THE MICROBIOLOGICAL DETERMINATION OF AMINO ACIDS · 2003-03-03 · THE MICROBIOLOGICAL DETERMINATION OF AMINO ACIDS III. METHIONINE BY CARL M. LYMAN, OLIVE MOSELEY, BETTY BUTLER,

THE MICROBIOLOGICAL DETERMINATION OF AMINO ACIDS

III. METHIONINE

BY CARL M. LYMAN, OLIVE MOSELEY, BETTY BUTLER, SUZANNE WOOD, AND FRED HALE

(From the Texas Agricultural Experiment Station, Agricultural and Mechanical College of Texas, College Station)

(Received for publication, July 22, 1946)

Simplification of the media for the microbiological determination of amino acids can be accomplished in some instances by replacing most of the amino acids with natural products. This can be done when means are available for preferentially destroying or removing one or more of the amino acids in the natural material. Thus, a medium containing aoid- hydrolyzed casein has been used by Greene and Black (1) for the deter- mination of tryptophane and the medium used by Lewis and Olcott (2) for the determination of glutamic acid contained a casein hydrolysate from which the glutamic acid had been removed by autoclaving at pH 2.9 and extracting with ethyl acetate.

The preferential oxidation of methionine with hydrogen peroxide was studied by Toennies and Callan (3). A method for removing not only methionine but also cystine, tryptophane, and tyrosine from peptone preparations by the use of hydrogen peroxide has been described by Lyman et al. (4). It was shown that this type of preparation was satisfactory for use in media for microbiological tests.

The present communication describes a method for the determination of methionine with Leuconostoc mesenteroides as the test organism and a medium in which most of the amino acid nitrogen is supplied by hydrogen peroxide-treated peptone.

One of the most useful methods for testing the reliability of assay values obtained by microbiological methods is to carry out the tests with more than one organism and, when practical, with more than one assay medium. In this investigation Streptococcus faecalis R was used as a second organ- ism. Although hydrogen peroxide-treated peptone can be used in media for use with Streptococcus faecalis R, pure amino acids were used instead in order that the medium as well as the organism should be different in the comparative tests.

Inasmuch as the methionine values obtained by Stokes (5) ,and co- workers with Streptococcus faecalis R are somewhat lower than most of the values obtained by chemical methods, it seemed desirable to determine methionine on the same hydrolysates with a chemical method as well as

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162 METHIONINE DETERMINATION

with the two microbiological methods. The calorimetric method of Mc- Carthy and Sullivan (6), as modified by Csonka and Denton (7), was chosen for this purpose.

EXPERIMENTAL

Determination of Methionine with Leuctinostoc Mesenteroides

Organ&m-Lewonostoc mesenteroides P-60 was maintained by weekly transfers as stabs in solid medium containing the following ingredients: peptoniaed milk 1 per cent, tryptone 1 per cent, filtered tomato juice 200 ml. per liter of medium, agar 1 per cent. Washed cells from 18 hour cultures grown on a liquid medium of the same composition as above, except for the omission of the agar, were used to inoculate the tests.

TABLE I Medium* for Determination of Methionine with Leuconostoc mesenteroides

HzOz-treated peptone .......... 15 gm. Thiamine ....................... 2 mg. Glucose “ ........................ 40 “ Pyridoxine ..................... 4 Sodium acetate ” “ ................ 24 Calcium pantothenate. ......... 4 Ammonium chloride.. .......... 12 “ Riboflavin ...................... 4 “ I(-)-Tryptophane 100 mg. Nicotinic acid 4 “ ............. .................. dl-Tyrosine. “ ................... 200 Biotin .......................... 10 y I(-)-Cystine.. “ “ ................. 200 Folk acid (synthetic). .......... 3 Adenine sulfate ................ 20 “ p-Aminobenzoic acid ............ 0.2 “ Guanine ....................... 20 “ Salt Solution lt ................ 10 ml. Uracil.. 20 “ “ “ ....................... 2$ 10 “ ................

