Abstract
The addition of calcium chloride to rat liver homogenates resulted in activation of phosphoenolpyruvate carboxykinase by as much as 50%. The enhanced activity was inhibited by quinolinic acid; it was not additive with activation by FeCl2, and stimulation was prevented by 1,10-phenanthroline. Activation by calcium was lost when the particulate fractions of liver were removed, but an activating system could be reconstituted with isolated mitochondria, purified P-enolpyruvate carboxykinase, and purified ferroactivator. Iron-loaded mitochondria were more responsive to calcium than controls. A release of Fe2+ from washed mitochondria could be detected spectrophotometrically when 25-75 nmol of Ca/mg of protein were added to the mitochondrial suspension. If Ca2+ was buffered with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, the threshold of Ca2+ necessary for release of Fe2+ was approximately 10(-7) M, with peak response between 5 X 10(-7) and 10(-6) M. Total Fe2+ detected was normally 20-30 pmol of Fe2+/mg of protein. The synthetic activator of P-enolpyruvate carboxykinase, 3-aminopicolinic acid, as well as other picolinic acid derivatives, is capable of withdrawing Fe2+ associated with the mitochondrial fraction; after incubation with mitochondria, 3-aminopicolinate will activate phosphoenolpyruvate carboxykinase in the absence of exogenous metal.
Highlights
25-75 m o l of Ca/mg of protein were added to the (8-ll),and will cause a lowering of blood glucosein the intact mitochondrial suspension
We describe the existence of a pool of Fe2+ associatedwith the mitochondrial fraction of rat liver that can be mobilized by several stimuli, including a range of concentrations ofCa", and 3-aminopicolinic acid
When ruthenium red was present in the low speed supernatant Iron efflux in response to calciumwasobserved whether fraction, the activation of P-enolpyruvate carboxykinase fol- mitochondria were prepared from liversof rats thathad been lowingaddition of calcium wasprevented (Fig.6 A ),but rather fed ad libitum, fasted 24 h, or had been made diabetic with high concentrations of the compound wererequired
Summary
Preparation of Fractions-Mitochondria were prepared from rat line sulfonate; 3AP, 3-aminopicolinic acid. Mitochondria (40 mg/ml) were incubated for 20 min on ice with 0.2 mg of digitonin/mg of mitochondrial protein in 0.3 M mannitol, 3 mM Hepes-Na, pH 7.4, 1m~ EGTA containing 1mg of bovine serum albumin/ml They were centrifuged at 20,000 x g for 5 min, washed with buffer minus digitonin or bovine serum albumin before use. At pH 7.0,log Keqapfpor the calcium EGTA complex was carboxykinase activity was measured in the supernatant fraction; taken as 6.68 [31]; free Ca2' under these conditions could be control activity = 0.61 pmol of P-enolpyruvate/min/ml of cytosol. 600 X g supernatant fraction, or 0.1 ml of cytosol plus 0.1 ml of mitochondria (6.9mg/ml) or microsomes (15.4 mg/ml)was incubated 3 min at 25 "C with or without calcium, assayed for P-enolpyruvate carboxykinase.
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
