Studies on lipoic acid uptake by bacteria

BIOCHIMICA ET BIOPHYSICA ACTA

41

BBA 4204

STUDIES ON LIPOIC ACID UPTAKE BY BACTERIA I. CHARACTERIZATION OF T H E REACTION DONALD C. SANDERS AND FRANKLIN R. LEACH Department of Biochemistry, Oklahoma State University, Stillwater, Okla. (U.S.A.) (Received March i8th, 1963) SUMMARY An energy-requiring, temperature-dependent system capable of concentrating lipoic acid in the cell pool exists in Streptococcus faecalis. This system is easily saturated with lipoic acid and is constitutive. Octanoic acid competes with lipoic acid while the analogues 8-methylthioctic acid, 1,2-dithiolane-3-caproic acid, and 1,2-dithiolane3-butyric acid are without effect. The cell wall does not influence uptake in any ratelimiting fashion. The system has been demonstrated in Escherichia coli, Staphylococcus aureus, and Aerobacter aerogenes. INTRODUCTION Transport mechanisms are essential in controlling both the extent and rate of bacterial cell growth. COHEN AND MONOD1 have suggested the generic name "permease" for the stereospecific, functionally distinct enzymes responsible for the transport process. Systems for the transport of carbohydrates 2, amino acids 3, 4, peptides s and vitamins e-9 have been demonstrated in bacterial cells and various properties of these systems have been documented. Certain of the permeases are under the control of inductionrepression while others appear to be present constitutively 1. Because of the widespread metabolism of most of the compounds used as substrates in permeation studies, the characterization of the actual transport complex has not been possible. The uptake systems for vitamins should be relatively specific, and it might be possible because of the limited metabolism of vitamins to isolate a transport complex. Although vitamin B12 (see ref. 6), biotin 7,s and folic acid 9 uptake have been studied in microorganisms, the unknown aspects of their metabolism and functioning might make interpretation of results difficult. Therefore, a system was selected which would catalyze the uptake of a vitamin that had limited and known metabolic functions. Lipoic acid was the vitamin of choice, since the reactions through which the free vitamin is converted to the protein-boundlO, n, enzymatically functional form are known. Also, the nature of its binding to the protein through the e-amino group of lysine12,13, and its sole role in S. faecalis and E. coli as an essential component of ,¢-keto acid oxidases have been establishedJ4,15. This paper reports properties of an uptake system present in S. favcalis 10CI for lipoic acid. Work by GONSALUS16 suggested that the uptake system should be constitutive in these cells. Two preliminary communications have appeared ~7,is. Bioehim. Biophys. Acta, 82 (1964) 41-49

42

D . C . SANDERS, F. R. LEACH MATERIALS AND METHODS

Synthesis of 35S-labeled lipoic acia Ethyl-DL-6,8-dichlorooctanoate was prepared from adipic acid by the procedure described by REEl) AND NIU t°. 3aS was introduced into DL-6,8-dichlorooctanoie acid using NazSx by the procedure of ACKER AND WAYNE20. The crude labeled lipoic acid was distilled as described by THOMAS AND REED 21. The crystalline product had a melting point of 57-5 80 and gave an absorption spectrum identical to authentic lipoic acid. Ascending paper chromatography in two solvent systems yielded a single nilroprusside-sodium cyanide-positive spot which contained 99 % of the radioactivity. The remaining radioactivity was found at the origin. Specific activity of the final product was 46/zC/mg.

Growth of cells Cells of S.faecalis l o C I were grown for 8-1o h at 37 ° in the lipoic acid-free medium described by GUNSALUS et al. 22. The cells were then harvested by centrifugation and washed twice with cold salts solution as described by LEACH AND SNELL5. The cell yield was determined using a standard curve of dry weight versus ahsorbancy.

Measurement of uptake A quantity of cells sufficient to give a final concentration of 300/zg (dry wt.) per ml was added to a tube containing the salts solution described previously. The cells were equilibrated to temperature for 15 rain. Glucose was added to a concentration TABLE 1 Rt~LEASE OF CELL CONTENTS F i v e 5-ml cell s u s p e n s i o n s c o n t a i n i n g i m g of cells a n d t m g of glucose pe r ml were t r e a t e d as s h o w n below, t h a w e d in cold (if frozen), centrifuged, a n d t h e a b s o r b a n c y of t h e s u p e r n a t a n t r e a d a t 260 m/*. A m e a s u r e of t o t a l release was o b t a i n e d b y s o n i c a t i o n of one a l i q u o t for 45 rain u s i n g a R a y t h e o n IO kC sonic oscillator. Treatment

