- Email: [email protected]
On the mechanism of malonyl-CoA independent fatty acid synthesis
BIOCHIMICA
BBA
ET BIOPHYSICA
235
ACTA
56123
ON THE
MECHANISM
OF
JfALONYL-CoA
IKDEPENDENT
FATTY
ACID
SYNTHESIS II.
ISOLATION,
PROPERTIES
z,3-HEXENOYL-CoA
E. R. PODACK*
April
AND SUBCELLULAR
LOCATION
TRANS-2,3-DECENOYL-CoA
OF TRANS-
REDUCTASE
AND W. SEUBERT
Physiologisch-chemisches (Received
AND
z8th,
Institut
der Univevsitiit,
Glittingen
(Germany)
1972)
SUMMARY I. The separation by zonal centrifugation of two enoyl-CoA reductases located in microsomes and mitochondria of rat liver is described. 2. The microsomal enzyme can use either NADH or NADPH as hydrogen donor. It shows optimal activity at pH 7.9 with trans-2,3-hexenoyl-CoA as substrate. 3. The mitochondrial enzyme shows an absolute requirement for NADPH as hydrogen donor with an optimum at pH 7.7. An investigation of the chain length specifity revealed a maximum with trans-2,3-decenoyl-CoA4. In contrast to the microsomal system, the enzymatic activity from mitochondria could be solubilized by treatment with acetone. 4. Varying effects of both NADH and NADPH on the rate of acetyl-CoAdependent chain elongation in crude mitochondrial preparations, reported by several groups, could be explained by varying degrees of microsomal contamination. 5. A possible physiological significance of the mitochondrial system in hydrogen transport and conservation of energy is discussed.
INTRODUCTION
Unidirectional reaction sequences involved in degradative processes are in most cases by-passed by nucleoside triphosphate dependent reactions, thus creating an irreversible synthetic process. This principle is verified by the degradation and synthesis of glucose, glycogen, fatty acids, lipids and other important constituents of the cell. In some cases, however, the redox difference between the NADH/NAD+ system and a substrate couple participating as hydrogen acceptor in a synthetic process helps to overcome an energy barrier. Examples of this principle are the conversion of * Essential GZjttingen.
part of the Doctor’s
Thesis of E. R. Podack
at the Medical
Riochim.
Biophys.
Faculty
Acta,
of the IJniversity
280
(1972)
235~247
E. R. PODACK, W. SEURERT
236
phosphoglycerate to glycerinaldehyde phosphate, or the conversion of P-hydroxymethylglutaryl-CoA to mevalonic acid. Chain elongation of fatty acids by reversal of the P-oxidation is another illustrative example for this principle. As is evident from the free energy of the reaction sequence summarized in Eqns I to 4 (Table I), reversal of the /?-oxidation, including the FAD-dependent dehydrogenase, would not be possible from a thermodynamic point of view (dF’, = +8,45 (kcal/mole)). Substitution of the FAD-dependent acyl-CoA dehydrogenase (Eqn 4) by a pyridinnucleotidedependent enoyl-CoA reductase (Eqn 5), first described by Langdonl and Seubert et a1.2J, shifts the equilibrium in favour of the synthetic process (W’, = -10.35 (kcal/mole))4-9. Experimental evidence for a reversal of /?-oxidation of fatty acids was first demonstrated by Seubert et al. 2. in 1957. According to the studies of numerous groups, this process is apparently restricted to the mitochondrial compartment under physiological conditions 1op1*. Reversal of p-oxidation in the cytoplasm, as reported for the combined action of enzymes of p-oxidation and fatty acid synthetase, needs further experimental support with respect to its physiological significance1g~20. T,\BLE
I
Thermodynamic
CAI,CU~ATIOS FOR THE REVERSAI. OF FiTTY ACID OXIDATION _~~~.
li ’
stefi
+ CH,-CO-SCo.4 + II-CO-CH,-CO-SCo.4 -t HSCoA 1.56. (thiolase)” K-CO-CH,-CO-SCoA + NADH + H+ R-CHOH-CH,-CO-SCoA + N4D-‘5.25 (P-hydroxyacyl-CbA dehydrogcnase)’ K-CHOH-CH,-CO-SCo_4 + R-CH = CH-CO-XXX + H,O ‘5.7 (enoyl-Co.4 hydratasc)j R-CH = CH-CO-SCo.4 + CoQH, + R-CH,-CH,-CC)-SCoA A CoQ, . (acyl-CoA dehydrogenase and B I F)6-9 R-CH=CH-CO-SCoiZ+NADPH(NADH) +H+ + R-CH,-CH,-CO-SCokt NADP+(NAD+) (enoyl-CoA reductase)le3
.!lE’,(m r-j
.4F’, (kcal~tnolr)
Eqn
ATO.
_____
KW3~S-CoA
Free energy of reversal Free energy of reversal
of B-oxidation of /j-oxidation
including including
IO-'
-
+
6.65
(I)
102
~ 81.9
-
3.75
(2)
t 0.75
(3)
I- 4.8
(4)
~ 14.0
(5)
_
im104
-305
acyl-Cob dehydrogenase enoyl-CoA reductase
+
X.4.5
--IO.jj
The physiological function of the mitochondrial system was originally restricted to chain elongation involved in the synthesis of polyunsaturated fatty acids. The discovery of a malonyl-Co&dependent chain elongation21-25 of fatty acids in the microsomal fraction of liver questioned such a role. According to Sugteren2” chain elongation in microsomes occurs no longer at the level of enzyme-bound derivatives of pantetheine, as reported for fatty acid synthetase 26, but rather at the level of coenzyme A derivatives. Enoyl-CoA reductase activity was detected in microsomes without further characterization of nucleotide and chainlength specifityZ2. Since most mitochondrial preparations employed for the study of acetyl-CoA dependent chain elongation in liver have been crude preparations, and have most likely been contaminated by microsomes, a possible “mitochondrial” chain elongation activity in liver preparations could have been imitated by a combined action of enoyl-CoA reductase from microsomes and the enzyme thiolase, I$-hydroxyacy-CoA-dehydrogenase and
MALONYL-CO.4
INDEPENDENT
FATTY
ACID
237
SYNTHESIS
enoyl-CoA hydratase of the oxidative sequence in mitochondria. This possibility seemed extremely likely, since in some cases optimal chain elongation activities were observed after sonic treatment or freezing and thawing of mitochondriar2~27. A reinvestigation of the subcellular localization of enoyl-CoA reductase was therefore initiated. These studies resulted unexpectedly in the localization and characterization of two different enoyl-CoA-reductases of liver in microsomes and mitochondria, respectively. MATERIAL
AND
METHODS
Enzymes and substmtes Preparation and analysis of enoyl-CoA hydratase (EC 4.2.1.17), @-ketothiolase (EC 2.3.1.9) and the various coenzyme A derivatives have been described in the preceeding paper3. !I-14C]Acetyl-CoA was prepared by reaction of IO pmoles 11-1K]acetic anhydride, dissolved in 1.5 ml peroxide-free tetrahydrofuran, with an equal amount of coenzyme A according to the method of Lynen and Wielandz8. Yield: 7o-8o0/o. Assays
of enzymes
and substrates
Acetyl-CoA and [14C]acetyl-CoA were assayed according to Buckel and Eggerer”’ by following the decrease in absorbance at 233 nm. Trans-2,3-enoyl-CoA derivatives and coenzyme A derivatives of saturated fatty acids were quantitated according to the method of Seubert et a1.3. Protein was determined according to the method of Lowry et aL30, and from the absorbance at 260 and 280 nm31. For enzyme assays, the mitochondrial fractions were dissolved with sodium deoxycholate or Triton X-100 at final concentrations of 0.1% and 0.5%, respectively. There was no inhibition of the various enzyme assays by these additions. Glutamate dehydrogenase was assayed according to Hogeboom and Schneide?, with the following exception: the assay mixture contained 0.1 M potassium phosphate buffer (pH 7.7) and 2 m&I ADP. Thiolase and enoyl-CoA hydratase were assayed according to the method of Seubert et a1.3, acid phosphatase according to the method of Appelmans and de Duve33. Assay of glucose-6-phosphatase. Contamination of mitochondria by microsomes is usually quantitated by assays of glucose-6-phosphatase, an enzyme exclusively located in the microsomal fraction (see ref. 34). Acid phosphatase, an enzyme located in the lysosomal fraction, also shows activity with glucose 6-phosphate. In order to restrict assays of glucose-6-phosphatase activity mainly to microsomal impurities in the mitochondrial fractions, glucose-6-phosphatase assays were carried out at pH 8.0. As is evident from the pH-dependence of acid phosphatase with /%glycerophosphate as the substrate (Fig. I), lysosomal acid phosphatase activity on cleavage of glucose 6-phosphate is negligible under these conditions. However, glucose-6-phosphatase still shows significant activity at pH 8.0 (Fig. I). The assay mixture for glucose-6-phosphatase contained in 0.4 ml: 50 pmoles saccharose; 0.1 pmole EDTA; IO pmoles Tris-HCl buffer, pH 8.0. The reaction IO pmoles glucose 6-phosphate; mixture is incubated for 15 min at 37 “C. Liberated phosphate is assayed according to Baganski
et a1.35. Biochim. &@hys.
Acta, 280
(1972)
235-247
E. K. POLMCK, IV. SIJI:REHT
238
06.
