A microsomal lipoamide dehydrogenase in the muscle of Ascaris lumbricoides var. suum

Comp. Biochem. Physiol., Vol. 66B, pp. 499 to 504

0305-0491/80/0801-0499502.00/0

© Pergamon Press Lid 1980. Printed in Great Britain

A M I C R O S O M A L L I P O A M I D E D E H Y D R O G E N A S E IN THE MUSCLE O F ASCARIS LUMBRICOIDES VAR. S U U M SADAYUKI MATUDA and FuJIO OBO Department of Biochemistry, Faculty of Medicine, Kagoshima University, Usuki, Kagoshima 890, Japan

(Received 20 November 1979) Abstract--1. Subceilular fractionation of Ascaris muscle was carried out by differential centrifugation. A large amount of lipoamide dehydrogenase was found exclusively in microsomal fraction which had no activities of oxidative decarboxylation of ct-ketoacids or glycine. 2. The lipoamide dehydrogenase was easily solubilized and purified to homogeneity from Ascaris muscle microsomes with trypsin, but was not solubilized with 1% Triton X-100. 3. The purified enzyme had a molecular weight of about 97,000 consisting of two subunits whose molecular weight was about 53,000. One molecular of enzyme contained 2 bound molecules of FAD. 4. The amino acid analysis revealed that the amino acid composition of the Asearis lipoamide dehydrogenase was similar to the lipoamide dehydrogenase from other sources, but tryptophan lacked in it.

INTRODUCTION The parasitic roundworm, Ascaris lumbricoides var. suum resides in the small intestine of swine where the oxygen tension is low. It has been reported that this nematode has a faintly functional tricarboxylic acid cycle in muscle mitochondria where a specific type of anaerobic energy metabolism occurs (Saz & Weil, 1960; Saz & Vidrine, 1957; Oya et al., 1965). As reported in previous papers, Matuda (1979) has suggested that Ascaris muscle microsomes possess an electron-transport system containing at least a b-type cytochrome and a NADH-ferricyanide reductase. In the course of the study on this electron-transport system, we have found in Ascaris muscle microsomes a large amount of a lipoamide dehydrogenase ( N A D H :lipoamide oxidoreductase EC 1.6.4.3) which is easily obtained in a pure state by solubilization with proteinase, such as trypsin and alkaline protease. In general the lipoamide dehydrogenase is known as a component of ~t-ketoacid dehydrogenase complexes, such as pyruvate and ~t-ketoglutarate dehydrogenases in mammalian, plant and bacteria (Williams, 1976), and is also known to function physiologically in the oxidative decarboxylation of glycine as reported in the anaerobic bacterium Peptococcus #lycinophilus (Robinson et al., 1973). This paper describes the exclusive location of the lipoamide dehydrogenase without definite functions mentioned above, in microsomes among the subcellular fractions of Ascaris muscle. A report is also made on the purification and some properties of the enzyme. MATERIALS AND METHODS

Ascaris muscle fractionation Adults of Ascaris suum were obtained at a public slaughterhouse. The muscle was dissected free of reproductive and digestive organs, washed and cut into pieces with a razor, then washed several times with cold saline solution. The tissues were homogenized in 9 vol of 0.25 M sucrose 499

with a Potter-Elvehjem-type homogenizer equipped with a Teflon pestle. The homogenate was centrifuged at 700 g for 7 min to remove unbroken cells, cell debris and the nuclear fraction. The supernatant was carefully collected by decantation and centrifuged at 10,000 O for 25 min. The resulting supernatant was again centrifuged at 20,000 g for 25 min. The residues from the two centrifugations were combined and washed several times with 0.25 M sucrose solution to remove glycogen and used as "P-I" (mitochondrial) fraction. The post-mitochondrial supernatant was centrifuged at 105,000 g for 60 min. The sediment obtained was designated as as "P-2" (microsomal) fraction. P-2 fraction was washed twice with 1.15% KCI solution. The final supernatant was named fraction "S".

