Domain exchange: characterization of a chimeric lipase of hepatic lipase and lipoprotein lipase.

Hepatic lipase and lipoprotein lipase hydrolyze fatty acids from triacylglycerols and are critical in the metabolism of circulating lipoproteins. The two lipases are similar in size and amino acid sequence but are distinguished by functional differences in substrate preference and cofactor requirement. Presumably, these distinctions result from structural differences in functional domains. To begin localization of these domains, a chimeric lipase was constructed composed of the N-terminal 329 residues of rat hepatic lipase linked to the C-terminal 136 residues of human lipoprotein lipase. The chimera hydrolyzed both monodisperse short-chain (esterase) and emulsified long-chain (lipase) triacylglycerol substrates with catalytic and kinetic properties closely resembling those of native hepatic lipase. However, monoclonal antibodies to lipoprotein lipase inhibited the lipase activity, but not the esterase function, of the chimera. Therefore, the chimeric molecule is a functional lipase and contains elements and characteristics from both parental enzymes. It is proposed that the N-terminal domain, containing the active center from hepatic lipase, governs the catalytic character of the chimera, and the C-terminal domain is essential for hydrolysis of long-chain substrates.     

Effect of aging on pancreatic lipolytic enzymes

Pancreatic lipolytic enzyme activities and plasma lipids were measured in three age groups of female Fisher 344 rats (3 months [young], 12 months [adult], and 27 months [old]) in order to evaluate age-related changes and a possible correlation of these parameters. Cholesterol esterase activity was measured in pancreas homogenate, while the lipase activity was further fractionated into heparin-Sepharose unretained (lipase I) and retained (lipase II) fractions. In analogy to the lipolytic enzymes of the human pancreas, lipase I corresponds to pancreatic lipase and lipase II corresponds to pancreatic carboxylesterase. Plasma triglyceride and cholesterol levels were significantly higher in old as compared with adult and young rats. There was no significant correlation between plasma lipid and pancreatic enzyme activity levels. Cholesterol esterase and pancreatic lipase (lipase I) activity did not show any consistent change with age. Pancreatic carboxylesterase (lipase II), on the other hand, was consistently lower in adult and old animals. Although the importance of pancreatic carboxylesterase for triglyceride hydrolysis remains to be established, our results suggest that this enzyme is under long-term metabolic control. We suggest that early in life, high levels of this enzyme may be needed to supplement pancreatic lipase in order to optimize digestion of dietary fat required for optimal growth.     

Effects of tocopherol deficiency on lipid metabolism in the arterial wall of rats on normal and high cholesterol diets

The effects of dietary tocopherol deficiency on arterial wall enzymes involved in lipid synthesis and hydrolysis were studied in rats receiving normal diets and diets supplemented with 1% cholesterol. Arterial wall lipase and cholesterol esterase were associated with both the lysosome and microsome fractions, whereas acyl CoA synthetase, triglyceride synthesizing activity, cholesteryl ester synthesizing activity and cytidine diphosphatecholine-1,2-diacyl glycerol choline phosphotransferase (CPT) were found mainly in the microsomal fraction. When tocopherol was depleted from either the normal or high cholesterol diets, the following changes occurred in the arterial wall: (1) increase in thiobarbituric acid reactive substances; (2) decrease in lysosomal acid lipase and acid cholesteryl esterase; (3) decrease in the microsomal enzymes, acyl CoA synthetase, triglyceride synthesizing activity, cholesteryl ester synthesizing activity, neutral lipase and neutral cholesteryl esterase; and (4) increase in microsomal CPT. The results of these studies suggest that dietary tocopherol plays an important role in both lipid synthesis and degradation in the arterial wall, and the results may account for the accumulation of lipids and lipoperoxides in atherosclerotic lesions.     

