191

So&mica et Biophysics Acta, 398 (1975) 191-203 @ Elsevier Scientific Publishing Company, Amsterdam

-- Printed in The Netherlands

BBA 56634

THE INTRACELLULAR LOCALIZATION OF LONG-CHAIN SYNTHETASE IN BROWN ADIPOSE TISSUE

JAN I. PEDERSEN,

ERIK SLINDE*,

BIRTHE GRYNNE

ACYL-CoA

and MAGNE AAS

Institute for Nutrition Research, University of Oslo, Blindern and *Department of Biochemistry, University of Bergen, Bergen, (Norway) (Received

December

24th, 1974)

Summary

1. The acyl-CoA synthetase activity in brown adipose tissue of coldexposed guinea pig has been studied by measuring the rate of p~mitoylc~nitine fo~ation in the presence of excess carnitine p~mitoyltr~sf~~e. 2. The rate of p~mitoylc~itine formation in the mitochondria was found to be 161 it 64 nmol =mg-’ * min-’ (n = 9). 3. In the absence of added palmitate and bovine serum albumin a total of 35 f 1 nmol endogenous fatty acids * mg-” were activated with three different mitochondrial preparations. 4. Three different experimental aeproaches have been used to study the subcellular localization of the enzyme: (a) conventional differential centrifugation (De Duve, C., Pressman, B.C., Gianetto, R., Wattiaux, R. and Appelmans, F. (1955) Biochem. J. 60, 604-617) (b) the determination of the sediterm of different marker enzymes (Slinde, E. and Flatmark, T. (1973) Anal. Biochem. 56, 324.-340) and (c) the de~rm~ation of the stoichiomet~ between the activities of these enzymes sedimen~d at higher centrifugal effects. 5. Throughout all fractionation procedures, the long-chain acyl-CoA synthetase follows strictly the amine oxidase generally considered to be exclusively located on the mitochondrial outer membrane.

Introduction

To fulfill its function as a thermogenic organ [l] brown adipose tissue is to a large extent dependent on.its stored lipids [2,3] mobilized and oxidized under situations of thermal stress [4]. This tissue is thus characterized both by a high capacity for fatty acid ]5] and ~acyl~ycerol synthesis 163 as well as a high capacity for fatty acid oxidation (for review see ref. 7) Fatty acids not only serve as oxidizable substrates during thermogenic activation of the tissue,

