The effect of bovine serum albumin on partial reactions of palmitoyl-CoA chain elongation by rat liver microsomes

44

Biochimica

et Biophysics

0 ElsPvier/North-Holland

Acta,

,531

Biomedical

(1978) Press

44-55

BBA 51238

THE EFFECT OF BOVINE SERUM ALBUMIN ON PARTIAL REACTIONS OF PALMITOYL-boa CHAIN ELONGATION BY RAT LIVER MI~ROSOMES

JOHN T. BERNERT,

Jr. and HOWARD

SPRECHER

*

~~par~~z~lz# o~~hys~ological Cfternisfr.y, College o~~~e~ici~e, 10th Avenue, Columbus, Ohio 43.220 (U.S.A.j (Received

February

28th,

Ohio State

~~zi~~~rsiiy, 333 Iti.

1978)

Summary In the absence of albumin, u/s curves for both condensation and overall chain elongation demonstrated that the specific activity for overall chain elongation was 3.7 times that of condensation. When the molar ratio of palmitoyl-CoA to albumin was greater than 2 : 1, the specific activity of chain elongation exceeded that of condensation. At these low albumin concentrations, in the absence of NADPH, the ~-ketostearoyl-CoA was converted back to palmitate. This cleavage reaction is inhibited by albumin in a concentrationdependent manner. When the palmitoyl-CoA to albumin molar ratio was less than 2 : 1, the specific activity for condensation exceeded that for overall chain elongation and some /3-ketostearate was shown to accumulate under chain elongation conditions. The specific activity for dehydration of /3-hydroxystearoyl-CoA was maximal when the acyl-CoA to albumin molar ratio was between 10 : 1 and 4 : 1 but the rate of this reaction was not markedly influenced by variations in albumin concentration. The specific activity for the NADPH-dependent reduction of Z-trans-octadecenoyl-CoA was 18 nmol . min-’ 1 mg-’ in the absence of albumin and increased to a maximum of 112 when the substrate to albumin molar ratio was 2 : 1. At higher albumin concentrations the reductase reaction was inhibited. Conversely, the specific activity for the reverse dehydrase was maximal at low albumin concentrations and the rate of this reaction declined as the albumin concentration increased. Our results demonstrate that albumin not only alleviates a substrate induced inhibition but also regulates the metabolic fate of 2truns-octadecenoyl-CoA and in this regard may possibly substitute for acyl-CoA binding proteins.

* To

whom

all correspondence

should

be addressed.

45

Introduction

In 1965, Nugteren ]I] demonstrated that the microsomal chain elongation of long chain acyl-CoA derivatives proceeded according to the following reaction sequence: 0 II R-C-SCoA

+ malonyl-CoA

R_-CT=?--C-S&A

NAD3

0 II 7 --f R-C-CH2-C-SCoA

0 ?” II NADpf! R-CH-CH2-C-SCoA

.-f

R-CH2-CH2-C-SCoA

H

Recently we demonstrated that the rates of both the condensation and CY$enoyl-CoA reductase reactions were elevated when albumin was included in the incubations in a 2 : 1 molar ratio (acyl-CoA : albumin) [Z J. Conversely, the rate of the dehydrase reaction was the same whether albumin was deleted from the incubation or included in a molar ratio of 2 : 1. In our previous investigation no systematic studies were carried out to further define how these reaction rates are influenced by varying the albumin concentration. Consequently, we have undertaken in this study to examine in greater detail how individual reaction rates in the conversion of palmitate to stearate are modified by varying the albumin concentration in the incubation. Experimental

