Assessing the Spectroscopic Properties and Enzyme Activity of Fluorescent Caspase Substrates

Abstract An enzyme associated with carthamin formation in Carthamus tinctorius L. (carthamin-synthe-sizing enzyme) was isolated from the soluble protein extract of the hypocotyl tips of the etiolated seedlings and purified up to 157-fold by the procedures applying (NH4)2SO4 fractionation, Ca(CH3CO2)2 precipitation, protamine sulfate treatment, Celite adsorption chromatography, and Sephadex G-100 gel filtration. Results from atomic absorption spectral analysis of the enzyme protein showed to contain K as a major component, and Ca and Mg as minor ones. Fe, Cu, and Mn could not be detected in the preparation. At pH 4.8 in 50.0 mᴍ acetate buffer, the partially purified enzyme reacted positively with a flame-coloured precarthamin to produce a reddish product in open cuvettes with incubation medium. The reaction product was identified as carthamin by examining its colour, chromatographic mobilities in different developing solvents and spectroscopic properties inclusive shifts, often by comparing with those of an authentic specimen. Anaerobic incubation reduced the enzyme activity, while exogenously applied O2 slightly enhanced the catalytic rate of carthamin formation. The enzyme was sensitive to phosphorus sub stances. Among those compounds tested at 1.2 mᴍ level, orthophosphate showed the most striking inhibitory action on the enzyme. Metal ions affected on the enzyme activity by different extents. Mn2+ stimulated the enzyme reaction, while Cu2+ and Mo6+ exhibited reverse effects. Fe2+, Fe3+, Zn2+, Mg2+, and Co2+ were also unfavourable to the enzyme catalyzed carthamin formation. The preparation of the carthamin-synthesizing enzyme showed no activity of polyphenol oxidase or peroxidase under the conditions specifically designed for detecting both enzyme activities.

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Catalytic and spectroscopic analysis of blue copper-containing nitrite reductase mutants altered in the environment of the type 2 copper centre: implications for substrate interaction

Biochemical Journal ◽

10.1042/bj3530259 ◽

2001 ◽

Vol 353 (2) ◽

pp. 259-266 ◽

Cited By ~ 7

Author(s):

Miguel PRUDÊNCIO ◽

Robert R. EADY ◽

Gary SAWERS

Keyword(s):

Electron Transfer ◽

Enzyme Activity ◽

Nitrite Reductase ◽

Methyl Viologen ◽

Spectroscopic Properties ◽

Sodium Dithionite ◽

Epr Spectrum ◽

Characteristic Type

The blue dissimilatory nitrite reductase (NiR) from Alcaligenes xylosoxidans is a trimer containing two types of Cu centre, three type 1 electron transfer centres and three type 2 centres. The latter have been implicated in the binding and reduction of nitrite. The Cu ion of the type 2 centre of the oxidized enzyme is ligated by three His residues, and additionally has a co-ordinated water molecule that is also hydrogen-bonded to the carboxyl of Asp92 [Dodd, Van Beeumen, Eady and Hasnain (1998), J. Mol. Biol. 282, 369Ő382]. Two mutations of this residue have been made, one to a glutamic acid residue and a second to an asparagine residue; the effects of both mutations on the spectroscopic and catalytic properties of the enzyme have been analysed. EPR spectroscopy revealed that both mutants retained intact type 1 Cu centres with g‖ = 2.12 (A‖ = 0mT) and g⊥ = 2.30 (A⊥ = 6.4mT), which was consistent with their blue colour, but differed in their activities and in the spectroscopic properties of the type 2 centres. The D92E mutant had an altered geometry of its type 2 centre such that nitrite was no longer capable of binding to elicit changes in the EPR parameters of this centre. Accordingly, this mutation resulted in a form of NiR that had very low enzyme activity with the artificial electron donors reduced Methyl Viologen and sodium dithionite. As isolated, the EPR spectrum of the Asp92 → Asn (D92N) mutant showed no characteristic type 2hyperfine lines. However, oxidation with iridium hexachloride partly restored a type 2 EPR signal, suggesting that type 2 copper is present in the enzyme but in a reduced, EPR-silent form. Like the Asp92 → Glu mutant, D92N had very low enzyme activities with either Methyl Viologen or dithionite. Remarkably, when the physiological electron donor reduced azurin I was used, both mutant proteins exhibited restoration of enzyme activity. The degree of restoration differed for the two mutants, with the D92N derivative exhibiting approx. 60% of the activity seen for the wild-type NiR. These findings suggest that on formation of an electron transfer complex with azurin, a conformational change in NiR occurs that returns the catalytic Cu centre to a functionally active state capable of binding and reducing nitrite.