192

but they also appear to regulate the rnito~~ond~~ enemy-transducing system [8] in such a way as to permit a major portion of the energy to be dissipated as heat (for review see ref. 7). Activation and transfer of a small portion of endogenous fatty acids between mitochondrial compartments markedly affect energy coupling [ 91 and ATPase activity [lo] as well as the structural organization of the mitochondrial inner membrane [lo] . Both a carnitinedependent and a less active carnitine-independent oxidation of fatty acids have been described in isolated brown adipose tissue mitochondria [ 111. Thus, for several reasons a study of the initial step in the utilization of fatty acids, the activation by the long-chain acyl-CoA synthetase (EC 6.2.1.3) in brown adipose tissue would be desirable. We here report data on the intracellular localization of this enzyme. A preliminary account of this work has already appeared (121. Materials and Methods Reagents El I-’ 4C] tyramine hydrochloride was obtained from The Radiochemical Centre, Amersham, England. L-[Me-3H]carnitine (a gift from Professor Bremer, Rikshospitalet, Oslo) was diluted with L-carnitine (Koch-Light Laboratories, Colnbrook, England) to give a spec. act. of 200 cpm~nmol as counted in the s~int~lation counter. Carnitine p~mitoyl~~sferase (EC 2.3.1.21) was prepared as described by Norum [13] with some modifications [14]. Other chemicals were commercial products of the highest purity. Animals and preparation of cellular subfractions 3 week old guinea pigs were cold exposed in an environment of 5-6°C for at least 10 days. The interscapular and parts of the cervical brown adipose tissue from 2-4 animals were cut into small pieces and rinsed in a medium consisting of 0.25 M sucrose, 2 mM HEPES buffer pH 7.2,0.2 mM EDTA and 0.5 mM ATP. The tissue mince was homogenized in a Potter-Elvehjem homogenizer at 465 rev.fmin and with two strokes of a loose-fitting teflon pestle. Sub~actions of the homogenate were prepared essentially according to de Duve et al. [15]. Some m~ifi~ations in the cent~fugation procedure were introduced because the average sedimentation coefficient of brown adipose tissue mitochondria (approx. 8000 S) is markedly lower than for rat liver mitochondria (approx. 12 000 S) [16]. Cell debris and nuclei were sedimented at a time integral of (rev./min)2 = 25 * lo7 min-’ (5000 rev./min, 10 min) in the Sorvall RCBB refrigerated centrifuge with the HB 4 rotor (Rmin = 5.8 cm, R, Bx = 14.2 cm). The supernatant was filtered through gauze and the residue rehomogenized and centrifuged as above. The cake of fat on the top of the supernatant was carefully removed and the combined filtered supernatants (the cytoplasmic extract) were centrifuged at Jk(rev./min)‘dt equal to 155 + 10’ min-’ (10 000 rev.fmin, 14 mm). The sediment was washed twice and finally suspended in 5 ml of the homoge~zation medium (mitochondri~ fraction). The combined supernatants resulting from the mitochon~i~ preparation were centrifuged at Ji(rev./minf2dt equal to 350 - lo7 min-’ (13 000 rev./min, 20 min). The

193

sediment was washed twice and suspended in 5 ml of the homogenization medium (light mitochondrial fraction). The combined supernatants resulting from this fraction were centrifuged in a Beckman ultracentrifuge type L2-65K with the SW 27 rotor at 27 000 rev./min for 50 min (R, in = 8.2 cm, R, Bx = 16.1 cm). The pellet was washed once and resuspended in 5 ml of the homogenization medium (the microsomal fraction). The combined supematants resulting from preparation of the microsomal fraction are termed the particle-free supematant. All procedures were performed at O-4’%. The different fractions were stored below -70°C until analyzed. Determination of sediterms*

The theoretical sediterm of intact brown adipose tissue mitochondria (Fig. 1) was calculated according to Slinde and Flatmark [17] from their aver-

1oc

5c

15c

ICU 2 E e %

5c

E a 0 8OC

6OC

4oc

. l

2oc

. .

*

1

Fig. 1. Sedimentation of enzymes as a function of J&ev./min)2dt when &in = 6.2 cm and R,,, = 14.4 cm. The solid line represents the saditerm of 8000 S as calculated from Eqns 1 and 2. The convergence values were chosen aa shown in Table II. (A) Cytochrome c oxidale. (B) Pahnitoyl-CoA synthetase (0) and amine oxidase (*). (C) NADPHcytocbrome c reductase. (note the change in unit interval on the ordinate).

* The sediterm [17] reprasents the distribution of a chosen S-particle between sediment and supernatant at different time integrals of (rev./mh# when R,, and Rh are piven.

194

age sedimentation coefficient equation,

(S) of 8000 f 480 S [16]

using the following

rev./min)‘dt R min = R,,,lO--O

3.5 * 1013

where R,i, and R,.. are the distances from the axis of rotation to the top and the bottom of the fluid column, respectively (in cm), t is the time in min. If S is constant and the time integral of (rev./mm)’ varies, the calculated value R, in = R, will give the quantity of the actual intact S-particle sedimented,

