349

Biochimica et Biophysica Acta, 497 (1977) 349--357 © Elsevier/North-Holland Biomedical Press

BBA 28209

RAT LIVER ~-TOCOPHEROL BINDING PROTEIN

GEORGE L. CATIGNANI and JOHN G. BIERI

Laboratory o f Nutrition and Endocrinology, Na tional Ins titu te of Arthritis, Me ta bolism and Digestive Diseases, National Institutes o f Health, Bethesda Md. 20014 (U.S.A.)

(Received November 15th, 1976)

Summary

1. The properties of rat liver cytoplasmic ~-tocopherol binding protein have been studied.

2. The binding protein sedimented in the 3 S region of sucrose density gra- dients, and gel filtration indicated an approximate molecular weight of 30 500.

3. Of the tissues examined by the present assay, binding was detectable only in the liver.

4. Optimal binding was achieved by incubation at 26°C for 4 h and was independent of pH between 7.4 and 9.0.

5. Pronase completely abolished binding. The binding protein was, however, almost completely resistant to trypsin, and unaffected by RNAase, DNAase, triacylglycerol lipase, and phospholipase C.

6. A variety of tocopherol analogues and other lipid-soluble compounds were tested for their ability to compete for binding. Only ~-tocopherol and to a lesser extent ~-tocotrienol and 7-tocopherol exhibited competition. ~-Tocoph- erol acetate, ~-tocopherol quinone and 6-hydroxy-2,5,7,8-tetramethylchroman- 2-carboxylic acid had no effect on binding.

7. Tocopherol binding was reversible, and the tocopherol was not metab- olized during incubation.

Introduction

A recent report from this laboratory demonstrated that rat liver cytoplasm contains a protein that binds ~-[3H]tocopherol in vitro with high affinity and specificity [1]. The binding protein sedimented in the 3 S region on sucrose gradients. Gel filtration resulted in a preliminary molecular weight estimate of 31 000. Competition for binding was demonstrated with an excess of unlabeled ~-tocopherol while an excess of either ~-tocopherol quinone or a-tocopherol acetate had no effect. Binding was undetectable following incubation of labeled cytosol with pronase.

350

The present report describes the optimal assay conditions and additional properties of a-tocopherol binding protein.

Materials and Methods

Materials RRR-a-[5-methyl-3H]tocopherol (3.8 Ci/mmol) was obtained from Amers-

ham/Searle Corp. (Arlington Heights, Ill.). All-rac-a-tocopherol, all-rac-7- tocopherol, REE-a-tocotrienol, 6-hydroxy-2,5,7,8-tetramethylchroman-2-car- boxylic acid (Trolox) and 1,25-dihydroxycholecalciferol were generous gifts of Hoffman-LaRoche Inc. (Nutley, N.J.). RNAase A(Type XII-A), DNAase 1, lipase (triacylglycerol, type VI), phospholipase C, trypsin, coenzyme Q9 and blue dextran were purchased from Sigma Chemical Co. (St. Louis, Mo.). Pronase was purchased from Calbiochem (La Jolla, Calif.). Vitamin K-l, oleic acid, and cholesterol were obtained from I.C.N. (Cleveland, Ohio). RRR-a- tocopherol quinone and RRR-a-tocopherol acetate were purchased from East- man Organic Chemicals (Rochester, N.Y.), and NJV'-diphenyl-p-phenylene- diamine from B.F. Goodrich Co. (Akron, Ohio). The molecular weight markers, myoglobin, chymotrypsinogen, ovalbumin and bovine serum were products of Schwartz-Mann (Orangeburg, N.Y.).

Methods Preparation of tissues. All tissues were collected from male Holtzman rats

maintained from weaning on a tocopherol-free diet as previously described [1]. Livers were perfused with ice cold 0.9% NaC1 unless stated otherwise. All tis- sues were homogenized in four volumes (w/v) of 0.05 M Tris • HC1 buffer (pH 7.5) using a glass-teflon homogenizer. Heart and skeletal muscle (gastroc- nemius) were first homogenized briefly with an Ultra Turrax (blade type) homogenizer. In some experiments the homogenizing buffer contained either 0.2 M KC1 or 0.05% (w/v) Triton X-100. The homogenates were centrifuged at 10 000 × g for 15 min and the supernatant removed and centrifuged at 105 000 × g for 1 h at 4°C. The high speed supernatant (cytosol) was collected free from floating lipid and used immediately. Serum was diluted 1 : 4 (v/v) with buffer before use.

