scholarly journals Characterization of the catalytic properties of the membrane-anchored metalloproteinase ADAM9 in cell-based assays

2017 ◽  
Vol 474 (9) ◽  
pp. 1467-1479 ◽  
Author(s):  
Thorsten Maretzky ◽  
Steven Swendeman ◽  
Elin Mogollon ◽  
Gisela Weskamp ◽  
Umut Sahin ◽  
...  
Keyword(s):  
Translational Regulation ◽  
Phorbol Esters ◽  
Catalytic Properties ◽  
Negative Control ◽  
Ectodomain Shedding ◽  
Molecular Fingerprint ◽  
Ephrin Receptor ◽  
New Information ◽  
Metalloprotease Inhibitors ◽  
20 Nm

ADAM9 (A Disintegrin And Metalloprotease 9) is a membrane-anchored metalloproteinase that has been implicated in pathological retinal neovascularization and in tumor progression. ADAM9 has constitutive catalytic activity in both biochemical and cell-based assays and can cleave several membrane proteins, including epidermal growth factor and Ephrin receptor B4; yet little is currently known about the catalytic properties of ADAM9 and its post-translational regulation and inhibitor profile in cell-based assays. To address this question, we monitored processing of the membrane-anchored Ephrin receptor B4 (EphB4) by co-expressing ADAM9, with the catalytically inactive ADAM9 E > A mutant serving as a negative control. We found that ADAM9-dependent shedding of EphB4 was not stimulated by three commonly employed activators of ADAM-dependent ectodomain shedding: phorbol esters, pervanadate or calcium ionophores. With respect to the inhibitor profile, we found that ADAM9 was inhibited by the hydroxamate-based metalloprotease inhibitors marimastat, TAPI-2, BB94, GM6001 and GW280264X, and by 10 nM of the tissue inhibitor of metalloproteinases (TIMP)-3, but not by up to 20 nM of TIMP-1 or -2. Additionally, we screened a non-hydroxamate small-molecule library for novel ADAM9 inhibitors and identified four compounds that selectively inhibited ADAM9-dependent proteolysis over ADAM10- or ADAM17-dependent processing. Taken together, the present study provides new information about the molecular fingerprint of ADAM9 in cell-based assays by showing that it is not stimulated by strong activators of ectodomain shedding and by defining a characteristic inhibitor profile. The identification of novel non-hydroxamate inhibitors of ADAM9 could provide the basis for designing more selective compounds that block the contribution of ADAM9 to pathological neovascularization and cancer.

Characterization of the catalytic activity of the membrane-anchored metalloproteinase ADAM15 in cell-based assays

2009 ◽  
Vol 420 (1) ◽  
pp. 105-113 ◽  
Author(s):  
Thorsten Maretzky ◽  
Guangli Yang ◽  
Ouathek Ouerfelli ◽  
Christopher M. Overall ◽  
Susanne Worpenberg ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Cancer Metastasis ◽  
Phorbol Esters ◽  
Catalytic Properties ◽  
Growth Factor Receptor ◽  
Ectodomain Shedding ◽  
Intact Cells ◽  
Peptide Substrates ◽  
Prostate Cancer Metastasis

ADAM15 (a disintegrin and metalloproteinase 15) is a membrane-anchored metalloproteinase, which is overexpressed in several human cancers and has been implicated in pathological neovascularization and prostate cancer metastasis. Yet, little is known about the catalytic properties of ADAM15. Here, we purified soluble recombinant ADAM15 to test for its ability to cleave a library of peptide substrates. However, we found no processing of any of the peptide substrates tested here, and therefore turned to cell-based assays to characterize the catalytic properties of ADAM15. Overexpression of full-length membrane-anchored ADAM15 or the catalytically inactive ADAM15E→A together with various membrane proteins resulted in increased release of the extracellular domain of the fibroblast growth factor receptor 2iiib (FGFR2iiib) by ADAM15, but not ADAM15E→A. This provided a robust assay for a characterization of the catalytic properties of ADAM15 in intact cells. We found that increased expression of ADAM15 resulted in increased FGFR2iiib shedding, but that ADAM15 was not stimulated by phorbol esters or calcium ionophores, two commonly used activators of ectodomain shedding. Moreover, ADAM15-dependent processing of FGFR2iiib was inhibited by the hydroxamate-based metalloproteinase inhibitors marimastat, TAPI-2 and GM6001, and by 50 nM TIMP-3 (tissue inhibitor of metalloproteinases 3), but not by 100 nM TIMP-1, and only weakly by 100 nM TIMP-2. These results define key catalytic properties of ADAM15 in cells and its response to stimulators and inhibitors of ectodomain shedding. A cell-based assay for the catalytic activity of ADAM15 could aid in identifying compounds, which could be used to block the function of ADAM15 in pathological neovascularization and cancer.

