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.     

Sugars as a metabolic regulator of storage protein mobilization in germinating seeds of yellow lupine (Lupinus luteus L.)

The intensity of protein reserve activation in yellow lupine (Lupinus luteus L.) organs cultured in vitro in the presence of saccharose and without sugar in the medium was studied. Isolated embryo axes, excised cotyledons and seeds deprived of their testae were grown on Heller medium: a) with 60 mM saccharose (+S), b) without sugar (−S) and c) for 72 hours without saccharose + for 24 hours in the presence of saccharose (−S→+S). Using nitroanilide substrates, exo- and endopeptidase proteolytic activity was investigated in enzymatic extracts. Proteolytic activity was examined in organs isolated from swollen seeds and also in organs cultured for 24, 48, 72 and 96 hours. The proteolytic activity was higher in organs cultured on medium without saccharose. Stimulation of the proteolytic activity on the first day of culture was not significant, but intensified in the successive days of culture. The organ subcultured onto a medium with saccharose (−S→+S) caused a significant limitation of proteolytic activity, even to a markedly lower level in comparison to that activity level in the material continuously supplemented with saccharose. Observations of ultrastructure in Transmission Electron Microscopy revealed increased protein body degradation in the absence of saccharose in the medium and an increased degree of cell vacuolization, which may be indicative of intensifying autophagic processes under conditions of carbohydrate deficit.     

The significance of the cotyledons for the formation of diamine oxidase in pea plants

The effect of the removal of cotyledons on the growth of etiolated pea plants and on the changes in diamine oxidase activity were followed. Plants with removed cotyledons showed higher diamine oxidase activity both in the shoots and in the roots when expressed per fresh weight unit. Higher diamine oxidase activity can be found also in the remaining cotyledon when one of the two cotyledons is removed (when expressed per fresh weight unit and per cotyledon). The plants belonging to those experimental variants in which a higher diamine oxidase activity was established (plants without cotyledons) had simultaneously a high content of its substrates. These plants at the same time contained in the shoots and in the roots smaller amounts of potassium. On the other hand, these findings cannot be applied when comparing individual organs (shoots and roots), as higher diamine oxidase activity can be found in the shoots than in the roots, but the content of its substrates is in the shoots lower. These data indicate that the relationship between the activity of diamine oxidase and the content of its substrates is probably rather complicated. In the connection with the fact that a high diamine oxidase activity can occur in the plant simultaneously with a high content of its substrates, it is possible to assume that (a) changes in the pattern of substrates may occur after the removing of the cotyledons, (b) the synthesis of diamine oxidase may be induced owing to a higher accumulation of substrates, (c) the enzyme may be spacially separated from the substrates in the cells.     

Enzymatic metabolism of 3-deoxyglucosone,a Maillard intermediate

Summary In order to verify the formation of endogenous 3-deoxyglucosone (3-DG), an intermediate compound in the Maillard reaction, we tried to detect 3-deoxyfructose (3-DF) which is main metabolite of 3-DG. Endogenous 3-DF was detected in the urine of normal and diabetic rats by the oral administration of 3-DG-free feed. Metabolizing activities of crude extracts prepared from porcine organs were examined using methylglyoxal (MG) and 3-DG as substrates. NAD- or NADP-dependent 2-oxoaldehyde dehydrogenase activity was detected in liver, kidney, small intestine and lung. On the other hand, NADH- or NADPH-dependent 2-oxoaldehyde reductase activity was detected in all porcine organs in which liver and kidney contained higher activity of NADPH-dependent enzyme than the other organs. The reductase which catalyzes the reduction of 3-DG to 3-DF and MG to acetol, was purified and characterized from porcine kidney. The enzyme was the same to NADPH-dependent-2-oxoaldehyde reductase from porcine liver, which is speculated to prevent the advanced stage of the Maillard reaction as a self-defense enzyme.     

