Ontogeny of enzymatic activities in fed and fasting turbot, Scophthalmus maximus L.

The presence and localization of acid and alkaline phosphatase, non-specific proteases, aminopeptidase, amylase, non-specific esterase and lipase was investigated by histoenzymologic methods in fed and fasting turbot from day 1 to day 40 post-hatching and compared with published data. Alkaline phosphatase and aminopeptidase activities were delected at day 1 in the distal region of the developing digestive tube. At day 3 (opening of the mouth) aminopeptidase and alkaline phosphatase activities were found all along the intestine. Sites of non-specific esterase and protease activities became apparent in the digestive tract at days 2 and 3 respectively. Amylase was present in the exocrine pancreas at day 3 and in the lumen of the intestine at day 4. Acid phosphatase was active in the cellular structure surrounding the yolk stores and in the lipid droplets at day 1 and in the intestinal epithelium at day 3. Lipase was found at day 15 when the larvae metamorphose into juveniles.
All the investigated enzymes were detected in fasting animals, except for lipase. However, the intensities of the enzymatic activities were weaker in the fasting specimens relative to the fed specimens between days 7 and 10.     

Regulation of JH epoxide hydrolase versus JH esterase activity in the cabbage looper, Trichoplusia ni, by juvenile hormone and xenobiotics

JH III esterase and JH III epoxide hydrolase (EH) in vitro activity was compared in whole body Trichoplusia ni homogenates at each stage of development (egg, larva, pupa and adult). While activity of both enzymes was detected at all ages tested, JH esterase was significantly higher than EH activity except for day three of the fifth (last) stadium (L5D3). For both enzymes, activity was highest in eggs. Adult virgin females had 4.6- and 4.0-fold higher JH esterase and EH activities, respectively, than adult virgin males. JH III metabolic activity also was measured in whole body homogenates of fifth stadium T. ni that were fed a nutritive diet (control) or starved on a non-nutritive diet of alphacel, agar and water. With larvae that were starved for 6, 28 and 52 h, EH activity per insect equivalent was 48%, 5% and 1%, respectively, of the control insects. At the same time points, JH esterase activity levels in starved T. ni were 29%, 4% and 3% of that of insects fed the nutritive diet. Selected insect hormones and xenobiotics were administered topically or orally to fifth stadium larvae for up to 52 h, and the effects on whole body EH and JH esterase activity analyzed. JH III increased the JH III esterase activity as high as 2.2-fold, but not the JH III EH activity. The JH analog, methoprene, increased both JH esterase and EH activity as high as 2.5-fold. The JH esterase inhibitor, 3-octylthio-1,1,1-trifluoropropan-2-one (OTFP), had no impact on EH activity. The epoxides trans- and cis-stilbene oxide (TSO and CSO) in separate experiments increased the EH activity approximately 2.0-fold. TSO did not alter JH esterase levels when topically applied, but oral administration reduced activity to 70% of the control at 28 h, and then increased the activity 1.8-fold at 52 h after the beginning of treatment. CSO had no effect on JH esterase activity. Phenobarbital increased EH activity by 1.9-fold, but did not change JH esterase levels. Clofibrate and cholesterol 5alpha,6alpha-epoxide had no effect on EH. JH esterase activity also was not affected by clofibrate, but cholesterol 5alpha,6alpha-epoxide reduced the JH esterase activity to 60-80% of the control. The biological significance of these results is discussed.     

Structural variations in the alanine-rich antifreeze proteins of the pleuronectinae

The sequence and activity of antifreeze proteins from two right eye flounder species were compared to assess the influence of structural variations on antifreeze capacity. The cDNA encoding the major serum antifreeze protein in the yellowtail flounder (Limanda ferruginea) was cloned from liver tissue. Its DNA sequence shows that the precursor to the antifreeze is a 97-residue preproportion. Edman degradation identified the N-terminus of the 48-amino-acid mature serum antifreeze protein and confirmed the sequence of the first 36 residues. A comparison with the previously determined winter flounder antifreeze protein and mRNA sequences shows strong homology through the 5' and 3' untranslated regions and in the peptide region. The mature protein section has the greatest sequence variation. Specifically, the yellowtail antifreeze protein, in contrast to that of the winter flounder, contains a fourth 11-amino-acid repeat and lacks several of the hydrophilic residues that have been postulated to aid in the binding of the protein to ice crystals. Intramolecular salt bridges are present in the antifreeze proteins from both species but in different registries with respect to the 11-amino-acid repeats. On a mass basis the yellowtail flounder antifreeze, though longer than that of the winter flounder, is only 80% as effective at depressing the freezing temperature of aqueous solutions. This lower activity might be due to the reduced number of hydrophilic ice-binding residues per molecule.     