PH Fig. I. pH-dependence of glucose+phosphatasc and acid phosphatase. c ---~7, cleavage of glucose h-phosphate by microsomes; O--O, cleavage of fi-glycerophosphatc by light mitochondria. Buffers employed: IO ~moles sodium acetate buffer, pH 5.0; IO /Lmolespotassium citrate, pH 6.5; IO ,uumoles Tris-HCl buffer from pH 6.X to 8.6. For additional components of assay mixtures see Material and Methods.
Acetyl-CoA-dependent
chain elongation
was assayed in a reaction
mixture
con-
taining in 0.5 ml: 25 pmoles glycyl glycine buffer, pH 8.0; 0.05 pmole [r-14CjacetyCoA (specific activity 5 to 2.5 mCi/mmole) ; 0.05 hLrnole decanoyl-CoA; 4 nmoles rotenone (IO+ 31 in 50% ethanol); 0.5 pmole NADPH; 0.5 pmole NADH; 20 to ZOO pg mitochondrial protein. Addition of enoyl-CoA hydratase, /%hydroxyacyl-CoA dehydrogenase and thiolase was not necessary because of sufficient amounts present in the preparations used. The reaction mixture was incubated for 5 to IO min at 37 “C. Incorporation in the fatty acid fraction is determined after hydrolysis with NaOH. I munit corresponds to the incorporation of I nmole [Xlacetate per min in the light petroleum-soluble fraction. Enoyl-CoA reductase assays contained in z ml: 180 kmoles potassium phosphate buffer (pH 7.1 or pH 8.0); 0.5 pmole NADPH; 0.1 to 0.2 ,umole enoyl-CoA of different chain lengths; 5 to 30 munits enoyl-CoA reductase. The reaction mixture is incubated at 30 “C. Oxidation of NADPH is followed at 334 nm. The reaction is started by the addition of substrate. To compensate for a substrate-independent oxidation of NADPH by the enzyme preparation, all assays had to be carried out against a control containing no substrate. For assays of an NADH-dependent enoyl-CoA reductase in subcellular fractions, the above reaction mixture is supplemented with 2 pmoles KCN and 20 nmoles rotenone to inhibit oxidation of NADH by enzymes of the respiratory chain. After solubilization of enoyl-CoA reductase, additions of KCN and rotenone are no longer necessarv. Fractionation of subcellular particles Fractionation of the homogenate from liver into nuclei (Fraction N), heavy mitochondria (Fraction M), light mitochondria (Fraction L), microsomes (Fraction P) and supernatant (Fraction S) was carried out in isotonic sucrose solution according to the method of de Duve et aZ.3”. Biochim.
Hiophys. Acta,
2X0
(1972)
235-247
XALOKYL-COA
INDEPENDEi’GT FATTY ACID SYNTHESIS
Subfractionation Beckman
of heavy
mitochondria
rotor was carried out according
239
by zonal centrifugation
in an Al 14
to the method of Poole et aZ.37with modifica-
tions (G. Weiss, B. Killer and W. Seubert, unpublished) : 500 ml of a sucrose density gradient ranging from 0.4 to 0.75 RI sucrose (0.02 $1 pottasium glpcylglycine pH 7.5) are introduced into the rotor, spinning at 3000 rev./min, through the edge line. The gradient is established by mixing both solutions in a Beckman gradient pump, Model 141. The remainder of the rotor volume is filled with a 150 ml cushion of 1.0 &l sucrose, supplemented with 0.02 M potassium gl~cylglycine (appro.~. 250 mg protein) of heavy mitochondria suspended
buffer, pH 7.5. 14 ml in 0.25 31 sucrose are
introduced by a syringe through the core line. An overlay of 50 ml 0.2 Rl glucose is then added to move the mitochondrial zone pass the core. The rotor is then accelerated to 6000 rev./min and run for 15 min. After deceleration to 3000 rev/min, the contents are collected from the coreline by displacement with 1.2 M sucrose, supplemented with 0.02 M potassium glycylglycine buffer, pH 7.5. The contents of the rotor including overlay, gradient and cushion are collected in 22 fractions each of 30 ml. The distribution of protein among the various fractions was determined from the absorbance at 575 nm. The distrit~ution of the various marker enzymes was determined after treatment of the individual fractions with deoxycholate or Triton SIOO. Possible contamination by remaining intact cells could be excluded by assays of lactic dehydrogenases.
For the results of zonal centrifugation
see Fig. 3 and Table II.
~~&~~Y~~~~~~, oj acetone ~o~~d~~~~~~ heavy ~~tocho~~~~ii~ and otk s~~c~~~,u~~~ j~a~t~o~~~ Acetone powders from subcellular fractions were prepared according to the method of Dahlen and Porter 15. Soluble decenoyl-CoA reductase is extracted from I g of acetone powder by homogenization in 20 ml of 0.1 M potassium phosphate buffer, pH 7.1. After aging for an additional 30 min at o “C,insoluble material is sedimented for 30 min at IO~OOOXR. Enoyl-Co.4 reductase can be assayed in the supernatant with NADPH (Table III). RESULTS
Separation of emyE-CoA reductases Studies of Van ToP4 on the distribution of the two microsomal marker enzymes glucose-6-phosphatase and NADPH-cytochrome c reductase in fractions from rat liver microsomes, isolated by continuous sucrose densitv gradient centrifugation, revealed glucose-6-phosphatase to be located at a site of higher density compared to NADPH-cytochrome c reductase. Van To1 concludes from this finding, that the microsomal contamination of mitochondria is due to the presence of “heavy” microsomes, contributing relatively more glucose-6-phosphatase activity than NADPH-cytochrome c reductase activity. Therefore, for a quantitation of microsomal impurities in submitochondrial fractions, assays of glucose-6-phosphatase were given preference to NADPH-cytochrome c reductase as the marker enzyme of microsomes in our studies. A mitochondrial location of glucose-6-phosphatase seems to be excluded on the basis of different distributions of the former enzyme and rotenone insensitive NADH-cytochrome c reductase among sublnitochondrial fractions mainly contained in the outer membrane of mitochondria34. -4s is evident from Fig. 2, after a carefully subfractionation of mitochondria by Biochim.
Biqbhys.
Acta,
280
(1972)
235y-217
E. R. PODACK, X’. SEUBERT
240
P
7o. EO50. x t: 5 40. ;; Q 2sXIM
20. 0
L
00
10
20
30
40
50
Fig. 2. Subcellular distribution of g’lucose-G-phosphatase (pW 8.0). j_* according to de Du\-e et aZ.J6 Nuclei (Fraction N) WW~ discarded.
D- of n
rat liver wvcre fractionated
Fig. 3, Distribution of protein and individua.1 enzymes after zonal centrifugation of heavy mitochondria. I.+ ml of heavy mitochondria, obtained from 20 g liver were fractionated as described in I I .. I , turbidity of individual fractions Material and Methods. -.-..--., sucrose density gradient; (Asi & : ----, distribution of glucose-6-phosphatase; -----, distribution of glutamate dchydrogenase; -, distribution of lactate dehydrogenase. differential
centrifugation
of glucosephosphatase A comparison
(35 munits/mg studied
somal the
of the specific
activity impurities
properties
of about
protein necessary from
of
in the heavy
activity
the activities
fractions
up to a factor
by the microsomal ductase
present
protein)3s25 with
in mitochondrial
differences
by de Duve 36, there is still considerable
as described
activity
would
and light mitochondrial
of enoyl-CoA
reductase
of the chain
(0.2 to 1.3 munits/mg IOO. Contamination thus be sufficient
mitochondria
acids.
As illustrated
in I@.
zonal
centrifugation
is achieved
thus seemed
only
of mitochondria to account
an absolute
separation in fractions
enzyme
system
protein)12~~4~25~3s~39~~o, shows
acetyl-Co.4-dependent
3, complete
in microsomes
elongation
in the last step of chain elongation.
mitochondrial
activity fractions.
Removal requisite
chain
in the
re-
of the microfor a study
elongation
of microsomal located
as low as 1%
for the enoyl-CoA
of
impurities region
of
fatty upon
of higher
XALONYL-COA TABLE
INDEPENDEKT
FATTY ACID SYNTHESIS
241
II
DISTRIBUTION ENOYL-CoA MITOCHONDRIA
OF
GLUCOSE-&PHOSPHATASE,
REDUCTASE, (&'f) AFTER
AND
GLUTAMATE
ACETYL-COA-DEPENDENT
ZONAL
DEHYDROGENASE, CHAIN
THIOLASE,
ELONGATION
AMONG
ENOYL-CO.4
HYDRATASE,
SUBFRACTIONS
OF
HEAVY
CENTRIFUGATION
14 ml of fraction M (260 ms) from 20 g rat liver were purified by zonal centrifugation as described in Material and Methods. Enoyl-CoA reductase and enoyl-CoA hydratase were assayed with 0.075 mM octenoyl-Co.4 at pH 7.1 and pH 8.9 respectively; the substrate of thiolase was ,%ketooctanoyl-CoA. For additional assays and components see Material and Methods. _. .-____ -..._ F?U&_WZ Glwose- 6~~u~a~la~~ Thiolase Enoyl-CoA Enoyl-CoA AcPtyl-CaA f&?sphatase drhydrogenase [ U~~its/mgj hydratase reductasr chain, (units/wag) (UnitSjmg) (atnits/vmg) (nztwitslwg} clonga.tio~a (wlu~a~its~mg) M (heavy mitochondria) 0.053 No. 3 (60-90 ml of rotor content) 0.162 No. 4 (go--120 ml of rotor content) 0.93 Nos. 7-g (180.-270 ml of rotor content) O.OI2 Nos. 12-14 (330-420 ml of rotor content) 0.0012
1.41
0.68
4.18
14.3
I.74
2.07
0.11
2.79
12.3
I.35
3.11
1.27
5.23
4.23
5.88
34.83
3.81
3.34
9.41
9.6 11.3
g.o
.-l"-l-- ~~___.
