Soluble and membrane-bound leucyl- and arginyl-aminopeptidase activities in subcellular fractions of young and adult rat brains

The subcellular distribution of soluble and membrane-bound leucyl- and arginyl-aminopeptidase activities were analyzed in one and five month old rat brains using Leu- and Arg-2-naphthylamide as substrates. Both soluble leucyl- and arginyl-aminopeptidase activities showed the highest levels in the synaptosomal fraction in the two groups of rats. The highest levels of membrane-bound leucyl- and arginyl-aminopeptidase activities were found in the microsomal fraction in the two ages studied. There were no differences between the two ages in soluble leucyl- and arginyl-aminopeptidase activities. However, a significant decrease in both membrane-bound enzymatic activities was evidenced in the synaptosomal fraction of older rats. Developmental changes of these aminopeptidase activities in a determined subcellular localization, may reflect modifications in their effect on neuropeptides susceptible to be hydrolyzed in this particular location.     

Calcium-dependent and calcium-independent gelatinolytic proteinase activities of the rat ventral prostate and its secretion: characterization and effect of castration and testosterone treatment

Calcium-dependent and calcium-independent proteinase activities were detected in extracts of rat ventral prostate and its secretion by use of gelatin-containing SDS-PAGE zymography. Ca(2+)-independent proteinase activities of 22, 26, and 73-79 kDa and Ca(2+)-dependent activities of 58, 63, and 66 kDa were found in the adult gland. The 26- (most intense activity of gland) and 22-kDa activities were present in secretion and were not expressed in the undifferentiated gland of the 10-day-old animal. The Ca(2+)-dependent activities were also present in the secretion, where the 63-kDa form was more prominently expressed than the 58- and 66-kDa bands. The Ca(2+)-dependent and Ca(2+)-independent proteinase activities both responded to a broad range of pH values in the incubation media. The 73-79-kDa Ca(2+)-independent activities were sensitive to benzamidine and the Ca(2+)-dependent activities were inhibited by EDTA and EGTA. Both Ca(2+)-dependent and Ca(2+)-independent proteinase activities responded to androgenic manipulations. Castration was followed by the appearance of a 35-kDa Ca(2+)-independent proteinase (at 2 days) and a 43-kDa Ca(2+)-dependent proteinase (at 4 days). In the Ca(2+)-independent proteinase group, the 73-79-kDa activities were increased somewhat and the 22- and 26-kDa activities decreased after castration. The Ca(2+)-dependent proteinases of 58, 63, and 66 kDa increased in activity with castration, but activity of the 58-kDa form decreased again at 7 days after castration. Treatment of animals upon castration for 4 days with hydrocortisone prevented these changes in proteinase activities whereas treatment with actinomycin D or tranexamic acid did not. Testosterone propionate replacement therapy of rats castrated for 16 days stimulated the activities of the 22- and 26-kDa and 73-79-kDa Ca(2+)-independent and the 58- and 63-kDa Ca(2+)-dependent proteinases with 4 days of therapy. The activities of the 35-kDa Ca(2+)-independent and the 43-kDa Ca(2+)-dependent proteinases were repressed with 8 days of testosterone treatment. Thus, individual proteinases show differential changes in activity during development and in response to androgenic manipulation: this suggests that in addition to proteinases which are secreted, others may be involved in intracellular functions or in mediating tissue organization changes.     

Epoxide hydrase and glutathione S-transferase activities with selected alkene and adrene oxides in several marine species.

Epoxide hydrase and glutathione (GSH) S-transferase activities were measured in subcellular fractions prepared from liver or hepatopancreas and some extrahepatic organs of a number of marine species common to Maine or Florida. These activities were easily detected in the species studied. In fish, hepatic GSH S-transferase activities were normally higher than hepatic epoxide hydrase activities for the alkene oxide (styrene oxide and octene oxide) and arene oxide (benzo[a]pyrene 4,5-oxide) substrates studied, whereas in crustacea, hepatopancreas epoxide hydrase activities were higher than hepatopancreas GSH S-transferase activities with the same substrates. Extrahepatic organs from fish and crustacea usually had higher GSH S-transferase activities than epoxide hydrase activities with the alkene and arene oxide substrates. GSH S-transferase activity was also found in liver or hepatopancreas of every aquatic species studied and in a number of extrahepatic organs, when 1,2-dichloro-4-nitrobenzene or 1-chloro-2,4-dinitrobenzene served as substrate.     

