FAT-SOLUBLE VITAMINS

XXXVIII. MICROORGANISMS AND THE SYNTHESIS OF CAROTENE AND VITAMIN A*

BY CARL A. BAUMANN ANII H. STEENBOCK

(From the Department of Agricultural Chemistry, University of Wisconkn, Madison)

4ND MARY A. INGRAHAM AND E. B. FRED

(From the Department of Agricultural Bacteriology, University of Wisconsin, Madison)

(Received for publication, September 11, 1933)

Synthesis of Carotene by Microorganisms

The presence of carotenoids in microorganisms has long been accepted, but the available evidence for this belief is meager. The earliest claims are based on the solubility of certain bac- terial pigments in fat solvents, and on the production of a blue color upon the addition of concentrated sulfuric acid (1). Later Tswett’s chromatographic method (2) was applied and the various fractions obtained were examined spectroscopically. By means of this technique the absorption bands of carotene and of lycopene were identified (3-5). Reader furthermore succeeded in isolating enough crystals of lycopene for identification under the micro- scope (3). Since carotene is converted by the animal into vitamin A (6), it was to be expected that carotene-containing microorgan- isms would be able to correct symptoms of vitamin A deficiency, but early attempts to demonstrate such activity in microorganisms by means of rat feeding experiments failed. Assays were made by Wolhnann and Vagliano (7), Slanetz (8), Cunningham (9), Bieling

* Published with the permission of the Director of the Wisconsin Agri- cultural Experiment Station.

Presented in part before the American Society of Biological Chemists at Cincinnati, April 10, 1933 (Baumann, C. A., Steenbock, H., and Ingraham, M. A., J. Biol. Chem., 109, xiii (1933); Proc. Am. Sot. Biol. Chcm.$, xiii (1933)).

339

by guest on June 16, 2020http://w

ww

.jbc.org/D

ownloaded from

340 Fat-Soluble Vitamins. XXXVIII

(lo), and Reader (3). Since these experiments were carried out before the importance of vitamin D for growth in rats was realized, and since the investigators did not always publish full experi- mental details, it is impossible to determine the value of these findings. That a certain yellow Corynebacterium could synthesize a vitamin A-active substance from an inactive substrate was first demonstrated by Skinner and Gunderson (11) in 1932. They did not determine, however, whether vitamin A itself, its pre- cursor carotene, or some other substance capable of functioning as vitamin A in the rat was produced. As vitamin A itself has not been found except in materials of animal origin, we have examined this Corynebacteriuml further to determine the nature of the biologically active material. In addition we have analyzed cultures of other yellow organisms for carotene, and have made confirmatory tests by feeding the dried crude cultures of four difYerent strains of these organisms to rats which showed symp- toms of vitamin A deficiency.

The organisms were grown on agar slants. A solid substrate was preferred to a liquid medium because these aerobic organisms were found to grow more rapidly and also to produce more pigment on agar than on broth. A yeast water medium was used which contained agar 20 gm., glucose 10 gm., KzHPOl 1 gm., MgSOr.- 7Hz0 1 gm., NaCl 1 gm., yeast water2 100 cc., and tap water 900 cc. 2800 gm. of agar were melted in 126 liters of water in a steam- kettle, and the other ingredients were added immediately before bottling. About 100 cc. were added to a 12 ounce signet bottle. The bottle was capped, autoclaved, and slanted to give a surface of 90 sq.cm. available for growth. Inoculation was made with compressed air which was forced through 3 feet of sterile cotton wadding in a metal gun. The air forced a water suspension of the organisms through an atomizer in a continuous spray. The bottles were quickly uncapped, momentarily held over the tip of the atomizer and then recapped. In this manner it was found

1 We wish to express our thanks to Dr. Skinner of the University of iMinnesota for giving us a culture of this organism for our experimental work.

* The yeast water was made from a 10 per cent suspension of starch-free and moist brewers’ yeast by steaming it for 2 to 3 hours, then sterilizing and allowing suspended matter to settle out.

by guest on June 16, 2020http://w

ww

.jbc.org/D

ownloaded from

Baumann, Steenbock, Ingraham, and Fred 341

possible to inoculate about 1000 bottles in an hour. About 350 bottles were used in each experiment,. The yield was between 0.1 and 0.3 gm. of dried cells per bottle. Contamination rarely re- sulted.