scholarly journals Indirect evidence for a strict negative control of S-adenosyl-l-methionine decarboxylase by spermidine in rat hepatoma cells

1981 ◽  
Vol 196 (2) ◽  
pp. 411-422 ◽  
Author(s):  
Pierre S. Mamont ◽  
Anne-Marie Joder-Ohlenbusch ◽  
Marlyse Nussli ◽  
Jeffrey Grove
Keyword(s):  
Actinomycin D ◽  
Catalytic Properties ◽  
Indirect Evidence ◽  
Negative Control ◽  
Regulatory Control ◽  
Decarboxylase Activity ◽  
Enzyme Protein ◽  
Site Of Action ◽  
Htc Cells ◽  
Rat Hepatoma

1. Direct or indirect inhibitors of l-ornithine decarboxylase (EC 4.1.1.17), structurally related or unrelated to l-ornithine, including dl-α-difluoromethylornithine, α-methylornithine and 1,3-diaminopropane, used alone or in combination, decreased polyamine concentrations in rat hepatoma tissue culture (HTC) cells and increased S-adenosyl-l-methionine decarboxylase activity (EC 4.1.1.50). 2. Comparison of the catalytic properties of S-adenosyl-l-methionine from cells with elevated and normal activities revealed no apparent modification of the catalytic site as judged by affinity for the substrate, stimulation by di- and tri-amines and inhibition by methylglyoxal bis-(guanylhydrazone). 3. Actinomycin D and cycloheximide, and RNA and a proteinsynthesis inhibitor respectively, blocked the increase of S-adenosyl-l-methionine decarboxylase activity elicited by α-difluoromethylornithine. In polyamine-depleted cells the apparent half-life of elevated S-adenosyl-l-methionine decarboxylase activity, determined by inhibition of protein synthesis, was 2.5-fold longer than in control cells. The present results suggest that elevation of S-adenosyl-l-methionine decarboxylase activity by α-difluoromethylornithine is due to stabilization of the enzyme. 4. Restoration of the normal intracellular putrescine content, by addition of putrescine to the medium of polyamine-deficient cells, transiently increased S-adenosyl-l-methionine decarboxylase activity. Thereafter, intracellular conversion of putrescine into spermidine was accompanied by inactivation of the enzyme at a rate that was similar to that found on addition of spermidine itself. No relationship between total intracellular spermine content and S-adenosyl-l-methionine decarboxylase activity could be established. 5. Addition of 1mm-1,3-diaminopropane to polyamine-deficient cells did not cause a decrease in the activity of S-adenosyl-l-methionine decarboxylase, whereas addition of 1,5-diaminopentane (cadaverine) did. 1,3-Diamino-N-(3-aminopropyl)propane did not accumulate in cells treated with α-difluoromethylornithine and 1,3-diaminopropane, whereas addition of 1,5-diaminopentane led to the accumulation of 1,5-diamino-N-(3-aminopropyl)pentane. 1,3-Diamino-N-(3-aminopropyl)propane (10μm) was as effective as spermidine in decreasing S-adenosyl-l-methionine decarboxylase activity. Thus effectiveness of a diamine in decreasing enzyme activity is related to its capability of being converted into a closely structurally related homologue of spermidine by spermidine synthase. 6. The spermidine site of action appears to be post-translational since (a) the spermidine-induced decrease of S-adenosyl-l-methionine activity was not prevented by actinomycin D and (b) spermidine in the presence of cycloheximide led to a synergistic inactivation of the enzyme with a decay rate that progressively approached control values. Altogether these results are indirect evidence for a strict negative control of S-adenosyl-l-methionine decarboxylase by spermidine and substantiate previous findings [Mamont, Duchesne, Grove & Tardif (1978) Exp. Cell Res.115, 387–393]. Spermidine appears to act on some processes involved in denaturation and/or degradation of the enzyme protein. Putrescine appears to decrease the rate of these processes. The physiological significance of the regulatory control of S-adenosyl-l-methionine decarboxylase is discussed.