Assay of enzyme activities The activities of NADH- and NADPH-cytochrome c reduetase, and that of succinate-cytochrome c reductase were measured by following the increase in absorbance at 550 nm, according to the methods of Imai et al. (1966), and Stotz (1955), respectively. NADH-ferricyanide and NADH-2,6-diehlorophenolindophenol reductase activities were measured by the method of Takesue & Omura (1970). Lipoamide dehydrogenase activity was measured using lipoic acid as substrate by following the decrease in absorbance at 340 nm, taking a miUimolar extinction coefficient of NADH as 6.2 (Horecker, 1948). Unless stated otherwise, the lipoamide dehydrogenase activity was assayed at pH 5.8. The absorbance change was also followed in the absence of lipoic acid to correct the spontaneous oxidation and breakdown of NADH at this pH. Ferricyanide-linked ct-ketoglutarate and pyruvate dehydrogenase activities were measured as described by Massey (1960) and Reed (1966). Analysis of flavin The enzyme was boiled for 2min and then quickly cooled. After the denatured enzyme was precipitated by centrifugation, aliquot samples of the supernatant were spotted on a cellulose sheet. 1-Butanol:acetic acid:water (12:3:5) or 5% Na2HPO4 was used as a solvent. Chromatography was performed in the dark, and the spots were visualized under ultraviolet lamp. Measurement of fluorescence The fluorescence of the detached flavin contained in the

500

SADAYUKI MATUDAand FuJlO OBO

yellowish supernatant described above was measured in quartz cuvettes (3 ml, 1-cm light path) at room temperature m a Hitachi 203 fluorescence spectrophot0meter.

Table 1. Enzymatic activities of subcellular fractions of Ascaris muscle

Polyacrylamide eel electrophoresis

P-I

P-2

S

The polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) was carried out by the method of Weber & Osborn (1969). The proteins used as standards were gamma-globulin (mol. wtl60,000), bovine serum albumin (mol. wt 67,000), ovalbumin (mol. wt 45,000), trypsin (mol. wt 23,000) and myoglobin (mol. wt 17,000).

Succinate-cytochrome c reductase

33

7

*

NADH-cytochrome c reductase

25

127

6

Amino acid analysis

~-ketoglutarate dehydrogenase (containing 0.1~ Triton X-100)

The protein was hydrolyzed for 22hr at ll0°C in 6 N HCI. The hydrolysates were analyzed with a Hitachi 835 high-speed amino acid analyzer. The content of tryptophan was estimated by the spectrophotometric method of Goodwin & Morton (1946).

lmmunolooical experiments A rabbit was immunized with purified lipoamide dehydrogenase in the following way. One mg of the purified enzyme in 1.0 ml of 50 mM Tris-HCl buffer (pH 8.0) was mixed with an equal volume of Freund's complete adjuvant (Difco Co.) and was injected in the footpads of the animal. 3 and 4 weeks later 0.5 mg of the enzyme was injected into the same animal, respectively. One week after the last injection, blood was obtained from the ear vein of the immunized animal. ?-globulin fractions were prepared by 50% saturation with ammonium sulfate from the sera of the immunized and the control animals. Double immunodiffusion analysis on 1% agar gel was run.

Protein determination Protein was determined by the method of Lowry et al. (1951), using bovine serum albumin as a standard.

Recudents and chemicals Sephadex and DEAE-23 cellulose were obtained from Pharmacia Fine Chemicals, Uppsala, and Whatman Biochemicals, Ltd., respectively. Rotenone, cytochrome c (type III), NADPH (type I) and trypsin (type III) were purchased from Sigma Chemical Co., and NADH (grade II) was from Boehringer Mannheim GmbH. Triton X-100, lipoic acid, ~-ketoglutarate and pyruvate were purchased from Wako Pure Chemical Industries, Ltd (Japan). 2,6-Dichlorophenolindophenol was obtained from Merk. Other chemicals used were commercial products of reagent grade.

