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Proteins can be lysine-acetylated both enzymatically, by lysine acetyltransferases (KATs), and non-enzymatically, by acetyl-CoA and/or acetyl-phosphate. Such modification can be reversed by lysine deacetylases classified as NAD+ -dependent sirtuins or by classical Zn2+ -dependent deacetylases (KDACs). The regulation of protein lysine acetylation events by KATs and sirtuins/KDACs, or by non-enzymatic processes, is often assessed only indirectly by mass spectrometry or by mutational studies in cells. Mutational approaches to study lysine acetylation are limited, as these often poorly mimic lysine acetylation. Here, we describe protocols to assess the direct regulation of protein lysine acetylation by both sirtuins/KDACs and KATs, as well as non-enzymatically. We first describe a protocol for the production of site-specific lysine-acetylated proteins using a synthetic biological approach, the genetic code expansion concept (GCEC). These natively folded, lysine-acetylated proteins can then be used as direct substrates for sirtuins and KDACs. This approach addresses various limitations encountered with other methods. First, results from sirtuin/KDAC-catalyzed deacetylation assays obtained using acetylated peptides as substrates can vary considerably compared to experiments using natively folded substrate proteins. In addition, producing lysine-acetylated proteins for deacetylation assays by using recombinantly expressed KATs is difficult, as these often do not yield proteins that are homogeneously and quantitatively lysine acetylated. Moreover, KATs are often huge multi-domain proteins, which are difficult to recombinantly express and purify in soluble form. We also describe protocols to study the direct regulation of protein lysine acetylation, both enzymatically, by sirtuins/KDACs and KATs, and non-enzymatically, by acetyl-CoA and/or acetyl-phosphate. The latter protocol also includes a section that explains how specific lysine acetylation sites can be detected by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). The protocols described here can be useful for providing a more detailed understanding of the enzymatic and non-enzymatic regulation of lysine acetylation sites, an important aspect to judge their physiological significance. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of N-(e)-lysine-acetylated proteins using the genetic code expansion concept (GCEC) Basic Protocol 2: In vitro sirtuin (SIRT)-catalyzed deacetylation of lysine-acetylated proteins prepared by the GCEC Basic Protocol 3: In vitro KDAC/HDAC-catalyzed deacetylation of lysine-acetylated proteins Basic Protocol 4: In vitro lysine acetylation of recombinantly expressed proteins by lysine acetyltransferases (KATs) Basic Protocol 5: In vitro non-enzymatic lysine acetylation of proteins by acetyl-CoA and/or acetyl-phosphate.

The acetylation/acylation (ac(et)ylation) of lysine side chains is a dynamic post-translational modification (PTM) regulating fundamental cellular processes with implications on the organisms’ ageing process: metabolism, transcription, translation, cell proliferation, regulation of the cytoskeleton and DNA damage repair. First identified to occur on histones, later studies revealed the presence of lysine ac(et)ylation in organisms of all kingdoms of life, in proteins covering all essential cellular processes. A remarkable finding showed that the NAD+-dependent sirtuin deacetylase Sir2 has an impact on replicative lifespan in Saccharomyces cerevisiae suggesting that lysine acetylation has a direct role in the ageing process. Later studies identified sirtuins as mediators for beneficial effects of caloric/dietary restriction on the organisms’ health- or lifespan. However, the molecular mechanisms underlying these effects are only incompletely understood. Progress in mass-spectrometry, structural biology, synthetic and semi-synthetic biology deepened our understanding of this PTM. This review summarizes recent developments in the research field. It shows how lysine ac(et)ylation regulates protein function, how it is regulated enzymatically and non-enzymatically, how a dysfunction in this post-translational machinery contributes to disease development. A focus is set on sirtuins and lysine acyltransferases as these are direct sensors and mediators of the cellular metabolic state. Finally, this review highlights technological advances to study lysine ac(et)ylation.

