An Extracellullar Nuclease from Bacillus Firmus VKPACU-1: Specificity and Mode of Action

An extracellular nuclease from Bacillus firmus VKPACU-1 was multifunctional enzyme, this nuclease hydrolyzed poly U rapidly and more preferentially than the other homopolyribonucleotides. Hydrolysis of RNA this enzyme released mononucleotides in the order 5′UMP > 5′AMP > 5′GMP where as in hydrolysis of DNA the mononucleotides in the order of 5′dAMP > 5′dGMP > 5′dTMP and oligonucleotides. Uridylic linkages in RNA and adenylic linkages in DNA were preferentially cleaved by the nuclease. Nuclease produced oligonucleotides having only 3’ hydroxyl and 5’ phosphate termini. Present nuclease hydrolyzed RNA and DNA released oligonucleotides as major end products and mononucleotides, suggesting an endo mode of action.     

Effect of amines as activators on the alcohol-oxidizing activity of pyrroloquinoline quinone-dependent quinoprotein alcohol dehydrogenase

Pyrroloquinoline quinone-dependent quinoprotein alcohol dehydrogenases (PQQ-ADH) require ammonia or primary amines as activators in in vitro assays with artificial electron acceptors. We found that PQQ-ADH from Pseudomonas putida KT2440 (PpADH) was activated by various primary amines, di-methylamine, and tri-methylamine. The alcohol oxidation activity of PpADH was strongly enhanced and the affinity for substrates was also improved by pentylamine as an activator.     

Differential responses to chronic hypoxia and dietary restriction of aerobic capacity and enzyme levels in the rat myocardium

Chronic exposure of mammals to hypoxia induces a state of anorexia. We aimed to determine the role played by diet restriction in the alterations of myocardial energy metabolism occurring under chronic hypoxia in order to detect the specific effects of hypoxia per se.Adult female rats were exposed to normobaric hypoxia (Fi O2 = 0.10) for three weeks; pair-fed rats, kept under normoxic conditions, received the same amount of food as hypoxic rats. The oxidative capacity of myocardial ventricles and some skeletal muscles was evaluated using permeabilized fibers. Several metabolic enzyme activities were measured on extracts from myocardium and soleus.Diet restriction increased the activity of lactate dehydrogenase in both ventricles while it augmented phosphofructokinase and pyruvate kinase activities only in the left ventricle and depressed the respiratory rate in the right ventricle only.Hypoxia per se induced a rise in hexokinase activity in all studied oxidative muscles and a fall of hydroxy-acyl CoA-dehydrogenase activity in both myocardial ventricles. The respiratory rate and the citrate synthase activities were unaffected by hypoxia.We conclude that chronic hypoxia per se leads to specific alterations in myocardial metabolism that could favor the use of exogenous glucose at the expense of free fatty acids without any change in the oxidative capacity.     

Enzymatic characterization of ancestral/group-IV clade xyloglucan endotransglycosylase/hydrolase enzymes reveals broad substrate specificities

Xyloglucan endotransglycosylase/hydrolase (XTH) enzymes play important roles in cell wall remodelling. Although previous studies have shown a pathway of evolution for XTH genes from bacterial licheninases, through plant endoglucanases (EG16), the order of development within the phylogenetic clades of true XTHs is yet to be elucidated. In addition, recent studies have revealed interesting and potentially useful patterns of transglycosylation beyond the standard xyloglucan–xyloglucan donor/acceptor substrate activities. To study evolutionary relationships and to search for enzymes with useful broad substrate specificities, genes from the ‘ancestral’ XTH clade of two monocots, Brachypodium distachyon and Triticum aestivum, and two eudicots, Arabidopsis thaliana and Populus tremula, were investigated. Specific activities of the heterologously produced enzymes showed remarkably broad substrate specificities. All the enzymes studied had high activity with the cellulose analogue HEC (hydroxyethyl cellulose) as well as with mixed-link β-glucan as donor substrates, when compared with the standard xyloglucan. Even more surprising was the wide range of acceptor substrates that these enzymes were able to catalyse reactions with, opening a broad range of possible roles for these enzymes, both within plants and in industrial, pharmaceutical and medical fields. Genome screening and expression analyses unexpectedly revealed that genes from this clade were found only in angiosperm genomes and were predominantly or solely expressed in reproductive tissues. We therefore posit that this phylogenetic group is significantly different and should be renamed as the group-IV clade.     

The insulin-degrading enzyme as a link between insulin and neuropeptides metabolism

We have applied a recently developed HPLC-MS enzymatic assay to investigate the cryptic peptides generated by the action of the insulin-degrading enzyme (IDE) on some neuropeptides (NPs) involved in the development of tolerance and dependence to opioids. Particularly, the tested NPs are generated from the NPFF precursor (pro-NPFF (A)): NPFF (FLFQPQRF) and NPAF (AGEGLSSPFWSLAAPQRF). The results show that IDE is able to cleave NPFF and NPAF, generating specific cryptic peptides. As IDE is also responsible for the processing of many other peptides in the brain (amyloid beta protein among the others), we have also performed competitive degradation assays using mixtures of insulin and the above mentioned NPs. Data show that insulin is able to slow down the degradation of both NPs tested, whereas, surprisingly, NPAF is able to accelerate insulin degradation, hinting IDE as the possible link responsible of the mutual influence between insulin and NPs metabolism.     