Studies on the enzymology of purified preparations of brush border from rabbit kidney

1. A method for the preparation of brush border from rabbit kidneys is described. Contamination by other organelles was checked by electron microscopy and by the assay of marker enzymes and was low. 2. Seven enzymes, all hydrolases, were substantially enriched in the brush-border preparation and are considered to be primarily located in this structure. They are: alkaline phosphatase, maltase, trehalase, aminopeptidase A, aminopeptidase M, gamma-glutamyl transpeptidase and a neutral peptidase assayed by its ability to hydrolyse [(125)I]iodoinsulin B chain. 3. Adenosine triphosphatases were also present in the preparation, but showed lower enrichments. 4. Alkaline phosphatase was the most active phosphatase present in the preparation. The weak hydrolysis of AMP may well have been due to this enzyme rather than a specific 5'-nucleotidase. 5. The two disaccharidases in brush border were distinguished by the relative heat-stability of trehalase compared with that of maltase. 6. The individuality of the four peptidases was established by several means. The neutral peptidase and aminopeptidase M, both of which can attack insulin B chain, differed not only in response to inhibitors and activators but also in the inhibitory effect of a guinea-pig antiserum raised to rabbit aminopeptidase M. This antiserum inhibited both the purified and the brush-border activities of aminopeptidase M. The neutral peptidase and gamma-glutamyl transpeptidase were unaffected but aminopeptidase A was weakly inhibited. The characteristic responses to Ca(2+) and serine with borate served to distinguish aminopeptidase A and gamma-glutamyl transpeptidase from other peptidases. 7. No dipeptidases, tripeptidases or carboxypeptidases were identified as brush-border enzymes. 8. Incubation of brush border with papain released almost all the aminopeptidase M activity but only about half the activities of maltase, gamma-glutamyl transpeptidase and aminopeptidase A. No release of alkaline phosphatase, trehalase or the neutral peptidase was observed.     

Comparison of routine oxygen consumption rates of three species of pleuronectids at three temperatures

The oxygen consumption rates of three species of pleuronectids, the yellowtail flounder, Pleuronectes ferrugineus (Storer), the winter flounder, Pleuronectes americanus (Walbaum), and the American plaice, Hippoglossoides platessoides (Fabricius), were examined under simulated, land-based, aquaculture conditions. Routine oxygen consumption (ROC) rates for groups of each species were measured simultaneously using single-pass, flow-through respirometry. This study was conducted over three seasons at temperatures from 2°C to 14°C. An analysis of variance identified a significant interaction between temperature and species on the oxygen consumption rates of these flounder. The analysis indicated that at each temperature, ROC rates were significantly different among the three species (P < 0.05). A subsequent test of each species'ROC rate across the three temperatures indicated that both the yellowtail flounder and the winter flounder had significantly different ROC rates at each temperature experiment (P < 0.05). The ROC of yellowtail and winter flounder responded similarly to changes in experimental temperatures.     

怀头鲇早期发育阶段消化酶及碱性磷酸酶活性变化(英文)

采用动物性饵料和人工饲料培育1～10日龄怀头鲇(Silurus soldatovi)仔稚鱼,分析测定了全鱼酸性、碱性蛋白酶、淀粉酶、脂肪酶以及碱性磷酸酶的活性。结果表明:孵化后3天开口期仔鱼已具有较高的碱性蛋白酶活性,5日龄时碱性蛋白酶比活力达到较高值,8日龄时出现低值,总体变化呈波动上升趋势;酸性蛋白酶活性在1～8日龄处于较低水平,8日龄后开始迅速升高;淀粉酶活性在5日龄左右达到最高值,随后酶活性开始下降至较低水平;脂肪酶活性变化波动较大,表现为双峰型,两个峰值分别出现在3～4日龄和6～8日龄。摄食动物性饵料仔稚鱼消化酶活性和碱性磷酸酶活性均高于摄食人工饲料。在整个早期发育过程中,碱性蛋白酶比酸性蛋白酶活性高,碱性蛋白酶、淀粉酶比活力在约8日龄仔稚鱼转变期明显下降,而酸性蛋白酶活性开始迅速升高,这说明消化酶活性的变化与仔稚鱼发育过程中消化机能转换具有相关性。怀头鲇在10日龄内碱性磷酸酶活性呈上升趋势,表明怀头鲇胃肠道功能的逐步发育完善。     