Cholinesterase activity in tissues of some crab species from the Japan Sea

The comparative study of the cholinesterase activity in some crab species was carried out for the first time with use of a set of thiocholine substrates. The substrate specificity was studied in stellar nerve, heart, and hemolymph of three crab species. The crab hemolymph was shown to be characterized by the highest enzyme activity. The enzyme from various crab organs has different structure o substrate specificity. Properties of crab enzymes were compared with acetylcholinesterase (AChE) of human blood erythrocytes, butyrylcholinesterase (BuChE) of horse blood serum, enzyme of squids and bivalve molluscs. The obtained data allow the conclusion to be made on differences in properties of enzymes both at the interspecies and at the tissue levels.     

STUDIES ON THE NATURAL SUBSTRATE FOR PROTEIN METHYLASE II IN MAMMALIAN BRAIN AND BLOOD

—The activity of protein methylase II (S-adenosylmethionine:protein-carboxyl methyltransferase, EC 2.1.1.24), which methylates (esterifies) free carboxyl groups in the substrate protein, was measured in several mammalian organs in an effort to elucidate the nature of the natural substrate for the enzyme. The highest endogenous substrate activity was found in posterior and anterior pituitary glands, possibly in association with neurosecretory granules. In other parts of the brain endogenous substrates are lacking, although the cytosol fractions contain high activity of the enzyme which methylates exogenously added substrates. Rat whole blood also contains endogenous substrate protein. The protein precipitated by 50% (NH₄)₂SO₄ contained active substrate protein whereas blood protein methylase II is localized exclusively in the erythrocytes. Cohn fractions I, II and III are more active as substrate for protein methylase II than fraction V.     

Are Z-Arg-Gly-Phe-Phe-Leu-MNA and Z-Arg-Gly-Phe-Phe-Pro-MNA suitable substrates for the demonstration of cathepsin D activity?

The suitability of Z-Arg-Gly-Phe-Phe-Leu-MNA and Z-Arg-Gly-Phe-Phe-Pro-MNA for the assessment of cathepsin D activity was tested in biochemical and histochemical experiments. Substrates were dissolved in dimethylformamide and used at 0.1-0.5 mM in various buffers over a pH range of 3.5-7.4. Homogenates of various rat organs and isolated purified enzymes [cathepsin D from bovine spleen, dipeptidyl peptidase (DPP) IV from porcine kidney and rat lung] were used as enzyme sources. Pepstatin, di-isopropylfluorophosphate (DFP), p-chloromercuribenzoate, o-phenanthroline and a series of DPP IV inhibitors were used in inhibitor experiments. At pH 3.5 and 5.0, substrates were used in a two-step postcoupling procedure with aminopeptidase M and dipeptidyl peptidase IV as auxiliary enzymes and Fast Blue BB as coupling agent. Results were compared with those obtained with haemoglobin. Above pH 5.0 substrates were used in a one-step postcoupling procedure. Cryostat sections of snap-frozen or cold aldehyde-fixed tissue pieces of various rat organs and biopsies of human jejunal mucosa were used in histochemical experiments. As in biochemical tests a two-step procedure was used in the pH range 3.5-5.0, but Fast Blue B was used in the second step for the simultaneous coupling. Above pH 5.0 a one-step simultaneous azo coupling procedure was used with Fast Blue B as coupling agent. At pH 3.5 the hydrolysis rate of both synthetic substrates was about 100x lower than that of haemoglobin when cathepsin D from bovine spleen was used.(ABSTRACT TRUNCATED AT 250 WORDS)     

Scanning Electron Microscopy of the Nematode-trapping Organs in Arthrobotrys oligospora

The three-dimensional capture organs of the nematodetrapping fungus Arthrobotrys oligospora are presented in scanning electron micrographs. The fungus was grown directly on agar substrates or on millipore filters or glass cover slips placed on agar substrates. To avoid shrinkage of the soft-walled mycelial material, a fixation in O_sO₄-vapour was performed, before freeze-drying and coating with Au-Pd alloy. When capture organs were prepared in this way, it was possible to visualize them in their erect position in the scanning electron microscope. Young capture organs, both spontaneously formed and nematode-induced, remained standing better than old ones. An adhesive substance on the capture organs, with the aid of which nematodes are trapped, seems to be visible in these pictures.     