“ “ 36 ................ 10 ‘6 Neutralize and dilute to 1 liter

* Medium for 200 cultures of 10 ml. final volume (5 ml. of the above medium per culture).

t Salt Solution 1, KzHPO* 25 gm., KHzPO* 25 gm., water 250 ml. $ Salt Solution 2, MgS01.7HzO 10.0 gm., NaCl 0.5 gm., MnSOa4HzO 0.5 gm.,

water 250 ml. $ Salt Solution 3, FeS0,.7Hz0 0.5 gm., water 250 ml.

Medium-The composition of the medium used for the determination of methionine with Leuconostoc mesenteroides is given in Table I. The glucose, ammonium chloride, and sodium acetate are added as solids and the rest of the ingredients from stock solutions preserved with a little tolu- ene and stored in the refrigerator.

The stock solution of hydrogen peroxide-treated peptone is prepared as follows: 50 gm. of Bacto-peptone are dissolved in 250 ml. of water and 250 ml. of 2 N HCl are added after the peptone is completely dissolved. 0.025 mole of hydrogen peroxide (2.8 gm. of 30 per cent HBOS) is added

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LYMAN, MOSBLEF, BUTLER, WOOD, AND HALE 163

and the solution allowed to stand overnight at room temperature. The material is then steamed for 30 minutes at atmospheric pressure, stirred while hot, cooled, neutralized with sodium hydroxide, and steamed again, this time for 1 hour. The purpose of the second steaming is to decompose any hydrogen peroxide which is not used up by the oxidative reactions. The preparation is ready for use after diluting to a final volume of 1 liter. A reagent grade of hydrogen peroxide which does not contain any preserva- tive should be used.

dl - METHIONINE - GAMMA PER TUBE

FIG. 1. Typical standard curve for the determination of methiouine with &eu- conostoc mesenteroides. Titration values are for 5 ml. aliquots from 10 ml. cultures.

In the preliminary phases of this investigation more hydrogen peroxide was used than is recommended here. An improvement in the medium was obtained when the treatment of the peptone was carried out as indi- cated above.

Assay Procedure-Titration of the acid produced after 4 days incubation was used as a measure of the growth of the organisms. The general pro- cedure for carrying out the assays was the same as previously used for amino acid assays with Lactobacillus arabinow (S), with the following exceptions: A constant temperature water bath at 35” was used instead of an incubator. The period of incubation was 4 days instead of 3 days.

Standard Curve-A typical standard curve is shown in Fig. 1. The slight

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164 METHIONINBl DPTERMlNATION

dip in the curve is not a matter of chance but always occurred with this medium.

Determination of Methionine with Streptococcus faecalis R

Medium-The composition of the medium used for the determination of methionine with Streptococcus faeculis R is given in Table II. In this medium a buffer consisting of a mixture of sodium succinate and sodium acetate is used instead of sodium acetate alone. Streptococcus faecalis is not very well adapted to growth in acid solution. Both sodium succinate

TABLE II Medium* for Determination of Methionine with Streptococcus faecalis R

Glucose ....................... 40 gm. Succinic acid 20 “ .................. Sodium acetate (anhydrous). .. 6 “ Adenine sulfate ............... 10 mg. Guanine ...................... 10 “ Uracil. ....................... 10 “ Xanthine ..................... 10 “ Riboflavin .................... 1 “ Niacin 2 “ ........................ Pyridoxamine ................. 0.8 I‘ Thiamine chloride ............ 0.4 “ Calcium pantothenate ......... 0.4 “ Biotin ........................ 27 -p-Aminobenzoic acid .......... 2 “ Folic acid (synthetic), ........ 10 “ Salt Solution 1.. .............. 10 ml.

“ “ 2 ................ 10 “ “ “ 3 10 ‘( ................

dl-Alanine ........... I(+)-Arginine ....... dl-Aspartic acid. .... Z(-)-Cystine ........ dl-Glutamic acid ..... Glycine. ............. I(-)-Histidine ....... dl-Isoleucine. ........ dl-Leucine. .......... Z(+)-Lysine ......... dl-Phenylalanine ..... I(-)-Proline ......... dl-Serine ............ dl-Threonine. ........ I(-)-Tryptophane ... dl-Tyrosine. ......... dl-Valine. ...........