1.6 I.33 I.08 ._
_~
density in a sucrose gradient (between 330 and 660 ml of the rotor content). Lactic dehydrogenase is restricted to specific light fractions with a maximum in Fraction 3, originally representing the mitochondrial suspension added on the gradient. The distribution of lactate dehydrogenase rather points to a cytosolic impurity introduced with the suspension of the heavy mitochondria than to contaminations by intact cells. Cytoplasmic fatty acid synthetase, which also could contribute enoyl-CoA reductase activities to a mitochondrial chain elongation, was thus excluded in our studies. In Table II the specific activities of glucose-6-phosphatase, glutamate dehydrogenase, enoyl-CoA reductase, thiolase, enoyl-CoA hydratase and the acetyl-CoAdependent chain elongation of representative fractions of the gradient from Fig. 3 are listed. The enzymes of /J-oxidation coincide with glutamate dehydrogenase, a marker enzyme of the mitochondrial matrix. Enoyl-CoA reductase activities show 2 maxima with distributions indicating the existence of two enoyl-CoA reductases in microsomes and mitochondria, respectively. Additional evidence for different enoyl-CoA reductases was obtained by a study of the solubility properties of microsomal and mitochondrial enoyl-CoA reductase. While showing high enoyl-CoA reductase activity in a suspension of purified microsome-, s acetone treatment of the fractions and suhsequent extraction of the dry powder with phosphate buffer resulted in a complete loss of activity. On the contrary, acetone treatment of the heavy mitochondria collected in Fractions 12 to 14 of the zonal centrifugation (Fig. 3) and subsequent extraction, resulted in an enrichment of enoyl-CoA reductase (Table III). The different solubilities of tnitochondrial and microsomal enoy-CoA reductases offered a simpIe way to separate these enzymes for a study of their kinetic properties. Further attempts at purification of the soluble form (fractionation with ammonium sulfate, heat inactivation, absorption of inactive protein on calcium phosphate gel, ion-exchange chromato~aphy and electrofocusing) resulted in all cases in inactivation of the enzyme. The kinetic properties of mitochondrial enoyl-CoA reductase were therefore studied in the crude extract from heavy mitochondria acetone powders. Biochim. Biofihys. Acta, 280 (1972) 235-247
E. R. PODACK,
242
SOLUBILITY PROPERTIES OF
EKOYL-CO.&
RED”CTASES
FROM
PIJRIFIEU
MITOCH09DRIA
ASD
\V. SEUHERT
M~CROSOMES
Microsomes were prepared from 40 g liver as described by Seubert et al. 3 for the isolation of the microsomal that the second centrifugation was run for zo min at 17oooxg. enovl-CoA reductase with the exception, Puiified mitochondria were obtained by zonal centrifugation as described. The preparation of acetone powder :~nt~ c traction :. described in Material and Methods. Enoyl-CoA reductase was assayed with octcnoyl-Co.1 (0.075 mM mltochondria; 0.1 mM microsomes) at pH 7. I. For additional components see Material and Methods. Treatment
Ilficrosomal
of
s1rbcrZlular fvaction
spec. act. (mzoaits/wzg)
Total activity (units)
Activity vecovevrd (“,,)
dlitochondrial rnoyl CoA rrductasc ~___. __~_. Total spec. act. Total .4ctivity pvotrin fmunitslnzg) activity rccovrvc~d (mits) (“,,) (q)
396
39
15.3
I00
240
12.7
3.0-l
100
60
0
II0
15
1.65
5-t ..~
Total pvofrin
enoyl-CoA
I%?! Deoxycholate (0. I I:)) Prepn of acetone dry powder, subsequent extract of soluble activity
rrductasc
0
0
The insoluble form of enoyl-CoX reductase was purified from a microsomal fraction by treatment with sodium cholate as described by Seubert et ad.3. Freeze-drying of this enzyme allowed storage of the enzyme preparations for at least four months. Khetic
$vofieYties of insoluble and soluble elzq??l-CoA-redtlctnse Final proof for the existence of different enoyl-CoA reductases in microsomes and mitochondria is evident from the kinetic properties illustrated in Figs. 4 to 8. The microsomal enzyme can use either NADH or NADPH (Fig. 4) (R, KADH = 2.0 ‘IO-~ 1\1; K, NADPH = 1.1 ‘IO-~ M). The mitochondrial preparations (Fig. 5) show an absolute XADPH specifity (K,, NADPH = 4.5 IO? M). Different affinities of both enzymes for octenoyl-CoA are evident from Fig. 6 (microsomal enoyl-CoA reductase: K, octenoyl-CoA = 4. IO-~ nl. Mitochondrial enoyl-CoA reductase : 1.6. IO@ 51). The relationship between pH and reaction rates show different maxima for the microsomal and mitochondrial enoyl-CoA reductase at pH 7.7 and 7.9, respectively (Fig. 7). According to Seubert et al.” microsomal enoyl-CoA reductase is most active bvith hexenoyl-Co.4 as substrate. \Vith the soluble enzyme from mitochondria the maximum is shifted to a chain length of C,, (Fig. 8).
40
80
120 NADH
160 xx) or NADPH
240
200
(JIM)
Fig. -I_, Nucleotide specificity of the microsomal NADH; inset: Lineweaver-Burk plots. Substrate: (freeze dried enzyme). For additional components Biochim.
Biqbhys.
Acta,
ZPO
(1972)
*3j-247
enoyl-CoA reductasc. ::---‘, NADPH; 0-o 0.075 mM octenoyl-CoA, pH 7.1; 0.44mg protein and assay conditions, see Material and Methods.
MALONYL-COi%
IKDEPENDENT
FATTY
0
NADH or NADPH
20
ACID
40 60 1 IS (mM_‘)
243
SYNTHESIS
60
(9M)
Fig. 5. Nucleotide specificity of the mitochondrial enoyl-CoA-reductasc. ~-c, NADI’H ; O-O, NADH. Inset: Lineweaver-Burk plots. Substrate: 0.1 mM octenoyl-Cob, pH 7.1; 1.46 mg protein (acetone powder extract) For additional components of the assay mixture see Material and Methods.
Octenoyi-CoH
( JJFS, 1
Fig. 6. Relation between reaction rate and substrate concentration. Microsomal (O-0) (o.gq mg freeze dried enzyme) and mitochondrial (O--O) (1.35 mg acetone powder extract) enoylCoA reductase; 90 mM potassium phosphate buffer, pH 7.r. For additional components see Material and Methods. Inset: Lineweaver-Burk plots.
The designations trnns-2,3-hexenoyl-CoA reductase and traas-z,3-decenoyl-CoA reductase are suggested for the microsomal and mitochond~al enzyme, respectively. DISCUSSION
The differentiation
of two enoyl-CoA
reductases
located
in microsomes
and
mitochondria of liver gives final proof for the existence of amitochond~al acetyl-CoAdependent chain elongation in this organ in addition toa malonyl-CoA-dependent pathway in microsomes. The distribution of the insoluble hexenoyl-CoA reductase among subcellular fractions (Table II) adds strong support to a participation of this enzyme in the microsomal malonyl-C.o~~-dependent chain elongation, involved in the synthesis of polyunsaturated fatty acids. The chain length specifity of this enzyme, showing a maximum with hexenoyl-CoA is not contradictory to such a role, since it is Biochim. Biophys. Acta,
280
(1972)
235-247
244
E.
0’
5.6
* 59
6.2
3 65
” 66
” 24
71
77
” 0.0
03
” 06
R.
PODACK,
M’.
SEUBEKT
6.9
PH
Fig. 7. pH-dependence of the microsomal and mitochondrial enoyl-CoA reductase. C,-reductase (;s--c) (0.53 mg protein) and C,,-reductase (O-O) (3.6 mg protein) both assayed with 0.05 ml\1 and 0,oT.j mill octenoyl-CoA, respectively. go mM potassium phosphate buffer, pH 5.6 to X.6 was employed. For additional components of assay mixtures see Material and Methods.
” c4 8. Chain length specificities of microsomal and mitochondrial enoyl-CoA reductase. Microsoma1 enoyl-Co-k-reductase(o-0) (from Seubert et ~1.~) and mitochondrial (~-‘--i’) (2.1 mg acetone powder extract) enoyl-CoA reductase. Composition of the reaction mixture for the mitochondrial enoyl-CoA reductase was: go mM potassium phosphate buffer, pH 7. I ; o. I mM enoyl-CoA derivatives shorter than dodecenoyl-CoA; 0.05 mM dodecenoyl-CoA and hcxadecenoyl-CoA. I’or addtional components of assay mixtures see Material and Methods. Fig.
most likely the result of an inhibitory effect, increasing with the chain length of the substrate as also reported for the saturated CoA-derivativesa. This interpretation is also in accord with an activation of the enzyme, observed with increasing desaturation of the acyl residue 22+r. Studies are in progress to give an answer to this question. A role of hexenoyl-CoA reductase in the microsomal malonyl-CoA-dependent chain elongation is also evident from a comparison of the pyridine nucleotide specifity of hexenoyl-Coil reductase and microsomal chain elongation reported by various authors. As summarized in Table IV, NADH can substitute for NADPH in the microsomal process. With respect to mitochondrial acetyl-CoA-dependent chain elongation, however, great discrepances concerning the requirement of NADH and NADPH are ~iocAim. Biophys.
Acta.
