A dot immunobinding assay for the rapid quantification of uncoupling ...

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A dot immunobinding assay for the rapid quantification of uncoupling protein in brown adipose tissue mitochondria Table 1. Measurement of uncoupling protein by do[ immunobinding Rats were caged singly and acclimated at either 22°C or 4°C for 3 weeks. Mice, housed at 22“C, were made diabetic by a single intraperitoneal injection of streptozotocin (175 mg/kg body wt.). After 12 days, they were infused with insulin (either 0 or 32 units/day per kg body wt.) for 12 days, using osmotic minipumps implanted subcutaneously. Values are means k s.E.M.,for the number of animals shown in parentheses.

RACHEL E. MILNER,* ALAlN GELOEN*t$ and PAUL TRAY HURN*$ * Nirtrition arid Metabolism Research Group, Departments of Medicine and Foods & Nutrition, 536 Newton Research Bi4ilding, University of Alberta, Edmonton, Alberta, Canada TbG 2C.2; t Laboratoire de Thermorigulation et Eneqitique de I’Exercise, URA 1341, 60373 Lyon, France and $Division of Hiochemical Sciences, Rowett Research Institute, Aberdeen AH2 0.SB, Scotland, U.K .

Uncoupling protein concentration (pglmg of mitochondrial protein)

The main function of brown adipose tissue (BAT) is to generate heat, the biochemical mechanism for which is centred on a tissue-specific mitochondrial protein, M , 32 000 [ 11. This protein, termed ‘uncoupling protein’, acts as a proton shortcircuit such that respiration is uncoupled from ATP synthesis [ I ] . The thermogenic capacity of BAT is determined by the amount of uncoupling protein in the tissue [ l , 21 and this shows major adaptive changes according to the physiological requirements for heat production (21. Several immunological methods have been described for the quantification of uncoupling protein. These include enzyme-linked immunosorbent assay (e.1.i.s.a.) [3-51,radioimmunoassay [6, 71 and immunoblotting [8, 91 procedures. Although these methods are sensitive and specific, the e.1.i.s.a. and radioimmunoassay techniques have not proved to be very robust, while immunoblotting is time consuming and only a limited number of samples can be handled at any one time. In view of these difficulties, we have now developed a dot immunoblotting (‘dot blot’) procedure for the quantification of uncoupling protein. Mitochondria were prepared from the axillary BAT of Richardson’s ground squirrel (Spermophilin richardsonii) and from interscapular BAT of rats or mice [9, lo]. Uncoupling protein was isolated from the mitochondria [9]and antibodies raised in rabbits [6].Separation of total mitochondrial proteins by SDS/PAGE, followed by immunoblotting on nitrocellulose [9], indicated that there was only one major band o f immunoreactivity and this was at the M, characteristic of uncoupling protein. Subsequently, mitochondrial proteins and purified uncoupling protein were applied directly to a nitrocellulose membrane housed in a 96-well ‘Bio-Dot’ microfiltration system (Bio-Rad, Richmond, U S A . ) , to determine whether uncoupling protein could be detected and quantified satisfactorily in a mixture of proteins without their prior separation. Spare binding sites on the nitrocellulose were blocked with bovine serum albumin (ln/n, w/v) and the membrane probed with rabbit anti-( uncoupling protein) serum. Antigen-antibody complexes were detected either with goat anti-rabbit IgG alkaline phosphatase conjugate (Bio-Rad)or 1251-proteinA (Amersham, Canada). When 12sl-proteinA was used, each ‘dot’ on the nitrocellulose membrane was cut out and its radioactivity measured. The colorimetric detection system indicated that uncoupling protein could be readily detected on nitrocellulose, when part of a mixture of mitochondrial proteins. Quantification was then examined using ‘%protein A. A linear relationship between d.p.m. and the amount of purified uncoupling protein was consistently found between 0 and 250 ng of the protein. There was also a linear relationship between d.p.m. and total mitochondrial protein over the range 0-3 ,ug of protein, although when more than 4-5 p g of mitochondrial protein

was used, saturation of antibody was noted. Below this quantity, the measured uncoupling protein concentration was independent of the amount of mitochondrial protein. The dot immunobinding procedure has been applied to the measurement of uncoupling protein in hibernating ground squirrels, cold-acclimated rats and diabetic mice. The results obtained indicate that the method can be SUCcessfully used with a variety of species (Table 1). An increased concentration of uncoupling protein is apparent on cold-acclimation, as previously reported [9, lo]. Streptozotocin-induced diabetes in mice led to a marked fall in the concentration of the protein, while the infusion of insulin into diabetic animals results in a partial restoration. We suggest that dot immunobinding provides a simple, sensitive (2-10 ng) and reproducible method for the rapid ( I day) quantification of uncoupling protein in BAT mitochondria. To date, the method has proved much more robust than the radioimmunoassay procedure [6, 101. The dot immunobinding assay should facilitate further studies on the regulation of the thermogenic activity and capacity of BAT. P. Trayhurn was a Scholar of the Alberta Heritage Foundation for Medical Research, and was supported by grants from the Foundation and from the Medical Research Council of Canada. 1. Nicholls, D. G., Cunningham, S. A. & Rial, E. (1986)in Brown Adipose 7issue (Trayhurn, P. & Nicholls, D. G., eds.), pp. 52-85, Edward Arnold, London 2. Trayhurn,P.(1989)I’roc. Nutr.Soc.48, 165-175 3. Cannon, B., Hedin, A. & Nedergaard, J. ( 1 982) FEBS Lett.

Abbreviations used: BAT, brown adipose tissue; e.i.s.a., enzymelinked immunosorbent assay.

Received 22 November I989

Vol. 18

Ground squirrels Hibernating Rats

Acclimated at 22°C Acclimated at 4°C Mice Control Diabetic (24 days) Diabetic +insulin

75.6 k 4.2( 10) 36.7 f 1 3 6 ) 70.5 2.2(5)

+

20.1 +2.3(10) 6.0 1 .5( 10) 15.0 f 1.9( 1 0 )

+

150,129-132 4. Hansen, E. S., Nedergaard, J., Cannon, B. & Knudsen, J. ( 1984) Comp. Biochem. Physiol. B79,441-445 5. Desautels, M. (1985)Am. J. I’hysiol. 249, E99-EI06 6. Lean, M. E. J., Branch, W. J., James, W. P. T., Jennings, G. & Ashwell,M.(1983)Biosci. Rep. 3,61-71 7. Peachey, T., French, R. R. & York, D. A. ( 1 988) Biochem. J . 249,45 1-457 8. Henningfield, M. F. & Swick, R. W. ( 1987) Biochem. Cell Bid. 65,245-25 1 9. Milner, R. E., Wang, L. C. H. & Trayhurn, P. (1989) Am. J. I’hysiol. 256, R42-R48 10. Trayhurn, P., Ashwell, M., Jennings, G., Richard, D. & Stirling, D. M. (1987)Am. J. I’hysiol. 252, E237-E243

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