Enzyme cytochemistry of rat organs after uremia with special reference to proteases

Wistar rat organs and tissues were investigated after acute and chronic uremia using enzyme cytochemical means whereby special attention was paid to plasma membrane and lysosomal proteases. Heart muscle, pancreas, spleen, stomach, duodenum, jejunum, colon and skeletal muscle did not show any clear-cut indications of alterations. After acute uremia activities of dipeptidyl peptidase IV, glutamyl aminopeptidase and microsomal alanyl aminopeptidase were decreased in the extraorbital gland and that of dipeptidyl peptidase IV in the submandibular gland. The thymus showed an increased staining for glutamyl aminopeptidase and lysosomal proteases. An activity increase of dipeptidyl peptidase IV, acid phosphatase and beta-N-acetyl-D-glucosaminidase occurred in bronchial lavage cells among which the alveolar macrophages predominated. In addition, their number was comparatively higher. Non-specific esterase activity was lowered in these cells. Alkaline phosphatase activity was drastically enhanced at the biliary pole of hepatocytes. Following chronic uremia all effects were less pronounced except for the lavage cells which were positive for glutamyl aminopeptidase, microsomal alanyl aminopeptidase and gamma-glutamyl transpeptidase and showed increased staining for lysosomal proteases, glycosidases and nonspecific phosphatases.     

Ontogenetic variation in habitat associations for four flatfish species in the Gulf of Maine-Georges Bank region

Multivariate ordination techniques were used to examine how size classes of four flatfish species, American plaice Hippoglossoides platessoides , winter flounder Pseudopleuronectes americanus , yellowtail flounder Limanda ferruginea and fourspot flounder Paralichthys oblongus , are related to bottom depth, bottom temperature, substratum grain size and temporal factors using a 35 year time series from autumn and spring in the Gulf of Maine-Georges Bank region. Depth had the most explanatory value during both seasons in most cases, with fish size generally increasing with depth. One exception was yellowtail flounder in the spring for which a temporal factor explained the most variance, reflecting an increase in size over the time series due to changes in fishing pressure. Temperature was secondarily important for yellowtail flounder in the autumn and for fourspot flounder in both seasons. Substratum type was secondarily important for winter and yellowtail flounders in the spring with smaller fish associated with larger substratum types. Seasonal associations with depth, temperature and substratum are related to seasonal spawning migrations, thermal preferences and other ecological factors.     

Differentiation of epithelial cells in human jejunum: Localization and quantification of aminopeptidase,alkaline phosphatase and aldolase isozymes in tissue sections

Summary Sections from human jejunum were stained histochemically for aminopeptidase and alkaline phosphatase and the aldolase isozymes were detected with the mixed aggregation immuno-cytochemical technique. All enzyme concentrations increased from the bottom to the upper part of the crypt. The concentration of aldolase-A per cell was the same in the upper part of the crypt and the villus, whereas the concentration of the other three enzymes was still higher. Therefore, high amounts of aldolase-B, aminopeptidase and alkaline phosphatase are present in cells highly active in absorption in a fashion similar to that found in the proximal tubule cells of kidney. The relatively undifferentiated cells of the crypts contained both aldolase-A and aldolase-B. Alkaline phosphatase gains its full activity later than aminopeptidase. The synthesis of microvillar membrane enzymes comes to an end earlier than that of the cytosol enzymes.     

Juvenile hormone titres and metabolism during starvation-induced supernumerary larval moulting of the tobacco hornworm, Manduca sexta L.

