Homogeneous distribution of phosphofructokinase in the rat liver acinus: a quantitative histochemical study.
A quantitative histochemical method was developed for the demonstration in rat liver of the activity of phosphofructokinase, one of the enzymes assumed to be rate-limiting for glycolysis. The procedure was based on the reduction of a tetrazolium salt as final electron acceptor and a multistep reaction using the exogenous or endogenous auxiliary enzymes aldolase, triosephosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase. The highest activity was found in unfixed cryostat sections of rat liver when the incubation medium contained 17% (wt/vol) polyvinyl alcohol, 100 mmol/L Tris-maleate buffer (pH 8.4), 20 mmol/L fructose-6-phosphate, 2 mmol/L ATP, 2 mmol/L MgCl2, 5.9 mmol/L NAD+, 0.47 mmol/L 1-methoxyphenazine methosulfate, 5 mmol/L sodium azide and 5 mmol/L Nitro BT. The addition of auxiliary enzymes was not necessary to demonstrate maximum activity in rat liver. The specificity of the reaction was proven by the absence of any specific (test minus control) reaction when the incubation was performed in the presence of 25 mmol/L phosphoenolpyruvate, a competitive inhibitor of phosphofructokinase. Cytophotometric analysis revealed that linear relationships exist between the amount of specific reaction product formed and incubation time and the section thickness. The Km values for fructose-6-phosphate and the Vmax values were not significantly different in periportal and pericentral areas of livers from either normally fed or 24-hr-fasted rats. The homogeneous distribution of phosphofructokinase activity in the liver acinus is in line with biochemical findings using hepatocytes isolated from the two different areas showing that these cells contained similar amounts of enzyme activity.(ABSTRACT TRUNCATED AT 250 WORDS)
Frederiks
W M
WM
Laboratory of Cell Biology and Histology, University of Amsterdam, The Netherlands.
Marx
F
F
van Noorden
C J
CJ
D000818:Animals; D002122:Calcium Chloride / Q000494:pharmacology; D005215:Fasting; D005636:Fructosephosphates / Q000378:metabolism; D006651:Histocytochemistry; D008099:Liver / Q000201:enzymology* / Q000378:metabolism; D008297:Male; D010728:Phosphoenolpyruvate / Q000494:pharmacology; D010732:Phosphofructokinase-1 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D012016:Reference Values; D014018:Tissue Distribution
D005636:Fructosephosphates; C027618:fructose-6-phosphate; D010728:Phosphoenolpyruvate; D010732:Phosphofructokinase-1; D002122:Calcium Chloride