Y=

R max -R, R max- Rmin

100

(2)

where Y is the percentage of the S-particle that has sedimented to the bottom at a certain time integral of (rev./min) 2 [ 171 . Sedimentation of different enzymic activities at increasing centrifugal effects Sedimentation experiments were performed at 4 f 1.5”C in the swingingbucket rotor HB-4 of Sorvall RC2-B centrifuge if not otherwise stated. To minimize timedependent loss of enzyme activity, four centrifuges were used simultaneously. When centrifugation time was less than 1 h an integrator with a precision greater than 3% [18] was used to determine the centrifugal effects. The maximal centrifugal effect was 12 hIat 10 300 rev./min. After homogenization of the tissue as described above (720 rev./min) the homogenate was transformed to a 50 ml centrifuge tube, and diluted with medium to 42 ml giving R, in = 6.2 cm, and centrifuged at jk(rev./min)2dt = 11.5 * 10’ min-’ (t = 10 min, Smi* = 100 000 S). The sediment was resuspended once and sedimented as above. The supernatants were filtered through gauze, combined and diluted to 170 ml and defined as “100% homogenate.” lo-ml samples of the 100% homogenate were transferred to 50-ml centri= 6.2 cm (42 fuge tubes R, Bx = 14.4 cm, and diluted with medium to R,i, ml), and subjected to different and increasing centrifugal effects. The supernatants were carefully decanted, and the pellets resuspended in 5 ml of the sedimentation medium. Resuspended sediments, to be analyzed for palmitoylCoA synthetase, were stored below -7O”C, others below -15°C until analyzed. Stoichiometric studies The homogenate was sedimented to give a nuclear fraction N (&in = 150 000 S) and from the supernatant thus obtained a mitochondrial fraction M (%n in = 3600 S) was generated. The remaining supernamnt was diluted to 230 ml and termed fraction So. 42-ml samples were transferred to four different centrifuge tubes and subjected to increasing centrifugal effects giving fractions = 2060 S), P2 (Smin = 820 S), Pa (Smin = 410 S) and Pa (Smin = 210 p1 (Gin S). A fifth fraction P5 (S, in = 15 S) was generated by sedimenting 38 ml of So at 25 000 rev./min for 20 h in a SW 27 rotor in the Beckman L2-65B ultracen-

195

trifuge. The sediments were treated as described above. The resulting supernatants were termed S1-Ss , respectively. Enzyme assays and other analytical methods

Long-chain acyl-CoA synthetase was measured according to Far&ad et al. [19] but with certain modifications. The method is based on the use of L-[Me3 Hlcarnitine and carnitine acyltransferase in excess to trap the acyl-CoA as acyl-I.,-[Me-3H] carnitine which can, be extracted from the reaction medium by n-butanol. The dependence of the rate of palmitoylcarnitine formation in brown adipose tissue mitochondria on various in vitro factors were tested and the final standard reaction medium worked out consisted of, in a volume of 1 ml: Tris/HCl buffer pH 7.9,lOO mM; KHZPO4 buffer pH 7.9,250 mM; ATP, 10 mM; MgClz , 10 mM; CoA, 0.2 mM; L-[Me-3H]carnitine, 4 mM; camitine palmitoyltransferase, 80 ,ug of protein; bovine serum albumin (fatty acid free), 4.2 mg; GSH, 5 mM; KCN, 5 mM. 1 mM palmitic acid (in ethanol) was used as substrate. Incubation temperature was 35°C and the reaction was run for 10 min. Enzymatic assays of amine oxidase (EC 1.4.3.4) [ 201, carnitine palmitoyltransferase [ 211, malate dehydrogenase (EC 1.1.1.37) [22], acid deoxyribonuclease (EC 3.1.4.6) [ 151, rotenone-insensitive NADPHcytochrome c reductase (EC 1.6.2.4) [23] and cytochrome c oxidase (EC 1.9.3.1) [17] were carried out as described. All spectrophotometric measurements were performed with a Shimadzu recording spectrophotometer (Model MPS50L). Radioactivity was counted in a Packard tricarb liquid scintillation spectrometer (Model 3385). Protein was determined by the Folin-Ciocalteu reagent [ 241.

