Development of leucine aminopeptidase activity during daylily flower growth and senescence

The possibility that exopeptidases, i.e. aminopeptidases and carboxypeptidases, in addition to the previously studied endopeptidase might also be developmentally regulated in daylily petals was examined. The level of leucine aminopeptidase and endopeptidase activities changed after the flower was fully open while that of carboxypeptidase activity remained relatively unchanged throughout senescence. Leucine aminopeptidase activity seemed to increase after the flower was fully open and peaked several hours earlier than endopeptidase did. Taken together, it is postulated that leucine aminopeptidase might play a role in protein turnover during flower opening and in the initiation of protein hydrolysis associated with petal senescence while the endopeptidase could be responsible for the breakdown of the bulk of proteins at the later stages. The drop in leucine aminopeptidase activity associated with the onset of daylily petal senescence was effectively halted by a cycloheximide treatment of cut daylily flowers for 24 h which was previously shown to prolong the vase life of the flowers and prevent protein loss from the petals. Apart from both being developmentally regulated in daylily petals, the leucine aminopeptidase activity and the previously studied endopeptidase are different in several aspects. They appear to have different pH optima, 8 for leucine aminopeptidase and 6.2 for endopeptidase. Unlike the endopeptidase activity, no new leucine aminopeptidase isozymes appeared during petal senescence, and the leucine aminopeptidase did not appear to belong to the cysteine class of proteolytic enzymes.     

Serum leucine aminopeptidase for monitoring viral infections with plasmacytoid reaction

Analysis of data on 9 cases with active cytomegalovirus infection in patients with kidney grafts showed a positive association of serum leucine aminopeptidase activity concentration with the appearance of plasmacytoid lymphocytes in blood. Additional studies indicate that like the liver, the lymphocytes contain leucine aminopeptidase in relatively large quantities and that this enzyme is increased about 3-fold in plasmacytoid lymphocytes when compared with the activity in normal lymphocytes. In contrast, the 'hepatic' enzyme alanine aminotransferase is practically absent in both lymphocytes and plasmacytoid cells. Therefore, the difference in serum between the relative increases of leucine aminopeptidase and alanine aminotransferase may be attributed to proliferating plasmacytoid lymphocytes. Earlier observations on a large number of cases of acute viral hepatitis A or B lend credence to this assumption. However, in this disease, the serum enzyme changes reflect the much greater involvement of the liver and the relatively slight, but significant, proliferation of plasmacytoid lymphocytes. Our hypothesis is confirmed by the recent observation of 3 cases of acute EBV infection (infectious mononucleosis) in otherwise healthy individuals showing greatly elevated leucine aminopeptidase in contrast to normal or slightly raised alanine aminotransferase in serum.     

Alteration in the regulation of plasma membrane glycoproteins of the hepatocyte during ontogeny

The expression of four integral membrane glycoproteins was examined in detail utilizing monospecific antibodies during liver development. These included asialoglycoprotein receptor, a hepatocyte glycoprotein residing in the sinusoidal domain, and three bile canalicular glycoproteins, leucine aminopeptidase, dipeptidyl peptidase IV, and a Mr 110,000 glycoprotein denoted GP 110. It was observed that asialoglycoprotein receptor, GP 110, and dipeptidyl peptidase IV were present in low amounts in fetal liver and reached adult levels between 1 to 3 weeks. In contrast, leucine aminopeptidase was present in nearly adult amounts in 18-day-old fetal livers. These observations were qualitatively confirmed by indirect immunofluorescent staining of frozen thin liver sections obtained from fetal and adult rats. Further, in fetal livers it was found that leucine aminopeptidase was not localized to typical bile canalicular areas. Immunoprecipitation studies performed in the presence of proteolytic inhibitors using detergent-solubilized extracts of metabolically labeled liver minces revealed that GP 110 was present in low amounts as Mr 110,000 and Mr 105,000 polypeptides in 17-day fetal livers but by 21 days of gestation the larger polypeptide was the major synthesis product. Conversely, the apparent molecular weights of leucine aminopeptidase and dipeptidyl peptidase IV were not altered during development. Experiments determining relative rates of synthesis using excess amounts of antibodies showed that the concentrations of the three bile canalicular glycoproteins in liver during ontogeny reflect their rates of synthesis. These results underscore that plasma membrane constituents of the hepatocyte undergo dramatic changes in expression and localization as the liver changes its physiological role at birth.     

Changes in plasma membrane enzyme activities during liver regeneration in the rat.