procedures

Materials. CoA(SH), NADPH and bovine serum albumin containing less than 0.005% free fatty acids were purchased from Sigma Chemical Co., St. Louis, MO. Malonyl-CoA was obtained from P-L Biochemicals, Milwaukee, Wise. The fat-free diet was a product of ICN-Pharmaceuticals, Chagrin Falls, Ohio. Palmitic acid, methyl palmitate and 2heptadecanone were obtained from Applied Science Laboratories, State College, Penn. [ l-‘4C]Palmitic acid was obtained from New England Nuclear, Boston, Mass. ~-[~e-3-‘4C]Ketostearate, DL-fl[ 3-14C]hydroxystearic acid and 2-truns-[3-‘4C]octadecenoic acid were synthesized from [ 1-14C Jpalmitic acid using the procedures described by Stoffel and Pruss [ 3 3. As previously described [ 21, a number of modifications of existing procedures were employed to make the CoA derivatives of [ 1-14C Jpalmitic acid, fl- [ 3-14C] ketostearic acid, DL-fi-[ 3-14C] hydroxystearic acid and 2-trans[ 3-14C] octadecenoic acid. Microsome preparation. Male weanling Sprague-Dawley rats were maintained on the fat-free diet for at least 6 weeks prior to use. Liver microsomes were prepared as previously described [4] and lyophilized. Protein was measured by the method of Lowry et al. [5] using albumin as standard. fncubations and analysis. Condensation reactions with palmitoyl-CoA and malonyl-CoA were performed for 2.5 mm in serum bottles in a total volume of 1.5 ml at 37°C in a metabolic shaker. Unless otherwise noted each vial contained 150 pmol potassium phosphate buffer (pH 7.4), 0.3 ymol malonyl-CoA, 2.5 mg microsomal protein and 100 nmol [ l-‘4C]palmitoyl-CoA. Overall chain

46

elongation reactions were measured under identical conditions except that each incubation also contained 2 pmol NADPH, and the serum vials were stoppered and flushed with Nz for 10 min prior to initiating the incubatioI1 in order to retard the aerobic desaturation of both the substrate and product. For dehydrase reactions each vial contained 150 pm01 phosphate buffer (pH 7.4), 180 nmol DL-/3-[3-‘4C]hydroxystearoyl-CoA and 0.625 mg protein. The incubations were carried out in a total volume of 1.5 ml at 37°C for 1.5 min. Reductase reactions were conducted with 136 nmol 2-trans-[3-14C]octadecenoyl-CoA. The protein concentration was 1.25 mg, 2 pmol NADPH were included and the incubation period was 0.5 min. The vials were flushed with N2 for 10 min prior to initiation of the reaction. The amount of albumin included in the incubations is provided in the figure legends. Concentrations were calculated assuming a molecular weight of 66 000 for the albumin. All incubations were initiated by addition of microsomes and terminated by adding 0.25 ml 4 M NaOH/5 ml CH30H. The serum bottles were replaced in the metabolic shaker for 1 h at 37°C to saponify the lipids. A Folch extract was then formed by addition of 0.25 ml 9 M HCl/lO ml CHClJl ml HzO. The top layer was discarded and the bottom layer taken to dryness under N,. For condensation reactions, the residue was taken up in CHC13/CH30H (2 : 1, v/v) and fractionated by thin-layer chromatography on silica gel G using light petroleum (boiling range 63-75”C)/diethyl ether/acetic acid (75 : 25 : 1, v/v). Internal standards of methyl palmitate, P-ketostearic acid, 2_heptadecanone, methyl-pketostearate and palmitic acid were added to each sample prior to thin-layer chromatography. Those standards were included since we have previously shown that even though the illcL~bations are subjected to saponificatio~~ conditions, small amounts of methyl palmitate and methyl /3-ketostearate may be recovered [2]. 2-Heptadecanone results from the decarboxylation of /3-ketostearic acid. Components were localized by spraying with a 0.2% solution of 2’, 7’-dichlorofluorescein in ethanol. The fractions were scraped into scintillation vials and 10 ml of a dioxane-based cocktail were added [6]. Radioactivity was measured in a Model 3380 Packard liquid scintillation spectrometer. In assays of overall chain elongation, dehydrase and reductase reactions, tile lipids from the Folch extract were converted to methyl esters by refluxing with 5% anhydrous HCl in methanol. Overall chain elongation was measured by fractionation of the methyl esters by radioactive gas-liquid chromatography as previously described [ 71. For dehydrase assays the methyl esters were applied to silica gel G plates and developed with light petroleum/diethyl ether/acetic acid (70 : 30 : 1, v/v). The hydroxy and non-hydroxy methyl esters were located by spraying with 2’,7’-dichlorofluorescein and scraped into scintillation vials for counting. When reductase reactions were assayed a combined thin-layer chromatography-gas-liquid chromato~aphy assay was used in order to measure the amount of /3-hydroxy acid produced by reversal of the dehydrase reaction, as well as the 01, P-saturated product. The methyl esters were first fractionated by Silica gel G thin-layer chromatography as outlined above except that only about 20% of the hydroxy and non-hydroxy methyl ester bands were scraped directly into scintillation vials. The remainder of the non-hydroxy methyl ester