The concept of biocatalytic PET degradation for industrial recycling processes had made a big step when the bacterium Ideonella sakaiensis was discovered to break PET down to its building blocks at ambient temperature. This process involves two enzymes: cleavage of ester bonds in PET by PETase and in MHET, the resulting intermediate, by MHETase. To understand and further improve this unique capability, structural analysis of the involved enzymes was aimed at from early on. We describe a repertoire of methods to this end, including protein expression and purification, crystallization of apo and substrate-bound enzymes, and modeling of PETase complexed with a ligand.

Cationic, phosphine-ligated Pt-Au cluster compounds, [Pt(AuPPh{sub 3}){sub 8}](NO{sub 3}){sub 2} (1) and [(PPh{sub 3})Pt(AuPPh{sub 3}){sub 6}](NO{sub 3}){sub 2} (2), have been immobilized on silica and alumina supports. Characterization of the supported clusters by {sup 31}P MAS NMR, IR, and UV-visible spectroscopy and study by chemisorption and temperature-programmed desorption of CO show that the clusters are immobilized intact without measurable fragmentation or irreversible transformation. This is confirmed by quantitative desorption of the clusters. The reactivity of the supported clusters with CO and H{sub 2} was found to be similar to the known reactivities in solution and molecular solid phases. The cluster [(PPh{sub 3})-Pt(H)(AuPPh{sub 3}){sub 7}](NO{sub 3}){sub 2} (3) was also immobilized on silica and found to be intact by MAS{sup 31}P NMR and desorption experiments. The turnover rate (TOR) for H{sub 2}-D{sub 2} equilibration was determined for these supported clusters at room temperature. The clusters 1 and 2 were good catalysts on silica and alumina supports after treatment at 135{degrees}C under vacuum (TOR = 10-20 s{sup -1}), but showed significant activation by treatment at 110{degrees}C under H{sub 2} (TOR = 85-220 s{sup -1}). Cluster 3/SiO{sub 2} gave a similar rate (TOR = 170 s{sup -1}) with no thermal activation. All samplesmore » subjected to the thermolysis conditions given above were found to be intact by {sup 31}P MAS NMR and could be quantitatively desorbed. The high activities of the supported Pt-Au clusters are believed to result from support-promoted, partial PPh{sub 3} dissociation. Desorption experiments shows that this dissociation is reversible.« less

Burying SiO 2 layers into silicon substrates by silicon direct bonding is one of the most promising substrate technologies for increasing the high-temperature capability of smart power devices for high-temperature applications. Leakage currents are suppressed and minority charge carriers are kept away from critical components. Temperature effects on semiconductor device parameters caused by the high ambient temperature and the dissipated power have to be considered in the circuit design phase. Measurement results show the effects of the reduced thermal conductivity and of the very small thermal capacitance of the active device region. In addition, the results of a simplified analytical calculation method will be presented in this paper.

The basic principles of wafer fusion bonding including pretreatment, room temperature mating, and thermal annealing are presented. Techniques for the characterization of the bond quality are reviewed. Results for fusion bonding of other materials such as silicon nitride and polysilicon are discussed with a view to bond quality and application. Examples of fusion processes for power device fabrication show the feasibility of the technique.

A novel LDMOS transistor structure with breakdown voltages above 100 V has been fabricated in silicon-on-insulator-on-silicon (SOIS). This structure has been fabrication by silicon direct bonding (SDB) and etch-back to a typical film thickness of 1 mu m. The silicon carrier layer (handle) serves as a back-gate electrode, which, under proper bias, improves the transistor characteristics significantly. The effective channel length or basewidth is 0.3 mu m. Under these conditions, the drift region becomes the current-limiting element. The physics in the drift region in thin silicon films ( >