Inelastic Relaxation in Tin Dioxide Thin Films

2006 ◽  
Vol 115 ◽  
pp. 275-278 ◽  
Author(s):  
V.I. Mitrokhin ◽  
S.I. Rembeza ◽  
E.S. Rembeza ◽  
A.A. Rudenko
Keyword(s):  
Thin Films ◽  
Internal Friction ◽  
Tin Dioxide ◽  
Water Solution ◽  
Relaxation Processes ◽  
Reactive Magnetron ◽  
Internal Friction Peak ◽  
New Information ◽  
Average Grain Size ◽  
20 Nm

Internal friction results obtained in thin SnO2 films produced by reactive magnetron sputtering (thickness of the film ~ 1 μm) and by the dehydration of water solution of tin salts (thickness of the film ~ 10 μm) are reported. It is suggested that internal friction peak observed in SnO2 films at around 170 oC is caused by relaxation processes on grain boundaries (average grain size is 20 nm). The investigation of internal friction in SnO2 films can yield new information about the structure of thin films.

scholarly journals Structural, Surface and Catalytic Properties of Nano-Sized Ceria Catalysts

2009 ◽  
Vol 27 (4) ◽  
pp. 413-422 ◽  
Author(s):  
N.M. Deraz ◽  
A. Alarifi
Keyword(s):  
Cerium Oxide ◽  
Active Sites ◽  
Catalytic Properties ◽  
Heating Temperature ◽  
Nitrogen Adsorption ◽  
Structural Surface ◽  
Oxide Particles ◽  
Ceria Catalysts ◽  
20 Nm ◽  
Xrd And Tem

Well-dispersed uniform spheres of crystalline CeO2 were prepared by calcining precursor particles obtained by heating ammonium cerium nitrate for 4 h. These spherical substrates were examined using XRD and TEM methods, and by nitrogen adsorption studies at −196 °C. Subsequently, such cerium oxide particles prepared by calcination at 400–600 °C were used as catalysts for the conversion of isopropanol at 250–450 °C, using a flow method. The results obtained showed that increasing the heating temperature of the system investigated from 400 °C to 600 °C stimulated the formation of a well-crystallized CeO2 phase having a crystallite size varying between 10 and 20 nm. Both the surface area and catalytic activity of cerium oxide were found to decrease on increasing the calcination temperature. All solids investigated behaved as dehydrogenation catalysts which were selective towards the formation of acetone. The heat treatment did not alter the mechanism of dehydrogenation of isopropanol, but changed the concentration of active sites involved in the catalyzed reaction without altering their energetic nature.

Subsidiary Components of the Flagella of Chlamydomonas Reinhardii

1970 ◽  
Vol 7 (3) ◽  
pp. 823-839
Author(s):  
J. M. HOPKINS
Keyword(s):  
Repeat Distance ◽  
Chlamydomonas Reinhardii ◽  
Central Pair ◽  
3 Dimensional ◽  
New Information ◽  
Radial Spokes ◽  
Axis Parallel ◽  
Outer Pair ◽  
Dispersed Material ◽  
20 Nm

Some subsidiary components of flagella from the alga Chlamydomonas reinhardii have been studied in the electron microscope using frayed and partially dispersed material, negatively stained. In all, 5 distinct subsidiary structures have been observed, 3 of which are associated with the 9 pairs of outer tubules and 2 with the central pair of tubules. 1. Radial spokes, about 33 nm long and 5 nm in diameter, are attached at right angles to the A tubule of each outer pair and extend into the lumen of the flagellum in the direction of the central pair of tubules, but do not reach them. The spokes usually occur in pairs along the length of each A tubule. The interval between pairs is about 70 nm and that between the 2 members of each pair about 30 nm. 2. ‘Secondary fibres’. The distal end of each spoke terminates in a hammerhead-like attachment some 10-20 nm by 5 nm with its axis parallel to the long axis of the flagellum. These hammerhead attachments are now identified with the so-called ‘secondary fibres’ previously deduced from micrographs of embedded and sectioned material. There is no evidence from the present work of a continuous secondary fibre throughout the length of the flagellum. 3. Side arms are found attached to the A tubule of each outer pair. These arms, which occur in pairs, are roughly at right angles to the radial spokes which are also attached to the A tubules. The side arm material is distributed along the tubule at regular intervals of about 14 nm. 4. The chemically more stable centre tubule has 2 longitudinal rows of projections, each projection being about 18 nm long with a repeat distance of about 16 nm. 5. Occasionally, on the chemically less stable centre tubule, there is observed one row of projections which are somewhat similar to those on the other tubule. New information has made it possible to reinterpret earlier work and to present a 3-dimensional picture of the external flagellum and its parts.