. . . . 400 mg. . . . 400 c;

. . . . 800 “

. . . . . 400 (‘

. . so0 “

. . . . . 400 “

. . . 400 “

. . . . . 400 “

. . 400 “

. . 400 ‘< . . . . 400 “

. 400 ‘( 400 “

. . 400 “ . . . 200 “

. 400 ‘l . . . . 400 “

Add 12 gm. of NaOH pellets and finish neutralizing with NaOH solution. Dilute to 1 liter.

* Medium for 200 cultures of 10 ml. final volume (5 ml. of the above medium per culture).

and sodium citrate exert a strong buffering action at a pH range closer to neutrality than the effective range of an acetate buffer. The result of using either of these substances as buffers in media for Streptococcus faecalis R is to increase markedly the amount of acid formed by the organism. So- dium succinate has the advantage of producing less caramelization upon sterilization of the medium.

Assay Procedure-With a few exceptions, the method of handling the organism and of conducting the tests with Streptococcus faeculis R was the same as for Leuwnostoc mesenttkdes. The period of autoclaving the tubes

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LYMAN, MOSELEY, BUTLER, WOOD, AND HALE 165

for sterilization was reduced to 10 minutes. The incubation time was 3 days.

Standard Curve-A typical standard curve is given in Fig. 2.

Determination of Metbionine by Colorimettic Method

The method used was that of McCarthy and Sullivan (6), as modified by Csonka and Denton (7). The phospho244ungstic acid used for the precipitation of the basic amino acids was prepared according to the method of Wu (9).

20 40 60 80 (\I-METHIONINE -GAMMA PER TUBE

FIG. 2. Typical standard curve for the determination of methionine with Strepto- COCCUS fuecalis R. Titration. values are for 5 ml. aliquots from 10 ml. cultures.

Hydrolysis of Proteins and Foodstu$s

Except when otherwise stated, the proteins and foodstuffs were hydro- lyzed by refluxing 0.5 to 2.0 gm. samples with 100 ml. of 6 N HCI for 24 hours. Most of the hydrochloric acid was removed by distillation at reduced pressure on the water bath. The hydrolysates were then neu- tralized and diluted to 100 ml. in a volumetric flask. If a precipitant was present, it was removed by titration after diluting to volume.

Preparation of Egg Albumin

Egg albumin was prepared from fresh eggs. A crystalline product was obtained by the method of Kekwick and Cannan (10). The material was

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166 METHIONINI DETERMINATION

then coagulated in hot water as described by Chibnall, Rees, and Wil- liams (11).

RESULTS AND DISCUSSION

Before the hydrogen peroxide-treated peptone was adapted for use in the Leuconostoc methionine medium, various modifications of a medium con- taining pure amino acids were tried in an attempt to improve the regularity of the standard curves and the reproducibility of the assay values. No modification of the pure amino acid medium was found which gave as con- sistently satisfactory results as the treated peptone medium.

Specificity tests with Leuconostoc mesenteroides and Streptococcus faecalis R showed that neither organism can use the non-natural or d form of methionine and that pure dl-methionine is a satisfactory standard since it is exactly one-half as active as I( -)-methionine. Homocystine and homocysteine were inactive for both organisms.

A study was made of the conditions necessary for the complete liberation of methionine from proteins by acid hydrolysis. The following three hydrolysis procedures gave methionine values which were in excellent agreement: (1) refluxing for 24 hours with 6 N hydrochloric acid; (2) re- fluxing for 24 hours with 6 N sulfuric acid; and (3) autoclaving at 15 pounds pressure for 6 to 8 hours with 3 N hydrochloric acid. Although autoclaving with 1 N hydrochloric acid has been successfully used for the liberation of certain other amino acids, this procedure proved to be unsatisfactory for methionine. Autoclaving for 8 hours or even longer with 1 N hydrochloric acid always gave low results.