N o t frozen Q u i c k frozen (N2) Slow freezing ( - - 2 4 ° ) M u s h y ice Sonicated

,]~n ml ~

0.086 o.121 o.131 0.062 I. 7 °

of I mg/ml and incubation continued for 15 min. Then labeled lipoic acid was added and aliquots were removed at appropriate intervals. The uptake reaction was stopped by ejecting the aliquot (0.5 ml) into mushy ice (I ml of frozen salts solution) or into a centrifuge tube previously cooled to the temperature of liquid nitrogen and suspended in a Dewar flask of the coolant. The aliquots were thawed slowly at 2 °. The cells were sedimented by centrifugation and washed twice with cold salts solution. Two washings were sufficient to remove essentially all of the radioactivity which could be washed away (o.I % of the total removed by extensive washing remained). After the final washing, the cell pellet was suspended in 0.5 ml of distilled water, transferred Biochim.

B i o p l ~ v s . ~lcta, 82 (19(~4) 4 f 4'~

STUDIES OF LIPOIC ACID UPTAKE BY BACTERIA. I.

43

to a planchette and then dried. Radioactivity was determined using a Baird-Atomic ultra-thin-window gas-flow Geiger tube and counted to a 2 % standard counting error. In each case a blank value obtained by incubation of radioactive lipoic acid with boiled cells was subtracted. Table I shows that the stopping of the reaction either by cooling with mushy ice or freezing in a liquid nitrogen cooled tube did not release excessive amounts of 260 m/z absorbing material from the cells. Either method was used to stop the reaction.

Fractionation of cells To determine the distribution of the lipoic acid in the cell, a modification of the fractionation procedure of PARK AND HANCOCKza was used. Fractionations by this procedure resulted in a high percentage of the total radioactivity in the lipid (alcoholsoluble) fraction and a low percentage in the pool (trichloroacetic acid-soluble) fraction. This artifact was found to be due to the insolubility of the lipoic acid in the acidic solution. In the modified procedure, the pool lipoic acid was extracted by boiling the cells in distilled water for 15 min and then the original procedure was followed. Table I I shows results of using both fractionation schemes. TABLE II A COMPARISON OF FRACTIONATION AND

BY THE

METHOD

A MODIFIED

METHOD

OF PARK

AND

HANCOCK

Two 5-ml suspensions of cells (i mg/ml) and glucose (I mg/ml) in neutral salts solution were exposed to radioactive lipoic acid (io/~g/ml) for 3 ° nlin at 2o ° as described u n d e r normal u p t a k e conditions. After washing, one cell pellet was fractionated b y the m e t h o d of PARK AND HANCOCK. The o t h e r pellet was first boiled in 5 ml of distilled w a t e r for 15 rain to remove the pool lipoic acid; the r e m a i n d e r of the fractionation was the same as described for the first pellet. Percent of total radioactivity Fraction

Method

of PARK

Modified

HANCOCK

m,elhod

W a t e r (boiling)

--

57.2

Cold trichloroacetic acid (5 ~o at o °)

o.36

AND

E t h a n o l - w a t e r , 3: I (v/v)

54.9

H o t trichloroacetic acid (5 ~o at 9 o°)

o.22

2.2 11.4 3.9

T r y p s i n soluble

18.2

9.4

Residue

2 i. I

14.6

RESULTS

Proportionality of uptake to cell concentration To determine the cell concentration range over which the uptake of lipoic acid would be proportional, varying concentrations of cells were incubated for 30 min with IO/zg labeled lipoic acid per ml as shown in Fig. I. The amount of uptake was proportional to cell concentration up to 500 /Lg/ml; thereafter increased cell concentration did not increase uptake. In most experiments a cell concentration of 300 /~g/ml was used. With o.5-ml samples taken, there was no requirement for self-absorption correction. Biochim. Biophys. Acta, 82 (1964) 41-49

44

D . C . SANDERS, F. R. LEACH

Effect of lipoic acid concentration on uptake Fig. 2 shows that the extent of uptake was proportional to the substrate concentration to about 20 /~g/ml. Above this concentration there was a slight linear I00C

80C "~ soc v

O

f

600C

1°

J

~4ooc

40C o

>

"~200C

g 2oc

.o_ ~o

°o

~,

~,

~

"Cell concentration

(mg/ml)

o IX

Fig. i. Effect of cell concentration on lipoic acid uptake. V a r y i n g a m o u n t s of cells, given in d r y weight, were incubated with io # g of radioactive lipoic acid per ml at 20 ° for 3 ° min; t h e n the reaction was s t o p p e d b y freezing the cells in liquid nitrogen, and t h e y were washed twice and plated.

