gel filtration in the absence and presence of 1% Triton X-100, respectively. This enzyme was different from human renal gamma-glutamyltransferase not only in apparent molecular masses, but also in amino acid compositions of both the subunits to each other. Experiments with the antisera raised against the purified enzyme revealed that the enzyme was different from the renal, hepatic and testicular enzymes in reactivity to the antibody though partially related to those enzymes. Ouchterlony double diffusion analysis indicated that both human seminal plasma and prostatic extract contained two types of gamma-glutamyltransferase, one is that we purified and the other the renal type. Hence, it is most likely that gamma-glutamyltransferase accounting for most of the enzyme activity in semen results from prostata followed by secretion to seminal plasma.     

Dependence of histochemical staining of leukocyte aminopeptidase upon its biochemical enzyme activity

The relationship between histochemical staining and biochemical activity of the enzyme was investigated using leukocytes with different aminopeptidase activities. In guinea-pig neutrophils and macrophages which have a relatively high enzyme activity, the histochemical staining correlated with the biochemical enzyme activity. On the other hand, guinea-pig lymphocytes and mouse neutrophils whose enzyme activities were 8.25 +/- 0.27 mU/10(7) cells and 6.18 +/- 0.87 mU/10(7) cells, respectively, were not stained by histochemical techniques. When guinea-pig neutrophils were modified chemically by diazotized sulfanilic acid at different concentrations, the histochemical staining of neutrophils decreased in proportion to the degree of inhibition of their biochemical enzyme activity and hardly became detectable below 10 mU/10(7) cells. However, guinea-pig neutrophils contained the soluble enzyme, corresponding to 5 mU/10(7) cells, which leaked out rapidly from cells during staining procedure, suggesting that the limit of visualization of the membrane-bound aminopeptidase activity by the histochemical techniques is about 5 mU/10(7) cells. The membrane-bound enzyme activities in guinea-pig lymphocytes and mouse neutrophils were 5 mU and 3 mU per 10(7) cells, respectively, and so it is possible that these leukocytes hardly stained histochemically.     

Nucleotidase activity in regenerating liver and in clonal strains of hepatoma and pituitary cells in culture.

A nucleotidase of the combined 3'- and 5'-type (nucleotide phosphohydrolase, E.C.3.1.3.31) was present in the cytosol of regenerating rat liver cells, and of rat hepatoma and pituitary cells in culture. The enzyme activity per milligram of cell protein was very similar in regenerating liver and in three of the different cell types. The hepatoma cell strain which showed the slowest growth rate had a three-fold higher basal enzyme activity. After the first days of regenerative growth in rat liver and during early plateau phase of cell growth, there was a 50-120% increase in specific enzyme activity. In the hepatoma cells, the enzyme activities were also compared to the cellular content and synthesis of RNA and DNA. The increase in enzyme activity occurred concomitantly with a reduced incorporation of 3H-thymidine into DNA. The possible physiological role of this nucleotidase in nucleic acid and nucleotide metabolism is discussed.