Red blood cell hemolysate was prepared by using saline-washed packed cells in two volumes of water. The resulting lysate was freed of cell debris by cen- trifugation at 10 000 × g for 15 min and diluted with an equal volume of 0.1 M Tris • HC1 buffer (pH 7.5). before use. Aliquots of cytosol, serum or hemoly- sate were incubated, except where otherwise indicated, for 4 h at 26°C in the presence of 100 nM a-[3H]tocopherol, added in 4--5 pl of ethanol per ml of cytosol. Where indicated, unlabeled competitors were added in 1--5 pl of ethanol per ml of cytosol. Ethanol alone was added to control samples.

Gel filtration and sucrose gradient centrifugation. Gel filtration was per- formed as previously described [1]. The molecular weight of the binding pro- tein was determined by comparing the elution volume to that of the markers myoglobin (17 830), chymotrypsinogen (25 000), ovalbumin (45 000), and bovine serum albumin (67 000). The void volume was determined with blue dextran. Sucrose gradient centrifugation and fractionation were also performed

351

as previously described [1] with the exception that the gradients were cen- trifuged in a Spinco SW 50.1 rotor at 45 000 rev./min for 18 h at 4°C (189 000 × gav).

Enzyme digestion studies. Following incubation of cytosols with [3H]- tocopherol for 4 h at 26°C, 200 pg of each enzyme, dissolved in 0.1 ml of 0.05 M Tris • HC1 buffer (pH 7.5), were added to 1 ml of cytosol and incubated for an additional 2 h at 26 ° C. Buffer alone was added to control samples.

Purification of [3H]tocopherol. Purification of [3H]tocopherol was per- formed by the method of Bieri [2] on silica gel G containing 0.004% sodium fluorescein with benzene/ethanol (99 : 1, v/v) as the developing solvent. The purified tocopherol was dissolved in ten times the volume of benzene/ethanol (9 : 1, v/v) as originally supplied, divided into aliquots and stored a t - -40°C. A radiochemical purity above 85% was maintained by periodic purifications (usually bimonthly).

Extraction and analysis of [3H] tocopherol. Radioactivity was extracted from either labeled cytosol or pooled Sephadex G-100 fractions by making the sample 50% in ethanol, adding 50 pg of unlabeled tocopherol and extracting twice with an equal volume of hexane. Approx. 98% of the total radioactivity was extracted. The hexane was removed by evaporation under a stream of N2, and the sample was redissolved in a convenient volume of benzene and ana- lyzed by thin-layer chromatography as above. The tocopherol was visualized under an ultraviolet lamp and the silica gel marked into equal segments. Each segment was scraped from the plate directly into a scintillation vial and the radioactivity measured.

Bincling assay. The assay of tocopherol binding protein is based on the total amount of radioactivity bound in the 3 S region of the sucrose gradient, minus the radioactivity bound in the presence of a 400-fold excess of unlabeled to- copherol (non-specific binding). If the radiochemical purity is maintained above approx. 85%, the amount of non-specific binding of tritium is less than 2% of the amount of tritium specifically bound. Protein was determined by the biuret method [3] and all radioactivity measurements were made in toluene- Triton X-100-based scintillation fluid as previously described [1].

Results

Sedimentation behavior. A typical gradient profile of liver cytosol incubated in the presence of 100 nM ~-[3H]tocopherol is shown in Fig. 1. It also illus- trates that the addition of a 400-fold excess of unlabeled tocopherol reduced the bound radioactivity to less than 1%. The bound tocopherol sedimented in the 3 S region of the gradient when compared to the markers chymotrypsino- gen (2.7 S) or ovalbumin (3.7 S).

Molecular weight. Gel filtration of labeled liver cytosol on Sephadex G-100 resulted in a molecular weight estimate of 30 500. Fig. 2 depicts a plot of Ve/ Vo (elution volume/void volume) versus log molecular weight for tocopherol binding protein and four marker proteins.

Tissue distribution. Following the incubation of [3H]tocopherol with tissue cytosols of rat liver, kidney, heart, lung, testes, intestinal mucosa, muscle, brain, serum or erythrocyte hemolysate, ~-tocopherol binding protein was

352

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2 4 6 8 10 12 14 16 18 20 Top FRACTION NUMBER

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- TOCOPHEROL NDING PROTEIN

ALBUMIN

BOVINE SERUM

LBUMIN BSA !MER

I I I I I I I I I I I I /

4.04.14.24.3 4.44.54.64.74.8 4.95.05.152.

