scholarly journals Chemical modification of aldehyde dehydrogenase by a vinyl ketone analogue of an insect pheromone

1990 ◽  
Vol 272 (2) ◽  
pp. 351-358 ◽  
Author(s):  
E E Blatter ◽  
M L Tasayco ◽  
G Prestwich ◽  
R Pietruszko
Keyword(s):  
Aldehyde Dehydrogenase ◽  
Vinyl Ketone ◽  
Insect Pheromone ◽  
Tobacco Budworm ◽  
Peptide Peak ◽  
General Utility ◽  
Enzyme Substrate ◽  
Affinity Labelling ◽  
Substrate Protection ◽  
Pheromone Metabolism

A major component of the sex pheromone from the tobacco budworm moth Heliothis virescens is a C16 straight-chain aldehyde with a single unsaturation at the eleventh position. The sex pheromones are inactivated when metabolized to their corresponding acids by insect aldehyde dehydrogenase. During this investigation it was demonstrated that the C16 aldehyde is a good substrate for human aldehyde dehydrogenase (EC 1.2.1.3) isoenzymes E1 and E2 with Km and Kcat. values at pH 7.0 of 2 microM and 0.4 mumol of NADH/min per mg and of 0.6 microM and 0.24 mumol of NADH/min per mg respectively. A vinyl ketone analogue of the pheromone inhibited insect pheromone metabolism; it also inactivated human aldehyde dehydrogenase. Total inactivation of both isoenzymes was achieved at stoichiometric (equal or less than the subunit number) concentrations of vinyl ketone, incorporating 2.1-2.6 molecules/molecule of enzyme. Substrate protection was observed in the presence of the parent aldehyde and 5′-AMP. Peptide maps of tryptic digests of the E2 isoenzyme modified with 3H-labelled vinyl ketone showed that incorporation occurred into a single peptide peak. The labelled peptide of E2 isoenzyme was further purified on h.p.l.c. and sequenced. The label was incorporated into cysteine-302 in the primary structure of E2 isoenzyme, thus indicating that cysteine-302 is located in the aldehyde substrate area of the active site of aldehyde dehydrogenase. Affinity labelling of aldehyde dehydrogenase with vinyl ketones may prove to be of general utility in biochemical studies of these enzymes.

Kinetic measurement of guanine deaminase in serum with a centrifugal analyzer.

1981 ◽  
Vol 27 (4) ◽  
pp. 560-561 ◽  
Author(s):  
Y Nishikawa ◽  
K Fukumoto
Keyword(s):  
Aldehyde Dehydrogenase ◽  
Kinetic Method ◽  
Reference Interval ◽  
Healthy Humans ◽  
Automated Method ◽  
Enzyme Substrate ◽  
Guanine Deaminase ◽  
One Step ◽  
Lag Period ◽  
Method R

Abstract We describe an enzymic, one-step kinetic method for determination of guanine deaminase (guanase, EC 3.5.4.3) in serum with a centrifugal analyzer. A combined enzyme-substrate system consists of the enzymes xanthine oxidase, catalase, and aldehyde dehydrogenase, the coenzyme NAD+, the substrate guanine, and ethanol in tris(hydroxymethyl)methylamine buffer, with KCl added as activator for aldehyde dehydrogenase. The method requires only 40 microL of sample. Guanase activity in 28 samples can be determined within 10 min by setting a 4-min lag period. The increase in absorbance at 340 nm is linearly proportional to the activity of guanase to 60 U/L. Within-run precision (CV) was 1.32 to 4.50% over the range studied. Day-to-day precision corresponds to CVs of 4.8 to 7.2% over the same range of guanase activity. The reference interval, as calculated from data on 25 healthy humans, was 0 to 1.02 U/L. The enzymic automated method shows good correlation with Caraway's (Clin. Chem. 12: 187, 1966) method (r = 0.949).

Oxidation of aromatic aldehydes and aliphatic secondary alcohols by Hyphomicrobium spp.

1982 ◽  
Vol 28 (1) ◽  
pp. 65-72 ◽  
Author(s):  
Jürgen Köhler ◽  
Arnold C. Schwartz
Keyword(s):  
Oxygen Consumption ◽  
Aldehyde Dehydrogenase ◽  
Secondary Alcohol ◽  
Aromatic Aldehydes ◽  
Polyacrylamide Gel ◽  
Secondary Alcohols ◽  
Multiple Forms ◽  
Aldehyde Dehydrogenases ◽  
Enzyme Substrate ◽  
Secondary Alcohol Dehydrogenase

Two of six tested strains of Hyphomicrobium respired on benzaldehyde with higher rates than on formaldehyde, and three strains with equal or lower rates, whereas one strain, Hyphomicrobium X, showed almost negligible respiration on benzaldehyde. Various substituted benzaldehydes stimulated oxygen consumption to lower rates than benzaldehyde itself in active strains. All strains contained multiple forms of dye-linked aldehyde dehydrogenase, as evident from polyacrylamide gel electropherograms of cell-free extracts and activity tests on the gels with nitroblue tetrazolium. All bands of this enzyme reacted more strongly with benzaldehyde than with formaldehyde in all strains. On gels of some strains additional bands appeared with benzaldehyde as the enzyme substrate. Hyphomicrobium X again displayed the lowest activity of this enzyme on the gels. A new band of this enzyme appeared on gels of strain ZV 580 after growth on methylamine, when tested in this respect. NAD-dependent secondary alcohol dehydrogenase was present on gels of three strains, which respired on 2-propanol.Difference spectra and observation of the degree of reduction of ubiquinone and cytochromes b and c of two strains indicated that the electrons from benzaldehyde oxidation were transferred to cytochrome c.The results are discussed with regard to the significance of dye-linked aldehyde dehydrogenases with broad substrate specificity for the catabolism and oligocarbophilic growth of Hyphomicrobium and the taxonomic relevance of the aldehyde dehydrogenase pattern observed on polyacrylamide gel electropherograms.