°o

/

o/° ~'o

2'o

3'o

Lipo~c ocid concn.(ptg/ml)

,~o

5o

Fig. 2. The effect of lipoic acid concentration on uptake. Aliquots of a cell suspension containing i m g (dry weight) of cells per ml were incubated for 3 ° m i n at 2o ° w i t h the indicated a m o u n t s of lipoic acid. The reaction was s t o p p e d b y freezing the cells in liquid nitrogen.

increase in the amount of lipoic acid taken up which was probably entrance by diffusion with high lipoic acid concentrations. In subsequent experiments 20 /~g of lipoic acid per ml was used which is sufficient to saturate the uptake system when 300 Fg of cells per ml was present. Note that this experiment was done with a cell concentration of I mg/ml. Calculations based on a density of I for the bacterial cells reveal that with lower concentrations of lipoic acid a ioo-fold concentration of lipoic acid in the interior of the cell occurred. The apparent K m for lipoic acid was 9.2. lO -6 M.

Effect of temperature on uptake The results shown in Fig. 3 demonstrate that the amount of lipoic acid uptake was proportional to the temperature of incubation. There was little uptake at o ° and a slightly increased uptake at IO ° while significant uptake occurred at both 200 and 37 ° . This type of temperature effect would be expected for an active process rather than diffusion, In all cases there was a rapid uptake which reached a maximum at approx. 3o sec followed by a slight decline in the amount of accumulated lipoic acid; this "overshoot" phenomenon might be due to the requirement of time and sufficient intracellular concentrations to activate an exit system. The lipoic acid which has been concentrated was not easily displaced by cold lipoic acid added to the medium.

Kinetics of uptake The rate of uptake of lipoic acid at normal biological temperatures made the measurement of the kinetics of the reaction difficult. Fig. 4 shows results obtained Biochim. Biophys. Acta, 82 (t964) 41 4 9

STUDIES OF LIPOIC ACID UPTAKE BY BACTERIA. I.

45

at 20 ° in which the rate is linear for about IO sec. There is a peak of uptake between 30 and 45 sec which is defined as the "overshoot" phenomenon. Then a slight decrease in the amount of uptake occurs followed by a constant amount remaining in the cells. 250

0

800

.E

600

E 3400 O0

"~ 200 o .o_ n,,-

50

~O

/

- - 0

q

O

'

'O I,

"I

1~

Time (rain)

2'4

3'2

I

%

2

~

i

Iame Cmin)

6

;

,b

Fig. 4. T h e kinetics of lipoic acid u p t a k e at 20 °. Fig. 3. T h e effect of t e m p e r a t u r e on lipoic acid u p t a k e . Aliquots of a cell s u s p e n s i o n c o n t a i n i n g A s a m p l e of cells c o n t a i n i n g 3oo/zg (dry weight) 300/zg (dry weight) per ml were i n c u b a t e d w i t h per ml was i n c u b a t e d for 15 rain a t 2o °. 2o /zg of r a d i o a c t i v e lipoic acid per ml a t t h e t e m p e r a t u r e s i n d i c a t e d a n d s a m p l e s were t a k e n at t h e i n d i c a t e d t i m e intervals. T h e r e a c t i o n w a s s t o p p e d b y freezing t h e cells in liquid nitrogen. 0 - - 0 , o°; O - - O , IO°; A - - A , 2o°; & - - & , 37 °.

Energy requirementfor uptake The demonstration of an energy requirement for uptake measured with lactic acid bacteria is difficult because of endogenous substrates; however, incubation of the cells for extended periods at 37 ° resulted in depletion of the internal sources so that an energy requirement could be demonstrated. Table I I I shows these results in which a 3-h incubation was required to demonstrate that glucose was required for lipoic acid uptake and that the uncoupling agent, 2,4-dinitrophenol, inhibited uptake even in the presence of glucose. TABLE III ENERGY