LOG M O L E C U L A R W E I G H T

Fig. 1. Sucrose d e n s i t y g rad ien t c e n t r i f u g a t i o n of ra t l iver c y to so l i n c u b a t e d wi th 100 nM c~- [3H] tocoph - erol fo r 4 h a t 26°C in the p resence (e e ) or ab sence (0 o) o f a 400 - fo ld mo la r excess (40 pM)

of un labe led c~-tocopherol. The s e d i m e n t a t i o n of c h y m o t r y p s i n o g e n (2.7 S) a n d o v a l b u m i n (3.7 S) is

i n d i c a t e d by a r rows .

Fig. 2. Molecular w e i g h t of ra t l iver c y t o p l a s m i c ~ - t o c o p h e r o l b i n d i n g p ro t e in e s t i m a t e d by gel f i l t r a t ion

on S e p h e d e x G-100. The s t a n d a r d p ro t e in s were a s s u m e d to have the m o l e c u l a r we igh t s l i s ted in Mater ia ls a n d M e t h o d s . The e lu t ion of ~ - t o c o p h e r o l b i n d i n g p ro te in , as i n d i c a t e d by the a r r o w , c o r r e s p o n d s to the

e lu t ion of a p r o t e i n w i t h a m o l e c u l a r we igh t of 30 500.

detectable by the present assay method only in the liver. Sucrose gradient assays of serum revealed a peak of bound radioactivity in

the 4.6 S region (fraction 11) of the gradient. This region corresponds to the sedimentation of serum albumin, which is known to bind tocopherol [4]. An occasional sample of unperfused liver also exhibited binding in the 4.6 S region of the gradient, rendering the assay unsuitable due to overlap with the 3 S bind- ing protein peak. Therefore all livers used in subsequent experiments were per- fused with ice-cold 0.9% NaC1.

Effects o f time and temperature. In order to establish optimum binding con- ditions for the assay of ~-tocopherol binding protein, liver cytosol was labeled with 100 nM ~-[3H]tocopherol and incubated for different times at various temperatures. The results of these experiments are illustrated in Fig. 3. Incuba- tion at 26°C for 4 h resulted in maximal binding. Some thermal denaturation of protein occurs at 37 and 45°C and removal of the precipitated protein resulted in a variable loss of radioactivity from the cytosol. Using lower tem- peratures, longer incubation times are needed for the reaction to reach comple- tion. Samples were routinely incubated 26°C for 4 h.

Estimate o f the tissue content of (~-tocopherol binding protein. A 1 : 4 (w/v) liver homogenate generally resulted in a cytosol containing from 11 to 13 mg protein per ml. Aliquots of 0.2 ml (2.2--2.6 mg protein) analyzed by sucrose density gradients usually resulted in a total of from 14 000 to 20 000 cpm specifically bound in the 3 S region of the gradient. These figures represent a range of approx. 3--5 pmol of bound tocopherol per ml of cytosol protein or approx. 165--325 pmol bound per g of liver. Assuming that the binding protein binds 1 mol of tocopherol per mol and has a molecular weight of 30 500, liver

353

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6

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o i i ~ I 215 ~ 315 0 L I

5 10 15 20 30 4 45 50 TEMPERATURE (°C)

Fig. 3. Effects of va ry ing the t i m e a nd t e m p e r a t u r e of i n c u b a t i o n o n t h e binding of (~ - [3H] tocophero l t o

i t s hepa t ic b ind ing p ro te in . Cytosols were i n c u b a t e d wi th 1 0 0 nM ~ - [ 3 H ] t o e o p h e r o l a t the t e m p e r a t u r e i n d i c a t e d for 1 h (o o), 2 h (e e ) or 4 h (~- ~) and assayed by sucrose dens i ty g rad ien t cen t r i fuga t ion .

would contain approx. 5--9 pg of tocopherol binding protein per g. The binding protein generally approached saturation at cytosol concentra-

tions of from 90 to 110 nM tocopherol. Each 0.2-ml aliquot of cytosol con- tained approx. 7--13 pmol of bound tocopherol, representing from 33 to 50% of the total added tocopherol. Attempts to accurately measure kinetic param- eters have been unsuccessful due to the non-specific binding of tocopherol to the surface of the reaction vessels used.

Effect of pH. Varying the pH of the incubation between 7.4 and 9.0 had little effect on the binding of [3H]tocopherol to the binding protein. Below 7.4, however, binding began to decrease rapidly, falling to 50% of maximal at pH 6.5.