Mitochondrial and Peroxisomal β-Oxidation of Stearic and Lignoceric Acids by Rat Brain

Crude subcellular fractions were prepared from adult rat brains by differential centrifugation of brain homogenates. Greater than 98% of the cellular mitochondrial marker enzyme activity sedimented in the heavy and light mitochondrial pellets, and less than 1% of the activity sedimented in microsomal pellets. Lysosomal marker enzyme activities mainly (71-78% of cellular activity) sedimented in the heavy and light mitochondrial pellets. Significant amounts of the lysosomal marker enzyme activity also sedimented in the crude microsomal pellets (9-13% of total) and high-speed supernatants (14-16% of total). The specific activities of microsomal and peroxisomal marker enzyme activities were highest in the crude microsomal pellets. Fractionation of the crude microsomal pellets on Nycodenz gradients resulted in the separation of the bulk of the remaining mitochondrial, lysosomal, and microsomal enzyme activities from peroxisomes. Fatty acyl-CoA synthetase activities separated on Nycodenz gradients as two distinct peaks, and the minor peak of the activities was in the peroxisomal enriched fraction. Fatty acid beta-oxidation activities also separated as two distinct peaks, and the activities were highest in the peroxisomal enriched fractions. Mitochondria were purified from the heavy mitochondrial pellets by Percoll density gradients. Fatty acyl-CoA synthetase and fatty acid beta-oxidation activities were present in both the purified mitochondrial and peroxisomal enriched fractions. Stearoyl-CoA synthetase activities were severalfold greater compared to lignoceroyl-CoA synthetase, and stearic acid beta-oxidation was severalfold greater compared to lignoceric acid beta-oxidation in purified mitochondrial and peroxisomal enriched fractions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Drug induction of hepatic glutathione S-transferases in male and female rats*.

The induction of the glutathione S-transferases by phenobarbital and polycyclic hydrocarbons was studied in male and female rats. Administration of phenobarbital resulted in 60-80% increase in S-aryl and S-aralkyl enzyme specific activities, whereas the S-epoxide and S-alkyl activities were increased by 30-40%. In following the sequence of induction, the former two activities were noted to reach peak activities before an increase in the latter two activities was observed. Both 3-methylcholanthrene and 3,4-benzopyrene were shown toi nduce these four enzymic activities, although without the discrimination between pairs of activities noted with phenobarbital. No change in Km accompanied the increase in Vmax. after induction by drugs, and no change occurred in Ki for sulphobromophthalein inhibition. Significantly lower enzyme specific activities were found for three of the activities studied in female rats but no difference was observed in the S-alkyltransferase activity. However, the proportional increase in the enzymic activities in response to phenobarbital was the same in males and females. These studies demonstrate the drug induction of a group of cytosolic drug-metabolizing enzymes as well as the identification of sex differences in these activities.     

The Metabolic and Phagocytic Activities of Leukocytes from Patients Receiving Corticosteroid and Radiation Therapy,and Patients with Bacterial Infections

Peripheral blood leukocytes from patients given corticosteroid or radiation therapy, as well as patients with bacterial or viral infections, were studied with regard to the selected enzyme activities of the hexose monophosphate shunt (HMS). Glucose-6-phosphate dehydrogenase (G-6-PD), 6-phosphogluconate dehydrogenase (6-PGD) and reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase were assayed spectrophotometrically on mixed leukocyte suspensions in isotonic glycerol. Enzyme activities of G-6-PD and NADPH oxidase in patients receiving corticosteroid or radiation therapy were significantly lower than the enzyme activity of 6-PGD. In patients with bacterial infections, activities of the three enzymes increased but in patients with viral infections, only the activities of NADPH oxidase and G-6-PD were slightly decreased. Nitroblue tetrazolium (NBT) dye-reducing activities of neutrophils from patients receiving corticosteroid or radiation therapy were attenuated which coincides with the reduced activities of HMS enzymes. From these results, it is likely that the reduced activities of intraleukocytic HMS enzymes of patients receiving corticosteroid or radiation therapy are correlated with intracellular bactericidal activities which might result from the attenuated level of hydrogen peroxide production.     