The organisms reached maximum growth and pigmentation in approximately 2 weeks, when they were removed from the agar slant by means of suction. The cellular material was then placed in evaporating dishes, dried at 50” in the dark, and stored at 4”. The product was analyzed spectroscopically for carotene and biologically for total vitamin A activity. The organisms fed were t,he diphtheroid used by Skinner and Gunderson, Organism G-101; a closely related diphtheroid obtained from lake water, Organism G-102; two non-sporulating rod forms, Organisms D-202 and D-205; and one spore-forming rod, Organism E104. These organisms, as well as related types, will be more fully discussed in another paper.

Carotene determinations were made as follows: The cultures were extracted with hot 95 per cent alcohol, the alcohol was dilmed with water to a concentration of 85 per cent, and the solution was then extract,ed with petroleum ether. Partition between these solvents separated the pigment into carotene and xanthophyll fractions. The yellow pigment content of each was determined with a Lovibond tintometer. The petroleum ether-soluble caro- t.ene fraction was then saponified by the addition of an equal vol- ume of 20 per cent alcoholic KOH and heated for 45 minutes at 60’ under a stream of nitrogen. In the process the petroleum ether was distilled off. The saponification mixture was t,hen diluted with water, extracted with ether, and submitted to the phase test as above. Xanthophyll esters, originally soluble in the petroleum ether, were thus liberated as free xanthophyll and appeared in the alcohol layer. The yellow units of both fractions were read in the tintometer, and the petroleum ether fraction was examined spectroscopically in chloroform solution. Both tinto- metric and spectrophotometric readings gave a measure of the amount of carotene present.

For the presence of vitamin A, spectral absorption at 323 rnp was t.aken as the criterion. Solutions for analysis were prepared as follows: The crude culture was extracted with hot 95 per cent alcohol, and the extract was saponified by the addition of enough concent,rated aqueous KOH solution to bring the concentration of

by guest on June 16, 2020http://w

ww

.jbc.org/D

ownloaded from

342 Fat-Soluble Vitamins. XXXVIII

the KOH to 10 per cent,. Saponification was continued for 30 min- utes under a reflux at 70” and under Nz. The alcohol solution was extracted with et,her, the ether washed, dried, and evaporated, and t,he residue dissolved in a small volume of absolute methyl alcohol. After freezing in a mixture of solid COZ and acetone, the methyl alcohol solution was filtered and analyzed with a Hilger quartz spectrophotometer.

The vitamin A. activity of the microorganisms was determined by feeding the dry cultures to rats on a standard low vitamin A ration. Rats were kept on our basal synthetic ration (12) which consists of casein 18, cooked corn-starch 69, agar 2, Salts 40 (13) 4, yeast 6.9, irradiated yeast 0.1. This ration is adequate for the purpose intended in all dietary factors except vitamin A. When the animals showed both a cessation of growth and definite symptoms of ophthalmia, the dry cultures were added to the diet. Restoration of growth and cure of ophthalmia were taken as indices of the amount of vitamin A activity present. Each culture was fed at four levels of supplement,, and three animals were placed upon each level. The absence of carot,ene and of vitamin A in t,he bacterial culture medium tias demonstrated not only by the nega- tive results obtained wit,h many organisms but also by the failure of rats to grow when t,hey received 3 gm. daily of the yeast from which the medium had been prepared.

Although to date we have not isolated carotene as such from our microorganisms, t.he method of preparation of the carot,ene fraction to our mind constitutes proof that carotene itself was present, The processes of saponification and solvent partition outlined above remove all known pigments from the petroleum ether solution except carotene and lycopene. These can readily be identified by their absorption spectra, since the bands of lyco- pene differ from those of carotene in that they are markedly dis- placed toward the red end of the spectrum (14,15). As measured on a Bausch and Lomb universal spectrophotometer, the absorp- tion spectrum of the carotene fraction was found to be identical with that of a solution of pure carotene prepared from carrots.” The carotene curve and that of bacterial carotene were of the same

S The carotene was crystalline, melted at HI”, and was part of the prep- aration sent by this laboratory to the British Medical Research Council for use as part of the international standard preparation of vitamin A.

by guest on June 16, 2020http://w

ww

.jbc.org/D

ownloaded from

Baumann, Steenbock, Ingraham, and Fred 343

general shape (Chart I). The maxima appeared at 460 and 485 rnp, and the ratio of the extinction coefficients h& and EdM) were in the ratio of 1.00: 1.19 in both cases. As did carotene from other sources, the bacterial carotene gave a blue color when treated with SbCh in chloroform. The ratio of the yellow to blue units, as

Carrot 8

Bacterial Carotene Fraction-

Wave Length.. . mp

450 500 550

CHART I. Absorption spectra of pure carotene and of the carotene frac- tion of Organism G-101 (in CHCS).

measured on a Lovibond tintometer was 6.2: 1.0, conforming to the properties of plant carotene. Furthermore our bacterial caro- tene in petroleum ether solution was not adsorbed by CaCQ, nor was the spectral absorption altered by the attempt.