+ rotenonei" Lipoamide dehydrogenase

Pyruvate dehydrogenase

Distribution of lipoamide dehydrooenase amon9 the subcellular fractions of Ascaris muscle As shown in Table 1, a high specific activity of lipoamide dehydrogenase was found predominantly in P-2. The activities of pyruvate and a-ketoglutarate dehydrogenase were very low in each fraction, particularly in P-2. In the previous paper, Matuda (1979) reported that P-I nearly all consisted of vesicular components morphologically. The mitochondrial contamination in this P-2 fraction was calculated to be about 10% on the basis of the content of succinatecytrochrome c reductase used as an enzymatic marker of mitochondria. At the present study, the high specific activity of succinate--cytochrome c reductase was also found in P-I, but that of the lipoamide dehydrogenase was localized mainly in microsomes, showing that it was not mitochondrial origin.

107

*

60

800

I1

4

0

5.5

13

1

6.7

* Not determined. t 5/al of an ethanolic solution of rotenone (4 mg rotenone/ml) was used. Enzymatic activities were assayed in total vol of 2.5 ml as described in Materials and Methods and were expressed as nmol of acceptors reduced/mg protein/min.

Solubilization and purification of the lipoamide dehydrogenase from Ascaris muscle microsomes All the column chromatographys were carried out at 4°C in a cold room. Step I. Solubilization of the enzyme. As shown in Table 2, the lipoamide dehydrogenase was easily solubilized from Ascaris microsomes by the low concentration of trypsin. About 50% the lipoamide dehydrogenase was solubilized with 1% deoxycholate from Ascaris microsomes. However, Triton X-100, E D T A and sodium chloride (1 M) could not solubilize the lipoamide dehydrogenase from microsomes. This fact suggests that the lipoamide dehydrogenase are bound to microsomal membrane. In the following use the microsomes suspended in 5 0 m M Tris-HCl buffer (pH 8.0) were solubilized with 0.1% trypsin for 20 min at 37°C and centrifuged at 105,000g for 60rain.

Table 2. Solubilization of lipoamide dehydrogenase from Ascaris muscle microsomes by various reagents Solubilization (~) Sup Ppt

Additions RESULTS

21

Trypsin

0.005 (%) 0.05 (%)

98 99

3 1

Deoxycholate

0.1 (%) 1(%)

2.5 44

99 41

Triton X-100

0.1 (~) 1 (~o)

0 0.5

99 99

NaCI

1 (M)

1

99

EDTA

I (mM) 10 (mM)

9 0

99 99

Microsomes (7 mg of protein per ml) suspended in 50 mM Tris-HC1 buffer (pH 8.0) were treated with various additions for 30 min at 4°C, except for trypsin (30 min at 37°C). Then the suspensions were centrifuged at 105,000g for 60 min. The lipoamide dehydrogenase activities in the precipitate and supernatant were assayed as described in Materials and Methods. Values are percentages of those for untreated microsomes.

501

Lipoamide dehydrogenase in Ascaris

E

1"5l

t-

O

O.Z~

1.o

--- O"1 e

o.s

8

,sina

1__ 2"S- Sb- 7"S

409-

00

I-2~o~ I-~4oo~ ~_

/~ 4~

o.ff

Fraction Number

F r a c t i o n Number Fig. 1. 1st Gel-filtration on a Sephadex G-200 column. Experimental procedures are described in the text. Fractions of 3.2 ml were collected. About 200 mg of protein were applied on a column. The enzyme activity is measured at pH 7.0. One unit is defined as an absorbance change of 0.1 per min.