Recovery tests with both assay organisms were carried out on a variety of different materials. In some of these tests the methionine was added to the materials before hydrolysis and in other tests the metbionine was

added to the neutralized hydrolysates. Satisfactory recoveries ranging from 97 to 102 per cent were obtained with both types of tests. Agreement between the values obtained at different test levels was a little more satis- factory with Leuconostoc mesenteroides than with Streptococcus faecalis R.

As a preliminary experiment in the comparison of the three methods for determining metbionine, two mixtures of pure amino acids were analyzed for their methionine content. With regard to the calorimetric method, evidence was desired concerning the following points: It is known that phosphotungstic acid precipitates carry down with them some of the mono- amino acids. Does the use of phosphotungstic acid for the removal of the basic amino acids, as recommended by Csonka and Denton, result in an appreciable loss of methionine? The second question is, does the glycine content of proteins and foodstuffs seriously interfere with the accuracy of this method?

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LYMAN, MOSRLEY, BUTLER, WOOD, AND HALE 167

Amino acid Test Mixture 1 (Table III) was patterned in a general way after the amino acid composition of casein. Since tryptophane is destroyed during acid hydrolysis, it was omitted. Amino acid Test Mixture 2 was the same as Test Mixture 1, except that the glycine was increased from 5 to 166 mg. The ratio by weight of the methionine content to the glycine content in Test Mixture 2 was therefore 1:4.

TABLE III

Amino Acid Test Mixture 1

trig. w. Alanine ............................ 55 Leucine ............................. 90 Arginine ........................... 40 Lysine .............................. 70 Aspartic acid ....................... 60 Methionine ......................... 25 Cystine ............................ 4 Phenylalanine. ..................... 50 Glutamic acid ...................... 230 Proline ............................. 80 Glycine ............................ 5 Serine .............................. 60 Histidine ........................... 25 Threonine ........................... 40 Hydroxyproline. ................... 20 Tyrosine ............................ 60 Isoleucine .......................... 60 Valine .............................. 60

TABLE IV Methionine Analyses of Amino Acid Test Mixtures

Test Mixture 1 Test Mixture 2

Adyticd methoa Me;tS&ne ReCOV~ Meftzdine Recovery

m. #CT cent mg. $61 cent

Chemical, McCarthy and Sullivan, modified by Csonka and Denton. . 25.3 101.2 24.3 97.2

Microbiological, Leuconostoc mesenter- aides................................. 24.8 99.2 25.0 100.0

Microbiological, Streptococcus faecalis R. 24.6 98.4 25.2 ldo.8

The results of the analyses given in Table IV indicate that the use of phosphotungstic acid for the removal of the basic amino acids does not result in a signilicant loss of methionine and that for the purpose of evalu- atingfoodstuffs a glycine content of 4 times that of methionine does not seriously interfere with the usefulness of the method. The figures given in Table IV are averages of values obtained at five different test levels.

Values for the methionine content of a few natural products as deter- mined by the three different procedures are given in Table V. Possible sources of errors would be expected to be quite different in the chemical and microbiological methods. Substantial agreement between the values

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168 MSTHIONINE DETERMINATION

obtained by the three methods was therefore considered as evidence in favor of the reliability of the methionine analyses.

In determining the methionine content of foodstuffs containing relatively large amounts of carbohydrates, one of the important problems is to make certain that the hydrolysis of the material is accomplished without undue loss of methionine as a result of humin formation or other causes. In this investigation an attempt was made to evaluate the effect of humin forma- tion on the loss of methionine by hydrolyzing casein in the presence of various carbohydrates. The casein carbohydrate mixtures were refluxed

TABLE V Comparison of Three Methods for Determining Methionine E#ect of Presence of Curbo-

hydrates during Hydrolysis of Protein

Material analyzed

Beef loin*................................. “ liver*.................................

Ceseint . . . . . . . . . . . . . . “ hydrolyzed in presence of equal

weight of sucrose.. :. . . . . . . . . . . . . . Casein hydrolyzed in presence of equal

weight of arabinose.. . . . . . . . . . . . . Casein hydrolyzed in presence of equal

weight of starch.. . . . . . . . . . . . . . . . -

Methionine found in 1 PI< stein

Microbiological method with Chemical method

-

. _

-

per cent 9.9 cent

2.52 2.52 2.34 2.24 2.72 2.58

2.42 2.42

2.49 2.55

2.45 2.46

psr cent

2.45 2.27 2.57

2.41

2.46

2.42

* Protein content calculated as nitrogen content X 6.25. t Difco isoelectric oasein; values not corrected for moisture and ash.

for 24 hours with 6 N hydrochloric acid. Methionine was then determined in the hydrolysates by three different methods.