scholarly journals Phorbol 12-myristate 13-acetate activates an electrogenic H+-conducting pathway in the membrane of neutrophils

1992 ◽  
Vol 281 (3) ◽  
pp. 697-701 ◽  
Author(s):  
A Kapus ◽  
K Szászi ◽  
E Ligeti
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Nadph Oxidase ◽  
Phorbol Esters ◽  
Charge Compensation ◽  
Kinase C ◽  
Low Concentrations ◽  
Conducting Pathway ◽  
20 Nm ◽  
Stimulation Of

The mode of activation of an H(+)-conducting pathway present in the membrane of neutrophils was investigated. (1) Resting neutrophils released protons through an electrogenic Cd(2+)-inhibitable (K0.5 approximately 20 microM) route when a pH gradient and appropriate charge compensation was provided. (2) The rate of H+ efflux was stimulated over 2.5-fold by 4 beta-phorbol 12-myristate 13-acetate (PMA; K0.5 approximately 0.7 nM) or by 4 beta-phorbol 12,13-dibutyrate (K0.5 approximately 20 nM) even when the NADPH oxidase was blocked by p-chloromercuribenzoate. (3) Staurosporine inhibited the effect of PMA. (4) The H+ egress was not enhanced by 4 alpha-phorbol 12,13-didecanoate. (5) Low concentrations of Cd2+ (less than 40 microM) inhibited the H+ flux without influencing the oxidase. The results raise the possibility that protein kinase C could be involved in the activation of an electrogenic H(+)-conducting pathway in the membrane of neutrophils. The activation of this route by phorbol esters seems to be independent of the stimulation of NADPH oxidase.

scholarly journals The N domain of somatic angiotensin-converting enzyme negatively regulates ectodomain shedding and catalytic activity

2005 ◽  
Vol 389 (3) ◽  
pp. 739-744 ◽  
Author(s):  
Zenda L. Woodman ◽  
Sylva L. U. Schwager ◽  
Pierre Redelinghuys ◽  
Adriana K. Carmona ◽  
Mario R. W. Ehlers ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Angiotensin Converting Enzyme ◽  
Catalytic Properties ◽  
Close Contact ◽  
Catalytic Efficiency ◽  
Ectodomain Shedding ◽  
Converting Enzyme ◽  
Peptide Substrates ◽  
Recognition Motif ◽  
Hydrolysis Of

sACE (somatic angiotensin-converting enzyme) consists of two homologous, N and C domains, whereas the testis isoenzyme [tACE (testis ACE)] consists of a single C domain. Both isoenzymes are shed from the cell surface by a sheddase activity, although sACE is shed much less efficiently than tACE. We hypothesize that the N domain of sACE plays a regulatory role, by occluding a recognition motif on the C domain required for ectodomain shedding and by influencing the catalytic efficiency. To test this, we constructed two mutants: CNdom-ACE and CCdom-ACE. CNdom-ACE was shed less efficiently than sACE, whereas CCdom-ACE was shed as efficiently as tACE. Notably, cleavage occurred both within the stalk and the interdomain bridge in both mutants, suggesting that a sheddase recognition motif resides within the C domain and is capable of directly cleaving at both positions. Analysis of the catalytic properties of the mutants and comparison with sACE and tACE revealed that the kcat for sACE and CNdom-ACE was less than or equal to the sum of the kcat values for tACE and the N-domain, suggesting negative co-operativity, whereas the kcat value for the CCdom-ACE suggested positive co-operativity between the two domains. Taken together, the results provide support for (i) the existence of a sheddase recognition motif in the C domain and (ii) molecular flexibility of the N and C domains in sACE, resulting in occlusion of the C-domain recognition motif by the N domain as well as close contact of the two domains during hydrolysis of peptide substrates.