Step 2. Fractionation with ammonium sulfate. To the mpernatant the solid ammonium sulfate was slowly added with constant stirring to 4 0 ~ saturation, followed centrifugation at 105,000 g for 20min, and the precipitate was discarded. The supernatant was brought up to 7 0 ~ saturation by adding solid ammonium sulfate. After stirring for 20 min, the suspension was centrifuged as above. The precipitate was dissolved in a minimum volume of 50raM Tris-HC1 buffer (pH 8.0). Step 3. Ist Gel-filtration on Sephadex G-200. The sample from step 2 was applied to a column of Sephadex G-200 (2.6 x 55cm) equilibrated with 50 m M Tris-HCl buffer. Elution was carried out with this buffer (Fig. 1). The fractions with the dehydrogenase activity were combined and concentrated on an Amicon PM 10 filter. Step 4. DEAE-23 cellulose column chromatography. Step 3 enzyme was applied to a DEAE-23 cellulose column (1 x 19cm) equilibrated with 50raM Tris-HC1 buffer (pH 8.0). The column was washed with the same buffer and the enzyme was eluted with a linear gradient formed from 100ml of 50ml Tris-HC1 buffer containing 500 mM NaC1. The fractions with the dehydrogenase activity were combined and concentrated.

Step 5. Rechromatography on a Sephadex G-200. The concentrated enzyme was applied to a column of

Fig. 2. 2nd Gel-filtration of DEAE-23 cellulose column eluate on a Sephadex G-200 column. Experimental procedures are described in the text. Fractions of 2.2 ml were collected. About 15 mg of protein were applied on a column. The enzyme activity is measured at pH 7.0. One unit is defined as an absorbance change of 0.1 per min. Sephadex G-200 (1.9 x 55cm) equilibrated with 50 mM Tris-HC1 buffer. Elution was carried out with the same buffer. The lipoamide dehydrogenase was eluted as a single peak, as shown in Fig. 2. The results of purification of the lipoamide dehydrogenase are summarized in Table 3. The lipoamide dehydrogenase was purified 30-fold over the microsomal fraction with a recovery of 18~o.

Properties of the purified lipoamide dehydrogenase Purity and molecular weight. As demonstrated in Fig. 3, SDS-polyacrylamide gel electrophoresis of the purified lipoamide dehydrogenase showed a single band. When the relative migration of the purified enzyme was compared to those of the molecular weight standards, the purified enzyme corresponded to the molecular weight of 53,000 (Fig. 4A). The molecular weight of the purified enzyme was also estimated by plotting log molecular weight vs elution volume according to the method of Andrews (1965). Molecular weight of 97,000 was found for the purified enzyme (Fig. 4B). These results suggest that the lipoamide dehydrogenase is composed of two subunits of identical molecular weight of about 53,000.

Optical absorption spectrum, the chromophore and fluorescence of the purified enzyme. Absorption maximum of the purified enzyme was observed at 455 nm with a shoulder at 430 and 480 nm. A broad absorption was observed from 360 to 370 nm. The chromophore detached by boiling the enzyme was found to be FAD using a paper chromatography. From the

Table 3. Summary of the purification of lipoamide dehydrogenase Steps Microsomes Supernatant

40-70~o (NH,,)2SO,, Sephadex G-200 DEAE-23 Cellulose Sephadex G-200

Protein (mg)

Specific activity (Units/mg)

Total activity (Units)

Yield (~o)

780 482 182 32 15 5

53* 73 185 716 1070 1500

41,340 35,186 33,670 22,912 16,050 7500

100 85 81 55 39 18

* Rotenone (10- s M) was added to the reaction mixture. The specific activity was expressed as units per mg of protein and one unit was defined as an absorbance change of 0.1 per min.