感染大链壶菌后蚊幼虫脂肪、酯酶和脂肪酶的变化

目的　从蚊虫生理生化代谢角度探讨大链壶菌(Lagenidiumgiganteum)灭蚊机制。　方法　采用组织化学方法检测正常致倦库蚊 (Culexquinquefasciatus)和白纹伊蚊 (Aedesalbopicus)幼虫于感染大链壶菌后不同时间两种蚊幼虫体内的脂肪含量、酯酶和脂肪酶活性,进行显微摄影和定量图像分析。　结果　感染后24h,致倦库蚊幼虫体内的脂肪含量低于对照组,而酯酶和脂肪酶活性高于对照组;感染后48h和72h,两种蚊幼虫体内的脂肪含量均明显低于对照组,酯酶和脂肪酶活性明显高于对照组。　结论　大链壶菌感染引起蚊幼虫体内脂肪含量减少,而与脂肪水解相关的酯酶和脂肪酶活性升高,导致体内脂肪代谢紊乱,是蚊幼虫致死的重要原因     

Hydrolase activities in the rat aorta. III. Effects of regular swimming activity and its cessation.

It is possible that one of the consequences of regular physical activity could be a change of vascular metabolism. We studied the effects of regular swimming activity on specific activities of aortic hydrolases of male rats. Enzymes included: neutral alpha-glucosidase and lysosomal beta-galactosidase, N-acetyl-beta-glucosaminidase, cathepsin C, acid alpha-glucosidase, and acid cholesteryl esterase. After 8 or 16 weeks of a 1-hour/day swimming protocol, specific activities of four of the six aortic enzymes studied were increased over control levels, increases ranging from 7 to more than 42%. Acid cholesteryl esterase was one of the enzymes most affected by the exercise, increasing 25-30% above control levels. An 8-week sedentary period, after 8 weeks of a swimming regimen, resulted in return of the activity of acid cholesteryl esterase, but not those of the other hydrolases, to control levels. Decreases in body weight, blood pressure, and serum lipid levels also occurred in the swimming rats. Weight reduction per se was excluded as an explanation for the increases in aortic enzymes or decrease in serum cholesterol found with swimming. These findings show that regular physical activity is yet another factor with discrete and significant effects on the catabolic activity of vascular tissue.     

The esterase patterns in the ovaries and the embryonated eggs of Aedes aegypti L.

The esterases of Aedes aegypti were studied in the ovary before and during a gonotrophic cycle and also in fully embryonated eggs by means of disc electrophoresis. During oogenesis no significacant changes can be observed besides a marked increase in the total esterase activity. A different esterase pattern is found in eggs after embryogenesis. The electrophoretic mobility of some esterase bands is highly increased in the ovary compared to other organs. One esterase fraction in the ovary and two in the fertilized egg were identified as acetylcholinesterases. All other enzymes are carboxylesterases. The results are compared to those of previous authors and are discussed in view of possible functions of esterases during reproduction.     

Pancreatic lipolytic enzymes in human duodenal contents. Radioimmunoassay compared with enzyme activity.

The total pancreatic lipolytic capacity was determined in duodenal contents in healthy humans 10-120 min after a liquid test meal, by estimating the amount of pancreatic lipase, colipase, carboxyl ester lipase, and phospholipase A2 by means of radioimmunoassays and enzymatic assays. The molar concentrations of the different proteins were of the same order of magnitude. The relative specific activity (enzyme activity/milligram immunoreactive protein expressed as a percentage of the specific activity of the respective pure protein) amounted to 75-120% for lipase, 45-80% for colipase, 30-70% for carboxyl ester lipase, and 45-120% for phospholipase A2. These varied, and sometimes low values can be explained by the fact that the enzymes are inhibited or partly inactivated in the duodenal contents by surface denaturation, in which cases the products are still immunoreactive. Also, the proforms of colipase and phospholipase A2 may not always be completely activated. Furthermore, the specific activities of the pure enzymes (and thus the relative specific activities) are related to the methods used, which are not specific enough to distinguish completely the three enzymes and the cofactor in duodenal contents.     

