Specific Activities Of Leucine Aminopeptidase Research Articles

Testing the adaptive plasticity of gypsy moth digestive enzymes in response to tannic acid using phenotypic selection analysis

The adaptive significance of plasticity of digestive enzyme responses to allelochemical stress was tested on 32 full-sib gypsy moth families from an oak forest (the Quercus population) and 26 families from a locust-tree forest (the Robinia population), reared on control or tannic acid-supplemented diets. By using the relative growth rate as a fitness measure in phenotypic selection analyses, we revealed that higher specific activity of leucine aminopeptidase in Quercus larvae and lower specific activity of trypsin in Robinia larvae were adaptive in the control environment. In Quercus larvae, elevated specific activities of leucine aminopeptidase and lipase were adaptive in the stressful environment. There were no plasticity costs for the enzyme activities in either experimental group. The obtained results suggest that adaptive plasticity of digestive enzyme activity in gypsy moth larvae contributes to optimal growth rate under various environmental conditions.

Adaptive phenotypic plasticity of gypsy moth digestive enzymes

Abstract The adaptiveness of plasticity of digestive enzyme responses to allelochemical stress was tested on 32 full-sib families of gypsy moth larvae from an oak forest population (the Quercus population) and 26 families from a locust-tree forest (the Robinia population), reared either on control diet, or on tannin-supplemented diet. Using the duration of larval development as an indirect measure of fitness, phenotypic selection analyses revealed that lower specific activities of total proteases and trypsin, and higher specific activity of leucine aminopeptidase were adaptive for both populations in the control environment. Plasticity was only shown to be costly for total proteases and trypsin activity in Quercus larvae. In a stressful environment, the most apparent adaptive response was a significant increase in lipase activity. There was no plasticity cost for lipase activity. The two populations differed in the direction of selection acting on α-glucosidase activity, which favoured decreased activity in Quercus larvae and increased activity in Robinia larvae in the control environment. α-glucosidase activity in Quercus larvae is characterized by cost of homeostasis, while cost of plasticity was shown for Robinia larvae. The results obtained on the plasticity of digestive enzyme activity indicate how this generalist species copes with variation in plant allelochemicals.

Effects of tannic acid on trypsin and leucine aminopeptidase activities in gypsy moth larval midgut

The effects of allelochemical stress on genetic variations in the specific activities of gypsy moth digestive enzymes (trypsin and leucine aminopeptidase) and relative midgut mass (indirect measure of food consumption), as well as variability in their plasticity, were investigated in fifth instar gypsy moths originating from two populations with different trophic adaptations (oak and locust-tree forests). Thirty-two full-sib families from the Quercus population and twenty-six full-sib families from the Robinia population were reared on an artificial diet with or without supplementation with tannic acid. Between population differences were observed as higher average specific activity of trypsin and relative midgut mass in larvae from the Robinia population. Significant broad-sense heritabilities were observed for the specific activity of trypsin in the control state, and for specific activity of leucine aminopeptidase in a stressful environment. Significantly lower heritability for relative midgut mass was recorded in larvae from the Robinia population reared under stressful conditions. Significant variability of trypsin plasticity in larvae from both populations and significant variability of leucine aminopeptidase plasticity in larvae from the Robinia population point to the potential for the evolution of enzyme adaptive plastic responses to the presence of stressor. Non-significant across-environment genetic correlations do not represent a constraint for the evolution of enzyme plasticity.

Ontogeny of the digestive tract and enzymatic activity in white seabass, Atractoscion nobilis, larvae

