Linked Life Data

2154228

Source:http://linkedlifedata.com/resource/pubmed/id/2154228

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
2
pubmed:dateCreated
1990-3-1
pubmed:abstractText
In the search for a phosphate acceptor to estimate cyclic AMP-dependent protein kinase (A-kinase) activity in respiratory tissues devoid of the disadvantages inherent with the use of histones, we compared and contrasted a conventional substrate, histone IIa, with that of a novel heptapeptide phosphate acceptor, Kemptide (Kemp et al., J Biol Chem 252: 4888-4894, 1977). The specific activities of soluble A-kinase isolated from guinea-pig lung parenchyma and from bovine tracheal smooth muscle were significantly (12-16-fold) greater when Kemptide was used as substrate when compared to histone IIa. Moreover, studies with a specific inhibitor of A-kinase demonstrated that whilst the cyclic AMP-stimulated phosphorylation of Kemptide was catalysed exclusively by A-kinase, only approximately 89% of the phosphate incorporated into histone IIa was attributed to the activity of this enzyme. The activation constant (Kact) of cyclic AMP for A-kinase was estimated to be 1.5 microM when Kemptide was used as substrate, 25-fold higher than that obtained using histone IIa (60 nM) under identical conditions. In a complementary series of experiments it was found that both the basal and isoprenaline-stimulated A-kinase activity ratios were significantly higher when histone IIa was used as substrate when compared with that obtained using Kemptide. Elevating the ionic strength of the homogenization buffer or assay cocktail with NaCl produced a reversible (following dialysis), concentration-dependent, mixed uncompetitive, inhibition of cyclic AMP-stimulated histone phosphorylation; phosphokemptide formation was unaffected. The ability of salt to inhibit A-kinase activity also affected the elution profile of A-kinase isoenzymes following their separation by DEAE-cellulose chromatography. With histone IIa as substrate the Type I:Type II isoenzyme ratio in guinea-pig lung and in bovine tracheal smooth muscle was 9:91 and 35:65, respectively. Changing the phosphate acceptor to Kemptide reduced these respective ratios to 5:95 and 29:71. Although the effect on the isoenzyme ratio was small, the specific activities of the two isoenzymes isolated from both tissues were markedly attentuated by ca. 9% (Type I) and 36% (Type II). It is concluded that Kemptide is without many of the disadvantages inherent with histone IIa and is, thus, a preferable phosphate acceptor for estimating soluble A-kinase activity and determining the isoenzyme ratio in guinea-pig lung and in bovine tracheal smooth muscle.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jan
pubmed:issn
0006-2952
pubmed:author
pubmed:issnType
Print
pubmed:day
15
pubmed:volume
39
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
271-83
pubmed:dateRevised
2009-11-19
pubmed:meshHeading
pubmed:year
1990
pubmed:articleTitle
Evaluation of kemptide, a synthetic serine-containing heptapeptide, as a phosphate acceptor for the estimation of cyclic AMP-dependent protein kinase activity in respiratory tissues.
pubmed:affiliation
Division of Pharmacology and Toxicology, Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, Canada.
pubmed:publicationType
Journal Article, Research Support, Non-U.S. Gov't