REQUIREMENTS

FOR

THE

UPTAKE

OF LIPOIC

ACID

Cells in a c o n c e n t r a t i o n of 3oo Hg/ml were i n c u b a t e d in t h e n e u t r a l salts solution for 3 h at 37 °. T h e cell s u s p e n s i o n was divided into 4 aliquots a n d t h e a d d i t i o n s i n d i c a t e d below (glucose, i m g / m l a n d 2,4-dinitrophenol, 5" lO-a M) were m a d e a n d t h e m i x t u r e was i n c u b a t e d for 15 min. R a d i o active ]ipoic acid (IO/~g/ml) was a d d e d a n d t h e u p t a k e a t 3 ° m i n was d e t e r m i n e d . Addition to cell suspension

None Glucose Dinitrophenol Glucose+dinitrophenol

Counts/rain in cells a~ 30 rain

25 23o 45 5o

Biochim. Biophys. Acta, 82 (1964) 4 1 - 4 9

46

D . C . SANDERS, F. R. LEACH

Specificity of the uptake reaction Structural analogues of lipoic acid were used to determine the specificity of the uptake reaction. The most effective biological inhibitor of lipoic acid 24, 8-methylthioctic acid (supplied by E. L. R. STOKSTAD), had little effect on the uptake reaction as shown in Table IV. The C7 and C 9 analogues of lipoic acid (supplied by L. J. REED) were also without effect on lipoic acid uptake. Octanoic acid was the only effective inhibitor found. T A B L E IV SPECIFICITY

OF

THE

UPTAKE REACTION

Cells (300/zg/ml) were i n c u b a t e d in n e u t r a l s a l t s s o l u t i o n for 15 m i n ; glucose (I m g / m l ) w a s a d d e d a n d i n c u b a t i o n c o n t i n u e d 15 m o r e m i n u t e s a t 2o °. T h e n t h e p o t a s s i u m s a l t of t h e i n h i b i t o r w a s a d d e d j u s t p r i o r to t h e a d d i t i o n of l a b e l e d lipoic acid. V a r i a t i o n i n c o n t r o l u p t a k e is due to d i f f e r e n t specific a c t i v i t i e s a n d ages of t h e lipoic a c i d s a mpl e s . Substrate concentration (,ug/ml)

2o

Inhibitor

8-Methylthioctic

Concentration (Ftg/ml)

Counts/rain taken up at 1o rain Control

Inhibited

2o

IOOO

82o

IO

1,2-Dithiolane-3-caproic

io

28o

295

I0

1,2-Dithiolane-3-butyric

io

280

255

io

Octanoic

io

51 o

275

Intracdlular form of the accumulated lipoie acid Paper chromatography and radioautography of the cell pool material obtained by boiling the cells revealed two radioactive substances. These had RF values identical to those of ~- and fl-lipoic acids. Paper chromatography and radioautography of the material obtained b y acid hydrolysis of the whole cells again revealed that ~- and fl-lipoic acids were the only radioactive substances detected. The sulfur from lipoic acid was not incorporated into other compounds in these experiments.

Distribution of radioactivity under different conditions of uptake Any structure in the cell through which lipoic acid was transported should be detectable by pulse labeling or by comparison of the distribution of radioactivity in cells that are actively taking up lipoic acid and those that are saturated with lipoic acid. The results of fractionation studies under three conditions are shown in Table V. Cells were grown under three conditions: (a) lipoic acid deficient, (b) unlabeled lipoic acid sufficient, and (c) labeled lipoic acid sufficient. In case (c) an aliquot of the cells was fractionated after harvesting and washing. In the other cases the cells were allowed to take up labeled lipoic acid for 15 rain. For exchange measurement in separate aliquots, unlabeled lipoic acid was added after 15 rain incubation with labeled lipoic acid and incubated for an additional 15 rain. With the other aliquot of case (c) unlabeled lipoic acid was added and the cell suspension incubated for 15 min, then fractionated. Under conditions of uptake by lipoic acid-deficient cells, it has not been possible to get exchange when unlabeled lipoic acid is added. The first two columns illustrate that. However, when lipoic acid is present during growth of the organisms, an exBiochim.

Biophys.

Acta,

82 (1964) 4 1 - 4 9

STUDIES OF LIPOIC ACID UPTAKE BY BACTERIA. I.