The metabolic and phagocytic activities of leukocytes from patients receiving corticosteroid and radiation therapy, and patients with bacterial infections.

Peripheral blood leukocytes from patients given corticosteroid or radiation therapy, as well as patients with bacterial or viral infections, were studied with regard to the selected enzyme activities of the hexose monophosphate shunt (HMS). Glucose-6-phosphate dehydrogenase (G-6-PD), 6-phosphogluconate dehydrogenase (6-PGD) and reduced nicotinamide-adenine dinucleotide phosphate (NADPH) oxidase were assayed spectrophotometrically on mixed leukocyte suspensions in isotonic glycerol. Enzyme activities of G-6PD and NADPH oxidase in patients receiving corticosteroid or radiation therapy were significantly lower than the enzyme activity of 6-PGD. In patients with bacterial infections, activities of the three enzymes increased but in patients with viral infections, only the activities of NADPH oxidase and G-6PD were slightly decreased. Nitroblue tetrazolium (NBT) dyereducing activities of neutrophils from patients receiving corticosteroid or radiation therapy were attenuated which coincides with the reduced activities of HMS enzymes. From these results, it is likely that the reduced activities of intraleukocytic HMS enzymes of patients receiving corticosteroid or radiation therapy are correlated with intracellular bactericidal activities which might result from the attenuated level of hydrogen peroxide production.     

X-chromosome inactivation and enzymatic activities in diploid and triploid fibroblasts

Diploid and triploid rabbit embryos obtained by artificial fertilization were cultured. No Barr's body in XXY and only one Barr's body in XXX triploid fibroblasts was observed. Six enzymatic activities were also determined. Two X-chromosome-bound enzymatic activities, glucose-6-phosphate dehydrogenase and phosphoglycerate kinase, were significantly increased in triploid fibroblasts, while another X-chromosome-bound activity (hypoxanthine phosphoribosyl transferase) was not modified. Among the autosome-bound activities studied, pyruvate kinase and adenine phosphoribosyl transferase were not modified, whereas in the triploid cells the 6-phosphogluconate dehydrogenase activity was decreased. The relationship between these modifications of enzymatic activities in triploids and the X-chromosome inactivation is discussed.     

Pyrophosphatase, Peroxidase and Polyphenoloxidase Activities during Leaf Development and Senescence

Inorganic pyrophosphatase, peroxidase, and polyphenoloxidase activities were studied as the function of leaf insertion level in eight monocotyledonous and eight dicotyledonous species. Alkaline inorganic pyrophosphatase shows a declining activity toward the end of senescence whereas no regular drift in either peroxidase or polyphenoloxidase activities was noticed during senescence of attached leaves. In the primary leaves of rice, peroxidase and polyphenoloxidase activities were high in the senescent leaves and there exists a correlation between chlorophyll content and peroxidase activity though not with polyphenoloxidase activity. Upon detachment leaves exhibit increasing peroxidase and polyphenoloxidase activities with time. The distribution of the enzyme activities during senescence of attached leaves is suggested to be species-specific, and an increase in peroxidase and polyphenoloxidase activities cannot be taken as an indicator of leaf senescence.     

Comparative distribution of glutamyl and aspartyl aminopeptidase activities in mouse organs.