band was eluted with diethyl ether and the methyl esters were fractionated by radioactive gas-liquid chromatography to separate methyl stearate from methyl 2-truns-octadecenoate. Carrier methyl-2-trans-octadecenoate was included as an internal standard. The rate of dehydrase reversal as well as the reductase reaction was then determined by calculating the percentage of radioactivity present in unreacted substrate, /3-hydroxy fatty acid and in the o(, P-saturated product. Results Previously, we demonstrated that when albumin is included in incubations with palmitoyl-CoA and malonyl-CoA, the rate of the condensation reaction is influenced in two ways. When the palmitoyl-CoA to albumin molar ratio was 2 : 1, a higher specific activity was obtained and the albumin alleviated a substrate induced inhibition which occurred when the palmitoyl-CoA concentration exceeded 20 E.IM [2]. We did not systematically examine how albumin influenced the rate of overall chain elongation however, in that all these reaction rates were measured with an acyl-CoA to albumin molar ratio of 2. The u/s curves in Fig. 1 for condensation and overall chain elongation were obtained in the absence of albumin. For both reactions there was a substrate induced inhibition when the concentration of palmitoyl-CoA exceed 20-30 PM. More importantly, the maximum specific activity of overall chain elongation was considerably greater than that of condensation. This is obviously physiologically impossible since an individual reaction cannot proceed more slowly than the overall metabolic process. In order to more precisely determine how albumin influences these two reac-

2.5 !3l E ‘; *

.

/\ 2.0

\

.-C E ;

. \

/

I.5

.

Y u 2 a

1.0

5 E c

05

.

.i

I

/-lo_ / 40

20 [I’*-C] Fig.

1.

absence

Kinetics of bovine

of

condensation serum

60

60

FY\LMlTOYL-CoA

albumin.

(0)

100

(pm1 and

overall

chain

elongation

(0)

of

[l-14C]palmitoyl-CoA

in

the

I 1.1

3:t

IO I

I I 32

2.64

3.96

5.26

6.60

mq ALBUMIN

Fig. 2. The effect of increasing concentrations of bovine serum albumin of the condensation (“) overall chain elongation (0) of palmitoyl-CoA. The concentration of palmitoyl-CoA was 65.9 PM.

and

tions we measured the rates of both condensation and chain elongation in the presence of increasing amounts of albumin. As shown in Fig. 2, and in accordance with our previous results 121, the rates of condensation and chain elongation were virtually identical when the palmitoyl-CoA to albumin molar ratio was 2 : 1. Conversely, at low albumin concentrations the apparent rate of condensation was less than that of chain elongation. In the condensation reaction fl-ketostearoyl-CoA is the end product, while in overall chain elongation the fl-ketostearoyl-CoA should be rapidly depleted as an intermediate substrate in the chain elongation process. The discrepancy in rates for chain elongation versus condensation could thus be explained if the condensation product which accumulated in the absence of NADPH was partially converted back to palmitoyl-CoA in a thiolytic reaction which in turn was influenced by the presence of albumin. This would be consistent with the observations of Seubert and Podack [ 81 that their microsomal preparations always contained /3-ketothiolase activity. In order to determine if albumin protected P-ketostearoyl-CoA from cleavage we measured the rate of conversion of /3-[3-‘4C]ketostearoyl-CoA to palmitic acid in the presence of increasing concentrations of albumin. As shown in Fig. 3, as the molar ratio of albumin to P-ketostearoyl-CoA increased, the conversion of fl-ketostearoyl-CoA to palmitic acid declined, confirming that albumin may protect fl-ketostearoyl-CoA from thiolytic activity during condensation assays. On the other hand, when overall chain elongation is measured in the presence of NADPH, the P-ketostearoyl-CoA appears to serve preferentially as a substrate for the chain elongation process thus reducing the rate of cleavage

49

IO .

7

a

0’

. %I

2.1

/

i:i AWL-CoA:

ALBUMIN

Fig, 3. Inhibition of the conversion of @-[3-‘4CJketostearoyl-CoA to pahnitic acid by albumin. Incubation vials contained 150 @mol potassium phosphate buffer (pH 7.4), 81.9 nmol P-[3-14CJketostearoy1CoA, 0.625 mg microsomal protein and albumin concentrations as indicated in a total volume of 1.5 ml. An incubation interval of 1.5 min was employed which was well within the linear range for the reaction in the absence of albumin. The reaction mixtures were assayed as described for the condensation reaction. and the fatty acid product was confirmed as being pahnitic acid by methylation and gas-liquid chromatography.