280
(1972)135-247
IV
NADH
* ** *** i
--
___.
__
.--.
NA DPH
MICROSOMAL
AND
_l_-~
36.5 15 40 44.5 40 30 24 -
28.5
--
NA DPti-
-
80
and -tetraenoic
-
18
29
53 100
acid.
-
II
3.’ 0 -_
-
57
73
ELONC;ATION
nucleotide
NO
CHAIN
100
__.
NA DH
MITOCHONI)RIAL
Substrate: endogcnous fatty acids. Decanoyl-Co-4 as primer. l’almitoyl-CoA as primer. Incorporation of y-[l%]linoleic acid in C,,-trienoic
____--.
Colli et al.%* 100 Colli et aZ.%** 94 Colli et a!.%*** 100 Quagliariello et LzZ.~% 100 WakillD 100 Barron12 IO0 Boone and WakiF IO0 Podack2j 100 Donaldson et a1.38 Guchait et al.“6 Stoffel and Ach*‘,f Aeberhard and Menkes 29 -Landriscina et aLz4 -
NADH+
ox
iVlitochondrial chain elongation relative a~t~~it9 (9;;) -. ..___-
AK~L) NADPH
.____I~.
RESPECTIVELY ..-
A &hors
CoA,
EPFECTSOF
TABLE ACIDS
xy
[~~C]MALOSYL-COX
9 IO0 100 85
IO0 ICI0
-
ZOO
95
100
100 100
-
-
-
___---
-
54
-
30
9.5 -
-
No nucleotide
[K]ACETYL-
13.5
-_
AK;D
93
92 12.5
7.5 100
-
-
-
Microsonaal chain elongation activily (%) _-_______ .-.-NA DPH NADH NA DH + NA DPH _.-___
relative
OFFATTY
(Table IV). A possible participation of a transhydrogenase has been excluded by Colli et aZ.ll. The discrepances plained by a differing degree of contamination by microsomal
evident
detergents
after treatment witlt can thus only he exenoyl-CoA reductase
activity in the various mitochondrial preparations employed. Only in fractions obtained by zonal centrifugation (Fractions 12-14, Fig. 3) or with the solubilized system, could we show an almost absolute requirement for NADPH in chain elongation. This result should be expected on the basis of the SADPH specificity of mitochondrial enoyl-CoA reductase. The low activity with XADPH alone is readily explained by the NADH specifity of P-113.‘drox~acyl-CoA dehydrogenase in liver4’. Incorpnration in the absence of NADPH may be due to chain elongation up to the stage of the a,p-unsaturated acyl-CoA. Different chain elongation systems in liver suggest different physiological roles for each system. As will be shown in a forthcoming communication, a participation of the microsomal enoyl-CoA reductase in the synthesis of polyunsaturated fatty acids is evident. With respect to the physiological significance of the mitochondrial system from liver, the authors prefer a possible role in hydrogen transfer from NADPH to the respiratory chain. As an alternate role, the transport of hydrogen from the cytosol to the mitochondrial compartment and conservation of energy, respectively, has to system from be considered, as suggested by Whereat d aE.43-4Bfor the mitochondrial heart muscle. Experiments are in progress to differentiate among these possibilities. ACKNOWLEDGEMENTS
This investigation was supported by the Deutsche Forschungsgemeinschaft. The Stiftung Volkswagenwerk provided us the scientific equipment. The excellent technical assistance of Sabine Buss is gratefully acknowledged.
9 IO I1 12 13 14 I5 I6 77 18 19 20 21
Larl~don, G. R. (195.5) J. .4?J'Z.~h'm. SOC. 77, jIg0 Seubert, XV., Greull, G. and Lynen, F. (1957) Angrw. Chum. 69, 359 Sellbert, W., Lamberts, I., Kramer, R. and Ohlp, B. (1968) Biochim. Biophys. ,4cta 164, 498 Lyncn, li. and Decker, K. (1957) EYgrb. f’hysiol. 49, 327 Stern, J. K., in Colowick, S. I’. and Kaplarl, N. 0. Methods in ITnzymology, Vol. T, :lcademic l’ress, New lTork, 1955, p. j-j9 C,rane, F. I,., pllii, S., Hauge, J. J., Green, D. 12. and Beinert, H. (1956) j. BioE. Chmzz. 118, 701 Crane, F. I,. and Beinert, H. (1956) J. Bid. Chrm. 2x8, 717 Slater, E.C., Colpa-Boonstra, J. P. and Links, 1. in Wolstcnholmc, G. E. 1%‘. and O’Connor, C. M., Ciba Foundations Symposizrm, Qztinows in Elrctvon Transport, Little Brown, Boston, 1961, p. 346 Hauge, G. (1956) .I. ATVZ.Chp~. SOC. 78, jZ60 Wakil, S. J. (1961) J. Lipid HFS. 2, I Christ, E. J. and Hiilsmann, W. C. (1962) Biochim. Riophys. Acta 60, 72 Barron, 1~. J. (1966) Biochirn. Biophys. Acta 116, 42.5 Christ, B. J. c’. J. (1968) Biochzm. Uiophys. Acta 152, 50 Colli, W., Hinkle, P. C. and Pullman, M. E., (1969) J. Viol. Chcwz. 214, 6432 Dahlcn, J, V. and Porter, J. 1%‘. (1968) Arch. Biochmz. Biofdzys. 127, 207 Quagliariello, E., Landriscina, C. and Coratelli, P. (1968) Biochirn. Riophys. Acta 161, IL Whereat, A. F. in Paoletti, K. and Kritchevsky, D. Adwanms in Lipid Research, Vol. 9, Academic Press, Sew York and London, 1971, p. 119 \Vit-I’eetcrs, E. M., Scholte, H. R. and Elenbaas, H. L. (1970) Bzochim. Biophys. Acta 210, 360 Nandekar, A. K. N. and Kumar, S. (1969) Arch. Biochmz. Riophys. 134, 563 Lin, c. \1. and Kumar, S. (1972) J. Viol. Chenz. 247, 604 Stoffel, W. and Schiitte, E., Lipoide, 16 Collog. deu GeseElschaft fiiv Physiologische Chemi~, Mosbachll?adrti 1965, Springer Verlag, Berlin, 1966, p. 64
Bzochim.
Hioph.ys.
-lcta,
280 (1971)
235.-247
MALONYL-CoA
22
23 24 25 26 27 2x 29 30 31 32 33 34 35 36 37 38 39 40 41 42
INDEPENDENT
FATTY
ACID SYNTHESIS
247
Nugteren, D. H. (1965) Biochim. Biophys. Acta 106, 280 Aeberhard, E. and Menkes, J. H. (1968) J. Biol. Chem. 243, 3834 Landriscina, C., Gnoni, G. V. and Quagliariello, E. (1970) Biochim. Biophys. Acta 202, 405 Podack, E. R., Doctoral Thesis, Medical Faculty Gijttingen 1971 Lynen, F. (1967) Biochem. J, 102, 3S1 Harlan, W. R. and Wakil, S. J. (1963) J. Biol. Chem. 238, 3216 Lynen, F. and Wieland, O., in Colowick, S. P. and Kaplan, N. O., Methods in Enzymology, Vol. I, Academic Press, New York, 1955, 1,. 566 Buckel, W. and Eggerer, H. (1965) B&hem. Z., 343, 29 Lowry, 0. H., Rosebrough, N. J.. Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem. 193, 265 Layne, E. in Colowick, S. P. and Kaplan, N. O., Methods in Enzymology, Vol. 3, Academic Press, New York, 1957, p. 447 Hogeboom, G. H. and Schneider, %‘. C. (1953) J. Biol. Chem. 204, 233 Appelmans, F. and de Duve, C. (1955) B&him. J. j9, 433 van Tol, A. (1970) Biochim. Biophys. Arta 219, 227 der cnzymatischen Baganski, E. S., Foa, I’. P. and Zak, B. in Bergmeyer, H. U., Xethoden Analyse, Vol. I, Verlag Chemie, Weinheim, 1970, p. 839 de Duve, C., Pressmann, C., Gianetto, K., Wattiaux, R. and .\ppelmans, F. (X955) B&hem. J. 60, 604 I’oole, B., Higashi, T. and de Duve, C. (1970) J. Cell. BioZ. 45, 408 Harlan, W. R. and Wakil, S. J. (1962) Biochem. Biophys. Rrs. Commuw. S, 131 Donaldson, W. E., Wit-Peeters, E. M. and Scholte, H. R. (1970) B&him. Biophys. 4cta 202, 35 Howard, C. F. (1968) B&him. Biophys. Acta 164, 448 Stoffel, W. and Ach, K. L. (1964) Hoppe Seyler’s Z. Physiol. Chem. 337, 123 Decker, K. in Pummerer-Erlangen, R., Die aktiviertr Essigstiure, Ferdinand Enke Verlag,
Stuttgart,
1959, p, 229
43 Whereat, A. F., Hull, F. E., Orishimo, 31. W. and Rabinowitz, J. L. (1967) J. Biol. Chem. 242, 40’3 4-l Hull, F. E. and Whereat, A. F. (1967) J, Biol. Chem. 242, 4023 45 Whereat, A. F., Orishimo, M. W., Nelson, J. and Phillips, S. J. (1969) J. Biol. Chem. 244, 6498 Biophys 117, 541 46 Guchhait, R. B., Putz, G. R. and Porter, J. W. (1966) Arch. B&hem. 9, 1470 47 Boone, S. C. and Wakil, S. J. (1970) Bzochemisfvy
Biuchinz. Biophys.