Effect of salt or detergent. The addition of either 0.2 M KC1 or 0.05% Triton X-100 to the homogenizing buffer had no effect on the binding of [3H]tocoph- erol, indicating that the binding protein is most likely a cytoplasmic protein.

Enzyme digestion studies. The effect of treatment by various hydrolytic enzymes on the binding of a-[3H]tocopherol was examined. As previously reported [1], pronase digestion results in complete loss of binding activity. The binding protein appeared to be almost completely resistant to trypsin, losing only 11% activity, and was unaffected by treatment with RNAase, DNAase, lipase or phospholipase C.

Binding specificity. Binding specificity was determined by the relative affin- ity of the binding protein for a-[3H]tocopherol in the presence of a 400-fold molar excess of either a-tocopherol or one of several unlabeled tocopherol analogues. Table I gives the results of these experiments.

A 400-fold excess of a-tocopherol reduced the binding of a-[3H]tocopherol to 2%. ~'-Tocopherol and a-tocotrienol, although less effective, reduced binding to approx. 40 and 30%, respectively, a-Tocopherol acetate, a-tocopherol quinone or Trolox had no effect on binding.

The addition of a 400-fold excess of any of a variety of other lipid-soluble compounds, including retinol, retinoic acid, 1,25 dihydroxycholecalciferol, cholesterol, oleic acid, vitamin K-l, coenzyme Q9, dexamethasone, and diphenylphenylenediamine had no effect on binding.

Dissociation of bound [3H]tocopherol. The reversibility of binding of [3H]-

3 5 4

T A B L E I

E F F E C T S O F T O C O P H E R O L A N A L O G U E S O N T H E B I N D I N G O F ( ~ - [ 3 H ] T O C O P H E R O L T O I T S H E P A T I C C Y T O S O L B I N D I N G P R O T E I N

A l i q u o t s o f l ive r c y t o s o l w e r e i n c u b a t e d w i t h 1 0 0 n M ( ~ - [ 3 H ] t o c o p h e r o l in t h e p r e s e n c e o r a b s e n c e o f a

4 0 0 - f o l d m o l a r e x c e s s o f u n l a b e l e d t o c o p h e r o l a n a l o g u e ( 4 0 p M ) f o r 4 h a t 2 6 ° C . B i n d i n g w a s d e t e r m i n e d b y s u c r o s e g r a d i e n t a s s a y .

A d d i t i o n B i n d i n g (%)

N o n e 1 0 0 ~ - T o c o p h e r o l 2 7 - T o c o p h e r o l 39 ( ~ - T o c o p h e r o l a c e t a t e 1 0 0 ~ - T o c o p h e r o l q u i n o n e 98 ( ~ - T o c o t r i e n o l 31 T r o l o x 1 0 0

tocopherol is illustrated in Fig. 4. As shown, the bound tocopherol could be effectively displaced from the binding protein by the excess unlabeled tocoph- erol. The addition of an equal volume of ethanol alone to control aliquots had no effect on binding.

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i 2 3 4 ° ° T

ORIGIN AUTHENTIC SOLVENT a-TOCOPHEROL FRONT

Fig . 4. D i s s o c i a t i o n o f b o u n d ~ - [ 3 H ] t o c o p h e r o l f r o m i t s h e p a t i c c y t o s o l b i n d i n g p r o t e i n b y t h e a d d i t i o n o f e x c e s s u n l a b e l e d ~ - t o c o p h e r o l . C y t o s o l w a s i n c u b a t e d w i t h 1 0 0 n M ~ - [ 3 H ] t o c o p h e r o l f o r v a r i o u s t i m e s u p t o 7 h a t 2 6 ° C ( e ~). M a x i m a l b i n d i n g w a s a c h i e v e d a t 4 h . A l i q u o t s w e r e t a k e n a t 4 h a n d e i t h e r a 4 0 0 - f o l d e x c e s s o f u n l a b e l e d ~ - t o c o p h e r o l i n e t h a n o l w a s a d d e d t o t h e i n c u b a t i o n m i x t u r e (© . . . . . . o) , o r e t h a n o l a l o n e ( e . . . . . . e ) , a n d i n c u b a t i o n c o n t i n u e d . A t t h e i n d i c a t e d t i m e a n a l i q u o t w a s r e m o v e d f r o m e a c h s a m p l e a n d a s s a y e d b y s u c r o s e d e n s i t y g r a d i e n t e e n t r i f u g a t i o n .