502

SADAYUKIMATUDAand FuI[o OBO

"~0 20" •10. t,,. ..0 3

~ ><

u _=

2'

o

I 11 II 50t 100 Elution Volume(ml)

._m

10

"U 5 ~

2

"6 IE

10

J=

Fig. 3. SDS-gel electrophoresis of the purified lipoamide dehydrogenase in 10~ polyacrylamide gel containing 0.1~o sodium dodecyl sulfate. i n t e n s i t y o f a b s o r p t i o n at 455 n m , t h e flavin c o n t e n t

in the purified enzyme was estimated to be 18.5 nmols per mg of protein, taking a millimolar extinction coefficient of F A D to be 11.3 (Beinert, 1956). This flavin content corresponds to a minimum molecular weight of 53,000. This result also suggests that the native lipoamide dehydrogenase is composed of two subunits, containing each 1 mol FAD. As shown in Fig. 5, the fluorescence intensity of the native enzyme was about 3 times that of the detached flavin.

5'

B.

!

I

0.5 1.0 Relative Mobility

Fig. 4. Estimation of the molecular weight of the purified lipoamide dehydrogenase. The experiments were performed as described in the text. A; Gel-filtration on Sephadex G-200 column (1.9 x 24cm) according to the method of Andrews (1965). B; SDS-polyacrylamide gel electrophoresis. Polyacrylamide gels were prepared with 10% polyacrylamide, a; gamma-globulin, b; bovine serum albumin, c; ovalbumin, d; trypsin, e; myoglobin, LDase; Ascaris lipoamide dehydrogenase. >,

~50

1

/

o \

Electron acceptors of the lipoamide dehydrogenase. As shown in Table 4, this lipoamide dehydrogenase catalyzed the reduction of lipoic acid, ferricyanide, DCPI, cytochrome c, but did not catalyze the reduction of glutathione. The reactivity of the lipoamide dehydrogenase with cytochrome c was negligible. The lipoamide dehydrogenase did not require N A D P H as an electron donor. Heat stability and optical pH. The lipoamide dehydrogenase activity was not lost by heating at 70°C for 40 min, but rapidly lost by heating at 85°C. As

41)0

I)UU

D~U

OUU

Wavelength(rim) Fig. 5. Fluorescence spectra of the native enzyme, detached flavin and authentic FAD. I. The native enzyme (6 x 10-6M), II. The authentic FAD (10-SM), III, The detached flavin; the detached flavin was obtained by the procedure described in Materials and Methods. The excitation wavelength was 360 nm.

Table 4. Specificity of the lipoamide dehydrogenase for electron donor and acceptor Donors

Acceptors

NADH (0.1 mM)

Lipoic Acid (2 raM) Ferricyanide (0.8 mM) DCPI (0.1 mM)* Cytochrome c (0.05 mM) Glutathione (Oxidized form, 1 raM)

NADPH (0.12 mM)

Lipoic Acid (2 raM) Ferricyanide (0.8 mM) DCPI (0.1 mM)* Cytochrome c (0.05 mM) Glutathione (Oxidized form, 1 mM)

Activity (/1tools reduced acceptors/mg protein/min)

* DCPI; 2,6-dichlorophenolindophenol. The assays were conducted as described in Materials and Methods.

23 9 3 0.02 0 0 0 0 0 0

~.=~

Lipoamide dehydrogenase in Ascaris

100~> 50 °

Z

5

6 7 pH

8

9

Fig. 6. Effect of pH on the lipoamide dehydrogenase activity. 0.1M citrate (ptt4-7.5); (O O), and 50raM Tris-HC1 (pH 7.5-8.5); (O e) were used as buffers. The maximal activity of the lipoamide dehydrogenase was taken as 100~.