Acta,
280 (1972) 235-247
BBA
ET BIOPHYSICA
235
ACTA
56123
ON THE
MECHANISM
OF
JfALONYL-CoA
IKDEPENDENT
FATTY
ACID
SYNTHESIS II.
ISOLATION,
PROPERTIES
z,3-HEXENOYL-CoA
E. R. PODACK*
April
AND SUBCELLULAR
LOCATION
TRANS-2,3-DECENOYL-CoA
OF TRANS-
REDUCTASE
AND W. SEUBERT
Physiologisch-chemisches (Received
AND
z8th,
Institut
der Univevsitiit,
Glittingen
(Germany)
1972)
SUMMARY I. The separation by zonal centrifugation of two enoyl-CoA reductases located in microsomes and mitochondria of rat liver is described. 2. The microsomal enzyme can use either NADH or NADPH as hydrogen donor. It shows optimal activity at pH 7.9 with trans-2,3-hexenoyl-CoA as substrate. 3. The mitochondrial enzyme shows an absolute requirement for NADPH as hydrogen donor with an optimum at pH 7.7. An investigation of the chain length specifity revealed a maximum with trans-2,3-decenoyl-CoA4. In contrast to the microsomal system, the enzymatic activity from mitochondria could be solubilized by treatment with acetone. 4. Varying effects of both NADH and NADPH on the rate of acetyl-CoAdependent chain elongation in crude mitochondrial preparations, reported by several groups, could be explained by varying degrees of microsomal contamination. 5. A possible physiological significance of the mitochondrial system in hydrogen transport and conservation of energy is discussed.
INTRODUCTION
Unidirectional reaction sequences involved in degradative processes are in most cases by-passed by nucleoside triphosphate dependent reactions, thus creating an irreversible synthetic process. This principle is verified by the degradation and synthesis of glucose, glycogen, fatty acids, lipids and other important constituents of the cell. In some cases, however, the redox difference between the NADH/NAD+ system and a substrate couple participating as hydrogen acceptor in a synthetic process helps to overcome an energy barrier. Examples of this principle are the conversion of * Essential GZjttingen.
part of the Doctor’s
Thesis of E. R. Podack
at the Medical
Riochim.
Biophys.
Faculty
Acta,
of the IJniversity
280
(1972)
235~247
E. R. PODACK, W. SEURERT
236
phosphoglycerate to glycerinaldehyde phosphate, or the conversion of P-hydroxymethylglutaryl-CoA to mevalonic acid. Chain elongation of fatty acids by reversal of the P-oxidation is another illustrative example for this principle. As is evident from the free energy of the reaction sequence summarized in Eqns I to 4 (Table I), reversal of the /?-oxidation, including the FAD-dependent dehydrogenase, would not be possible from a thermodynamic point of view (dF’, = +8,45 (kcal/mole)). Substitution of the FAD-dependent acyl-CoA dehydrogenase (Eqn 4) by a pyridinnucleotidedependent enoyl-CoA reductase (Eqn 5), first described by Langdonl and Seubert et a1.2J, shifts the equilibrium in favour of the synthetic process (W’, = -10.35 (kcal/mole))4-9. Experimental evidence for a reversal of /?-oxidation of fatty acids was first demonstrated by Seubert et al. 2. in 1957. According to the studies of numerous groups, this process is apparently restricted to the mitochondrial compartment under physiological conditions 1op1*. Reversal of p-oxidation in the cytoplasm, as reported for the combined action of enzymes of p-oxidation and fatty acid synthetase, needs further experimental support with respect to its physiological significance1g~20. T,\BLE
I
Thermodynamic
CAI,CU~ATIOS FOR THE REVERSAI. OF FiTTY ACID OXIDATION _~~~.
li ’
stefi
+ CH,-CO-SCo.4 + II-CO-CH,-CO-SCo.4 -t HSCoA 1.56. (thiolase)” K-CO-CH,-CO-SCoA + NADH + H+ R-CHOH-CH,-CO-SCoA + N4D-‘5.25 (P-hydroxyacyl-CbA dehydrogcnase)’ K-CHOH-CH,-CO-SCo_4 + R-CH = CH-CO-XXX + H,O ‘5.7 (enoyl-Co.4 hydratasc)j R-CH = CH-CO-SCo.4 + CoQH, + R-CH,-CH,-CC)-SCoA A CoQ, . (acyl-CoA dehydrogenase and B I F)6-9 R-CH=CH-CO-SCoiZ+NADPH(NADH) +H+ + R-CH,-CH,-CO-SCokt NADP+(NAD+) (enoyl-CoA reductase)le3
.!lE’,(m r-j
.4F’, (kcal~tnolr)
Eqn
ATO.
_____
KW3~S-CoA
Free energy of reversal Free energy of reversal
of B-oxidation of /j-oxidation
including including
IO-'
-
+
6.65
(I)
102
~ 81.9
-
3.75
(2)
t 0.75
(3)
I- 4.8
(4)
~ 14.0
(5)
_
im104
-305
acyl-Cob dehydrogenase enoyl-CoA reductase
+
X.4.5
--IO.jj
The physiological function of the mitochondrial system was originally restricted to chain elongation involved in the synthesis of polyunsaturated fatty acids. The discovery of a malonyl-Co&dependent chain elongation21-25 of fatty acids in the microsomal fraction of liver questioned such a role. According to Sugteren2” chain elongation in microsomes occurs no longer at the level of enzyme-bound derivatives of pantetheine, as reported for fatty acid synthetase 26, but rather at the level of coenzyme A derivatives. Enoyl-CoA reductase activity was detected in microsomes without further characterization of nucleotide and chainlength specifityZ2. Since most mitochondrial preparations employed for the study of acetyl-CoA dependent chain elongation in liver have been crude preparations, and have most likely been contaminated by microsomes, a possible “mitochondrial” chain elongation activity in liver preparations could have been imitated by a combined action of enoyl-CoA reductase from microsomes and the enzyme thiolase, I$-hydroxyacy-CoA-dehydrogenase and
MALONYL-CO.4
INDEPENDENT
FATTY
ACID
237
SYNTHESIS
enoyl-CoA hydratase of the oxidative sequence in mitochondria. This possibility seemed extremely likely, since in some cases optimal chain elongation activities were observed after sonic treatment or freezing and thawing of mitochondriar2~27. A reinvestigation of the subcellular localization of enoyl-CoA reductase was therefore initiated. These studies resulted unexpectedly in the localization and characterization of two different enoyl-CoA-reductases of liver in microsomes and mitochondria, respectively. MATERIAL
AND
METHODS
Enzymes and substmtes Preparation and analysis of enoyl-CoA hydratase (EC 4.2.1.17), @-ketothiolase (EC 2.3.1.9) and the various coenzyme A derivatives have been described in the preceeding paper3. !I-14C]Acetyl-CoA was prepared by reaction of IO pmoles 11-1K]acetic anhydride, dissolved in 1.5 ml peroxide-free tetrahydrofuran, with an equal amount of coenzyme A according to the method of Lynen and Wielandz8. Yield: 7o-8o0/o. Assays
of enzymes
and substrates
Acetyl-CoA and [14C]acetyl-CoA were assayed according to Buckel and Eggerer”’ by following the decrease in absorbance at 233 nm. Trans-2,3-enoyl-CoA derivatives and coenzyme A derivatives of saturated fatty acids were quantitated according to the method of Seubert et a1.3. Protein was determined according to the method of Lowry et aL30, and from the absorbance at 260 and 280 nm31. For enzyme assays, the mitochondrial fractions were dissolved with sodium deoxycholate or Triton X-100 at final concentrations of 0.1% and 0.5%, respectively. There was no inhibition of the various enzyme assays by these additions. Glutamate dehydrogenase was assayed according to Hogeboom and Schneide?, with the following exception: the assay mixture contained 0.1 M potassium phosphate buffer (pH 7.7) and 2 m&I ADP. Thiolase and enoyl-CoA hydratase were assayed according to the method of Seubert et a1.3, acid phosphatase according to the method of Appelmans and de Duve33. Assay of glucose-6-phosphatase. Contamination of mitochondria by microsomes is usually quantitated by assays of glucose-6-phosphatase, an enzyme exclusively located in the microsomal fraction (see ref. 34). Acid phosphatase, an enzyme located in the lysosomal fraction, also shows activity with glucose 6-phosphate. In order to restrict assays of glucose-6-phosphatase activity mainly to microsomal impurities in the mitochondrial fractions, glucose-6-phosphatase assays were carried out at pH 8.0. As is evident from the pH-dependence of acid phosphatase with /%glycerophosphate as the substrate (Fig. I), lysosomal acid phosphatase activity on cleavage of glucose 6-phosphate is negligible under these conditions. However, glucose-6-phosphatase still shows significant activity at pH 8.0 (Fig. I). The assay mixture for glucose-6-phosphatase contained in 0.4 ml: 50 pmoles saccharose; 0.1 pmole EDTA; IO pmoles Tris-HCl buffer, pH 8.0. The reaction IO pmoles glucose 6-phosphate; mixture is incubated for 15 min at 37 “C. Liberated phosphate is assayed according to Baganski
et a1.35. Biochim. &@hys.
Acta, 280
(1972)
235-247
E. K. POLMCK, IV. SIJI:REHT
238
06.
PH Fig. I. pH-dependence of glucose+phosphatasc and acid phosphatase. c ---~7, cleavage of glucose h-phosphate by microsomes; O--O, cleavage of fi-glycerophosphatc by light mitochondria. Buffers employed: IO ~moles sodium acetate buffer, pH 5.0; IO /Lmolespotassium citrate, pH 6.5; IO ,uumoles Tris-HCl buffer from pH 6.X to 8.6. For additional components of assay mixtures see Material and Methods.
