Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
19
pubmed:dateCreated
1994-6-16
pubmed:abstractText
In contrast to liver and heart 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases, the testis isozyme lacks a phosphorylation site for cAMP-dependent protein kinase. In order to determine the effect of phosphorylation site location for the protein kinase on rat testis bifunctional enzyme, consensus amino acid sequences (RRXS) were added at different distances from the N-terminus by site-directed mutagenesis. The expressed wild-type enzyme (WT) and mutant enzymes containing a phosphorylation site at Ser7 (mutant enzyme RT2KS7, where RT2K = rat testis 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase), Ser15 (RT2KS15), or Ser30 (RT2KS30) were purified to apparent homogeneity. All the mutant enzymes served as substrates for the protein kinase, and the phosphate incorporation was over 90%. The Km values of protein kinase A for RT2KS7, RT2KS15, and RT2KS30 were 250 microM, 110 microM, and 50 microM, respectively, and the relative rates were 1, 8, and 23. Various kinetic parameters of dephospho and phospho forms of these enzymes were determined. The kinetic constants of the dephospho form of RT2KS30 were similar to those of WT, but those of RT2KS15 and RT2KS7 showed an 8-fold increase in KmFru6P, an approximately 30% decrease in the Fru-6-P,2-kinase activity, and a 3-fold increase in fructose-2,6-bisphosphatase activity. Phosphorylation of RT2KS30 resulted in a shift in the Fru-6-P saturation curve from Michaelis-Menten kinetics to sigmoidal, with increased KmFru6P and activation of fructose-2,6-bisphosphatase. The kinetic constants of RT2KS15 and RT2KS7 were not altered by phosphorylation. All the mutant enzymes were more sensitive to heat inactivation than was WT.(ABSTRACT TRUNCATED AT 250 WORDS)
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
May
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
17
pubmed:volume
33
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
5766-71
pubmed:dateRevised
2007-11-15
pubmed:meshHeading
pubmed-meshheading:8180203-Amino Acid Sequence, pubmed-meshheading:8180203-Animals, pubmed-meshheading:8180203-Base Sequence, pubmed-meshheading:8180203-Cyclic AMP-Dependent Protein Kinases, pubmed-meshheading:8180203-Enzyme Stability, pubmed-meshheading:8180203-Fluorescence, pubmed-meshheading:8180203-Iodides, pubmed-meshheading:8180203-Kinetics, pubmed-meshheading:8180203-Male, pubmed-meshheading:8180203-Molecular Sequence Data, pubmed-meshheading:8180203-Mutagenesis, Site-Directed, pubmed-meshheading:8180203-Phosphofructokinase-2, pubmed-meshheading:8180203-Phosphoric Monoester Hydrolases, pubmed-meshheading:8180203-Phosphorylation, pubmed-meshheading:8180203-Phosphotransferases (Alcohol Group Acceptor), pubmed-meshheading:8180203-Rats, pubmed-meshheading:8180203-Substrate Specificity, pubmed-meshheading:8180203-Testis
pubmed:year
1994
pubmed:articleTitle
Effect of adding phosphorylation sites for cAMP-dependent protein kinase to rat testis 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.
pubmed:affiliation
Research Service, Department of Veterans Affairs Medical Center, Dallas, Texas.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S., Research Support, U.S. Gov't, Non-P.H.S.