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Rat liver guanidinoacetate methyltransferase. Proximity of cysteine residues at positions 15, 90 and 219 as revealed by site-directed mutagenesis and chemical modification

Biochemical Journal ◽

10.1042/bj2770399 ◽

1991 ◽

Vol 277 (2) ◽

pp. 399-406 ◽

Cited By ~ 10

Author(s):

Y Takata ◽

T Date ◽

M Fujioka

Keyword(s):

Enzyme Activity ◽

Chemical Modification ◽

Rat Liver ◽

Large Change ◽

Spectroscopic Properties ◽

Site Directed Mutagenesis ◽

Cross Linking ◽

Directed Mutagenesis ◽

Wild Type ◽

Nitrobenzoic Acid

Cys-90 of rat liver guanidinoacetate methyltransferase is a very reactive residue, and chemical modification of this residue results in a large decrease in activity [Fujioka, Konishi & Takata (1988) Biochemistry 27, 7658-7664]. To understand better the role of Cys-90 in catalysis, this residue was replaced with alanine by oligonucleotide-directed mutagenesis. The mutant is active and has kinetic constants similar to those of wild-type, indicating that Cys-90 is not involved in catalysis and substrate binding. The u.v.-absorption, fluorescence and c.d. spectra are also unchanged. Reaction of the mutant with an equimolar amount of 5,5′-dithiobis-(2-nitrobenzoic acid) or 2-nitro-5-thiocyanobenzoic acid results in an almost quantitative disulphide cross-linking between Cys-15 and Cys-21). The same treatment effects disulphide bond formation between Cys-15 and Cys-90 in wild type [Fujioka, Konishi & Takata (1988) Biochemistry 27, 7658-7664]. Since the mutant and wild-type enzymes appear to have similar secondary and tertiary structures, these results suggest that Cys-15, Cys-90 and Cys-219 of the methyltransferase occur spatially close together. The mutant cross-linked between Cys-15 and Cys-219 and the wild-type cross-linked between Cys-15 and Cys-90 show very similar spectroscopic properties. Although treatment of the mutant and wild-type enzymes with equimolar concentrations of 5,5′dithiobis-(2-nitrobenzoic acid) causes a large loss of enzyme activity in each case, kinetic analyses with the modified enzymes suggest that cross-linking of Cys-15 with Cys-90 or Cys-219 does not abolish activity and does not result in a large change in the Michaelis constants. Incubation of the mutant enzyme with excess 2-nitro-5-thiocyanobenzoic acid leads to modification of Cys-207 in addition to Cys-15 and Cys-219. Retention of considerable enzyme activity in the modified enzyme indicates that Cys-207 is also not an essential residue.

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Morphological and biochemical analyses of changes in rat hepatocytes related to wine and ethanol consumption

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100111276 ◽

1984 ◽

Vol 42 ◽

pp. 232-233

Author(s):

S.M. Geyer ◽

C.L. Mendenhall ◽

J.T. Hung ◽

E.L. Cardell ◽

R.L. Drake ◽

...

Keyword(s):

Electron Microscopy ◽

Endoplasmic Reticulum ◽

Enzyme Activity ◽

Malic Enzyme ◽

Smooth Endoplasmic Reticulum ◽

Rat Hepatocytes ◽

Mature Male ◽

Treatment Groups ◽

Malic Enzyme Activity ◽

Biochemical Analyses

Thirty-three mature male Holtzman rats were randomly placed in 3 treatment groups: Controls (C); Ethanolics (E); and Wine drinkers (W). The animals were fed synthetic diets (Lieber type) with ethanol or wine substituted isocalorically for carbohydrates in the diet of E and W groups, respectively. W received a volume of wine which provided the same gram quantity of alcohol consumed by E. The animals were sacrificed by decapitation after 6 weeks and the livers processed for quantitative triglycerides (T3), proteins, malic enzyme activity (MEA), light microscopy (LM) and electron microscopy (EM). Morphometric analysis of randomly selected LM and EM micrographs was performed to determine organellar changes in centrilobular (CV) and periportal (PV) regions of the liver. This analysis (Table 1) showed that hepatocytes from E were larger than those in C and W groups. Smooth endoplasmic reticulum decreased in E and increased in W compared to C values.

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