Substrate specificity of monoamine oxidase from the brain and liver of the human fetus during antenatal ontogenesis

Dynamics of changes in the monoaminooxidase activity with respect to different substrates is characterized by a number of peculiarities and varies essentially in the process of ripening of the organs. Both in the brain and in the liver tyramine (a substrate of enzymes of types A and B), serotonin and norepinephrine (substrates of type A) and benzyl amine (substrate of type B) are subjected to oxidative deamination: tyramine--most intensely, serotonin and norepinephrine--less intensely and benzyl amine--least intensely.     

Distributions of Globotriaosylceramide Isoforms,and Globotriaosylsphingosine and Its Analogues in an α-Galactosidase A Knockout Mouse,a Model of Fabry Disease

Fabry disease is caused by deficient activity of α-galactosidase A (GLA) and characterized by systemic accumulation of glycosphingolipids, substrates of the enzyme. To gain insight into the pathogenesis of Fabry disease based on accumulated substrates, we examined the tissue and plasma distributions of globotriaosylceramide (Gb3) isoforms, and globotriaosylsphingosine (lyso-Gb3) and its analogues in a GLA knockout mouse, a model of Fabry disease, by means of liquid chromatography-mass spectrometry and nano-liquid chromatography-tandem mass spectrometry, respectively. The results revealed that the contents of these substrates in the liver, kidneys, heart, and plasma of GLA knockout mice were apparently higher than in those of wild-type ones, and organ specificity in the accumulation of Gb3 isoforms was found. Especially in the kidneys, accumulation of a large amount of Gb3 isoforms including hydroxylated residues was found. In the GLA knockout mice, the proportion of hydrophobic Gb3 isoforms was apparently higher than that in the wild-type mice. On the other hand, hydrophilic residues were abundant in plasma. Unlike that of Gb3, the concentration of lyso-Gb3 was high in the liver, and the lyso-Gb3/Gb3 ratio in plasma was significantly higher than those in the organs. The concentration of lyso-Gb3 was apparently higher than those of its analogues in the organs and plasma from both the GLA knockout and wild-type mice. This information will be useful for elucidating the basis of Fabry disease.     

Comparative analysis of enzyme activities and mRNA levels of peptidyl prolyl cis/trans isomerases in various organs of wild type and Pin1-/- mice

We investigated the enzyme activity of peptidyl prolyl cis/trans isomerases (PPIases) in brain, testis, lung, liver, and mouse embryonic fibroblasts (MEF) of Pin1+/+ and Pin1-/- mice. The aim of this study is to determine if other PPIases can substitute for the loss of Pin1 activity in Pin1-/- mice and what influence Pin1 depletion has on the activities of other PPIases members. The results show that high PPIase activities of Pin1 are found in organs that have the tendency to develop Pin1 knockout phenotypes and, therefore, provide for the first time an enzymological basis for these observations. Furthermore we determined the specific activity (k(cat)/K(M)) of endogenous Pin1 and found that it is strongly reduced as compared with the recombinant protein in all investigated organs. These results suggest that posttranslational modifications may influence the PPIase activity in vivo. The activities originating from cyclophilin and FKBP are not influenced by the Pin1 knockout, but a basal enzymatic activity towards phosphorylated substrates could be found in Pin1-/- lysates. Real time PCR experiments of all PPIases in different mouse organs and MEF of Pin1+/+ and Pin1-/- mice support the finding and reveal the specific expression profiles of PPIases in mice.     

Evidence of two enzymes performing the de-N-glycosylation of proteins in barley: expression during germination, localization within the grain and set-up during grain formation