The incorporation of image sensors with parallel signal-processing components would increase image-processing speed dramatically. With the arrangement of the sensor elements in a separate silicon layer above the signal-processing circuits, the fill factor would be optimized as well. In this paper we report the fabrication of silicon photodiodes for application in such a three-dimensional integrated image sensor. The silicon for the sensor elements is grown with selective epitaxy, starting at seed windows (etched openings on an oxidized silicon surface). In the course of the chemical vapour deposition (CVD) process, the silicon grows laterally over the surrounding silicon dioxide, even at larger steps. The silicon crystals are polished for height reduction and planarization. Photodiodes with defined thickness and hexagonal shape determined with a plasma oxide layer are produced and various parameters measured. The photodiodes have their maximum responsivity in the visible range. Stacking faults originating at the border of the seed windows cause distinct leakage currents. Investigations of the cleaning procedure and the edge geometry of the seed windows to reduce these crystal defects are under study.

The feasibility of a retinal prosthesis for the blind has been discussed since the 1950s. Early attempts to realize a subretinal implantable prosthesis suffered from technical and microsurgical limitations at that time. Nowadays these problems can be solved because of the amazing progress that has been made in the fields of intraocular surgery, silicon microfabrication and our knowledge of the silicon/neuron junction. Therefore, a new attempt to develop a retinal prosthesis seems justified. While other research groups1, 2, 3 have concentrated their efforts on the development of an epiretinal visual prosthesis, which receives energy and preprocessed visual information via a relatively complex telemetric system, Chow and coworkers4, 5, 6, 7 published the concept of a retinal implant consisting of thousands of microphotodiodes. The principle of this concept is based on an implant that is placed in subretinal space and stimulates the underlying retinal cells with the photocurrents generated solely from the incident light (Fig. 1).

The article contains sections titled: 1. History 2. Properties 3. Occurrence; Ore Extraction and Beneficiation 3.1. Minerals 3.2. Deposits 3.3. Mining 3.4. Ore Beneficiation 4. Smelting 4.1. Fundamental Theory of Smelting 4.2. Special Aspects of the Winning of Tin from its Ores 4.3. Production of Crude Tin 4.3.1. General Aspects 4.3.2. Ore Preparation prior to Reduction 4.3.2.1. Pyrometallurgical Enrichment of Low-Grade Concentrates 4.3.2.2. Roasting 4.3.2.3. Leaching 4.3.3. Reduction 4.3.3.1. Reduction in a Shaft Kiln 4.3.3.2. Reduction in a Reverberatory Furnace 4.3.3.3. Reduction in Rotary Kilns 4.3.3.4. Reduction in an Electric Furnace 4.3.3.5. Other Reduction Processes 4.3.4. Slag Processing 5. Refining 5.1. Pyrometallurgical Refining 5.1.1. Removal of Iron 5.1.2. Removal of Copper 5.1.3. Removal of Arsenic 5.1.4. Removal of Lead 5.1.5. Removal of Bismuth 5.2. Electrorefining 5.2.1. Electrorefining in Acid Medium 5.2.2. Electrorefining in an Alkaline Medium 5.2.3. Other Methods of Electrorefining 6. Recovery of Tin from Scrap Materials and Residues 7. Analysis 7.1. Analysis of Ores and Concentrates 7.2. Analysis of Metallic Tin 8. Economic Aspects 9. Tin Alloys and Coatings 10. Inorganic Tin Compounds 10.1. Tin(II) Compounds 10.2. Tin(IV) Compounds 11. Organic Compounds of Tin 11.1. Properties of Organotin Compounds 11.2. Production of Organotin Compounds 11.3. Industrially Important Compounds 11.4. Analysis of Organotin Compounds 11.5. Storage and Shipping of Organotin Compounds 11.6. Pattern of Production and Consumption 12. Toxicology

Thermal bonding of oxidized wafers is used to produce dielectrically insulated silicon for device fabrication. The two step bonding process is described and experiments with different wafer surfaces including prestructured and nitride coated wafers are presented. Thinning techniques for Siliconon-Insulator (SOI) fabrication are compared regarding film uniformity and material quality. SOI films in the thickness of 1–10 microns have been obtained with an etchback process using a p+-etchstop layer.