In no organism examined was carotene the only yellow pigment

by guest on June 16, 2020http://w

ww

.jbc.org/D

ownloaded from

344 Fat-Soluble Vitamins. XXXVIII

present. In fact the maximum proportion of yellow units due to carotene was 50 per cent. In certain strains such as our Organism

75 mge. Or@?dam G-102 Daily

CHART II. Growth of rats on dried cultures of microorganisms

D-202, an encapsulated bacterium, only 6 per cent of the total yellow pigmentation was attributable to carotene. The ratio of

by guest on June 16, 2020http://w

ww

.jbc.org/D

ownloaded from

Baumann, Steenbock, Ingraham, and Fred 345

carotene to the other yellow pigments appeared to vary not only with the strain of organism, but also with age, with the rate of growth, with the freshness of the culture, and with the composition of the culture medium. No attempts were made to study pig- ments other than carotene. Cursory examination, however, indi- cated that there were present xanthophyll and xanthophyll esters, and also other pigments soluble in alcohol, which turned blue or a brilliant red on the addition of alkali.

In each of the organisms which gave a positive growth response when fed to rats, enough carotene was found to be present in the amounts fed to account for the observed vitamin A activity. For Organism G-101 the minimum daily dose of dried bacterial growth required to restore growth and cure ophthalmia was 60 mg. (Chart II). This contained 1.37 of carotene. For Organism G-102, a closely related form, the minimum dose was 75 mg., equivalent to 2.6~ carotene. For Organism D-202, an encapsulated form, the minimum daily dose was 55 mg. which contained 37 of carotene. Although within the scope of our experiments the amounts of bacterial carotene found necessary were not in exact agreement, they were equal to or in excess of the amount of pure carotene, namely, 1.37, necessary for a similar response. Hence it is unnecessary to assume in these microorganisms the presence of vitamin A itself. Organism D-205 which contained large amounts of alcohol-soluble pigment, but none soluble in petroleum ether, was inactive as a source of vitamin A. Organism E104, a yellow type, the pigment of which was insoluble in alcohol, was likewise inactive.

That the biological activity of the organisms resides in the puri- fied petroleum ether fraction was demonstrated by feeding such a fraction prepared from Organism G-102 in cottonseed oil to rats in daily amounts equivalent to 12 and 48 Lovibond yellow units respectively. 12 yellow units = 6-y carotene. Both levels gave a growth response. The corresponding alcohol fractions were devoid of activity in daily doses equivalent to 96 yellow units.

The biological activity of the microorganisms is not due to vita- mm A itself, since absorption studies showed no absorption in the region of 320 to 330 rnp in addition to that which could be attrib- uted to the carotenoid pigments present. Another argument against the presence of vitamin A as such is the fact that the

by guest on June 16, 2020http://w

ww

.jbc.org/D

ownloaded from

346 Fat-Soluble Vitamins. XXXVIII

Carr-Price (16) yellow to blue ratio, 6.2:1.0, which was obtained on the active fraction of microorganisms, is within the normal range for the carotenoid pigments. If vitamin A were present in sufficient amounts to account for the biological activity, the yellow to blue ratio.of the bacterial extracts would be abnormally low for carotenoid pigments, since the pure vitamin shows a yellow to blue ratio of 1: 200.

Alleged Transformation of Carotene into Vitamin A by Microorganisms

The close chemical relationship between carotene and vitamin A suggested the possibility that the transformation of the pigment into the vitamin by animals (12) might be duplicated with micro- organisms. In an attempt to effect such a transformation we exposed samples of carotene to the action of many types of micro- organisms on various media and then examined the products for vitamin A by means of the Carr-Price SbCh reaction (16).