The occurrence of two enzymes performing de-N-glycosylation of glycoproteins, namely, endo-N-acetyl-beta-D-glucosaminidase (ENGase, EC 3.2.1.96) and peptide-N(4)-(N-acetyl-beta-D-glucosaminyl) asparagine amidase (PNGase, EC 3.5.1.52) was investigated in barley, cv. Plaisant (a winter six rowed variety). The dry grain showed both activities according to the HPLC detection of the hydrolysis of fluorescent resorufin-labelled substrates. However, PNGase activity was 16-fold higher than ENGase activity. During germination, both activities increased, PNGase by only 1.5-fold compared to nearly 4.8-fold for ENGase over the 4 d following imbibition. The localization of these activities within the grain showed that the major contribution of PNGase was due to the endosperm, typically representing over 90% of the whole grain activity. In contrast, ENGase activity was especially high in the embryo and, later, in the developing plantlet (10-fold higher than in the endosperm), particularly in the rootlets and scutellum. In developing spikes, PNGase activity was 5.6-fold higher than in the leaves, but similar ENGase activity was measured in both organs. During grain formation, PNGase activity followed dry matter increase together with endosperm development. In contrast, ENGase activity dropped by 66% at the beginning of grain filling before stabilizing until harvest. The occurrence of de-N-glycosylation-performing enzymes throughout the development of barley raises the question of the nature of their natural substrates. Moreover, the prevalence of one of these enzymes over the other depending on the organ and the developmental stage, could represent the first evidence of specific functions for each enzyme.     

Proteolytic activity in relationship to senescence and cotyledonary development in Pisum sativum L.

Changes in the weight and in the chlorophyll, free amino-acid and protein content of developing and senescing, vegetative and reproductive organs of Pisum sativum L. (cv. Burpeeana) were measured, and the proteolytic activity in extracts from the senescing leaf and the subtended pod was followed in relation to these changes. Protein content decreased in the ageing leaf and pod while it increased in the developing cotyledon. The proteolytic activity of the leaf did not increase as the leaf protein content decreased. In contrast, proteolytic activity in the subtended pod increased while the protein level decreased. The proteolytic activity in the extracts from the ageing organs was greater than the rates of protein loss. The proteolytic activity of leaf and pod extracts was greater on protein prepared from the respective organ than on non-physiological substrates. Proteolysis was increased by 2-mercaptoethanol and ethylenediaminetetraacetate but was not influenced by addition of ATP to the reaction mixture. The pH optimum was at 5.0. Free amino acids did not accumulate in the senescing leaf or pod when protein was degraded in each organ. It is suggested that these amino acids were quickly metabolized in situ or translocated to sink areas in the plant, especially to the developing seeds.     

Pharmacology and physiology of monoamine oxidase activity in vertebrates--a comparative study

Monoamine oxidase (MAO) activity is high in brain, where it regulates neurotransmitter levels, and in the 'detoxifying' organs. Two MAO isoenzymes (A and B) apparently exist in terrestrial tetrapods, but only one form (type A-like) can be detected in teleosts and in aquatic amphibia. MAO activity is regulated by both endogenous (hormones, substrates) and exogenous (daylength, temperature) factors.     

Specificity and multiple forms of beta-galactosidase in the rat.

1. Ten rat tissues and organs have been assayed for beta-galactosidase with phenyl beta-d-galactoside, p-nitrophenyl beta-d-galactoside, p-aminophenyl beta-d-galactoside and 4-methylumbelliferyl beta-d-galactoside as substrates. 2. The relative activities of these tissues are independent of the mode of assay, and maximum rates of hydrolysis are not greatly affected by the nature of the substrate. 3. Inhibition studies suggest the liver enzyme has no associated beta-glucosidase activity. 4. There is no cellular localization of preferential activity towards any of the four substrates in liver, kidney or spleen. 5. Evidence suggesting the non-destructive penetration of liver lysosomal membranes by p-nitrophenyl beta-d-galactoside is presented. 6. Liver lysosomal beta-galactosidase exists in multiple forms that can be separated on DEAE-cellulose, and the enzyme components that are bound to the membrane appear to be similar to those of the lysosome sap. 7. The chromatographic pattern of enzyme excreted in the urine is compared with those from the kidney, intestine, spleen and liver.     