Acetyl-CoA-dependent
chain elongation
was assayed in a reaction
mixture
con-
taining in 0.5 ml: 25 pmoles glycyl glycine buffer, pH 8.0; 0.05 pmole [r-14CjacetyCoA (specific activity 5 to 2.5 mCi/mmole) ; 0.05 hLrnole decanoyl-CoA; 4 nmoles rotenone (IO+ 31 in 50% ethanol); 0.5 pmole NADPH; 0.5 pmole NADH; 20 to ZOO pg mitochondrial protein. Addition of enoyl-CoA hydratase, /%hydroxyacyl-CoA dehydrogenase and thiolase was not necessary because of sufficient amounts present in the preparations used. The reaction mixture was incubated for 5 to IO min at 37 “C. Incorporation in the fatty acid fraction is determined after hydrolysis with NaOH. I munit corresponds to the incorporation of I nmole [Xlacetate per min in the light petroleum-soluble fraction. Enoyl-CoA reductase assays contained in z ml: 180 kmoles potassium phosphate buffer (pH 7.1 or pH 8.0); 0.5 pmole NADPH; 0.1 to 0.2 ,umole enoyl-CoA of different chain lengths; 5 to 30 munits enoyl-CoA reductase. The reaction mixture is incubated at 30 “C. Oxidation of NADPH is followed at 334 nm. The reaction is started by the addition of substrate. To compensate for a substrate-independent oxidation of NADPH by the enzyme preparation, all assays had to be carried out against a control containing no substrate. For assays of an NADH-dependent enoyl-CoA reductase in subcellular fractions, the above reaction mixture is supplemented with 2 pmoles KCN and 20 nmoles rotenone to inhibit oxidation of NADH by enzymes of the respiratory chain. After solubilization of enoyl-CoA reductase, additions of KCN and rotenone are no longer necessarv. Fractionation of subcellular particles Fractionation of the homogenate from liver into nuclei (Fraction N), heavy mitochondria (Fraction M), light mitochondria (Fraction L), microsomes (Fraction P) and supernatant (Fraction S) was carried out in isotonic sucrose solution according to the method of de Duve et aZ.3”. Biochim.
Hiophys. Acta,
2X0
(1972)
235-247
XALOKYL-COA
INDEPENDEi’GT FATTY ACID SYNTHESIS
Subfractionation Beckman
of heavy
mitochondria
rotor was carried out according
239
by zonal centrifugation
in an Al 14
to the method of Poole et aZ.37with modifica-
tions (G. Weiss, B. Killer and W. Seubert, unpublished) : 500 ml of a sucrose density gradient ranging from 0.4 to 0.75 RI sucrose (0.02 $1 pottasium glpcylglycine pH 7.5) are introduced into the rotor, spinning at 3000 rev./min, through the edge line. The gradient is established by mixing both solutions in a Beckman gradient pump, Model 141. The remainder of the rotor volume is filled with a 150 ml cushion of 1.0 &l sucrose, supplemented with 0.02 M potassium gl~cylglycine (appro.~. 250 mg protein) of heavy mitochondria suspended
buffer, pH 7.5. 14 ml in 0.25 31 sucrose are
introduced by a syringe through the core line. An overlay of 50 ml 0.2 Rl glucose is then added to move the mitochondrial zone pass the core. The rotor is then accelerated to 6000 rev./min and run for 15 min. After deceleration to 3000 rev/min, the contents are collected from the coreline by displacement with 1.2 M sucrose, supplemented with 0.02 M potassium glycylglycine buffer, pH 7.5. The contents of the rotor including overlay, gradient and cushion are collected in 22 fractions each of 30 ml. The distribution of protein among the various fractions was determined from the absorbance at 575 nm. The distrit~ution of the various marker enzymes was determined after treatment of the individual fractions with deoxycholate or Triton SIOO. Possible contamination by remaining intact cells could be excluded by assays of lactic dehydrogenases.
For the results of zonal centrifugation
see Fig. 3 and Table II.
~~&~~Y~~~~~~, oj acetone ~o~~d~~~~~~ heavy ~~tocho~~~~ii~ and otk s~~c~~~,u~~~ j~a~t~o~~~ Acetone powders from subcellular fractions were prepared according to the method of Dahlen and Porter 15. Soluble decenoyl-CoA reductase is extracted from I g of acetone powder by homogenization in 20 ml of 0.1 M potassium phosphate buffer, pH 7.1. After aging for an additional 30 min at o “C,insoluble material is sedimented for 30 min at IO~OOOXR. Enoyl-Co.4 reductase can be assayed in the supernatant with NADPH (Table III). RESULTS
Separation of emyE-CoA reductases Studies of Van ToP4 on the distribution of the two microsomal marker enzymes glucose-6-phosphatase and NADPH-cytochrome c reductase in fractions from rat liver microsomes, isolated by continuous sucrose densitv gradient centrifugation, revealed glucose-6-phosphatase to be located at a site of higher density compared to NADPH-cytochrome c reductase. Van To1 concludes from this finding, that the microsomal contamination of mitochondria is due to the presence of “heavy” microsomes, contributing relatively more glucose-6-phosphatase activity than NADPH-cytochrome c reductase activity. Therefore, for a quantitation of microsomal impurities in submitochondrial fractions, assays of glucose-6-phosphatase were given preference to NADPH-cytochrome c reductase as the marker enzyme of microsomes in our studies. A mitochondrial location of glucose-6-phosphatase seems to be excluded on the basis of different distributions of the former enzyme and rotenone insensitive NADH-cytochrome c reductase among sublnitochondrial fractions mainly contained in the outer membrane of mitochondria34. -4s is evident from Fig. 2, after a carefully subfractionation of mitochondria by Biochim.
Biqbhys.
Acta,
280
(1972)
235y-217
E. R. PODACK, X’. SEUBERT
240
P
7o. EO50. x t: 5 40. ;; Q 2sXIM
20. 0
L
00
10
20
30
40
50
Fig. 2. Subcellular distribution of g’lucose-G-phosphatase (pW 8.0). j_* according to de Du\-e et aZ.J6 Nuclei (Fraction N) WW~ discarded.
D- of n
rat liver wvcre fractionated
Fig. 3, Distribution of protein and individua.1 enzymes after zonal centrifugation of heavy mitochondria. I.+ ml of heavy mitochondria, obtained from 20 g liver were fractionated as described in I I .. I , turbidity of individual fractions Material and Methods. -.-..--., sucrose density gradient; (Asi & : ----, distribution of glucose-6-phosphatase; -----, distribution of glutamate dchydrogenase; -, distribution of lactate dehydrogenase. differential
centrifugation
of glucosephosphatase A comparison
(35 munits/mg studied
somal the
of the specific
activity impurities
properties
of about
protein necessary from
of
in the heavy
activity
the activities
fractions
up to a factor
by the microsomal ductase
present
protein)3s25 with
in mitochondrial
differences
by de Duve 36, there is still considerable
as described
activity
would
and light mitochondrial
of enoyl-CoA
reductase
of the chain
(0.2 to 1.3 munits/mg IOO. Contamination thus be sufficient
mitochondria
acids.
As illustrated
in I@.
zonal
centrifugation
is achieved
thus seemed
only
of mitochondria to account
an absolute
separation in fractions
enzyme
system
protein)12~~4~25~3s~39~~o, shows
acetyl-Co.4-dependent
3, complete
in microsomes
elongation
in the last step of chain elongation.
mitochondrial
activity fractions.
Removal requisite
chain
in the
re-
of the microfor a study
elongation
of microsomal located
as low as 1%
for the enoyl-CoA
of
impurities region
of
fatty upon
of higher
XALONYL-COA TABLE
INDEPENDEKT
FATTY ACID SYNTHESIS
241
II
DISTRIBUTION ENOYL-CoA MITOCHONDRIA
OF
GLUCOSE-&PHOSPHATASE,
REDUCTASE, (&'f) AFTER
AND
GLUTAMATE
ACETYL-COA-DEPENDENT
ZONAL
DEHYDROGENASE, CHAIN
THIOLASE,
ELONGATION
AMONG
ENOYL-CO.4
HYDRATASE,
SUBFRACTIONS
OF
HEAVY
CENTRIFUGATION
14 ml of fraction M (260 ms) from 20 g rat liver were purified by zonal centrifugation as described in Material and Methods. Enoyl-CoA reductase and enoyl-CoA hydratase were assayed with 0.075 mM octenoyl-Co.4 at pH 7.1 and pH 8.9 respectively; the substrate of thiolase was ,%ketooctanoyl-CoA. For additional assays and components see Material and Methods. _. .-____ -..._ F?U&_WZ Glwose- 6~~u~a~la~~ Thiolase Enoyl-CoA Enoyl-CoA AcPtyl-CaA f&?sphatase drhydrogenase [ U~~its/mgj hydratase reductasr chain, (units/wag) (UnitSjmg) (atnits/vmg) (nztwitslwg} clonga.tio~a (wlu~a~its~mg) M (heavy mitochondria) 0.053 No. 3 (60-90 ml of rotor content) 0.162 No. 4 (go--120 ml of rotor content) 0.93 Nos. 7-g (180.-270 ml of rotor content) O.OI2 Nos. 12-14 (330-420 ml of rotor content) 0.0012
1.41
0.68
4.18
14.3
I.74
2.07
0.11
2.79
12.3
I.35
3.11
1.27
5.23
4.23
5.88
34.83
3.81
3.34
9.41
9.6 11.3
g.o
.-l"-l-- ~~___.