To evaluate the functional role of glutamyl and aspartyl aminopeptidases, their soluble and membrane-bound activities were measured simultaneously in several tissues of normal mice using arylamide derivatives as substrates. Although the soluble aspartyl aminopeptidase activity showed its highest levels in the testicle, the rest of the activities presented their highest levels in the kidney. Different patterns of distribution were observed for glutamyl and aspartyl aminopeptidase activities and also for soluble and membrane-bound aspartyl aminopeptidase activities. However no major differences were observed between soluble and membrane-bound glutamyl aminopeptidase activities. This unequal distribution suggests that the use of arylamide derivatives as substrates is a sensitive method that distinguishes between these enzymatic activities. The results also suggest different functions for soluble and membrane-bound aspartyl aminopeptidase activities, and for glutamyl and aspartyl aminopeptidase activities.     

Age- and diapause-related acid and alkaline phosphatase activities in the intestine and malpighian tubules of the Colorado potato beetle, Leptinotarsa decemlineata (Say)

Specific activities for soluble (s) and membrane (m)-bound acid (ACP) and alkaline phosphatases (ALP) were determined in the midgut, hindgut, and Malpighian tubules for developing, prediapausing, and diapausing adult Colorado potato beetles, Leptinotarsa decemlineata (Say). High ACP activities were found in the hindgut and Malpighian tubules while high ALP activities were found in the Malpighian tubules. Variation in both ACP and ALP activities in each tissue reflects fluctuation in protein synthesis and secretion involved with digestion, excretion, and other unknown functions. Phosphatase activities in the tissues examined show the dynamic nature of diapause in this insect. Diapausing beetles showed increases in phosphatase activity after hormone treatments. JHA treatments increased s-ACP and m-ACP activities in all tissues but 20-HE did not increase activity in any tissue. Allatotropin tended to mimic the effects of JHA treatment. The s-ALP activity was also increased in all tissues whereas m-ALP was increased in the midgut and hindgut by JHA treatment. Malpighian tubule m-ALP activity was only increased by 20-HE treatments. Allatotropin was not as effective in increasing ALP activities as it was with ACP activities.     

Serum activities of dipeptidyl-aminopeptidase II and dipeptidyl-aminopeptidase IV in tumor-bearing animals and in cancer patients

Serum activities of dipeptidyl-aminopeptidases (DAP) II and IV were measured in tumor-bearing animals and in patients with blood and solid cancers using highly sensitive and specific fluorometric methods. In mice with intraperitoneal or subcutaneous implantation of Ehrlich ascites tumor cells, serum DAP II activity was increased and serum DAP IV activity was decreased, resulting in a significant increase in the ratio of serum DAP II and DAP IV activities. The increase in the ratio of these two activities paralleled the size of the subcutaneous tumors. However, both serum DAP II and DAP IV activities were increased in rats with experimental hepatic cancer induced by 3'-methyl-4-dimethylaminoazobenzene, and the increase in the ratio of the two activities was not significant. In cancer patients, as compared with healthy subjects, serum DAP II activity was increased and serum DAP IV activity was decreased, the ratio of serum DAP II and DAP IV activities being markedly increased in cancer patients. Both serum DAP II and DAP IV activities were increased in patients with hepatic cancer as were those in rats with hepatic cancer, but the increase in DAP II was greater than that of DAP IV; thus the ratio of the two activities increased significantly. These data suggest that the increase of the serum DAP II/DAP IV ratio could be a biochemical index of cancer.     

Characterization of alpha-amylase and pullulanase activities of Clostridium thermohydrosulfuricum. Evidence for a novel thermostable amylase.

Thermostable extracellular alpha-amylase and pullulanase activities of Clostridium thermohydrosulfuricum E 101-69 were characterized in a crude enzyme preparation. The activities responded similarly to temperature and pH, with optima at 85-90 degrees C and pH 5.6. The activities were stable at 65 degrees C, but were inactivated gradually in an identical manner at higher temperatures in the absence of Ca2+ and substrate. Ca2+ stabilized both activities similarly at high temperatures. Ca2+ also stimulated both activities, whereas EDTA reversed this stimulation. The activities were similarly inactivated at pH extremes. The two activities distributed in the same way during isoelectric focusing. The results suggest that the two activities are properties of the same protein, representing a novel, thermostable, amylase.     