The secretory nature of the excretory gland cells of Strephanurus dentatus. 3. Proteinase inhibitors

A proteinase inhibitor(s) was found in extracts of the excretory gland cells, intestines, esophagi, reproductive organs, and body walls from Stephanurus dentatus adults. The specific activity of the inhibitor(s) in the excretory gland cell extract was 45–175 times greater than in the other tissues. It is heat stable at pH 5.0 and inhibits the esterolytic activity of trypsin and chymotrypsin using p-toluenesulfonyl-l-arginine methyl ester hydrochloride (TAME) and benzoyl-l-tyrosine ethyl ester (BTEE) as the substrates, respectively, and also the proteolytic activity of both chymotrypsin and trypsin using casein as the substrate. S. dentatus adults maintained in NCTC 109 medium, secreted a trypsin inhibitor.     

Are Z-Arg-Gly-Phe-Phe-Leu-MNA and Z-Arg-Gly-Phe-Phe-Pro-MNA suitable substrates for the demonstration of cathepsin D activity?

Summary The suitability of Z-Arg-Gly-Phe-Phe-Leu-MNA and Z-Arg-Gly-Phe-Phe-Pro-MNA for the assessment of cathepsin D activity was tested in biochemical and histochemical experiments. Substrates were dissolved in dimethylformamide and used at 0.1–0.5 mM in various buffers over a pH range of 3.5–7.4. Homogenates of various rat organs and isolated purified enzymes [cathepsin D from bovine spleen, dipeptidyl peptidase (DPP) I.V from porcine kidney and rat lung] were used as enzyme sources. Pepstatin, di-isopropylfluorophosphate (DFP),p-chloromercuribenzoate,o-phenanthroline and a series of DPP IV inhibitors were used in inhibitor experiments. At pH 3.5 and 5.0, substrates were used in a two-step postcoupling procedure with aminopeptidase M and dipeptidyl peptidase IV as auxiliary enzymes and Fast Blue BB as coupling agent. Results were compared with those obtained with haemoglobin. Above pH 5.0 substrates were used in a one-step postcoupling procedure.Cryostat sections of snap-frozen or cold aldehyde-fixed tissue pieces of various rat organs and biopsies of human jejunal mucosa were used in histochemical experiments. As in biochemical tests a two-step procedure was used in the pH range 3.5–5.0, but Fast Blue B was used in the second step for the simultancous coupling. Above pH 5.0 a onestep simultaneous azo coupling procedure was used with Fast Blue B as coupling agent.At pH 3.5 the hydrolysis rate of both synthetic substrates was about 100 x lower than that of haemoglobin when cathepsin D from bovine spleen was used. The activity was inhibited by pepstatin. With increasing pH the hydrolysis rate of Z-Arg-Gly-Phe-Phe-Pro-MNA increased, while that of Z-Arg-Gly-Phe-Phe-Leu-MNA decreased when organ homogenates were used as enzyme sources. However, the activity was not inhibited by pepstatin. It was inhibited by DFP. The extent of the inhibition with other substances was species and organ dependent. Z-Arg-Gly-Phe-Phe-Pro-MNA was also cleaved by isolated and purified DPP IV of porcine kidney and rat lung and the activity was inhibited by DFP and DPP IV inhibitors.In histochemical experiments the staining obtained with both synthetic substrates at pH 3.5 was weak and rather diffuse, with only slight accentuation or none at all in the lysosomal region of cells. In the pH range 5.5–7.4 a very distinct reaction was observed with Z-Arg-Gly-Phe-Phe-Pro-MNA only. The reaction product was localized in the brush border of enterocytes and of cells of the proximal kidney tubules. Endothelial cells of glomeruli and capillaries of the propria of the human jejunum also displayed a positive reaction. Lymphocytes in the propria of rat small intestine reacted to some extent. The reaction was inhibited by DFP. The extent of the inhibition with other substances varied.Z-Arg-Gly-Phe-Phe-Leu-MNA and Z-Arg-Gly-Phe-Phe-Pro-MNA are not efficient substrates for the assessment of cathepsin D activity. In histochemical studies diffusion artifacts must always be considered. In the pH range 5.5–7.4, Z-Arg-Gly-Phe-Phe-Pro-MNA is cleaved by a serine endopeptidase and by a metalloendopeptidase. It remains to be established whether prolyl endopeptidase or DPP IV (or both) and which metalloendopeptidase are responsible for the cleavage. In the evaluation of enterobiopsies the demonstration of this activity is a sensitive means for the assessment of the state of the brush border.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday.     