30 [3-“C]

60

90

150

120

p-KETOSTEAROYL-CoA

(@Ml

Fig. 4. Kinetics of the P-ketostearoyl-CoA reductase reaction analyzed at a constant acvl-CoA to albumin molar ratio of 2 : 1, and in the presence of 1.33 miM NADPH: stearic acid (0). palmitic acid (of. The reaction was analyzed in the same manner as the condensation reactions, except that part of the fatty acid band on thin-layer chromatography was eluted. methylated and fractionated by gas-liquid-cbromatography to separate palmitate and stearate.

50

back to palmitic acid even at low albumin concentrations. This suggestion is consistent with the results of Fig. 4 which demonstrate that at an acyl-CoA to albumin molar ratio of 2 : 1 and in the presence of NADPH, the conversion of /3-ketostearoyl-CoA to stearic acid was always much greater than its conversion back to palmitic acid. Thus inclusion of NADPH in the incubation results in the preferential removal of fl-ketostearoyl-CoA for continuation of the chain elongation process. When the palmitoyl-CoA to albumin molar ratio is less than 2 : 1 then, as shown in Fig. 2, the rate of condensation exceeds that of overall chain elongation. These findings suggest that one or more of the intermediates produced during chain elongation must accumulate under these conditions. To evaluate this possibility a chain elongation reaction was run with a palmitoyl-CoA to albumin molar ratio of 1.25 : 1. Following saponification, the reaction mixture was analyzed by thin-layer chromatography and small amounts of radioactive components were recovered which had RF values identical to those of /3-ketostearic acid, 2-heptadecanone and methyl-fl-ketostearate. In order to more completely characterize these radioactive components, ten incubations were analyzed under the above conditions for chain elongation. The lipid extracts were pooled, taken to dryness, dissolved in 4% KOH in 90% ethanol and refluxed for 3 h to convert the fl-ketostearic acid and methyl-(3-ketostearate to 2-heptadecanone. The extract was acidified, extracted and analyzed by thinlayer chromatography using the conditions normally employed for assaying the condensation reaction. Portions of the silica gel were transferred to scintillation vials and counted. As shown in Fig. 5 the major radioactive component had an RF identical to that of the free fatty acid standard, while a minor radioactive with 2-iieptacomponent was produced which had an RF value identical decanone. The remainder of each of these two radioactive zones was eluted with ether and the radioactive component which had an RF value identical with 2-heptadecanone was shown to possess a retention time identical to that of carrier 2-heptadecanone when analyzed by radioactive gas-liquid chromatography. The radioactive components which had an RF value identical to that of a free fatty acid were refluxed with 5% anhydrous HCl in methanol and analyzed by radioactive gas-liquid chromatography. A summary of the radioactive compounds produced during the incubation, as fractionated by the combined thin-layer chromatography-gas-liquid chromatography assay, is presented in Table I. Although these incubations were carried out after flushing the vials with N,, total anaerobic conditions were never obtained. The radioactive palmitoleate is formed by desaturation of palmitoyl-CoA while the 18 : 1 probably is a mixture of 11-18 : 1 formed by chain elongation of palmitoleate as well as oleate which is produced by desaturation of stearate. In calculating overall rates of chain elongation the radioactive palmitoleate was always included as substrate and the radioactive 18 : 1 as product. The calculated specific activity for chain elongation, i.e. nmol stearate plus 18 : 1, is thus 3.3 nmol * mm-’ . mg-i. Likewise, the specific activity for condensation, i.e. nmol stearate plus 18 : 1 plus P-ketostearate, is 3.9 nmol . min-’ . mg-‘. The corresponding specific activities from Fig. 2 are 3.3 and 4.1. These latter values were obtained when both chain elongation and condensation were measured by using the conventional assay procedures.

51

oR’G’N 0, o2 Fig.

5.

Thin-layer

elongation

chromatogram

reaction

described

in the

heptadecanone

p4

(4)

of

conducted

text.

at

Standards

and

0,

methyl

the

an

radioactive

acyl-CoA

are @ketostearic palmitate

products

to

albumin

acid

(1).

recovered ratio

free

of

fatty

following

:

1.25 acid

(2).

1.

a palmitoyl-CoA

Conditions

methyl

of

chain

analysis

0-ketostearate

(3).

are 2-

(5).