F i g . 5. T h i n - l a y e r c h r o m a t o g r a m o f t h e h e x a n e e x t r a c t o f r a t l i ve r c y t o s o l p r e v i o u s l y i n c u b a t e d in t h e p r e s e n c e o f 1 0 0 n M a - [ 3 H ] t o c o p h e r o l f o r 4 h a t 2 6 ° C . , h e x a n e e x t r a c t o f l i ve r , , a u t h e n - t ic c ~ - [ 3 H ] t o c o p h e r o l . T h e o r i g i n , s o l v e n t f r o n t a n d s e g m e n t c o n t a i n i n g a u t h e n t i c ( ~ - t o c o p h e r o l a r e i n d i c a t e d b y a r r o w s .

355

Iden tifica tion o f incu ba ted and bound tritium as [3H] tocopherol. In order to determine if tocopherol was metabolized or destroyed during incubation of liver cytosols, the radioactivity was extracted from the cytosol and analyzed by thin-layer chromatography. As shown in Fig. 5, 92% of the radioactivity was recovered as unchanged tocopherol. Thin-layer analysis of a sample of the authentic a-[3H]tocopherol used in the incubation showed that the radiopuri ty was 93.5% as shown in Fig. 5.

The peak fractions from Sephadex G-100 were also extracted and analyzed to determine if the bound radioactivity was unchanged a-tocopherol. These fractions represent specifically bound counts since Sephadex chromatography effectively separates a-tocopherol binding protein from both the high molec- ular lipoprotein-associated tocopherol (void volume of Sephadex G-100) and unbound tocopherol [1]. Analysis of the extract by thin-layer chromatography revealed 96.5% of the bound counts were extracted and recovered as a-tocoph- erol.

Discussion

The presence of a specific ~-tocopherol binding protein in rat liver cytoplasm has been demonstrated. The binding activity is detectable as protein bound radioactivity by sucrose density gradient centrifugation or gel filtration of rat liver cytosol labeled in vitro with a-[ 3H] tocopherol.

The binding protein sediments in the 3 S region of sucrose gradients (Fig. 1) and has the molecular weight of 30 500 as judged by gel filtration (Fig. 2).

Other properties of the binding protein have been examined and optimal assay conditions established. Using a sucrose density gradient assay the only tissue with detectable levels of binding protein is the liver. It should also be mentioned that the binding protein is detectable in female rat liver but not in uterus. It is likewise present in the livers of several small laboratory animals including mouse, guinea pig, hamster, rabbit and chicken. Quantitative com- parisons have not been made, since each animal had been raised on a complete diet and endogenous tissue tocopherol competes for binding of [3H] tocopherol [1].

Incubation of liver cytosol at 26°C for 4 h was found to result in maximal binding. The gradual increase in binding as temperature increases eliminates the possibility of an activation mechanism at some critical temperature necessary for optimal binding. It should be noted that the thermal denaturation that takes place at 46°C for 4 h results in a loss of only about 8% of the total bind- ing activity (Fig. 3) (compared to 26°C for 4 h) while approx. 46% of the total protein is lost (data not shown).

In addition to its relative resistance to thermal denaturation, the tocopherol binding protein is relatively stable to t reatment with trypsin. The fact that some activity is lost, however, and that all binding is destroyed by pronase, points to the protein nature of the binding site. Other enzyme treatment sug- gests that the binding protein is not a nucleoprotein or a l ipoprotein or that, if so, such components are not necessary for binding.

The competi t ion studies illustrated in Table I revealed certain structural requirements of a molecule as essential for its interaction with the binding

356

protein. The necessity of a free chroman hydroxy group, an intact chromanol ring structure and the presence of a side chain, all absolute requirements for biological activity are demonstrated by the complete inability of a-tocopherol acetate, a-tocopherol quinone and Trolox, respectively, to compete with a-to- copherol for binding. Even minor modifications, such as removal of a chroman methyl group (3,-tocopherol) or replacement of the saturated side chain with an unsaturated side chain (a-tocotrienol), result in a molecule capable of only limited competit ion with a-tocopherol.

It should be mentioned that, with the exception of a-tocopherol acetate, which is rapidly hydrolyzed in vivo but which is not hydrolyzed by the liver cytosol preparation used in this study, the relative activities of the compounds listed in Table I to compete for binding parallel the relative biological activities of these compounds in vivo, i.e. a-tocopherol > 7-tocopherol or a-toco- trienol > a-tocopherol quinone or Trolox. Whether there is any biological sig- nificance to this observation remains to be determined.