Silicon on insulator on silicon (SOIS) has been produced with silicon direct bonding (SDB). Within a silicon film of 15- mu m thickness, islands with ubiquitous oxide isolation have been formed for the simultaneous integration of 150-V power VDMOS transistors, CMOS circuits in a channelless sea-of-gates array with 2- mu m gates, and bipolar transistors. The up-drain VDMOS transistors with 2- Omega -mm/sup 2/ specific on-resistance allow multiple isolated outputs, so high-voltage push-pull drivers can be fabricated in a single chip. The bipolar transistors are comparable to those of a 60-V standard process with vertical n-p-n and lateral p-n-p current gains of 80. >

Sedimentation of an autumn &atom bloom and its effect on sediment was simulated in the laboratory. Benthic response was similar to that observed in previous field studies: (1) a response within hours, (2) a rise of the chemocline, and (3) a change to pronounced anaerobiosis. Benthic metabolism was affected down to at least 7 cm sediment depth. Significantly more organic matter was consunled than was added by &atoms. Sediment oxygen consumption explained < 3 O/ O of the organic matter used, while heat production explained 89 % of the burned material integrated over 7 cm sediment depth. The geochernical model of oxidizing organic matter is criticized Development of ATP-blomass was in phase with the observed pattern of heat production. Efficiency of benthic response (P/C) was 50 to 60 % in the surface and

Summary: Resume: Zusammenfassung Thirty-eight different agronomic and horticultural crop plants were tested for aryl acylamidase enzymes which could hydrolyse the amide bond of the herbicide, 3′,4′-dichloropropion-anilide (propanil). More than half of these plants, from ten different plant families, covering thirty genera, possessed enzymes capable of degrading propanil in this manner. Enzyme preparations from leaf lettuce (Lactuca sativa L.) appeared to be the richest source of aryl acylamidase activity. The total enzyme units present, as well as specific activities of enzymes from these plant tissue preparations, varied widely in the different plants. Hydrotyse du 3′,4′-dichtoropropionanitide par des aryt acylamytases vegetates. Trente-huit cultures differentes parmi lesquelles des plantes horticoles ont ete essayees quant a la production d'aryl acylamylases, enzymes qui pourraient hydrolyser la liaison amidede l'herbicide 3′,4′-dichloropropionanilide (propanil). Plus de la moitie de ces plantes appartenant 4 10 families differentes et representant 30 genres possedent des enzymes capables de degrader le propanil de cette maniere. Des preparations enzymatiques effectuees a partir de la feuille de laitue (Lactuca sativa L.) se sont revelees etre les sources les plus abondantes d'activite aryl acylamylase. Le total des unites enzymatiques presentes, aussi bien que les activites specifiques des enzymes extraites de ces preparations de tissus vegetaux ont varie de facon importante selon les especes vegetales. Hydrolyse von 3′,4′-Dichlorpropionanilid durch pflanzliche Arytacytamidasen Es wurden 38 verschiedene landwirtschaftliche und gartnerische Kulturpflanzen auf Arylacylamidase Enzyme untersucht, die die Amidbindung des Herbizids 3′,4′-Dichlor-propionanilid (Propanil) hydrolysieren konnten. Mehr als die Halfte dieser Pflanzen, aus zehn verschiedenen Pflanzenfamilien, 30 Gattungen einschliessend, enthielten Enzyme, die Propanil auf diese Weise abzubauen vermochten. Enzympraparationen von Kopfsalat (Lactuca sativa L.) schienen die ergiebigste Quelle fur die Arylacylamidase-Aktivitat zu sein. Die insgesamt vorhandenen Enzymeinheiten, als auch die spezifischen Aktivitaten der Enzyme der Gewebepraparationen, schwankten bei den verschiedenen Pflanzen ziemlich stark.