Changes in activities of plasma membrane enzymes during liver regeneration may be related to the maintenance of hepatic function or to the regulation of cell proliferation. Plasma membranes were isolated from rat livers at various times after partial hepatectomy, and the specific activities of alkaline phosphatase, (Na+ + K+)-ATPase, leucine aminopeptidase, 5'-nucleotidase, and adenylate cyclase (basal and with glucagon or epinephrine) were measured. Alkaline phosphatase and (Na+ + K+)-ATPase activity increased 3.6-fold and 2-fold respectively, during the first 48 h after partial hepatectomy. The time of onset and duration of change suggest that these increases in activity are involved in the maintenance of bile secretion. Decreases in leucine aminopeptidase activity at 48--108 h and in 5'-nucleotidase activity at 12--24 h were observed, which may be involved in the restoration of protein and accumulation of RNA. The basal activity of adenylate cyclase increased after partial hepatectomy. The response of adenylate cyclase to epinephrine showed a transitory increase between 36 and 108 h after surgery, while the response to glucagon was decreased by approximately 50% at all time points through 324 h after surgery. These changes in the hormone responsiveness of adenylate cyclase are similar to those previously observed in fetal and preneoplastic liver.     

Possible exposure of leucine aminopeptidase on the cell surface of rabbit blood neutrophils by digitonin treatment

The sensitivity of leucine aminopeptidase to diazotized sulfanilic acid (DSA) was compared between neutrophils from blood and peritoneal exudates of rabbit. The leucine aminopeptidase activity of peritoneal neutrophils was inhibited about 40% by DSA, whereas that of blood neutrophils was not inhibited at all by the reagent. However, pretreatment of blood neutrophils with digitonin in the presence and in the absence of divalent cations rendered leucine aminopeptidase sensitive to DSA to the same extent as peritoneal neutrophils, without affecting the cell viability and lactate dehydrogenase activity. These findings seem to indicate that the leucine aminopeptidase of blood neutrophils, which is normally inaccessible to DSA, was exposed on the cell surface by digitonin treatment.     

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.     

Na+,K+-ATPase activity and potassium, sodium and calcium levels in the rat myocardium and brain during pain-induced emotional stress

An emotional-algesic stress in the period of its development and after-effect causes a different-directed influence on the state of the ionic transport in the heart and brain of rats. The Na+, K+-ATPase activity in the left ventricle of the heart decreases with a simultaneous increase in the sodium content and decrease in the calcium and potassium content. The Na+, K+-ATPase activation is observed in the cortex of cerebral hemispheres with a simultaneous increase in the content of mentioned ions.     

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.     

Bacterial Colonization and Ectoenzymatic Activity in Phytoplankton-Derived Model Particles: Cleavage of Peptides and Uptake of Amino Acids

Abstract Phytoplankton-derived model particles were created in laboratory from a mixture of autoclaved diatom cultures. These particles were colonized by a marine bacterial community and incubated in rolling tanks in order to examine the relationship between aminopeptidase activity and leucine uptake. Bacteria inhabiting particles and ambient water were characterized for abundance, biovolume, aminopeptidase activity, leucine uptake, and growth rate. Particles were a less favorable habitat than ambient water for bacterial growth since growth rates of particle-attached bacteria were similar or even lower than those of free-living bacteria. During the first ∼100 h of the particle decomposition process, there were not statistically significant differences in the aminopeptidase activity:leucine uptake ratio between attached and free-living bacteria. From ∼100 h to ∼200 h, this ratio was higher for attached bacteria than for free-living bacteria. This indicates an uncoupling of aminopeptidase activity and leucine uptake. During this period, attached and free-living bacteria showed similar hydrolytic activities on a cell-specific basis. In the free-living bacterial community, variations in aminopeptidase activity per cell were associated with variations in leucine uptake per cell and growth rates. However, in the attached bacterial community, when leucine uptake and growth rates decreased, aminopeptidase activity remained constant. Thus, after ∼100 h, particle-attached bacteria were not taking advantage of their high aminopeptidase activity; consequently the hydrolysed amino acids were released into the ambient water, supporting the growth of free-living bacteria. These results demonstrate that over the particle decomposition process, the relationship between hydrolysis and uptake of the protein fraction shows different patterns of variation for attached and free-living bacterial communities. However, in our experiments, this uncoupling was not based on a hyperproduction of enzymes by attached bacteria, but on lower uptake rates when compared to the free-living bacteria. Received: 4 February 1997; Accepted: 9 May 1997     

Intramolecularly-quenched fluorescent peptides as fluorogenic substrates ofleucine aminopeptidase and inhibitors of clostridial aminopeptidase.