When tobacco hornworm (manduca sexta) larvae are starved for 5 days immediately after ecdysis to the 5th instar, then fed normal diet, they undergo a supernumerary moult instead of metamorphosis. During starvation the titre of juvenile hormone in the haemolymph increased to a maximum of 3 ng juvenile hormone I equivalents/ml (determined by the black Manduca larval bioassay) on the fourth day of starvation, then began a decline which continued through the subsequent feeding period. The changes in juvenile hormone titre were not attributable to changes in haemolymph volume during starvation (only a 5% decrease) and subsequent feeding. During starvation the esterase activity of the haemolymph declined 4-fold with a 2-fold larger decrease in the DFP-insensitive, presumably juvenile hormone specific, esterase activity. Both the total and the juvenile hormone-specific esterase activity then increased as a function of larval weight during the subsequent feeding period. As growth was slow in the prolongedly starved larvae, sufficient juvenile hormone was present at the time of prothoracicotropic hormone (PTTH) and ecdysteroid release at the beginning of the fourth day of feeding to prevent metamorphosis.     

Substrate specificity in the control of digestive enzymes in larvae of the black carpet beetle

When starved larvae of the black carpet beetle, Attagenus megatoma, were fed selected diets, increases in proteolytic, trypsin, and chymotrypsin activity were correlated with total midgut protein and not with the amount of food consumed. Although larvae initially consumed more of a starch diet than of 2 diets that contained added protein, total protease activity in these larvae was minimal. Starch-fed larvae and larvae fed a casein-sucrose diet had a consistently higher level of sucrase activity than larvae fed an all-casein diet. These total results support a secretagogue mechanism for control of digestive enzyme synthesis in insects. In addition, the absence of parallel stimulation of different digestive enzymes by a single substrate (starch) indicated nutrient class specificity in the control of inducible midgut enzymes in this species.     

Differential amplification of antifreeze protein genes in the pleuronectinae

Summary The organization of antifreeze protein (AFP) genes in the yellowtail flounder was investigated by Southern blotting and the characterization of clones from a genomic library. This flounder, like the closely related winter flounder, has a set of 10–12 linked but irregularly spaced AFP genes. However, it lacks the tandemly amplified set of 20 such genes that are present in the winter flounder. DNA sequence analysis of a tandemly repeated gene from winter flounder showed that it can code for one of the two most abundant AFP components in the serum. Consistent with this higher AFP gene dosage, the peak serum AFP level in midwinter was 9 mg/ml in the winter flounder and only 4 mg/ml in the yellowtail flounder. A recent amplification of the AFP gene in the winter flounder lineage might be responsible for the higher serum AFP levels in this fish. This increase in gene dosage might have helped the winter flounder colonize the ice-laden, shallow-water niche that it currently occupies along the east coast of North America. Genomic Southern blotting of two other righteye flounders, the smooth flounder and the American plaice, illustrates another example of a differential amplification of AFP genes that correlates with a species' exposure to ice.     

Enzymatic histochemistry of mouse kidney in plastic

Two-micrometer sections of methacrylate-embedded kidney were used to investigate the enzymatic activities of mouse kidney where the proximal tubule and Bowman's capsule from the same corpuscle were viewed in the same section. Alkaline phosphatase, acid phosphatase, 5'-nucleotidase, gamma-glutamyl transpeptidase, N-acetyl-beta-glucosaminidase, leucine aminopeptidase, alpha-naphthyl butyrate esterase, and adenosine triphosphatase activities were observed in the proximal tubule, but only 5'-nucleotidase, alpha-naphthyl butyrate esterase, and alkaline phosphatase were observed in the squamous portion of the parietal epithelium of Bowman's capsule. The use of methacrylate-embedded tissue allowed more precise localization of enzymatic activity than is possible with most frozen sections. This may provide interesting applications not only for characterization of kidney diseases but also for characterization of other normal and abnormal tissues.     