The carotene used in the majority of these experiments was a crude carotene made from carrots by petroleum ether extraction. It was brought into the colloidal state as follows: The petroleum ether extract was evaporated, the residue dissolved in hot acetone, and the acetone solution dropped into a large volume of water at 80” at such a rate that most of the acetone was immediately vola- tilized. During this procedure the water was agitated continu- ously to aid in the dispersion. After the addition of the carotene solution, the aqueous mixture was distilled to one-half its original volume under reduced pressure in the presence of nitrogen.

In the 6rst cultures the colloidal carotene was added directly to the media, which were then sterilized and incubated with the appropriate organisms. Later it was deemed advisable to defer the addition of carotene until the fermentation was proceeding vigorously. This latter procedure had the advantage that it avoided changes in the carotene which might have been caused by autoclaving. In some cases cultures were also made with pure carotene in the colloidal as well as in the crystalline condition. The following media were used: (1) a crude corn mash, (2) a glu- cose peptone medium, (3) yeast water prepared from autoclaved brewers’ yeast, and (4) an inorganic medium.

The organisms were grown under conditions of temperature,

by guest on June 16, 2020http://w

ww

.jbc.org/D

ownloaded from

Baumann, Steenbock, Ingraham, and Fred 347

pH, and available nutrient known to be favorable for their develop ment. They are listed in Table I. They include seven species of bacteria, thirteen species of yeasts, twelve species of molds, and thirteen mixed cultures obtained from soil, manure, air, water,

TABLE I

Organisms with Which Fermentation of Carotene was Attempted

Bacterial species

Staphylococcus aureus Streptococcus lactis Sarcina lutea Bacillus subtilis Leuconostoc mesenterioides (2 strains) Bacterium dioxyacetonicum Acetobacter aceticum Escherichia coli Aerobacter aerogenes Serratia marcescens

‘6 pokwy~ Rhizobium meliloti (2 strains)

Chromobacterium violaceum Clostridium acetobutylicum Thermophilic cellulose-fermenting “ acetoethylicum

culture “ saccharobutylicum

Yeasts

Saccharomyces cerewisis (3 strains) ‘I thermantitorum “ carlsbergensis

Monilia psilosis Endomyces vernalis Zygosaccharomyces (from pollen)

Torula pulcherrima L‘ dattila “ alactosa “ mono58

Willia anomala 3 unknown species

Molds

Aspergillus jischeri “ fumigatus ‘I niger ‘I sydowi &I nidulans

Syncephalin

Penicillium chrysogenum “ purpurogenum

Pzecilomyces varioti Cunninghamella elegans Homodendron

Also natural mixed flora of air, peat, manure, soil, water, milk, and sewage cultured at 20”, 30”, 37”, and 55”

milk, sewage, and peat. Incubations were carried out for periods varying from 2 days to 3 weeks. Samples were usually run in triplicate with a positive control of carotene plus medium, and a negative control of organism plus medium. Most of the experi- ments were run at at least two different temperatures.

by guest on June 16, 2020http://w

ww

.jbc.org/D

ownloaded from

348 Fat-Soluble Vitamins, XXXVIII

Vitamin A determinations were carried out as follows: The medium was treated with an equal volume of 95 per cent alcohol to facilitate extraction. It was then extracted with ether, the ether evaporated under reduced pressure with nitrogen, and the residue dissolved in chloroform. The Carr-Price test was t,hen carried out, a solution of SbCh being used in chloroform saturated at 0”. Since both carotene and vitamin A are known to produce a blue color with SbCl,, and since intermediates in the transformation of

TABLE II

Changes in Yellow to Blue Ratio on Addition of Vitamin A to Carotene

Lovibond yellow units

0.4 co. burbot liver oil*. 5 “ colloidal carotene + 1 drop

burbot liver oil. 5 cc. colloidal carotene + 2 drops

burbot liver oil. . . 5 cc. colloidal carotene + 4 drops

burbot liver oil.. . . 5 cc. colloidal carotene + 8 drops

burbot liver oil. . .