Hormone-sensitive lipase in differentiated 3T3-L1 cells and its activation by cyclic AMP-dependent protein kinase

Differentiation of 3T3-L1 fibroblasts to adipocyte-like cells was accompanied by a 19-fold increase in neutral triglyceride lipase activity, a 12-fold increase in diglyceride lipase activity, a 10-fold increase in monoglyceride lipase activity, and a 280-fold increase in cholesterol esterase activity. In contrast, acid acylhydrolase activities did not increase during differentiation. The rate of glycerol release from unstimulated intact cells increased by more than 1 order of magnitude upon differentiation. Isoproterenol (1 microM) and 1-methyl-3-isobutylxanthine (0.1 mM) further stimulated this rate of glycerol release 3-fold. The neutral triglyceride lipase activity in cell-free preparations of differentiated cells was activated 105% by cyclic AMP-dependent protein kinase. Neutral cholesterol esterase, diglyceride lipase, and monoglyceride lipase were also activated (117%, 10%, and 37+, respectively) by cyclic AMP-dependent protein kinase. In contrast, protein kinase had no effect on any of the four lysosomal acid acylhydrolase activities. Thus, hormone-sensitive lipase, the most characteristic and functionally important enzyme of adipose tissue, has been characterized in differentiated 3T3-L1 cells. The 3T3-L1 cell should be a valuable model system in which to study regulation of hormone-sensitive lipase, particularly its long-term regulation.     

Relationship between activity of hydrolases in fungi isolated from vagina of pregnant women and selected symptoms

Pregnancy is considered a factor of vulvovaginal mycosis. Secretion of hydrolases is an important determinant of Candida virulence. Thus, the aim of the study was to found the relationship between activity of 19 hydrolases in fungi isolated from vagina of pregnant women and symptoms of mycosis. 251 pregnant women were examined. Samples were collected from vagina and cultured on Sabouraud media. Activity of hydrolytic enzymes was evaluated using API ZYM (bioMerieux) test Fungi were found in 20.1% of vaginal samples. Symptoms were detected in 45.8% of women. Only 32.1% of women with discharge and 26.4% with pruritus had mycosis. Out of the 19 examined hydrolazes, 13 active enzymes were detected in fungal strains. We found for the first time the relationship between activity of fungal esterase lipase and the presence of vaginal discharge in pregnant women.     

Determination of elastase-1 serum levels in post ERCP/EST pancreatic damage.

Recently an ELISA using specific antibodies to detect elastase-1 in serum has become available. Earlier studies using a radioimmunoassay reported a prolonged elevation of serum elastase as compared to other pancreatic enzymes in acute pancreatitis. The aim of the present study was to compare the changes of serum levels of ELISA-elastase-1, lipase and amylase in acute pancreatic damage following ERCP. Blood samples were prospectively collected at five time points before and after the endoscopic procedures in 212 patients. Samples were analyzed for pancreatic serum enzymes, acute phase proteins and routine parameters. A pain score was used for clinical evaluation. Relevant post ERCP pancreatic damage was defined as CRP elevation from < 10 mg/l to > 10 mg/l in the presence of persistent abdominal pain without laboratory evidence of cholangitis and without clinical or laboratory signs of pancreatitis before the endoscopic procedures. Elastase-1 time course paralleled the courses of lipase and amylase peeking at six hrs. There was no prolonged elevation of elastase-1. Ten out of 204 patients (4.9%) were found to have relevant pancreatic damage. Depending on the cut off point used, sensitivity/specificity were as follows: lipase 80-100%/30.9-71.6%; amylase 70-90%/44.3-88.7%; elastase-1 60-90%/64.9-81.4%. In conclusion ELISA-elastase-1 is a marker of acute pancreatic damage similar to lipase and amylase. Although elastase-1 may show a better specificity than the other enzymes, this seems to be a matter of definition of the normal range. The determination of serum ELISA-elastase-1 does not provide additional information in acute pancreatic damage as compared to a combination of lipase and amylase.     

Human granulocyte lysosomal elastase activity using t-butyloxycarbonyl-L-alanine p-nitrophenyl ester and elastin-rhodamine as substrates.