The data given in Table V show that a small but measurable loss of methionine did take place. These data suggest that the loss of methionine, which takes place when such products as wheat and corn are hydrolyzed with strong mineral acids, is probably not large enough seriously to interfere with the usefulness of the methionine values.

Further work is needed to find a method of eliminating this loss. Hydrol- ysis with hydriodic acid reduces the amount of humin formation but is unsatisfactory when microbiological methods are to be used for the deter- mination of methionine because methionine is demethylated by hydriodic acid. It is known that basic hydrolysis results in the destruction of methionine.

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LYMAN, MOmLEY, BUTLIQIR, WOOD, AND HALD 169

Table VI gives the methionine content of some proteins as determined by the use of Leucorwstoc mesenteroides and 8treptococcus faecalis R. The values obtained by the two organisms are in substantial agreement, al- though most of the values obtained with Leucowstoc axe a few per cent higher than the values obtained with the other organism. Because of better agreement at different test levels, the values obtained with Leu- umostoc were considered to be the more accurate.

The ash content of, the casein sample was high, but the nitrogen content indicates that the sample was probably reasonably free from other im- purities. The methionine value for casein obtained with Leuconostoc

TABLE VI Methionine Content of Some Proteins As Determined by Two Microbiological Methods

Nitrogen and methionine values corrected for moisture and ash.

Protein an&&

Crystalline egg albumin.. Casein, Difco isoelectric. Bovine plasma albumin*. Blood fibrin, Wilson.. . . . Zeint . . . . . . Gelatin?. . . . . . Hemoglobint . . . . . . . . . .

-

-

-

Ash Nitrogen content content

psr cmt 0.17 1.33 0.53 1.08 0.34 1.06 2.28

par ten: 15.56 15.50 16.14 16.06 15.20 17.84 15.65$

T Methlonlne wntent

)I

-

p.s? csnt 4.54 2.96 0.81 2.18 1.65 0.88 1.51$

-

s

-

#.sr cmt 4.48 2.81 0.77 2.14 1.58 0.83 1.53f:

* Crystalline product obtained from the Armour Laboratories. t Commercial products. $ Corrected for moisture but not for ash.

mesenteroides, when calculated to a’moisture- and ash-free basis, becomes 2.96 per cent. Recently, Dunn (12) and coworkers have described a micro- biological method for the dete rmination of methionine in which La.ctob&llus fermenti is used as the test organism. The methionine content of casein reported by these workers was 3.03 per cent (corrected for moisture and ash). These values are in good agreement with the methionine content of casein as determined by a number of different chemical procedures (13-17). In general, the values obtained by the volatile iodide method (18) are somewhat higher.

Using their modification of the calorimetric method, Csonka and Denton (7) obtained a value of 2.72 (corrected for moisture and ash) for the methi- onine content of casein. The value of 2.57 (uncorrected) obtained by the

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170 METHIONINE DETERMINATION

present authors by this same method becomes 2.80 when corrected for moisture and ash.

The methionine content of crystalline egg albumin as reported here is in good agreement with the values obtained by Lavine (17) by both the periodide titration method and the homocystine method. The figures previously reported by Kassell and Brand (14) are substantially higher. The literature values for the methionine value of crystalline egg albumin show considerably less agreement than is the case with casein. For ex- ample, Csonka and Denton (7) reported a value of 3.73 for the methionine content of their sample of egg albumin.

TABLE VII Methionine Content of Some Foodstufls

Wheat. ................................ Corn .................................. Kafir .................................. Oatmeal ............................... Dried skim milk ....................... Lamb chop ............................. Pork liver .............................. Peanut meal ........................... Cottonseed meal ....................... Soy bean meal ......................... Alfalfa leaf meal .......................