The development of the digestive system and digestive enzyme activity in white seabass larvae, Atractoscion nobilis, were analyzed from hatching until 40 days post hatch (dph) using histological and biochemical approaches. The development of the digestive system in A. nobilis larvae was similar to that reported for other marine fish species. Larvae at 3 dph (0.55 ± 0.001 mg wet weight and 3.6 ± 0.02 mm total length), cultured at 18 °C in seawater, presented all the structures (i.e. differentiation of the alimentary canal into the buccopharynx, esophagus, anterior and posterior intestines, pancreas with zymogen granules, liver, gall bladder and open mouth) necessary for the digestion and absorption of nutrients such as proteins and lipids (primarily). At this time, the larvae had fully-developed digestive systems that allowed them to digest inert feed and to absorb nutrients throughout the intestine walls. On the other hand, most digestive enzyme activities were detected at the moment of hatching. Trypsin activity was 0.80 ± 0.16 mU/mg protein at 1 dph (0.51 ± 0.001 mg wet weight larvae), and increased gradually during the following days, but most notably after the initial exogenous feeding at 4 dph. The specific activity of chymotrypsin was 7.21 ± 1.29 mU × 10 − 4 /mg protein at 1 dph and reached peak level (15.9 ± 1.02 mU × 10 − 4 /mg protein) at 18 dph (6.6 ± 0.003 mg wet weight larvae). The specific activity of leucine aminopeptidase increased continuously from 1.31 ± 0.05 mU × 10 − 3 /mg protein at 1 dph to 15.91 ± 0.40 mU × 10 − 3 /mg protein at 18 dph. The activity of α-amylase at 1 dph was 1.35 ± 0.09 U/mg protein, increasing to 8.07 ± 0.98 U/mg protein at 16 dph. The activity of pepsin was detected at a very low level (0.71 ± 0.53 U/mg protein) at 10 dph, and a stepwise increase in activity was observed between 16 and 20 dph, reaching maximum level (13.92 ± 0.09 U/mg protein) at 40 dph. These results indicate that the digestive tract develops rapidly in this species and that the stomach becomes functional between 16 and 18 dph. It should, therefore, be possible to start weaning the fish at this young age.

Purification and Identification of a Novel Leucine Aminopeptidase from Bacillus thuringiensis israelensis

A novel leucine aminopeptidase was purified from a Bacillus thuringiensis israelensis (Bti) culture. The purification stages included heating the concentrated supernatant to 65 degrees 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 degrees 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.

Nutritive stress effects on growth and digestive physiology of Lymantria dispar larvae

The effects of parental and offspring diet on larval growth, food consumption and utilization, and activities of three digestive enzymes (a-amylase trypsin, leucine aminopeptidase) were examined in extremely polyphagous insect, the gypsy moth (Lymantria dispar). In parental generation, gypsy moth larvae were reared on oak (Quercus cerris) leaves as optimal host or beech (Fagus silvatica) leaves which contains flavonoids and alkaloids. In offspring generation, after molting into the 4th instar, they were either switched from oak to beech or remained on oak leaves. Decreased growth and food utilization efficiency, increased assimilation efficiency and activities of a-amylase and trypsin were recorded in larvae switched to beech leaves. Significant parental effects were demonstrated for fourth instar duration, weight of fifth instar larvae and specific activity of leucine aminopeptidase. Physiological, ecological and evolutionary context of the obtained results were stressed in the present paper.

Isolation and characterization of canalicular and basolateral plasma membrane fractions from human liver

A method is described for the isolation of subfractions from human liver plasma membranes, enriched in canalicular domains (cLPM) and basolateral domains (blLPM), respectively, and the results are compared to those obtained with rat liver. The studies were performed in 18 human livers. The cLPM (isolated at densities 1.103–1.127 for human and 1.036–1.127 for rat cLPM) from human as well as rat liver showed a lower density than the blLPM (1.141–1.161 for human and 1.151–1.172 for rat biLPM). Human and rat blLPM were characterized by increased levels of (Na +/K +)-ATPase (relative enrichment 33 and 21, respectively). Both human and rat cLPM showed high specific activities of leucine aminopeptidase; relative enrichment factors were 42 and 31, respectively. Mg 2+-ATPase and alkaline phosphatase, specific canalicular enzymes in rat liver, were only sligtly enriched in the cLPM of human liver, which indicates that these enzymes are not suitable as marker enzymes for human liver cLPM. Both cLPM and bILPM of human and rat origin were only slightly contaminated with mitochondria, lyposomes, Golgi membranes and endoplasmic reticulum. Total recoveries of cLPM and blLPM were 0.02 mg protein/g liver each for the human membrane preparations, compared to 0.07 and 0.16 mg protein/g liver for the membranes prepared from rat liver. Analysis of membrane fluidity revealed that the human liver cLPM were more rigid than blLPM (mean difference in fluorescence polarization P DPH 0.024). They contained more cholesterol (0.43 vs. 0.30 μmol/mg protein) and phospholipids (0.54 vs. 0.39 μmol/mg protein, respectively), which was compatible to rat liver plasma membrane fractions. This study shows that besides similarities, there are several differences between human and rat liver plasma membrane fractions.