scholarly journals Mechanisms whereby insulin increases diacylglycerol in BC3H-1 myocytes

1988 ◽  
Vol 256 (1) ◽  
pp. 175-184 ◽  
Author(s):  
R V Farese ◽  
D R Cooper ◽  
T S Konda ◽  
G Nair ◽  
M L Standaert ◽  
...  
Keyword(s):  
Insulin Treatment ◽  
Phorbol Esters ◽  
De Novo ◽  
Specific Radioactivity ◽  
Ionophore A23187 ◽  
Insulin Effect ◽  
Kinase C ◽  
20 Nm ◽  
Hydrolysis Of ◽  
Protein Kinase C Activation

We previously suggested that insulin increases diacylglycerol (DAG) in BC3H-1 myocytes, both by increases in synthesis de novo of phosphatidic acid (PA) and by hydrolysis of non-inositol-containing phospholipids, such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE). We have now evaluated these insulin effects more thoroughly, and several potential mechanisms for their induction. In studies of the effect on PA synthesis de novo, insulin stimulated [2-3H]glycerol incorporation into PA, DAG, PC/PE and total glycerolipids of BC3H-1 myocytes, regardless of whether insulin was added simultaneously with, or after 2 h or 3 or 10 days of prelabelling with, [2-3H]glycerol. In prelabelled cells, time-related changes in [2-3H]glycerol labelling of DAG correlated well with increases in DAG content: both were maximal in 30-60 s and persisted for 20-30 min. [2-3H]Glycerol labelling of glycerol 3-phosphate, on the other hand, was decreased by insulin, presumably reflecting increased utilization for PA synthesis. Glycerol 3-phosphate concentrations were 0.36 and 0.38 mM before and 1 min after insulin treatment, and insulin effects could not be explained by increases in glycerol 3-phosphate specific radioactivity. In addition to that of [2-3H]glycerol, insulin increased [U-14C]glucose and [1,2,3-3H]glycerol incorporation into DAG and other glycerolipids. Effects of insulin on [2-3H]glycerol incorporation into DAG and other glycerolipids were half-maximal and maximal at 2 nM- and 20 nM-insulin respectively, and were not dependent on glucose concentration in the medium, extracellular Ca2+ or protein synthesis. Despite good correlation between [3H]DAG and DAG content, calculated increases in DAG content from glycerol 3-phosphate specific radioactivity (i.e. via the pathway of PA synthesis de novo) could account for only 15-30% of the observed increases in DAG content. In addition to increases in [3H]glycerol labelling of PC/PE, insulin rapidly (within 30 s) increased PC/PE labelling by [3H]arachidonic acid, [3H]myristic acid, and [14C]choline. Phenylephrine, ionophore A23187 and phorbol esters did not increase [2-3H]glycerol incorporation into DAG or other glycerolipids in 2-h-prelabelling experiments; thus activation of the phospholipase C which hydrolyses phosphatidylinositol, its mono- and bis-phosphate, Ca2+ mobilization, and protein kinase C activation, appear to be ruled out as mechanisms to explain the insulin effect on synthesis de novo of PA, DAG and PC.(ABSTRACT TRUNCATED AT 400 WORDS)

Lattice Imaging of Twin, Lath and α/Martensite Boundaries in Duplex Low Carbon Steels

1977 ◽  
Vol 35 ◽  
pp. 118-119
Author(s):  
J. Y. Koo ◽  
G. Thomas
Keyword(s):  
High Resolution Electron Microscopy ◽  
Ferrite Matrix ◽  
Carbon Steels ◽  
Bright Field Image ◽  
Low Carbon ◽  
Lattice Imaging ◽  
Bright Field ◽  
Lattice Image ◽  
New Information ◽  
Resolution Electron

High resolution electron microscopy has been shown to give new information on defects(1) and phase transformations in solids (2,3). In a continuing program of lattice fringe imaging of alloys, we have applied this technique to the martensitic transformation in steels in order to characterize the atomic environments near twin, lath and αmartensite boundaries. This paper describes current progress in this program.Figures A and B show lattice image and conventional bright field image of the same area of a duplex Fe/2Si/0.1C steel described elsewhere(4). The microstructure consists of internally twinned martensite (M) embedded in a ferrite matrix (F). Use of the 2-beam tilted illumination technique incorporating a twin reflection produced {110} fringes across the microtwins.

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