Mechanism of a long-chain fatty aldehyde dehydrogenase induced during the development of bioluminescence in Beneckea harveyi

1983 ◽  
Vol 61 (5) ◽  
pp. 301-306 ◽  
Author(s):  
Andrew Bognar ◽  
Edward Meighen
Keyword(s):  
Aldehyde Dehydrogenase ◽  
Substrate Inhibition ◽  
Kinetic Mechanism ◽  
Fatty Aldehyde ◽  
Rate Limiting Step ◽  
Dead End ◽  
Sequential Mechanism ◽  
High Concentrations ◽  
Substrate Protection ◽  
Long Chain

The kinetic mechanism of a long-chain aldehyde dehydrogenase that is induced during the development of bioluminescence in Beneckea harveyi has been investigated. Parallel lines were obtained in Lineweaver–Burk plots with NAD+ and long-chain aldehydes (heptaldehyde, nonylaldehyde). However, product and dead-end inhibitor studies, substrate protection (NAD+, aldehyde) against inactivation with N-ethylmaleimide, and in particular, a noncompetitive substrate inhibition pattern with aldehyde at high concentrations showed that aldehyde dehydrogenase had a sequential mechanism. The data were consistent with a nonrapid equilibrium random mechanism with a preferred order of addition of substrates (NAD+, aldehyde) and an ordered release of products with NADH release being the last and rate-limiting step in the reaction, a mechanism very similar to that found for short-chain mammalian aldehyde dehydrogenases. The present experiments emphasize the caution that must be taken in interpreting parallel patterns in initial velocity experiments, as well as the difficulty in classifying sequential enzyme mechanisms as either strictly ordered or random.

Microstructure of laser-irradiated, conducting Kapton polyimide

1995 ◽  
Vol 53 ◽  
pp. 502-503
Author(s):  
D. L. Callahan ◽  
Z. Ball ◽  
H. M. Phillips ◽  
R. Sauerbrey
Keyword(s):  
Electron Microscopy ◽  
Phase Transition ◽  
Transmission Electron Microscopy ◽  
Cross Section ◽  
Film Surface ◽  
Cross Sectional ◽  
General Utility ◽  
Transmission Electron ◽  
Considerable Debate ◽  
Potential Applications

Ultraviolet laser-irradiation can be used to induce an insulator-to-conductor phase transition on the surface of Kapton polyimide. Such structures have potential applications as resistors or conductors for VLSI applications as well as general utility electrodes. Although the percolative nature of the phase transformation has been well-established, there has been little definitive work on the mechanism or extent of transformation. In particular, there has been considerable debate about whether or not the transition is primarily photothermal in nature, as we propose, or photochemical. In this study, cross-sectional optical microscopy and transmission electron microscopy are utilized to characterize the nature of microstructural changes associated with the laser-induced pyrolysis of polyimide.Laser-modified polyimide samples initially 12 μm thick were prepared in cross-section by standard ultramicrotomy. Resulting contraction in parallel to the film surface has led to distortions in apparent magnification. The scale bars shown are calibrated for the direction normal to the film surface only.

Cell-Specific Chemical Delivery Using a Selective Nitroreductase–Nitroaryl Pair

2018 ◽  
Author(s):  
Todd D. Gruber ◽  
Chithra Krishnamurthy ◽  
Jonathan B. Grimm ◽  
Michael R. Tadross ◽  
Laura M. Wysocki ◽  
...  
Keyword(s):  
Small Molecules ◽  
Mammalian Cells ◽  
Target Cells ◽  
Specific Chemical ◽  
E Coli ◽  
Enzyme Substrate ◽  
Cell Specificity ◽  
General Chemical ◽  
Increased Activity ◽  
Enzyme Variant

<p>The utility of<b> </b>small molecules to probe or perturb biological systems is limited by the lack of cell-specificity. ‘Masking’ the activity of small molecules using a general chemical modification and ‘unmasking’ it only within target cells could overcome this limitation. To this end, we have developed a selective enzyme–substrate pair consisting of engineered variants of <i>E. coli</i> nitroreductase (NTR) and a 2‑nitro-<i>N</i>-methylimidazolyl (NM) masking group. To discover and optimize this NTR–NM system, we synthesized a series of fluorogenic substrates containing different nitroaromatic masking groups, confirmed their stability in cells, and identified the best substrate for NTR. We then engineered the enzyme for improved activity in mammalian cells, ultimately yielding an enzyme variant (enhanced NTR, or eNTR) that possesses up to 100-fold increased activity over wild-type NTR. These improved NTR enzymes combined with the optimal NM masking group enable rapid, selective unmasking of dyes, indicators, and drugs to genetically defined populations of cells.</p>

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