Mitochondrial activities of palmitoyl-CoA synthetase in brown adipose tissue

Under incubation conditions ensuring maximal formation of palmitoylcarnitine a rate of 161 + 64 nmol - mg-’ - min-’ (mean + S.D.) was found for 9 different mitochondrial preparations. A value of 84 f 13 nmol - mg-’ - min-’ (n = 4) has been observed with rat liver mitochondria under similar conditions 1201. This rate exceeds by a factor of 10-15 the capacity for fatty acid oxidation as observed with isolated brown adipose tissue mitochondria [7,25]. Hence, the activation reaction probably does not represent any limiting step in the oxidation of fatty acids in thermogenically activated brown adipose tissue. In the absence of added substrate (and bovine serum albumin) a total of 35 f 1 nmol fatty acids - mg protein-’ was found to be activated with 3 different mitochondrial preparations. This constitutes a major fraction of the total amount of endogenous, tit&able free fatty acids in these mitochondria, viz. 40 * 30 nmol - mg-l [ 261. This result indicates that during the process of recoupling of brown @ipose tissue mitochondria by ATP, carnitine and CoA [9] , although only a small amount of fatty acids is oxidized [9] a considerable portion of the endogenous fatty acids may be transformed into their activated forms.

196

The subcellular distribution of palmitoyl-CoA synthetase activity in brown ad ipose tissue The distribution of palmitoyl-CoA synthetase and different marker enzymes between different cellular subfractions is shown in Table I. The recovery both of enzymes and protein is in the range 85-105%. About one half of the activity of the mitochondrial inner membrane marker carnitine palmitoyltransferase [21] is found in the mitochondrial fraction. About the same proportion of the NADPH-cytochrome c reductase is recovered in the microsomal fraction. The highest relative specific activities of the two enzymes are found in the mitochondrial and microsomal fractions, respectively. Although low but significant levels of NADPH-cytochrome c reductase have been detected in the outer membrane of both rat liver [27] and pig heart mitochondria [28], by far the highest activities are associated with the microsomes [ 29,301. The very low relative specific activity of this enzyme in the mitochondrial, as compared to the microsomal fraction (Table I) suggests that it can be used as a marker for the .microsomes in the tissue under study. Palmitoyl-CoA synthetase shows a distribution very similar to amine oxidase. About 30% of the activity of both enzymes is recovered in the mitochondrial fraction and slightly less than this in the microsomal fraction. The small differences in relative specific activity between the two enzymes in the mitochondrial and microsomal fraction respectively are within the experimental error.

Sidimentation profile of marker enzymes and protein as a function of increasing centrifugal effect Samples of the 100% homogenate were prepared as described in Materials and Methods. From Table II and Fig. 1 it is seen how protein and enzymic activities are distributed as a function of increasing centrifugal effects. The cytochrome c oxidase localized to the inner mitochondrial membrane [30] showed (within experimental error) identical activities for sediment Nos 6, 7, 8 and 9 (Table II). This level was therefore defined as the convergence level (100%) which means that all intact mitochondria have sedimented [17] . From Fig. 1A it is seen that the cytochrome c oxidase activity follows the theoretically calculated sediterm of 8000 S, and reaches the convergence level. At this level the amount of cytochrome c oxidase sedimented was 76% of the 100% homogenate value. The fraction not sedimented at this centrifugal effect, i.e. 24%, has to be present as lighter particles since cytochrome c oxidase is a typical integral membrane protein. The amine oxidase, as well as the palmitoylCoA synthetase showed convergence at sediment Nos 7 and 8 (Table II). The sedimentation profile of the amine oxidase and the palmitoyl-CoA synthetase activities is shown in Fig. lB, and it is seen that both activities follow the mitochondrial sediterm, and the convergence levels are reached when 45% of the palmitoyl-CoA synthetase activity and 41% of the amine oxidase activity have sedimented. To make the NADPHcytochrome c reductase activity comparable to the other enzyme activities, sediment No. 6 was defined as 100% (Table II). At this centrifugal effect the convergence level of the theoretical 8000 S particle is reached. It was not possible, however, to calculate a theoreti-

