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  • Analytical Chemistry and Spectroscopy  (11)
  • Phenylacetate  (2)
  • *Muscle Relaxation
  • 1990-1994  (3)
  • 1980-1984  (11)
  • 1
    ISSN: 1432-072X
    Keywords: Phenylacetate ; CoA ligase ; Phenylacetyl ; CoA ; Anaerobic aromatic metabolism ; Pseudomonas ; Alpha ; Oxidation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The enzyme catalysing the first step in the anaerobic degradation pathway of phenylacetate was purified from a denitrifying Pseudomonas strain KB 740. It catalyses the reaction phenylacetate+CoA+ATP → phenylacetyl-CoA+AMP+PPi and requires Mg2+. Phenylacetate-CoA ligase (AMP forming) was found in cells grown anaerobically with phenylacetate and nitrate. Maximal specific enzyme activity was 0.048 μmol min-1 x mg-1 protein in the mid-exponential growth phase. After 640-fold purification with 18% yield, a specific activity of 24.4 μmol min-1 mg-1 protein was achieved. The enzyme is a single polypeptide with Mr of 52 ±2 kDa. The purified enzyme shows high specificity towards the aromatic inducer substrate phenylacetate and uses ATP preferentially; Mn2+ can substitute for Mg2+. The apparent K m values for phenylacetate, CoA, and ATP are 60, 150, and 290 μM, respectively. The soluble enzyme has an optimum pH of 8.5, is insensitive to oxygen, but is rather labile and requires the presence of glycerol and/or phenylacetate for stabilization. The N-terminal amino acid sequence showed no homology to other reported CoA-ligases. The expression of the enzye was studied by immunodetection. It is present in cells grown anaerobically with phenylacetate, but not with mandelate, phenylglyoxylate, benzoate; small amounts were detected in cells grown aerobically with phenylacetate.
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  • 2
    ISSN: 1432-072X
    Keywords: Phenylacetate ; 4-Hydroxyphenylacetate ; Phenylglyoxylate ; Alpha-Oxidation ; Pseudomonas ; Oxidoreductase ; CoA ligase ; Benzoyl-CoA ; Anaerobic aromatic metabolism
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Anaerobic degradation of (4-hydroxy)phenylacetate in denitrifying Pseudomonas sp. was investigated. Evidence is presented for α-oxidation of the coenzyme A (CoA)-activated carboxymethyl side chain, a reaction which has not been described. The C6−C2 compounds are degraded to benzoyl-CoA and furtheron to CO2 via the following intermediates: Phenylacetyl-CoA, phenylglyoxylate, benzoyl-CoA plus CO2; 4-hydroxyphenylacetyl-CoA, 4-hydroxyphenylglyoxylate, 4-hydroxybenzoyl-CoA plus CO2, benzoyl-CoA. Trace amounts of mandelate possibly derived from mandelyl-CoA were detected during phenylacetate degradation in vitro. The reactions are catalyzed by (i) phenylacetate-CoA ligase which converts phenylacetate to phenylacetyl-CoA and by a second enzyme for 4-hydroxyphenylacetate; (ii) a (4-hydroxy)-phenylacetyl-CoA dehydrogenase system which oxidizes phenylacetyl-CoA to (4-hydroxy)phenylglyoxylate plus CoA; and (iii) (4-hydroxy)phenylglyoxylate: acceptor oxidoreductase (CoA acylating) which catalyzes the oxidative decarboxylation of (4-hydroxy)phenylglyoxylate to (4-hydroxy)benzoyl-CoA and CO2. (iv) The degradation of 4-hydroxyphenylacetate in addition requires the reductive dehydroxylation of 4-hydroxybenzoyl-CoA to benzoyl-CoA, catalyzed by 4-hydroxybenzoyl-CoA reductase (dehydroxylating). The whole cell regulation of these enzyme activities supports the proposed pathway. An ionic mechanism for anaerobic α-oxidation of the CoA-activated carboxymethyl side chain is proposed. Phenylacetic acids are plant constituents and in addition are formed from a large variety of natural aromatic compounds by microorganisms; their degradation therefore plays a significant role in nature, as illustrated in the preceding paper (Mohamed and Fuchs 1993). We have investigated and purified an enzyme which catalyzes the first step in the anaerobic degradation of phenylacetate in a denitrifying Pseudomonas sp. Phenylacetate is converted to phenylacetyl-CoA by phenylacetate-CoA ligase (AMP forming). The postulated function of this enzyme is corroborated by the strict regulation of its expression. 4-Hydroxyphenylacetate appears to be similarly activated by an independent enzyme prior to further degradation. We have suggested before that phenylacetyl-CoA is anaerobically converted by α-oxidation of the side chain to phenylglyoxylate1, which is oxidatively decarboxylated to benzoyl-CoA plus CO2 (Seyfried et al. 1991; Dangel et al. 1991). 4-Hydroxyphenylacetate was proposed to be similarly oxidized to 4-hydroxybenzoyl-CoA plus CO2, followed by reductive dehydroxylation to benzoyl-CoA. The evidence was not presented in full, and the crucial α-oxidation was not demonstrated in vitro. We present here ample evidence for this pathway. A hypothetical mechanism is proposed by which the oxidation of the α-methylene group to an α-carbonyl group may occur.