Age-related change in brush borders of rat kidney cortex

Age-related change of rat renal brush borders was examined with electron microscopy and biochemical procedures. Total activity of renal brush borders enzymes, such as alkaline phosphatase and leucine aminopeptidase in the homogenate, was significantly decreased wiht age. Acid phosphatase activity and protein content were not significantly changed with age. Specific activity of leucine aminopeptidase in brush border fraction was significantly decreased at a later stage of age. Protein content of brush border fraction was decreased significantly with age. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, some of the proteins disappeared during aging. Electron microscopic observations of kidney cortex showed that microvilli of renal brush borders from old rats were observed to be fewer than those from the young; epithelial cells in young rats have more densely packed brush border projections than those in old rats. From these results, it is suggested that during aging renal brush borders are degraded, and that protein components of the brush borders were different between old and young.

Regulation of bile salt transport in rat liver. Evidence that increased maximum bile salt secretory capacity is due to increased cholic acid receptors.

Expansion of the bile salt pool size in rats increases maximum excretory capacity for taurocholate. We examined whether increased bile salt transport is due to recruitment of centrolobular transport units or rather to adaptive changes in the hepatocyte. Daily sodium cholate (100 mg/100 g body wt) was administered orally to rats. This treatment was well tolerated for at least 4 d and produced an 8.2-fold expansion of the bile salt pool. This expanded pool consisted predominently (99%) of cholic and deoxycholic acids. Significantly increased bile salt transport was not observed until 16 h after bile acid loading, and maximum elevations of transport capacity to 2.3-fold of control required approximately 2 d. In contrast, maximum sulfobromophthalein excretion rates increased 2.2-fold as early as 4 h and actually fell to 1.5-fold increase at 4 d. We studied the possibility that this adaptive increase in bile salt secretory transport was due to changes in canalicular surface membrane area, lipid composition, or increased number of putative carriers. Canalicular membrane protein recovery and the specific activities of leucine aminopeptidase, Mg(++)-ATPase and 5'-nucleotidase activities were unaltered by bile salt pool expansion. The content of free and esterified cholesterol and total phospholipids was unchanged in liver surface membrane fractions compared with control values. In contrast, sodium cholate administration selectively increased specific [(14)C]cholic acid binding sites twofold in liver surface membrane fractions. Increased numbers of [(14)C]cholic acid receptors (a) was associated with the time-dependent increase in bile salt transport, and (b) was selective for the taurine conjugate of cholate and (c) was reduced by chenodeoxycholate. Changes in bile acid binding sites 16 h following taurocholate and chenodeoxycholate and the lack of change with glycocholate was associated with comparable changes in bile salt transport. In conclusion, selective bile salts increase bile salt transport in the liver through an adaptive increase in the density of putative bile acid carriers in liver surface membrane.

Cell membrane enzymes: L-gamma-glutamyl transpeptidase, leucine aminopeptidase, maltase and trehalase in normal and leukaemic lymphocytes.

Several cell-membrane enzymes, which serve functions in amino acid and sugar transport, were measured in peripheral blood lymphocytes from chronic B and T lymphocytic disorders, blast cells in acute leukaemias, and in normal lymphocytes from cord blood, peripheral blood of adults, tonsils and bone-marrow plasma cells in myelomatosis. The specific activities of L-gamma-glutamyl transpeptidase, maltase and trehalase were low, as compared with those measured in normal blood lymphocytes, in the acute leukaemias and in the chronic B-cell disorders. In myelomatosis and in the chronic T-cell disorders, the specific activity of these three enzymes was in the normal range or above normal. The specific activity of leucine aminopeptidase was low in all the chronic B-cell disorders and in some of the lymphoblastic leukaemias. It was elevated in Sézary syndrome cells but low in T-chronic lymphocytic leukaemia. All four enzymes were lower than normal in cord blood lymphocytes and higher than normal in tonsils. These findings are discussed in relation to the patterns of lymphoid cell differentiation and maturation in normal tissues and in leukaemic states.