47

TABLE V DISTRIBUTION OF RADIOACTIVITY IN FRACTIONS OF S. faecalis Cells were g r o w n for 12 h in the lipoic acid-deficient m e d i u m containing: (a) no lipoic acid for lipoic acid-deficient cells, (b) 5 ° /zg unlabeled lipoic acid per ml for lipoic acid-sufficient cells, and (c) 50 # g 35S-labeled lipoic acid per ml for [35S~lipoic acid-sufficient cells. Cell samples were harvested a n d w a s h e d b y centrifugation. Aliquots containing 5 m g d r y weight of ceils were fractionated b y the modified PARK AND HANCOCK procedure described in MATERIALSAND METHODS. Lipoio acid-deficient cells

Fraction

Uptake counts[rain

Water

265°

Exchange

Uptake

Exchange

% countslmin

% countslmin %

39

36

270°

[*sS]Lipoic acid-su~cient cells

Lipoio acid-suO~cient cells

counts/rain

7450

85

3290

Uptake

Exchange

% counts~rain % 72

175o

45

counts~rain % lO8O

36

Cold trichloroacetic acid

23 °

3

39 °

5

5°

i

20

i

3°

i

20

i

Alcohol-water

221o

32

2980

4°

52o

6

26o

6

5oo

13

43 °

14

H o t trichloroacetic acid

5° o

7

67o

9

9o

i

i io

2

3o

I

3°

i

Protein

1232

18

768

IO

66o

8

93 °

20

165o

42

142o

48

Total

6822

lO9

75o8

IOO

8770

ioi

461o

IOi

3960

lO2

2980

IOO

change occurs. Either labeled or unlabeled lipoic acid allows this exchange as is shown in the other columns of the Table V. It appears that the transport system functions uni-directionally and that the exchange system is inducible. The greatest difference in distribution of radioactivity appears in the alcoholwater fraction when all conditions are compared. In cells which are actively accumu-

200

\

° 0

o

lOC

/,,

:_~

.£ "u

'

ltO ' Time (rain)

20

'

30

Fig. 5. A comparison of rates of u p t a k e of L35S]lipoic acid b y whole cells and protoplasts. Whole cells ( O - - O ) and protoplasts ( A - - A ) derived from 3oo/~g cells (dry weight) per ml were incubated for 15 min in the salts m e d i u m containing 0.6 M sucrose. Glucose (o.I mg/ml) was added a n d incubation continued for I5 m i n at 37 °. 35S-Labeled lipoic acid was added at a concentration of 20/~g/ml and samples were t a k e n at the indicated time intervals w i t h the reaction being s t o p p e d b y the mushy-ice technique. All washings were done in the presence of o.6 M sucrose to p r e v e n t lysis of the protoplasts. U p t a k e occurs to a lesser e x t e n t in older cells used in this experiment as contrasted to the usual experiment. Biochim. Biophys. Acta, 82 (1964) 41-49

48

D . C . SANDERS, F. R. LEACH

lating lipoic acid for the first time this fraction is significantly labeled both in terms of total counts and percent of the radioactivity in the cells.

Uptake of lipoic acid by protoplasts A comparison of the ability of whole cells and protoplasts to take up lipoic acid is shown in Fig. 5- The protoplasts were prepared from cells 12 h old by lysozyme treatment in the presence of the neutral-salts solution in 0.6 M sucrose and the washings were also made with the sucrose-salts solution. There is little difference in the time course of uptake.

Uptake by other organisms E. coli, S. aureus, and A. aerogenes all were capable of taking up lipoic acid when exposed to IO/~g of labeled lipoic acid per ml at 20 °. It is known that the lipoic acid attached to the c~-keto acid oxidase does not exchange during catalysis of the reaction ; therefore, most of the uptake shown above must be accumulated in tile cell pool. DISCUSSION

The overall characteristics of the lipoic acid uptake system are similar to those obtained when amino acids, peptides, or carbohydrates have been the substrate. Many different models have been postulated for transport phenomena and confusion has arisen in trying to make all systems meet the same parameters. One major variation in the lipoic acid uptake system and those previously examined is the difficulty in obtaining exchange of the labeled intracellular material with unlabeled material present in the external medium. This occurs in spite of the fact that 5o % of the intracellular material is extractable with boiling water. It might be that there is no reversal of the uptake system and no way that exit by the independent system can occur at such a rate as to make an exchange reaction demonstrable. However, the "overshoot" phenomenon is consistent with the presence of an exit system. Specificity studies reported here do not allow distinction between competition for the same entry system or for limiting energy sources. Previous work by REED 1:~ demonstrated that caprylyl adenylate was an effective inhibitor of the conversion of lipoic acid to the protein-bound form. The C 7 and C 9 analogues of lipoic acid were not very effective in inhibiting either the whole-cell or cell-free-extract lipoic aciddependent reactions and, thus, it is not too surprising that they fail to give significant inhibition in the uptake studies 2a. Transport systems are presumed to be located in the cytoplasmic membrane and be composed of lipoprotein. One should, therefore, observe a flow of radioactivity through a lipid-soluble fraction during the uptake process. Evidence obtained by fractionation studies reported in this paper makes this appear to be the case. Extraction of the cells with boiling water is designed to reduce any effect of the apparent distribution of lipoic acid caused by its insolubility in acid. The low values obtained with two sets of conditions suggest that this artifact was eliminated (see Table V). A significant uptake into the lipid-soluble fraction occurred which does not remain associated with this fraction after longer incubations. All these findings suggest that at least a portion of the transport system is located in or must pass through the lipoprotein of the cell membrane. Biochim. Biophys. Acta, 82 (t9~)4) 41 49

