Optimal assay conditions for enzymatic characterization of homozygous and heterozygous twitcher mouse

1986 ◽  
Vol 877 (1) ◽  
pp. 1-8 ◽  
Author(s):  
Srinivasa Raghavan ◽  
Allan Krusell
Keyword(s):  
Enzymatic Characterization ◽  
Twitcher Mouse ◽  
Assay Conditions

scholarly journals Molecular and Enzymatic Characterization of Flavonoid 3′-Hydroxylase of Malus × domestica

Plants ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 1956
Author(s):  
Julia Weissensteiner ◽  
Christian Molitor ◽  
Silvija Marinovic ◽  
Lisa Führer ◽  
Syed Waqas Hassan ◽  
...  
Keyword(s):  
Malus Domestica ◽  
Site Directed Mutagenesis ◽  
Cdna Clones ◽  
Enzymatic Characterization ◽  
Recombinant Enzymes ◽  
Polyphenol Oxidases ◽  
Apple Leaves ◽  
Ring Hydroxylation ◽  
Assay Conditions

Malus × domestica (apple) accumulates particularly high amounts of dihydrochalcones in various tissues, with phloridzin (phloretin 2′-O-glucoside) being prevalent, although small amounts of 3-hydroxyphloretin and 3-hydroxyphloridzin are also constitutively present. The latter was shown to correlate with increased disease resistance of transgenic M. × domestica plants. Two types of enzymes could be involved in 3-hydroxylation of dihydrochalcones: polyphenol oxidases or the flavonoid 3′-hydroxylase (F3′H), which catalyzes B-ring hydroxylation of flavonoids. We isolated two F3′H cDNA clones from apple leaves and tested recombinant Malus F3′Hs for their substrate specificity. From the two isolated cDNA clones, only F3′HII encoded a functionally active enzyme. In the F3′HI sequence, we identified two putatively relevant amino acids that were exchanged in comparison to that of a previously published F3′HI. Site directed mutagenesis, which exchanged an isoleucine into methionine in position 211 restored the functional activity, which is probably because it is located in an area involved in interaction with the substrate. In contrast to high activity with various flavonoid substrates, the recombinant enzymes did not accept phloretin under assay conditions, making an involvement in the dihydrochalcone biosynthesis unlikely.

scholarly journals Enzymatic characterization of interferon-induced antiviral GTPases murine Mx1 and human MxA proteins.

1994 ◽  
Vol 269 (3) ◽  
pp. 2009-2015 ◽  
Author(s):  
K. Melén ◽  
T. Ronni ◽  
T. Lotta ◽  
I. Julkunen
Keyword(s):  
Enzymatic Characterization

scholarly journals Enzymatic Characterization of the N-Acetylation of 3-Phosphoglyceraldehyde Dehydrogenase by Acetyl Phosphate

1970 ◽  
Vol 245 (11) ◽  
pp. 2946-2953
Author(s):  
Jane Harting Park ◽  
Denis C. Shaw ◽  
Elizabeth Mathew ◽  
Blanche P. Meriwether
Keyword(s):  
Acetyl Phosphate ◽  
Enzymatic Characterization

scholarly journals Characterization of adenosine deaminase (ADA) in Hemolymph of Biomphalaria glabrata

2005 ◽  
Vol 65 (2) ◽  
pp. 371-376 ◽  
Author(s):  
M. R. Vale ◽  
R. V. Pereira ◽  
S. M. Almeida ◽  
Y. M. Almeida ◽  
S. F. L. C. Nunes
Keyword(s):  
Adenosine Deaminase ◽  
Incubation Temperature ◽  
Small Variation ◽  
Healthy Human ◽  
Ph Variation ◽  
Cellular Events ◽  
Low Concentrations ◽  
Key Enzyme ◽  
Assay Conditions

Adenosine is an important signaling molecule for many cellular events. Adenosine deaminase (ADA) is a key enzyme for the control of extra- and intra-cellular levels of adenosine. Activity of ADA was detected in hemolymph of B. glabrata and its optimum assay conditions were determined experimentally. The pH variation from 6.2 to 7.8 caused no significant change in ADA activity. Using adenosine as a substrate, the apparent Km at pH 6.8 was 734 µmols.L-1. Highest activity was found at 37ºC. Standard assay conditions were established as being 15 minutes of incubation time, 0.4 µL of pure hemolymph per assay, pH 6.8, and 37ºC. This enzyme showed activities of 834 ± 67 µmol.min-1.L-1 (25ºC) and 2029 ± 74 µmol.min-1.L-1 (37ºC), exceeding those in healthy human serum by 40 and 100 times, respectively. Higher incubation temperature caused a decrease in activity of 20% at 43ºC or 70% at 50ºC for 15 minutes. The ADA lost from 26 to 78% of its activity when hemolymph was pre-incubated at 50ºC for 2 or 15 minutes, respectively. Since the ADA from hemolymph presented high levels, it can be concluded that in healthy and fed animals, adenosine is maintained at low concentrations. In addition, the small variation in activity over the 6.2 to 7.8 range of pH suggests that adenosine is maintained at low levels in hemolymph even under adverse conditions, in which the pH is altered.

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