1.6 I.33 I.08 ._
_~
density in a sucrose gradient (between 330 and 660 ml of the rotor content). Lactic dehydrogenase is restricted to specific light fractions with a maximum in Fraction 3, originally representing the mitochondrial suspension added on the gradient. The distribution of lactate dehydrogenase rather points to a cytosolic impurity introduced with the suspension of the heavy mitochondria than to contaminations by intact cells. Cytoplasmic fatty acid synthetase, which also could contribute enoyl-CoA reductase activities to a mitochondrial chain elongation, was thus excluded in our studies. In Table II the specific activities of glucose-6-phosphatase, glutamate dehydrogenase, enoyl-CoA reductase, thiolase, enoyl-CoA hydratase and the acetyl-CoAdependent chain elongation of representative fractions of the gradient from Fig. 3 are listed. The enzymes of /J-oxidation coincide with glutamate dehydrogenase, a marker enzyme of the mitochondrial matrix. Enoyl-CoA reductase activities show 2 maxima with distributions indicating the existence of two enoyl-CoA reductases in microsomes and mitochondria, respectively. Additional evidence for different enoyl-CoA reductases was obtained by a study of the solubility properties of microsomal and mitochondrial enoyl-CoA reductase. While showing high enoyl-CoA reductase activity in a suspension of purified microsome-, s acetone treatment of the fractions and suhsequent extraction of the dry powder with phosphate buffer resulted in a complete loss of activity. On the contrary, acetone treatment of the heavy mitochondria collected in Fractions 12 to 14 of the zonal centrifugation (Fig. 3) and subsequent extraction, resulted in an enrichment of enoyl-CoA reductase (Table III). The different solubilities of tnitochondrial and microsomal enoy-CoA reductases offered a simpIe way to separate these enzymes for a study of their kinetic properties. Further attempts at purification of the soluble form (fractionation with ammonium sulfate, heat inactivation, absorption of inactive protein on calcium phosphate gel, ion-exchange chromato~aphy and electrofocusing) resulted in all cases in inactivation of the enzyme. The kinetic properties of mitochondrial enoyl-CoA reductase were therefore studied in the crude extract from heavy mitochondria acetone powders. Biochim. Biofihys. Acta, 280 (1972) 235-247
E. R. PODACK,
242
SOLUBILITY PROPERTIES OF
EKOYL-CO.&
RED”CTASES
FROM
PIJRIFIEU
MITOCH09DRIA
ASD
\V. SEUHERT
M~CROSOMES
Microsomes were prepared from 40 g liver as described by Seubert et al. 3 for the isolation of the microsomal that the second centrifugation was run for zo min at 17oooxg. enovl-CoA reductase with the exception, Puiified mitochondria were obtained by zonal centrifugation as described. The preparation of acetone powder :~nt~ c traction :. described in Material and Methods. Enoyl-CoA reductase was assayed with octcnoyl-Co.1 (0.075 mM mltochondria; 0.1 mM microsomes) at pH 7. I. For additional components see Material and Methods. Treatment
Ilficrosomal
of
s1rbcrZlular fvaction
spec. act. (mzoaits/wzg)
Total activity (units)
Activity vecovevrd (“,,)
dlitochondrial rnoyl CoA rrductasc ~___. __~_. Total spec. act. Total .4ctivity pvotrin fmunitslnzg) activity rccovrvc~d (mits) (“,,) (q)
396
39
15.3
I00
240
12.7
3.0-l
100
60
0
II0
15
1.65
5-t ..~
Total pvofrin
enoyl-CoA
I%?! Deoxycholate (0. I I:)) Prepn of acetone dry powder, subsequent extract of soluble activity
rrductasc
0
0
The insoluble form of enoyl-CoX reductase was purified from a microsomal fraction by treatment with sodium cholate as described by Seubert et ad.3. Freeze-drying of this enzyme allowed storage of the enzyme preparations for at least four months. Khetic
$vofieYties of insoluble and soluble elzq??l-CoA-redtlctnse Final proof for the existence of different enoyl-CoA reductases in microsomes and mitochondria is evident from the kinetic properties illustrated in Figs. 4 to 8. The microsomal enzyme can use either NADH or NADPH (Fig. 4) (R, KADH = 2.0 ‘IO-~ 1\1; K, NADPH = 1.1 ‘IO-~ M). The mitochondrial preparations (Fig. 5) show an absolute XADPH specifity (K,, NADPH = 4.5 IO? M). Different affinities of both enzymes for octenoyl-CoA are evident from Fig. 6 (microsomal enoyl-CoA reductase: K, octenoyl-CoA = 4. IO-~ nl. Mitochondrial enoyl-CoA reductase : 1.6. IO@ 51). The relationship between pH and reaction rates show different maxima for the microsomal and mitochondrial enoyl-CoA reductase at pH 7.7 and 7.9, respectively (Fig. 7). According to Seubert et al.” microsomal enoyl-CoA reductase is most active bvith hexenoyl-Co.4 as substrate. \Vith the soluble enzyme from mitochondria the maximum is shifted to a chain length of C,, (Fig. 8).
40
80
120 NADH
160 xx) or NADPH
240
200
(JIM)
Fig. -I_, Nucleotide specificity of the microsomal NADH; inset: Lineweaver-Burk plots. Substrate: (freeze dried enzyme). For additional components Biochim.
Biqbhys.
Acta,
ZPO
(1972)
*3j-247
enoyl-CoA reductasc. ::---‘, NADPH; 0-o 0.075 mM octenoyl-CoA, pH 7.1; 0.44mg protein and assay conditions, see Material and Methods.
MALONYL-COi%
IKDEPENDENT
FATTY
0
NADH or NADPH
20
ACID
40 60 1 IS (mM_‘)
243
SYNTHESIS
60
(9M)
Fig. 5. Nucleotide specificity of the mitochondrial enoyl-CoA-reductasc. ~-c, NADI’H ; O-O, NADH. Inset: Lineweaver-Burk plots. Substrate: 0.1 mM octenoyl-Cob, pH 7.1; 1.46 mg protein (acetone powder extract) For additional components of the assay mixture see Material and Methods.
Octenoyi-CoH
( JJFS, 1
Fig. 6. Relation between reaction rate and substrate concentration. Microsomal (O-0) (o.gq mg freeze dried enzyme) and mitochondrial (O--O) (1.35 mg acetone powder extract) enoylCoA reductase; 90 mM potassium phosphate buffer, pH 7.r. For additional components see Material and Methods. Inset: Lineweaver-Burk plots.
The designations trnns-2,3-hexenoyl-CoA reductase and traas-z,3-decenoyl-CoA reductase are suggested for the microsomal and mitochond~al enzyme, respectively. DISCUSSION
The differentiation
of two enoyl-CoA
reductases
located
in microsomes
and
mitochondria of liver gives final proof for the existence of amitochond~al acetyl-CoAdependent chain elongation in this organ in addition toa malonyl-CoA-dependent pathway in microsomes. The distribution of the insoluble hexenoyl-CoA reductase among subcellular fractions (Table II) adds strong support to a participation of this enzyme in the microsomal malonyl-C.o~~-dependent chain elongation, involved in the synthesis of polyunsaturated fatty acids. The chain length specifity of this enzyme, showing a maximum with hexenoyl-CoA is not contradictory to such a role, since it is Biochim. Biophys. Acta,
280
(1972)
235-247
244
E.
0’
5.6
* 59
6.2
3 65
” 66
” 24
71
77
” 0.0
03
” 06
R.
PODACK,
M’.
SEUBEKT
6.9
PH
Fig. 7. pH-dependence of the microsomal and mitochondrial enoyl-CoA reductase. C,-reductase (;s--c) (0.53 mg protein) and C,,-reductase (O-O) (3.6 mg protein) both assayed with 0.05 ml\1 and 0,oT.j mill octenoyl-CoA, respectively. go mM potassium phosphate buffer, pH 5.6 to X.6 was employed. For additional components of assay mixtures see Material and Methods.
” c4 8. Chain length specificities of microsomal and mitochondrial enoyl-CoA reductase. Microsoma1 enoyl-Co-k-reductase(o-0) (from Seubert et ~1.~) and mitochondrial (~-‘--i’) (2.1 mg acetone powder extract) enoyl-CoA reductase. Composition of the reaction mixture for the mitochondrial enoyl-CoA reductase was: go mM potassium phosphate buffer, pH 7. I ; o. I mM enoyl-CoA derivatives shorter than dodecenoyl-CoA; 0.05 mM dodecenoyl-CoA and hcxadecenoyl-CoA. I’or addtional components of assay mixtures see Material and Methods. Fig.
most likely the result of an inhibitory effect, increasing with the chain length of the substrate as also reported for the saturated CoA-derivativesa. This interpretation is also in accord with an activation of the enzyme, observed with increasing desaturation of the acyl residue 22+r. Studies are in progress to give an answer to this question. A role of hexenoyl-CoA reductase in the microsomal malonyl-CoA-dependent chain elongation is also evident from a comparison of the pyridine nucleotide specifity of hexenoyl-Coil reductase and microsomal chain elongation reported by various authors. As summarized in Table IV, NADH can substitute for NADPH in the microsomal process. With respect to mitochondrial acetyl-CoA-dependent chain elongation, however, great discrepances concerning the requirement of NADH and NADPH are ~iocAim. Biophys.
Acta.
280
(1972)135-247
IV
NADH
* ** *** i
--
___.
__
.--.