STUDIES OF LIPOIC ACID UPTAKE BY BACTERIA. I.

49

ACKNOWLEDGEMENTS

Taken in part from a thesis submitted as partial requirement for Master of Science degree by D. C. SANDERS, Oklahoma State University. This work was sapported in part by grants from the National Vitamin Foundation and the National Science Foundation. Preliminary communications have appeared17, TM. This investigation was supported in part by a Public Health Service research career program award CA-K3-6487 from the National Cancer Institute. REFERENCES 1G.N. 2G.N. 3R.J. 4G.N. 5F.R. 6E.L. 7H.C. sH.C.

COHEN AND J. MONOD, Bacteriol. Rev., 21 (1957) 169. COHEN AND H. V. RICKENB~RG, Compt. Rend., 240 (1955) 466. BRITTEN, R. B. ROBERTS AND E. F. FRENCH, Proc. Natl. Acad. Sci. U.S., 41 (1955) 863. COHEN AND H. V. RICKENBERG, Ann. Inst. Pasteur, 91 (1956) 693. LEACH AND E. E. SNELL, J. Biol. Chem., 235 (196o) 3523 . OGINSKY, Arch. Biochem. Biophys., 36 (1952) 71. LICHSTEIN AND R. B. FERGUSON, J. Biol. Chem., 233 (1958) 243. LICHSTEIN AND J. R. WALLER,J. Bacteriol., 81 (1961) 65. 9 R . C. WOOD AND G. H . HITCHINGS, J . Biol. Chem., 234 (1959) 2381. 10 L. J. REED, F. R. LEACH AND M. KOIKE, J. Biol. Chem., 232 (1958) 123. 11 L. J. REED, M. KOIKE, M. E. LEVlTCH AND F. R. LEACH, J. Biol. Chem., 232 (1958) 143. 12 H. NAWA, W. T. BRADY, M. KOlK]S AND L. J. REED, J. Am. Chem. Soc., 81 (I959) 29o8. 13 H. NAWA, W. T. BRADY, M. KOIK~ AND L. J. REED, J. Am. Chem. Soc., 82 (196o) 896. 14 I. C. GUNSALUS,in W. D. MCELROY AND B. GLASS, The Mechanism of Enzyme Action, J o h n s H o p k i n s Press, Baltimore, 1954, p. 545. 15 M. KOIKE AND L. J. REED, J. Biol. Chem., 235 (196o) 1931. is I. C. GUNSALUS, Jr. Cellular Comp. Physiol., 41 (1953) suppl. I, 113. 17 D. C. SANDERS AND F. R. LEACH, Biochim. Biophys. Acta, 62 (1962) 604. 18 D. C. SANDERS AND F. R. LEACH, Federation Proc., 21 (1962) 47f. 19 L. J. REED AND C-I. NIU, J. Am. Chem. Soc., 77 (1955) 416. 2o D. S. ACKER AND W. J. WAYNE, J. Am. Chem. Soc., 79 (1957) 6483. 21 R. C. THOMAS AND L. J. REED, J. Am. Chem. Soc., 77 (1955) 5446. 22 I. C. GUNSALUS, M. I. DOLIN AND L. STRUGLIA, J. Biol. Chem., 194 (1952) 849. 2a j . T. PARK AND R. HANCOCK, J. Gen. Microbiol., 22 (196o) 249. 24 E. L. R. STOKSTAD, Federation Proc., 13 (1951) 712. 25 R. C. THOMAS AND L. J. REED, J. Am. Chem. Soc., 78 (1956) 6151.

Biochim. Biophys. Acta, 82 (1964) 41-49