ISSN:
1573-5168
Keywords:
caffeine
;
catecholamines
;
fish
;
glucose production
;
liver
;
method/assay
;
phosphorylation
;
prostaglandin E2
Source:
Springer Online Journal Archives 1860-2000
Topics:
Biology
Notes:
Abstract The absence of a reproducible method for the assay of glycogen phosphorylase (GPase) in isolated fish hepatocytes has made the interpretation of hormone-induced glycogenolysis data difficult. This study presents such an assay and demonstrates its sensitivity to hormonal activation. The enzyme is assayed in the reverse direction using glucose 1-phosphate (G1-P) and glycogen as substrates and uses standard methods for the quantification of the liberated inorganic phosphate. The assay is highly reproducible, sensitive, and provides an excellent means to follow small and rapid changes in enzyme phosphorylation status following the addition of hormones. We show for hepatocytes isolated from rockfish (Sebastes caurinus) and brown bullhead (Ameiurus (Ictalurus) nebulosus) that small concentrations of three model hormones, namely epinephrine (catfish), norepinephrine, and prostaglandin E2 (rockfish), lead to the rapid, concentration and time-dependent conversion of existing GPase into the active GPase a form. Some of the enzyme seems to be impervious to hormonal activation, as the highest %GPase a never reaches 100%. We provide evidence that changes in enzyme phosphorylation status provide a better short-term insight into hormone-dependent activation than estimates of glucose or some other end products, that usually must accumulate for long periods before detection is possible. Our data also show that GPase in freshly isolated hepatocytes is already in an activated state and cells should be given a period of ‘rest’ for several hours before hormonal studies involving glycogen breakdown or the cAMP cascade are initiated.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1023/A:1007762229093
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