View metadata, citation and similar papers at core.ac.uk brought to you by CORE

provided by Elsevier - Publisher Connector

Volume 28, number 2 FEBS LETTERS December 1972

GUANOSINE-DIPHOSPHATE CAUJlNG CHANGES 1N THE PHOSPHORYLATION PATTERN OF ADENl& NUCLEOTIDES IN MlTOCHONDRIA FROM BROWN ADIPOSE TISSUE

J. RAFAEL, H.W. HELDT and H.-J. HOHORST Institut fiir Physiologische Chemie, Klinikum der Universitiit Frankfurt, 6 Frankfurt (Main), Germany

and

Institut ftir Physiologische Chemie und Physikalische Biochemie, Universitiit Miinchen, 8 Miinchen, Germany

Received 20 October 1972

1. Introduction energy state of the mitochondria; recoupling of the oxidative phosphorylation stated earlier in polaro- Oxidative phosphorylation of mitochondria graphic experiments [ 1,6] should be reflected in a isolated from brown adipose tissue (BAT) was found rise of the ATP/ADP ratio in the mitochondria. to be more or less loosely coupled in the sense of a decreased or even missing ADP dependent respiratory control and reduced P/O ratios [ 1, 21. The “tightness” 2. Materials and methods of the coupling of mitochondrial respiration and phosphorylation appears to be correlated to the Mitochondria from BAT of newborn guinea pigs thermogenetic activity of the tissue [ 1,3,4]. Respira- were prepared as described previously [7]. The mito- tory control and ATP production of BAT mitochon- chondrial protein was determined by a modified dria can be restored in vitro by incubating the mito- biuret method [ 11. chondria in a medium containing 2% bovine serum The intramitochondrial adenine nucleotides were albumin [5] or certain nucleoside di- and triphos- determined by radioactivity measurements. For this phates, especially guanosine-di-phosphate [ 1, 3,6]. purpose the mitochondrial adenine nucleotides were Attempting to analyse this recoupling mechanism in labelled by incubating the stock suspension (10 mg more detail we determined the intramitochondrial protein/ml) for 30 min at 2’ with 4 PM [14C]ADP adenine nucleotide levels in BAT mitochondria from (specific activity 500 &i/mole). After careful newborn guinea pigs during respiratory experiments washing (3 times) the mitochondria (1 mg protein/ under the influence of serum albumin, guanosine- ml) were aerobically incubated in the presence of diphosphate and other nucleotides. The relative 3 mM a-oxoglutarate, 2 mM EDTA, 2 mM MgCI,, amounts of adenosine phosphates should indicate the 5 mM potassium phosphate, 0.25 M sucrose and 10 mM tri-ethanolamine*HCl buffer, pH 7.1 at 25’. At varying times samples were taken from the reac- Enzymes: tion mixture and deproteinized in 0.6 M HClO,. In Hexokinase = ATP:D-hexose 6-phosphotransferase the neutralized extract the adenine nucleotides were (EC 2.7.1.1). separated by small column ion exchange chromato- Pyruvate kinase = ATP:pyruvate phosphotransferase graphy [8, 93, and the radioactivity was measured (EC 2.7.1.40). My.okinase = ATP:AMP phosphotransferase by liquid scintillation [9]. It was checked by (EC 2.7.4.3). enzymatic determination of ATP with hexokinase

North-Holland Publishing Company - Amsterdam 125 Volume 28, number 2 FEBS LETTERS December 1912

[lo] that the mitochondrial adenine nucleotides adenine nucleotides are present as AMP, the ATP/ were equally labelled, and the specific activity of ADP ratio being below 0.5 (see figs. 1 and 2). the internal adenine nucleotides was evaluated from Fig. 1 demonstrates typical changes of the these measurements. adenosine phosphate levels in BAT mitochondria The amount of adenine nucleotides that leaked oxidizing a-oxoglutarate at 25” with no ADP out from the mitochondria into the medium during present in the medium. If no serum albumin is the experiment was controlled by separating the added to the reaction mixture (experiment on the organelles from the medium by silicone layer filter- left side) the ATPjADP ratio remains low; this ing centrifugation [ 111. The leakage under the used result is in accord with the largely uncoupled experimental conditions was found to be less than oxidative phosphorylation of BAT mitochondria 8% of the total mitochondrial adenine nucleotide which has been confirmed in polarographic experi- pool. ments under similar experimental conditions [ 12, Enzymes and nucleotides as well as o-oxoglutarate 131. The remarkable phosphorylation of AMP is were obtained from Boehringer Man~eim GmbH very likely to be caused by GTP produced in sub- (Mannheim, Germany), bovine serum albumin from strate level phosphorylation [ 141: as reported earlier Serva Entwicklungslabor (Heidelberg, Germany); [I] BAT mitochondria from newborn guinea pigs other chemicals were from Merck (Darmstadt, oxidizing cr-oxoglutarate exhibit P/O ratios of about Germany); “C-Iabelled adenine nucleotides were 1.0 thus indicating a fully coupled substrate level obtained from Amersham Buchler GmbH (Braun- phosphorylation. schweig, Germany). If BAT mitochondria oxidize ~-oxoglutarate in

3. Results and discussion

BAT mitochondria from newborn guinea-pigs when isolated conventionally appear to be in a state of low energy if the intramitochondrial ATP/ADP ratio is taken as a criterion for the energy state of the mitochondria. Usually 60-80% of the mitochondrial

no albumin 2% albumin

A' A' ,I _*___,___"--,----_'- .- 60 120 180 set

Fig. 1. Adenosine phosphates and ATP/ADP ratio (m--m--e) in isolated BAT mitochondria from newborn guinea pigs during respiration with a-oxoglutarate; no albumin and 2% albumin, resp., were present in the me~um; in neither experiment was ADP added. Experimental conditions and reaction medium are described in Material and methods.