Pyruvate dehydrogenase activities and rates of lipogenesis during the fed-to-starved transition in liver and brown adipose tissue of the rat.

The percentages of pyruvate dehydrogenase complex (PDH) in the active form (PDHa) in two lipogenic tissues (liver and brown adipose tissue) in the fed state were 12.0% and 13.4% respectively. After acute (0.5 h) insulin treatment, PDHa activities had increased by 77% in liver and by 234% in brown fat. Significant decreases in PDHa activities were observed in both tissues by 5 h after the removal of food. The patterns of decline in PDHa activities in the two lipogenic tissues were similar in that the major decreases in activities were observed within the first 7 h of starvation. The significant decreases in PDHa activities observed after starvation for 6 h were accompanied by decreased rates of lipogenesis. Hepatic and brown-fat PDHa activities after acute (30 min) exposure to exogenous insulin were less in 6 h-starved than in fed rats, but the absolute increases in PDHa activities over the 30 min exposure period were similar in fed and 6 h-starved rats. Increases in PDHa activities were paralleled by increases in lipid synthesis in both tissues. Re-activation of PDH in response to insulin treatment or chow re-feeding after 48 h starvation occurred more rapidly in brown adipose tissue than in liver. The results are discussed in relation to the importance of the activity of the PDH complex as a determinant of the total rate of lipogenesis during the fed-to-starved transition and after insulin challenge or re-feeding.     

Hormone, inorganic phosphate concentrations and enzyme activity in hemodialyzed and kidney-transplanted children

Several serum hormone concentrations, enzyme activities, and inorganic phosphate complexes were investigated in 13 hemodialyzed children, 7 kidney-transplanted children, and in 15 healthy controls. Prior to kidney transplantation 10 of the 14 tested hormone levels of hemodialyzed children differed significantly from those of healthy controls; however, after kidney transplantation most of them normalized, only the angiotensin-converting enzyme and alkaline phosphatase activities were significantly elevated in comparison with the control group. Among the different inorganic phosphate complexes, dialysis had the least effect on the CaHPO4 complex. In the hemodialyzed group the plasma renin activities were decreased and the amylase and lipase activities were increased.     

Control and location of acyl-hydrolysing phospholipase activity in pathogenic mycobacteria.

Phospholipase activities releasing fatty acyl moieties from phosphatidylcholine and phosphatidylethanolamine and lysophospholipase activity releasing fatty acid from lyso-phosphatidylcholine were detected in both Mycobacterium microti and Mycobacterium avium. Fatty acyl groups were released from both the 1- and 2-positions of phosphatidylcholine. Generally, phospholipase activities of M. avium were cryptic while phospholipase activities of M. microti were located on the bacterial surface. However, intact M. microti did not release fatty acids from phospholipids faster than M. avium. Neither Mycobacterium secreted acyl-hydrolysing phospholipase activity. All phospholipase activities were stimulated by including phospholipids in growth media: generally, cell extracts contained 6- to 15-fold higher specific activities than extracts from mycobacteria grown in media without added phospholipid. However, not all phospholipase activities were stimulated to the same degree in any given set of conditions, suggesting the existence of more than one phospholipase gene in each Mycobacterium.     

铜陵铜矿区凤丹根际和非根际土壤酶活性

对铜陵铜矿区凤丹根际和非根际土壤酶活性特征进行了研究.结果表明,凤丹根际土壤各种酶活性显著大于非根际土壤.凤丹非根际土壤过氧化氢酶、磷酸酶和脲酶活性能敏感地反映土壤重金属复合污染状况;根际环境对土壤酶活性的影响表现为: 磷酸酶＞脲酶＞过氧化氢酶＞蔗糖酶＞多酚氧化酶,影响率分别为131.562%、92.492%、87.557%、59.673%34.076%;土壤各种酶活性与重金属复合污染程度呈显著负相关,相关系数均-0.868以上,表现出重金属复合污染对土壤酶活性的抑制效应.凤丹可有效地改善土壤环境,提高土壤各种酶的活性.     