5

365

402

384

378

-

_

-

Lovibond Yellow to blue units blue ratio

829 1:172

193 1.84:l

236 1.7:1

351 l.l:l

512 1:1.4

* The burbot oil was an oil extracted from the livers of the fish and was obtained through the courtesy of the Smith Brothers Fisheries, Port Washington, Wisconsin. Since 0.4 cc. = 829 blue units, 1 cc. = 2070 blue units. The pipette delivered 44 drops per cc.; therefore 1 drop = 46 blue units. The recovery of vitamin A when added to colloidal carotene can be calculated as follows: Carotene + 1 drop oil = 193 blue units; carotene + 8 drops oil = 512 blue units. 7 drops of oil increase the total blue units by 512 to 193 = 319 blue units. 319 blue units -I- 7 = 45 blue units per drop, which agrees with the direct determination.

the one form into the other would probably also form a blue color, this transformation can be detected with the SbCla reaction only when the quantitative relations of the colors formed are considered. This fact is neglected in a recent claim that carotene was trans- formed into vitamin A by means of liver enzymes because, after incubation and treatment with SbCls, “the appearance of a blue color is interpreted as due to the presence of vitamin A” (17).

The reaction of carotenoids with SbC& is admittedly open to certain objections. The intensity of the blue color is affected by

by guest on June 16, 2020http://w

ww

.jbc.org/D

ownloaded from

Baumann, Steenbock, Ingraham, and Fred 349

such factors as the temperature, the concentration of the reagent, the presence of inhibitors, and even the intensity of illumination (18, 19). However, Moore (20) has shown that carotene and vitamin A have such widely divergent yellow to blue ratios, that the SbCh test is of value in distinguishing between these sub- stances. Carotene shows a yellow to blue rat,io of 6: 1; vitamin A, of 1: 200. The addition of vitamin A to carotene therefore results in a marked increase in the blue values, with a corresponding fall in the yellow to blue ratio, since t,he total number of yellow units is not materially altered by vitamin A additions (see Table II). If, on the other hand, carotene is transformed into vitamin A, there will result a destruction of carotene, and hence the total number of yellow units will be decreased at a more rapid rate than the total blue units. The formation of vitamin A will cause a relatively large increase in the blue values, and hence the yellow to blue rat,io will fall markedly, even when the proportion of carotene transformed into vitamin A is small. A drop in the yellow to blue ratio wit.h an increase in total blue values is therefore a neces- sary accompaniment of the transformation of carotene into vitamin A, but such a drop in ratio, as Woolf and Moore have pointed out (21), does not in itself constitute a proof of the formation of vit.amin A. The absence of these changes in color, however, de& nitely indicates the absence of t,he transformation.

In none of t,he organisms examined was the synthesis of vit,amin A demonstrated. Usually there was a decrease in the number of tota. yellow units as a result of exposure to the conditions of the fermentation, and this decrease was accompanied by a Iesser decrease in the number of blue units. The yellow to blue ratio was therefore lowered. However, similar changes occurred in the cont,rol samples of colloidal carotene. In fact, many organisms appeared to exert a protective effect upon the carotene, since fre- quently the rate of fading of the pigment proceeded most rapidly in t,he control sample. In no case was the total number of blue units increased, and hence it appears that under t,he conditions of the experiment carotene is not transformed into vitamin A by these microorganisms. This is in harmony with the observation of Ahmad and Drummond (22) who reported that organisms of the digestive tract do not transform carotene into vitamin A,

by guest on June 16, 2020http://w

ww

.jbc.org/D

ownloaded from

350 Fat-Soluble Vitamins. XXXVIII

DISCUSSION

The presence of carotene in microorganisms naturally raises the question of the value of that carot.ene to the organism, but on the basis of available data it is impossible to attribute any definite function to this pigment. The amount, of pigment in dried cellular matter appears to vary with the nature of the medium, with the pH of the medium, with the rate of growth of the organism, with the age of the culture, and, in certain cases, with the length of time which has elapsed since the organism was removed from its natural habitat.

It is known that some organisms have the power to absorb pig- ment from the surrounding medium. Thus a routine method of distinguishing the avian from the human tubercle bacillus consists of placing the organism in old blood serum from which the human form absorbs pigment whereas the avian variety does not. Our medium was free from carotene, as was demonstrated by feeding trials, but there remains the possibility that some samples of yeast could have been contaminated with carotene-containing organisms. Such an assumption might possibly explain the find- ing of Honeywell, Dutcher, and Ely (23) that certain yeast sam- ples possessed vitamin A activity, a finding which, however, has not yet been duplicated by other laboratories.