Release of alkaline phosphodiesterase I from rat kidney plasma membrane produced by the phosphatidylinositol-specific phospholipase C of Bacillus thuringiensis

The release of plasma-membrane-bound enzymes by phosphatidylinositol-specific phospholipase C obtained from Bacillus thuringiensis was investigated. Among the ectoenzymes of plasma membrane tested, alkaline phosphodiesterase I was released markedly from rat kidney cortex slices, in addition to alkaline phosphatase and 5'-nucleotidase. Other membrane-bound enzymes; alanine aminopeptidase, leucine aminopeptidase, dipeptidyl peptidase, leucine aminopeptidase, dipeptidyl peptidase IV, esterase and gamma-glutamyl transpeptidase could not be liberated from the treated slices. Alkaline phosphodiesterase I was released linearly from rat kidney slices with the concentration of phosphatidylinositol-specific phospholipase C, but little enzyme was released from rat liver slices. Alkaline phosphodiesterase I separated from kidney tissue with n-butanol still retained phosphatidylinositol and was transformed into a lower molecular weight form by phosphatidylinositol-specific phospholipase C. This suggests an important function for phosphatidylinositol in the binding of alkaline phosphodiesterase I to the plasma membrane of rat kidney cells. The alkaline phosphodiesterase I released from rat kidney had a molecular weight of about 240,000 and an isoelectric point (pI) of 5.4. The enzyme hydrolyzed the phosphodiester linkage of p-nitrophenyl-thymidine 5'-monophosphate at pH 8.9 and had a Km value of 0.3 mM. The enzyme was activated by Mg2+ and Ca2+, but was inhibited by EDTA. Strong inhibition took place on the addition of adenosine 5'-phosphosulfate or the nucleotide pyrophosphates, i.e., UDP-galactose and alpha, beta-methylene ATP.     

Activity of gut alkaline phosphatase,proteases and esterase in relation to diapause of pharate first instar larvae of the gypsy moth,Lymantria dispar

Two distinctly different patterns of gut enzyme activity were noted in relation to diapause in pharate first instar larvae of the gypsy moth, Lymantria dispar. Trypsin, chymotrypsin, elastase, aminopeptidase and esterase activities were low at the initiation of diapause and through the period of chilling needed to terminate diapause. At the completion of a 150 day chilling period, activity of each of these enzymes quickly increased when the pharate larvae were transferred to 25°C. By contrast, activity of alkaline phosphatase (ALP) increased rapidly at the onset of diapause, remained elevated throughout diapause, increased again during postdiapause, and then dropped at the time of hatching. In addition, zymogram patterns of ALP activity differed qualitatively in relation to diapause: several bands were detectable during the pre- and postdiapause periods, but only one band, a band of high mobility, was visible during diapause. The ALP isozyme present in diapausing pharate larvae had a pH optimum of 10.6. Diapause in the gypsy moth can be averted by application of an imidazole derivative, KK-42, and pharate larvae treated with KK-42 showed elevated protease and esterase activity, low ALP activity, and expressed ALP isozymes with low mobility. Thus the overall patterns of gut enzyme activity and the ALP zymogram in KK-42 treated individuals were similar to those observed in untreated individuals at the termination of diapause. Our results suggest a unique pattern of enzyme activity in the gut that is regulated by the diapause program. Arch. Insect Biochem. Physiol. 37:197–205, 1998. © 1998 Wiley-Liss, Inc.     

Lytic enzyme activity in autolysing mycelium of Aspergillus niger

We have studied changes in the activity of some lytic enzymes contained in mycelium of Aspergillus niger in cultures relative to the autolytic phase of growth. Acid phosphatase, polygalacturonidase and alpha-amylase activity reached its highest level (40.7, and 8 U/sample, respectively) at the initiation of the autolytic phase of growth. 1.3-beta-Glucanase and beta-N-acetylglucosaminidase reached its highest level (3.5 and 2 U/sample, respectively) during the first days of autolysis. Alkaline phosphatase, cellulase, invertase, esterase, chitinase and proteolytic activity is also present in autolysing mycelium of A. niger, though comparatively low. Their maximum activity coincided with the beginning of the autolytic phase of growth. In all enzymes studied here, as autolyis proceeded, enzyme activity decreased by about 90%. Only esterase activity remained nearly constant throughout the whole period of autolysis described here.     