盐城海滨湿地盐沼植被及农作物下土壤酶活性特征

在盐城海滨湿地盐沼植被和农田内采集土壤样品,测定了4种土壤酶(脲酶、转化酶、过氧化氢酶和碱性磷酸酶)的活性,分析了盐沼植被、农作物及土壤理化因子对土壤酶活性的影响。结果表明:由海滨湿地滩涂围垦形成的各类农田其土壤酶活性较高,且均高于湿地盐沼植被;湿地盐沼植被下4种土壤酶活性均高于无植被生长的光滩,且不同类型植被间土壤酶活性差异显著;4种酶的活性大小总体表现为大豆(Glycine max)地棉花(Gossypium hirsutum)地玉米(Zea mays)地互花米草(Spartina alterniflora)滩白茅(Imperata cylindrica var.major)滩碱蓬(Suaeda salsa)滩光滩。相关分析表明,4种土壤酶之间及其与土壤有机碳、全氮均表现出显著正相关,而与土壤盐分、pH值之间存在显著负相关。海滨湿地盐沼植被的发育扩展不仅增加了土壤中的养分含量,也提高了土壤酶活性。     

Enzymatic formation of inosine 3',5'-monophosphate and of 2'-deoxyguanosine 3',5'-monophosphate. Inosinate and deoxyguanylate cyclase activity.

Enzymes in particulate fractions from sea urchin sperm and in soluble fractions from rat lung were shown to catalyze the formation of inosine 3',5'-monophosphate (cyclic IMP) and of 2'-deoxyguanosine 3',5'-monophosphate (cyclic dGMP) from ITP and dGTP, respectively. With sea urchin sperm particulate fractions, Mn2+ was an essential metal cofactor for inosinate, deoxyguanylate, guanylate and adenylate cyclase activities. Heat-inactivation studies differentiated inosinate and deoxyguanylate cyclase activities from adenylate cyclase, but indicated an association of these activities with guanylate cyclase. Preincubation of sea urchin sperm particulate fractions with trypsin altered in a very similar manner guanylate, inosinate, and deoxyguanylate cyclase activities, and various metals and metal-nucleotide combinations protected the three cyclase activities to comparable degrees against trypsin. The relative guanylate, deoxyguanylate and inosinate cyclase activities at 0.1 mM nucleoside triphosphate were 1.0, 0.5 and 0.08, respectively. With these three cyclase activities, plots of reciprocal velocities against reciprocal Mn2+-nucleoside triphosphate concentrations were concave upward, suggesting positive homotropic effects. With rat lung soluble preparations, relative guanylate, deoxyguanylate, inosinate and adenylate cyclase activities at 0.09 mM nucleoside triphosphate were 1.0, 1.7, 0.1 and 0, respectively. MnGTP was a competitive inhibitor of deoxyguanylate cyclase activity (Ki equals 12.2 muM) and MndGTP was a competitive inhibitor of guanylate cyclase activity (Ki equals 16.2 muM). Inhibition studies using ITP were not conducted. When soluble fractions from rat lung were applied to Bio-Gel A 1.5 m columns, elution profiles of guanylate, deoxyguanylate and inosinate cyclase activities were similar. These results suggest that deoxyguanylate, guanylate and inosinate cyclase activities reside within the same protein molecule.     

Disaccharidase activities in pregnant and lactating rats

The changes in intestinal disaccharidase activities in pregnancy and lactation could be explained as a result of an adaptation to the energy demands of each reproductive stage. To examine these changes, disaccharidase activities (lactase, sucrase, maltase, and trehalase) were measured in the jejunum and ileum of virgin, pregnant and lactating Wistar rats. Minor changes in disaccharidase activities were observed in pregnancy, whereas an increase of disaccharidase activities per small intestine length normalized to body mass was observed in lactation.