Adenosine-5-phosphates (ATP, ADP, AMP) are adsorbed by clay minerals at very low concen- trations (~

The interaction between different lattice defects and domain walls yields different temperature dependence of the coercive force H c . This paper presents an experimental study of the temperature variation of the coercive field of plastically deformed nickel single crystals during recovery within the recovery stages III and IV. H c is found to be a very sensitive quantity for the indication of recovery processes, if it is measured at low temperatures (78° K). A total recovery rate \DeltaH_{c}/H_{c} in these stages between 6 and 15 percent is observed, depending on orientation. It is very difficult to separate stages III and IV, which show a complicated substructure. The temperature dependence of H c is expressed in the form H_{c} = H_{co} \cdot K_{1}^{-n} (K 1 is the first constant of magnetocrystalline energy). The power n is found to lie near one for \langle100\rangle crystals, between 1/4 and 1/2 for \langle111\rangle and between 1/12 and 1/8 for \langle110\rangle and \langlemedium\rangle orientation. The variation of n with annealing temperature indicates a great influence of the dislocation network on the recovery process in stage IV.

An aryl acylamidase which hydrolyzes the herbicide propanil (3′,4′-dichloropropionanilide), has been isolated from tulip bulbs and partially purified and characterized. Several chlorinated ring-substituted propionanilide analogs as well as 3,4-dichloroacetanilide and 3′,4′-dichloro-2-methacrylanilide (dicryl) were hydrolyzed by the enzyme. The partially purified enzyme lacked sensitive-SH groups and possessed a broad pH optimum between 6·8 and 7·8. The enzyme was relatively stable up to about 50°, but lost activity rapidly on exposure to temperatures above this. The optimum temperature of assay with 3′,4′-dichloropropionanilide was 53°. The apparent activation energy of the enzyme was 10·3 kcal/mole and the apparent Km was 2·50 × 10−3m, with 3′,4′-dichloropropionanilide as substrate. The properties of this acylamidase from tulip were compared with aryl acylamidases from rice and liver.

A soluble enzyme with carbonic anhydrase activity was isolated from pea leaves and purified 200-fold by ammonium sulfate fractionation, gel filtration and preparative polyacrylamide gel electrophoresis. The purified enzyme behaved as a single component in analytical polyacrylamide gel disc electrophoresis at pH 8·9. Its molecular weight was 194,000 as determined by gel filtration and analytical ultracentrifugation. When the enzyme was electrophorized in the presence of sodium dodecyl sulfate, a molecular weight of 30,000 was observed. The specific activity for the dehydration reaction was approximately 1000 U/mg protein at 20° and the Km was 30 mM. The enzymatic activity was inhibited by acetazolamide, p-chloromercuribenzoate and o-iodosobenzoate. No esterase activity was detected with p-nitrophenyl acetate as the substrate. The amino acid composition of the enzyme has been determined. Neutron activation analysis indicated the presence of zinc in the ratio of six moles of zinc per one mole of enzyme.

Anomalous water samples were prepared by condensing water vapor into glass and quartz capillaries. The transfer of microliter quantities of anomalous water/water mixtures into larger capillaries suitable for further studies was accomplished by centrifugation. Some of the droplets showed optical anisotropy when viewed under a polarizing microscope. A density gradient method was developed to measure the specific gravity of anomalous water droplets. In anomalous water the rate of trypsincatalyzed hydrolysis of the chromogenic substrate Nα-benzoyl-DL-arginine p-nitroanilide was less than 10% of the rate of hydrolysis observed in normal water. As reported earlier, the state of water associated with the enzyme carbonate hydro-lyase (EC 4.2.1.1) was studied by means of γ-γ directional correlation. With the use of the same technique and 133Ba tracer, a method was developed to measure and compare relaxation times and field symmetries in normal water, protein-bound water, and anomalous water condensed in capillaries.