Polymorphonuclear leukocyte lysosomal esterolytic activity on the synthetic substrate, t-butyloxycarbonyl-L-alanine p-nitrophenyl ester was observed to correlate well with polymorphonuclear leukocyte granule elastase activity measured on the natural substrate, elastin, bound to rhodamine. In addition, the effect of highly specific, irreversible chloromethyl ketone elastase inhibitors on leukocyte lysosomal elastase activity was similar, using t-butyloxycarbonyl-L-alanine p-nitrophenyl ester or elastin-rhodamine as substrate. Whether polymorphonuclear leukocyte lysosomal granules contain two different enzymes, a true elastase with esterase activity and a similar esterase without elastase activity, as found in the human pancreas, is, as yet, unknown. Both enzyme activities have been identified in isoenzymes of purified human polymorphonuclear leukocyte lysosomal elastase. The correlations observed between the two enzymes, if present in polymorphonuclear leukocytes, are sufficiently strong to use the esterase assay for clinical purposes.     

Peritoneal absorption of pancreatic enzymes in dogs.

To elucidate peritoneal absorption of pancreatic enzymes, plasma levels of amylase, lipase, and trypsinogen were measured after the intraperitoneal injection of 10 ml human pancreatic juice in dogs. Plasma pancreatic amylase, lipase, and trypsinogen were determined using the immunoassay specific to the corresponding human pancreatic enzyme to exclude cross-reaction with the endogenous enzyme activities of the canine plasma. Plasma immunoreactivity of human amylase persistently rose during 24 h after the injection, whereas elevation of plasma amylase enzyme activity become significant only at 24 h. Increase of plasma lipase was not remarkable. Significant increase of the immunoreactivity was observed only at 24 h but there was no significant increase of the enzyme activity during this period. Plasma trypsinogen immunoreactivity peaked at 2 h remained significantly elevated during 24 h. The transperitoneal absorption of pancreatic enzymes in dogs was confirmed using intraperitoneal injection of human pancreatic juice combined with immunoassay specific to human pancreatic enzymes.     

Enzymatic abnormalities in erythroleukemia.

Enzymatic studies were performed on erythroblasts obtained from marrows of 2 patients with untreated erythroleukemia. Cytochemically, erythroblasts showed abnormalities of several enzymes involved in carbohydrate metabolism, as well as abnormalities of specific and nonspecific esterases. Electrophoretic analysis of esterases extracted from predominatly erythroid marrows showed strong moderately fluoride-resistant nonspecific esterase activity with alpha-naphthyl acetate, and weak activity with alpha-naphthyl butyrate. Isoenzymatic patterns of specific esterase activity in erythroleukemia were indistinguishable from those found in myeloblastic leukemia. The results are consistent with the concept of the Di Guglielmo syndrome in which a preleukemic erythroid disorder may precede the emergence of acute myeloblastic or myelomonocytic leukemia.     

A sialic acid-specific O-acetylesterase in human erythrocytes: possible identity with esterase D, the genetic marker of retinoblastomas and Wilson disease.

The "nonspecific" esterases are a family of enzymes that were originally identified because of their reaction with synthetic O-acetyl ester substrates. While the electrophoretic polymorphisms of these enzymes have been extremely useful for genetic studies, their biological functions have remained completely unknown. Esterase D is characterized by its reactivity with 4-methylumbelliferyl acetate. This enzyme has recently been of particular interest because of its tight linkage to the putative recessive gene causing retinoblastomas, and to the recessive gene causing Wilson disease. We describe here the partial purification of a human erythrocyte esterase that appears to be highly specific for O-acetylated sialic acids. We next present evidence that suggests that esterase D is identical to this sialic acid-specific O-acetylesterase. First, both activities copurify from human erythrocyte lysates through several different purification steps, each of which use different principles of separation. Second, both activities show a remarkably similar profile of inhibition with a variety of different agents. Third, they both show a nearly identical heat-inactivation profile. This cytosolic sialic acid-specific O-acetylesterase appears to be involved in the "recycling" of O-acetylated sialic acid molecules. Thus, esterase D may be the first nonspecific esterase for which a specific biological role can be predicted.     

Effects of prolonged ethanol intake and malnutrition on rat pancreas.