Protein

Qcr cent 15.36 8.03 9.88

14.76 33.44 20.91 20.44 38.69 43.19 43.32 21.32

- I

T Moisture

#Jeer u?M

10.94 12.74 12.40 11.44 6.80

7.26 9.50

12.02 5.85

Methionine n foodstuffs

per cent gsr cenf 0.197 1.28 0.167 2.08 0.150 1.52 0.236 1.60 0.872 2.61 0.512 2.45 0.447 2.19 0.297 0.77 0.615 1.42 0.574 1.33 0.255 1.20

Methionine in protein

The methionine values obtained in the present investigation by the use of Streptococcus faecdis R are significantly and consistently higher than those obtained by Stokes et al. (5) with the same organism but a different medium.

The methionine content of some foodstuffs is given in Table VII. These data were obtained by the use of Leuumostoc mesenteroides.

This investigation was supported in part by a grant from the American Meat Institute.

The authors wish to express their appreciation to the Lederle Labora- tories, Inc., for the synthetic folic acid used in this investigation.

SUMMARY

A method for determining methionine in proteins and food&u& by the use of Leuconostoc mesenteroides is described. In this method a simplified

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LYMAN, MOSELEY, BUTLER, WOOD, AND HALE 171

medium is used in which most of the ammo acid nitrogen is supplied by hydrogen peroxide-treated peptone.

Methionine was determined in a variety of natural products by the use of Streptococcus fat~~lis R as well as with Leuconostoc mesenteroides. The two microbiological methods were checked against each other and also against a calorimetric method for determining methionine. Substantially the same values were obtained with all three methods.

Tests in which casein was hydrolyzed with acid in the presence of various carbohydrates showed that the presence of the carbohydrates during hydrolysis resulted in a small but measurable loss of methionine.

The methionine content of some proteins and foodstuffs is given.

BIBLIOGRAPHY

1. ,Greene, R. D., and Black, A., Proc. Sot. Exp. Biol. and Med., 54, 322 (1943). 2. Lewis, J. C., and Olcott, H. S., J. Biol. Chem., 167, 265 (1945). 3. Toennies, G., and Callan, T. P., J. Bio.?. Chem., 129, 481 (1939). 4. Lyman, C. M., Moseley, O., Wood, S., and Hale, F., Arch. Biochem., 10,427 (1946). 5. Stokes, J. L., Gunness, M., Dwyer, I. M., and Caswell, M. C., J. Biol. Chem., 166,

35 (1945). 6. McCarthy, T. E., and Sullivan, M. X., J. Biol. Chem., 141,871 (1941). 7. Csonka, F. A., and Denton, C. A., J. Biol. Chem., 163,329 (1946). 8. Kuiken, K. A., Norman, W. H., Lyman, C. M., Hale, F., and Blotter, L., J. Biol.

Chem., 161, 616 (1943). 9. Wu, H., J. Biol. Chem., 43, 189 (1926).

10. Kekwick, R. A., and Cannan, R. K., Biochem. J., 30,277 (1936). 11. Chibnall, A. C., Rees, M. W., and Williams, E. F., B&hem. J., 37, 354 (1943). 12. Dunn, M. S., Camien, M. N., Shankman, S., and Block, H., J. Biol. Chem., 163,

577 (1946). 13. Baernatein, H. D., J. Biol. Chem., 116, 25,33 (1936). 14. Kassell, B., and Brand, E., J. Bid. Chem., 125, 145 (1938). 15. Kuhn, R., Birkofer, L., and Quackenbush, F. W., Ber. them. Ges., 72,407 (1939). 16. Beach, E. F., and Teague, D. M., J. Biol. Chem., 142,277 (1942). 17. Lavine, T. F., J. Biol. Chem., 161,231 (1943). 18. Baernstein, H. D., J. Biol. Chem., 97, 669 (1932).

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Suzanne Wood and Fred HaleCarl M. Lyman, Olive Moseley, Betty Butler,

III. METHIONINEDETERMINATION OF AMINO ACIDS:

THE MICROBIOLOGICAL

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