Measurements of the perturbed gamma-gamma directional correlation were made for the 173-247 keV cascade in 111Cd at the In ion site in dipalmitoylphosphatidylcholine liposomes. A time-dependent electric quadrupole interaction was observed. Below the order in equilibrium fluid phase transition temperature the exponential damping coefficient was lambda 2 = 0.033 +/- 0.007 ns-1. Above the transition temperature the coefficient was lambda 2 = 0.015 +/- 0.006 ns-1 indicating a twofold increase in molecular mobility in the disordered phase. Absolute values for the motional correlation time, tau c, were estimated to be on the order of a few nanoseconds. The presence of cholesterol was found to cause no significant difference in the molecular mobility of the phosphatidylcholine head groups in the ordered and fluid states.

Nineteen different genera of weeds encompassing twelve plant families were surveyed for hydrolytic enzymes which were able to metabolize the amide bonds in several groups of herbicides. Propanil (3′,4′-dichloropropionanilide), 1,1-dimethyl-3-phenylurea (fenuron), and isopropyl carbanilate (propham) were employed as enzyme substrates, representing the amide, urea, and carbamate classes. Propanil was hydrolyzed at widely diverse rates by approximately 70% of the plants tested. Only one plant showed a hydrolysis of propham and fenuron. The study demonstrates a widespread distribution of hydrolytic enzymes capable of degrading the herbicide, propanil. An apparent lack of hydrolytic enzymes able to metabolize propham and fenuron was also noted.

The structural features of carbonic anhydrase (carbonate hydro-lyase; EC 4.2.1.1) in aqueous solution were probed by the positron annihilation technique. The data obtained under varying conditions of temperature, pH, and enzyme concentration were interpreted in terms of the free volume model. The change of enzymic activity with temperature is accompanied by a change in free volume of the protein. Upon thermal denaturation an irreversible change in free volume of the molecule occurred. At low temperatures the protein-water interactions were investigated. These results are discussed in terms of current concepts of structure-function relationships in proteins. This study shows the sensitivity of the positron annihilation method toward the structure of proteins related to their overall conformation and to the nature of bound water.

Publisher Summary Many organisms require nickel for growth. This chapter discusses the function and properties of three nickel enzymes. Methanogenic bacteria are dependent on nickel for growth. In an earlier study, by using the radioactive isotope 63Nickel, it was shown that about 50% of the nickel taken up by these organisms was incorporated into factor F430. All methanogenic bacteria are able to grow on H2 and CO2 as sole energy sources. These organisms contain two uptake hydrogenases. The name uptake hydrogenase points out that the physiological role of these enzymes is to catalyze the uptake of hydrogen rather than its formation. The synthesis of both hydrogenases is dependent on nickel, which indicates that nickel is an essential component of both enzymes.

Continuous fermentation of sulphite waste liquor by Propionibacterium arabinosum was accomplished in a system with bacterial population maintained in suspension (3.0 × 108 cells per ml) in circulating liquor, and partially as visible colonies on lump limestone. The limestone served to maintain optimum pH in the fermenting waste liquor. When the population was established, no growth substances were necessary in waste liquor feed. Recycling a portion of the fermented liquor was beneficial in obtaining rapid adjustment of pH through reaction of calcium salts of organic acids with acidic components of fresh waste liquor. The calcium lactate present in recycle liquid stimulated rate of conversion of fermentable sugar to volatile acids and aided in pH control. Conversion of fermentable sugars to volatile acids was obtained at 83.0 to 86.5% efficiency in 55 hours retention time. The volatile acids were propionic and acetic acids in 2:1 mole ratio.

The feasibility of using enzymatic reactions for carbon dioxide control of closed atmospheres was investigated in two directions. The hydration and dehydration of carbon dioxide by bovine carbonate hydro-lyase (EC 4.2.1.1) was developed to a continuous laboratory scale operation. The multienzyme system, pyruvate: CO2 ligase (EC 6.4.1.1), L-malate: NAD oxidoreductase (EC 1.1.1.37), and L-malate: NADP oidoreductase (decarboxylating) (EC 1.1.1.40), was tested for carbon dioxide exchange under static conditions.