Nutritional factors, especially the protein and fat content of the diet, may change pancreatic morphology after ethanol induced injury. This study was performed to delineate the combined effects of a low fat diet and longterm ethanol ingestion on the rat pancreas. Male Sprague-Dawley rats were maintained with five different diets for 12 weeks and the pancreas removed on the day they were killed. Rats fed a very low fat diet without ethanol (5% of total calories as lipid) developed malnutrition, pancreatic steatosis, and reduction in zymogen granules content. Animals fed a 35% lipid diet with ethanol also developed pancreatic steatosis but changes in zymogen granules content were not detected. Both malnutrition and longterm ethanol consumption increased pancreatic cholesterol ester content, and their effects were additive. Pancreatic steatosis was accompanied with hypercholesterolaemia. Amylase, lipase, and cholesterol esterase content were reduced in malnourished rats; but longterm ethanol ingestion, regardless of the nutritional state, increased lipase content and decreased amylase. It is suggested that high serum cholesterol concentrations and increased pancreatic lipase activity could cause accumulation of cholesterol esters in acinar cells. Fat accumulation in the pancreas has been reported as the earliest histopathological feature in alcoholic patients and may be responsible for cytotoxic effects on the acinar cells at the level of the cell membrane. Although it is difficult to extrapolate results in this animal study to the human situation, the results presented in this work might explain the higher incidence of pancreatitis is malnourished populations as well as in alcoholic subjects that is reported in dietary surveys.     

Lipase and unspecific esterase activity in the fat body of Aedes aegypti L.

In the fat body of Aedes aegypti a very high unspecific esterase activity and a low lipolytic activity was found. The electrophoretic isozyme patterns of the unspecific esterases show only few changes in the different physiological stages. The activity of the unspecific esterases as well as of the lipase is especially high in young sugar fed and in blood fed mosquitoes which points to special energy requirements in these stages. The role of the unspecific esterases is discussed.     

Histochemical patterns in the tadpole tail during normal and thyroxine-induced metamorphosis. I. Alkaline phosphatase, acid phosphatase, esterase, and aminopeptidase

The distribution of alkaline phosphatase, acid phosphatase, esterase, and aminopeptidase was demonstrated by histochemical methods in sections of both growing and resorbing tails of Rana pipiens tadpoles. In general, main sites of localization of each of the enzymes were the epidermis and connective tissue, but not striated muscle. In addition, alkaline phosphatase, acid phosphatase, and esterase were present in the ependyma of the spinal cord and the endothelium of various blood vessels. The latter two enzymes were also found in spinal ganglia and, along with aminopeptidase, in the notochord. As the tail shortened during both normal and thyroxine-induced metamorphosis, activity of these enzymes increased; it became particularly prominent in the epidermis and in phagocytes in the connective tissue. These data on the differential distribution of four hydrolytic enzymes in the R. pipiens tail support histochemical and biochemical studies in other species.     

Unexplained elevated serum pancreatic enzymes: a reason to suspect celiac disease.

BACKGROUND & AIMS: The frequency of elevated serum pancreatic enzymes in patients with celiac disease (CD) is unknown. The aim of this study was to evaluate the serum levels of pancreatic enzymes in CD patients. METHODS: Serum pancreatic isoamylase and lipase levels were assayed in 90 adult and 112 pediatric consecutive CD patients at diagnosis and after 12 months of gluten-free diet (GFD). Serum elastase and trypsin levels were assayed in a subgroup of adult CD patients. Pancreatic ultrasonography was also performed. RESULTS: Twenty-six adult (29%) and 29 pediatric (26%) CD patients exhibited elevated values of serum pancreatic amylase and/or lipase; trypsin was elevated in 69% and elastase in 19%. The frequency of elevated serum pancreatic enzymes observed was identical in the patients with "typical" and "atypical" CD symptoms and in the asymptomatic patients. Most of the elevated values were lower than 2-fold the threshold limits. Elevated pancreatic enzymes were not associated with alcohol consumption, drug use, presence of abdominal pain, or diabetes mellitus. Abdominal ultrasound scan showed no abnormal findings in the pancreatic region in any of the CD patients. After 12 months of GFD, pancreatic amylase was elevated in 3 cases and lipase in 2 cases; these patients had not strictly adhered to the GFD. CONCLUSIONS: We demonstrated a frequency of about 25% of elevated pancreatic enzymes values in CD patients, including subjects without gastrointestinal manifestations and apparently asymptomatic subjects. The finding of elevated serum amylase or lipase level, in the absence of signs of pancreatic disease, would appear to suggest a need to screen for celiac disease.     