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  • 3
    Electronic Resource
    Electronic Resource
    Weinheim : Wiley-Blackwell
    Journal of High Resolution Chromatography 3 (1980), S. 575-582 
    ISSN: 0935-6304
    Keywords: Numerical examples ; Constant volume Varigrad ; Hydrostatic Varigrad ; Incremental method of gradient elution ; Modified incremental method ; Non-homogeneous Varigrad ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: Part 2 deals with the application of the equations derived in Part 1 to the systematic calculations of the constant volume and hydrostatic Varigrads. Examples are given which illustrate the advantage of using the modified incremental method of gradient elution. The non-homogeneous Varigrad, where mixing chambers may vary in volume, is also treated.
    Additional Material: 9 Ill.
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  • 4
    Electronic Resource
    Electronic Resource
    Weinheim : Wiley-Blackwell
    Journal of High Resolution Chromatography 4 (1981), S. 477-478 
    ISSN: 0935-6304
    Keywords: Square relationship ; Effect of separation efficiency ; Simple equation for plate comparison ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Additional Material: 2 Tab.
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  • 5
    Electronic Resource
    Electronic Resource
    Weinheim : Wiley-Blackwell
    Journal of High Resolution Chromatography 3 (1980), S. 507-515 
    ISSN: 0935-6304
    Keywords: Constant volume Varigrad ; Hydrostatic Varigrad ; Incremental method of gradient elution ; Modified incremental method, advantages of Poisson distribution ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: Different methods for producing a pre-assigned gradient in liquid chromatography are presented. A systematic approach to the calculations involved is given. The derived equations are mostly Poisson and Poisson summation distributions which are tabulated in the literature. The incremental method of gradient elution developed by Scott has been modified. In the modified apparatus two mixing chambers are used instead of one. This leads to an appreciable decrease in the number of reservoirs needed for the same precision in fitting a desired gradient. The application of the derived equations, together with other Varigrad modifications, will be given in Part 2 of this paper (to be published shortly in this Journal).
    Additional Material: 4 Ill.
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  • 6
    Electronic Resource
    Electronic Resource
    Weinheim : Wiley-Blackwell
    Journal of High Resolution Chromatography 4 (1981), S. 474-476 
    ISSN: 0935-6304
    Keywords: Non-linear isotherm ; From extraction to chromatography ; Numerical solution ; Poisson distribution fit ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Additional Material: 1 Ill.
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  • 7
    Electronic Resource
    Electronic Resource
    Weinheim : Wiley-Blackwell
    Journal of High Resolution Chromatography 5 (1982), S. 115-116 
    ISSN: 0935-6304
    Keywords: Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
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  • 8
    Electronic Resource
    Electronic Resource
    Weinheim : Wiley-Blackwell
    Journal of High Resolution Chromatography 5 (1982), S. 306-310 
    ISSN: 0935-6304
    Keywords: Moments, skew, and excess ; Continuous vs. discrete distribution ; Modified normal and Poisson distributions ; Distribution fit for non-linear isotherms ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: Several distributions of interest in chromatography were studied. In each case, the moments up to 4th moment were evaluated and the skew and the excess were deduced. The distributions studied in this article are the normal, continuous Poisson, discrete Poisson, exponentially modified normal, and exponentially modified Poisson distributions.
    Additional Material: 2 Ill.
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  • 9
    Electronic Resource
    Electronic Resource
    Weinheim : Wiley-Blackwell
    Journal of High Resolution Chromatography 5 (1982), S. 441-443 
    ISSN: 0935-6304
    Keywords: Kovats' index ; Simple linear index ; Relative volatility determination ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Additional Material: 3 Ill.
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  • 10
    ISSN: 0935-6304
    Keywords: Theory ; Plate theory ; Non-linear isotherms ; Quasi-Gaussian distribution ; Poisson and Poisson summation distributions ; Washing and deposition ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: The treatment of the non-linear isotherm in chromatography by one of the authors [1] is extended to other cases of practical interest. The original paper dealt with the case of an initially thin zone resulting in a Poisson distribution the asymmetry of which is a function of the plate number and the non-linearity constant. The present work shows that the same relations apply to initially thick zones and to the cases of washing and deposition leading to simple relations for breakthrough curves. For significantly non-linear isotherms other quasi-Gaussian distributions can give a better fit.
    Additional Material: 9 Ill.
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