These results suggest that at high albumin concentrations the rate of condensation exceeds that of overall chain elongation because not all of the P-ketostearate is used as a substrate for subsequent reactions. This result would be expected if /3-ketostearoyl-CoA was hydrolyzed to free /3-ketostearic acid and thus rendered inactive as a substrate. Additionally or alternatively, some of the

TABLE

I

RADIOACTIVE WITH

RAT

COMPONENTS LIVER

: ALBUMIN

MITOYL-CoA component

16

: 0

16

:

18: 18

1

:

OF

WHEN

THE 1.25

PRESENCE

:

Radioactivity

Amount (nmol)

89

65.6

745

: 0 *

:

Unidentified * Assayed * * Represents

1 **

[1-‘4CIPALMITOYL-CoA OF

MALONYL-CoA

WAS AND

1.

(dpm)

23

1

2-trans-18

RATIO

IN

6 951

0

(3-k&o-18

PRODUCED

MICROSOMES

5.0

769

17.4

4 111

3.0

5 467

4.0

<150


5 141

as 2-heptadecanone. counts

obtained

from

the

origin

to the

free

fatty

acid

band

on TLC

NADPH

INCUBATED AT

A PAL-

.

01 IO I

41

21 ACYL-COP,

L

. 2

I5

~ 4

1I

I

ALBUMIN . 6

6

.

I

10

mq ALBUMIN

Fig. 6. The effect of increasing concentrations of albumin tion. Conditions of analysis were as stated in Methods.

on the il-hydroxystearoyl-CoA

dehydrase

reac-

P-ketostearoyl-CoA may have so tightly bound to albumin that it did not serve as a substrate for reduction to the fl-hydroxystearoyl-CoA. Our results suggest that the only intermediate which accumulated was fl-ketostearate. As noted previously, the specific activity for condensation (Table I) was the same in this analysis as when condensation was measured in the conventional manner with a palmitoyl-CoA to albumin molar ratio of 1.25 : 1 (Fig. 2). Since condensation is rate limiting [ 21, this type of agreement could only be obtained if there was no significant accumulation of either /3-hydroxystearate or 2-truns-octadecenoate. Although we did not attempt to measure the levels of P-hydroxystearate when overall chain elongation was assayed, we were able to demonstrate that no 2-truns-octadecenoate accumulated (Table I). The curve in Fig. 6 was obtained when we measured the effect that increasing concentrations of albumin have on the rate of dehydration of P-hydroxystearoyl-CoA. As previously reported, the rate of the dehydrase reaction is the same in the absence of albumin as when the substrate to albumin molar ratio is 2 : 1. Albumin does exert a moderate influence on this reaction however, since at low albumin concentrations there is a small elevation in reaction rate, and when the substrate to albumin molar ratio exceeds 4 the reaction rate progressively declines. As shown in Fig. 7, albumin markedly influenced the rate of the cu,jSenoylCoA reductase reaction. In the absence of albumin the specific activity of the reductase reaction was only 18. As the amount of albumin in the incubation was increased the specific activity of the reductase reaction rose to attain a maximum value of 112 which coincides with a substrate to albumin molar ratio of 2 : 1. At albumin concentrations above this level the rate of the reductase reaction was again inhibited. Conversely, the rate of reversal for the dehydrase reaction was maximum at high substrate to albumin ratios and steadily declined as the amount of albumin in the incubation was increased.

53

0.

%I

2:1 ACYL-CoA

2

4

1.5:1

I:1

: ALBUMIN

6

8

mg ALBUMIN

Fig. 7. The effect of increasing concentrations of albumin on both the reduction and hydration of Z-tram[3-14C]octadecenoyl-CoA in the presence of 1.33 mM NADPH: steak acid (,‘), fl-hydroxystearic acid(=).