126 Volume 28, number 2 FEBS LETTERS December 1972

Table 1 Influence of nucleotides on the ATP/ADP ratio in BAT ~tochondria from newborn guinea pigs.

Addition GDP GTP UDP CTP (mM) - 0.1 2 0.1 2 0.1 2 0.1 2

ATPJADP 0.82 2.90 3.21 0.98 3.31 0.72 2.69 0.91 2.92

ATP/ADP ratios were determined after 60 set of respiration at 25”; substrate was ol-oxoglutarate; concentrations of nucleotides present in the reaction mixture as indicated; the medium contained no albumin. Experimental conditions are described in Materials and methods. the presence of 2% serum albumin (fig. I, experi- This effect is quite specific for GDP as shown ment on the right) the ATP level as well as the from the results of table 1. With other nucleoside ATP/ADP ratio increases constantly. This rise of the di- and tri-phosphates, 20 times higher concentrations energy state of BAT mitochondria accords with the are needed in order to obtain a similar rise of the influence of serum albumin on the mitochondrial ATP/ADP ratio. oxygen consumption which has been observed ear- The rapid increase of the ATP/ADP ratio in the lier [S]: during the first minutes of incubation in the mitochondria caused by low concentrations of GDP presence of albumin the extremely high oxidation corresponds to the results which have been obtained of ol-oxoglutarate is reduced and ADP dependent in polarographic experiments [6, 151: 5-10 set respiratory control can be demonstrated. after the addition of 100 PM GDP to the reaction As demonstrated in fig. 2 addition of 100 PM mixture the Ioosely coupled oxidative phospho~la- GDP to the reaction mixture causes a very rapid tion of BAT mitochondria appears to be fully change of the mitochondrial ATP and ADP levels; restored. Other nucleoside di- and tri-phosphates the ATP/ADP ratio rises extensively. The AMP level must be used in lo-20 times higher concentrations is relatively little influenced by GDP. The experi- (l-2 mM) to yield a similar recoupling activity. ment shows that recoupling of BAT mitochondria It appears from the results that the phosphoryla- by GDP is not necessarily subject to the presence tion pattern of the internal adenine nucleotides is a of albumin in the reaction medium although albumin very sensitive and fast reacting indicator for the increases the GDP effect additionally.

5 no albumin 5 2% atbumin AMP AMP

60 120 180 set

Fig. 2. Changes in the mitochond~al adenosine phosphate pool and ATP/ADP ratio (=--m--m) of isolated BAT mitochond~ from newborn guinea pigs during respiration with cu-oxoglutarate before and after the addition of 100 MM GDP to the reaction mixture. Experimental details were as in fig. 1. 127 Volume 28, number 2 FEBS LETTERS December 1972 coupling state of the respiratory chain phosphoryla- I61 H.-J. Hohorst and J. Rafael, Hoppe-Seyler’s 2. Physiol. tion in BAT mitochondria. Measurement of this Chem. 349 (1968) 268. phosphorylating pattern may prove as a valuable ]71 J. Rafael, M. Hiisch, D. Stratmann and H.-J. Hohorst, Hoppe-Seyler’s Z. Physiol. Chem. 351 (1970) 1513. tool for elucidating the remarkable effect of GDP 181 E. Pfaff, Doctor’s thesis, Ph~ps-Universit~t, Marburg in recoupling the oxidative phosphorylation in brown 1965. fat mitochondria. 191 H.W. Heldt, M. Klingenberg and M. Milovancev, Euro- pean J. Biochem., in print. [lOI H.-J. Hohorst, F.H. Kreutz and Th. Biicher, Biochem Z. 332 (1959) 18. References [Ill M. Klingenberg and E. Pfaff, Methods Enzymol. 10 (1967) 680. [I] J. Rafael, D. Klaas and H.-J. Hohorst, Hoppe Seyler’s 1121 0. Lindberg, J. DePierre, E. Rylander and B.A. 2. Physiol. Chem. 349 (1968) 1711. Afzelius, .I. Cell Biol. 34 (1967) 293. (21 .J.L Pedersen and M.J. Grav, European J. Biochem. 25 I131 G.A. Trusler, J.E. McBirnie, F.G. Pearson, A.G. GornaJl (1972) 75. and W.G. Bigelow, Surg. Forum 4 (1953) 7 2, [ 31 H.J. Grav, J.I. Pedersen and EN. Christiansen, Euro- iI41 H.W. Heldt and K. Schwalbach, European J. Biochem. peanJ. Biochem. 12 (1970) 11. 1 (1967) 199. \4] M.T. Andersen, E.N. Christiansen, M.J. Grav a& J.1. 1151 J. Rafael, D. Klaas and H.-J. Hohorst, Abstr. V. FEBS Pedersen, Acta Physiol. Stand. 80 (1970) I. Meeting, Prague, 1968. 151 H.-J.Hohorst and D. Stratmann, Abstr. IV. FEBS Meeting, Oslo, 1967.

128