shown in Fig. 6, the maximum activity of the lipoamide dehydrogenase was observed at about pH 5.7. Kinetics of the lipoamide dehydrogenase. The lipoamide dehydrogenase had a K , of 0.3 mM for lipoic acid as the electron acceptor and a K= of 17 #M for NADH as the electron donor. The Vmax(#mols oxidized donor/mg protein/min) for the NADH was 24. Immunological studies. Upon immunodiffusion (Fig. 7) crude and purified lipoamide dehydrogenase from Ascaris muscle yielded each a single precipitation line with the antiserum against the purified lipoamide dehydrogenase and these lines fused together, but that from the pig heart did not yield the precipitation line. Amino acid composition. The amino acid composition of Ascaris lipoamide dehydrogenase is given in Table 5, Ascaris lipoamide dehydrogenase had an amino acid composition similar to the enzymes from other sources (Matthews et al., 1974; Burleigh & Williams, 1972; Williams et al., 1967), but no tryptophan was found in it. As shown in Table 5, A28o/E4s5 ratio

Fig. 7. Double diffusion reaction of Ascaris lipoamide dehydrogenase with anti-Ascaris lipoamide dehydrogenase serum. 1, 2, 3 and 4 of well contained purified Ascaris lipoamide dehydrogenase, crude Ascaris lipoamide dehydrogenase, beef heart muscle lipoamide dehydrogenase and anti-Ascaris lipoamide dehydrogenase serum, respectively.

503

Table 5. Amino acid composition of Ascaris lipoamide dehydrogenase Amino Acid Asp Thr Ser Glu Gly Ala 1/2 Cys Val Met ILe Leu Tyr Phe Lys His Arg Pro Try* Total amino acid residues FAD

No. of residues No. of residues per histidine per 53,000 4.15 2.8 1.98 4.5 5.06 4.18 0.336 3.6 1.06 2.4 3.9 0.98 1.014 3.38 1.0 1.25 2.04 0

46 31 22 50 57 47 4 40 12 27 43 11 12 37 11 14 22 0 486 1 4.7

A2so/E4~5

*Determined on separate samples as described in Materials and Methods. of the purified lipoamide dehydrogenase was 4.7 and was lower than those of other sources. This low value may be due to the absence of tryptophan in Ascaris lipoamide dehydrogenase. DISCUSSION In the previous paper, Matuda (1979) reported that the microsomal fraction prepared from the Ascaris muscle nearly all consisted of vesicular components morphologically, and the mitochondrial contamination in it was calculated to be about 10~ on the basis of the content of the succinate-cytochrome c reductase used as an enzymatic marker of mitochondria. In the present study, a very high specific activity of lipoamide dehydrogenase was found in microsomal fraction which had no activities of pyruvate and ~t-ketoglutarate dehydrogenase and had very low specific activity of succinate-cytochrome c reductase. The protein of the lipoamide dehydrogenase amounted to 15-20~o of the total microsomal protein (from Table 3). As shown in Table 1, little or no activities of pyruvate and ~t-detoglutarate dehydrogenase resided in mitochondrial and microsomal fractions. Furthermore the mixing of both fractions had no effect on these dehydrogenase activities, therefore, it appears that the lipoamide dehydrogenase in Ascaris microsomes does not play any role in the oxidative of ct-ketoacids. It has been also reported that lipoamide dehydrogenase functions physiologically in the oxidative decarboxylation of glycine in the anaerobic bacterium Peptococcus glycinophilus (Robinson et al., 1973). Our experiments showed that Ascaris muscle lipoamide dehydrogenase does not play any role in the oxidative decarboxylation of glycine (data not shown). Although the basic catalytic and spectral properties, and identification of coenzyme form of

504

SADAYUKI MATUDA and PuJto OBO

Ascaris microsomal lipoamide dehydrogenase did not differ appreciably from those of lipoamide dehydrogenase from other species, it is very interesting that in Ascaris a large a m o u n t of lipoamide dehydrogenase is localized in microsomes rather than m i t o c h o n d r i a contrary to mammalian. Acknowledgement--We wish to thank Miss H. Tomeki for the amino acid analysis. REFERENCES