If we accept our evidence that carotene is actually synthesized by the organisms in question, then in this respect, these organisms resemble plants more closely than animals, since carotene when present in animals is of exogenous origin. The absence of vitamin A from microorganisms and their failure to transform carotene into vitamin A likewise associate these organisms with plants, since vitamin A as such has not been demonstrat,ed in a plant material. The similarity in structure of the carot,ene and the phytol mole- cules, as well as the fact that carotene always accompanies chloro- phyll in plants, has led to the belief t,hat carotene synthesis is in some way associated with chlorophyll activity. In microorgan- isms, however, we have an illustration of the fact that the forma- tion of carotene is not dependent upon the presence of chlorophyll.

SUMMARY

Certain microorganisms were found to synthesize carotene. Whenever an organism showed vitamin A activity as determined

by guest on June 16, 2020http://w

ww

.jbc.org/D

ownloaded from

Baumann, Steenbock, Ingraham, and Fred 351

by feeding experiments, enough carotene was found to be present to account for that activity. The vitamin A activity of microorgan- isms did not appear to be affected by the presence of yellow pig- ment,s other than carotene. Since spectrographic determination failed t,o reveal an absorption band at 328 rnp, it is exceedingly improbable that vitamin A as such is generally present in bacteria. Attempk to effect the transformat,ion of carotene int,o vitamin A by microorganisms failed.

BIBLIOGRAPHY

1. Zopf, W., Bat. Zig., 49,53,09,35 (1389) ; 2. tissensch. mikr., 6,172 (1889). 2. Tswett, M,, Ber. hot. Ges., 24,316 (1906). 3. Reader, V., Biochem. J., 19, 1039 (1925). 4. Chargaff, E., Centr. Bald., 1. Abt., 119,121 (1930). 5. Stone, F. M., and Coulter, G. B., J. Gen. Physfol., 16,629 (1932). 6. Moore, T., Lancet, 2,330 (1929). 7. Wollmann, E., and Vagliano, H., Corn@. rend. Sot. biol., 66, 832 (1922). 8. Slanetz, E. J., A&t. Bat., 7, 352 (1923). 9. Cunningham, R. L., Am. Rev. Tuberc., 9,487 (1924).

10. Bieling, R., 2. Hyg. u. Infektionskr., 194,347 (1925). 11. Skinner, C. E., and Gunderson, M. F., J. Biol. Chem., 97,53 (1932). 12. Steenbock, H., Nelson, M. T., and Black, A., J. Biol. Chem., 62, 275

(1924-25). 13. Steenbock, H., and Nelson, E. M., J. BioZ. Chem., 66,355 (1923). 14. Willstatter, R., and Stall, A., Untersuchungen fiber Chlorophyll, Berlin

(1913). 15. Monteverde, N. A., and Lubimenko, N. W., BUZZ. acad. SC., Petrograd,

series 6, 7, 1105 (1913), cited by Palmer, L. S., Carotenoids and re- lated pigments, American Chemical Society monograph series, 222 (1922).

16. Carr, F. H., and Price, E. A., Biochem. J., 29,497 (1926). 17. Pariente, A. C., and Ralli, E. P., Proc. Sot. Ezp. BioZ. and Med., 29,

1209 (1932). 18. Norris, E. R., and Church, A. E., J. BioZ. Chem., 69,421 (1930). 19. von Euler, B., and Karrer, P., HeZv. chim. acta, 16,496 (1932). 20. Moore, T., Biochem. J., 24,692 (1930). 21. Woolf, B., and Moore, T., Lancet, 2, 13 (1932). 22. Ahmad, B., and Drummond, J. C., J. Sot. Chem. Znd., 59, 133 T (1931). 23. Honeywell, H. E., Dutcher, R. A., and Ely, J., J. Nutrition, 3,491(1931).

by guest on June 16, 2020http://w

ww

.jbc.org/D

ownloaded from

Ingraham and E. B. FredCarl A. Baumann, H. Steenbock, Mary A.

VITAMIN ASYNTHESIS OF CAROTENE AND

MICROORGANISMS AND THE FAT-SOLUBLE VITAMINS: XXXVIII.

1933, 103:339-351.J. Biol. Chem. 

  http://www.jbc.org/content/103/2/339.citation

Access the most updated version of this article at

 Alerts:

  When a correction for this article is posted• 

When this article is cited• 

alerts to choose from all of JBC's e-mailClick here

  tml#ref-list-1

http://www.jbc.org/content/103/2/339.citation.full.haccessed free atThis article cites 0 references, 0 of which can be

by guest on June 16, 2020http://w

ww

.jbc.org/D

ownloaded from