The competit ion studies also illustrated that diphenylphenylenediamine, known to prevent certain vitamin E-deficient diseases [5], has no effect on the binding of a- [ 3H] tocopherol.

The displacement of bound a-[3H]tocopherol from the binding protein by the addition of a 400-fold excess of unlabeled a-tocopherol illustrates that binding is non-covalent (Fig. 4). Extraction and analysis of radioactivity follow- ing incubation of cytosol with a-[3H]tocopherol reveals that tocopherol is virtually unchanged during incubation.

The small distributional differences (1.5%) between the incubated and authentic tocopherol on thin-layer chromatography are wholly accounted for in the fraction which corresponds to the migration of a-tocopherol quinone (Fig. 5, Fraction 2). The quinone is a known oxidation product of a-tocoph- erol. It may be produced both as an artifact during chromatography of a-to- copherol on silica gel [6] and as an in vivo metabolite of a-tocopherol [7].

The present study indicates that the incubation of a-tocopherol with liver cytosol in vitro results in the production of very little a-tocopherol quinone and no other detectable metabolites. If other metabolites exist, their concen- tration would be of the order of 10 -1° mol or less/ml cytosol.

The observation that the binding protein is detectable only in the liver may serve in the resolve of its function. Bieri and Anderson [8] have reported that the peroxidation observed in vitamin E-deficient chicken liver homogenates is inhibited from 70 to 100% by addition of vitamin E-sufficient homogenates. The inhibiting factor was found to be heat labile and non-diffusible. Inhibition was specific for liver in that neither vitamin E-sufficient muscle homogenate would inhibit peroxidation in deficient liver or deficient muscle, nor would vitamin E-sufficient liver homogenate inhibit peroxidation in deficient muscle or brain. Similar observations were made using rat liver although the effect was usually not as complete.

In preliminary experiments (unpublished), the addition of an antiserum to rat serum had no effect on binding. This, coupled with the fact that no 3 S peak can be demonstrated in serum by sucrose gradient centrifugation, strongly suggests that the binding protein is not released into the circulation.

There is presently no evidence for or against the possibility of a catalytic

3 5 7

function for the protein. It is likewise unknown if the binding protein plays a role in mediating the action of ~-tocopherol in vivo. Delineating its physiolo- gical role remains for future investigation.

References

1 Ca t i gnan i , G .L . ( 1 9 7 5 ) B i o c h e m . B i o p h y s . Res . C o m m u n . 67 , 6 6 - - 7 2 2 Bieri, J .G . ( 1 9 6 9 ) in L ip id C h r o m a t o g r a p h i c A n a l y s i s (Mar ine t t i , G .U. , ed . ) , Vol . 2, pp . 4 5 9 - - 4 7 8 ,

Marce l D e k k e r Inc . , N e w Y o r k 3 L a y n e , E. ( 1 9 5 7 ) in M e t h o d s in E n z y m o l o g y ( C o l o w i c k , S.P. a n d K a p l a n , N .O. , eds.) , Vol . III, pp.

4 5 0 - - 4 5 1 , A c a d e m i c Press, Inc . , N e w Y o r k 4 V o t h , O .L . a n d Miller, R .C. ( 1 9 5 8 ) A r c h . B i o c h e m . B i o p h y s . 7 7 , 1 9 1 - - 2 0 5 5 S c o t t , M.L. ( 1 9 7 0 ) in T h e F a t So lub le V i t a m i n s ( D e L u c a , H .F . a n d Su t t i e , J .W. , eds . ) , pp . 3 5 5 - - 3 6 8 ,

T h e Un ive r s i t y o f Wiscons in Press, M a d i s o n 6 P lack , P .A. a n d Bieri , J . G . ( 1 9 6 4 ) B i o c h i m . B i o p h y s . A c t a 84 , 7 2 9 - - 7 3 8 7 Csa l l any , A.S. , Drape r , H .H . a n d S h a h , S .N. ( 1 9 6 2 ) A r c h . B i o c h e m . B i o p h y s . 9 8 , 1 4 2 - - 1 4 5 8 Bieri, J .G . a n d A n d e r s o n , A .A . ( 1 9 6 0 ) A r c h . B i o c h e m . B i o p h y s . 9 0 , 1 0 5 - - 1 1 0