Selective detection of UCP 3 expression in skeletal muscle: effect of thyroid status and temperature acclimation.
A novel peptide antibody to UCP 3 is characterized which is sensitive and discriminatory for UCP 3 over UCP 2, UCP 1 and other mitochondrial transporters. The peptide antibody detects UCP 3 expression in E. coli, COS cells and yeast expression systems. The peptide antibody detects a single approximately 33 kDa protein band in mitochondria from isolated rat skeletal muscle, mouse and rat brown adipose tissue, and in whole muscle groups (soleus and extensor digitorum longus) from mice. No 33 kDa band is detectable in isolated mitochondria from liver, heart, brain, kidney and lungs of rats, or gastrocnemius mitochondria from UCP 3 knock-out mice. From our data, we conclude that the peptide antibody is detecting UCP 3 in skeletal muscle, skeletal muscle mitochondria and brown adipose tissue mitochondria. It is also noteworthy that the peptide antibody can detect human, mouse and rat forms of UCP 3. Using the UCP 3 peptide antibody, we confirm and quantify the increased (2.8-fold) UCP 3 expression observed in skeletal muscle mitochondria isolated from 48-h-starved rats. We show that UCP 3 expression is increased (1.6-fold) in skeletal muscle of rats acclimated over 8 weeks to 8 degrees C and that UCP 3 expression is decreased (1.4-fold) in rats acclimated to 30 degrees C. Furthermore, UCP 3 expression is increased (2.3-fold) in skeletal muscle from hyperthyroid rats compared to euthyroid controls. In addition, we show that UCP 3 expression is only coincident with the mitochondrial fraction of skeletal muscle homogenates and not peroxisomal, nuclear or cytosolic and microsomal fractions.
Cunningham
O
O
Department of Biochemistry, Trinity College Dublin, Biotechnology Building, Room 0.16, Dublin 2, Ireland.
McElligott
A M
AM
Carroll
A M
AM
Breen
E
E
Reguenga
C
C
Oliveira
M E M
ME
Azevedo
J E
JE
Porter
R K
RK
D000064:Acclimatization / Q000502:physiology*; D002001:Adipose Tissue, Brown / Q000737:chemistry; D000818:Animals; D019556:COS Cells; D002352:Carrier Proteins / Q000032:analysis*; D007473:Ion Channels; D024101:Mitochondrial Proteins; D018482:Muscle, Skeletal / Q000737:chemistry*; D020675:Peroxisomes / Q000737:chemistry; D051381:Rats; D013963:Thyroid Hormones / Q000494:pharmacology*; D000071247:Uncoupling Protein 3
D002352:Carrier Proteins; D007473:Ion Channels; D024101:Mitochondrial Proteins; D013963:Thyroid Hormones; C000604392:UCP3 protein, human; C000604393:Ucp3 protein, mouse; C000604394:Ucp3 protein, rat; D000071247:Uncoupling Protein 3