Isolation and characterization of Brush border fragments from mosquito mesenterons

Brush border fragments were isolated from homogenates of mesenterons from the mosquito, Culex tarsalis, by a combination of Ca²⁺ precipitation and differential centrifugation. These preparations were routinely enriched seven- to eightfold for the brush border marker enzyme, leucine aminopeptidase. Alkaline phosphatase, a putative brush border marker for both vertebrate and invertebrate brush borders, was found to be unsuitable for Cx. tarsalis. Isoelectric focusing electrophoresis coupled with histochemical enzyme detection was used to enumerate isozymic species of nonspecific esterases [3], leucine aminopeptidase [1], and alkaline phosphatase [1] in isolated brush border fragments. Leucine aminopeptidase activity was solubilized by papain digestion, suggesting an extrinsic active site for this membrane-bound enzyme. The predominant nonspecific esterase isozyme remained membrane-bound. Conventional staining (ie, Coomassie Blue and silver) of proteins separated by isoelectric focusing, sodium dodecylsulfate, and two-dimensional electrophoresis indicated a simple pattern for brush border fragments, with two proteins predominating among the 11–14 routinely detected.     

A new fluorescence method for alkaline phosphatase histochemistry.

We have developed a new fluorescence method for the histochemical localization of alkaline phosphatase activity. Calcium phosphate deposited at the sites of alkaline phosphatase activity in a Gomori-type reaction are identified by calcium binding fluorochromes. The calcium binding fluorochromes calcein, calcein blue, and xylenol orange were investigated, with each fluorochrome being included in the alkaline phosphatase incubating medium and used in a single-step procedure. Alkaline phosphatase activity was studied in freeze-substituted, resin-embedded human liver and jejunal biopsies, and each fluorochrome produced intense fluorescence of different colors at sites of alkaline phosphatase activity. Calcein, calcein blue, and xylenol orange produced green, blue, and red fluorescence, respectively. Sites of enzyme activity were accurately localized without evidence of diffusion, and there was an absence of non-enzyme-catalyzed binding of any of the fluorochromes to tissue. This fluorescence method, which is particularly suited to investigating the localization and distribution of the activity of different enzymes in the same section, was used to investigate the distribution and co-localization of alkaline phosphatase and aminopeptidase M in human liver and jejunum.     

Effect of DIMBOA on growth and digestive physiology of Sesamia nonagrioides (Lepidoptera: noctuidae) larvae

The hydroxamic acid 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) was assessed for its effect on growth and digestive physiology of larvae of the stalk corn borer Sesamia nonagrioides Lef. Nutritional indices and activities of some digestive and detoxification enzymes were determined for larvae feeding on a DIMBOA-containing diet for the first two days of the third instar (short-term feeding assays), and from neonates to third instar (long-term feeding assays). DIMBOA reduced the relative growth rate and the efficiency of conversion of ingested food without affecting the relative consumption rate in long-term feeding assays, but it had no effect in short-term assays. Moreover, elastase-like activity was significantly increased by DIMBOA in short-term feeding assays, whereas microsomal oxidase activity was increased and esterase activity was reduced in long-term feeding assays. In vitro, DIMBOA inhibited the activities of carboxypeptidases, aminopeptidase, glutathione S-transferase and esterase, but it had no effect on trypsin, chymotrypsin and elastase. The implications of the altered levels of proteases and detoxification enzyme activities on the digestive physiology of larvae feeding on DIMBOA-containing diets are discussed.     

Digestive enzymes in larval Coregonus lavaretus L.

Using histochemical methods, morphofunctional aspects of the alimentary tract of larval coregonids were investigated. Larvae of Coregonus lavaretus were reared for 34 days with either zooplankton or one of two dry diets. Ontogeny, localization and diet-related modifications of the following enzymes were examined: trypsin (luminal digestion), aminopeptidase, maltase, alkaline phosphatase (brush border-bound digestion) and unspecific esterase (intracellular nutrient processing). All of the enzymes studied were present in 13-day-old larvae. Except for the intracellularly located unspecific esterase, there was an ontogenetic enhancement of enzyme staining intensities accompanied by a significant increase in the volume of the intestinal mucosa. Enzyme activities differed within and between intestinal regions. This finding suggests that a spatial gradient of nutrient breakdown and absorption already exists in the morphologically and physiologically incompletely developed digestive system of larval coregonids. Digestive enzyme activities were modified in response to the dietary regimen. There was no obvious correlation between enzymic response and growth performance of the larvae.