I

OF PALMITOYL-CoA

SYNTHETASE

AND SOME MARKER

ENZYMES

IN SUBFRACTIONS

OF BROWN

ADIPOSE

TISSUE

HOMOGENATES

ribonuclease NADPW-cytochrome reductasa Protein

Malate dehydrogenase Acid deoxy-

PahnitoylCoA synthetase Carnitine pahnitoyitransferase Amine ,oxidase

c

k

1.79

f

0.709 f

114.7

0.076

0.42

5.7

8.5

0.427

0.067

+ 18.2

0.161 f

52.0

0.190 f

+

9.8

Absolute values. total homogenate 12.7 + 1.04 20.0 f 1.64 14.3 2 1.17 16.3 t 1.26 13.0 i: 1.07 4.0 f 0.33 12.2 f. 3.5

1.0

1.7

0.6

2.3

1.7

2.3

Nuclear fraction

0.69 3.0 f 0.25 12.0 f 5.8

1.0

28.3 f 1.0 2.36 48.3 f 10.0 4.03 32.0 f 6.1 2.67 38.3 f 4.9 3.19 8.3 f 3.8

Mitocbondriai fraction

1.7

2.9

2.6

3.6

4.3

2.35 4.0 + 2.6 1.18 3.4 f 1.0

8.0 f 2.35 10.0 i 2.94 10.0 f 2.94 10.3 t 3.03 8.0 f

Light mitochondriaI fraction

Percentage values and relative specific activities

1.76 43.3 f 4.5 5.85 7.4 1: 2.1

26.0 k 2.6 3.51 10.3 f 7.0 1.39 27.7 f 12.1 3.74 4.7 f 0.6 0.64 13.0 ?r 3.6

Microsomai fraction

101-105

87-100

97

96

7.6

97 85-100

92-

4.4

95

98

9.2

90-

92-

ViJ)

2.9

0.80 42.3 f 10.7 0.63 67.0 + 13.7

0.33 6.3 i 0.09 12.0 f 0.18 26.7 f 0.40 53.3 +

22.0 f 12.6

Particle-free supernatant

Recovery

The absolute values for enzyme activities in total homogenate are expressed in @mol. min-’ except for csrnitine paimitoyltransferase and acid deoxyribonuclease which are given in arbitrary units Proteii is given in mg. The enzyme activities and the protein content in the subfractions are expressed as percent of the totaIm whole homogenate (i.e. cytoplamnic extract + nuclear fraction). The percentage values are given as the mean f S.D. of three fxactionations. The relative specific activity represents the ratio between the percentage of total enzymic activity and the percentage of total protein in the various fractions. The recovery is only given for two frsctionations since values for the cytoplasmic extract were lost in one experiment.

DISTRIBUTION

TABLE

OF PROTEIN AND DIFFERENT

ENZYMES

OF BROWN ADIPOSE TISSUE HOMOGENATE

* The convergence

-

6.93

--

131.4

-

14.1 37.4 68.8 77.6 90.4 93.8 102.9 9 7.5 105.8 92.8 114.4 105.8 105.8 113.4 113.4

0.17 1.10 1.30 1.45 1‘61 1.75 1.82 1.97 2.06 2.10 2.13 2.17 2.20 2.28 2.60

(me)

Cytochrome c oxidase (relative activity)*

Protein in sediment

values (Y = 100%) represent the mean of the numbers in italics,

20.4 44.1 71.9 104 155 217 309 464 638 962 1230 1820 3010 5850 7660

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15

100% homogenate

Time integral of (ra”./mirI)2

The intracellular localization of long-chain acyl-CoA synthetase in brown adipose tissue.

191 So&mica et Biophysics Acta, 398 (1975) 191-203 @ Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands BBA 56634 THE...
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