Mobilization of Nitrogen in Fruiting Plants of a Cultivar of Cowpea

Patterns of flow of nitrogen were constructed for the post-anthesisdevelopment of symbiotically-dependent cowpea (Vigna unguiculataWalp. cv. Vita 3-Rhizobium CB756). Nitrogen fixed after floweringcontributed 40% of the fruits' total intake of N, mobilizationof N fixed before flowering the remaining 60%. Leaflets, nodulatedroot, stem plus petioles, and peduncles contributed mobilizedN in the approximate proportions 5: 2: 1: 1 respectively. Eachfruit drew on all available current sources of N, but N fromleaves was distributed preferentially to closest fruit(s), andlower fruits monopolized the N exported from nodulated rootsduring late fruiting. Rates of nitrogen fixation declined parallel with decreasingnet photosynthesis of shoots. Leaflets at upper reproductivenodes mobilized 70–77% of their N and declined steeplyin net photosynthesis rate per unit chlorophyll or per unitribulose-l, 5-bisphosphate carboxylase (RuBPCase)² before abscisingduring mid- to late fruiting, whereas leaflets at lower vegetativenodes (1–3) mostly failed to abscise, lost 44–57%of their N and maintained photosynthetic activity throughoutfruiting. Peptide hydrolase activity was examined in extracts of leaflets,roots and nodules, by autodigestion of extracts, or in assaysusing bovine haemoglobin and purified RuBPCase isolated fromcowpea as substrates. Hydrolase activities during fruiting werebroadly related to N loss from plant organs, but asynchronyin peaks of activity against different protein substrates indicateddistinct groups of hydrolases within single organs. Hydrolaseactivity of leaflet extracts against RuBPCase was highly andpositively correlated with in vivo rates of loss of RuBPCasefrom the same leaflets, and preferential degradation of thisprotein occurred during leaflet senescence. Key words: Nitrogen, Mobilization: Cowpea     

Sensitive detection of transglycosylating activity of xyloglucan endotransglycosylase/hydrolase (XTH) after isoelectric focusing in polyacrylamide gels.

The paper describes a sensitive and rapid zymogram technique for detection of transglycosylating activity (XET) of xyloglucan endotransglycosylase/hydrolase (XTH; EC 2.4.1.207) in polyacrylamide isoelectric focusing gels. After the electrophoresis, the separating gel was overlaid and incubated with an agarose detection gel containing XET substrates: tamarind-seed xyloglucan as the glycosyl donor and sulphorhodamine-labeled xyloglucan-derived oligosaccharides (XGO-SRs) as the glycosyl acceptors. The transglycosylation catalyzed by XTH caused incorporation of the fluorescent label into the high-M(r) polysaccharide. Selective removal of unreacted XGO-SRs from the agarose replicas by washing with organic solvents revealed the zones corresponding to XET activity as bright pink fluorescent spots under UV-light. The method appears suitable for a number of purposes such as analysis of the isoenzyme composition of XTHs with XET activity in crude extracts from various plants and plant organs, monitoring the enzyme expression at various stages of plant development and/or for checking enzyme purity in the course of its isolation procedure.     

The antihypertensive drug carvedilol inhibits the activity of mitochondrial NADH-ubiquinone oxidoreductase

A study is presented on the interaction of carvedilol with mitochondria isolated from several rat organs. It is shown that carvedilol causes a moderate uncoupling effect under non phosphorylating succinate supported respiration of intact mitochondria, as well as a marked inhibition of coupled respiration with NAD-dependent substrates. The inhibitory effect was also found in the bovine heart purified Complex I as well as in experiments with mitochondrial particles, where the individual redox segments of the respiratory chain were analysed. It is also shown that carvedilol, though exhibiting an intrinsic scavenger activity, caused reactive oxygen species to be produced as a consequence of its inhibitory effect on the steady-state respiration. Under these conditions the pro-oxidant activity of carvedilol appears to prevail over its scavenging activity, and a net generation of ROS is promoted.