A sensitive fluorigenic substrate for selective in vitro and in vivo assay of leukotriene A4 hydrolase activity

Leukotriene A4 hydrolase (LTA4H) is a bifunctional zinc-dependent metalloprotease bearing both an epoxide hydrolase, producing the pro-inflammatory LTB4 leukotriene, and an aminopeptidase activity, whose physiological relevance has long been ignored. Distinct substrates are commonly used for each activity, although none is completely satisfactory; LTA4, substrate for the hydrolase activity, is unstable and inactivates the enzyme, whereas aminoacids β-naphthylamide and para-nitroanilide, used as aminopeptidase substrates, are poor and nonselective. Based on the three-dimensional structure of LTA4H, we describe a new, specific, and high-affinity fluorigenic substrate, PL553 [l-(4-benzoyl)phenylalanyl-β-naphthylamide], with both in vitro and in vivo applications. PL553 possesses a catalytic efficiency (k_cat/K_m) of 3.8 ± 0.5 × 10⁴ M⁻¹ s⁻¹ using human recombinant LTA4H and a limit of detection and quantification of less than 1 to 2 ng. The PL553 assay was validated by measuring the inhibitory potency of known LTA4H inhibitors and used to characterize new specific amino-phosphinic inhibitors. The LTA4H inhibition measured with PL553 in mouse tissues, after intravenous administration of inhibitors, was also correlated with a reduction in LTB4 levels. This authenticates the assay as the first allowing the easy measurement of endogenous LTA4H activity and in vitro specific screening of new LTA4H inhibitors.     

痛泻要方对“肝气乘脾”泄泻小鼠肠黏膜及肠内容物消化酶活性的影响

目的探讨痛泻要方对"肝气乘脾"泄泻小鼠肠道酶活性的影响。方法采用"番泻叶-离心管束缚夹尾法"进行"肝气乘脾"泄泻造模,造模成功后以痛泻要方治疗,造模和治疗后分别分析小鼠肠道酶活性。结果造模后,模型组小鼠肠道内容物的淀粉酶活性显著降低(t=4.007,P=0.015),纤维素酶、蛋白酶、蔗糖酶活性下降不显著。模型组小鼠肠黏膜蛋白酶、淀粉酶、蔗糖酶活性显著下降(t_蛋=5.652,P=0.005;Z_淀=-1.964,P=0.050;t_蔗=4.737,P=0.009)。痛泻要方治疗后,中药干预组小鼠肠道内容物蛋白酶、纤维素酶、乳糖酶和蔗糖酶活性变化不显著;自然恢复组的淀粉酶活性显著高于正常组(t=-7.497,P=0.002)。中药干预组小鼠肠道前段黏膜蛋白酶、乳糖酶、淀粉酶、蔗糖酶活性恢复不显著;中药干预组小鼠肠道黏膜中段乳糖酶、蔗糖酶活性显著低于自然恢复组(t_乳=4.074,P=0.013;t_蔗=8.072,P0.001),而蛋白酶、淀粉酶及纤维素酶活性均高于自然恢复组;中药干预组小鼠肠道后段黏膜乳糖酶、蔗糖酶及淀粉酶活性显著高于正常组(t_乳=-7.962,P0.001;t_蔗=-15.921,P0.001;Z_淀=6.489,P=0.034),蛋白酶与纤维素酶活性均有所升高。结论 "肝气乘脾"泄泻肠道内容物及黏膜淀粉酶活性显著降低,痛泻要方对"肝气乘脾"泄泻小鼠肠黏膜乳糖酶、蔗糖酶及淀粉酶活性作用显著。     

Partial isolation from intact cells of a cell surface-exposed lysophosphatidylinositol-phospholipase C

A novel cell surface phosphoinositide-cleaving phospholipase C (ecto-PLC) activity was isolated from cultured cells by exploiting its presumed external exposure. Biotinylation of intact cells followed by solubilization of the biotinylated proteins from a membrane fraction and recovery onto immobilized-avidin beads, allowed assay of this cell surface enzyme activity apart from the background of the substantial family of intracellular PLCs. Several cell lines of differing ecto-PLC expression were examined as well as cells stably transfected to overexpress the glycosylphosphatidylinositol (GPI)-anchored protein human placental alkaline phosphatase (PLAP) as a cell surface enzyme marker. The resulting bead preparations from ecto-PLC positive cells possessed calcium-dependent PLC activity with preference for lysophosphatidylinositol (lysoPI) rather than phosphatidylinositol (PI). The function of ecto-PLC of intact cells evidently is not to release GPI-anchored proteins at the cell surface, as no detectable Ca²⁺-dependent release of overexpressed PLAP from ecto-PLC-positive cells was observed. To investigate the cell surface linkage of the ecto-PLC itself, intact cells were treated with bacterial PI-PLC to cleave simple GPI anchors, but no decrease in ecto-PLC activity was observed. High ionic strength washes of biotinylated membranes prior to the generation of bead preparations did not substantially reduce the lysoPI-PLC activity. The results verify that the ecto-PLC is truly cell surface-exposed, and unlike other members of the PLC family that are thought to be peripheral membrane proteins, this novel lysoPI-PLC is most likely a true membrane protein. J. Cell. Biochem. 65:550–564. © 1997 Wiley-Liss Inc.     

Conservation of Dynamics Associated with Biological Function in an Enzyme Superfamily

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Protective Effect of Betaine on Respiratory Enzymes

Effects of betaine and NaC1 in various concentrations on the activities of enzymes in tricarboxylic acid cycle (isocitric dehydrogenase, malic dehydrogenase, succinic dehydrogenase and fumarase), terminal oxidation (cytochrome oxidase) and photorespiratory pathway (glycolate oxidase and hydroxypyruvate reductase) have been studied. Betaine, in contrast to electolyte NaC1 was non-inhibitory to these enzymes up to 500 mmol/L. Partial protection against NaC1 inhibition to the activities of these enzymes were afforded by betaine. These results were consistent with the postulated role of betaine in cytoplasmic osmoregulation. These results showed that betaine was a superior compatible solute.