Discussion

The results obtained in this study suggest that albumin influences the rates of partial reactions in the microsomal chain elongation of palmitate to stearate in different ways. In the absence of albumin, the maximum specific activity for condensation and overall chain elongation occurs when the palmitoyl-CoA concentration is 20-30 FM. Previously, we demonstrated that in the absence of albumin, the cu,P-enoyl-CoA reductase reaction proceeded at a maximum rate when the concentration of 2-trans-octadecenoyl-CoA was 20 PM [ 21. All three reactions were inhibited when higher levels of substrate were employed, and in each case this probably reflected the formation of micelles by the substrate. Gatt and his colleagues [9,10] have noted that a number of enzymes acting on amphipathic substrates use only the monomeric form. When the substrate concentration exceeds the critical micelle concentration the micelles may inhibit the enzyme, or alternatively they may bind to the enzyme but be acted upon at a slower rate than is the monomeric form. The critical micelle concentration of acyl-CoA derivatives is 2-10 FM [II]. Although inhibition of all three reactions first occurred at a substrate concentration well above this level, the studies of Lamb et al. [12] demonstrated that fatty acyl-CoA derivatives bind to microsomal proteins thus lowering the amount of acyl-CoA available in the bulk phase for micelle formation. Similarly, when albumin is included in the incubation it may bind the substrate and prevent or retard micelle formation.

In the condensation reaction albumin not only prevents a substrate induced inhibition, but in a concentration-dependent manner inhibits breakdown of pketostearoyl-CoA to palmitate. This effect of albumin may result through a direct binding of the substrate, or indirectly through an inhibition of deacylase activity. Studies of Jeffcoat et al. 1131 with liver microsomes and by Brophy and Vance [ 141 with brain microsomes have demonstrated that albumin inhibits the activity of thiolesterases acting on acyl-CoA derivatives. Although the CoASH required for thiolytic cleavage of P-ketostearoyl-CoA would be produced when l~almitoyl-CoA condenses with malonyl-CoA, additional CoASH would be made available by hydrolysis of palmitoyl-CoA. Inhibition of microsomal thiolesterases by albumin would thus decrease the amount of CoASH made available as a substrate for fl-ketothiolase. We previously reported that the specific activity of the dehydrase reaction was unaffected when the molar ratio of P-hydroxystearoyl-CoA to albumin was 2 : 1 [Z]. A more thorough analysis of the effect of albumin demonstrated that the specific activity of this reaction was maximum when the substrate to albumin molar ratio was 10 : 1. The reason for this increase in rate is not apparent, however, it is unlikely that it can be attributed to the alleviation of micelle inhibition. Although the critical micelle concentration of j3-hydroxyacyl-CoA derivatives is not known, a u/s curve measured in the absence of albumin did not show any inhibition even when the substrate concentration was 270 PM. There was a progressive decline in specific activity when the phydroxystearoyl-CoA to albumin molar ratio was less than 6 : 1. At these higher albumin concentrations the dehydrase and albumin may compete for the substrate. If the albumin-bound substrate is less accessible to the enzyme, then one would expect to observe a decline in specific activity. The roles played by albumin in mediating the rate of the a$-enoyl-CoA reductase reaction may well be more complex than for the other reactions. As already pointed out, albumin probably prevents a micelle-induced inhibition occurring when the concentration of 2-trans-octadecenoyl-CoA exceeds 20---30 @‘I [2]. In addition, the concentration of albumin in the incubations regulates the metabolic fate of Z-beaks-o~tade~enoyl-CoA. In the absence of albumin or when the molar ratio of 2-Pans-octadecenoyl-CoA to albumin is greater than 3 : 1, the substrate is preferentially converted back to phydroxystearate. As the molar ratio of 2-trans-octadecenoyl-CoA to albumin decreases, the specific activity of the reductase increases until a maximum is reached when the molar ratio of substrate to albumin is between 2 : 1 and 1.5 : 1. At these molar ratios the specific activity of the reverse dehydrase reaction is minimal. When the molar ratio of 2-Pans-octadecenoyl-CoA to albumin is 1 : 1, the specific activity of the reductase declines and there is no increase in rate for the reverse dehydrase reaction. At high albumin concentrations it would appear that the substrate binds with albumin in such a way that the resulting complex is less accessible to either the reductase or the reverse dehydrase. Our results also suggest that albumin may possibly substitute for in vivo acyl-CoA carrier proteins. Kawashima et al. [15] have shown that stearoyl-CoA inhibits the overall chain elongation of palmitoyl-CoA to stearate. It would appear that the most effective type of inhibition would be a feed-back interaction in which stearoyl-CoA inhibits the rate limiting step, i.e. condensation. In

5.5

addition to those roles already discussed for albumin our results do not preclude the possibility that albumin substitutes for an in vivo acyl-CoA carrier protein and prevents inhibition by binding stearoyl-CoA. Acknowledgement This study was supported by Grant Number AM20387 Institutes of Health, United States Public Health Service.

from the National

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3

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4

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5

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P.J.

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Cleland,

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Sprecher,

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