Fluorogenic oligopeptide derivatives of the type Lys(ABz)-ONBzl, where ABz iso-aminobenzoyl (anthraniloyl), X stands for Ala Phe, or Ala-Ala, and ONBzlis p-nitrobenzyloxy, were synthesized and shown to be hydrolyzed by leucine aminopeptidase. The hydrolysis is accompanied by an increase in fluorescence due to disruptionof the intramolecular quenching of the fluorescent anthraniloyl moiety by the nitrobenzyester group. The spectral characteristics of the compounds are not consistent withan energy transfer mechanism according to F?rster, therefore the quenching isassumed to be caused by a direct encouter between the quenching and the fluorecentgroups. The change in fluorescence that accompanies the enzymic hydrolysis ofthe first peptide bound was used for quantitative measurement of the activity ofthe activity of leucine aminopeptidase and for the determination of some of itskinetic parameters. A bacterial aminopeptidase from Clostrdium histolyticumthat is very similar to leucine aminopeptidase in its substrate specificity inits substrate specificity did not hydrolyze the above peptidederivatives. Thehydrolysis of leucine p-nitroanilide by this enzyme was found to be inhibitedby the three peptides and the corresponding inhibition constants were determined.     

beta-Endorphin: structure-activity relationships in the guinea pig ileum and opiate receptor binding assays.

The opiate activities of some derivatives and enzymatic digests of camel and human β-endorphin were determined in the guinea pig ileum and rat brain opiate receptor binding assays. Derivatives of β-endorphins altered within the amino-terminal five residues showed pronounced losses in activity. Anisylation of the C-terminal glutamic acid residue of β_h-endorphin produced only small reductions in activity. Chymotryptic digestion greatly weakened the opiate activities of β_h-endorphin, whereas carboxypeptidase A, tryptic and leucine aminopeptidase digests showed only small losses in potency. The C-terminus of β-endorphin appears to contribute little directly to opiate activity. Amino acid analysis and assay of the leucine aminopeptidase digests suggest that the larger potency of β-endorphin relative to Met-enkephalin may be a consequence of its greater resistance to exopeptidase attack.     

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.     

Dexamethasone selectively regulates the activity of enzymatic markers of cerebral endothelial cell lines

Summary Two endothelial cell lines were derived from grafts of the central nervous system using retrovirus mediated gene transfer to introduce the polyoma middle-T oncogene into fetal rat brain endothelial cells and transplantation of these cells into adult rat brain. In this report, we further characterize these cells and the effect of dexamethasone on the expression of specific enzymatic markers. These cells take up acetylated low density lipoprotein, leucine, and glucose, and express Factor VIII-related antigen, angiotensin converting enzyme, alkaline phosphatase, gamma-glutamyltranspeptidase, and as yet undescribed aminopeptidase A and B-like enzymes. When grown on semi-permeable membranes, these transformed cells do not spontaneously retain small hydrophilic molecules. In culture, one of the lines (EC 193) forms a confluent monolayer of spindle-shaped cells homogenously expressing gamma-glutamyltranspeptidase at a level comparable to primary cells. The other cell line (EC 219) grows as clusters of elongated cells, and gamma-glutamyltranspeptidase activity is expressed mainly in cells forming the clusters. This clustered pattern changes to a confluent one after culture on type-I collagen. Dexamethasone increases angiotensin-converting enzyme activity, and decreases the expression of gamma-glutamyltranspeptidase and aminopeptidase A, whereas the aminopeptidase B activity is little modified. Inhibition of aminopeptidase A activity by amastatin, potentiates angiotensin II effects on DNA synthesis. These results indicate that retrovirally transformed brain endothelial cells are a useful model for studying the blood-brain barrier in vitro and that dexamethasone, an agent with the potential to reduce brain edema, directly affects some blood-brain barrier properties in these endothelial cell lines.     

Purification and Identification of a Novel Leucine Aminopeptidase from <Emphasis Type="Italic">Bacillus thuringiensis israelensis</Emphasis>

A novel leucine aminopeptidase was purified from a Bacillus thuringiensis israelensis (Bti) culture. The purification stages included heating the concentrated supernatant to 65°C for 90 min, anion-exchange chromatography by DEAE cellulose, and hydrophobic chromatography by phenyl Sepharose. The specific activity of leucine aminopeptidase after the hydrophobic chromatography increased by 215.5-fold and the yield was 16%. The molecular weight of the active enzyme was 59 kDa. Mass spectrometry analysis of the 59-kDa leucine aminopeptidase revealed that this protein has at least 41% homology with the cytosol leucine aminopeptidase produced by Bacillus cereus. Maximal leucine aminopeptidase activity occurred at 65°C, pH 10 toward leucine as the amino acid terminus. The enzyme was strongly inhibited by bestatin, dithiothreitol, and 1,10-phenanthroline, indicating that the enzyme might be considered as a metallo-aminopeptidase that has disulfide bonds at the catalytic site or at a region that influences its configuration. Examination of the purified leucine aminopeptidase’s effect on the activation of the protoxin Cyt1Aa from Bti revealed that when it acts synergistically with Bti endogenous proteases, it has only a minor role in the processing of Cyt1Aa into an active toxin.     