Activation of hormone-sensitive lipase and phosphorylase kinase by purified cyclic GMP-dependent protein kinase

Cyclic GMP-dependent protein kinase, purified to homogeneity from bovine lung, was shown to activate hormone-sensitive lipase partially purified from chicken adipose tissue. The degree of activation was the same as that effected by cyclic AMP-dependent protein kinase although higher concentrations of the cyclic GMP-dependent enzyme were required (relative activities expressed in terms of histone H2b phosphorylation units). Activation by cyclic AMP-dependent protein kinase was completely blocked by the heat-stable protein kinase inhibitor protein from skeletal muscle but activation by the cyclic GMP enzyme was not inhibited. Lipase fully activated by cyclic AMP-dependent protein kinase showed no further change in activity when treated with cyclic GMP-dependent protein kinase. Lipase activated by cyclic GMP-dependent protein kinase was reversibly deactivated by purified phosphorylase phosphatase (from bovine heart); full activity was restored by reincubation with cyclic GMP and cyclic GMP-dependent protein kinase. Cholesterol esterase activity in the chicken adipose tissue fraction, previously shown to be activated along with the triglyceride lipase by cyclic AMP-dependent protein kinase, was also activated by cyclic GMP-dependent protein kinase. Crude preparations of hormone-sensitive triglyceride lipase from human or rat adipose tissue and cholesterol esterase from rat adrenal were also activated by cyclic GMP-dependent protein kinase. Purified phosphorylase kinase (rabbit skeletal muscle) was also shown to be activated by cyclic GMP-dependent protein kinase. The present results, together with those of other workers on histone phosphorylation, suggest that the substrate specificities of cyclic GMP-dependent and cyclic AMP-dependent protein kinase may be similar. This is discussed in the light of a model recently proposed with regard to the relationship between the subunit structures of the two kinases. The physiologic significance of the findings remains to be established.     

Receptor-like function of heparin in the binding and uptake of neutral lipids

Molecular mechanisms regulating the binding, amphipathic stabilization, and metabolism of the major neutral lipids (e.g., cholesteryl esters, triglycerides, and fatty acids) are well studied, but the details of their movement from a binding compartment to a metabolic compartment deserve further attention. Since all neutral lipids must cross hydrophilic segments of plasma membranes during such movement, we postulate that a critical receptor-like site exists on the plasma membrane to mediate a step between binding and metabolism and that membrane-associated heparin is a key part of this mediator. For example, intestinal brush border membranes containing heparin bind homogeneous human pancreatic 125I-labeled cholesterol esterase (100 kDa) and 125I-labeled triglyceride lipase (52 kDa). This interaction is enzyme concentration-dependent, specific, and saturable and is reversed upon addition of soluble heparin. Scatchard analysis demonstrates a single class of receptors with a Kd of 100 nM and a Bmax of approximately 50-60 pmol per mg of vesicle protein. In contrast, enzymes associated with the hydrolysis of hydrophilic compounds such as amylase, phospholipase A2, and deoxyribonuclease do not bind to intestinal membranes in this manner. Human pancreatic cholesterol esterase also binds specifically and saturably to cultured intestinal epithelial cells (CaCo-2), and soluble heparin significantly diminishes the cellular uptake of the resultant hydrophobic reaction products (cholesterol and free fatty acids). We conclude that a physiological role for intestinal heparin is that of a mediator to bind neutral lipolytic enzymes at the brush border and thus promote absorption of the subsequent hydrolyzed nutrients in the intestine. This mechanism may be a generalizable pathway for transport of neutral lipids into endothelial and other cells.