NA DPH
MICROSOMAL
AND
_l_-~
36.5 15 40 44.5 40 30 24 -
28.5
--
NA DPti-
-
80
and -tetraenoic
-
18
29
53 100
acid.
-
II
3.’ 0 -_
-
57
73
ELONC;ATION
nucleotide
NO
CHAIN
100
__.
NA DH
MITOCHONI)RIAL
Substrate: endogcnous fatty acids. Decanoyl-Co-4 as primer. l’almitoyl-CoA as primer. Incorporation of y-[l%]linoleic acid in C,,-trienoic
____--.
Colli et al.%* 100 Colli et aZ.%** 94 Colli et a!.%*** 100 Quagliariello et LzZ.~% 100 WakillD 100 Barron12 IO0 Boone and WakiF IO0 Podack2j 100 Donaldson et a1.38 Guchait et al.“6 Stoffel and Ach*‘,f Aeberhard and Menkes 29 -Landriscina et aLz4 -
NADH+
ox
iVlitochondrial chain elongation relative a~t~~it9 (9;;) -. ..___-
AK~L) NADPH
.____I~.
RESPECTIVELY ..-
A &hors
CoA,
EPFECTSOF
TABLE ACIDS
xy
[~~C]MALOSYL-COX
9 IO0 100 85
IO0 ICI0
-
ZOO
95
100
100 100
-
-
-
___---
-
54
-
30
9.5 -
-
No nucleotide
[K]ACETYL-
13.5
-_
AK;D
93
92 12.5
7.5 100
-
-
-
Microsonaal chain elongation activily (%) _-_______ .-.-NA DPH NADH NA DH + NA DPH _.-___
relative
OFFATTY
(Table IV). A possible participation of a transhydrogenase has been excluded by Colli et aZ.ll. The discrepances plained by a differing degree of contamination by microsomal
evident
detergents
after treatment witlt can thus only he exenoyl-CoA reductase
activity in the various mitochondrial preparations employed. Only in fractions obtained by zonal centrifugation (Fractions 12-14, Fig. 3) or with the solubilized system, could we show an almost absolute requirement for NADPH in chain elongation. This result should be expected on the basis of the SADPH specificity of mitochondrial enoyl-CoA reductase. The low activity with XADPH alone is readily explained by the NADH specifity of P-113.‘drox~acyl-CoA dehydrogenase in liver4’. Incorpnration in the absence of NADPH may be due to chain elongation up to the stage of the a,p-unsaturated acyl-CoA. Different chain elongation systems in liver suggest different physiological roles for each system. As will be shown in a forthcoming communication, a participation of the microsomal enoyl-CoA reductase in the synthesis of polyunsaturated fatty acids is evident. With respect to the physiological significance of the mitochondrial system from liver, the authors prefer a possible role in hydrogen transfer from NADPH to the respiratory chain. As an alternate role, the transport of hydrogen from the cytosol to the mitochondrial compartment and conservation of energy, respectively, has to system from be considered, as suggested by Whereat d aE.43-4Bfor the mitochondrial heart muscle. Experiments are in progress to differentiate among these possibilities. ACKNOWLEDGEMENTS
This investigation was supported by the Deutsche Forschungsgemeinschaft. The Stiftung Volkswagenwerk provided us the scientific equipment. The excellent technical assistance of Sabine Buss is gratefully acknowledged.
9 IO I1 12 13 14 I5 I6 77 18 19 20 21
Larl~don, G. R. (195.5) J. .4?J'Z.~h'm. SOC. 77, jIg0 Seubert, XV., Greull, G. and Lynen, F. (1957) Angrw. Chum. 69, 359 Sellbert, W., Lamberts, I., Kramer, R. and Ohlp, B. (1968) Biochim. Biophys. ,4cta 164, 498 Lyncn, li. and Decker, K. (1957) EYgrb. f’hysiol. 49, 327 Stern, J. K., in Colowick, S. I’. and Kaplarl, N. 0. Methods in ITnzymology, Vol. T, :lcademic l’ress, New lTork, 1955, p. j-j9 C,rane, F. I,., pllii, S., Hauge, J. J., Green, D. 12. and Beinert, H. (1956) j. BioE. Chmzz. 118, 701 Crane, F. I,. and Beinert, H. (1956) J. Bid. Chrm. 2x8, 717 Slater, E.C., Colpa-Boonstra, J. P. and Links, 1. in Wolstcnholmc, G. E. 1%‘. and O’Connor, C. M., Ciba Foundations Symposizrm, Qztinows in Elrctvon Transport, Little Brown, Boston, 1961, p. 346 Hauge, G. (1956) .I. ATVZ.Chp~. SOC. 78, jZ60 Wakil, S. J. (1961) J. Lipid HFS. 2, I Christ, E. J. and Hiilsmann, W. C. (1962) Biochim. Riophys. Acta 60, 72 Barron, 1~. J. (1966) Biochirn. Biophys. Acta 116, 42.5 Christ, B. J. c’. J. (1968) Biochzm. Uiophys. Acta 152, 50 Colli, W., Hinkle, P. C. and Pullman, M. E., (1969) J. Viol. Chcwz. 214, 6432 Dahlcn, J, V. and Porter, J. 1%‘. (1968) Arch. Biochmz. Biofdzys. 127, 207 Quagliariello, E., Landriscina, C. and Coratelli, P. (1968) Biochirn. Riophys. Acta 161, IL Whereat, A. F. in Paoletti, K. and Kritchevsky, D. Adwanms in Lipid Research, Vol. 9, Academic Press, Sew York and London, 1971, p. 119 \Vit-I’eetcrs, E. M., Scholte, H. R. and Elenbaas, H. L. (1970) Bzochim. Biophys. Acta 210, 360 Nandekar, A. K. N. and Kumar, S. (1969) Arch. Biochmz. Riophys. 134, 563 Lin, c. \1. and Kumar, S. (1972) J. Viol. Chenz. 247, 604 Stoffel, W. and Schiitte, E., Lipoide, 16 Collog. deu GeseElschaft fiiv Physiologische Chemi~, Mosbachll?adrti 1965, Springer Verlag, Berlin, 1966, p. 64
Bzochim.
Hioph.ys.
-lcta,
280 (1971)
235.-247
MALONYL-CoA
22
23 24 25 26 27 2x 29 30 31 32 33 34 35 36 37 38 39 40 41 42
INDEPENDENT
FATTY
ACID SYNTHESIS
247
Nugteren, D. H. (1965) Biochim. Biophys. Acta 106, 280 Aeberhard, E. and Menkes, J. H. (1968) J. Biol. Chem. 243, 3834 Landriscina, C., Gnoni, G. V. and Quagliariello, E. (1970) Biochim. Biophys. Acta 202, 405 Podack, E. R., Doctoral Thesis, Medical Faculty Gijttingen 1971 Lynen, F. (1967) Biochem. J, 102, 3S1 Harlan, W. R. and Wakil, S. J. (1963) J. Biol. Chem. 238, 3216 Lynen, F. and Wieland, O., in Colowick, S. P. and Kaplan, N. O., Methods in Enzymology, Vol. I, Academic Press, New York, 1955, 1,. 566 Buckel, W. and Eggerer, H. (1965) B&hem. Z., 343, 29 Lowry, 0. H., Rosebrough, N. J.. Farr, A. L. and Randall, R. J. (1951) J. Biol. Chem. 193, 265 Layne, E. in Colowick, S. P. and Kaplan, N. O., Methods in Enzymology, Vol. 3, Academic Press, New York, 1957, p. 447 Hogeboom, G. H. and Schneider, %‘. C. (1953) J. Biol. Chem. 204, 233 Appelmans, F. and de Duve, C. (1955) B&him. J. j9, 433 van Tol, A. (1970) Biochim. Biophys. Arta 219, 227 der cnzymatischen Baganski, E. S., Foa, I’. P. and Zak, B. in Bergmeyer, H. U., Xethoden Analyse, Vol. I, Verlag Chemie, Weinheim, 1970, p. 839 de Duve, C., Pressmann, C., Gianetto, K., Wattiaux, R. and .\ppelmans, F. (X955) B&hem. J. 60, 604 I’oole, B., Higashi, T. and de Duve, C. (1970) J. Cell. BioZ. 45, 408 Harlan, W. R. and Wakil, S. J. (1962) Biochem. Biophys. Rrs. Commuw. S, 131 Donaldson, W. E., Wit-Peeters, E. M. and Scholte, H. R. (1970) B&him. Biophys. 4cta 202, 35 Howard, C. F. (1968) B&him. Biophys. Acta 164, 448 Stoffel, W. and Ach, K. L. (1964) Hoppe Seyler’s Z. Physiol. Chem. 337, 123 Decker, K. in Pummerer-Erlangen, R., Die aktiviertr Essigstiure, Ferdinand Enke Verlag,
Stuttgart,
1959, p, 229
43 Whereat, A. F., Hull, F. E., Orishimo, 31. W. and Rabinowitz, J. L. (1967) J. Biol. Chem. 242, 40’3 4-l Hull, F. E. and Whereat, A. F. (1967) J, Biol. Chem. 242, 4023 45 Whereat, A. F., Orishimo, M. W., Nelson, J. and Phillips, S. J. (1969) J. Biol. Chem. 244, 6498 Biophys 117, 541 46 Guchhait, R. B., Putz, G. R. and Porter, J. W. (1966) Arch. B&hem. 9, 1470 47 Boone, S. C. and Wakil, S. J. (1970) Bzochemisfvy
Biuchinz. Biophys.
Acta,
280 (1972) 235-247