ANDREWS P. (1965) The gel-filtration behaviour of proteins related to their molecular weights over a wild range. Biochem. J. 96, 595 606. BEINERT H. (1956) In The Enzymes (Edited by BOYER P. D., LARDY H. A. & MYRBACK K.) Vol. II, pp. 339-416, Academic Press, New York. BURLEIGH B. D. JR & WILLIAMSC. H. JR. (1972) The isolation and primary structure of a peptide containing the oxidation-reduction active cystine of Escherichia coil lipoamide dehydrogenase. J. biol. Chem. 247, 2077 2082. GOODWIN T. W. & MORTON R. A. (1946) The spectrophotometric determination of tyrosine and tryptophan in proteins. Biochem. J. 40, 628-632. HORECKER B. L. & KORNBERG A. (1948) The extinction coefficients of the reduced band of pyridine nucleotides. J. biol. Chem. 175, 385-390. IMAI K., OMURA T. & SATO R. (1966) Biochemical characterization of microsomes isolated from heart and skeletal muscles. J. Biochem. Tokyo 60, 274-285. LOWRY O. H., ROSEBROUGHN. J., FARR A. L. & RANDALL R. J. (1951) Protein measurement with the folin phenol reagent. J. biol. Chem. 193, 265-275. MASSEY V. (1960) The composition of the ketoglutarate dehydrogenase complex. Biochim. biophys. Acta 38, 447-460. MATTHEWS R. G., ARSCOTT L. D. & WILLIAMS C. H. JR. (1974) Isolation, characterization and partial sequencing of cystine and thiol peptides of pig heart lipoamide dehydrogenase. Biochim. biophys. Acta 370, 26-38.

MATUDA S. (1979) Biochemical studies on the muscle microsomes of Ascaris lumbricoides vat. suum. I. Biochemical characterization and electron transport of Ascaris microsomes. J. Biochem., Tokyo 85, 343-350. MATUDA S. (1979) Biochemical studies on the muscle microsomes of Ascaris lumbricoides var. suum. II. Purification and characterization of b-type cytochrome and NADH-ferricyanide reductase from Ascaris muscle microsomes. J. Biochem. Tokyo 85, 351-358. OYA H., KIKUCHI G., BANDO T. & HAYASHI H. (1965) Muscle tricarboxylic acid cycle in Ascaris lumbricoides var. suis. Exp. Parasit. 17, 229. REED J. L. & WILLIAMSC. R. (1966) In Methods in Enzymology (Edited by WOOD A. W.) Vol. 9, pp. 247-265. Academic Press, New York. ROBINSON J. R., KLEIN S. M. & lAGERS R. D. (1973) Glycine metabolism J. biol. Chem. 248, 5319-5323. SAZ n. J. • WEIL A. (1960) The mechanism of the formation of ct-methylhutyrate from carbohydrate by Ascaris lumbricoides muscle. J. biol. Chem. 235, 914-918. SAZ H. J. & VIDRINE A. (1957) The mechanism of formation of succinate and propionate by Ascaris lumbricoides muscle. J. biol. Chem. 234, 2001-2005. STOTZ E. (1955) In Methods in Enzymology (Edited by COLOWICK S. P. & KAPLAN N. O.) Vol. 2, pp. 740-744. Academic Press, New York. TAKESUE S. & OMURA T. (1970) Purification and properties of NADH-cytochrome b5 reductase solubilized by lysosomes from rat liver microsomes. J. Biochem. Tokyo 67, 267-276. WEBER K. & OSBORN M. (1969) The reliability of molecular weight determinations by dodecyl sulfatepolyacrylamide gel electrophoresis. J. biol. Chem. 244, 4406-4412. WILLIAMS C. H. JR. (1976) In The Enzymes (Edited by BOYER P. D.) Vol. 13, pp. 106-129. Academic Press, New York. WILLIAMS C. H. JR., ZANETTI G., ARSCOTT L. D. & MCALLISTER J. K. (1967) Lipoamide dehydrogenase, glutathione reductase, thioredoxin reductase, mid thioredoxin. J. biol. Chem. 242, 5226-523 I.