Aminopeptidase activity in the livers of rats with experimental chronic renal failure

Membrane-bound aminopeptidase activities in livers of rats with experimental renal failure were assayed. Only aminopeptidase A activity was decreased with the reduction in renal function, but aminopeptidase B and Leu-aminopeptidase activity did not change. The liver membrane-bound aminopeptidase A activity was inhibited by the addition of angiotensin I or -II in the enzyme assay system. From these results, it is expected that a decrease in liver membrane-bound aminopeptidase A activity may play a role in increasing angiotensin II during renal failure.     

Expression of proteolytic enzymes during Dictyostelium discoideum spore germination

The specific activity of cathepsin B-like, cathepsin D-like, and leucine aminopeptidase enzymes was measured in dormant, aging, and germinating spores of wild-type and mutant Dictyostelium discoideum.The activity of leucine aminopeptidase was relatively constant during spore aging and spore germination. The level of cathepsin D-like activity was highest in young dormant spores but decreased during germination or aging.The level of cathepsin B-like activity remained constant in wild-type spores which were aged for 13 days. The dormant spores of spontaneous germination mutants initially contained low levels of cathepsin B-like activity which increased during aging. Thus, there was no correlation between the level of endogenous cathepsin B activity and the ability to be autoactivated or heat-activated. The level of cathepsin B-like activity does not have a role in the generation of energy for the swelling stage of germination. Finally, the combined level of endogenous and exogenous cathepsin B activity increased more than 20-fold during the emergence of myxamoebae suggesting that the enzyme(s) may play a role at this development stage of germination.     

Degradation of Melanotropin Inhibiting Factor by Brain

Degradation of melanotropin inhibiting factor (MIF) was measured by fluorometry, using pareptide as an internal standard, following the separation of the dansyl derivatives of MIF and its metabolites by HPLc. MIF was not split by carboxypeptidases A and B, prolidase, or pyroglutamate aminopeptidase. It was hydrolyzed by leucine aminopeptidase, aminopeptidase M, and carboxypeptidase Y. Rat brain hydrolyzed 159 nmol of MIF per mg of protein per h; the activity was linear with enzyme concentration. Hydrolysis start from the N-terminal end, as shown by the appearance of proline as the first metabolite of the MIF degradation, followed by leucine, glycinamide, leucylglycine, and glycine. Activity in the rat brain regions was in the order striatum, medulla oblongata > cortex, hippocampus, midbrain > hypothalamus, cerebellum, and pituitary. The enzyme was mostly in the supernatant, with significant amounts in the myelin and synaptosomal fractions. MIF aminopeptidase could be separated from carboxypeptidase by centrifugation at 30,000 x g for 20 min and precipitation with 45--75% (NH4)2SO4. It showed pH optima in the alkaline range (8.25 and 8.75) and was inhibited by EDTA, EGTA, SQ 14,225, puromycin, bacitracin, and bestatin.     

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.     

Enzymatic and protein patterns at different developmental stages in the cherry fruit fly Rhagoletis cerasi L.

Protein patterns and two isoenzyme systems (leucine aminopeptidase, LAP, and alanine aminopeptidase, AAP) were examined in the cherry fruit fly Rhagoletis cerasi L. at different stages of ontogeny LAP activity was quantitatively determined.Differences were observed in protein patterns for puparia in diapause, post-diapause and at adult emergence. Some LAP and AAP isoenzymes appear in post-diapause which had not been observed during diapause. It is suggested that there is a direct link between variations in protein patterns and proteolytic activity.     

Changes in plasma membrane enzyme activities during liver regeneration in the rat

Changes in activities of plasma membrane enzymes during liver regeneration may be related to the maintenance of hepatic function or to the regulation of cell proliferation. Plasma membranes were isolated from rat livers at various times after partial hepatectomy, and the specific activities of alkaline phosphatase, (Na⁺ + K⁺)-ATPase, leucine aminopeptidase, 5′-nucleotidase, and adenylate cyclase (basal and with glucagon or epinephrine) were measured. Alkaline phosphatase and (Na⁺ + K⁺)-ATPase activity increased 3.6-fold and 2-fold respectively, during the first 48 h after partial hepatectomy. The time of onset and duration of change suggest that these increases in activity are involved in the maintenance of bile secretion. Decreases in leucine aminopeptidase activity at 48–108 h and in 5′-nucleotidase activity at 12–24 h were observed, which may be involved in the restoration of protein and accumulation of RNA. The basal activity of adenylate cyclase increased after partial hepatectomy. The response of adenylate cyclase to epinephrine showed a transitory increase between 36 and 108 h after surgery, while the response to glucagon was decreased by approximately 50% at all time points through 324 h after surgery. These changes in the hormone responsiveness of adenylate cyclase are similar to those previously observed in fetal and preneoplastic liver.